Simultaneous Qualitative and Quantitative Study of Main Compounds in Commelina communis Linn. by UHPLC–Q-TOF-MS-MS and HPLC–ESI-MS-MS

Simultaneous Qualitative and Quantitative Study of Main Compounds in Commelina communis Linn. by... Abstract This study reported the identification and determination of the main components of Commelina communis Linn. A total of 62 compounds were identified in C. communis Linn. extract, which included 29 flavonoids and flavonoid glycosides, 17 phenolic acids, 4 alkaloids, 1 pyrimidine alkaloids, 3 sterols and 8 fatty acids and others by ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry. Moreover, a specific, simple, rapid and sensitive high performance liquid chromatography with tandem mass spectrometry method was developed and validated for determination of 13 components of C. communis Linn., which included orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin. All calibration curves showed good linearity (r ≥ 0.9991) within the test range. The intra- and inter-day precisions (relative standard deviation%, RSD%) were within 1.04 and 0.92%, and the recoveries ranged from 98.64 to 100.8%. These results may contribute to the further study and quality control for C. communis Linn. Introduction Commelina communis Linn., a species of the Commelina genus, is widely distributed in the world, especially in the tropics and subtropics, which include farmland, forest edge, street, valley, hillside grass, hillside wetlands and so on (1). It has long been used as a febrifuge or a diuretic in Chinese folk medicine (2). Commelina communis Linn. is commonly applied to common cold, high fever, sore throat, edema oliguria, hot shower astringent pain, bloated boils poison (3–6), and it was recorded by Chinese pharmacopoeia 2015 (CP 2015) (7). Moreover, it has potential developing value because of habitat complexity and extensive resources (8, 9). Commelina communis Linn. is not only edible value but also has medicinal value, which can extensively served as potherb for soup or fry, and also be made into dried vegetable to prevent from getting cold, or has the effect on treating hypertensive as tea (10–13). Commelina communis Linn. mainly contains flavonoids and flavonoid glycosides, phenolic acid, alkaloids, pyrimidine alkaloids, sterols, polysaccharides, fatty acids and others (14–17), which owes pharmacological effects of antibacterial (18, 19), anti-inflammatory (17), anti-oxidation (20), analgesic (3), hypoglycemic (21) and so on. This study made research on chemical compositions of C. communis Linn. using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS) for the first time. Assay of C. communis Linn. was not recorded in CP 2015. Moreover, HPLC was mainly used to control the quality of C. communis Linn. in previous studies (9), which is more tedious. Therefore, a sensitive, rapid and simple method (HPLC–ESI-MS-MS) was established for determination main components in C. communis Linn., which also attributed to quality control for C. communis Linn. Experimental Chemicals and reagents Orientin (15102309) and vitexin (15120611) with 98% purity were purchased by Shanghai Shifeng Biological Technology Co., Ltd. Iso-orientin and isovitexin (MUST-16042001) with 98.88% purity were purchased by Chengdu Institute of Biology, Chinese Academy of Sciences. Rutin (100080–201408) with 92.8% purity, ferulic acid (110773–201313) with 99.6% purity, quercetin (100081–201408), isorhamnetin (110860–201410), kaempferol (110861–201310), chlorogenic acid (110753–201415), vanillic acid (110776–200402) and caffeic acid (110885–200102) with 98% purity were purchased by National Institutes for Food and Drug Control. Apigenin, protocatechuate (20151021), luteolin, luteolin-7-O-β-d-glucoside (20151204), р-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde (20151228) with 98% purity were purchased by Solarbio. Acacetin (100929) and diosmetin (101216) were purchased by Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. Isoquercitrin and hyperin were made by laboyatry. Methanol and acetonitrile (HPLC grade) were purchased from J.T.-BAKER Company (USA), and formic acid (HPLC grade) was purchased from DIKMA Company (USA). Ethanol (Analytical grade) was provided by Tianjin Kemiou Chemical Reagent Co., Ltd. (China). Purified water was obtained from Wahaha (Hangzhou Wahaha Group Co., Ltd.). The crude wild C. communis Linn. was collected from Qinhuangdao and identified by Mingde Bu, Professor of Hebei Medical Qigong Hospital. The sources of C. communis Linn. included SD-1 (End of August Beidaihe, Insolation), SD-2 (August Beidaihe, Insolation), SD-3 (End of August Beidaihe, Dried in shade) and SD-4 (End of August Shanhaiguan, Dried in shade). UHPLC–Q-TOF-MS-MS instruments for qualitative analysis UHPLC experiments were performed on a Shimadzu UHPLC system (Kyoto, Japan), which was coupled with a triple TOFTM 5600+ MS/MS system (AB SCIEX, CA, USA). The chromatographic separation was carried on Poroshell 120 SB-C18 (2.1 mm × 100 mm, 2.7 μm). The column temperature was maintained at 25°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). The gradient elution program was optimized for the separation, and the program was as follows: 10–10% B from 0 to 1 min, 10–13% B from 1 to 3 min, 13–17% B from 3 to 13 min, 17–95% B from 13 to 21 min, 95–95% B from 21 to 26 min. After holding the composition of 95% solvent B for 5 min, the column returned to its starting conditions with 1 min and maintained the composition of 10% solvent B for 5 min for column balance. The mobile phase flow rate was set to 0.3 mL min−1, and the injection volume was 1 μL. A Triple TOFTM 5600+ system with Duo-SprayTM ion sources operating in the positive and negative electrospray ionization (ESI) mode was used for the detection. The following MS/MS conditions were used: ion spray voltage, +5.5 and −4.5 kV; the turbo spray temperature, 550°C and declustering potential (DP), 60 and −60 V. Nitrogen was used as the nebulizer and the auxiliary gas, and the nebulizer gas (gas 1), the heater gas (gas 2) and the curtain gas were set to 55, 55 and 35 psi, respectively. The IDA (information dependent acquisition) criteria were provided to the ions that matched the mass defect window to obtain the MS/MS spectra. The experiments were run with the scan of 100–1,000 and 50–1,000 amu for the full MS and MS/MS experiments, respectively. And the MS/MS experiments were run with 200 and 70 ms of accumulation time for the full MS and MS/MS experiments, respectively. The collision energy (CE) was set to 35 and −35 eV, and the collision energy spread (CES) was 15 eV. HPLC–ESI-MS-MS instruments for determination analysis Liquid chromatography system (Agilent 1200, USA) equipped with a quaternary solvent delivery system, an autosampler, an automatic degasser and a column compartment was used for determination analysis. Mass spectrometric detection was composed of a 3200 QTRAPTM system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion equipped with a Turbo V source and Turbo Ionspray interface. Instrument control and data acquisition were carried out Analyst software (version 1.6.2) from Applied Biosystems/MDS Sciex. Chromatographic separation was performed on a Column Wonda Cract ODS-2C18 (150 mm × 4.6 mm, 5 μm); mobile phase A (0.1% formic acid: 2 mmol L−1 ammonium formate)-B (methanol), gradient elution program: 0–2 min (28–30% B), 2–5 min (30–33% B), 5–17 min (33–47% B), 17–19 min (47–85% B), 19–25 min (85–95% B), and then returning to the original 28% B. Gradient elution was carried out after pre-equilibration for 6 min, and flow rate was set to 0.8 mL min−1. Injection volume was 10 μL. The typical extract ions chromatograms (XIC) of MRM chromatograms of standards and sample obtained were shown in Figure 1. Figure 1. View largeDownload slide View largeDownload slide Representative extraction chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin standards (A), monitored MRM transitions of the standards (B), and Commelina communis Linn. samples (C). Figure 1. View largeDownload slide View largeDownload slide Representative extraction chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin standards (A), monitored MRM transitions of the standards (B), and Commelina communis Linn. samples (C). The operating conditions for the ESI interface were as follows: the ion spray voltage was set to −4.5 kV; the turbo spray temperature was 650°C; nebuliser gas (gas 1) and heater gas (gas 2) were set to 60 and 65 arbitrary units, respectively; the curtain gas was kept at arbitrary units and interface heater was on. Nitrogen was used in all cases. The retention time, characteristic MS/MS fragment ions data, DP and CE for each analyte were listed in Table I. Instrument control, data acquisition and evaluation were performed with Analyst 1.6.2 software (AB SCIEX, Ontario, Canada). Table I. HPLC–ESI-MS-MS Data of 13 Components From Commelina communis Linn. Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Table I. HPLC–ESI-MS-MS Data of 13 Components From Commelina communis Linn. Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Sample and standard solution preparations The dry C. communis Linn. powder (60 mesh, 1 g) was accurately weighed and ultrasonicated for 40 min with 25 mL 70% ethanol, continuous ultrasound twice (8, 9, 22). The extracted solution was adjusted to the original weight by 70% ethanol. And the filtrate was combined and then concentrated to dryness in vacuo at 50°C. The residue was dissolved with methanol into a 5 mL volumetric flask for qualitative and quantitative analysis. The resultant was filtered through a 0.22 μm millipore filter. The injection volume was 10 μL for the analysis of HPLC–ESI-MS-MS, but UHPLC–Q-TOF-MS-MS was 1 μL. For qualitative analysis of the UHPLC–Q-TOF-MS-MS, all standards were respectively weighted appropriate amount, which were dissolved with 80% methanol to prepare mixture solution. For quantitative analysis, each standard (orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin) weighted accurately were dissolved in 80% methanol to prepare stock solutions. The concentrations of the stock solution of each standard were 0.91, 1.05, 0.985, 1.09, 0.96, 0.023, 0.95, 0.91, 0.214, 1.015, 0.107, 0.137 and 0.0234 mg mL−1, respectively, which was diluted to appropriate concentrations with methanol–water (80:20, v/v) for constructing calibration curves. All solutions were stored at 4°C for UHPLC–Q-TOF-MS-MS and HPLC–ESI-MS-MS analysis. Results and Discussion Analytical methods In this study, the analysis of chemical compositions in C. communis Linn. was based on a Triple TOF instrument with on-line data acquisition. The detail analytic strategy was as follows (23, 24): (i) On-line data were acquired by full-scan, and accurate MS/MS data were obtained. (ii) Chemical component database was established according to the investigation of chemical constituents of C. communis Linn. (3–6, 14–16, 18, 19, 25). (iii) The compounds of small error were filtrated. (iv) Compounds were estimated on the basis of the characteristic fragment ions and retention time. Data acquisition and procession were performed with PeakView 1.2 software. The determination method of 13 components for C. communis Linn. was mainly adjusting the gradient of the mobile phase to separate isomers. Then method validation and determination were getting on. A series of analysis such as the linearity, accuracy, matrix effect, stability, precision, limits of detection (LOD) and quantification (LOQ) were conducted to validate the feasibility of method. A total of 62 compounds were detected in C. communis Linn. extract, which included 29 flavonoids and flavonoid glycosides, 17 phenolic acids, 4 alkaloids, 1 pyrimidine alkaloids, 3 sterols and 8 fatty acids and others. Among of them, 38 compounds were detected in the positive ESI (ESI+) mode, and 24 compounds were found in the negative ESI (ESI−) mode. What is more, 22 compounds were identified by comparing with related reference substances. This study also made research on simultaneous determination of 13 constituents in C. communis Linn. by HPLC–ESI-MS-MS. It is extremely troublesome to distinguish and separate isomers in the process of identification because of identical molecular formulae, small difference in retention time, MS/MS spectra and chemical polarity. Thus, an important parameter Clog P, which was calculated using the program Chemdraw Ultra 12.0, was introduced to distinguish isomers. The compound with the smaller Clog P value commonly has shorter retention time in a reversed phase liquid chromatography system (23, 24). The structures of 62 compounds were showed in Figure S1. The detected compounds were listed in Table II. UHPLC–Q-TOF-MS positive (A) and negative (B) total ion chromatogram of C. communis Linn. were shown in Figure 2. The MS/MS of detected compounds and reference substances were shown in Figures S2 and S3, respectively. HPLC–MS-MS total ion chromatogram of C. communis Linn. (A) and mixture reference substance (B) were shown in Figure S4. Table II. Analysis on Chemical Constituents of Commelina communis Linn. Extract Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Table II. Analysis on Chemical Constituents of Commelina communis Linn. Extract Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of positive (A) and negative (B) of Commelina communis Linn. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of positive (A) and negative (B) of Commelina communis Linn. Identification of 62 Compounds by UHPLC–Q-TOF-MS-MS The positive ESI+ mode Flavonoids and flavonoid glycosides Compounds 1–5 turned up at 7.80, 8.25, 12.20, 14.75 and 15.09 min, respectively. The protonated molecular ions [M + H]+ were at m/z 449.1084, 449.1082, 449.1077, 449.1073 and 449.1079, and they had same fragment ion at m/z 287.0553 ([M-C6H10O5+H]+). Compounds 1, 2 and 3 were identified as iso-orientin, orientin and luteolin-7-O-β-d-glucoside through comparison with relevant reference substances (15–17, 25). However, compounds 4 and 5 were concluded as luteolin-5-O-β-d-glucoside and luteolin-4′-O-β-d-glucoside on the basis of the MS/MS data at m/z 287.0565, which were firstly detected in C. communis Linn. Compounds 6–8 had identifical protonated molecule ions [M + H]+ at m/z 463.1244, 463.1247 and 463.1236, which were detected at 8.36, 12.32 and 15.49 min. They were judged as chrysoeriol-7-O-β-d-glucoside (3), flavocommelitin (25) and luteolin-7-methylether-4′-O-glucoside in accordance with the secondary fragment ions at m/z 301.0122 ([M-C6H10O5+H]+) and 287.0605 ([M-C6H10O5-CH2+H]+). Among of them, luteolin-7-methylether-4′-O-glucoside was firstly isolated from C. communis Linn. Compounds 9 and 10 were eluted at 10.34 and 10.92 min, showing the protonated molecule ions [M + H]+ at m/z 433.1132 and 433.1131. And their fragment ions were same as that of reference substances. So they were confirmed to be vetexin (15, 16, 25) and iso-vetexin (15, 16), and the characteristic fragment ions all were detected at m/z 397.0927, 379.0795, 351.0884, 313.0708 and 283.0605. Compounds 11 and 12 eluted at 11.15 and 11.94 min were observed in the XIC at m/z 465.1029 and 465.1032 ([M + H]+). They had parallel characteristic fragment ion at m/z 303.0507 ([M-C6H10O5+H]+), which was consistent with MS/MS data of reference substance. Therefore, they were ensured to be hyperin and isoquercitrin (3). Among of them, hyperin was firstly gained in C. communis Linn. Compound 13 was eluted at 11.15 min with the prontonated molecule ion of m/z 611.1610 ([M + H]+). The characteristic product ions at m/z 465.1020 ([M-C6H10O4+H]+) and 303.0517 ([M-C12H20O9+H]+) appeared in the MS/MS spectrum, which were the same as the fragment ions of reference substance. As a consequence, it was ascertain as rutin (25). The protonated molecule of m/z 447.1288 ([M + H]+, compound 14), eluted at 11.90 min, showed a series of distinctive fragment ions at m/z 285.1330 and 267.0654 by loss of C6H10O5 and H2O. According to these characteristic fragments, it was identified to be swertisin (17). The protonated molecule ion of m/z 343.1182 (compound 15) was eluted at 13.59 min. It showed a series of distinctive fragment ions at m/z 301.1374 and 283.1034 by loss of CO, CH2 and 2CH2O. Then it was deduced as 3,3′,4′,7-tetramethoxyflavone (14). Compounds 16 and 17 with the protonated molecule ions at m/z 479.1191 and 479.1189 ([M + H])+ were detected 15.09 and 15.30 min. The MS/MS were found at m/z 317.0665([M-C6H10O5+H]+) and 302.0454 ([M-C6H10O5-CH3+H]+) in the XIC of compounds 16 and 17. According to the characteristic fragments, compounds 16 and 17 were deduced as isorhamnetin-3-O-β-d-glucoside (3) and isorhamnetin-7-O-β-d-glucoside. The compound 17 was deduced on the basis of the MS/MS information. Compounds 18–20 were detected at m/z 303.0501, 301.0710 and 317.0656 ([M + H]+), eluted at 16.56, 17.25 and 17.35 min, respectively. They were determined as quercetin, diosmetin and isorhamnetin (3) according to the fact that their MS/MS fragment ions were well matched with the corresponding standard substances. What is more, quercetin and disometin were found under the experiment conditions for the first time. Phenolic acids Compounds 21, 29 appeared at 1.12 and 22.18 min with protonated molecule ions ([M + H]+) at m/z 185.0439 and 179.0703. The distinctive fragments of compound 21 were observed at m/z 170.1081 and 142.0798 by loss of CH3 and CO. So it was inferred as methyl gallate (17). The MS/MS of compound 29 were gained at m/z 161.0590, 133.0659, 118.0412 and 90.0475 through loss of H2O, CH2O, O, CO, CH3 and CH = CH. Therefore, compound 29 was concluded as methyl 4-hydroxycinnamate (15, 16, 19). Compounds 22, 26–28 were showed at 3.57, 4.30, 5.06 and 8.79 min. The protonated molecule ions ([M + H]+) were showed at m/z 355.1028, 169.0493, 123.0442 and 195.0653, respectively. Their fragment ions were same as that of reference substances. So they were ascertained as chlorogenic acid, vanillic acid (25), р-hydroxybenzaldehyde and ferulic acid. Moreover, compounds 22, 27 and 28 were recognized for the first time. The protonated molecule ions ([M + H]+) of compounds 23–25 (14, 25) were detected at m/z 199.0602, 199.0599 and 199.0602, eluted at 1.75, 3.09 and 3.83 min. The MS/MS of compound 23 were found at m/z 181.0489, 155.0733, 140.0468, 125.0236, 111.0330, 95.0506 and 77.0404. The secondary fragment ions of compound 24 were detected at m/z 184.0332, 153.0615, 141.0724 and 109.0667. The typical fragment ions of compound 25 were observed at m/z 153.0556, 139.0393, 125.0594, 111.0445, 95.0499 and 79.0551, so it was deduced as syringic acid. The structures of compounds 23 and 24 were shown in the Figure S1. The fragmentation pathways of compounds 23, 24 and 25 were same, getting the fragment ions by successful loss of H2O, CO2, CH3, CH2, CH2O and O. Alkaloids The alkaloidal compounds were detected in ESI+ mode. The compounds 30–33 with protonated molecule ions ([M + H]+) at m/z 356.1566, 194.1023, 164.0920 and 169.0758 were inferred as 7-O-β-d-glucopyranosyl-α-homonojirmycin (6, 15, 16), α-homonojirmycin (6, 15, 16), 1-deoxynojirimycin (6, 15, 16) and norharman (15, 16), respectively, and they were showed at 0.73, 0.76, 0.78 and 5.61 min. The MS/MS of compound 30 were received at m/z 194.1032, 176.0924, 158.0817 and 140.0817, which lost C6H10O5, H2O, H2O and H2O. The secondary fragments of compound 31 were gained at m/z 176.0909 ([M-H2O+H]+), 158.0823 ([M-2H2O+H]+) and 140.0720 ([M-3H2O+H]+). The characteristic fragment ions of compound 32 were observed at m/z 146.0814, 128.0709, 110.0607 and 82.0664 by loss of H2O, H2O, H2O and CO. The typical fragment ions of compound 33 were acquired at m/z 142.0649 ([M-CHN + H]+) and 115.0546 ([M-2CHN+H]+). Pyrimidine alkaloids Compound 34 was detected with precursor ion at m/z 268.1043 ([M + H]+), and was eluted at 1.05 min. A series of distinctive fragment ions (26) were received at m/z 152.0566 ([M-C5H8O3+H]+), 136.0623 ([M-C5H8O4+H]+) and 119.0353 ([M-C5H8O4-NH2+H]+). The compound 34 was named as adenosine (15, 16). Sterols Sterols compounds were detected in the positive ESI mode, which included stigmast-5-ene-3β, 7α-diol (compound 35) (15, 16) and fridelin (compound 36) (15, 16). Their protonated molecule ions ([M + H]+) were detected at m/z 431.3887 and 427.3931, and they were eluted at 21.99 and 23.13 min, respectively. The MS/MS of compound 35 were showed at m/z 317.2086, 233.1533 and 215.1428 by loss of C8H18, C5H6, H2O and H2O, and the fragment ions of compound 36 were obtained at m/z 409.1083 ([M-H2O+H]+) and 331.0528 ([M-H2O-C6H5+H]+). Fatty acid and others Compound 37 was detected at 14.90 min with precursor ions at m/z 316.1550 ([M + H]+). The major fragment ions were showed at m/z 138.0917 ([M-C10H10O3+H]+) and 121.0658 ([M-C10H10O3-OH + H]+). And it was inferred as commelinins A (15, 16). Compound 38 was judged as (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide) (17) in light of the distinctive fragment ions at m/z 164.0632, 147.0447, 119.0497 and 103.0552 by missing C8H9N, OH, CO and O. The precursor ion was at m/z 284.1291 ([M + H]+), and it was eluted at 16.00 min. The negative ESI mode Flavonoids and flavonoid glycosides Compounds 39 and 40 eluted at 4.37 and 14.96 min, respectively, showed deprotonated molecule ions [M−H]– at m/z 623.1647 and 623.1647. The typical fragments of compound 39 appeared at m/z 479.1477 and 315.0516 by loss of C6H8O4 and C12H20O9. Moreover, the characteristic fragment ions of compound 40 were gained at m/z 461.1120 and 299.0539 by loss of C6H10O5 and C12H20O10. Therefore, they were inferred to be isorhamnetin-3-O-rutinoside (3) and flavocommelin (15, 16). Compound 41 with deprotonated molecular ion [M−H]− at m/z 607.1694 was detected at 4.44 min. The characteristic fragment ions were acquired at m/z 445.1138 and 283.1032 by loss of C6H10O5 and C12H20O10. The compound 41 was deduced as flavocommelinin A (15, 16) based on the secondary fragments. Compound 42 was eluted at 16.14 min with a deprotonated molecule ion of m/z 591.1729 ([M−H]−). A series of product ions of compound 42 were detected at m/z 445.2241 ([M-C6H10O4-H]−) and 283.0117 ([M-C12H20O9-H]−), so it was deduced as 2′′-O-α-l-rhamnosylswertisin (16, 17). Compound 43 gave a deprotonated molecule [M−H]– at m/z 415.1039, which was observed at a retention time of 16.16 min. It was judged as isofurcatain (3) in light of product ion at m/z 269.0446 gained through loss of C6H10O4. Compounds 44–47 with deprotonated molecule ions ([M−H]−) at m/z 285.0407, 269.0461, 285.0406 and 283.0622 had the retention time of 16.49, 17.10, 17.20 and 18.47 min, respectively. The chromatogram retention time and MS/MS fragment ions kept consistent with that of reference substances. Their characteristic fragments were showed in the Table II. Hence, it were confirmed that they were luteolin (14, 15), apigenin (14, 15), kaempferol and acacetin according the secondary ions and retention times. Compounds 46 and 47 were observed for the first time. Phenolic acids Compounds 48, 51, 53–55 were showed at 1.08, 3.42, 4.91, 6.98 and 13.93 min. The precursor ions ([M−H]−) were showed at m/z 139.0041, 137.0249, 121.0299, 163.0407 and 187.0981. Secondary fragments of compound 48 were detected at m/z 94.0312 ([M-COOH-H]−) and 67.0218 ([M-COO-CO-H]−). Characteristic fragment ions of compound 51 were acquired at m/z 93.0363 ([M-CO2-H]−) and 75.0098 ([M-CO2-H2O-H]−). MS/MS data of compound 53 were observed at m/z 93.0347 and 75.0138 missing CO and H2O. Typical product ions of compound 54 were received at m/z 119.0513, 103.0109 and 93.0356 losing CO2, O and CH = CH. Distinctive fragment ions of compound 55 were showed at m/z 142.0039 ([M-COOH-H]−), 125.0972 ([M-COOH-OH-H]−) and 97.0673 ([M-2COOH]−). According to fragmentation information, compounds 48, 51, 53–55 were deduced as cumaric acid (14), р-hydroxybenzoic acid (3), benzoic acid (25), р-hydroxycinnamic acid (3) and azelaic acid (25), respectively. Compounds 49, 50 and 52 turned up at 2.07, 3.14 and 4.42 min with the precursor ions ([M−H]−) at m/z 153.0199, 137.0250 and 179.0357. The product ions of compound 49, 50 and 52 were well matched with the related reference substances. Hence, the compound 49, 50 and 52 were confirmed as protocatechuate (25), 3,4-dihydroxybenzaldehyde and caffeic acid (3). Sterols Compound 56 was found at 24.66 min with a deprotonated molecule ion ([M−H]−) at m/z 411.3628. The characteristic fragment ions were detected at m/z 393.3393 ([M-H2O-H]−) and 349.3472 ([M-H2O-C3H8-H]−). According to the fragmentation information, the compound 56 was deduced as stigmasterol (14). Fatty acid and others The deprotonated molecule ions ([M−H]−) of compounds 57–59 were showed at m/z 181.0720, 160.0411 and 419.1343 from the XIC, which were eluted at 0.79, 9.99 and 13.21 min. The compound 57 (27) had the MS/MS at m/z 163.0560 ([M-H2O-H]−) and 101.0241 ([M-H2O-C2H6O2-H]−). Compound 58 owed the typical fragment ion at m/z 116.0509 ([M-CO2-H]−). Compound 59 possessed the characteristic fragment ions at m/z 373.0822 and 341.1208, which were received by loss of CH2O, O, O and O. On the basis of the fragment informant, compound 57–59 were inferred as d-mannose (4, 15, 16), 3-carboxy-indole (17) and rhaponticin (17). Compounds 60–62 with the deprontonated molecule ions at m/z 739.2176 ([M+HCOOH-H]−), 283.1181 ([M−H]−) and 683.2644 ([M−H]−) were eluted at 14.02, 15.92 and 16.66 min. Compound 60 showed the MS/MS at m/z 693.2124 and 387.0905 by loss of HCOOH and C12H18O9. Compound 61 had the secondary fragments ion at m/z 121.1021 ([M-C6H10O5-H]−). Compound 62 owed the characteristic fragment ions at m/z 520.2025, 504.1712 and 352.1187 by loss of C8H9N, CH2, CH2O, O, C8H9N and CH3O. Therefore, they were inferred as 4,4′-dihydroxy-3,3′-dimethoxy-β-truxinic acid (17), 2-phenylethyl-β-d-glucoside (17) and commelinin B (15, 16). Quantitative study for C. communis Linn. samples by HPLC–MS-MS Linearity, limit of detection and limit of quantification Stock solution containing 13 reference compounds were diluted to appropriate concentrations with methanol–water (80:20, v:v) for establishment of calibration curves. A total of seven concentrations of the solution were analyzed, and then the calibration curves were constructed by plotting the peak area (Y) versus the concentration (X, ng mL−1) for each analyte. Limit of detection (LOD) and limit of quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of ~3 and 10, respectively. The results were list in Table III. Table III. Regression, LODs and LOQs for the Investigated Compounds Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  aY: peak area; X: concentration of compound (ng mL−1). bLOD, limit of detection. cLOQ, limit of quantification. Table III. Regression, LODs and LOQs for the Investigated Compounds Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  aY: peak area; X: concentration of compound (ng mL−1). bLOD, limit of detection. cLOQ, limit of quantification. Precision The precision of method was validated by analyzing C. communis Linn. sample, which included intra- and inter-day precision. The intra-day precision was performed by repeating six times within 1 day, while the inter-day precision was consecutively performed on three days. The determination of 13 ingredients investigated was performed from corresponding calibration curve. Relative standard deviation (RSD) values of peak area for each of compound were calculated respectively. The results showed that the RSD values of intra- and inter-day precision were <1.04 and 0.92%, respectively. Accuracy Accuracy of the method was received by spiking the mixed standard solutions with three different concentration levels (low, medium and high) to prepare known amount of C. communis Linn. samples, which were dealt with through the above proposed method. Moreover, triplicate samples were conducted for each level. The result showed that the average recoveries of 13 components measured were in range of 98.64–100.8% and RSD values were in range of 0.11–0.78%. Stability The stability of the sample was analyzed at 0, 2, 4, 8, 12 and 24 h at room temperature, respectively. The results showed that RSD values of all measured peak area were <0.59%, indicating a good stability. Matrix effects The evaluation of matrix effects is a vital issue for the analysis of botanical extracts by HPLC–ESI-MS-MS. Co-eluting compounds originating from matrix can lead to signal enhancement or suppression (28). The matrix effects of 13 components were quantitatively investigated, which was performed by adding known amounts of the mixed standard solution at three levels (low, medium and high) to half of the extraction samples and recording the analyte peak areas of the spiked sample matrix (A), the other half of the extraction samples (B) and the standard solutions (C) (28, 29). Moreover, triplicate samples were prepared at each level. Matrix effects were calculated using the following equation: matrix effect (%) = (A–B)/C × 100%. The matrix effects of 13 components ranged from 93.4 to 103.1% and the values of RSD were in range of 0.34–1.31%, which indicated that the matrix effects had no influence. Repeatability The repeatability experiment was performed by preparing for six independent sample solutions of C. communis Linn. in parallel. The RSD values of 13 compounds were not more than 0.97%, which showed a good reproducibility. The results of precision, repeatability, accuracy, stability and matrix effect were showed in Table S1 and Table IV, respectively. Table IV. Matrix Effects of 13 Components in Commelina communis Linn. Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Table IV. Matrix Effects of 13 Components in Commelina communis Linn. Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Sample determination Commelina communis Linn. samples (SD-1, SD-2, SD-3 and SD-4) in Qinhuangdao (China) were determined under the above conditions. Representative chromatograms were shown in Figure 1. Peak identify was established by comparing retention time of sample with that of reference compounds. The calibration curves were applied for the quantitative analysis of 13 compounds. The results of 13 analytes in C. communis Linn. samples were summarized in Table V. The results showed that the content of orientin, iso-orientin, vitexin, isovitexin, rutin in SD-3 and SD-4 were higher than that of in SD-1 and SD-2, which indicated that the content of 13 analytes were different according to the different dried methods and source. Table V. Contents of 13 Active Components in Commelina communis Linn. Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Table V. Contents of 13 Active Components in Commelina communis Linn. Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Experiments compared with 50% methanol, 70% methanol, 95% methanol, 100% methanol, 50% ethanol, 70% ethanol, 95% ethanol and 100% ethanol as extraction solvent for C. communis Linn. sample, which showed that the extraction efficiency of 70% ethanol was highest. Afterwards comparing ultrasonic extraction and reflux extraction, the extraction efficiency was similar in two ways. Ultrasonic extraction was chose in this study, because ultrasonic was relatively quick and easy. It also examined ultrasonic frequency (once, twice and three times) and ultrasonic time (30, 40, 60 and 90 min). The results showed that continuous ultrasonic twice, 40 min every time, respectively, had highest extraction efficiency. Methanol–water and acetonitrile–water were chose as mobile phase for gradient eluting during the qualitative and quantitative analysis. The results showed that more compounds were observed using acetonitrile as eluent by UHPLC–Q-TOF-MS-MS. However, elutive power of two different mobile phases was similar for the quantitative analysis by HPLC–MS-MS. Because methanol was cheaper and smaller toxicity than acetonitrile, it was applied for determination of sample. Therefore, acetonitrile–water and methanol–water were served as eluent of qualitative and quantitative analysis, respectively. To improve peak shape, different concentrations of formic acid (0.05, 0.1, 0.2 and 0.3%) in water for qualitative and quantitative analysis study were compared, which showed that peak shape of 0.1% formic acid was best. Moreover, different concentrations of ammonium formate (1, 2 and 3 mmol L−1) and ammonium acetate (1, 2 and 3 mmol L−1) were compared. So acetonitrile–water (0.1% formic acid) was used for qualitative study, and methanol–water (0.1% formic acid, 2 mmol L−1 ammonium acetate) was the mobile phase for quantitative analysis. Conclusion In the present study, a powerful analytical strategy dependent on-line data acquisition combining with multiple post acquisition data processing was employed for fast screening and identification the chemical compositions of C. communis Linn. What is more, luteolin-5-O-β-d-glucoside (compound 4), luteolin-4′-O-β-d-glucoside (compound 5), luteolin-7-methylether-4′-O-glucoside (compound 8), hyperin (compound 11), hyperin (compound 11), isorhamnetin-7-O-β-d-glucoside (compound 17), diosmetin (compound 19), chlorogenic acid (compound 22), 4-hydroxy-2,6-dimethoxyl-benzoic acid (compound 23), 3-hydroxy-4,5-dimethoxy-benzoic acid (compound 24), р-hydroxybenzaldehyde (compound 27), ferulic acid (compound 28), methyl 4-hydroxycinnamate (compound 29), kaempferol (compound 46), acacetin (compound 47) and 3,4-dihydroxybenzaldehyde (compound 50) were firstly gained in C. communis Linn. The fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid were shown in Figure 3. And 13 compounds exiting in C. communis Linn were firstly determined by HPLC–MS-MS. Many isomers were found in C. communis Linn., which were distinguish through Clog P values This is the first report of simultaneous qualitative and quantitate study of main chemical components of C. communis Linn. by UHPLC–Q-TOF-MS-MS and HPLC–MS-MS. Furthermore, the application of this approach provided essential data for further studies of C. communis Linn. Figure 3. View largeDownload slide The predominant fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid. Figure 3. View largeDownload slide The predominant fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid. Supplementary Data Supplementary data are available at Journal of Chromatographic Science online. Acknowledgments Thanks for the instrument support of the Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University. Conflict of interest statement The authors have declared no conflict of interest. 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Google Scholar CrossRef Search ADS   29 Shi, X.W., Wu, Y.B., Lv, T., Wang, Y.f., Fu, Y., Sun, M.M., et al.  .; A chemometric-assisted LC-MS/MS method for the simultaneous determination of 17 limonoids from different parts of Xylocarpus granatum fruit; Analytical and Bioanalytical Chemistry , ( 2017); 409: 4669– 4679. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Simultaneous Qualitative and Quantitative Study of Main Compounds in Commelina communis Linn. by UHPLC–Q-TOF-MS-MS and HPLC–ESI-MS-MS

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Abstract

Abstract This study reported the identification and determination of the main components of Commelina communis Linn. A total of 62 compounds were identified in C. communis Linn. extract, which included 29 flavonoids and flavonoid glycosides, 17 phenolic acids, 4 alkaloids, 1 pyrimidine alkaloids, 3 sterols and 8 fatty acids and others by ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry. Moreover, a specific, simple, rapid and sensitive high performance liquid chromatography with tandem mass spectrometry method was developed and validated for determination of 13 components of C. communis Linn., which included orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin. All calibration curves showed good linearity (r ≥ 0.9991) within the test range. The intra- and inter-day precisions (relative standard deviation%, RSD%) were within 1.04 and 0.92%, and the recoveries ranged from 98.64 to 100.8%. These results may contribute to the further study and quality control for C. communis Linn. Introduction Commelina communis Linn., a species of the Commelina genus, is widely distributed in the world, especially in the tropics and subtropics, which include farmland, forest edge, street, valley, hillside grass, hillside wetlands and so on (1). It has long been used as a febrifuge or a diuretic in Chinese folk medicine (2). Commelina communis Linn. is commonly applied to common cold, high fever, sore throat, edema oliguria, hot shower astringent pain, bloated boils poison (3–6), and it was recorded by Chinese pharmacopoeia 2015 (CP 2015) (7). Moreover, it has potential developing value because of habitat complexity and extensive resources (8, 9). Commelina communis Linn. is not only edible value but also has medicinal value, which can extensively served as potherb for soup or fry, and also be made into dried vegetable to prevent from getting cold, or has the effect on treating hypertensive as tea (10–13). Commelina communis Linn. mainly contains flavonoids and flavonoid glycosides, phenolic acid, alkaloids, pyrimidine alkaloids, sterols, polysaccharides, fatty acids and others (14–17), which owes pharmacological effects of antibacterial (18, 19), anti-inflammatory (17), anti-oxidation (20), analgesic (3), hypoglycemic (21) and so on. This study made research on chemical compositions of C. communis Linn. using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS) for the first time. Assay of C. communis Linn. was not recorded in CP 2015. Moreover, HPLC was mainly used to control the quality of C. communis Linn. in previous studies (9), which is more tedious. Therefore, a sensitive, rapid and simple method (HPLC–ESI-MS-MS) was established for determination main components in C. communis Linn., which also attributed to quality control for C. communis Linn. Experimental Chemicals and reagents Orientin (15102309) and vitexin (15120611) with 98% purity were purchased by Shanghai Shifeng Biological Technology Co., Ltd. Iso-orientin and isovitexin (MUST-16042001) with 98.88% purity were purchased by Chengdu Institute of Biology, Chinese Academy of Sciences. Rutin (100080–201408) with 92.8% purity, ferulic acid (110773–201313) with 99.6% purity, quercetin (100081–201408), isorhamnetin (110860–201410), kaempferol (110861–201310), chlorogenic acid (110753–201415), vanillic acid (110776–200402) and caffeic acid (110885–200102) with 98% purity were purchased by National Institutes for Food and Drug Control. Apigenin, protocatechuate (20151021), luteolin, luteolin-7-O-β-d-glucoside (20151204), р-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde (20151228) with 98% purity were purchased by Solarbio. Acacetin (100929) and diosmetin (101216) were purchased by Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. Isoquercitrin and hyperin were made by laboyatry. Methanol and acetonitrile (HPLC grade) were purchased from J.T.-BAKER Company (USA), and formic acid (HPLC grade) was purchased from DIKMA Company (USA). Ethanol (Analytical grade) was provided by Tianjin Kemiou Chemical Reagent Co., Ltd. (China). Purified water was obtained from Wahaha (Hangzhou Wahaha Group Co., Ltd.). The crude wild C. communis Linn. was collected from Qinhuangdao and identified by Mingde Bu, Professor of Hebei Medical Qigong Hospital. The sources of C. communis Linn. included SD-1 (End of August Beidaihe, Insolation), SD-2 (August Beidaihe, Insolation), SD-3 (End of August Beidaihe, Dried in shade) and SD-4 (End of August Shanhaiguan, Dried in shade). UHPLC–Q-TOF-MS-MS instruments for qualitative analysis UHPLC experiments were performed on a Shimadzu UHPLC system (Kyoto, Japan), which was coupled with a triple TOFTM 5600+ MS/MS system (AB SCIEX, CA, USA). The chromatographic separation was carried on Poroshell 120 SB-C18 (2.1 mm × 100 mm, 2.7 μm). The column temperature was maintained at 25°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). The gradient elution program was optimized for the separation, and the program was as follows: 10–10% B from 0 to 1 min, 10–13% B from 1 to 3 min, 13–17% B from 3 to 13 min, 17–95% B from 13 to 21 min, 95–95% B from 21 to 26 min. After holding the composition of 95% solvent B for 5 min, the column returned to its starting conditions with 1 min and maintained the composition of 10% solvent B for 5 min for column balance. The mobile phase flow rate was set to 0.3 mL min−1, and the injection volume was 1 μL. A Triple TOFTM 5600+ system with Duo-SprayTM ion sources operating in the positive and negative electrospray ionization (ESI) mode was used for the detection. The following MS/MS conditions were used: ion spray voltage, +5.5 and −4.5 kV; the turbo spray temperature, 550°C and declustering potential (DP), 60 and −60 V. Nitrogen was used as the nebulizer and the auxiliary gas, and the nebulizer gas (gas 1), the heater gas (gas 2) and the curtain gas were set to 55, 55 and 35 psi, respectively. The IDA (information dependent acquisition) criteria were provided to the ions that matched the mass defect window to obtain the MS/MS spectra. The experiments were run with the scan of 100–1,000 and 50–1,000 amu for the full MS and MS/MS experiments, respectively. And the MS/MS experiments were run with 200 and 70 ms of accumulation time for the full MS and MS/MS experiments, respectively. The collision energy (CE) was set to 35 and −35 eV, and the collision energy spread (CES) was 15 eV. HPLC–ESI-MS-MS instruments for determination analysis Liquid chromatography system (Agilent 1200, USA) equipped with a quaternary solvent delivery system, an autosampler, an automatic degasser and a column compartment was used for determination analysis. Mass spectrometric detection was composed of a 3200 QTRAPTM system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion equipped with a Turbo V source and Turbo Ionspray interface. Instrument control and data acquisition were carried out Analyst software (version 1.6.2) from Applied Biosystems/MDS Sciex. Chromatographic separation was performed on a Column Wonda Cract ODS-2C18 (150 mm × 4.6 mm, 5 μm); mobile phase A (0.1% formic acid: 2 mmol L−1 ammonium formate)-B (methanol), gradient elution program: 0–2 min (28–30% B), 2–5 min (30–33% B), 5–17 min (33–47% B), 17–19 min (47–85% B), 19–25 min (85–95% B), and then returning to the original 28% B. Gradient elution was carried out after pre-equilibration for 6 min, and flow rate was set to 0.8 mL min−1. Injection volume was 10 μL. The typical extract ions chromatograms (XIC) of MRM chromatograms of standards and sample obtained were shown in Figure 1. Figure 1. View largeDownload slide View largeDownload slide Representative extraction chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin standards (A), monitored MRM transitions of the standards (B), and Commelina communis Linn. samples (C). Figure 1. View largeDownload slide View largeDownload slide Representative extraction chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin standards (A), monitored MRM transitions of the standards (B), and Commelina communis Linn. samples (C). The operating conditions for the ESI interface were as follows: the ion spray voltage was set to −4.5 kV; the turbo spray temperature was 650°C; nebuliser gas (gas 1) and heater gas (gas 2) were set to 60 and 65 arbitrary units, respectively; the curtain gas was kept at arbitrary units and interface heater was on. Nitrogen was used in all cases. The retention time, characteristic MS/MS fragment ions data, DP and CE for each analyte were listed in Table I. Instrument control, data acquisition and evaluation were performed with Analyst 1.6.2 software (AB SCIEX, Ontario, Canada). Table I. HPLC–ESI-MS-MS Data of 13 Components From Commelina communis Linn. Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Table I. HPLC–ESI-MS-MS Data of 13 Components From Commelina communis Linn. Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Component  Relative molecular mass  tR (min)  Ion source model  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  Orientin  448.4  15.44  ESI−  447.3  327.2  −64  −31  357.0  −30  299.1  −48  Iso-orientin  448.4  16.15  ESI−  446.9  327.0  −58  −34  357.1  −30  297.3  −46  Vitexin  432.4  17.40  ESI−  431.0  311.1  −74  −32  283.0  −46  Isovitexin  432.4  19.59  ESI−  431.3  311.0  −67  −31  283.1  −31  341.2  −47  Rutin  610.5  21.12  ESI−  609.3  300.1  −87  −48  Apigenin  270.2  23.22  ESI−  269.0  116.9  −64  −51  Protocatechuate  154.1  6.13  ESI−  153.0  108.9  −34  −22  Vanillic acid  168.2  10.94  ESI−  167.0  151.9  −27  −19  107.9  −25  Caffeic acid  180.2  11.04  ESI−  179.0  135.0  −47  −23  Ferulic acid  194.2  16.83  ESI−  193.0  134.0  −32  −24  Luteolin  286.2  22.69  ESI−  285.0  132.9  −51  −47  Quercetin  302.2  22.42  ESI−  301.0  150.9  −75  −31  Isorhamnetin  316.3  23.07  ESI−  315.0  300.0  −70  −30  151.0  −39  Sample and standard solution preparations The dry C. communis Linn. powder (60 mesh, 1 g) was accurately weighed and ultrasonicated for 40 min with 25 mL 70% ethanol, continuous ultrasound twice (8, 9, 22). The extracted solution was adjusted to the original weight by 70% ethanol. And the filtrate was combined and then concentrated to dryness in vacuo at 50°C. The residue was dissolved with methanol into a 5 mL volumetric flask for qualitative and quantitative analysis. The resultant was filtered through a 0.22 μm millipore filter. The injection volume was 10 μL for the analysis of HPLC–ESI-MS-MS, but UHPLC–Q-TOF-MS-MS was 1 μL. For qualitative analysis of the UHPLC–Q-TOF-MS-MS, all standards were respectively weighted appropriate amount, which were dissolved with 80% methanol to prepare mixture solution. For quantitative analysis, each standard (orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin) weighted accurately were dissolved in 80% methanol to prepare stock solutions. The concentrations of the stock solution of each standard were 0.91, 1.05, 0.985, 1.09, 0.96, 0.023, 0.95, 0.91, 0.214, 1.015, 0.107, 0.137 and 0.0234 mg mL−1, respectively, which was diluted to appropriate concentrations with methanol–water (80:20, v/v) for constructing calibration curves. All solutions were stored at 4°C for UHPLC–Q-TOF-MS-MS and HPLC–ESI-MS-MS analysis. Results and Discussion Analytical methods In this study, the analysis of chemical compositions in C. communis Linn. was based on a Triple TOF instrument with on-line data acquisition. The detail analytic strategy was as follows (23, 24): (i) On-line data were acquired by full-scan, and accurate MS/MS data were obtained. (ii) Chemical component database was established according to the investigation of chemical constituents of C. communis Linn. (3–6, 14–16, 18, 19, 25). (iii) The compounds of small error were filtrated. (iv) Compounds were estimated on the basis of the characteristic fragment ions and retention time. Data acquisition and procession were performed with PeakView 1.2 software. The determination method of 13 components for C. communis Linn. was mainly adjusting the gradient of the mobile phase to separate isomers. Then method validation and determination were getting on. A series of analysis such as the linearity, accuracy, matrix effect, stability, precision, limits of detection (LOD) and quantification (LOQ) were conducted to validate the feasibility of method. A total of 62 compounds were detected in C. communis Linn. extract, which included 29 flavonoids and flavonoid glycosides, 17 phenolic acids, 4 alkaloids, 1 pyrimidine alkaloids, 3 sterols and 8 fatty acids and others. Among of them, 38 compounds were detected in the positive ESI (ESI+) mode, and 24 compounds were found in the negative ESI (ESI−) mode. What is more, 22 compounds were identified by comparing with related reference substances. This study also made research on simultaneous determination of 13 constituents in C. communis Linn. by HPLC–ESI-MS-MS. It is extremely troublesome to distinguish and separate isomers in the process of identification because of identical molecular formulae, small difference in retention time, MS/MS spectra and chemical polarity. Thus, an important parameter Clog P, which was calculated using the program Chemdraw Ultra 12.0, was introduced to distinguish isomers. The compound with the smaller Clog P value commonly has shorter retention time in a reversed phase liquid chromatography system (23, 24). The structures of 62 compounds were showed in Figure S1. The detected compounds were listed in Table II. UHPLC–Q-TOF-MS positive (A) and negative (B) total ion chromatogram of C. communis Linn. were shown in Figure 2. The MS/MS of detected compounds and reference substances were shown in Figures S2 and S3, respectively. HPLC–MS-MS total ion chromatogram of C. communis Linn. (A) and mixture reference substance (B) were shown in Figure S4. Table II. Analysis on Chemical Constituents of Commelina communis Linn. Extract Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Table II. Analysis on Chemical Constituents of Commelina communis Linn. Extract Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Peak no.  Formula  Name  TR (min)  Error (ppm)  Clog P  Electrospray ionization mode  Extraction mass (Da)  Found at mass (Da)  MS/MS  1  C21H20O11  Iso-orientin  7.80  1.3  0.2098  +  449.1078  449.1084  431.0982, 413.0879, 395.0775, 353.0666, 329.0667, 299.0560, 287.0553  2  C21H20O11  Orientin  8.25  0.9  0.2598  +  449.1078  449.1082  431.0981, 413.0862, 395.0756, 383.0768, 353.0672, 329.0662, 299.0549, 287.1056, 243.0282, 137.0332  3  C21H20O11  Luteolin-7-O-β-d-glucoside  12.20  −0.2  0.8148  +  449.1078  449.1077  287.0553  4  C21H20O11  Luteolin-5-O-β-d-glucoside  14.75  −1.1  −0.08523  +  449.1078  449.1073  287.0565  5  C21H20O11  Luteolin-4′-O-β-d-glucoside  15.09  0.2  0.6672  +  449.1078  449.1079  287.0548  6  C22H22O11  Chrysoeriol-7-O-β-d-glucoside  8.36  1.9  0.7019  +  463.1235  463.1244  445.1344, 427.1322, 367.0823, 343.0766, 313.1264, 301.0719, 286.0477  7  C22H22O11  Flavocommelitin  12.32  2.6  0.7780  +  463.1235  463.1247  445.1028, 397.0918, 367.0836, 343.1066, 313.0966, 285.1092  8  C22H22O11  Luteolin-7-methylether-4′-O-glucoside  15.49  0.2  1.253  +  463.1235  463.1236  445.1344, 427.1322, 409.1033, 367.0832, 343.0766, 313.1264 301.0122, 287.0605  9  C21H20O10  Vitexin  10.34  0.7  0.8540  +  433.1129  433.1132  415.1030, 397.0926, 379.0813, 367.0818, 351.0866, 323.0935, 313.0715, 283.0612, 121.0284  10  C21H20O10  Iso-vitexin  10.92  0.5  0.8040  +  433.1129  433.1131  415.1018, 397.0927, 379.0795, 367.1211, 351.0884, 349.1022, 337.0704, 313.0708, 283.0605  11  C21H20O12  Hyperin  11.15  0.2  −0.3355  +  465.1028  465.1029  303.0507  12  C21H20O12  Isoquercitrin  11.94  0.9  −0.3355  +  465.1028  465.1032  303.0507  13  C27H30O16  Rutin  11.15  0.5  −1.361  +  611.1607  611.1610  465.1020, 303.0517  14  C22H22O10  Swertisin  11.90  0.4  0.9163  +  447.1286  447.1288  429.1066, 411.1089, 393.0824, 351.0871, 327.0870, 297.0732, 285.1330, 267.0654  15  C19H18O6  3,3′,4′,7-tetramethoxyflavone  13.59  1.7  2.770  +  343.1176  343.1182  302.1431, 301.1374, 283.1034, 215.0128, 148.0391  16  C22H22O12  Isorhamnetin-3-O-β-d-glucoside  15.09  1.5  0.1092  +  479.1184  479.1191  317.0665, 302.0454  17  C22H22O12  Isorhamnetin-7-O-β-d-glucoside  15.30  1.0  0.5109  +  479.1184  479.1189  317.0655  18  C15H10O7  Quercetin  16.56  0.6  1.504  +  303.0499  303.0501  257.0449, 229.0491, 201.0543, 165.0191, 153.0182, 137.0224  19  C16H12O6  Diosmetin  17.25  1.0  2.750  +  301.0707  301.0710  286.0490, 258.0534, 229.0521, 153.0193  20  C16H12O7  Isorhamnetin  17.35  0  1.951  +  317.0656  317.0656  302.0413, 285.0363, 274.0431, 273.0409, 257.0482, 245.0457, 229.0451, 217.0596, 153.0174, 121.0459  21  C8H8O5  Methyl gallate  1.12  −3.2  0.9314  +  185.0445  185.0439  170.1081, 142.0798, 141.0322, 97.0303  22  C16H18O9  Chlorogenic acid  3.57  1.1  −1.433  +  355.1024  355.1028  193.0462, 192.0390, 185.0413, 163.0329, 145.0288, 135.0445, 117.0340  23  C9H10O5  4-Hydroxy-2,6-dimethoxyl-benzoic acid  1.75  0.5  0.7078  +  199.0601  199.0602  181.0489, 155.0733, 140.0468, 136.0021, 125.0236, 111.0330, 95.0506, 77.0404  24  C9H10O5  3-Hydroxy-4,5-dimethoxy-benzoic acid  3.09  −1.0  0.9678  +  199.0601  199.0599  184.0332, 153.0615, 141.0724, 116.9284, 109.0667, 105.0693  25  C9H10O5  Syringic acid  3.83  0.5  1.068  +  199.0601  199.0602  153.0556, 139.0393, 137.0608, 125.0594, 121.0278, 111.0445, 95.0499, 79.0551  26  C8H8O4  Vanillic acid  4.30  −1.2  1.355  +  169.0495  169.0493  151.0399, 141.0778, 136.0158, 126.0396, 125.0624, 123.0435, 110.0379, 93.0359, 65.0413  27  C7H6O2  р-Hydroxybenzaldehyde  5.06  0.8  1.885  +  123.0441  123.0442  95.0516, 77.0402, 51.0265  28  C10H10O4  Ferulic acid  8.79  0.5  1.421  +  195.0652  195.0653  177.1155, 149.0588, 145.0300, 134.0362, 117.0440, 89.0418, 77.0385  29  C10H10O3  Methyl 4-hydroxycinnamate  22.18  0  1.798  +  179.0703  179.0703  161.0590, 133.0659, 118.0412, 90.0475  30  C13H25NO10  7-O-β-d-glucopyranosyl-α-homonojirmycin  0.73  4.2  −3.028  +  356.1551  356.1566  194.1032, 176.0924, 158.0817, 140.0817, 127.0396, 85.0299  31  C7H15NO5  α-Homonojirmycin  0.76  0  −1.451  +  194.1023  194.1023  176.0909, 158.0823, 140.0720, 85.0399, 82.0666  32  C6H13NO4  1-Deoxynojirimycin  0.78  1.8  −1.424  +  164.0917  164.0920  146.0814, 128.0709, 110.0607, 82.0664, 80.0551, 69.0355, 68.0516  33  C11H8N2  Norharman  5.61  −1.2  2.559  +  169.0760  169.0758  142.0649, 115.0546  34  C10H13N5O4  Adenosine  1.05  1.1  −2.158  +  268.1040  268.1043  152.0566, 136.0623, 119.0353  35  C29H50O2  Stigmast-5-ene-3β, 7α-diol  21.99  0.7  8.040  +  431.3884  431.3887  317.2086, 233.1533, 215.1428, 191.1072, 187.1471, 177.0905, 173.0963, 159.0804, 145.0999, 131.0853, 97.0655  36  C30H50O  Fridelin  23.13  −0.7  10.74  +  427.3934  427.3931  409.1083, 331.0528  37  C18H21NO4  Commelinins A  14.90  2.2  1.631  +  316.1543  316.1550  138.0917, 137.0604, 121.0658  38  C17H17NO3  (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide)  16.00  3.5  2.188  +  284.1281  284.1291  164.0632, 147.0447, 121.0651, 119.0497, 103.0552, 91.0553  39  C28H32O16  Isorhamnetin-3-O-rutinoside  4.37  4.7  −0.9160  −  623.1618  623.1647  479.1477, 315.0516, 300.0291, 284.0376  40  C28H32O16  Flavocommelin  14.96  4.7  −0.06143  −  623.1618  623.1647  461.1120, 299.0539, 284.0428  41  C28H32O15  Flavocommelinin A  4.44  4.3  −0.08969  −  607.1668  607.1694  487.1245, 445.1138, 283.1032  42  C28H32O14  2′′-O-α-l-rhamnosylswertisin  16.14  1.7  0.4616  −  591.1719  591.1729  545.1798, 445.2241, 427.1032, 412.0905, 283.0117  43  C21H20O9  Isofurcatain  16.16  1.0  1.355  −  415.1035  415.1039  341.0663, 323.0214, 311.0574, 283.0621, 269.0446  44  C15H10O6  Luteolin  16.49  0.7  2.311  −  285.0405  285.0407  241.0503, 217.0503, 199.0393, 174.9560, 151.0036, 149.0248, 133.0289, 121.0294, 112.9236, 107.0135, 61.9033  45  C15H10O5  Apigenin  17.10  1.8  2.905  −  269.0456  269.0461  225.0449, 201.0532, 151.0030, 149.0248, 117.0346, 112.9854  46  C15H10O6  Kaempferol  17.20  0.4  2.100  −  285.0405  285.0406  284.0334, 256.0374, 229.1443, 211.1022, 183.1382, 171.0833, 151.0455, 139.1124, 117.0238, 112.9858, 93.0358  47  C16H12O5  Acacetin  18.47  3.5  3.486  −  283.0612  283.0622  268.0356, 240.0398, 236.1052, 221.1540, 211.0425, 151.0233, 112.9857  48  C6H4O4  Cumaric acid  1.08  2.9  0.1578  −  139.0037  139.0041  111.0100, 94.0312, 67.0218  49  C7H6O4  Protocatechuate  2.07  3.9  1.062  −  153.0193  153.0199  110.0328, 109.0300, 108.0213, 91.0192  50  C7H6O3  3,4-Dihydroxybenzaldehyde  3.14  4.4  1.030  −  137.0244  137.0250  119.0148, 109.0301, 108.0225, 92.0277, 81.0358, 61.9912  51  C7H6O3  р-Hydroxybenzoic acid  3.42  3.6  1.557  −  137.0244  137.0249  112.9851, 93.0363, 75.0098, 61.9911  52  C9H8O4  Caffeic acid  4.42  3.9  0.9750  −  179.0350  179.0357  136.0497, 135.0460, 134.0371, 121.0301, 112.9870, 107.0404, 88.9892, 72.9950, 61.9909  53  C7H6O2  Benzoic acid  4.91  3.3  1.885  −  121.0295  121.0299  120.0214, 93.0347, 92.0278, 75.0138, 61.9912  54  C9H8O3  р-Hydroxycinnamic acid  6.98  3.7  1.572  −  163.0401  163.0407  119.0513, 103.0109, 93.0356, 61.9910  55  C9H16O4  Azelaic acid  13.93  2.7  1.563  −  187.0976  187.0981  142.0039, 141.0923, 125.0972, 112.9861, 97.0673, 61.9909  56  C29H48O  Stigmasterol  24.66  −1.0  9.445  −  411.3632  411.3628  393.3393, 349.3472  57  C6H14O6  d-Mannose  0.79  1.1  −2.046  −  181.0718  181.0720  163.0560, 101.0241, 89.0247, 85.0308, 75.0109, 61.9913  58  C9H7NO2  3-Carboxyl-indole  9.99  4.4  2.131  −  160.0404  160.0411  155.1074, 116.0509, 112.9854, 61.9906  59  C21H24O9  Rhaponticin  13.21  −1.2  1.366  −  419.1348  419.1343  373.0822, 341.1208, 297.0628, 208.0933  60  C32H38O17  4,4′-Dihydroxy-3,3′-dimethoxy-β-truxinic acid  14.02  −1.2  −2.243  −  739.2185  739.2176  693.2124, 387.0905, 369.0991, 323.0988, 271.0980  61  C14H20O6  2-Phenylethyl-β-d-glycoside  15.92  −2.1  0.3710  −  283.1187  283.1181  147.1172, 121.1021, 95.0877  62  C38H40N2O10  Commelinin B  16.66  5.0  3.472  −  683.2610  683.2644  520.2025, 504.1712, 352.1187  Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of positive (A) and negative (B) of Commelina communis Linn. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of positive (A) and negative (B) of Commelina communis Linn. Identification of 62 Compounds by UHPLC–Q-TOF-MS-MS The positive ESI+ mode Flavonoids and flavonoid glycosides Compounds 1–5 turned up at 7.80, 8.25, 12.20, 14.75 and 15.09 min, respectively. The protonated molecular ions [M + H]+ were at m/z 449.1084, 449.1082, 449.1077, 449.1073 and 449.1079, and they had same fragment ion at m/z 287.0553 ([M-C6H10O5+H]+). Compounds 1, 2 and 3 were identified as iso-orientin, orientin and luteolin-7-O-β-d-glucoside through comparison with relevant reference substances (15–17, 25). However, compounds 4 and 5 were concluded as luteolin-5-O-β-d-glucoside and luteolin-4′-O-β-d-glucoside on the basis of the MS/MS data at m/z 287.0565, which were firstly detected in C. communis Linn. Compounds 6–8 had identifical protonated molecule ions [M + H]+ at m/z 463.1244, 463.1247 and 463.1236, which were detected at 8.36, 12.32 and 15.49 min. They were judged as chrysoeriol-7-O-β-d-glucoside (3), flavocommelitin (25) and luteolin-7-methylether-4′-O-glucoside in accordance with the secondary fragment ions at m/z 301.0122 ([M-C6H10O5+H]+) and 287.0605 ([M-C6H10O5-CH2+H]+). Among of them, luteolin-7-methylether-4′-O-glucoside was firstly isolated from C. communis Linn. Compounds 9 and 10 were eluted at 10.34 and 10.92 min, showing the protonated molecule ions [M + H]+ at m/z 433.1132 and 433.1131. And their fragment ions were same as that of reference substances. So they were confirmed to be vetexin (15, 16, 25) and iso-vetexin (15, 16), and the characteristic fragment ions all were detected at m/z 397.0927, 379.0795, 351.0884, 313.0708 and 283.0605. Compounds 11 and 12 eluted at 11.15 and 11.94 min were observed in the XIC at m/z 465.1029 and 465.1032 ([M + H]+). They had parallel characteristic fragment ion at m/z 303.0507 ([M-C6H10O5+H]+), which was consistent with MS/MS data of reference substance. Therefore, they were ensured to be hyperin and isoquercitrin (3). Among of them, hyperin was firstly gained in C. communis Linn. Compound 13 was eluted at 11.15 min with the prontonated molecule ion of m/z 611.1610 ([M + H]+). The characteristic product ions at m/z 465.1020 ([M-C6H10O4+H]+) and 303.0517 ([M-C12H20O9+H]+) appeared in the MS/MS spectrum, which were the same as the fragment ions of reference substance. As a consequence, it was ascertain as rutin (25). The protonated molecule of m/z 447.1288 ([M + H]+, compound 14), eluted at 11.90 min, showed a series of distinctive fragment ions at m/z 285.1330 and 267.0654 by loss of C6H10O5 and H2O. According to these characteristic fragments, it was identified to be swertisin (17). The protonated molecule ion of m/z 343.1182 (compound 15) was eluted at 13.59 min. It showed a series of distinctive fragment ions at m/z 301.1374 and 283.1034 by loss of CO, CH2 and 2CH2O. Then it was deduced as 3,3′,4′,7-tetramethoxyflavone (14). Compounds 16 and 17 with the protonated molecule ions at m/z 479.1191 and 479.1189 ([M + H])+ were detected 15.09 and 15.30 min. The MS/MS were found at m/z 317.0665([M-C6H10O5+H]+) and 302.0454 ([M-C6H10O5-CH3+H]+) in the XIC of compounds 16 and 17. According to the characteristic fragments, compounds 16 and 17 were deduced as isorhamnetin-3-O-β-d-glucoside (3) and isorhamnetin-7-O-β-d-glucoside. The compound 17 was deduced on the basis of the MS/MS information. Compounds 18–20 were detected at m/z 303.0501, 301.0710 and 317.0656 ([M + H]+), eluted at 16.56, 17.25 and 17.35 min, respectively. They were determined as quercetin, diosmetin and isorhamnetin (3) according to the fact that their MS/MS fragment ions were well matched with the corresponding standard substances. What is more, quercetin and disometin were found under the experiment conditions for the first time. Phenolic acids Compounds 21, 29 appeared at 1.12 and 22.18 min with protonated molecule ions ([M + H]+) at m/z 185.0439 and 179.0703. The distinctive fragments of compound 21 were observed at m/z 170.1081 and 142.0798 by loss of CH3 and CO. So it was inferred as methyl gallate (17). The MS/MS of compound 29 were gained at m/z 161.0590, 133.0659, 118.0412 and 90.0475 through loss of H2O, CH2O, O, CO, CH3 and CH = CH. Therefore, compound 29 was concluded as methyl 4-hydroxycinnamate (15, 16, 19). Compounds 22, 26–28 were showed at 3.57, 4.30, 5.06 and 8.79 min. The protonated molecule ions ([M + H]+) were showed at m/z 355.1028, 169.0493, 123.0442 and 195.0653, respectively. Their fragment ions were same as that of reference substances. So they were ascertained as chlorogenic acid, vanillic acid (25), р-hydroxybenzaldehyde and ferulic acid. Moreover, compounds 22, 27 and 28 were recognized for the first time. The protonated molecule ions ([M + H]+) of compounds 23–25 (14, 25) were detected at m/z 199.0602, 199.0599 and 199.0602, eluted at 1.75, 3.09 and 3.83 min. The MS/MS of compound 23 were found at m/z 181.0489, 155.0733, 140.0468, 125.0236, 111.0330, 95.0506 and 77.0404. The secondary fragment ions of compound 24 were detected at m/z 184.0332, 153.0615, 141.0724 and 109.0667. The typical fragment ions of compound 25 were observed at m/z 153.0556, 139.0393, 125.0594, 111.0445, 95.0499 and 79.0551, so it was deduced as syringic acid. The structures of compounds 23 and 24 were shown in the Figure S1. The fragmentation pathways of compounds 23, 24 and 25 were same, getting the fragment ions by successful loss of H2O, CO2, CH3, CH2, CH2O and O. Alkaloids The alkaloidal compounds were detected in ESI+ mode. The compounds 30–33 with protonated molecule ions ([M + H]+) at m/z 356.1566, 194.1023, 164.0920 and 169.0758 were inferred as 7-O-β-d-glucopyranosyl-α-homonojirmycin (6, 15, 16), α-homonojirmycin (6, 15, 16), 1-deoxynojirimycin (6, 15, 16) and norharman (15, 16), respectively, and they were showed at 0.73, 0.76, 0.78 and 5.61 min. The MS/MS of compound 30 were received at m/z 194.1032, 176.0924, 158.0817 and 140.0817, which lost C6H10O5, H2O, H2O and H2O. The secondary fragments of compound 31 were gained at m/z 176.0909 ([M-H2O+H]+), 158.0823 ([M-2H2O+H]+) and 140.0720 ([M-3H2O+H]+). The characteristic fragment ions of compound 32 were observed at m/z 146.0814, 128.0709, 110.0607 and 82.0664 by loss of H2O, H2O, H2O and CO. The typical fragment ions of compound 33 were acquired at m/z 142.0649 ([M-CHN + H]+) and 115.0546 ([M-2CHN+H]+). Pyrimidine alkaloids Compound 34 was detected with precursor ion at m/z 268.1043 ([M + H]+), and was eluted at 1.05 min. A series of distinctive fragment ions (26) were received at m/z 152.0566 ([M-C5H8O3+H]+), 136.0623 ([M-C5H8O4+H]+) and 119.0353 ([M-C5H8O4-NH2+H]+). The compound 34 was named as adenosine (15, 16). Sterols Sterols compounds were detected in the positive ESI mode, which included stigmast-5-ene-3β, 7α-diol (compound 35) (15, 16) and fridelin (compound 36) (15, 16). Their protonated molecule ions ([M + H]+) were detected at m/z 431.3887 and 427.3931, and they were eluted at 21.99 and 23.13 min, respectively. The MS/MS of compound 35 were showed at m/z 317.2086, 233.1533 and 215.1428 by loss of C8H18, C5H6, H2O and H2O, and the fragment ions of compound 36 were obtained at m/z 409.1083 ([M-H2O+H]+) and 331.0528 ([M-H2O-C6H5+H]+). Fatty acid and others Compound 37 was detected at 14.90 min with precursor ions at m/z 316.1550 ([M + H]+). The major fragment ions were showed at m/z 138.0917 ([M-C10H10O3+H]+) and 121.0658 ([M-C10H10O3-OH + H]+). And it was inferred as commelinins A (15, 16). Compound 38 was judged as (E-N [4-(2-hydroxyethyl) phenyl]-3-(4-hydroxy-phenyl)-acrylamide) (17) in light of the distinctive fragment ions at m/z 164.0632, 147.0447, 119.0497 and 103.0552 by missing C8H9N, OH, CO and O. The precursor ion was at m/z 284.1291 ([M + H]+), and it was eluted at 16.00 min. The negative ESI mode Flavonoids and flavonoid glycosides Compounds 39 and 40 eluted at 4.37 and 14.96 min, respectively, showed deprotonated molecule ions [M−H]– at m/z 623.1647 and 623.1647. The typical fragments of compound 39 appeared at m/z 479.1477 and 315.0516 by loss of C6H8O4 and C12H20O9. Moreover, the characteristic fragment ions of compound 40 were gained at m/z 461.1120 and 299.0539 by loss of C6H10O5 and C12H20O10. Therefore, they were inferred to be isorhamnetin-3-O-rutinoside (3) and flavocommelin (15, 16). Compound 41 with deprotonated molecular ion [M−H]− at m/z 607.1694 was detected at 4.44 min. The characteristic fragment ions were acquired at m/z 445.1138 and 283.1032 by loss of C6H10O5 and C12H20O10. The compound 41 was deduced as flavocommelinin A (15, 16) based on the secondary fragments. Compound 42 was eluted at 16.14 min with a deprotonated molecule ion of m/z 591.1729 ([M−H]−). A series of product ions of compound 42 were detected at m/z 445.2241 ([M-C6H10O4-H]−) and 283.0117 ([M-C12H20O9-H]−), so it was deduced as 2′′-O-α-l-rhamnosylswertisin (16, 17). Compound 43 gave a deprotonated molecule [M−H]– at m/z 415.1039, which was observed at a retention time of 16.16 min. It was judged as isofurcatain (3) in light of product ion at m/z 269.0446 gained through loss of C6H10O4. Compounds 44–47 with deprotonated molecule ions ([M−H]−) at m/z 285.0407, 269.0461, 285.0406 and 283.0622 had the retention time of 16.49, 17.10, 17.20 and 18.47 min, respectively. The chromatogram retention time and MS/MS fragment ions kept consistent with that of reference substances. Their characteristic fragments were showed in the Table II. Hence, it were confirmed that they were luteolin (14, 15), apigenin (14, 15), kaempferol and acacetin according the secondary ions and retention times. Compounds 46 and 47 were observed for the first time. Phenolic acids Compounds 48, 51, 53–55 were showed at 1.08, 3.42, 4.91, 6.98 and 13.93 min. The precursor ions ([M−H]−) were showed at m/z 139.0041, 137.0249, 121.0299, 163.0407 and 187.0981. Secondary fragments of compound 48 were detected at m/z 94.0312 ([M-COOH-H]−) and 67.0218 ([M-COO-CO-H]−). Characteristic fragment ions of compound 51 were acquired at m/z 93.0363 ([M-CO2-H]−) and 75.0098 ([M-CO2-H2O-H]−). MS/MS data of compound 53 were observed at m/z 93.0347 and 75.0138 missing CO and H2O. Typical product ions of compound 54 were received at m/z 119.0513, 103.0109 and 93.0356 losing CO2, O and CH = CH. Distinctive fragment ions of compound 55 were showed at m/z 142.0039 ([M-COOH-H]−), 125.0972 ([M-COOH-OH-H]−) and 97.0673 ([M-2COOH]−). According to fragmentation information, compounds 48, 51, 53–55 were deduced as cumaric acid (14), р-hydroxybenzoic acid (3), benzoic acid (25), р-hydroxycinnamic acid (3) and azelaic acid (25), respectively. Compounds 49, 50 and 52 turned up at 2.07, 3.14 and 4.42 min with the precursor ions ([M−H]−) at m/z 153.0199, 137.0250 and 179.0357. The product ions of compound 49, 50 and 52 were well matched with the related reference substances. Hence, the compound 49, 50 and 52 were confirmed as protocatechuate (25), 3,4-dihydroxybenzaldehyde and caffeic acid (3). Sterols Compound 56 was found at 24.66 min with a deprotonated molecule ion ([M−H]−) at m/z 411.3628. The characteristic fragment ions were detected at m/z 393.3393 ([M-H2O-H]−) and 349.3472 ([M-H2O-C3H8-H]−). According to the fragmentation information, the compound 56 was deduced as stigmasterol (14). Fatty acid and others The deprotonated molecule ions ([M−H]−) of compounds 57–59 were showed at m/z 181.0720, 160.0411 and 419.1343 from the XIC, which were eluted at 0.79, 9.99 and 13.21 min. The compound 57 (27) had the MS/MS at m/z 163.0560 ([M-H2O-H]−) and 101.0241 ([M-H2O-C2H6O2-H]−). Compound 58 owed the typical fragment ion at m/z 116.0509 ([M-CO2-H]−). Compound 59 possessed the characteristic fragment ions at m/z 373.0822 and 341.1208, which were received by loss of CH2O, O, O and O. On the basis of the fragment informant, compound 57–59 were inferred as d-mannose (4, 15, 16), 3-carboxy-indole (17) and rhaponticin (17). Compounds 60–62 with the deprontonated molecule ions at m/z 739.2176 ([M+HCOOH-H]−), 283.1181 ([M−H]−) and 683.2644 ([M−H]−) were eluted at 14.02, 15.92 and 16.66 min. Compound 60 showed the MS/MS at m/z 693.2124 and 387.0905 by loss of HCOOH and C12H18O9. Compound 61 had the secondary fragments ion at m/z 121.1021 ([M-C6H10O5-H]−). Compound 62 owed the characteristic fragment ions at m/z 520.2025, 504.1712 and 352.1187 by loss of C8H9N, CH2, CH2O, O, C8H9N and CH3O. Therefore, they were inferred as 4,4′-dihydroxy-3,3′-dimethoxy-β-truxinic acid (17), 2-phenylethyl-β-d-glucoside (17) and commelinin B (15, 16). Quantitative study for C. communis Linn. samples by HPLC–MS-MS Linearity, limit of detection and limit of quantification Stock solution containing 13 reference compounds were diluted to appropriate concentrations with methanol–water (80:20, v:v) for establishment of calibration curves. A total of seven concentrations of the solution were analyzed, and then the calibration curves were constructed by plotting the peak area (Y) versus the concentration (X, ng mL−1) for each analyte. Limit of detection (LOD) and limit of quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of ~3 and 10, respectively. The results were list in Table III. Table III. Regression, LODs and LOQs for the Investigated Compounds Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  aY: peak area; X: concentration of compound (ng mL−1). bLOD, limit of detection. cLOQ, limit of quantification. Table III. Regression, LODs and LOQs for the Investigated Compounds Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  Component  Regression equationa  r  Linear range (μg mL−1)  LODb (ng mL−1)  LOQc (ng mL−1)  Orientin  Y = 6.394 × 104x− 2.010 × 104  0.999 6  0.9775–62.56  1.57  6.32  Iso-orientin  Y = 3.790 × 104x+2.264 × 105  0.999 1  2.050–131.2  0.881  3.52  Vitexin  Y = 1.068 × 105x+9.162 × 102  0.999 7  1.212–77.57  7.51  30.3  Isovitexin  Y = 6.674 × 104x+1.199 × 103  0.999 5  0.1278–8.175  3.78  15.1  Rutin  Y = 5.696 × 104x+1.394 × 105  0.999 2  2.438–156.0  1.67  6.67  Apigenin  Y = 3.517 × 106x+5.876 × 103  0.999 4  0.0003595–0.05750  0.00685  0.0264  Protocatechuate  Y = 1.019 × 105x−7.882 × 103  0.999 8  0.2040–13.06  13.2  52.8  Vanillic acid  Y = 2.188 × 104x−2.386 × 103  0.999 8  0.1778–11.38  27.1  81.2  Caffeic acid  Y = 8.547 × 105x−5.968 × 103  0.999 7  0.05015–3.210  3.72  7.43  Ferulic acid  Y = 2.642 × 104x−1.030 × 103  0.999 9  0.1882–12.05  7.05  28.2  Luteolin  Y = 2.207 × 106x+1.714 × 104  0.999 1  0.008360–0.5350  0.00661  0.0264  Quercetin  Y = 2.798 × 106x+8.874 × 103  0.999 7  0.001874–0.1199  0.0132  0.846  Isorhamnetin  Y = 2.173 × 106x+6.767 × 102  0.999 7  0.0008225–0.05265  0.0296  0.599  aY: peak area; X: concentration of compound (ng mL−1). bLOD, limit of detection. cLOQ, limit of quantification. Precision The precision of method was validated by analyzing C. communis Linn. sample, which included intra- and inter-day precision. The intra-day precision was performed by repeating six times within 1 day, while the inter-day precision was consecutively performed on three days. The determination of 13 ingredients investigated was performed from corresponding calibration curve. Relative standard deviation (RSD) values of peak area for each of compound were calculated respectively. The results showed that the RSD values of intra- and inter-day precision were <1.04 and 0.92%, respectively. Accuracy Accuracy of the method was received by spiking the mixed standard solutions with three different concentration levels (low, medium and high) to prepare known amount of C. communis Linn. samples, which were dealt with through the above proposed method. Moreover, triplicate samples were conducted for each level. The result showed that the average recoveries of 13 components measured were in range of 98.64–100.8% and RSD values were in range of 0.11–0.78%. Stability The stability of the sample was analyzed at 0, 2, 4, 8, 12 and 24 h at room temperature, respectively. The results showed that RSD values of all measured peak area were <0.59%, indicating a good stability. Matrix effects The evaluation of matrix effects is a vital issue for the analysis of botanical extracts by HPLC–ESI-MS-MS. Co-eluting compounds originating from matrix can lead to signal enhancement or suppression (28). The matrix effects of 13 components were quantitatively investigated, which was performed by adding known amounts of the mixed standard solution at three levels (low, medium and high) to half of the extraction samples and recording the analyte peak areas of the spiked sample matrix (A), the other half of the extraction samples (B) and the standard solutions (C) (28, 29). Moreover, triplicate samples were prepared at each level. Matrix effects were calculated using the following equation: matrix effect (%) = (A–B)/C × 100%. The matrix effects of 13 components ranged from 93.4 to 103.1% and the values of RSD were in range of 0.34–1.31%, which indicated that the matrix effects had no influence. Repeatability The repeatability experiment was performed by preparing for six independent sample solutions of C. communis Linn. in parallel. The RSD values of 13 compounds were not more than 0.97%, which showed a good reproducibility. The results of precision, repeatability, accuracy, stability and matrix effect were showed in Table S1 and Table IV, respectively. Table IV. Matrix Effects of 13 Components in Commelina communis Linn. Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Table IV. Matrix Effects of 13 Components in Commelina communis Linn. Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Compound  Spiked (μg)  Matrix effect (%)  RSD (%)  Orientin  10.33  95.3  0.82  Vanillic acid  4.613  98.3  1.09  13.37  97.1  1.03  5.785  95.9  0.94  15.92  94.3  0.79  6.936  99.6  1.03  Iso-orientin  33.48  96.2  0.48  Caffeic acid  0.4684  97.9  0.39  41.92  94.6  0.34  0.5844  95.1  0.84  50.33  93.4  0.56  0.7003  98.3  0.64  Vitexin  18.45  99.2  1.17  Ferulic acid  3.142  100.5  0.55  23.12  96.6  0.98  3.915  98.9  1.07  27.71  103.1  0.64  4.694  99.2  0.74  Isovitexin  3.170  97.8  1.12  Luteolin  0.2242  96.3  1.19  3.986  99.5  1.01  0.2796  95.8  1.11  4.785  95.6  0.89  0.3349  97.4  0.99  Rutin  40.10  98.4  0.72  Quercetin  0.01615  98.3  0.59  50.06  100.2  0.54  0.02006  96.2  0.48  60.07  99.1  0.76  0.02396  97.5  0.73  Apigenin  0.01922  97.4  0.64  Isorhamnetin  0.01616  96.9  1.27  0.02415  98.3  1.18  0.02003  98.1  1.31  0.02892  99.5  0.92  0.02395  96.3  0.59  Protocatechuate  3.251  102.8  0.76          4.072  101.3  0.91          4.887  99.7  0.85          Sample determination Commelina communis Linn. samples (SD-1, SD-2, SD-3 and SD-4) in Qinhuangdao (China) were determined under the above conditions. Representative chromatograms were shown in Figure 1. Peak identify was established by comparing retention time of sample with that of reference compounds. The calibration curves were applied for the quantitative analysis of 13 compounds. The results of 13 analytes in C. communis Linn. samples were summarized in Table V. The results showed that the content of orientin, iso-orientin, vitexin, isovitexin, rutin in SD-3 and SD-4 were higher than that of in SD-1 and SD-2, which indicated that the content of 13 analytes were different according to the different dried methods and source. Table V. Contents of 13 Active Components in Commelina communis Linn. Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Table V. Contents of 13 Active Components in Commelina communis Linn. Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Sample ID  Mass fraction (μg g−1)  OrientIn  Iso-orientin  Vitexin  Isovitexin  Rutin  Apigenin  Protocatechuate  Vanillic acid  Caffeic acid  Ferulic acid  Luteolin  Quercetin  Isorhamnetin  SD-1  24.02  79.26  44.12  5.229  137.4  0.03605  12.32  12.18  1.458  5.364  0.1948  0.06122  0.03720  SD-2  26.64  83.37  45.91  8.032  101.8  0.04835  8.132  11.67  1.169  7.822  0.5639  0.04183  0.04002  SD-3  190.0  344.8  109.1  15.00  641.0  0.02218  7.185  5.680  1.648  4.422  0.6065  0.2626  0.04404  SD-4  120.1  210.0  107.3  23.99  163.7  0.04434  8.152  5.784  1.950  6.194  0.1599  0.06953  0.02544  Experiments compared with 50% methanol, 70% methanol, 95% methanol, 100% methanol, 50% ethanol, 70% ethanol, 95% ethanol and 100% ethanol as extraction solvent for C. communis Linn. sample, which showed that the extraction efficiency of 70% ethanol was highest. Afterwards comparing ultrasonic extraction and reflux extraction, the extraction efficiency was similar in two ways. Ultrasonic extraction was chose in this study, because ultrasonic was relatively quick and easy. It also examined ultrasonic frequency (once, twice and three times) and ultrasonic time (30, 40, 60 and 90 min). The results showed that continuous ultrasonic twice, 40 min every time, respectively, had highest extraction efficiency. Methanol–water and acetonitrile–water were chose as mobile phase for gradient eluting during the qualitative and quantitative analysis. The results showed that more compounds were observed using acetonitrile as eluent by UHPLC–Q-TOF-MS-MS. However, elutive power of two different mobile phases was similar for the quantitative analysis by HPLC–MS-MS. Because methanol was cheaper and smaller toxicity than acetonitrile, it was applied for determination of sample. Therefore, acetonitrile–water and methanol–water were served as eluent of qualitative and quantitative analysis, respectively. To improve peak shape, different concentrations of formic acid (0.05, 0.1, 0.2 and 0.3%) in water for qualitative and quantitative analysis study were compared, which showed that peak shape of 0.1% formic acid was best. Moreover, different concentrations of ammonium formate (1, 2 and 3 mmol L−1) and ammonium acetate (1, 2 and 3 mmol L−1) were compared. So acetonitrile–water (0.1% formic acid) was used for qualitative study, and methanol–water (0.1% formic acid, 2 mmol L−1 ammonium acetate) was the mobile phase for quantitative analysis. Conclusion In the present study, a powerful analytical strategy dependent on-line data acquisition combining with multiple post acquisition data processing was employed for fast screening and identification the chemical compositions of C. communis Linn. What is more, luteolin-5-O-β-d-glucoside (compound 4), luteolin-4′-O-β-d-glucoside (compound 5), luteolin-7-methylether-4′-O-glucoside (compound 8), hyperin (compound 11), hyperin (compound 11), isorhamnetin-7-O-β-d-glucoside (compound 17), diosmetin (compound 19), chlorogenic acid (compound 22), 4-hydroxy-2,6-dimethoxyl-benzoic acid (compound 23), 3-hydroxy-4,5-dimethoxy-benzoic acid (compound 24), р-hydroxybenzaldehyde (compound 27), ferulic acid (compound 28), methyl 4-hydroxycinnamate (compound 29), kaempferol (compound 46), acacetin (compound 47) and 3,4-dihydroxybenzaldehyde (compound 50) were firstly gained in C. communis Linn. The fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid were shown in Figure 3. And 13 compounds exiting in C. communis Linn were firstly determined by HPLC–MS-MS. Many isomers were found in C. communis Linn., which were distinguish through Clog P values This is the first report of simultaneous qualitative and quantitate study of main chemical components of C. communis Linn. by UHPLC–Q-TOF-MS-MS and HPLC–MS-MS. Furthermore, the application of this approach provided essential data for further studies of C. communis Linn. Figure 3. View largeDownload slide The predominant fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid. Figure 3. View largeDownload slide The predominant fragmentation pathways of flavonoids, alkaloids, sterols and phenolic acid. Supplementary Data Supplementary data are available at Journal of Chromatographic Science online. Acknowledgments Thanks for the instrument support of the Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University. Conflict of interest statement The authors have declared no conflict of interest. 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Published: Apr 11, 2018

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