UPLC Orbitrap HRMS Analysis of Panax quinquefolium L. for Authentication of Panax Genus with Chemometric Methods

UPLC Orbitrap HRMS Analysis of Panax quinquefolium L. for Authentication of Panax Genus with... Abstract Ginsenosides in Panax quinquefolium L. were determined using developed ultra-performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS) method with electrospray ionization and orbitrap MS analyzer in negative ionization mode. Optimal UPLC separation was achieved using a mixture of acetonitrile and water with 0.1% formic acid as the mobile phase in linear gradient elution. The MS parameters were optimized for reliable detection with enhanced selectivity and sensitivity, and improved identification and quantification of ginsenosides. The applicability of this method was demonstrated on ginsenosides from Panax quinquefolium L. (American ginseng), Panax ginseng (Chinese ginseng) and Panax notoginseng (Sanchi) roots and products. The differences between Chinese and Northern American Panax quinquefolium L., main roots and hair roots, and products from different pharmacy were investigated. The results were also confirmed by principal component analysis and partial least squares discriminatory analysis. It indicated that the strategy can be extended to rapid and accurate authentication of Panax genus. Introduction American ginseng, the root of Panax quinquefolium L. (Araliaceae) originally grown in southeast of Canada and northern USA, was used by native North Americans long before the arrival of Asian. It has been cultivated in China for decades and used in Chinese medicine for more than 200 years. And American ginseng is also used as functional food available on the commercial market to help improve the quality of life (1–3). It has been demonstrated that Panax quinquefolium. L cultivated in China is different from that grown in USA due to the compositional differences (4, 5). Panax quinquefolium L. (American ginseng), Panax ginseng (Chinese ginseng) and Panax notoginseng (Sanchi) are all highly valuable and important tonic plant in China (6). Although they all belong to Panax genus, the efficacy, pharmacological effects and clinical indications of them are different due to the significant differences in the types and quantities of ginsenosides in each root (7). Ginsenosides are the major pharmacologically active constituents of Panax genus. According to the differences of aglycone skeletons, protopanaxadiol (PPD), protopanaxatriol (PPT), oleanolic acid and ocotillol types of ginsenosides have been identified from ginseng roots and related products. The ocotillol type ginsenoside is the chemical marker to distinguish American ginseng from Chinese ginseng and Sanchi. With the increasing interests in ginseng for health care, the need to identify and quantify the chemical composition to ensure the quality, safety and efficacy is necessary. Several different methods have been developed to determine ginsenosides in ginseng plants. The most commonly used are high performance liquid chromatography (HPLC) with photodiode array (8–11) or evaporative light-scattering detector (12–15). Capillary electrophoresis techniques have also been reported and the sensitivity is similar to the HPLC methods (16). With the development of mass spectrometry (MS) interface technology, HPLC coupled to MS (HPLC-MS) has gained its popularity and been shown to be a powerful tool for quality control and consistency of both the chemical markers and active substances of traditional Chinese medicines. It has been applied to determine ginsenosides in ginseng plants (4, 17–27) and yields the information on molecular weights, structures and contents. Among the various LC, ultra-performance LC (UPLC) is considered suitable for metabolite and metabolomics research, due to its reduced analysis time, enhanced reproducibility and increased sample throughput, resolution and sensitivity (28–32). MS is an effective tool for the analysis of ginsenosides with different ionization techniques including matrix-assisted laser desorption ionization (33), electrospray ionization (ESI) (4, 19, 21–32, 34–36) and atmospheric pressure chemical ionization (37, 38). Different MS analyzers have been used in metabolite fingerprint including triple quadrupole (19, 23–25, 36–39), ion trap (21, 22) and quadrupole Time of Flight (TOF) MS (26–34). Orbitrap MS analyzer has not been reported in American ginseng research. The triple quadrupole was generally employed to search for metabolites using neutral loss scan and product ion scan, while ion trap allows structure elucidation of metabolite by MSn. However, these two types of analyzers can only provide nominal mass accuracy. Quadrupole TOF could generate high resolution mass accuracy. Orbitrap, on the other hand, provides much better accuracy, precision and much higher resolution of mass information generated. This study presents the development of an UPLC orbitrap HRMS method to efficiently and simultaneously determine ginsenosides in American ginseng. Gradient elution in UPLC and optimization of MS detection parameters were adjusted to obtain clear resolution of the peaks and maximum signal in the MS detector. Finally, we applied the UPLC orbitrap HRMS method developed to the identification and quantification of ginsenosides in American ginseng, Chinese ginseng and Sanchi roots and commercial products from different origins. With the principal component analysis (PCA) and partial least squares discriminatory analysis (PLS-DA) analysis, Panax genus root materials were distinguished and American ginseng products were authenticated and evaluated. The results demonstrated that the strategy is reliable for the authentication of botanical origin and can also be useful for the quality control of Chinese medicinal herbs. Experimental Chemicals HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Waltham, MA), ultrapure water (18 MΩ/cm) was prepared by a Milli-Q water system (Millipore, Bedford, USA) and HPLC grade formic acid from Sigma-Aldrich. Analytical grade methanol, n-butanol and petroleum ether were from Beijing Chemical Works. Reference ginsenosides, including ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, Ro, F1, F2, F3, pseudoginsenoside F11, notoginsenosides R1 and R2 were obtained from Jilin University (Changchun, JL, China). Reference solution preparation Reference solutions for 19 ginsenosides were prepared individually, with 1 mg of each compound dissolved in 10 mL of 50% (v/v) methanol–water to final concentration of 0.1 mg/mL. Combined each of the individual reference solutions and diluted to obtain final concentrations appropriate for different samples to prepare mixed reference solutions. Calibration curves with 6 dilutions of initial mixture reference solution were used to quantify 19 ginsenosides in different ginseng roots and products. Plant materials and sample preparation The information of all Panax samples was shown in Table I. The Panax samples including American ginseng, Chinese ginseng and Sanchi roots materials and American Ginseng products were collected from Jilin province and Wisconsin, and purchased from local pharmacy. The botanical origin was identified by Prof. Shumin Wang and deposited at the Jilin Ginseng Academy of the Changchun University of Chinese Medicine. All roots samples were dried under sunlight. About 100 g of each root were powdered using a pulverizer and sieved before extraction, assure the analyzed samples well-distributed and typical. Table I. The Origin Information of Different Ginseng Samples   Samples  Origins  S1(1–3)  American Ginseng main roots  Wisconsin, USA  S2(1–3)  American Ginseng main roots  Fusong, Jilin, China  S3(1–3)  American Ginseng main roots  Jingyu, Jilin, China  S4(1–3)  American Ginseng hair roots  Jingyu, Jilin, China  S5(1–3)  Chinese Ginseng main roots  Fusong, Jilin, China  S6(1–3)  Chinese Ginseng main roots  Jingyu, Jilin, China  S7(1–3)  Sanchi main roots  Yanshan, Wenshan, Yunnan, China  S8(1–3)  Sanchi main roots  Maguan, Wenshan, Yunnan, China  S9  American Ginseng main roots  Jilin Pharmacy, Changchun, China  S10  American Ginseng main roots  Changbai mountain pharmaceutical co., LTD  S11  American Ginseng root slices  Fubaicao Pharmacy, Changchun, China  S12  American Ginseng root slices  Jilin honored star pharmaceutical co., LTD  S13  American Ginseng root slices  Changbai mountain ginseng base, Jilin, China  S14  American Ginseng roots total saponin extracts  Jingyu, Jilin, China    Samples  Origins  S1(1–3)  American Ginseng main roots  Wisconsin, USA  S2(1–3)  American Ginseng main roots  Fusong, Jilin, China  S3(1–3)  American Ginseng main roots  Jingyu, Jilin, China  S4(1–3)  American Ginseng hair roots  Jingyu, Jilin, China  S5(1–3)  Chinese Ginseng main roots  Fusong, Jilin, China  S6(1–3)  Chinese Ginseng main roots  Jingyu, Jilin, China  S7(1–3)  Sanchi main roots  Yanshan, Wenshan, Yunnan, China  S8(1–3)  Sanchi main roots  Maguan, Wenshan, Yunnan, China  S9  American Ginseng main roots  Jilin Pharmacy, Changchun, China  S10  American Ginseng main roots  Changbai mountain pharmaceutical co., LTD  S11  American Ginseng root slices  Fubaicao Pharmacy, Changchun, China  S12  American Ginseng root slices  Jilin honored star pharmaceutical co., LTD  S13  American Ginseng root slices  Changbai mountain ginseng base, Jilin, China  S14  American Ginseng roots total saponin extracts  Jingyu, Jilin, China  Pulverized ginseng roots powders were extracted using petroleum ether degreasing with methanol and then water saturated n-butanol extractions. For each extraction, a 1.0 g aliquot of the powdered sample was soxhlet extracted with petroleum ether for 2 h. Then the residue was immersed with methanol for 12 h and soxhlet extracted with methanol for twice. After cooling, the methanol extractions were combined, filtered and concentrated under vacuum. The concentrate was dissolved with water and then extracted with water saturated n-butanol for 3 times. The n-butanol extractions were combined and dried under vacuum. The residue was redissolved in 1 mL 80% (v/v) methanol–water and the supernatant was passed through a 0.22 μm filter before UPLC-HRMS analysis. The extraction of five replicated samples and blank samples were prepared under the same procedure. Instrumentation and conditions The LC analysis was carried out using an UPLC system (Dionex Ultimate 3000, Thermo Scientific, USA) equipped with a quaternary gradient pump, an autosampler, a thermostatically column compartment and a photodiode array detector. The separations were performed using Thermo Scientific Syncronis C18 UPLC column (100 × 2.0 mm, 1.7 μm) at 35°C column temperature. Formic acid (0.1%, v/v, A) and acetonitrile (B) were used as the mobile phase. The gradient elution was programmed at a flow rate of 0.2 mL/min as follows: initial conditions of 15% (v/v) B held for 5 min, increased linearly to 90% (v/v) B in 30 min, increased linearly to 100% (v/v) B in 5 min, held at 100% (v/v) B for 5 min. The temperature of the autosampler was set at 15°C and the injection volume was 5 μL. The UPLC system coupled to Thermo Q-Exactive orbitrap high resolution mass spectrometer (Thermo Scientific, USA) with ESI in negative ion mode and using full mass scan type. The scan range was m/z 150.0–2000.0, scan rate was set at normal in centroid mode. The MS resolution was set at 70,000 and AGC target was 1e6. Maximum inject time was at 250 ms. In addition, the spray voltage was set at 2.5 kV, the capillary temperature was 320°C and S-lens RF level was fixed at 50 to get the best experimental conditions. N2 was used as the Sheath gas with flow rate at 40 and Aux gas with flow rate at 10. The aux gas heater temp was at 300 to achieve the highest signal intensity of detection. The UPLC-HRMS system was controlled with Xcalibur 3.0 data system software. Validation The UPLC orbitrap HRMS method was validated using the following parameters: coefficient of correlation (R2); linear range; intraday and interday repeatability; recovery and limits of detection (LOD) and quantification (LOQ). The linearity was established by analytical curves constructed. The LOD and LOQ were measured on the basis of the response at signal-to-noise ratio (S/N) of 3:1 and 10:1. Intraday repeatability was studied on a single day with five parallel experiments and interday repeatability was measured by the same procedure on three separate days. The recoveries were assessed with known amounts of references spiked into samples. The determination was performed in five repetitions. UPLC orbitrap HRMS data handling by chemometric methods All 19 ginsenosides contents data analyzed by PCA and PLS-DA were performed by running the R-3.2.1 statistical analysis software. Results Development of sample preparation method This sample preparation procedure was optimized using published methods for ginsenosides in Chinese ginseng (1), with some modifications. The conventional method uses heat-reflux, soxhlet and ultrasound-assisted extraction. Extraction solvent includes water, methanol and ethanol. In the procedure, the extraction time and times were also optimized. Different sample preparation differs in extraction efficiency, affecting the relative abundance of the extractable ginsenosides. Thus, we selected soxhlet with methanol to extract ginseng and then characterized diverse ginsenosides. Development of UPLC orbitrap HRMS method The UPLC system provided a rapid, effective and convenient analytical method for a wide range of ginsenosides present in Panax genus. Theoretically, a higher flow rate within the permitted range promises a good separation on the UPLC column whereas the tolerant flow rate for electrospray ion source is below 1.0 mL/min. Meanwhile, flow rate into the electrospray ion source should guarantee the best ionization efficiency and minimize ion suppression which may influence sensitivity. Taking sensitivity and resolution into consideration together, the ultimate flow rate was optimized at 0.2 mL/min throughout this study. Formic acid with different concentrations (0.1, 0.05 and 0.01%) were tested, the best peak shape and resolution was obtained by 0.1% formic acid. Column temperature was set at 35°C to alleviate the column pressure resulting from a higher flow rate, which can improve chromatographic separation and peak shape. The MS parameters of ginsenosides (PPD, PPT, oleanane and ocotillol types) were optimized by tuning each reference with directly infusing individual standard solutions at 5 μL/min using syringe pump. The spray voltage was determined in the range 2.0–4.5 kV, the capillary temperature was studied from 250 to 350°C and S-lens RF level was checked in the range 30–70 by manual setting. And the sheath gas flow rate, aux gas flow rate and aux gas heater temp were also optimized manually to achieve the highest signal intensity. The structures of four types of ginsenosides (PPD, PPT, oleanane and ocotillol) were shown in Table II. The deprotonated molecular ion [M–H]− presented higher intensity than the protonated molecular ion [M+H]+, thus, the negative ion mode was chosen for the determination of ginsenosides. Figure 1 shows the total ion current chromatograms of ginsenosides in three different Panax genus. Peak identifications were made by comparing retention time and MS spectra of the chromatographic peaks with the individual standard solutions to provide unambiguous results. Simultaneous monitoring of the 19 precursor ions can therefore be performed without sacrificing specificity and sensitivity. The overlapping peaks can be resolved by using the appropriate chromatographic gradient and by measuring selected ions characteristics through Extracted Ion Chromatography. The third important consideration is the high resolution MS allowed each analyte can be scanned under its accurate mass during the UPLC orbitrap HRMS determination. Table II. Structures of Protopanaxadiol, Protopanaxatriol, Oleanane and Ocotillol Types of Ginsenosides Structures  Ginsenosides  R1  R2  Molecular formula  Calculated mass [M–H]−  Measured mass [M–H]−  Mass error (ppm)    Ginsenosides Rb1  -Glc2-Glc  -Glc6-Glc  C54H92O23  1107.5952  1107.5972  1.81  Ginsenosides Rb2  -Glc2-Glc  -Glc6-Ara(p)  C53H90O22  1077.5846  1077.5875  2.69  Ginsenosides Rb3  -Glc2-Glc  -Glc6-Xyl  C53H90O22  1077.5846  1077.5868  2.04  Ginsenosides Rc  -Glc2-Glc  -Glc6-Ara(f)  C53H90O22  1077.5846  1077.5871  2.32  Ginsenosides Rd  -Glc2-Glc  -Glc  C48H82O18  945.5423  945.5446  2.43  Ginsenosides Rg3  -Glc2-Glc  -H  C42H72O13  783.4895  783.5096  2.57  Ginsenosides Rh2  -Glc  -H  C36H62O8  621.4367  621.4379  1.93  Ginsenosides F2  -Glc  -Glc  C42H72O13  783.4895  783.5084  2.41    Ginsenosides Re  -Glc2-Rha  -Glc  C48H82O18  945.5423  945.5451  2.96  Ginsenosides Rf  -Glc2-Glc  -H  C42H72O14  799.4844  799.4858  1.75  Ginsenosides Rg1  -Glc  -Glc  C42H72O14  799.4844  799.4859  1.88  Ginsenosides Rg2  -Glc2-Rha  -H  C42H72O13  783.4895  783.5066  2.18  Ginsenosides Rh1  -Glc  -H  C36H62O9  637.4316  637.4332  2.51  Ginsenosides F1  -H  -Glc  C36H62O9  637.4316  637.4329  2.04  Ginsenosides F3  -H  -Glc6-Ara(p)  C47H70O13  841.4742  841.4759  2.02  Notoginsenosides R1  -Glc2-Xyl  -Glc  C47H80O18  931.5267  931.5288  2.25  Notoginsenosides R2  -Glc2-Xyl  -H  C41H70O13  769.4739  769.4756  2.21    Ginsenosides Ro  -Glc2-Glc  -Glc  C48H76O19  955.4904  955.4926  2.30    Pseudoginsenoside F11  -Glc2-Rha  -H  C42H72O14  799.4844  799.4861  2.13  Structures  Ginsenosides  R1  R2  Molecular formula  Calculated mass [M–H]−  Measured mass [M–H]−  Mass error (ppm)    Ginsenosides Rb1  -Glc2-Glc  -Glc6-Glc  C54H92O23  1107.5952  1107.5972  1.81  Ginsenosides Rb2  -Glc2-Glc  -Glc6-Ara(p)  C53H90O22  1077.5846  1077.5875  2.69  Ginsenosides Rb3  -Glc2-Glc  -Glc6-Xyl  C53H90O22  1077.5846  1077.5868  2.04  Ginsenosides Rc  -Glc2-Glc  -Glc6-Ara(f)  C53H90O22  1077.5846  1077.5871  2.32  Ginsenosides Rd  -Glc2-Glc  -Glc  C48H82O18  945.5423  945.5446  2.43  Ginsenosides Rg3  -Glc2-Glc  -H  C42H72O13  783.4895  783.5096  2.57  Ginsenosides Rh2  -Glc  -H  C36H62O8  621.4367  621.4379  1.93  Ginsenosides F2  -Glc  -Glc  C42H72O13  783.4895  783.5084  2.41    Ginsenosides Re  -Glc2-Rha  -Glc  C48H82O18  945.5423  945.5451  2.96  Ginsenosides Rf  -Glc2-Glc  -H  C42H72O14  799.4844  799.4858  1.75  Ginsenosides Rg1  -Glc  -Glc  C42H72O14  799.4844  799.4859  1.88  Ginsenosides Rg2  -Glc2-Rha  -H  C42H72O13  783.4895  783.5066  2.18  Ginsenosides Rh1  -Glc  -H  C36H62O9  637.4316  637.4332  2.51  Ginsenosides F1  -H  -Glc  C36H62O9  637.4316  637.4329  2.04  Ginsenosides F3  -H  -Glc6-Ara(p)  C47H70O13  841.4742  841.4759  2.02  Notoginsenosides R1  -Glc2-Xyl  -Glc  C47H80O18  931.5267  931.5288  2.25  Notoginsenosides R2  -Glc2-Xyl  -H  C41H70O13  769.4739  769.4756  2.21    Ginsenosides Ro  -Glc2-Glc  -Glc  C48H76O19  955.4904  955.4926  2.30    Pseudoginsenoside F11  -Glc2-Rha  -H  C42H72O14  799.4844  799.4861  2.13  Figure 1. View largeDownload slide Total ion current chromatograms of ginsenosides. (A) American ginseng from Jingyu, Jilin, China; (B) American ginseng from Wisconsin, USA; (C) Chinese ginseng from Jingyu, Jilin, China; (D) Sanchi from Wenshan, Yunnan, China. Figure 1. View largeDownload slide Total ion current chromatograms of ginsenosides. (A) American ginseng from Jingyu, Jilin, China; (B) American ginseng from Wisconsin, USA; (C) Chinese ginseng from Jingyu, Jilin, China; (D) Sanchi from Wenshan, Yunnan, China. The method validation results were shown in Table III. The retention times were with SD around 0.02 min (n = 10). The relative standard deviation (RSD) of intraday and interday repeatability ranged between 0.2 and 1.9%. The LOD and LOQ of 19 ginsenosides were assayed. Calibration curves were constructed from the peak areas and the concentration of each ginsenosides and showed good linearity in the range of studied concentrations. The recovery of the ginsenosides ranged from 95.3 to 108.1%. These results indicate that the UPLC orbitrap HRMS detection methods are precise, accurate and sensitive for simultaneously quantitative evaluation of ginsenosides in Panax genus studied. The results also demonstrate that the sample preparation method used could extract ginsenosides simultaneously from the root materials. Table III. Performance of the UPLC-HRMS Method for Ginsenosides Detection Standards  LOD (fg/μL)  LOQ (fg/μL)  Intraday repeatability RSD (%)  Interday repeatability RSD (%)  Correlation coefficient (R2)  Linear range (ng/μL)  Regression equations  Added concentration (ng/μL)  Recovery (%)  Ginsenosides Rb1  0.025  0.075  0.2  0.8  0.9995  0.5–30  Y = 54.01X − 3.301  0.75  100.9 ± 0.2  2.5  95.3 ± 0.3  10  96.4 ± 0.2  Ginsenosides Rb2  0.02  0.06  0.2  0.7  0.9999  1–20  Y = 83.63X − 1.842  2  101.3 ± 0.2  10  104.3 ± 0.4  20  98.3 ± 0.3  Ginsenosides Rb3  0.17  0.5  1.3  1.3  0.9998  1–40  Y = 65.78X − 4.365  1  102.2 ± 1.8  10  96.9 ± 1.0  40  101.8 ± 0.9  Ginsenosides Rc  0.13  0.4  0.8  0.7  0.9999  0.5–25  Y = 81.40X − 6.990  0.5  100.1 ± 0.4  2.5  98.6 ± 0.8  15  98.1 ± 0.5  Ginsenosides Rd  0.007  0.02  0.6  1.8  0.9999  1–20  Y = 39.89X − 2.201  1  99.6 ± 1.5  7.5  98.2 ± 1.0  20  103.3 ± 0.5  Ginsenosides Rg3  0.015  0.05  1.1  1.5  0.9994  1–20  Y = 0.4529X + 8.106  1  99.8 ± 0.9  7.5  103.4 ± 0.6  20  104.6 ± 0.5  Ginsenosides Rh2  0.02  0.06  0.7  0.9  0.9995  1–20  Y = 3642X − 92.08  1  99.9 ± 0.8  7.5  102.5 ± 0.7  20  99.4 ± 0.5  Ginsenosides F2  0.003  0.01  0.9  0.7  0.9992  0.2–4  Y = 424.7X − 3.902  0.2  100.7 ± 0.9  1  98.6 ± 0.4  4  105.4 ± 0.2  Ginsenosides Re  0.007  0.02  0.5  1.4  0.9998  2–40  Y = 16.20X + 0.03311  2  99.9 ± 0.5  10  96.4 ± 0.6  40  98.4 ± 0.3  Ginsenosides Rf  0.01  0.03  0.4  1.0  0.9995  1–20  Y = 89.24X − 11.466  1  100.4 ± 0.8  5  104.4 ± 0.6  20  105.1 ± 0.2  Ginsenosides Rg1  0.07  0.2  0.6  1.3  0.9996  0.5–10  Y = 39.23X − 3.908  0.5  101.9 ± 0.6  1.5  103.3 ± 0.5  10  106.1 ± 0.9  Ginsenosides Rg2  0.1  0.4  1.0  1.9  0.9992  1–80  Y = 0.2531X + 1.723  1  101.6 ± 0.7  10  98.5 ± 0.9  80  102.1 ± 0.7  Ginsenosides Rh1  0.16  0.5  0.9  1.4  0.9996  1–80  Y = 439.21X + 8317  1  101.5 ± 1.2  10  108.1 ± 1.0  80  106.6 ± 0.5  Ginsenosides F1  0.002  0.005  0.4  1.0  0.9997  0.01–0.2  Y = 529.2X − 7.706  0.01  100.5 ± 1.0  0.05  104.2 ± 0.6  1.5  99.9 ± 0.4  Ginsenosides F3  0.03  0.1  1.1  1.3  0.9996  0.5–15  Y = 236.3X + 5.644  0.5  101.3 ± 0.7  2.5  99.6 ± 0.6  10  99.4 ± 0.8  Notoginsenosides R1  0.2  0.6  0.6  1.4  0.9994  0.1–3  Y = 12.91X − 0.547  0.1  99.7 ± 0.5  0.5  101.2 ± 1.0  2.5  103.9 ± 0.9  Notoginsenosides R2  0.008  0.025  0.7  1.2  0.9997  0.05–1.5  Y= 26.14X − 0.231  0.05  99.9 ± 1.2  0.25  100.0 ± 1.1  1.5  99.8 ± 0.7  Ginsenosides Ro  0.01  0.03  0.5  0.9  0.9993  0.5–20  Y = 19.61X − 0.03604  0.5  101.0 ± 1.1  2.5  98.1 ± 1.0  10  98.9 ± 0.7  Pseudoginsenoside F11  0.007  0.02  1.2  1.8  0.9995  1–20  Y = 203.0X − 1.474  1  101.7 ± 0.8  5  100.0 ± 0.7  20  100.9 ± 1.1  Standards  LOD (fg/μL)  LOQ (fg/μL)  Intraday repeatability RSD (%)  Interday repeatability RSD (%)  Correlation coefficient (R2)  Linear range (ng/μL)  Regression equations  Added concentration (ng/μL)  Recovery (%)  Ginsenosides Rb1  0.025  0.075  0.2  0.8  0.9995  0.5–30  Y = 54.01X − 3.301  0.75  100.9 ± 0.2  2.5  95.3 ± 0.3  10  96.4 ± 0.2  Ginsenosides Rb2  0.02  0.06  0.2  0.7  0.9999  1–20  Y = 83.63X − 1.842  2  101.3 ± 0.2  10  104.3 ± 0.4  20  98.3 ± 0.3  Ginsenosides Rb3  0.17  0.5  1.3  1.3  0.9998  1–40  Y = 65.78X − 4.365  1  102.2 ± 1.8  10  96.9 ± 1.0  40  101.8 ± 0.9  Ginsenosides Rc  0.13  0.4  0.8  0.7  0.9999  0.5–25  Y = 81.40X − 6.990  0.5  100.1 ± 0.4  2.5  98.6 ± 0.8  15  98.1 ± 0.5  Ginsenosides Rd  0.007  0.02  0.6  1.8  0.9999  1–20  Y = 39.89X − 2.201  1  99.6 ± 1.5  7.5  98.2 ± 1.0  20  103.3 ± 0.5  Ginsenosides Rg3  0.015  0.05  1.1  1.5  0.9994  1–20  Y = 0.4529X + 8.106  1  99.8 ± 0.9  7.5  103.4 ± 0.6  20  104.6 ± 0.5  Ginsenosides Rh2  0.02  0.06  0.7  0.9  0.9995  1–20  Y = 3642X − 92.08  1  99.9 ± 0.8  7.5  102.5 ± 0.7  20  99.4 ± 0.5  Ginsenosides F2  0.003  0.01  0.9  0.7  0.9992  0.2–4  Y = 424.7X − 3.902  0.2  100.7 ± 0.9  1  98.6 ± 0.4  4  105.4 ± 0.2  Ginsenosides Re  0.007  0.02  0.5  1.4  0.9998  2–40  Y = 16.20X + 0.03311  2  99.9 ± 0.5  10  96.4 ± 0.6  40  98.4 ± 0.3  Ginsenosides Rf  0.01  0.03  0.4  1.0  0.9995  1–20  Y = 89.24X − 11.466  1  100.4 ± 0.8  5  104.4 ± 0.6  20  105.1 ± 0.2  Ginsenosides Rg1  0.07  0.2  0.6  1.3  0.9996  0.5–10  Y = 39.23X − 3.908  0.5  101.9 ± 0.6  1.5  103.3 ± 0.5  10  106.1 ± 0.9  Ginsenosides Rg2  0.1  0.4  1.0  1.9  0.9992  1–80  Y = 0.2531X + 1.723  1  101.6 ± 0.7  10  98.5 ± 0.9  80  102.1 ± 0.7  Ginsenosides Rh1  0.16  0.5  0.9  1.4  0.9996  1–80  Y = 439.21X + 8317  1  101.5 ± 1.2  10  108.1 ± 1.0  80  106.6 ± 0.5  Ginsenosides F1  0.002  0.005  0.4  1.0  0.9997  0.01–0.2  Y = 529.2X − 7.706  0.01  100.5 ± 1.0  0.05  104.2 ± 0.6  1.5  99.9 ± 0.4  Ginsenosides F3  0.03  0.1  1.1  1.3  0.9996  0.5–15  Y = 236.3X + 5.644  0.5  101.3 ± 0.7  2.5  99.6 ± 0.6  10  99.4 ± 0.8  Notoginsenosides R1  0.2  0.6  0.6  1.4  0.9994  0.1–3  Y = 12.91X − 0.547  0.1  99.7 ± 0.5  0.5  101.2 ± 1.0  2.5  103.9 ± 0.9  Notoginsenosides R2  0.008  0.025  0.7  1.2  0.9997  0.05–1.5  Y= 26.14X − 0.231  0.05  99.9 ± 1.2  0.25  100.0 ± 1.1  1.5  99.8 ± 0.7  Ginsenosides Ro  0.01  0.03  0.5  0.9  0.9993  0.5–20  Y = 19.61X − 0.03604  0.5  101.0 ± 1.1  2.5  98.1 ± 1.0  10  98.9 ± 0.7  Pseudoginsenoside F11  0.007  0.02  1.2  1.8  0.9995  1–20  Y = 203.0X − 1.474  1  101.7 ± 0.8  5  100.0 ± 0.7  20  100.9 ± 1.1  Performance of UPLC orbitrap HRMS detection The three kinds of ginseng roots, 19 ginsenosides were analyzed and compared in 24 samples (S1-S8) (Table IV). The values (mean ± SD, n = 10) are expressed on a dry weight basis. Ginsenosides were identified by comparing accurate mass of molecular ions and retention times of the samples against standards. The ginsenosides contents of some samples exceeded the linear range, so dilution before injection is needed for quantitation. The recovery values of different compounds were taken into account when the contents were calculated. The results showed an obvious chemical variety of 19 ginsenosides in the three Panax genus, which suggested that each medicinal plant had its own chemical characteristics. Table IV. Application of the UPLC Orbitrap HRMS Method Developed to Some Panax Genus Samples Sample  PPD (μg/mL)  OLE (μg/mL)  OCT (μg/mL)  G-Rb1  G-Rb2  G-Rb3  G-Rc  G-Rd  G-Rg3  G-Rh2  G-F2  G-Ro  P-F11  S1(1)  5.35 ± 0.55  0.42 ± 0.05  0.35 ± 0.03  2.09 ± 0.35  3.73 ± 0.29  BQ  -  (31.38 ± 2.42) × 10−3  1.79 ± 0.24  1.86 ± 0.15  S1(2)  5.90 ± 0.21  0.45 ± 0.10  0.35 ± 0.08  2.14 ± 0.29  3.83 ± 0.25  BQ  -  (24.54 ± 2.50) × 10−3  1.60 ± 0.12  1.90 ± 0.20  S1(3)  5.27 ± 0.33  0.44 ± 0.08  0.33 ± 0.06  2.10 ± 0.20  3.70 ± 0.21  BQ  -  (30.51 ± 2.01) × 10−3  1.90 ± 0.18  2.00 ± 0.14  S2(1)  5.25 ± 0.57  0.35 ± 0.05  0.48 ± 0.08  1.52 ± 0.15  2.79 ± 0.23  BQ  -  0.25 ± 0.01  0.90 ± 0.04  0.71 ± 0.02  S2(2)  5.36 ± 0.49  0.34 ± 0.01  0.45 ± 0.04  1.59 ± 0.20  2.85 ± 0.24  BQ  -  0.19 ± 0.02  0.84 ± 0.03  0.90 ± 0.04  S2(3)  5.74 ± 0.27  0.35 ± 0.03  0.47 ± 0.05  1.52 ± 0.07  3.00 ± 0.30  BQ  -  0.26 ± 0.01  0.77 ± 0.03  0.86 ± 0.05  S3(1)  5.63 ± 0.41  0.40 ± 0.02  0.67 ± 0.06  2.11 ± 0.19  2.76 ± 0.17  BQ  -  (17.44 ± 1.27) × 10−3  1.49 ± 0.09  1.49 ± 0.11  S3(2)  5.81 ± 0.33  0.41 ± 0.02  0.66 ± 0.05  2.14 ± 0.25  2.95 ± 0.26  BQ  -  (18.22 ± 1.37) × 10−3  1.30 ± 0.10  1.56 ± 0.15  S3(3)  5.41 ± 0.39  0.37 ± 0.01  0.61 ± 0.06  2.00 ± 0.22  2.84 ± 0.24  BQ  -  (17.99 ± 1.64) × 10−3  1.56 ± 0.11  1.34 ± 0.14  S4(1)  10.34 ± 0.90  3.12 ± 0.15  1.15 ± 0.09  9.64 ± 0.80  9.77 ± 0.70  (16.78 ± 1.01) × 10−3  -  1.19 ± 0.20  4.29 ± 0.20  4.65 ± 0.20  S4(2)  9.99 ± 1.0  3.18 ± 0.17  1.01 ± 0.04  9.50 ± 0.77  9.69 ± 0.71  (17.00 ± 1.46) × 10−3  -  1.30 ± 0.10  4.60 ± 0.24  4.90 ± 0.21  S4(3)  10.11 ± 0.94  3.09 ± 0.11  1.10 ± 0.08  9.60 ± 0.70  9.81 ± 0.84  (16.20 ± 1.00) × 10−3  -  1.00 ± 0.09  4.90 ± 0.26  4.65 ± 0.25  S5(1)  1.62 ± 0.10  1.90 ± 0.19  1.99 ± 0.10  0.12 ± 0.01  1.76 ± 0.07  BQ  (13.96 ± 1.12) × 10−3  (10.52 ± 0.83) × 10−3  0.94 ± 0.02  -  S5(2)  1.55 ± 0.12  1.68 ± 0.13  1.89 ± 0.07  0.11 ± 0.01  1.56 ± 0.05  BQ  (13.01 ± 1.51) × 10−3  (10.88 ± 0.75) × 10−3  0.99 ± 0.02  -  S5(3)  1.89 ± 0.11  1.82 ± 0.12  2.00 ± 0.10  0.12 ± 0.01  1.39 ± 0.07  BQ  (14.56 ± 0.84) × 10−3  (9.74 ± 1.21) × 10−3  1.10 ± 0.04  -  S6(1)  1.59 ± 0.21  1.77 ± 0.20  2.11 ± 0.09  0.13 ± 0.01  1.44 ± 0.06  BQ  (13.74 ± 0.99) × 10−3  (10.15 ± 0.94) × 10−3  1.01 ± 0.09  -  S6(2)  1.65 ± 0.16  1.91 ± 0.18  1.97 ± 0.11  0.13 ± 0.01  1.50 ± 0.05  BQ  (14.10 ± 1.11) × 10−3  (10.47 ± 0.81) × 10−3  1.20 ± 0.05  -  S6(3)  1.78 ± 0.11  1.80 ± 0.14  1.89 ± 0.12  0.10 ± 0.01  1.67 ± 0.05  BQ  (13.06 ± 1.14) × 10−3  (9.99 ± 0.89) × 10−3  0.98 ± 0.03  -  S7(1)  7.29 ± 0.20  0.16 ± 0.01  0.72 ± 0.08  1.73 ± 0.05  3.86 ± 0.19  (15.33 ± 1.32) × 10−3  (19.28 ± 1.01) × 10−3  0.33 ± 0.02  -  -  S7(2)  8.00 ± 0.38  0.16 ± 0.01  0.81 ± 0.07  1.69 ± 0.06  3.66 ± 0.18  (15.90 ± 1.20) × 10−3  (14.95 ± 1.17) × 10−3  0.30 ± 0.02  -  -  S7(3)  7.63 ± 0.54  0.15 ± 0.01  0.74 ± 0.07  1.73 ± 0.08  3.57 ± 0.20  (14.78 ± 1.40) × 10−3  (16.99 ± 0.89) × 10−3  0.40 ± 0.01  -  -  S8(1)  7.54 ± 0.40  0.17 ± 0.01  0.79 ± 0.06  1.77 ± 0.06  3.66 ± 0.19  (15.26 ± 1.25) × 10−3  (17.26 ± 0.96) × 10−3  0.44 ± 0.02  -  -  S8(2)  8.11 ± 0.53  0.18 ± 0.01  0.70 ± 0.05  1.75 ± 0.07  3.89 ± 0.18  (15.74 ± 1.13) × 10−3  (18.37 ± 1.04) × 10−3  0.50 ± 0.01  -  -  S8(3)  7.96 ± 0.47  0.17 ± 0.01  0.82 ± 0.06  1.78 ± 0.06  3.50 ± 0.14  (15.07 ± 1.20) × 10−3  (19.15 ± 1.10) × 10−3  0.40 ± 0.01  -  -  S9  3.51 ± 0.23  0.22 ± 0.01  0.30 ± 0.06  1.10 ± 0.09  1.97 ± 0.10  BQ  -  0.38 ± 0.01  1.58 ± 0.04  0.19 ± 0.01  S10  4.68 ± 0.30  0.28 ± 0.02  0.22 ± 0.04  1.55 ± 0.07  1.43 ± 0.11  BQ  -  0.05 ± 0.001  2.61 ± 0.05  0.54 ± 0.01  S11  4.40 ± 0.30  0.16 ± 0.009  0.53 ± 0.04  0.91 ± 0.05  1.97 ± 0.10  (13.85 ± 1.27) × 10−3  -  0.24 ± 0.01  2.24 ± 0.04  1.07 ± 0.05  S12  4.30 ± 0.40  0.22 ± 0.01  0.39 ± 0.04  1.12 ± 0.07  1.76 ± 0.09  BQ  -  0.14 ± 0.01  2.16 ± 0.08  1.15 ± 0.04  S13  6.15 ± 0.59  0.51 ± 0.02  0.54 ± 0.05  2.46 ± 0.19  3.55 ± 0.21  (12.73 ± 0.85) × 10−3  -  0.21 ± 0.02  3.74 ± 0.08  2.33 ± 0.05  S14  0.03 ± 0.002  0.12 ± 0.01  0.44 ± 0.06  0.04 ± 0.001  0.30 ± 0.11  BQ  -  0.05 ± 0.001  (4.66 ± 0.71) × 10−3  0.34 ± 0.01  Sample  PPD (μg/mL)  OLE (μg/mL)  OCT (μg/mL)  G-Rb1  G-Rb2  G-Rb3  G-Rc  G-Rd  G-Rg3  G-Rh2  G-F2  G-Ro  P-F11  S1(1)  5.35 ± 0.55  0.42 ± 0.05  0.35 ± 0.03  2.09 ± 0.35  3.73 ± 0.29  BQ  -  (31.38 ± 2.42) × 10−3  1.79 ± 0.24  1.86 ± 0.15  S1(2)  5.90 ± 0.21  0.45 ± 0.10  0.35 ± 0.08  2.14 ± 0.29  3.83 ± 0.25  BQ  -  (24.54 ± 2.50) × 10−3  1.60 ± 0.12  1.90 ± 0.20  S1(3)  5.27 ± 0.33  0.44 ± 0.08  0.33 ± 0.06  2.10 ± 0.20  3.70 ± 0.21  BQ  -  (30.51 ± 2.01) × 10−3  1.90 ± 0.18  2.00 ± 0.14  S2(1)  5.25 ± 0.57  0.35 ± 0.05  0.48 ± 0.08  1.52 ± 0.15  2.79 ± 0.23  BQ  -  0.25 ± 0.01  0.90 ± 0.04  0.71 ± 0.02  S2(2)  5.36 ± 0.49  0.34 ± 0.01  0.45 ± 0.04  1.59 ± 0.20  2.85 ± 0.24  BQ  -  0.19 ± 0.02  0.84 ± 0.03  0.90 ± 0.04  S2(3)  5.74 ± 0.27  0.35 ± 0.03  0.47 ± 0.05  1.52 ± 0.07  3.00 ± 0.30  BQ  -  0.26 ± 0.01  0.77 ± 0.03  0.86 ± 0.05  S3(1)  5.63 ± 0.41  0.40 ± 0.02  0.67 ± 0.06  2.11 ± 0.19  2.76 ± 0.17  BQ  -  (17.44 ± 1.27) × 10−3  1.49 ± 0.09  1.49 ± 0.11  S3(2)  5.81 ± 0.33  0.41 ± 0.02  0.66 ± 0.05  2.14 ± 0.25  2.95 ± 0.26  BQ  -  (18.22 ± 1.37) × 10−3  1.30 ± 0.10  1.56 ± 0.15  S3(3)  5.41 ± 0.39  0.37 ± 0.01  0.61 ± 0.06  2.00 ± 0.22  2.84 ± 0.24  BQ  -  (17.99 ± 1.64) × 10−3  1.56 ± 0.11  1.34 ± 0.14  S4(1)  10.34 ± 0.90  3.12 ± 0.15  1.15 ± 0.09  9.64 ± 0.80  9.77 ± 0.70  (16.78 ± 1.01) × 10−3  -  1.19 ± 0.20  4.29 ± 0.20  4.65 ± 0.20  S4(2)  9.99 ± 1.0  3.18 ± 0.17  1.01 ± 0.04  9.50 ± 0.77  9.69 ± 0.71  (17.00 ± 1.46) × 10−3  -  1.30 ± 0.10  4.60 ± 0.24  4.90 ± 0.21  S4(3)  10.11 ± 0.94  3.09 ± 0.11  1.10 ± 0.08  9.60 ± 0.70  9.81 ± 0.84  (16.20 ± 1.00) × 10−3  -  1.00 ± 0.09  4.90 ± 0.26  4.65 ± 0.25  S5(1)  1.62 ± 0.10  1.90 ± 0.19  1.99 ± 0.10  0.12 ± 0.01  1.76 ± 0.07  BQ  (13.96 ± 1.12) × 10−3  (10.52 ± 0.83) × 10−3  0.94 ± 0.02  -  S5(2)  1.55 ± 0.12  1.68 ± 0.13  1.89 ± 0.07  0.11 ± 0.01  1.56 ± 0.05  BQ  (13.01 ± 1.51) × 10−3  (10.88 ± 0.75) × 10−3  0.99 ± 0.02  -  S5(3)  1.89 ± 0.11  1.82 ± 0.12  2.00 ± 0.10  0.12 ± 0.01  1.39 ± 0.07  BQ  (14.56 ± 0.84) × 10−3  (9.74 ± 1.21) × 10−3  1.10 ± 0.04  -  S6(1)  1.59 ± 0.21  1.77 ± 0.20  2.11 ± 0.09  0.13 ± 0.01  1.44 ± 0.06  BQ  (13.74 ± 0.99) × 10−3  (10.15 ± 0.94) × 10−3  1.01 ± 0.09  -  S6(2)  1.65 ± 0.16  1.91 ± 0.18  1.97 ± 0.11  0.13 ± 0.01  1.50 ± 0.05  BQ  (14.10 ± 1.11) × 10−3  (10.47 ± 0.81) × 10−3  1.20 ± 0.05  -  S6(3)  1.78 ± 0.11  1.80 ± 0.14  1.89 ± 0.12  0.10 ± 0.01  1.67 ± 0.05  BQ  (13.06 ± 1.14) × 10−3  (9.99 ± 0.89) × 10−3  0.98 ± 0.03  -  S7(1)  7.29 ± 0.20  0.16 ± 0.01  0.72 ± 0.08  1.73 ± 0.05  3.86 ± 0.19  (15.33 ± 1.32) × 10−3  (19.28 ± 1.01) × 10−3  0.33 ± 0.02  -  -  S7(2)  8.00 ± 0.38  0.16 ± 0.01  0.81 ± 0.07  1.69 ± 0.06  3.66 ± 0.18  (15.90 ± 1.20) × 10−3  (14.95 ± 1.17) × 10−3  0.30 ± 0.02  -  -  S7(3)  7.63 ± 0.54  0.15 ± 0.01  0.74 ± 0.07  1.73 ± 0.08  3.57 ± 0.20  (14.78 ± 1.40) × 10−3  (16.99 ± 0.89) × 10−3  0.40 ± 0.01  -  -  S8(1)  7.54 ± 0.40  0.17 ± 0.01  0.79 ± 0.06  1.77 ± 0.06  3.66 ± 0.19  (15.26 ± 1.25) × 10−3  (17.26 ± 0.96) × 10−3  0.44 ± 0.02  -  -  S8(2)  8.11 ± 0.53  0.18 ± 0.01  0.70 ± 0.05  1.75 ± 0.07  3.89 ± 0.18  (15.74 ± 1.13) × 10−3  (18.37 ± 1.04) × 10−3  0.50 ± 0.01  -  -  S8(3)  7.96 ± 0.47  0.17 ± 0.01  0.82 ± 0.06  1.78 ± 0.06  3.50 ± 0.14  (15.07 ± 1.20) × 10−3  (19.15 ± 1.10) × 10−3  0.40 ± 0.01  -  -  S9  3.51 ± 0.23  0.22 ± 0.01  0.30 ± 0.06  1.10 ± 0.09  1.97 ± 0.10  BQ  -  0.38 ± 0.01  1.58 ± 0.04  0.19 ± 0.01  S10  4.68 ± 0.30  0.28 ± 0.02  0.22 ± 0.04  1.55 ± 0.07  1.43 ± 0.11  BQ  -  0.05 ± 0.001  2.61 ± 0.05  0.54 ± 0.01  S11  4.40 ± 0.30  0.16 ± 0.009  0.53 ± 0.04  0.91 ± 0.05  1.97 ± 0.10  (13.85 ± 1.27) × 10−3  -  0.24 ± 0.01  2.24 ± 0.04  1.07 ± 0.05  S12  4.30 ± 0.40  0.22 ± 0.01  0.39 ± 0.04  1.12 ± 0.07  1.76 ± 0.09  BQ  -  0.14 ± 0.01  2.16 ± 0.08  1.15 ± 0.04  S13  6.15 ± 0.59  0.51 ± 0.02  0.54 ± 0.05  2.46 ± 0.19  3.55 ± 0.21  (12.73 ± 0.85) × 10−3  -  0.21 ± 0.02  3.74 ± 0.08  2.33 ± 0.05  S14  0.03 ± 0.002  0.12 ± 0.01  0.44 ± 0.06  0.04 ± 0.001  0.30 ± 0.11  BQ  -  0.05 ± 0.001  (4.66 ± 0.71) × 10−3  0.34 ± 0.01  Sample  PPT (μg/mL)  PPD/PPT  G-Re  G-Rf  G-Rg1  G-Rg2  G-Rh1  G-F1  G-F3  N-R1  N-R2  S1(1)  3.38 ± 0.46  -  0.61 ± 0.05  -  (39.41 ± 1.99) × 10−3  (10.48 ± 1.95) × 10−3  -  -  -  2.97  S1(2)  3.46 ± 0.33  -  0.80 ± 0.04  -  (46.82 ± 1.74) × 10−3  (11.00 ± 2.38) × 10−3  -  -  -  2.94  S1(3)  3.29 ± 0.41  -  0.75 ± 0.05  -  (30.43 ± 1.54) × 10−3  (9.45 ± 1.77) × 10−3  -  -  -  2.94  S2(1)  3.23 ± 0.29  -  0.27 ± 0.06  -  (29.28 ± 1.48) × 10−3  (14.34 ± 2.18) × 10−3  -  -  -  3.01  S2(2)  3.09 ± 0.30  -  0.20 ± 0.06  -  (27.34 ± 1.73) × 10−3  (13.76 ± 2.48) × 10−3  -  -  -  3.24  S2(3)  3.15 ± 0.34  -  0.33 ± 0.05  -  (14.99 ± 2.00) × 10−3  (13.42 ± 2.33) × 10−3  -  -  -  3.24  S3(1)  3.62 ± 0.33  -  0.31 ± 0.04  -  (45.28 ± 1.38) × 10−3  (11.54 ± 1.81) × 10−3  -  -  -  2.91  S3(2)  3.55 ± 0.47  -  0.30 ± 0.02  -  (89.00 ± 2.33) × 10−3  (11.59 ± 1.94) × 10−3  -  -  -  3.04  S3(3)  3.28 ± 0.44  -  0.21 ± 0.23  -  (65.34 ± 2.85) × 10−3  (10.57 ± 2.00) × 10−3  -  -  -  3.21  S4(1)  9.94 ± 0.69  -  1.38 ± 0.11  -  0.21 ± 0.02  (40.44 ± 3.00) × 10−3  -  -  -  3.05  S4(2)  9.80 ± 0.67  -  1.40 ± 0.12  -  0.30 ± 0.04  (34.11 ± 2.30) × 10−3  -  -  -  3.01  S4(3)  10.02 ± 1.20  -  1.50 ± 0.12  -  0.10 ± 0.01  (38.62 ± 3.11) × 10−3  -  -  -  2.98  S5(1)  1.69 ± 0.21  0.73 ± 0.05  0.70 ± 0.01  0.86 ± 0.02  1.40 ± 0.10  (12.58 ± 2.55) × 10−3  (35.19 ± 3.38) × 10−3  BQ  BQ  1.37  S5(2)  1.98 ± 0.23  0.80 ± 0.05  0.61 ± 0.04  0.80 ± 0.03  1.31 ± 0.10  (15.98 ± 2.88) × 10−3  (36.07 ± 2.04) × 10−3  BQ  BQ  1.22  S5(3)  1.69 ± 0.22  0.79 ± 0.04  0.68 ± 0.02  0.95 ± 0.03  0.50 ± 0.02  (15.01 ± 2.43) × 10−3  (24.88 ± 3.24) × 10−3  BQ  BQ  1.55  S6(1)  1.88 ± 0.20  0.77 ± 0.04  0.72 ± 0.03  0.88 ± 0.02  1.44 ± 0.09  (14.39 ± 1.84) × 10−3  (32.45 ± 1.89) × 10−3  BQ  BQ  1.22  S6(2)  1.82 ± 0.19  0.84 ± 0.03  0.69 ± 0.02  0.89 ± 0.03  0.90 ± 0.03  (13.93 ± 2.18) × 10−3  (34.52 ± 2.23) × 10−3  BQ  BQ  1.37  S6(3)  1.79 ± 0.21  0.82 ± 0.04  0.67 ± 0.03  0.90 ± 0.02  1.19 ± 0.05  (13.57 ± 2.00) × 10−3  (37.57 ± 2.74) × 10−3  BQ  BQ  1.33  S7(1)  1.59 ± 0.22  -  16.02 ± 1.55  2.30 ± 0.20  4.50 ± 0.15  (55.46 ± 2.55) × 10−3  (25.54 ± 2.54) × 10−3  11.79 ± 2.33  0.84 ± 0.05  0.38  S7(2)  1.60 ± 0.21  -  15.09 ± 1.56  2.51 ± 0.25  3.00 ± 0.12  (51.39 ± 2.64) × 10−3  (24.37 ± 3.01) × 10−3  12.22 ± 3.10  0.66 ± 0.04  0.42  S7(3)  1.44 ± 0.28  -  15.66 ± 1.70  2.49 ± 0.24  4.20 ± 0.14  (52.77 ± 3.00) × 10−3  (30.22 ± 3.20) × 10−3  12.05 ± 3.52  0.79 ± 0.05  0.39  S8(1)  1.66 ± 0.23  -  15.90 ± 1.58  2.53 ± 0.22  4.56 ± 0.13  (54.81 ± 2.22) × 10−3  (28.77 ± 2.15) × 10−3  12.24 ± 2.99  0.69 ± 0.03  0.40  S8(2)  1.59 ± 0.24  -  15.67 ± 1.65  2.54 ± 0.20  4.35 ± 0.11  (55.53 ± 2.50) × 10−3  (29.30 ± 2.84) × 10−3  11.99 ± 3.00  0.89 ± 0.04  0.43  S8(3)  1.51 ± 0.25  -  15.74 ± 1.60  2.60 ± 0.24  4.25 ± 0.15  (50.43 ± 2.90) × 10−3  (25.90 ± 3.12) × 10−3  12.20 ± 3.27  0.75 ± 0.05  0.41  S9  2.17 ± 0.40  -  1.01 ± 0.11  -  (15.07 ± 2.30) × 10−3  (8.68 ± 1.38) × 10−3  -  -  -  2.34  S10  3.05 ± 0.39  -  0.78 ± 0.08  -  0.12 ± 0.01  (9.43 ± 2.00) × 10−3  -  -  -  2.07  S11  3.42 ± 0.38  -  0.35 ± 0.04  -  0.19 ± 0.01  (7.99 ± 1.83) × 10−3  -  -  -  2.07  S12  3.40 ± 0.38  -  0.33 ± 0.05  -  0.20 ± 0.02  (6.84 ± 1.99) × 10−3  -  -  -  2.01  S13  3.66 ± 0.40  -  0.68 ± 0.06  -  0.16 ± 0.01  (15.40 ± 2.48) × 10−3  -  -  -  2.98  S14  0.29 ± 0.08  -  0.03 ± 0.000  -  0.03 ± 0.001  (5.52 ± 1.40) × 10−3  -  -  -  2.68  Sample  PPT (μg/mL)  PPD/PPT  G-Re  G-Rf  G-Rg1  G-Rg2  G-Rh1  G-F1  G-F3  N-R1  N-R2  S1(1)  3.38 ± 0.46  -  0.61 ± 0.05  -  (39.41 ± 1.99) × 10−3  (10.48 ± 1.95) × 10−3  -  -  -  2.97  S1(2)  3.46 ± 0.33  -  0.80 ± 0.04  -  (46.82 ± 1.74) × 10−3  (11.00 ± 2.38) × 10−3  -  -  -  2.94  S1(3)  3.29 ± 0.41  -  0.75 ± 0.05  -  (30.43 ± 1.54) × 10−3  (9.45 ± 1.77) × 10−3  -  -  -  2.94  S2(1)  3.23 ± 0.29  -  0.27 ± 0.06  -  (29.28 ± 1.48) × 10−3  (14.34 ± 2.18) × 10−3  -  -  -  3.01  S2(2)  3.09 ± 0.30  -  0.20 ± 0.06  -  (27.34 ± 1.73) × 10−3  (13.76 ± 2.48) × 10−3  -  -  -  3.24  S2(3)  3.15 ± 0.34  -  0.33 ± 0.05  -  (14.99 ± 2.00) × 10−3  (13.42 ± 2.33) × 10−3  -  -  -  3.24  S3(1)  3.62 ± 0.33  -  0.31 ± 0.04  -  (45.28 ± 1.38) × 10−3  (11.54 ± 1.81) × 10−3  -  -  -  2.91  S3(2)  3.55 ± 0.47  -  0.30 ± 0.02  -  (89.00 ± 2.33) × 10−3  (11.59 ± 1.94) × 10−3  -  -  -  3.04  S3(3)  3.28 ± 0.44  -  0.21 ± 0.23  -  (65.34 ± 2.85) × 10−3  (10.57 ± 2.00) × 10−3  -  -  -  3.21  S4(1)  9.94 ± 0.69  -  1.38 ± 0.11  -  0.21 ± 0.02  (40.44 ± 3.00) × 10−3  -  -  -  3.05  S4(2)  9.80 ± 0.67  -  1.40 ± 0.12  -  0.30 ± 0.04  (34.11 ± 2.30) × 10−3  -  -  -  3.01  S4(3)  10.02 ± 1.20  -  1.50 ± 0.12  -  0.10 ± 0.01  (38.62 ± 3.11) × 10−3  -  -  -  2.98  S5(1)  1.69 ± 0.21  0.73 ± 0.05  0.70 ± 0.01  0.86 ± 0.02  1.40 ± 0.10  (12.58 ± 2.55) × 10−3  (35.19 ± 3.38) × 10−3  BQ  BQ  1.37  S5(2)  1.98 ± 0.23  0.80 ± 0.05  0.61 ± 0.04  0.80 ± 0.03  1.31 ± 0.10  (15.98 ± 2.88) × 10−3  (36.07 ± 2.04) × 10−3  BQ  BQ  1.22  S5(3)  1.69 ± 0.22  0.79 ± 0.04  0.68 ± 0.02  0.95 ± 0.03  0.50 ± 0.02  (15.01 ± 2.43) × 10−3  (24.88 ± 3.24) × 10−3  BQ  BQ  1.55  S6(1)  1.88 ± 0.20  0.77 ± 0.04  0.72 ± 0.03  0.88 ± 0.02  1.44 ± 0.09  (14.39 ± 1.84) × 10−3  (32.45 ± 1.89) × 10−3  BQ  BQ  1.22  S6(2)  1.82 ± 0.19  0.84 ± 0.03  0.69 ± 0.02  0.89 ± 0.03  0.90 ± 0.03  (13.93 ± 2.18) × 10−3  (34.52 ± 2.23) × 10−3  BQ  BQ  1.37  S6(3)  1.79 ± 0.21  0.82 ± 0.04  0.67 ± 0.03  0.90 ± 0.02  1.19 ± 0.05  (13.57 ± 2.00) × 10−3  (37.57 ± 2.74) × 10−3  BQ  BQ  1.33  S7(1)  1.59 ± 0.22  -  16.02 ± 1.55  2.30 ± 0.20  4.50 ± 0.15  (55.46 ± 2.55) × 10−3  (25.54 ± 2.54) × 10−3  11.79 ± 2.33  0.84 ± 0.05  0.38  S7(2)  1.60 ± 0.21  -  15.09 ± 1.56  2.51 ± 0.25  3.00 ± 0.12  (51.39 ± 2.64) × 10−3  (24.37 ± 3.01) × 10−3  12.22 ± 3.10  0.66 ± 0.04  0.42  S7(3)  1.44 ± 0.28  -  15.66 ± 1.70  2.49 ± 0.24  4.20 ± 0.14  (52.77 ± 3.00) × 10−3  (30.22 ± 3.20) × 10−3  12.05 ± 3.52  0.79 ± 0.05  0.39  S8(1)  1.66 ± 0.23  -  15.90 ± 1.58  2.53 ± 0.22  4.56 ± 0.13  (54.81 ± 2.22) × 10−3  (28.77 ± 2.15) × 10−3  12.24 ± 2.99  0.69 ± 0.03  0.40  S8(2)  1.59 ± 0.24  -  15.67 ± 1.65  2.54 ± 0.20  4.35 ± 0.11  (55.53 ± 2.50) × 10−3  (29.30 ± 2.84) × 10−3  11.99 ± 3.00  0.89 ± 0.04  0.43  S8(3)  1.51 ± 0.25  -  15.74 ± 1.60  2.60 ± 0.24  4.25 ± 0.15  (50.43 ± 2.90) × 10−3  (25.90 ± 3.12) × 10−3  12.20 ± 3.27  0.75 ± 0.05  0.41  S9  2.17 ± 0.40  -  1.01 ± 0.11  -  (15.07 ± 2.30) × 10−3  (8.68 ± 1.38) × 10−3  -  -  -  2.34  S10  3.05 ± 0.39  -  0.78 ± 0.08  -  0.12 ± 0.01  (9.43 ± 2.00) × 10−3  -  -  -  2.07  S11  3.42 ± 0.38  -  0.35 ± 0.04  -  0.19 ± 0.01  (7.99 ± 1.83) × 10−3  -  -  -  2.07  S12  3.40 ± 0.38  -  0.33 ± 0.05  -  0.20 ± 0.02  (6.84 ± 1.99) × 10−3  -  -  -  2.01  S13  3.66 ± 0.40  -  0.68 ± 0.06  -  0.16 ± 0.01  (15.40 ± 2.48) × 10−3  -  -  -  2.98  S14  0.29 ± 0.08  -  0.03 ± 0.000  -  0.03 ± 0.001  (5.52 ± 1.40) × 10−3  -  -  -  2.68  Values are means (n = 10) of dry weight. “-”: not detected; BQ: below the limit of quantitation. Determination of ginsenosides in American Ginseng from China and Northern America Each of the three roots of American ginseng from Wisconsin (S1(1–3)), Fusong (S2(1–3)) and Jingyu (S3(1–3)) were found to contain significant amounts of pseudoginsenoside F11 at 0.71–2.00 μg/mg, but no ginsenoside Rf, notoginsenoside R1 and notoginsenoside R2 were detected. Among the 19 ginsenosides, Rb1, Re and Rd are the most abundant ginsenosides in S1-3. However, contents of ginsenoside Ro and pseudoginsenoside F11 in S2 appear to be lower significantly than those in S1 and S3, while the reverse is true for ginsenoside F2. But no significant differences between S1 and S3 were observed. Variations may occur due to various factors, such as geographical source, cultivation, harvest, storage and processing of the roots. However, we could not find obvious differences in the ginsenoside profiles of genetically same American ginseng from grown in Northern America or China. By comparing the ratios of PPD and PPT type ginsenosides’ total contents (PPD/PPT in Table IV) in extracts of American ginseng roots from China and North America, it was found that the American ginseng grown in China and North America show no major differences. Difference of ginsenosides in American ginseng main roots and hair roots from Jingyu, Jilin, China The investigation of different sections of American ginseng, main roots (S3(1–3)) and hair roots (S4(1–3)), showed that the contents of ginsenosides depending on the root type/diameter. The significantly higher contents of ginsenosides found in hair roots compared to main roots have led to the proposal that ginsenosides are mainly distributed in the periderm and cortex root tissues. As shown in Table IV, the total contents of PPD and PPT type ginsenosides were three times higher in root hairs. By comparing the ratios of PPD and PPT, no significant difference was found between main roots and root hairs. Authentication of American ginseng with Chinese ginseng and Sanchi The variation in the amount of 19 investigated ginsenosides in three Panax species was up to 50-folds. Peudoginsenoside F11 was only present in American ginseng (S1, S2, S3), whereas ginsenoside Rf is only present in Chinese ginseng (S5, S6). However, the situation is far more complex for the species Sanchi. The typical ginsenosides in Sanchi (S7, S8) is notoginsenoside R1 and notoginsenoside R2. Although these two ginsenosides were totally absent from American ginseng, it can still be found in trace amounts in Chinese ginseng. Fortunately, Sanchi does not contain ginsenoside Rf, which is the typical in Chinese ginseng. This can be conveniently employed in the differentiation among the three Panax species. It was also found that oleanane type ginsenoside Ro is present in American ginseng and Chinese ginseng, and absent in Sanchi. Therefore, the characteristics of ingredients can be used as markers for the discrimination of three Panax species. Meanwhile, the ratio of PPD/PPT showed certain regularity. A higher PPD/PPT ratio with value around 3 was usually indicative of American ginseng, while PPD/PPT value between 1 and 3 were usually characteristic for Chinese ginseng. Sanchi presented the lowest ratio of PPD/PPT less than 1. The differences in ginsenosides contents were probably a result of genetic differences among the three kinds of ginsengs. And these differences may explain their different medicinal efficacy. PCA of Panax genus The ginsenosides contents of 10 batches of each root sample were studied by PCA, and the differences resulting from botanical origin of American ginseng, Chinese ginseng and Sanchi were displayed. Importances of principal components and the contribution rate is the basis to choose principal component. After elimination of spectral outliers, PCA was applied to eliminate the data collinearity and to reduce the number of variables. The variances explained were shown in Table V. The results demonstrated that 81.795% contribution of the total variances is from the first two factors. A two factors model could explain the 81.795% experimental data. The projection display analysis of PCA was applied. The PCA factor scores obtained for the coordinate axis. The three-dimensional scatter of S1-S8 was shown in Figure 2. The American ginseng main roots from Wisconsin (S1), Fusong (S2) and Jingyu (S3) were clustered as one type, it also demonstrated that the American ginseng grown in China and North America show no major differences. And while Chinese ginseng (S5, S6) and Sanchi (S7, S8) were clustered as the other two types, respectively. So the distinction of similar species was obtained. The American ginseng hair roots were clustered as one type. And each type was with high clustered degree, respectively. Table V. The Variances Explained of Panax Genus Comp.  Standard deviation  Variance proportion (%)  Cumulative proportion (%)  1  2.946  45.680  45.680  2  2.620  36.115  81.795  3  1.768  16.453  98.248  Comp.  Standard deviation  Variance proportion (%)  Cumulative proportion (%)  1  2.946  45.680  45.680  2  2.620  36.115  81.795  3  1.768  16.453  98.248  Figure 2. View largeDownload slide PCA three-dimensional scatter of Panax genus. Figure 2. View largeDownload slide PCA three-dimensional scatter of Panax genus. Evaluation of commercial American ginseng products from different pharmacy Using the methods we developed, 19 ginsenosides levels were determined for 6 commercial American ginseng products samples (S9-S14) (Table IV), including 2 main roots, 3 main roots slices and 1 powder sample of extracted total ginsenosides. Applying the PLS-DA procedures in evaluation of the six commercial products, the principal components were screened out using the leave-one-out method. Error rate detection with centroids distance, max distance and mahalanobis distance were compared, all of them made the least error rate when using three principal components. The high rate of correct discrimination (87.30%) for American ginseng was considered by 10-fold-cross validation. Two brands of American ginseng main roots (S9, S10) and two brands of American ginseng main root slices (S11, S12) were found to contain pseudoginsenoside F11 without ginsenoside Rf, notoginsenoside R1 and notoginsenoside R2. It demonstrated that these four products are true for American ginseng as described. However ginsenosides detected were less than those described above, and the contents of PPD and PPT ginsenosides and the ratio of PPD/PPT were also less than samples S1, S2, S3. The PLS-DA results indicated that S9, S10 and S12 were classified as American ginseng from Fusong, and S11 was classified as American ginseng from Jingyu. Among these products, one brand of American ginseng slices (S13) that claimed to be made from American ginseng, whereas the contents of pseudoginsenoside F11, PPD and PPT types ginsenosides and the ratio of PPD/PPT were similar with the American ginseng from Wisconsin (S1). The PLS-DA results also indicated that S13 was classified as American ginseng from Wisconsin. For the extracted powder product (S14), it was actually derived from American ginseng. The values of PPD/PPT ratio was almost 3, whereas the contents of PPD and PPT types ginsenosides were much less than the ones in American ginseng main roots from Jingyu. The PLS-DA results also indicated that S14 was classified as American ginseng from Fusong. This result demonstrates that the extract procedure for this product with lower extraction efficiency, we could speculate that the purity of this product might be not enough for clinical usage. Discussion One aim of this work was to develop and validate an UPLC orbitrap HRMS method with high sensitivity and selectivity in order to simultaneously detect and quantify ginsenosides in Panax genus root materials. Since root tissues contain a variety of primary and secondary metabolites, the extraction of the compounds from samples of diverse origin needed to be taken into account for their simultaneous analysis. The matrix did not significantly affect the detection and the recoveries obtained were applicable to all the samples. Ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg3, Rh1, F1 and F2 were detected in most of the samples, while ginsenosides Rg1, Rh1, F1 and F2 have a wide range of concentrations. Our results not only confirm some published findings (19, 23–25) but also provide evidence that American ginseng contain detectable and quantifiable amounts of ginsenosides Rg3, Rh1, F1, F2. Because of the great similarity in chemical constituents, the difference within the same American ginseng species cultivated in different geographical locations was not significant. The differences among species in the same Panax genus are much more significant than those within the species. According to respective and total contents of PPD and PPT and the ratio of PPD/PPT, three species of Panax genus were distinguished. American ginseng, Chinese ginseng and Sanchi were clustered as three types by PCA. Thus the differentiation of cultivation regions as part of the quality control process is more difficult than that among different species of the Panax genus. The PLS-DA results reported here demonstrated that the quality of some brands of American ginseng products might have some defects. The use of UPLC orbitrap HRMS analysis allowed improvement in identification and quantification of ginsenosides, only simple full scan and accurate mass are enough, without any tedious previous optimization when develop determination procedure. But for general quantification MS (triple quadrupole MS) the previous optimization before quantification is required. The ions pairs, collision energy and proportional relation of each compound should be optimized before detection, which is time and solvents consuming. Meanwhile, the UPLC orbitrap HRMS method is not required the complicated sample pretreatment of complex matrix. And it presents higher selectivity, higher sensitivity, lower detection limit, higher reliability and accuracy in quantification. Conclusions This is the report of screening of ginsenosides from Panax genus using advanced UPLC orbitrap HRMS. The characteristic ginsenosides in specified type of ginsengs were confirmed. The use of UPLC orbitrap HRMS analysis allowed improvement in identification and quantification of ginsenosides. The PCA results also demonstrated that the American ginseng grown in China and North America showed no major differences. The quality and quantity evaluation of commercial American ginseng products could be preliminary obtained, which was confirmed by PLS-DA. The new analytical method we developed proved to be accurate, sensitive and reliable, and has the potential for application in further research on the occurrence of ginsenosides in many types of plant materials. Funding This work was supported by the Science and Technology Development Plan Project of Jilin Province (20160520181JH), the “13th Five-Year” Science and Technology Research Project of Jilin Province Education Department (2016 No. 30) and the Special Scientific Research Fund of Agricultural Public Welfare Profession of China (20130311106). 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UPLC Orbitrap HRMS Analysis of Panax quinquefolium L. for Authentication of Panax Genus with Chemometric Methods

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Oxford University Press
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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmx077
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Abstract

Abstract Ginsenosides in Panax quinquefolium L. were determined using developed ultra-performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS) method with electrospray ionization and orbitrap MS analyzer in negative ionization mode. Optimal UPLC separation was achieved using a mixture of acetonitrile and water with 0.1% formic acid as the mobile phase in linear gradient elution. The MS parameters were optimized for reliable detection with enhanced selectivity and sensitivity, and improved identification and quantification of ginsenosides. The applicability of this method was demonstrated on ginsenosides from Panax quinquefolium L. (American ginseng), Panax ginseng (Chinese ginseng) and Panax notoginseng (Sanchi) roots and products. The differences between Chinese and Northern American Panax quinquefolium L., main roots and hair roots, and products from different pharmacy were investigated. The results were also confirmed by principal component analysis and partial least squares discriminatory analysis. It indicated that the strategy can be extended to rapid and accurate authentication of Panax genus. Introduction American ginseng, the root of Panax quinquefolium L. (Araliaceae) originally grown in southeast of Canada and northern USA, was used by native North Americans long before the arrival of Asian. It has been cultivated in China for decades and used in Chinese medicine for more than 200 years. And American ginseng is also used as functional food available on the commercial market to help improve the quality of life (1–3). It has been demonstrated that Panax quinquefolium. L cultivated in China is different from that grown in USA due to the compositional differences (4, 5). Panax quinquefolium L. (American ginseng), Panax ginseng (Chinese ginseng) and Panax notoginseng (Sanchi) are all highly valuable and important tonic plant in China (6). Although they all belong to Panax genus, the efficacy, pharmacological effects and clinical indications of them are different due to the significant differences in the types and quantities of ginsenosides in each root (7). Ginsenosides are the major pharmacologically active constituents of Panax genus. According to the differences of aglycone skeletons, protopanaxadiol (PPD), protopanaxatriol (PPT), oleanolic acid and ocotillol types of ginsenosides have been identified from ginseng roots and related products. The ocotillol type ginsenoside is the chemical marker to distinguish American ginseng from Chinese ginseng and Sanchi. With the increasing interests in ginseng for health care, the need to identify and quantify the chemical composition to ensure the quality, safety and efficacy is necessary. Several different methods have been developed to determine ginsenosides in ginseng plants. The most commonly used are high performance liquid chromatography (HPLC) with photodiode array (8–11) or evaporative light-scattering detector (12–15). Capillary electrophoresis techniques have also been reported and the sensitivity is similar to the HPLC methods (16). With the development of mass spectrometry (MS) interface technology, HPLC coupled to MS (HPLC-MS) has gained its popularity and been shown to be a powerful tool for quality control and consistency of both the chemical markers and active substances of traditional Chinese medicines. It has been applied to determine ginsenosides in ginseng plants (4, 17–27) and yields the information on molecular weights, structures and contents. Among the various LC, ultra-performance LC (UPLC) is considered suitable for metabolite and metabolomics research, due to its reduced analysis time, enhanced reproducibility and increased sample throughput, resolution and sensitivity (28–32). MS is an effective tool for the analysis of ginsenosides with different ionization techniques including matrix-assisted laser desorption ionization (33), electrospray ionization (ESI) (4, 19, 21–32, 34–36) and atmospheric pressure chemical ionization (37, 38). Different MS analyzers have been used in metabolite fingerprint including triple quadrupole (19, 23–25, 36–39), ion trap (21, 22) and quadrupole Time of Flight (TOF) MS (26–34). Orbitrap MS analyzer has not been reported in American ginseng research. The triple quadrupole was generally employed to search for metabolites using neutral loss scan and product ion scan, while ion trap allows structure elucidation of metabolite by MSn. However, these two types of analyzers can only provide nominal mass accuracy. Quadrupole TOF could generate high resolution mass accuracy. Orbitrap, on the other hand, provides much better accuracy, precision and much higher resolution of mass information generated. This study presents the development of an UPLC orbitrap HRMS method to efficiently and simultaneously determine ginsenosides in American ginseng. Gradient elution in UPLC and optimization of MS detection parameters were adjusted to obtain clear resolution of the peaks and maximum signal in the MS detector. Finally, we applied the UPLC orbitrap HRMS method developed to the identification and quantification of ginsenosides in American ginseng, Chinese ginseng and Sanchi roots and commercial products from different origins. With the principal component analysis (PCA) and partial least squares discriminatory analysis (PLS-DA) analysis, Panax genus root materials were distinguished and American ginseng products were authenticated and evaluated. The results demonstrated that the strategy is reliable for the authentication of botanical origin and can also be useful for the quality control of Chinese medicinal herbs. Experimental Chemicals HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Waltham, MA), ultrapure water (18 MΩ/cm) was prepared by a Milli-Q water system (Millipore, Bedford, USA) and HPLC grade formic acid from Sigma-Aldrich. Analytical grade methanol, n-butanol and petroleum ether were from Beijing Chemical Works. Reference ginsenosides, including ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, Ro, F1, F2, F3, pseudoginsenoside F11, notoginsenosides R1 and R2 were obtained from Jilin University (Changchun, JL, China). Reference solution preparation Reference solutions for 19 ginsenosides were prepared individually, with 1 mg of each compound dissolved in 10 mL of 50% (v/v) methanol–water to final concentration of 0.1 mg/mL. Combined each of the individual reference solutions and diluted to obtain final concentrations appropriate for different samples to prepare mixed reference solutions. Calibration curves with 6 dilutions of initial mixture reference solution were used to quantify 19 ginsenosides in different ginseng roots and products. Plant materials and sample preparation The information of all Panax samples was shown in Table I. The Panax samples including American ginseng, Chinese ginseng and Sanchi roots materials and American Ginseng products were collected from Jilin province and Wisconsin, and purchased from local pharmacy. The botanical origin was identified by Prof. Shumin Wang and deposited at the Jilin Ginseng Academy of the Changchun University of Chinese Medicine. All roots samples were dried under sunlight. About 100 g of each root were powdered using a pulverizer and sieved before extraction, assure the analyzed samples well-distributed and typical. Table I. The Origin Information of Different Ginseng Samples   Samples  Origins  S1(1–3)  American Ginseng main roots  Wisconsin, USA  S2(1–3)  American Ginseng main roots  Fusong, Jilin, China  S3(1–3)  American Ginseng main roots  Jingyu, Jilin, China  S4(1–3)  American Ginseng hair roots  Jingyu, Jilin, China  S5(1–3)  Chinese Ginseng main roots  Fusong, Jilin, China  S6(1–3)  Chinese Ginseng main roots  Jingyu, Jilin, China  S7(1–3)  Sanchi main roots  Yanshan, Wenshan, Yunnan, China  S8(1–3)  Sanchi main roots  Maguan, Wenshan, Yunnan, China  S9  American Ginseng main roots  Jilin Pharmacy, Changchun, China  S10  American Ginseng main roots  Changbai mountain pharmaceutical co., LTD  S11  American Ginseng root slices  Fubaicao Pharmacy, Changchun, China  S12  American Ginseng root slices  Jilin honored star pharmaceutical co., LTD  S13  American Ginseng root slices  Changbai mountain ginseng base, Jilin, China  S14  American Ginseng roots total saponin extracts  Jingyu, Jilin, China    Samples  Origins  S1(1–3)  American Ginseng main roots  Wisconsin, USA  S2(1–3)  American Ginseng main roots  Fusong, Jilin, China  S3(1–3)  American Ginseng main roots  Jingyu, Jilin, China  S4(1–3)  American Ginseng hair roots  Jingyu, Jilin, China  S5(1–3)  Chinese Ginseng main roots  Fusong, Jilin, China  S6(1–3)  Chinese Ginseng main roots  Jingyu, Jilin, China  S7(1–3)  Sanchi main roots  Yanshan, Wenshan, Yunnan, China  S8(1–3)  Sanchi main roots  Maguan, Wenshan, Yunnan, China  S9  American Ginseng main roots  Jilin Pharmacy, Changchun, China  S10  American Ginseng main roots  Changbai mountain pharmaceutical co., LTD  S11  American Ginseng root slices  Fubaicao Pharmacy, Changchun, China  S12  American Ginseng root slices  Jilin honored star pharmaceutical co., LTD  S13  American Ginseng root slices  Changbai mountain ginseng base, Jilin, China  S14  American Ginseng roots total saponin extracts  Jingyu, Jilin, China  Pulverized ginseng roots powders were extracted using petroleum ether degreasing with methanol and then water saturated n-butanol extractions. For each extraction, a 1.0 g aliquot of the powdered sample was soxhlet extracted with petroleum ether for 2 h. Then the residue was immersed with methanol for 12 h and soxhlet extracted with methanol for twice. After cooling, the methanol extractions were combined, filtered and concentrated under vacuum. The concentrate was dissolved with water and then extracted with water saturated n-butanol for 3 times. The n-butanol extractions were combined and dried under vacuum. The residue was redissolved in 1 mL 80% (v/v) methanol–water and the supernatant was passed through a 0.22 μm filter before UPLC-HRMS analysis. The extraction of five replicated samples and blank samples were prepared under the same procedure. Instrumentation and conditions The LC analysis was carried out using an UPLC system (Dionex Ultimate 3000, Thermo Scientific, USA) equipped with a quaternary gradient pump, an autosampler, a thermostatically column compartment and a photodiode array detector. The separations were performed using Thermo Scientific Syncronis C18 UPLC column (100 × 2.0 mm, 1.7 μm) at 35°C column temperature. Formic acid (0.1%, v/v, A) and acetonitrile (B) were used as the mobile phase. The gradient elution was programmed at a flow rate of 0.2 mL/min as follows: initial conditions of 15% (v/v) B held for 5 min, increased linearly to 90% (v/v) B in 30 min, increased linearly to 100% (v/v) B in 5 min, held at 100% (v/v) B for 5 min. The temperature of the autosampler was set at 15°C and the injection volume was 5 μL. The UPLC system coupled to Thermo Q-Exactive orbitrap high resolution mass spectrometer (Thermo Scientific, USA) with ESI in negative ion mode and using full mass scan type. The scan range was m/z 150.0–2000.0, scan rate was set at normal in centroid mode. The MS resolution was set at 70,000 and AGC target was 1e6. Maximum inject time was at 250 ms. In addition, the spray voltage was set at 2.5 kV, the capillary temperature was 320°C and S-lens RF level was fixed at 50 to get the best experimental conditions. N2 was used as the Sheath gas with flow rate at 40 and Aux gas with flow rate at 10. The aux gas heater temp was at 300 to achieve the highest signal intensity of detection. The UPLC-HRMS system was controlled with Xcalibur 3.0 data system software. Validation The UPLC orbitrap HRMS method was validated using the following parameters: coefficient of correlation (R2); linear range; intraday and interday repeatability; recovery and limits of detection (LOD) and quantification (LOQ). The linearity was established by analytical curves constructed. The LOD and LOQ were measured on the basis of the response at signal-to-noise ratio (S/N) of 3:1 and 10:1. Intraday repeatability was studied on a single day with five parallel experiments and interday repeatability was measured by the same procedure on three separate days. The recoveries were assessed with known amounts of references spiked into samples. The determination was performed in five repetitions. UPLC orbitrap HRMS data handling by chemometric methods All 19 ginsenosides contents data analyzed by PCA and PLS-DA were performed by running the R-3.2.1 statistical analysis software. Results Development of sample preparation method This sample preparation procedure was optimized using published methods for ginsenosides in Chinese ginseng (1), with some modifications. The conventional method uses heat-reflux, soxhlet and ultrasound-assisted extraction. Extraction solvent includes water, methanol and ethanol. In the procedure, the extraction time and times were also optimized. Different sample preparation differs in extraction efficiency, affecting the relative abundance of the extractable ginsenosides. Thus, we selected soxhlet with methanol to extract ginseng and then characterized diverse ginsenosides. Development of UPLC orbitrap HRMS method The UPLC system provided a rapid, effective and convenient analytical method for a wide range of ginsenosides present in Panax genus. Theoretically, a higher flow rate within the permitted range promises a good separation on the UPLC column whereas the tolerant flow rate for electrospray ion source is below 1.0 mL/min. Meanwhile, flow rate into the electrospray ion source should guarantee the best ionization efficiency and minimize ion suppression which may influence sensitivity. Taking sensitivity and resolution into consideration together, the ultimate flow rate was optimized at 0.2 mL/min throughout this study. Formic acid with different concentrations (0.1, 0.05 and 0.01%) were tested, the best peak shape and resolution was obtained by 0.1% formic acid. Column temperature was set at 35°C to alleviate the column pressure resulting from a higher flow rate, which can improve chromatographic separation and peak shape. The MS parameters of ginsenosides (PPD, PPT, oleanane and ocotillol types) were optimized by tuning each reference with directly infusing individual standard solutions at 5 μL/min using syringe pump. The spray voltage was determined in the range 2.0–4.5 kV, the capillary temperature was studied from 250 to 350°C and S-lens RF level was checked in the range 30–70 by manual setting. And the sheath gas flow rate, aux gas flow rate and aux gas heater temp were also optimized manually to achieve the highest signal intensity. The structures of four types of ginsenosides (PPD, PPT, oleanane and ocotillol) were shown in Table II. The deprotonated molecular ion [M–H]− presented higher intensity than the protonated molecular ion [M+H]+, thus, the negative ion mode was chosen for the determination of ginsenosides. Figure 1 shows the total ion current chromatograms of ginsenosides in three different Panax genus. Peak identifications were made by comparing retention time and MS spectra of the chromatographic peaks with the individual standard solutions to provide unambiguous results. Simultaneous monitoring of the 19 precursor ions can therefore be performed without sacrificing specificity and sensitivity. The overlapping peaks can be resolved by using the appropriate chromatographic gradient and by measuring selected ions characteristics through Extracted Ion Chromatography. The third important consideration is the high resolution MS allowed each analyte can be scanned under its accurate mass during the UPLC orbitrap HRMS determination. Table II. Structures of Protopanaxadiol, Protopanaxatriol, Oleanane and Ocotillol Types of Ginsenosides Structures  Ginsenosides  R1  R2  Molecular formula  Calculated mass [M–H]−  Measured mass [M–H]−  Mass error (ppm)    Ginsenosides Rb1  -Glc2-Glc  -Glc6-Glc  C54H92O23  1107.5952  1107.5972  1.81  Ginsenosides Rb2  -Glc2-Glc  -Glc6-Ara(p)  C53H90O22  1077.5846  1077.5875  2.69  Ginsenosides Rb3  -Glc2-Glc  -Glc6-Xyl  C53H90O22  1077.5846  1077.5868  2.04  Ginsenosides Rc  -Glc2-Glc  -Glc6-Ara(f)  C53H90O22  1077.5846  1077.5871  2.32  Ginsenosides Rd  -Glc2-Glc  -Glc  C48H82O18  945.5423  945.5446  2.43  Ginsenosides Rg3  -Glc2-Glc  -H  C42H72O13  783.4895  783.5096  2.57  Ginsenosides Rh2  -Glc  -H  C36H62O8  621.4367  621.4379  1.93  Ginsenosides F2  -Glc  -Glc  C42H72O13  783.4895  783.5084  2.41    Ginsenosides Re  -Glc2-Rha  -Glc  C48H82O18  945.5423  945.5451  2.96  Ginsenosides Rf  -Glc2-Glc  -H  C42H72O14  799.4844  799.4858  1.75  Ginsenosides Rg1  -Glc  -Glc  C42H72O14  799.4844  799.4859  1.88  Ginsenosides Rg2  -Glc2-Rha  -H  C42H72O13  783.4895  783.5066  2.18  Ginsenosides Rh1  -Glc  -H  C36H62O9  637.4316  637.4332  2.51  Ginsenosides F1  -H  -Glc  C36H62O9  637.4316  637.4329  2.04  Ginsenosides F3  -H  -Glc6-Ara(p)  C47H70O13  841.4742  841.4759  2.02  Notoginsenosides R1  -Glc2-Xyl  -Glc  C47H80O18  931.5267  931.5288  2.25  Notoginsenosides R2  -Glc2-Xyl  -H  C41H70O13  769.4739  769.4756  2.21    Ginsenosides Ro  -Glc2-Glc  -Glc  C48H76O19  955.4904  955.4926  2.30    Pseudoginsenoside F11  -Glc2-Rha  -H  C42H72O14  799.4844  799.4861  2.13  Structures  Ginsenosides  R1  R2  Molecular formula  Calculated mass [M–H]−  Measured mass [M–H]−  Mass error (ppm)    Ginsenosides Rb1  -Glc2-Glc  -Glc6-Glc  C54H92O23  1107.5952  1107.5972  1.81  Ginsenosides Rb2  -Glc2-Glc  -Glc6-Ara(p)  C53H90O22  1077.5846  1077.5875  2.69  Ginsenosides Rb3  -Glc2-Glc  -Glc6-Xyl  C53H90O22  1077.5846  1077.5868  2.04  Ginsenosides Rc  -Glc2-Glc  -Glc6-Ara(f)  C53H90O22  1077.5846  1077.5871  2.32  Ginsenosides Rd  -Glc2-Glc  -Glc  C48H82O18  945.5423  945.5446  2.43  Ginsenosides Rg3  -Glc2-Glc  -H  C42H72O13  783.4895  783.5096  2.57  Ginsenosides Rh2  -Glc  -H  C36H62O8  621.4367  621.4379  1.93  Ginsenosides F2  -Glc  -Glc  C42H72O13  783.4895  783.5084  2.41    Ginsenosides Re  -Glc2-Rha  -Glc  C48H82O18  945.5423  945.5451  2.96  Ginsenosides Rf  -Glc2-Glc  -H  C42H72O14  799.4844  799.4858  1.75  Ginsenosides Rg1  -Glc  -Glc  C42H72O14  799.4844  799.4859  1.88  Ginsenosides Rg2  -Glc2-Rha  -H  C42H72O13  783.4895  783.5066  2.18  Ginsenosides Rh1  -Glc  -H  C36H62O9  637.4316  637.4332  2.51  Ginsenosides F1  -H  -Glc  C36H62O9  637.4316  637.4329  2.04  Ginsenosides F3  -H  -Glc6-Ara(p)  C47H70O13  841.4742  841.4759  2.02  Notoginsenosides R1  -Glc2-Xyl  -Glc  C47H80O18  931.5267  931.5288  2.25  Notoginsenosides R2  -Glc2-Xyl  -H  C41H70O13  769.4739  769.4756  2.21    Ginsenosides Ro  -Glc2-Glc  -Glc  C48H76O19  955.4904  955.4926  2.30    Pseudoginsenoside F11  -Glc2-Rha  -H  C42H72O14  799.4844  799.4861  2.13  Figure 1. View largeDownload slide Total ion current chromatograms of ginsenosides. (A) American ginseng from Jingyu, Jilin, China; (B) American ginseng from Wisconsin, USA; (C) Chinese ginseng from Jingyu, Jilin, China; (D) Sanchi from Wenshan, Yunnan, China. Figure 1. View largeDownload slide Total ion current chromatograms of ginsenosides. (A) American ginseng from Jingyu, Jilin, China; (B) American ginseng from Wisconsin, USA; (C) Chinese ginseng from Jingyu, Jilin, China; (D) Sanchi from Wenshan, Yunnan, China. The method validation results were shown in Table III. The retention times were with SD around 0.02 min (n = 10). The relative standard deviation (RSD) of intraday and interday repeatability ranged between 0.2 and 1.9%. The LOD and LOQ of 19 ginsenosides were assayed. Calibration curves were constructed from the peak areas and the concentration of each ginsenosides and showed good linearity in the range of studied concentrations. The recovery of the ginsenosides ranged from 95.3 to 108.1%. These results indicate that the UPLC orbitrap HRMS detection methods are precise, accurate and sensitive for simultaneously quantitative evaluation of ginsenosides in Panax genus studied. The results also demonstrate that the sample preparation method used could extract ginsenosides simultaneously from the root materials. Table III. Performance of the UPLC-HRMS Method for Ginsenosides Detection Standards  LOD (fg/μL)  LOQ (fg/μL)  Intraday repeatability RSD (%)  Interday repeatability RSD (%)  Correlation coefficient (R2)  Linear range (ng/μL)  Regression equations  Added concentration (ng/μL)  Recovery (%)  Ginsenosides Rb1  0.025  0.075  0.2  0.8  0.9995  0.5–30  Y = 54.01X − 3.301  0.75  100.9 ± 0.2  2.5  95.3 ± 0.3  10  96.4 ± 0.2  Ginsenosides Rb2  0.02  0.06  0.2  0.7  0.9999  1–20  Y = 83.63X − 1.842  2  101.3 ± 0.2  10  104.3 ± 0.4  20  98.3 ± 0.3  Ginsenosides Rb3  0.17  0.5  1.3  1.3  0.9998  1–40  Y = 65.78X − 4.365  1  102.2 ± 1.8  10  96.9 ± 1.0  40  101.8 ± 0.9  Ginsenosides Rc  0.13  0.4  0.8  0.7  0.9999  0.5–25  Y = 81.40X − 6.990  0.5  100.1 ± 0.4  2.5  98.6 ± 0.8  15  98.1 ± 0.5  Ginsenosides Rd  0.007  0.02  0.6  1.8  0.9999  1–20  Y = 39.89X − 2.201  1  99.6 ± 1.5  7.5  98.2 ± 1.0  20  103.3 ± 0.5  Ginsenosides Rg3  0.015  0.05  1.1  1.5  0.9994  1–20  Y = 0.4529X + 8.106  1  99.8 ± 0.9  7.5  103.4 ± 0.6  20  104.6 ± 0.5  Ginsenosides Rh2  0.02  0.06  0.7  0.9  0.9995  1–20  Y = 3642X − 92.08  1  99.9 ± 0.8  7.5  102.5 ± 0.7  20  99.4 ± 0.5  Ginsenosides F2  0.003  0.01  0.9  0.7  0.9992  0.2–4  Y = 424.7X − 3.902  0.2  100.7 ± 0.9  1  98.6 ± 0.4  4  105.4 ± 0.2  Ginsenosides Re  0.007  0.02  0.5  1.4  0.9998  2–40  Y = 16.20X + 0.03311  2  99.9 ± 0.5  10  96.4 ± 0.6  40  98.4 ± 0.3  Ginsenosides Rf  0.01  0.03  0.4  1.0  0.9995  1–20  Y = 89.24X − 11.466  1  100.4 ± 0.8  5  104.4 ± 0.6  20  105.1 ± 0.2  Ginsenosides Rg1  0.07  0.2  0.6  1.3  0.9996  0.5–10  Y = 39.23X − 3.908  0.5  101.9 ± 0.6  1.5  103.3 ± 0.5  10  106.1 ± 0.9  Ginsenosides Rg2  0.1  0.4  1.0  1.9  0.9992  1–80  Y = 0.2531X + 1.723  1  101.6 ± 0.7  10  98.5 ± 0.9  80  102.1 ± 0.7  Ginsenosides Rh1  0.16  0.5  0.9  1.4  0.9996  1–80  Y = 439.21X + 8317  1  101.5 ± 1.2  10  108.1 ± 1.0  80  106.6 ± 0.5  Ginsenosides F1  0.002  0.005  0.4  1.0  0.9997  0.01–0.2  Y = 529.2X − 7.706  0.01  100.5 ± 1.0  0.05  104.2 ± 0.6  1.5  99.9 ± 0.4  Ginsenosides F3  0.03  0.1  1.1  1.3  0.9996  0.5–15  Y = 236.3X + 5.644  0.5  101.3 ± 0.7  2.5  99.6 ± 0.6  10  99.4 ± 0.8  Notoginsenosides R1  0.2  0.6  0.6  1.4  0.9994  0.1–3  Y = 12.91X − 0.547  0.1  99.7 ± 0.5  0.5  101.2 ± 1.0  2.5  103.9 ± 0.9  Notoginsenosides R2  0.008  0.025  0.7  1.2  0.9997  0.05–1.5  Y= 26.14X − 0.231  0.05  99.9 ± 1.2  0.25  100.0 ± 1.1  1.5  99.8 ± 0.7  Ginsenosides Ro  0.01  0.03  0.5  0.9  0.9993  0.5–20  Y = 19.61X − 0.03604  0.5  101.0 ± 1.1  2.5  98.1 ± 1.0  10  98.9 ± 0.7  Pseudoginsenoside F11  0.007  0.02  1.2  1.8  0.9995  1–20  Y = 203.0X − 1.474  1  101.7 ± 0.8  5  100.0 ± 0.7  20  100.9 ± 1.1  Standards  LOD (fg/μL)  LOQ (fg/μL)  Intraday repeatability RSD (%)  Interday repeatability RSD (%)  Correlation coefficient (R2)  Linear range (ng/μL)  Regression equations  Added concentration (ng/μL)  Recovery (%)  Ginsenosides Rb1  0.025  0.075  0.2  0.8  0.9995  0.5–30  Y = 54.01X − 3.301  0.75  100.9 ± 0.2  2.5  95.3 ± 0.3  10  96.4 ± 0.2  Ginsenosides Rb2  0.02  0.06  0.2  0.7  0.9999  1–20  Y = 83.63X − 1.842  2  101.3 ± 0.2  10  104.3 ± 0.4  20  98.3 ± 0.3  Ginsenosides Rb3  0.17  0.5  1.3  1.3  0.9998  1–40  Y = 65.78X − 4.365  1  102.2 ± 1.8  10  96.9 ± 1.0  40  101.8 ± 0.9  Ginsenosides Rc  0.13  0.4  0.8  0.7  0.9999  0.5–25  Y = 81.40X − 6.990  0.5  100.1 ± 0.4  2.5  98.6 ± 0.8  15  98.1 ± 0.5  Ginsenosides Rd  0.007  0.02  0.6  1.8  0.9999  1–20  Y = 39.89X − 2.201  1  99.6 ± 1.5  7.5  98.2 ± 1.0  20  103.3 ± 0.5  Ginsenosides Rg3  0.015  0.05  1.1  1.5  0.9994  1–20  Y = 0.4529X + 8.106  1  99.8 ± 0.9  7.5  103.4 ± 0.6  20  104.6 ± 0.5  Ginsenosides Rh2  0.02  0.06  0.7  0.9  0.9995  1–20  Y = 3642X − 92.08  1  99.9 ± 0.8  7.5  102.5 ± 0.7  20  99.4 ± 0.5  Ginsenosides F2  0.003  0.01  0.9  0.7  0.9992  0.2–4  Y = 424.7X − 3.902  0.2  100.7 ± 0.9  1  98.6 ± 0.4  4  105.4 ± 0.2  Ginsenosides Re  0.007  0.02  0.5  1.4  0.9998  2–40  Y = 16.20X + 0.03311  2  99.9 ± 0.5  10  96.4 ± 0.6  40  98.4 ± 0.3  Ginsenosides Rf  0.01  0.03  0.4  1.0  0.9995  1–20  Y = 89.24X − 11.466  1  100.4 ± 0.8  5  104.4 ± 0.6  20  105.1 ± 0.2  Ginsenosides Rg1  0.07  0.2  0.6  1.3  0.9996  0.5–10  Y = 39.23X − 3.908  0.5  101.9 ± 0.6  1.5  103.3 ± 0.5  10  106.1 ± 0.9  Ginsenosides Rg2  0.1  0.4  1.0  1.9  0.9992  1–80  Y = 0.2531X + 1.723  1  101.6 ± 0.7  10  98.5 ± 0.9  80  102.1 ± 0.7  Ginsenosides Rh1  0.16  0.5  0.9  1.4  0.9996  1–80  Y = 439.21X + 8317  1  101.5 ± 1.2  10  108.1 ± 1.0  80  106.6 ± 0.5  Ginsenosides F1  0.002  0.005  0.4  1.0  0.9997  0.01–0.2  Y = 529.2X − 7.706  0.01  100.5 ± 1.0  0.05  104.2 ± 0.6  1.5  99.9 ± 0.4  Ginsenosides F3  0.03  0.1  1.1  1.3  0.9996  0.5–15  Y = 236.3X + 5.644  0.5  101.3 ± 0.7  2.5  99.6 ± 0.6  10  99.4 ± 0.8  Notoginsenosides R1  0.2  0.6  0.6  1.4  0.9994  0.1–3  Y = 12.91X − 0.547  0.1  99.7 ± 0.5  0.5  101.2 ± 1.0  2.5  103.9 ± 0.9  Notoginsenosides R2  0.008  0.025  0.7  1.2  0.9997  0.05–1.5  Y= 26.14X − 0.231  0.05  99.9 ± 1.2  0.25  100.0 ± 1.1  1.5  99.8 ± 0.7  Ginsenosides Ro  0.01  0.03  0.5  0.9  0.9993  0.5–20  Y = 19.61X − 0.03604  0.5  101.0 ± 1.1  2.5  98.1 ± 1.0  10  98.9 ± 0.7  Pseudoginsenoside F11  0.007  0.02  1.2  1.8  0.9995  1–20  Y = 203.0X − 1.474  1  101.7 ± 0.8  5  100.0 ± 0.7  20  100.9 ± 1.1  Performance of UPLC orbitrap HRMS detection The three kinds of ginseng roots, 19 ginsenosides were analyzed and compared in 24 samples (S1-S8) (Table IV). The values (mean ± SD, n = 10) are expressed on a dry weight basis. Ginsenosides were identified by comparing accurate mass of molecular ions and retention times of the samples against standards. The ginsenosides contents of some samples exceeded the linear range, so dilution before injection is needed for quantitation. The recovery values of different compounds were taken into account when the contents were calculated. The results showed an obvious chemical variety of 19 ginsenosides in the three Panax genus, which suggested that each medicinal plant had its own chemical characteristics. Table IV. Application of the UPLC Orbitrap HRMS Method Developed to Some Panax Genus Samples Sample  PPD (μg/mL)  OLE (μg/mL)  OCT (μg/mL)  G-Rb1  G-Rb2  G-Rb3  G-Rc  G-Rd  G-Rg3  G-Rh2  G-F2  G-Ro  P-F11  S1(1)  5.35 ± 0.55  0.42 ± 0.05  0.35 ± 0.03  2.09 ± 0.35  3.73 ± 0.29  BQ  -  (31.38 ± 2.42) × 10−3  1.79 ± 0.24  1.86 ± 0.15  S1(2)  5.90 ± 0.21  0.45 ± 0.10  0.35 ± 0.08  2.14 ± 0.29  3.83 ± 0.25  BQ  -  (24.54 ± 2.50) × 10−3  1.60 ± 0.12  1.90 ± 0.20  S1(3)  5.27 ± 0.33  0.44 ± 0.08  0.33 ± 0.06  2.10 ± 0.20  3.70 ± 0.21  BQ  -  (30.51 ± 2.01) × 10−3  1.90 ± 0.18  2.00 ± 0.14  S2(1)  5.25 ± 0.57  0.35 ± 0.05  0.48 ± 0.08  1.52 ± 0.15  2.79 ± 0.23  BQ  -  0.25 ± 0.01  0.90 ± 0.04  0.71 ± 0.02  S2(2)  5.36 ± 0.49  0.34 ± 0.01  0.45 ± 0.04  1.59 ± 0.20  2.85 ± 0.24  BQ  -  0.19 ± 0.02  0.84 ± 0.03  0.90 ± 0.04  S2(3)  5.74 ± 0.27  0.35 ± 0.03  0.47 ± 0.05  1.52 ± 0.07  3.00 ± 0.30  BQ  -  0.26 ± 0.01  0.77 ± 0.03  0.86 ± 0.05  S3(1)  5.63 ± 0.41  0.40 ± 0.02  0.67 ± 0.06  2.11 ± 0.19  2.76 ± 0.17  BQ  -  (17.44 ± 1.27) × 10−3  1.49 ± 0.09  1.49 ± 0.11  S3(2)  5.81 ± 0.33  0.41 ± 0.02  0.66 ± 0.05  2.14 ± 0.25  2.95 ± 0.26  BQ  -  (18.22 ± 1.37) × 10−3  1.30 ± 0.10  1.56 ± 0.15  S3(3)  5.41 ± 0.39  0.37 ± 0.01  0.61 ± 0.06  2.00 ± 0.22  2.84 ± 0.24  BQ  -  (17.99 ± 1.64) × 10−3  1.56 ± 0.11  1.34 ± 0.14  S4(1)  10.34 ± 0.90  3.12 ± 0.15  1.15 ± 0.09  9.64 ± 0.80  9.77 ± 0.70  (16.78 ± 1.01) × 10−3  -  1.19 ± 0.20  4.29 ± 0.20  4.65 ± 0.20  S4(2)  9.99 ± 1.0  3.18 ± 0.17  1.01 ± 0.04  9.50 ± 0.77  9.69 ± 0.71  (17.00 ± 1.46) × 10−3  -  1.30 ± 0.10  4.60 ± 0.24  4.90 ± 0.21  S4(3)  10.11 ± 0.94  3.09 ± 0.11  1.10 ± 0.08  9.60 ± 0.70  9.81 ± 0.84  (16.20 ± 1.00) × 10−3  -  1.00 ± 0.09  4.90 ± 0.26  4.65 ± 0.25  S5(1)  1.62 ± 0.10  1.90 ± 0.19  1.99 ± 0.10  0.12 ± 0.01  1.76 ± 0.07  BQ  (13.96 ± 1.12) × 10−3  (10.52 ± 0.83) × 10−3  0.94 ± 0.02  -  S5(2)  1.55 ± 0.12  1.68 ± 0.13  1.89 ± 0.07  0.11 ± 0.01  1.56 ± 0.05  BQ  (13.01 ± 1.51) × 10−3  (10.88 ± 0.75) × 10−3  0.99 ± 0.02  -  S5(3)  1.89 ± 0.11  1.82 ± 0.12  2.00 ± 0.10  0.12 ± 0.01  1.39 ± 0.07  BQ  (14.56 ± 0.84) × 10−3  (9.74 ± 1.21) × 10−3  1.10 ± 0.04  -  S6(1)  1.59 ± 0.21  1.77 ± 0.20  2.11 ± 0.09  0.13 ± 0.01  1.44 ± 0.06  BQ  (13.74 ± 0.99) × 10−3  (10.15 ± 0.94) × 10−3  1.01 ± 0.09  -  S6(2)  1.65 ± 0.16  1.91 ± 0.18  1.97 ± 0.11  0.13 ± 0.01  1.50 ± 0.05  BQ  (14.10 ± 1.11) × 10−3  (10.47 ± 0.81) × 10−3  1.20 ± 0.05  -  S6(3)  1.78 ± 0.11  1.80 ± 0.14  1.89 ± 0.12  0.10 ± 0.01  1.67 ± 0.05  BQ  (13.06 ± 1.14) × 10−3  (9.99 ± 0.89) × 10−3  0.98 ± 0.03  -  S7(1)  7.29 ± 0.20  0.16 ± 0.01  0.72 ± 0.08  1.73 ± 0.05  3.86 ± 0.19  (15.33 ± 1.32) × 10−3  (19.28 ± 1.01) × 10−3  0.33 ± 0.02  -  -  S7(2)  8.00 ± 0.38  0.16 ± 0.01  0.81 ± 0.07  1.69 ± 0.06  3.66 ± 0.18  (15.90 ± 1.20) × 10−3  (14.95 ± 1.17) × 10−3  0.30 ± 0.02  -  -  S7(3)  7.63 ± 0.54  0.15 ± 0.01  0.74 ± 0.07  1.73 ± 0.08  3.57 ± 0.20  (14.78 ± 1.40) × 10−3  (16.99 ± 0.89) × 10−3  0.40 ± 0.01  -  -  S8(1)  7.54 ± 0.40  0.17 ± 0.01  0.79 ± 0.06  1.77 ± 0.06  3.66 ± 0.19  (15.26 ± 1.25) × 10−3  (17.26 ± 0.96) × 10−3  0.44 ± 0.02  -  -  S8(2)  8.11 ± 0.53  0.18 ± 0.01  0.70 ± 0.05  1.75 ± 0.07  3.89 ± 0.18  (15.74 ± 1.13) × 10−3  (18.37 ± 1.04) × 10−3  0.50 ± 0.01  -  -  S8(3)  7.96 ± 0.47  0.17 ± 0.01  0.82 ± 0.06  1.78 ± 0.06  3.50 ± 0.14  (15.07 ± 1.20) × 10−3  (19.15 ± 1.10) × 10−3  0.40 ± 0.01  -  -  S9  3.51 ± 0.23  0.22 ± 0.01  0.30 ± 0.06  1.10 ± 0.09  1.97 ± 0.10  BQ  -  0.38 ± 0.01  1.58 ± 0.04  0.19 ± 0.01  S10  4.68 ± 0.30  0.28 ± 0.02  0.22 ± 0.04  1.55 ± 0.07  1.43 ± 0.11  BQ  -  0.05 ± 0.001  2.61 ± 0.05  0.54 ± 0.01  S11  4.40 ± 0.30  0.16 ± 0.009  0.53 ± 0.04  0.91 ± 0.05  1.97 ± 0.10  (13.85 ± 1.27) × 10−3  -  0.24 ± 0.01  2.24 ± 0.04  1.07 ± 0.05  S12  4.30 ± 0.40  0.22 ± 0.01  0.39 ± 0.04  1.12 ± 0.07  1.76 ± 0.09  BQ  -  0.14 ± 0.01  2.16 ± 0.08  1.15 ± 0.04  S13  6.15 ± 0.59  0.51 ± 0.02  0.54 ± 0.05  2.46 ± 0.19  3.55 ± 0.21  (12.73 ± 0.85) × 10−3  -  0.21 ± 0.02  3.74 ± 0.08  2.33 ± 0.05  S14  0.03 ± 0.002  0.12 ± 0.01  0.44 ± 0.06  0.04 ± 0.001  0.30 ± 0.11  BQ  -  0.05 ± 0.001  (4.66 ± 0.71) × 10−3  0.34 ± 0.01  Sample  PPD (μg/mL)  OLE (μg/mL)  OCT (μg/mL)  G-Rb1  G-Rb2  G-Rb3  G-Rc  G-Rd  G-Rg3  G-Rh2  G-F2  G-Ro  P-F11  S1(1)  5.35 ± 0.55  0.42 ± 0.05  0.35 ± 0.03  2.09 ± 0.35  3.73 ± 0.29  BQ  -  (31.38 ± 2.42) × 10−3  1.79 ± 0.24  1.86 ± 0.15  S1(2)  5.90 ± 0.21  0.45 ± 0.10  0.35 ± 0.08  2.14 ± 0.29  3.83 ± 0.25  BQ  -  (24.54 ± 2.50) × 10−3  1.60 ± 0.12  1.90 ± 0.20  S1(3)  5.27 ± 0.33  0.44 ± 0.08  0.33 ± 0.06  2.10 ± 0.20  3.70 ± 0.21  BQ  -  (30.51 ± 2.01) × 10−3  1.90 ± 0.18  2.00 ± 0.14  S2(1)  5.25 ± 0.57  0.35 ± 0.05  0.48 ± 0.08  1.52 ± 0.15  2.79 ± 0.23  BQ  -  0.25 ± 0.01  0.90 ± 0.04  0.71 ± 0.02  S2(2)  5.36 ± 0.49  0.34 ± 0.01  0.45 ± 0.04  1.59 ± 0.20  2.85 ± 0.24  BQ  -  0.19 ± 0.02  0.84 ± 0.03  0.90 ± 0.04  S2(3)  5.74 ± 0.27  0.35 ± 0.03  0.47 ± 0.05  1.52 ± 0.07  3.00 ± 0.30  BQ  -  0.26 ± 0.01  0.77 ± 0.03  0.86 ± 0.05  S3(1)  5.63 ± 0.41  0.40 ± 0.02  0.67 ± 0.06  2.11 ± 0.19  2.76 ± 0.17  BQ  -  (17.44 ± 1.27) × 10−3  1.49 ± 0.09  1.49 ± 0.11  S3(2)  5.81 ± 0.33  0.41 ± 0.02  0.66 ± 0.05  2.14 ± 0.25  2.95 ± 0.26  BQ  -  (18.22 ± 1.37) × 10−3  1.30 ± 0.10  1.56 ± 0.15  S3(3)  5.41 ± 0.39  0.37 ± 0.01  0.61 ± 0.06  2.00 ± 0.22  2.84 ± 0.24  BQ  -  (17.99 ± 1.64) × 10−3  1.56 ± 0.11  1.34 ± 0.14  S4(1)  10.34 ± 0.90  3.12 ± 0.15  1.15 ± 0.09  9.64 ± 0.80  9.77 ± 0.70  (16.78 ± 1.01) × 10−3  -  1.19 ± 0.20  4.29 ± 0.20  4.65 ± 0.20  S4(2)  9.99 ± 1.0  3.18 ± 0.17  1.01 ± 0.04  9.50 ± 0.77  9.69 ± 0.71  (17.00 ± 1.46) × 10−3  -  1.30 ± 0.10  4.60 ± 0.24  4.90 ± 0.21  S4(3)  10.11 ± 0.94  3.09 ± 0.11  1.10 ± 0.08  9.60 ± 0.70  9.81 ± 0.84  (16.20 ± 1.00) × 10−3  -  1.00 ± 0.09  4.90 ± 0.26  4.65 ± 0.25  S5(1)  1.62 ± 0.10  1.90 ± 0.19  1.99 ± 0.10  0.12 ± 0.01  1.76 ± 0.07  BQ  (13.96 ± 1.12) × 10−3  (10.52 ± 0.83) × 10−3  0.94 ± 0.02  -  S5(2)  1.55 ± 0.12  1.68 ± 0.13  1.89 ± 0.07  0.11 ± 0.01  1.56 ± 0.05  BQ  (13.01 ± 1.51) × 10−3  (10.88 ± 0.75) × 10−3  0.99 ± 0.02  -  S5(3)  1.89 ± 0.11  1.82 ± 0.12  2.00 ± 0.10  0.12 ± 0.01  1.39 ± 0.07  BQ  (14.56 ± 0.84) × 10−3  (9.74 ± 1.21) × 10−3  1.10 ± 0.04  -  S6(1)  1.59 ± 0.21  1.77 ± 0.20  2.11 ± 0.09  0.13 ± 0.01  1.44 ± 0.06  BQ  (13.74 ± 0.99) × 10−3  (10.15 ± 0.94) × 10−3  1.01 ± 0.09  -  S6(2)  1.65 ± 0.16  1.91 ± 0.18  1.97 ± 0.11  0.13 ± 0.01  1.50 ± 0.05  BQ  (14.10 ± 1.11) × 10−3  (10.47 ± 0.81) × 10−3  1.20 ± 0.05  -  S6(3)  1.78 ± 0.11  1.80 ± 0.14  1.89 ± 0.12  0.10 ± 0.01  1.67 ± 0.05  BQ  (13.06 ± 1.14) × 10−3  (9.99 ± 0.89) × 10−3  0.98 ± 0.03  -  S7(1)  7.29 ± 0.20  0.16 ± 0.01  0.72 ± 0.08  1.73 ± 0.05  3.86 ± 0.19  (15.33 ± 1.32) × 10−3  (19.28 ± 1.01) × 10−3  0.33 ± 0.02  -  -  S7(2)  8.00 ± 0.38  0.16 ± 0.01  0.81 ± 0.07  1.69 ± 0.06  3.66 ± 0.18  (15.90 ± 1.20) × 10−3  (14.95 ± 1.17) × 10−3  0.30 ± 0.02  -  -  S7(3)  7.63 ± 0.54  0.15 ± 0.01  0.74 ± 0.07  1.73 ± 0.08  3.57 ± 0.20  (14.78 ± 1.40) × 10−3  (16.99 ± 0.89) × 10−3  0.40 ± 0.01  -  -  S8(1)  7.54 ± 0.40  0.17 ± 0.01  0.79 ± 0.06  1.77 ± 0.06  3.66 ± 0.19  (15.26 ± 1.25) × 10−3  (17.26 ± 0.96) × 10−3  0.44 ± 0.02  -  -  S8(2)  8.11 ± 0.53  0.18 ± 0.01  0.70 ± 0.05  1.75 ± 0.07  3.89 ± 0.18  (15.74 ± 1.13) × 10−3  (18.37 ± 1.04) × 10−3  0.50 ± 0.01  -  -  S8(3)  7.96 ± 0.47  0.17 ± 0.01  0.82 ± 0.06  1.78 ± 0.06  3.50 ± 0.14  (15.07 ± 1.20) × 10−3  (19.15 ± 1.10) × 10−3  0.40 ± 0.01  -  -  S9  3.51 ± 0.23  0.22 ± 0.01  0.30 ± 0.06  1.10 ± 0.09  1.97 ± 0.10  BQ  -  0.38 ± 0.01  1.58 ± 0.04  0.19 ± 0.01  S10  4.68 ± 0.30  0.28 ± 0.02  0.22 ± 0.04  1.55 ± 0.07  1.43 ± 0.11  BQ  -  0.05 ± 0.001  2.61 ± 0.05  0.54 ± 0.01  S11  4.40 ± 0.30  0.16 ± 0.009  0.53 ± 0.04  0.91 ± 0.05  1.97 ± 0.10  (13.85 ± 1.27) × 10−3  -  0.24 ± 0.01  2.24 ± 0.04  1.07 ± 0.05  S12  4.30 ± 0.40  0.22 ± 0.01  0.39 ± 0.04  1.12 ± 0.07  1.76 ± 0.09  BQ  -  0.14 ± 0.01  2.16 ± 0.08  1.15 ± 0.04  S13  6.15 ± 0.59  0.51 ± 0.02  0.54 ± 0.05  2.46 ± 0.19  3.55 ± 0.21  (12.73 ± 0.85) × 10−3  -  0.21 ± 0.02  3.74 ± 0.08  2.33 ± 0.05  S14  0.03 ± 0.002  0.12 ± 0.01  0.44 ± 0.06  0.04 ± 0.001  0.30 ± 0.11  BQ  -  0.05 ± 0.001  (4.66 ± 0.71) × 10−3  0.34 ± 0.01  Sample  PPT (μg/mL)  PPD/PPT  G-Re  G-Rf  G-Rg1  G-Rg2  G-Rh1  G-F1  G-F3  N-R1  N-R2  S1(1)  3.38 ± 0.46  -  0.61 ± 0.05  -  (39.41 ± 1.99) × 10−3  (10.48 ± 1.95) × 10−3  -  -  -  2.97  S1(2)  3.46 ± 0.33  -  0.80 ± 0.04  -  (46.82 ± 1.74) × 10−3  (11.00 ± 2.38) × 10−3  -  -  -  2.94  S1(3)  3.29 ± 0.41  -  0.75 ± 0.05  -  (30.43 ± 1.54) × 10−3  (9.45 ± 1.77) × 10−3  -  -  -  2.94  S2(1)  3.23 ± 0.29  -  0.27 ± 0.06  -  (29.28 ± 1.48) × 10−3  (14.34 ± 2.18) × 10−3  -  -  -  3.01  S2(2)  3.09 ± 0.30  -  0.20 ± 0.06  -  (27.34 ± 1.73) × 10−3  (13.76 ± 2.48) × 10−3  -  -  -  3.24  S2(3)  3.15 ± 0.34  -  0.33 ± 0.05  -  (14.99 ± 2.00) × 10−3  (13.42 ± 2.33) × 10−3  -  -  -  3.24  S3(1)  3.62 ± 0.33  -  0.31 ± 0.04  -  (45.28 ± 1.38) × 10−3  (11.54 ± 1.81) × 10−3  -  -  -  2.91  S3(2)  3.55 ± 0.47  -  0.30 ± 0.02  -  (89.00 ± 2.33) × 10−3  (11.59 ± 1.94) × 10−3  -  -  -  3.04  S3(3)  3.28 ± 0.44  -  0.21 ± 0.23  -  (65.34 ± 2.85) × 10−3  (10.57 ± 2.00) × 10−3  -  -  -  3.21  S4(1)  9.94 ± 0.69  -  1.38 ± 0.11  -  0.21 ± 0.02  (40.44 ± 3.00) × 10−3  -  -  -  3.05  S4(2)  9.80 ± 0.67  -  1.40 ± 0.12  -  0.30 ± 0.04  (34.11 ± 2.30) × 10−3  -  -  -  3.01  S4(3)  10.02 ± 1.20  -  1.50 ± 0.12  -  0.10 ± 0.01  (38.62 ± 3.11) × 10−3  -  -  -  2.98  S5(1)  1.69 ± 0.21  0.73 ± 0.05  0.70 ± 0.01  0.86 ± 0.02  1.40 ± 0.10  (12.58 ± 2.55) × 10−3  (35.19 ± 3.38) × 10−3  BQ  BQ  1.37  S5(2)  1.98 ± 0.23  0.80 ± 0.05  0.61 ± 0.04  0.80 ± 0.03  1.31 ± 0.10  (15.98 ± 2.88) × 10−3  (36.07 ± 2.04) × 10−3  BQ  BQ  1.22  S5(3)  1.69 ± 0.22  0.79 ± 0.04  0.68 ± 0.02  0.95 ± 0.03  0.50 ± 0.02  (15.01 ± 2.43) × 10−3  (24.88 ± 3.24) × 10−3  BQ  BQ  1.55  S6(1)  1.88 ± 0.20  0.77 ± 0.04  0.72 ± 0.03  0.88 ± 0.02  1.44 ± 0.09  (14.39 ± 1.84) × 10−3  (32.45 ± 1.89) × 10−3  BQ  BQ  1.22  S6(2)  1.82 ± 0.19  0.84 ± 0.03  0.69 ± 0.02  0.89 ± 0.03  0.90 ± 0.03  (13.93 ± 2.18) × 10−3  (34.52 ± 2.23) × 10−3  BQ  BQ  1.37  S6(3)  1.79 ± 0.21  0.82 ± 0.04  0.67 ± 0.03  0.90 ± 0.02  1.19 ± 0.05  (13.57 ± 2.00) × 10−3  (37.57 ± 2.74) × 10−3  BQ  BQ  1.33  S7(1)  1.59 ± 0.22  -  16.02 ± 1.55  2.30 ± 0.20  4.50 ± 0.15  (55.46 ± 2.55) × 10−3  (25.54 ± 2.54) × 10−3  11.79 ± 2.33  0.84 ± 0.05  0.38  S7(2)  1.60 ± 0.21  -  15.09 ± 1.56  2.51 ± 0.25  3.00 ± 0.12  (51.39 ± 2.64) × 10−3  (24.37 ± 3.01) × 10−3  12.22 ± 3.10  0.66 ± 0.04  0.42  S7(3)  1.44 ± 0.28  -  15.66 ± 1.70  2.49 ± 0.24  4.20 ± 0.14  (52.77 ± 3.00) × 10−3  (30.22 ± 3.20) × 10−3  12.05 ± 3.52  0.79 ± 0.05  0.39  S8(1)  1.66 ± 0.23  -  15.90 ± 1.58  2.53 ± 0.22  4.56 ± 0.13  (54.81 ± 2.22) × 10−3  (28.77 ± 2.15) × 10−3  12.24 ± 2.99  0.69 ± 0.03  0.40  S8(2)  1.59 ± 0.24  -  15.67 ± 1.65  2.54 ± 0.20  4.35 ± 0.11  (55.53 ± 2.50) × 10−3  (29.30 ± 2.84) × 10−3  11.99 ± 3.00  0.89 ± 0.04  0.43  S8(3)  1.51 ± 0.25  -  15.74 ± 1.60  2.60 ± 0.24  4.25 ± 0.15  (50.43 ± 2.90) × 10−3  (25.90 ± 3.12) × 10−3  12.20 ± 3.27  0.75 ± 0.05  0.41  S9  2.17 ± 0.40  -  1.01 ± 0.11  -  (15.07 ± 2.30) × 10−3  (8.68 ± 1.38) × 10−3  -  -  -  2.34  S10  3.05 ± 0.39  -  0.78 ± 0.08  -  0.12 ± 0.01  (9.43 ± 2.00) × 10−3  -  -  -  2.07  S11  3.42 ± 0.38  -  0.35 ± 0.04  -  0.19 ± 0.01  (7.99 ± 1.83) × 10−3  -  -  -  2.07  S12  3.40 ± 0.38  -  0.33 ± 0.05  -  0.20 ± 0.02  (6.84 ± 1.99) × 10−3  -  -  -  2.01  S13  3.66 ± 0.40  -  0.68 ± 0.06  -  0.16 ± 0.01  (15.40 ± 2.48) × 10−3  -  -  -  2.98  S14  0.29 ± 0.08  -  0.03 ± 0.000  -  0.03 ± 0.001  (5.52 ± 1.40) × 10−3  -  -  -  2.68  Sample  PPT (μg/mL)  PPD/PPT  G-Re  G-Rf  G-Rg1  G-Rg2  G-Rh1  G-F1  G-F3  N-R1  N-R2  S1(1)  3.38 ± 0.46  -  0.61 ± 0.05  -  (39.41 ± 1.99) × 10−3  (10.48 ± 1.95) × 10−3  -  -  -  2.97  S1(2)  3.46 ± 0.33  -  0.80 ± 0.04  -  (46.82 ± 1.74) × 10−3  (11.00 ± 2.38) × 10−3  -  -  -  2.94  S1(3)  3.29 ± 0.41  -  0.75 ± 0.05  -  (30.43 ± 1.54) × 10−3  (9.45 ± 1.77) × 10−3  -  -  -  2.94  S2(1)  3.23 ± 0.29  -  0.27 ± 0.06  -  (29.28 ± 1.48) × 10−3  (14.34 ± 2.18) × 10−3  -  -  -  3.01  S2(2)  3.09 ± 0.30  -  0.20 ± 0.06  -  (27.34 ± 1.73) × 10−3  (13.76 ± 2.48) × 10−3  -  -  -  3.24  S2(3)  3.15 ± 0.34  -  0.33 ± 0.05  -  (14.99 ± 2.00) × 10−3  (13.42 ± 2.33) × 10−3  -  -  -  3.24  S3(1)  3.62 ± 0.33  -  0.31 ± 0.04  -  (45.28 ± 1.38) × 10−3  (11.54 ± 1.81) × 10−3  -  -  -  2.91  S3(2)  3.55 ± 0.47  -  0.30 ± 0.02  -  (89.00 ± 2.33) × 10−3  (11.59 ± 1.94) × 10−3  -  -  -  3.04  S3(3)  3.28 ± 0.44  -  0.21 ± 0.23  -  (65.34 ± 2.85) × 10−3  (10.57 ± 2.00) × 10−3  -  -  -  3.21  S4(1)  9.94 ± 0.69  -  1.38 ± 0.11  -  0.21 ± 0.02  (40.44 ± 3.00) × 10−3  -  -  -  3.05  S4(2)  9.80 ± 0.67  -  1.40 ± 0.12  -  0.30 ± 0.04  (34.11 ± 2.30) × 10−3  -  -  -  3.01  S4(3)  10.02 ± 1.20  -  1.50 ± 0.12  -  0.10 ± 0.01  (38.62 ± 3.11) × 10−3  -  -  -  2.98  S5(1)  1.69 ± 0.21  0.73 ± 0.05  0.70 ± 0.01  0.86 ± 0.02  1.40 ± 0.10  (12.58 ± 2.55) × 10−3  (35.19 ± 3.38) × 10−3  BQ  BQ  1.37  S5(2)  1.98 ± 0.23  0.80 ± 0.05  0.61 ± 0.04  0.80 ± 0.03  1.31 ± 0.10  (15.98 ± 2.88) × 10−3  (36.07 ± 2.04) × 10−3  BQ  BQ  1.22  S5(3)  1.69 ± 0.22  0.79 ± 0.04  0.68 ± 0.02  0.95 ± 0.03  0.50 ± 0.02  (15.01 ± 2.43) × 10−3  (24.88 ± 3.24) × 10−3  BQ  BQ  1.55  S6(1)  1.88 ± 0.20  0.77 ± 0.04  0.72 ± 0.03  0.88 ± 0.02  1.44 ± 0.09  (14.39 ± 1.84) × 10−3  (32.45 ± 1.89) × 10−3  BQ  BQ  1.22  S6(2)  1.82 ± 0.19  0.84 ± 0.03  0.69 ± 0.02  0.89 ± 0.03  0.90 ± 0.03  (13.93 ± 2.18) × 10−3  (34.52 ± 2.23) × 10−3  BQ  BQ  1.37  S6(3)  1.79 ± 0.21  0.82 ± 0.04  0.67 ± 0.03  0.90 ± 0.02  1.19 ± 0.05  (13.57 ± 2.00) × 10−3  (37.57 ± 2.74) × 10−3  BQ  BQ  1.33  S7(1)  1.59 ± 0.22  -  16.02 ± 1.55  2.30 ± 0.20  4.50 ± 0.15  (55.46 ± 2.55) × 10−3  (25.54 ± 2.54) × 10−3  11.79 ± 2.33  0.84 ± 0.05  0.38  S7(2)  1.60 ± 0.21  -  15.09 ± 1.56  2.51 ± 0.25  3.00 ± 0.12  (51.39 ± 2.64) × 10−3  (24.37 ± 3.01) × 10−3  12.22 ± 3.10  0.66 ± 0.04  0.42  S7(3)  1.44 ± 0.28  -  15.66 ± 1.70  2.49 ± 0.24  4.20 ± 0.14  (52.77 ± 3.00) × 10−3  (30.22 ± 3.20) × 10−3  12.05 ± 3.52  0.79 ± 0.05  0.39  S8(1)  1.66 ± 0.23  -  15.90 ± 1.58  2.53 ± 0.22  4.56 ± 0.13  (54.81 ± 2.22) × 10−3  (28.77 ± 2.15) × 10−3  12.24 ± 2.99  0.69 ± 0.03  0.40  S8(2)  1.59 ± 0.24  -  15.67 ± 1.65  2.54 ± 0.20  4.35 ± 0.11  (55.53 ± 2.50) × 10−3  (29.30 ± 2.84) × 10−3  11.99 ± 3.00  0.89 ± 0.04  0.43  S8(3)  1.51 ± 0.25  -  15.74 ± 1.60  2.60 ± 0.24  4.25 ± 0.15  (50.43 ± 2.90) × 10−3  (25.90 ± 3.12) × 10−3  12.20 ± 3.27  0.75 ± 0.05  0.41  S9  2.17 ± 0.40  -  1.01 ± 0.11  -  (15.07 ± 2.30) × 10−3  (8.68 ± 1.38) × 10−3  -  -  -  2.34  S10  3.05 ± 0.39  -  0.78 ± 0.08  -  0.12 ± 0.01  (9.43 ± 2.00) × 10−3  -  -  -  2.07  S11  3.42 ± 0.38  -  0.35 ± 0.04  -  0.19 ± 0.01  (7.99 ± 1.83) × 10−3  -  -  -  2.07  S12  3.40 ± 0.38  -  0.33 ± 0.05  -  0.20 ± 0.02  (6.84 ± 1.99) × 10−3  -  -  -  2.01  S13  3.66 ± 0.40  -  0.68 ± 0.06  -  0.16 ± 0.01  (15.40 ± 2.48) × 10−3  -  -  -  2.98  S14  0.29 ± 0.08  -  0.03 ± 0.000  -  0.03 ± 0.001  (5.52 ± 1.40) × 10−3  -  -  -  2.68  Values are means (n = 10) of dry weight. “-”: not detected; BQ: below the limit of quantitation. Determination of ginsenosides in American Ginseng from China and Northern America Each of the three roots of American ginseng from Wisconsin (S1(1–3)), Fusong (S2(1–3)) and Jingyu (S3(1–3)) were found to contain significant amounts of pseudoginsenoside F11 at 0.71–2.00 μg/mg, but no ginsenoside Rf, notoginsenoside R1 and notoginsenoside R2 were detected. Among the 19 ginsenosides, Rb1, Re and Rd are the most abundant ginsenosides in S1-3. However, contents of ginsenoside Ro and pseudoginsenoside F11 in S2 appear to be lower significantly than those in S1 and S3, while the reverse is true for ginsenoside F2. But no significant differences between S1 and S3 were observed. Variations may occur due to various factors, such as geographical source, cultivation, harvest, storage and processing of the roots. However, we could not find obvious differences in the ginsenoside profiles of genetically same American ginseng from grown in Northern America or China. By comparing the ratios of PPD and PPT type ginsenosides’ total contents (PPD/PPT in Table IV) in extracts of American ginseng roots from China and North America, it was found that the American ginseng grown in China and North America show no major differences. Difference of ginsenosides in American ginseng main roots and hair roots from Jingyu, Jilin, China The investigation of different sections of American ginseng, main roots (S3(1–3)) and hair roots (S4(1–3)), showed that the contents of ginsenosides depending on the root type/diameter. The significantly higher contents of ginsenosides found in hair roots compared to main roots have led to the proposal that ginsenosides are mainly distributed in the periderm and cortex root tissues. As shown in Table IV, the total contents of PPD and PPT type ginsenosides were three times higher in root hairs. By comparing the ratios of PPD and PPT, no significant difference was found between main roots and root hairs. Authentication of American ginseng with Chinese ginseng and Sanchi The variation in the amount of 19 investigated ginsenosides in three Panax species was up to 50-folds. Peudoginsenoside F11 was only present in American ginseng (S1, S2, S3), whereas ginsenoside Rf is only present in Chinese ginseng (S5, S6). However, the situation is far more complex for the species Sanchi. The typical ginsenosides in Sanchi (S7, S8) is notoginsenoside R1 and notoginsenoside R2. Although these two ginsenosides were totally absent from American ginseng, it can still be found in trace amounts in Chinese ginseng. Fortunately, Sanchi does not contain ginsenoside Rf, which is the typical in Chinese ginseng. This can be conveniently employed in the differentiation among the three Panax species. It was also found that oleanane type ginsenoside Ro is present in American ginseng and Chinese ginseng, and absent in Sanchi. Therefore, the characteristics of ingredients can be used as markers for the discrimination of three Panax species. Meanwhile, the ratio of PPD/PPT showed certain regularity. A higher PPD/PPT ratio with value around 3 was usually indicative of American ginseng, while PPD/PPT value between 1 and 3 were usually characteristic for Chinese ginseng. Sanchi presented the lowest ratio of PPD/PPT less than 1. The differences in ginsenosides contents were probably a result of genetic differences among the three kinds of ginsengs. And these differences may explain their different medicinal efficacy. PCA of Panax genus The ginsenosides contents of 10 batches of each root sample were studied by PCA, and the differences resulting from botanical origin of American ginseng, Chinese ginseng and Sanchi were displayed. Importances of principal components and the contribution rate is the basis to choose principal component. After elimination of spectral outliers, PCA was applied to eliminate the data collinearity and to reduce the number of variables. The variances explained were shown in Table V. The results demonstrated that 81.795% contribution of the total variances is from the first two factors. A two factors model could explain the 81.795% experimental data. The projection display analysis of PCA was applied. The PCA factor scores obtained for the coordinate axis. The three-dimensional scatter of S1-S8 was shown in Figure 2. The American ginseng main roots from Wisconsin (S1), Fusong (S2) and Jingyu (S3) were clustered as one type, it also demonstrated that the American ginseng grown in China and North America show no major differences. And while Chinese ginseng (S5, S6) and Sanchi (S7, S8) were clustered as the other two types, respectively. So the distinction of similar species was obtained. The American ginseng hair roots were clustered as one type. And each type was with high clustered degree, respectively. Table V. The Variances Explained of Panax Genus Comp.  Standard deviation  Variance proportion (%)  Cumulative proportion (%)  1  2.946  45.680  45.680  2  2.620  36.115  81.795  3  1.768  16.453  98.248  Comp.  Standard deviation  Variance proportion (%)  Cumulative proportion (%)  1  2.946  45.680  45.680  2  2.620  36.115  81.795  3  1.768  16.453  98.248  Figure 2. View largeDownload slide PCA three-dimensional scatter of Panax genus. Figure 2. View largeDownload slide PCA three-dimensional scatter of Panax genus. Evaluation of commercial American ginseng products from different pharmacy Using the methods we developed, 19 ginsenosides levels were determined for 6 commercial American ginseng products samples (S9-S14) (Table IV), including 2 main roots, 3 main roots slices and 1 powder sample of extracted total ginsenosides. Applying the PLS-DA procedures in evaluation of the six commercial products, the principal components were screened out using the leave-one-out method. Error rate detection with centroids distance, max distance and mahalanobis distance were compared, all of them made the least error rate when using three principal components. The high rate of correct discrimination (87.30%) for American ginseng was considered by 10-fold-cross validation. Two brands of American ginseng main roots (S9, S10) and two brands of American ginseng main root slices (S11, S12) were found to contain pseudoginsenoside F11 without ginsenoside Rf, notoginsenoside R1 and notoginsenoside R2. It demonstrated that these four products are true for American ginseng as described. However ginsenosides detected were less than those described above, and the contents of PPD and PPT ginsenosides and the ratio of PPD/PPT were also less than samples S1, S2, S3. The PLS-DA results indicated that S9, S10 and S12 were classified as American ginseng from Fusong, and S11 was classified as American ginseng from Jingyu. Among these products, one brand of American ginseng slices (S13) that claimed to be made from American ginseng, whereas the contents of pseudoginsenoside F11, PPD and PPT types ginsenosides and the ratio of PPD/PPT were similar with the American ginseng from Wisconsin (S1). The PLS-DA results also indicated that S13 was classified as American ginseng from Wisconsin. For the extracted powder product (S14), it was actually derived from American ginseng. The values of PPD/PPT ratio was almost 3, whereas the contents of PPD and PPT types ginsenosides were much less than the ones in American ginseng main roots from Jingyu. The PLS-DA results also indicated that S14 was classified as American ginseng from Fusong. This result demonstrates that the extract procedure for this product with lower extraction efficiency, we could speculate that the purity of this product might be not enough for clinical usage. Discussion One aim of this work was to develop and validate an UPLC orbitrap HRMS method with high sensitivity and selectivity in order to simultaneously detect and quantify ginsenosides in Panax genus root materials. Since root tissues contain a variety of primary and secondary metabolites, the extraction of the compounds from samples of diverse origin needed to be taken into account for their simultaneous analysis. The matrix did not significantly affect the detection and the recoveries obtained were applicable to all the samples. Ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg3, Rh1, F1 and F2 were detected in most of the samples, while ginsenosides Rg1, Rh1, F1 and F2 have a wide range of concentrations. Our results not only confirm some published findings (19, 23–25) but also provide evidence that American ginseng contain detectable and quantifiable amounts of ginsenosides Rg3, Rh1, F1, F2. Because of the great similarity in chemical constituents, the difference within the same American ginseng species cultivated in different geographical locations was not significant. The differences among species in the same Panax genus are much more significant than those within the species. According to respective and total contents of PPD and PPT and the ratio of PPD/PPT, three species of Panax genus were distinguished. American ginseng, Chinese ginseng and Sanchi were clustered as three types by PCA. Thus the differentiation of cultivation regions as part of the quality control process is more difficult than that among different species of the Panax genus. The PLS-DA results reported here demonstrated that the quality of some brands of American ginseng products might have some defects. The use of UPLC orbitrap HRMS analysis allowed improvement in identification and quantification of ginsenosides, only simple full scan and accurate mass are enough, without any tedious previous optimization when develop determination procedure. But for general quantification MS (triple quadrupole MS) the previous optimization before quantification is required. The ions pairs, collision energy and proportional relation of each compound should be optimized before detection, which is time and solvents consuming. Meanwhile, the UPLC orbitrap HRMS method is not required the complicated sample pretreatment of complex matrix. And it presents higher selectivity, higher sensitivity, lower detection limit, higher reliability and accuracy in quantification. Conclusions This is the report of screening of ginsenosides from Panax genus using advanced UPLC orbitrap HRMS. The characteristic ginsenosides in specified type of ginsengs were confirmed. The use of UPLC orbitrap HRMS analysis allowed improvement in identification and quantification of ginsenosides. The PCA results also demonstrated that the American ginseng grown in China and North America showed no major differences. The quality and quantity evaluation of commercial American ginseng products could be preliminary obtained, which was confirmed by PLS-DA. The new analytical method we developed proved to be accurate, sensitive and reliable, and has the potential for application in further research on the occurrence of ginsenosides in many types of plant materials. Funding This work was supported by the Science and Technology Development Plan Project of Jilin Province (20160520181JH), the “13th Five-Year” Science and Technology Research Project of Jilin Province Education Department (2016 No. 30) and the Special Scientific Research Fund of Agricultural Public Welfare Profession of China (20130311106). 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Published: Jan 1, 2018

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