Pharmacokinetic Assessments of Liquiritin, Protocatechuic Aldehyde and Rosmarinic Acid in Rat Plasma by UPLC–MS-MS After Administration of ZibuPiyin Recipe

Pharmacokinetic Assessments of Liquiritin, Protocatechuic Aldehyde and Rosmarinic Acid in Rat... Abstract The three analytes of the Traditional Chinese Medicine ZibuPiyin Recipe (ZBPYR), namely, liquiritin, protocatechuic aldehyde and rosmarinic acid, may synergistically play an important role in regulating memory and learning. However, the pharmacokinetic behaviors of these compounds after their co-administration remain unclear. To this end, a selective and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated in rat plasma for the study of these three major bioactive ingredients in ZBPYR. The analytes in the plasma samples were separated on a Shiseido Capcell core C18 column using bendrofluazide as an internal standard, with a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. Electrospray ionization in the negative-ion mode and multiple reaction monitoring were used to identify and quantify the three analytes. All of the calibration curves showed good linearity (r > 0.992) over the concentration range, with a lower limit of quantification of 5 ng/mL. The precision of the analytical method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation (SD) was within 15%. Satisfactory extraction efficiency (between 83.4 and 99.4%) and matrix effects (76.4–107.4) were obtained by liquid–liquid extraction. The pharmacokinetic results showed that the three bioactive ingredients were rapidly absorbed and had a short terminal half-life in rats after oral administration of ZibuPiyin recipe. This UPLC–MS-MS study method used in this study may be useful for assessing the pharmacokinetic characteristics of various compounds, which would be helpful in determining their clinical potential. Introduction Traditional Chinese Medicines (TCMs) usually yield therapeutic effects as they have multiple components and multiple targets (1–5). A typical TCM prescription consists of a complex mixture of many different herbal and mineral ingredients, and is often prescribed in combinations that afford synergistic effects or reduce possible adverse reactions (6). Distinct from chemical drugs, the pharmacokinetic features of individual constituents do not represent those of the whole. Pharmacokinetic studies of herbal medicines have been performed according to the procedures used for chemical drugs, but these studies have only monitored a few marker compounds (7). Those results may only partly reveal the pharmacokinetic behaviors of herbal medicines; therefore, pharmacokinetic studies on multibioactive constituents or marker compounds of TCMs are needed. ZibuPiyin Recipe (ZBPYR), a TCM that has been prescribed for amnesia, was shown to afford neuronal protection and improve learning and memory in animals through its antioxidant effects and regulation of energy metabolism (8–10). High-performance liquid chromatography (HPLC) fingerprinting showed three peaks that were considered to be the main active compounds in ZBPYR due to their specific pharmacological activities. The flavonoid liquiritin (C21H22O9, 418.394 g/mol) is a potent constituent of Glycyrrhiza uralensis, which is also known as Chinese liquorice. The effects of liquiritin on learning and memory in rats are related to the enhancement of antioxidase activity, clearance of oxygen radicals, and inhibition of neural apoptosis (11, 12). The phenolic compound protocatechuic aldehyde (PAL; C7H6O3, 138.12 g/mol) is a water-soluble bioactive component of Salvia miltiorrhiza, a deciduous perennial. PAL can efficiently protect dopaminergic neurons against neurotoxin injury both in vitro and in vivo, possibly via its ability to increase levels of the multifunctional DJ-1 protein, and decrease α-synuclein expression and its growth-promoting effects on spine density (13). Rosmarinic acid (C18H16O8, 360.32 g/mol) is a natural phenol carboxylic acid and an active constituent of S. miltiorrhiza. Its administration dose dependently prevented cognitive impairment induced by chronic ethanol in passive avoidance learning and memory, and disturbed oxidant/antioxidant status as a possible mechanism (14). In addition, rosmarinic acid protected aortic endothelial cell function and ultrastructure against diabetes-induced damage, possibly through its antioxidant and anti-inflammatory effects (15). Furthermore, the administration of rosmarinic acid to diabetic rats ameliorated water consumption and urination (16). Thus, these three analytes of ZBPYR appear to play important roles in regulating memory and learning, and as such, may have clinical utility in the treatment of amnesia, so it is necessary to study the pharmacokinetics of these compounds in vivo. To date, there have been no simultaneous studies on these three compounds, despite the fact that several studies have reported the pharmacokinetics of liquiritin, PAL or rosmarinic acid (17–19). However, these studies only focused on one or two constituents, so the pharmacokinetic behaviors of these compounds after their co-administration remain unclear. In addition, the composition of the compound prescriptions may vary with different herbal combinations and extraction processes (20). Furthermore, due to the likely herb–herb and drug–drug interactions, the pharmacokinetics of multi-ingredient preparations may differ from single ingredient preparations (21, 22). Thus, the ultra-performance liquid chromatography–tandem mass spectrometry method (UPLC–MS-MS) method, which has high sensitivity, selectivity and specificity and is highly robust for compound separation, was applied to study the pharmacokinetics of liquiritin, PAL and rosmarinic acid after administration of ZBPYR in rats. This method may be useful for assessing the pharmacokinetic characteristics of various compounds, which would be helpful in determining their clinical potential. Materials and Methods Chemicals, reagents and sample ZBPYR consists of 12 herbs including Panax ginseng C.A. Mey., Dioscorea opposita Thunb., Poriacocos (Schw.) Wolf, Paeonia lactiflora Pall., S. miltiorrhiza, Dolichos lablab L., Nelumbo nucifera Gaertn, Acorus tatarinowii Schott, Polygala tenuifolia Willd, Santalum album L., Citrus reticulate Blanco and G. uralensis Fisch., which were purchased from the Yunnan hay sources Pharmaceutical Co., Ltd. (Yunnan, China) and were authenticated by Dr Yunpeng Diao (College of Pharmacy, Dalian Medical University, Liaoning, China). The reference standards of liquiritin, PAL, rosmarinic acid and the internal standard (IS) bendrofluazide had a purity >98%, and were all purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade acetonitrile and methanol were obtained from Fisher Scientific (Fair Lawn, NJ, USA), and HPLC-grade formic acid was purchased from Waters (Milford, MA, USA). Deionized water was used throughout the study. Other reagents were of analytical grade. Instrumentation and chromatography Liquid chromatography For fast, selective and sensitive analyses of analytes, a Waters UPLC–MS-MS system (Waters) consisting of a Waters ACQUITYTM UPLC and Xevo TQ mass spectrometer with a triple quadruple mass analyzer and electrospray ionization (ESI) interface in a negative ion mode were used in this assay. UPLC–MS-MS and data analysis were performed with the MassLynxTM NT4.1 workstation (Waters). The separation of three analytes and the IS from endogenous substances was performed on a Capcell core C18 column (2.1 mm × 100 mm, 2.6 μm; Shiseido, Japan). The column and autosampler tray temperatures were kept constant at 40 and 10°C, respectively. The mobile phase consisting of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) was run at 10–90% A over 0–3.0 min, 90% A over 3.0–3.2 min, 90–10% A over 3.2–3.5 min and 10%A over 3.5–4.0 min in a gradient elution mode at a flow rate of 0.2 mL/min. MS conditions The optimal MS parameters were as follows: capillary voltage set at 2.0 kV, source temperature set at 150°C, desolvation gas (nitrogen) at 400°C at a flow rate of 400 L/h, and cone gas flow rate at 50 L/h. Multiple reaction monitoring (MRM) was used to quantitate the three analytes and the IS. The MRM mode was conducted by monitoring the transitions of the precursor ion and product ion. The dwell time was set at 0.2 s to observe a good peak shape. Argon was used as the collision gas. The cone voltage, collision energy and MRM transitions of three analytes and the IS are listed in Table I. Table I. Mass Parameters for Liquiritin (1), Protocatechualdehyde (2), Rosmarinic Acid (3) and Bendroflumethiazide (IS) Compound  MRM (m/z)  Cone (V)  Collision (kV)  Dwell (s)  1  417.2 → 255.1  40  25  0.2  2  137.0 → 108.1  40  25  0.2  3  359.0 → 161.2  30  20  0.2  IS  420.1 → 289.0  40  30  0.2  Compound  MRM (m/z)  Cone (V)  Collision (kV)  Dwell (s)  1  417.2 → 255.1  40  25  0.2  2  137.0 → 108.1  40  25  0.2  3  359.0 → 161.2  30  20  0.2  IS  420.1 → 289.0  40  30  0.2  Preparation of standard samples and quality control samples Stock solutions of liquiritin, PAL and rosmarinic acid (200 μg/mL) and the IS (40 ng/mL) were prepared in methanol. A set of standard solutions and quality control (QC) solutions for the three bioactive constituents were prepared by diluting the stock solutions (50 ng/mL) successively with methanol. All of the solutions were stored at −20°C. Plasma standards were prepared as follows: 20 μL of standard solutions containing liquiritin, PAL and rosmarinic acid and 20 μL IS were added to Eppendorf microtubes, dried under air flow at 37°C, and mixed with 100 μL blank plasma and 10 μL hydrochloric acid followed by vortexing for 30 s. The final concentrations of the plasma standards were 5.00, 10.0, 25.0, 50.0, 100, 200 and 400 ng/mL for liquiritin and rosmarinic acid; and 5.00, 10.0, 25.0, 50.0, 100 and 200 ng/mL for PAL. The QC samples were prepared at concentrations of 10.0, 50.0, 320.0 ng/mL for liquiritin and rosmarinic acid, and at 10.0, 40.0, 160.0 ng/mL for PAL. The samples were processed with liquid–liquid extraction by adding 2 mL ethyl acetate. The extraction procedure included vortexing the mixture for 3 min, centrifuging at 13,000 rpm (4°C) for 5 min, blowing to dry the transferred supernatant, and reconstituting with the initial mobile phase. After vortexing for 1 min and centrifuging at 13,000 rpm (4°C) for 5 min, an aliquot of 5 μL supernatant was injected into the UPLC–MS-MS system. Method validation The assay was validated using plasma from healthy Sprague Dawley rats, following the guidelines for bioanalytical method validation issued by the US Food and Drug Administration Center for Drug Evaluation and Research (23). Specificity and selectivity Interference by the endogenous matrix was evaluated using six Sprague Dawley rats by comparing the chromatograms of blank plasma, plasma spiked with the three analytes and the IS, and plasma after oral administration of ZBPYR. Sensitivity The lower limit of quantification (LLOQ) was defined as the lowest concentration measurable with precision <20%, and accuracy between 80 and 120% of the theoretical value. The signal-to-noise ratio for the LLOQ must be ≥10 (23). Linearity The linearity of the assay was assessed using three analyte plasma standards over a concentration range of 5–400 ng/mL for liquiritin and rosmarinic acid and 5–200 ng/mL for PAL in three consecutive patches. Calibration curves were constructed by the weighted (1/x2) linear regression of three analytes/IS peak area ratios versus the spiked concentrations. Two calibration curves were determined and fitted to one curve with a coefficient that not <0.990 in one patch. Precision and accuracy The intra- and inter-day precision and accuracy were assessed by quantitating three concentration levels of QC samples (six samples for each concentration level) on the same day and on three consecutive validation days. The precision was evaluated by relative standard deviation (RSD, %) and accuracy by the relative error (RE, %) of measured concentration versus the spiked concentration. The extraction recovery and matrix effect The recoveries of three analytes and the IS were determined at three QC levels with six replicates for each level by comparing the peak areas from extracted samples with those from post-extracted blank plasma samples spiked with the three analytes and IS. The matrix effect was measured at three QC levels by comparing the peak area of post-extracted blank plasma samples spiked with the three analytes and IS (A) with that of pure standard solution containing equivalent amounts of the compounds (B). The ratio (A/B × 100)% was used to evaluate the matrix effect. Stability Freeze–thaw stability, post-preparative stability, short-term stability and long-term stability were evaluated at three QC levels to assess the stability of three analytes and the IS in rat plasma. QC samples at concentrations of 10.0, 50.0 and 320 ng/mL for liquiritin and rosmarinic acid, and at concentrations of 10.0, 40.0 and 160 for PAL were frozen at −80°C, and then thawed at room temperature for each cycle. The three freeze–thaw cycles were evaluated. The post-preparative stability of the processed samples in an autosampler at 10°C was assessed to determine whether a delay in analysis could lead to instability of the analytes over 8 h. The short-term stability was assessed by putting the QC samples on the benchtop for 4 h before analysis. The long-term stability of the analytes in rat plasma at −80°C was evaluated by analyzing the QC samples over 4 weeks. The analyte was considered stable if the response of the measured analyte concentration to its corresponding theoretical value differed by <15%. Pharmacokinetic study Animals Male Sprague Dawley Rats weighing 220–250 g were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China). Before this assay, the animals were raised with free access to normal standard rodent chow and water in specific-pathogen free (SPF) conditions for 1 week, and were then fasted with access to only water for 12 h prior to the experiment. The animal study was performed in accordance with the guidelines for the use of experimental animals of Dalian Medical University and was approved by the Institutional Review Committee on Animal Care. The protocol was approved by the Animal Ethics Committee of the institution. Preparation of ZBPYR, dosing and sampling ZBPYR consists of 12 crude drugs: Panax ginseng C.A. Mey. (30.0 g), Dioscorea opposita Thunb. (15.0 g), Poriacocos (Schw.) Wolf (15.0 g), Paeonia lactiflora Pall.(15.0 g), S. miltiorrhiza (12.0 g), Dolichos lablab L. (15.0 g), Nelumbo nucifera Gaertn (20.0 g), Acoru statarinowii Schott (10.0 g), Polygala tenuifolia Willd (10.0 g), Santalum album L. (4.5 g), Citrus reticulate Blanco (9.0 g) and G.uralensis Fisch. (9.0 g) (8). According to the constituents of ZBPYR, 12 herbs (164.5 g) were subjected to reflux extraction with water for 2 h. The water decoction was evaporated under vacuum to obtain a final concentration of 34.2 g extract. The extract was accurately weighed and then dissolved in water to prepare the 0.3591 g/mL prescription solution. The concentrations of liquiritin, PAL and rosmarinic acid in ZBPYR were 1.74, 0.05 and 0.03 mg/g, respectively. The pharmacokinetic study in the Sprague Dawley rats was a single-dose, randomized study. The rats were orally administered ZBPYR extract at a dose of 3.591 g/kg (equivalent to 17.2725 g/kg crude drug). About 0.3 mL blood samples were collected from the orbital venous plexus in heparinized centrifuge tubes at 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 12, 20, 24, 30 h after oral administration of 1 mL/100 g ZBPYR extract (the concentrations of liquiritin, PAL and rosmarinic acid in ZBPYR were 1.74, 0.05 and 0.03 mg/g, respectively). Plasma samples were obtained following centrifugation at 12,000 rpm for 5 min, and stored at −80°C until subsequent use. Data analysis The pharmacokinetic parameters (AUC0−∞, AUC0−t, T1/2) of the three analytes rats were calculated with DAS2.1 software supplied by the Pharmacological Society of China (Beijing, China). The maximum concentration (Cmax) and time to reach the maximum concentration (Tmax) were directly obtained from the experimental data. All of the data are expressed as mean ± SD. Results Method development Optimization of sample extraction method We selected acetonitrile and methanol as the protein precipitation (PPT) solvents, and ethyl acetate, methyl tert-butyl ether, and diethyl ether as the liquid–liquid extraction solvents (data not shown). The optimization extraction method was evaluated by the recovery and matrix effect using QC samples. According to the data, the PPT method showed severe endogenous compound interference, which caused a large matrix effect. In addition, the protein residue can contaminate the LC–MS-MS system. Meanwhile, ethyl acetate yielded excellent results for PAL and liquiritin. On the other hand, the recovery was low (<50%) for rosmarinic acid, possibly due to its acidity; thus, an additive (hydrochloric acid, formic acid, acetic acid) was added to increase its yield. Finally, hydrochloric acid was used for optimization in peak shape, recovery and matrix effects of the analytes. Optimization of LC–MS-MS conditions MRM mode was selected to monitor the analytes. The presence of the proton adduction [M − H]− was more abundant than that of [M + H]+. The collision energy and fragment voltage were adjusted to produce the maximum response and optimal MRM transition (417.2 → 255.1 for liquiritin, 137.0 → 108.1 for PAL, 359.0 → 161.2 for rosmarinic acid and 420.1 → 289.0 for IS). The full-scan product ion spectra of three analytes and the IS are shown in Figure 1. The concentration of IS was chosen based on the response of the IS, which should be equal to the response intensity of three analytes with middle concentration. The constitution of the mobile phase was investigated to ensure the stability of the analytes. A Capcellcore C18 column (2.1 mm × 100 mm, 2.6 μm; Shiseido, Japan) was achieved better separation and shorter analytical time by comparing with Kinetex C18 column (100 mm × 4.6 mm, 2.6 um; Phenomenex, American) and ZORBAX SB-C18 column (150 mm × 4.6 mm, 3.5 um; Agilent, American). In the binary gradient system, acetonitrile–water led a higher response than methanol-water, as well as peak tailing. Compared with ammonium acetate, 0.1% formic acid promoted symmetry of the chromatographic peak and led to a robust response, so it was eventually chosen as the mobile phase additive. Figure 1. View largeDownload slide Full scan product ions of [M − H]− of liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS). Figure 1. View largeDownload slide Full scan product ions of [M − H]− of liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS). Method validation Specificity and selectivity Typical chromatograms obtained from rat plasma (Figure 2A), rat plasma spiked with the three analytes and IS (Figure 2B), and rat plasma sample obtained after oral administration of ZBPYR (Figure 2C) are shown in Figure 2. There were no endogenous component interference at the retention time of liquiritin (1.14 min), PAL (1.37 min), rosmarinic acid (1.84 min) and the IS (1.58 min) at their transitions. The optimized extraction method and LC–MS-MS conditions allowed good separation of the analytes from interference components. Thus, the specificity of this method was adequate for studying these three analytes in rat plasma. Figure 2. View largeDownload slide MRM chromatograms of blank rat plasma sample (A); blank rat plasma spiked with analytes (LLOQ) and IS (B); and rat plasma (spiked with IS) after administration of Zibupiyin Recipe at 2 h (C) for liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS, 4). Figure 2. View largeDownload slide MRM chromatograms of blank rat plasma sample (A); blank rat plasma spiked with analytes (LLOQ) and IS (B); and rat plasma (spiked with IS) after administration of Zibupiyin Recipe at 2 h (C) for liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS, 4). Linearity and LLOQ The calibration curves showed good linearity over the concentration range of 5–400 ng/mL for liquiritin and rosmarinic acid, and 5–200 ng/mL for PAL. Typical linear regression equations for the calibration curves (weighted factor 1/x2) were Y = 3.24E−3X + 4.18E−4 (r = 0.9928) for liquiritin, Y = 2.06E−2X + 1.80E−4 (r = 0.9946) for PAL, and Y = 4.82E−3X + 2.11E−5 (r = 0.9941) for rosmarinic acid, where X represents the concentration of three analytes in plasma and Y represents the peak area ratio. These results indicate that the LC–MS-MS response was proportional to the plasma concentration of the analytes, and that the assay was linear. The LLOQs for the three analytes were all set at 5 ng/mL with accuracy and intra- and inter-day precisions within 12.5%, which was stable and sufficient for this assay. Accuracy, precision, recovery and matrix effects Intra- and inter-day precision, accuracy, extraction recovery, and matrix effects for the three analytes are summarized in Table II. All of the intra- and inter-day, precision and accuracy results were within the acceptable criteria of ±15%, which indicated that this preprocessing method was consistent, precise and reproducible for the analytes at different concentrations. The extraction recovery and matrix effects ranged from 83.4–89.5% to 76.4–81.2% for liquiritin, 87.4–93.1% to 81.7–84.5% for PAL, and 95.7–99.4% to 99.9–107.4% for rosmarinic acid (Table II). The mean recovery and matrix effects of the IS were 92.3 and 102.9%, respectively, at a concentration of 40 ng/mL. These results indicate that the recovery and matrix effect for the three analytes were within the recommended values for assay validation, as stated in the “Guidance for Industry: Bioanalytical Method Validation” (23). Table II. Precision, Accuracy, Extraction Recovery and Matrix Effect of Liquiritin (1), Protocatechualdehyde (2), and Rosmarinic Acid (3) and IS in Rat Plasma Analytes  Spike concentration (ng mL−1)  Accuracy, RE (%)  Intra-day  Inter-day  Recovery (%, Mean ± SD)  Matrix effect (%, Mean ± SD)  RSD (%)  RSD (%)  1  10  −3.0  12.8  12.9  83.4 ± 7.2  78.9 ± 4.3  50  −4.4  11.8  12.6  89.5 ± 5.1  76.4 ± 3.7  320  3.2  9.4  9.2  88.1 ± 4.3  81.2 ± 4.9  2  10  −4.4  4.1  5.7  89.8 ± 3.6  82.7 ± 1.8  40  8.8  8.8  4.9  87.4 ± 2.8  84.5 ± 4.9  160  −5.2  5.3  6.4  93.1 ± 5.0  81.7 ± 3.8  3  10  −4.3  9.9  11.0  95.7 ± 8.4  102.1 ± 2.1  50  2.6  12.2  10.3  99.4 ± 6.7  99.9 ± 9.1  320  −3.0  8.6  9.2  96.1 ± 7.8  107.4 ± 3.8  IS  40  –  –  –  92.3 ± 3.7  102.9 ± 4.1  Analytes  Spike concentration (ng mL−1)  Accuracy, RE (%)  Intra-day  Inter-day  Recovery (%, Mean ± SD)  Matrix effect (%, Mean ± SD)  RSD (%)  RSD (%)  1  10  −3.0  12.8  12.9  83.4 ± 7.2  78.9 ± 4.3  50  −4.4  11.8  12.6  89.5 ± 5.1  76.4 ± 3.7  320  3.2  9.4  9.2  88.1 ± 4.3  81.2 ± 4.9  2  10  −4.4  4.1  5.7  89.8 ± 3.6  82.7 ± 1.8  40  8.8  8.8  4.9  87.4 ± 2.8  84.5 ± 4.9  160  −5.2  5.3  6.4  93.1 ± 5.0  81.7 ± 3.8  3  10  −4.3  9.9  11.0  95.7 ± 8.4  102.1 ± 2.1  50  2.6  12.2  10.3  99.4 ± 6.7  99.9 ± 9.1  320  −3.0  8.6  9.2  96.1 ± 7.8  107.4 ± 3.8  IS  40  –  –  –  92.3 ± 3.7  102.9 ± 4.1  Stability The stability of liquiritin, PAL and rosmarinic acid in rat plasma under different conditions is summarized in Table III, in which the precision and accuracy were within the acceptable criteria of ±15%. These results indicate that liquiritin, PAL and rosmarinic acid were stable in plasma at room temperature for 4 h, at −80°C for at least 30 days, after three freeze–thaw cycles, and at 10°C in an autosampler for 8 h in processed samples. Table III. Stability of Liquiritin (1), Protocatechualdehyde (2), and Rosmarinic Acid (3) in Rat Plasma At Different Conditions Determined by UPLC–MS-MS Analytes  Spike concentration (ng mL−1)  30 d, −80°C  4 h, room temperature  Three freeze–thaw circles  8 h,10°C  RE (%)  RSD (%)  RE(%)  RSD (%)  RE (%)  RSD (%)  RE (%)  RSD (%)  1  10  9.4  6.1  8.4  0.6  7.8  6.3  8.3  0.8  50  1.2  0.4  3.8  6.8  −3.8  3.4  0.3  0.6  320  −8.1  0.7  8.0  7.3  −4.4  10.2  6.3  6.1  2  10  −2.5  7.9  −0.7  4.7  1.5  7.5  7.2  5.5  40  12.9  1.5  −9.9  3.9  −8.6  10.0  7.8  −9.9  160  9.1  1.9  9.5  4.2  10.0  4.0  2.7  6.6  3  10  12.5  5.7  2.1  5.4  2.7  7.8  3.6  4.3  50  10.9  −3.4  8.7  4.4  9.9  2.9  12.6  5.9  320  6.7  3.2  −10.5  7.8  11.6  9.0  8.8  11.2  Analytes  Spike concentration (ng mL−1)  30 d, −80°C  4 h, room temperature  Three freeze–thaw circles  8 h,10°C  RE (%)  RSD (%)  RE(%)  RSD (%)  RE (%)  RSD (%)  RE (%)  RSD (%)  1  10  9.4  6.1  8.4  0.6  7.8  6.3  8.3  0.8  50  1.2  0.4  3.8  6.8  −3.8  3.4  0.3  0.6  320  −8.1  0.7  8.0  7.3  −4.4  10.2  6.3  6.1  2  10  −2.5  7.9  −0.7  4.7  1.5  7.5  7.2  5.5  40  12.9  1.5  −9.9  3.9  −8.6  10.0  7.8  −9.9  160  9.1  1.9  9.5  4.2  10.0  4.0  2.7  6.6  3  10  12.5  5.7  2.1  5.4  2.7  7.8  3.6  4.3  50  10.9  −3.4  8.7  4.4  9.9  2.9  12.6  5.9  320  6.7  3.2  −10.5  7.8  11.6  9.0  8.8  11.2  Pharmacokinetic study The developed method was successfully applied to the pharmacokinetic study of ZBPYR in rat plasma after oral administration of 0.3591 g/mL ZBPYR. The concentration–time curves (mean ± SD) are presented in Figure 3 and the corresponding pharmacokinetic parameters are listed in Table IV. The concentration of the drug was readily measurable up to 30 h. By observing the pharmacokinetic time profile curves, the mean Cmax of liquiritin, PAL and rosmarinic acid in male rats were 216, 124 and 223 ng/mL, respectively, with analogous concentration trends. Table IV. Pharmacokinetic Parameters of Liquiritin, Protocatechualdehyde and Rosmarinic Acid in Rat Plasma After Oral Administration of ZibuPiyin Recipe (ZBPYR) Parameters  Units  Liquiritin  Protocatechualdehyde  Rosmarinic acid  AUC0−∞  ug/L h  1447.4 ± 440.4  1425.5 ± 396.5  3099.4 ± 782.7  AUC0−t  ug/L h  1569.4 ± 494.4  1640.4 ± 325.6  3640.6 ± 1189.4  Cmax  ug/L  215.9 ± 24.9  123.7 ± 36.9  222.7 ± 86.7  Tmax  h  0.6 ± 0.2  1.2 ± 0.4  1.7 ± 0.7  T1/2  h  7.8 ± 0.8  9.7 ± 4.4  10.0 ± 2.8  Parameters  Units  Liquiritin  Protocatechualdehyde  Rosmarinic acid  AUC0−∞  ug/L h  1447.4 ± 440.4  1425.5 ± 396.5  3099.4 ± 782.7  AUC0−t  ug/L h  1569.4 ± 494.4  1640.4 ± 325.6  3640.6 ± 1189.4  Cmax  ug/L  215.9 ± 24.9  123.7 ± 36.9  222.7 ± 86.7  Tmax  h  0.6 ± 0.2  1.2 ± 0.4  1.7 ± 0.7  T1/2  h  7.8 ± 0.8  9.7 ± 4.4  10.0 ± 2.8  Figure 3. View largeDownload slide Rat plasma concentration (μg/L)–time (h) curves of liquiritin (1), protocatechuic aldehyde (2) and rosmarinic acid (3) after administration of ZBPYR. Figure 3. View largeDownload slide Rat plasma concentration (μg/L)–time (h) curves of liquiritin (1), protocatechuic aldehyde (2) and rosmarinic acid (3) after administration of ZBPYR. Discussion In a previous report, the pharmacokinetic profiles of PAL in rat serum after oral administration of Radix Salviae miltiorrhizae extract showed a double peak (18), which was different from our results, possibly because of the different pharmacokinetic profiles in different herbs. The mean Tmax of male rats for liquiritin, PAL and rosmarinic acid were ~0.6, 1.2 and 1.7 h, respectively, showing that the absorption of liquiritin was more rapid than that of PAL and rosmarinic acid, which is in accordance with previous studies (17–19). The data from our pharmacokinetic study demonstrated that liquiritin, PAL and rosmarinic acid in rat plasma had a rapid absorption and short terminal half-life, which may illustrate a short dosing interval and reveal the pharmacokinetic features of these constituents of ZBPYR in rats. The pharmacokinetic profile of these three analytes in rats after administration of ZBPYR was characterized for the first time in this study. These data may provide a foundation for future pharmacological studies on ZBPYR. Conclusions A rapid and specific UPLC–ESI–MS-MS method was established for the simultaneous determination of liquiritin, PAL and rosmarinic acid in rat plasma, which was highly sensitive and accurate. The method was successfully applied to the pharmacokinetic study of liquiritin, PAL and rosmarinic acid in rats after oral administration of ZBPYR extract, and the pharmacokinetic parameters may provide a suitable reference for clinical application. Funding This research was supported by National Natural Science Foundation of China (No. 81230084 and 81403306) and the Specialized Research Fund for the Doctoral Program of Higher Education of China from Ministry of Education, Science and Technology Development Center (20132105130001). References 1 Liu, D., Jia, X.B.; Analysis and discussion about current development of relevant studies on” traditional Chinese medicine components; China Journal of Chinese Material Medica , ( 2014); 39: 171– 174. 2 Sun, E., Xu, F.J., Zhang, Z.H.; Discussion about research progress and ideas on processing mechanism of traditional Chinese medicine; China Journal of Chinese Material Medica , ( 2014); 39: 363– 369. 3 Lao, Y., Wang, X., Xu, N.; Application of proteomics to determine the mechanism of action of traditional Chinese medicine remedies; Journal of Ethnopharmacology , ( 2014); 155: 1– 8. Google Scholar CrossRef Search ADS PubMed  4 Zhang, J., Gao, W., Hu, X.; The influence of compatibility of traditional Chinese medicine on the pharmacokinetic of main components in Fructusaurantii; Journal of Ethnopharmacology , ( 2012); 144: 277– 283. Google Scholar CrossRef Search ADS PubMed  5 Sun, H., Dong, T., Zhang, A.; Pharmacokinetics of hesperetin and naringenin in the Zhi Zhu Wan, a traditional Chinese medicinal formulae, and its pharmacodynamics study; Phytotherapy Research , ( 2013); 27: 1345– 1351. Google Scholar CrossRef Search ADS PubMed  6 Ma, W., Peng, Y., Wang, W., Bian, Q., Wang, N., Lee, D.Y.-W., et al.  .; Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC-MS/MS; Biomedical Chromatography , ( 2016); 30( 10): 1573– 1581. Google Scholar CrossRef Search ADS PubMed  7 He, S.M., Chan, E., Zhou, S.F.; ADME properties of herbal medicines in humans: evidence, challenges and strategies; Current Pharmaceutical Design , ( 2011); 17: 357– 407. Google Scholar CrossRef Search ADS PubMed  8 Shi, X., Lu, X.G., Zhan, L.B.; The effects of the Chinese medicine ZiBuPiYin recipe on the hippocampus in a rat model of diabetes-associated cognitive decline: a proteomic analysis; Diabetologia , ( 2011); 54: 1888– 1899. Google Scholar CrossRef Search ADS PubMed  9 Zhan, L., Jiang, W., Lu, X.; Mechanism of protective effect of spleen yin nourishing recipe on dendritic spines in rats; Journal—Beijing University of Traditional Chinese Medicine , ( 2007); 30: 597. 10 Zhan, L.B., Lin, H.Y., Gong, X.Y.; Effects of recipe on endoplasmic reticulum stress in brain tissue of spleen-yin deficiency Alzheimer’s Disease rats; World Science and Technology (Modernization of Traditional Chinese Medicine and Materia Medica) , ( 2011); 13: 993– 998. 11 Jia, S.L., Wu, X.L., Li, X.X., Dai, X.L., Gao, Z.L., Lu, Z., et al.  .; Neuroprotective effects of liquiritin on cognitive deficits induced by soluble amyloid-β1-42 oligomers injected into the hippocampus; Journal of Asian Natural Products Research , ( 2016); 18( 12): 1186– 1199. Google Scholar CrossRef Search ADS PubMed  12 Huang, X., Wang, Y., Ren, K.; Protective effects of liquiritin on the brain of rats with Alzheimer’s disease; The West Indian Medical Journal , ( 2016). 10.7727/wimj.2016.058. 13 Zhao, X., Zhai, S., An, M.S., Wang, Y.H., Yang, Y.F., Ge, H.Q., et al.  .; Neuroprotective effects of protocatechuic aldehyde against neurotoxin-induced cellular and animal models of Parkinson’s disease; PLoS One , ( 2013); 8( 10): e78220. Google Scholar CrossRef Search ADS PubMed  14 Hasanein, P., Seifi, R., Hajinezhad, M.R., Emamjomeh, A.; Rosmarinic acid protects against chronic ethanol-induced learning and memory deficits in rats; Nutritional Neuroscience , ( 2016). 10.1080/1028415X.2016.1203125. 15 Sotnikova, R., Okruhlicova, L., Vlkovicova, J., Navarova, J., Gajdacova, B., Pivackova, L., et al.  .; Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta; The Journal of Pharmacy and Pharmacology , ( 2013); 65( 5): 713– 723. Google Scholar CrossRef Search ADS PubMed  16 Sotnikova, R., Kaprinay, B., Navarova, J.; Rosmarinic acid mitigates signs of systemic oxidative stress in streptozotocin-induced diabetes in rats; General Physiology and Biophysics , ( 2015); 34( 4): 449– 452. Google Scholar PubMed  17 Yan, Y., Chai, C.Z., Wang, D.W.; Simultaneous determination of puerarin, daidzin, daidzein, paeoniflorin, albiflorin, liquiritin and liquiritigenin in rat plasma and its application to a pharmacokinetic study of Ge-Gen Decoction by a liquid chromatography–electrospray ionization-tandem mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2014); 95: 76– 84. Google Scholar CrossRef Search ADS PubMed  18 Ye, G., Wang, C.S., Li, Y.Y.; Simultaneous determination and pharmacokinetic studies on (3, 4-dihydroxyphenyl)-lactic acid and protocatechuic aldehyde in rat serum after oral administration of Radix Salviae miltiorrhizae extract; Journal of Chromatographic Science , ( 2003); 41: 327– 330. Google Scholar CrossRef Search ADS PubMed  19 Ma, B., Wang, Y., Zhang, Q.; Simultaneous determination of oridonin, ponicidin and rosmarinic acid from herbaisodi rubescentis extract by LC–MS-MS in rat plasma; Journal of Chromatographic Science , ( 2013); 51: 910– 918. Google Scholar CrossRef Search ADS PubMed  20 Bi, X., Gong, M., Di, L.; Review on prescription compatibility of shaoyaogancao decoction and reflection on pharmacokinetic compatibility mechanism of traditional chinese medicine prescription based on in vivo drug interaction of main efficacious components; Evidence-Based Complementary and Alternative Medicine , ( 2014); 2014: 208129. Google Scholar CrossRef Search ADS PubMed  21 Chen, Y., Ouyang, D.S., Kang, Z.; Effect of a traditional Chinese medicine Liu Wei Di Huang Wan on the activities of CYP2C19, CYP2D6 and CYP3A4 in healthy volunteers; Xenobiotica; the Fate of Foreign Compounds in Biological Systems , ( 2012); 42: 596– 602. Google Scholar CrossRef Search ADS PubMed  22 Tang, J., Song, X., Zhu, M.; Study on the pharmacokinetics drug–drug interaction potential of Glycyrrhiza uralensis, a traditional Chinese medicine, with lidocaine in rats; Phytotherapy Research , ( 2009); 23: 603– 607. Google Scholar CrossRef Search ADS PubMed  23 U.S. Food and Drug Administration. http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm64964.htm ( 2013). © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Pharmacokinetic Assessments of Liquiritin, Protocatechuic Aldehyde and Rosmarinic Acid in Rat Plasma by UPLC–MS-MS After Administration of ZibuPiyin Recipe

Loading next page...
 
/lp/ou_press/pharmacokinetic-assessments-of-liquiritin-protocatechuic-aldehyde-and-7tQ7feSoJw
Publisher
Oxford University Press
Copyright
© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
ISSN
0021-9665
eISSN
1945-239X
D.O.I.
10.1093/chromsci/bmx093
Publisher site
See Article on Publisher Site

Abstract

Abstract The three analytes of the Traditional Chinese Medicine ZibuPiyin Recipe (ZBPYR), namely, liquiritin, protocatechuic aldehyde and rosmarinic acid, may synergistically play an important role in regulating memory and learning. However, the pharmacokinetic behaviors of these compounds after their co-administration remain unclear. To this end, a selective and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated in rat plasma for the study of these three major bioactive ingredients in ZBPYR. The analytes in the plasma samples were separated on a Shiseido Capcell core C18 column using bendrofluazide as an internal standard, with a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. Electrospray ionization in the negative-ion mode and multiple reaction monitoring were used to identify and quantify the three analytes. All of the calibration curves showed good linearity (r > 0.992) over the concentration range, with a lower limit of quantification of 5 ng/mL. The precision of the analytical method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation (SD) was within 15%. Satisfactory extraction efficiency (between 83.4 and 99.4%) and matrix effects (76.4–107.4) were obtained by liquid–liquid extraction. The pharmacokinetic results showed that the three bioactive ingredients were rapidly absorbed and had a short terminal half-life in rats after oral administration of ZibuPiyin recipe. This UPLC–MS-MS study method used in this study may be useful for assessing the pharmacokinetic characteristics of various compounds, which would be helpful in determining their clinical potential. Introduction Traditional Chinese Medicines (TCMs) usually yield therapeutic effects as they have multiple components and multiple targets (1–5). A typical TCM prescription consists of a complex mixture of many different herbal and mineral ingredients, and is often prescribed in combinations that afford synergistic effects or reduce possible adverse reactions (6). Distinct from chemical drugs, the pharmacokinetic features of individual constituents do not represent those of the whole. Pharmacokinetic studies of herbal medicines have been performed according to the procedures used for chemical drugs, but these studies have only monitored a few marker compounds (7). Those results may only partly reveal the pharmacokinetic behaviors of herbal medicines; therefore, pharmacokinetic studies on multibioactive constituents or marker compounds of TCMs are needed. ZibuPiyin Recipe (ZBPYR), a TCM that has been prescribed for amnesia, was shown to afford neuronal protection and improve learning and memory in animals through its antioxidant effects and regulation of energy metabolism (8–10). High-performance liquid chromatography (HPLC) fingerprinting showed three peaks that were considered to be the main active compounds in ZBPYR due to their specific pharmacological activities. The flavonoid liquiritin (C21H22O9, 418.394 g/mol) is a potent constituent of Glycyrrhiza uralensis, which is also known as Chinese liquorice. The effects of liquiritin on learning and memory in rats are related to the enhancement of antioxidase activity, clearance of oxygen radicals, and inhibition of neural apoptosis (11, 12). The phenolic compound protocatechuic aldehyde (PAL; C7H6O3, 138.12 g/mol) is a water-soluble bioactive component of Salvia miltiorrhiza, a deciduous perennial. PAL can efficiently protect dopaminergic neurons against neurotoxin injury both in vitro and in vivo, possibly via its ability to increase levels of the multifunctional DJ-1 protein, and decrease α-synuclein expression and its growth-promoting effects on spine density (13). Rosmarinic acid (C18H16O8, 360.32 g/mol) is a natural phenol carboxylic acid and an active constituent of S. miltiorrhiza. Its administration dose dependently prevented cognitive impairment induced by chronic ethanol in passive avoidance learning and memory, and disturbed oxidant/antioxidant status as a possible mechanism (14). In addition, rosmarinic acid protected aortic endothelial cell function and ultrastructure against diabetes-induced damage, possibly through its antioxidant and anti-inflammatory effects (15). Furthermore, the administration of rosmarinic acid to diabetic rats ameliorated water consumption and urination (16). Thus, these three analytes of ZBPYR appear to play important roles in regulating memory and learning, and as such, may have clinical utility in the treatment of amnesia, so it is necessary to study the pharmacokinetics of these compounds in vivo. To date, there have been no simultaneous studies on these three compounds, despite the fact that several studies have reported the pharmacokinetics of liquiritin, PAL or rosmarinic acid (17–19). However, these studies only focused on one or two constituents, so the pharmacokinetic behaviors of these compounds after their co-administration remain unclear. In addition, the composition of the compound prescriptions may vary with different herbal combinations and extraction processes (20). Furthermore, due to the likely herb–herb and drug–drug interactions, the pharmacokinetics of multi-ingredient preparations may differ from single ingredient preparations (21, 22). Thus, the ultra-performance liquid chromatography–tandem mass spectrometry method (UPLC–MS-MS) method, which has high sensitivity, selectivity and specificity and is highly robust for compound separation, was applied to study the pharmacokinetics of liquiritin, PAL and rosmarinic acid after administration of ZBPYR in rats. This method may be useful for assessing the pharmacokinetic characteristics of various compounds, which would be helpful in determining their clinical potential. Materials and Methods Chemicals, reagents and sample ZBPYR consists of 12 herbs including Panax ginseng C.A. Mey., Dioscorea opposita Thunb., Poriacocos (Schw.) Wolf, Paeonia lactiflora Pall., S. miltiorrhiza, Dolichos lablab L., Nelumbo nucifera Gaertn, Acorus tatarinowii Schott, Polygala tenuifolia Willd, Santalum album L., Citrus reticulate Blanco and G. uralensis Fisch., which were purchased from the Yunnan hay sources Pharmaceutical Co., Ltd. (Yunnan, China) and were authenticated by Dr Yunpeng Diao (College of Pharmacy, Dalian Medical University, Liaoning, China). The reference standards of liquiritin, PAL, rosmarinic acid and the internal standard (IS) bendrofluazide had a purity >98%, and were all purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade acetonitrile and methanol were obtained from Fisher Scientific (Fair Lawn, NJ, USA), and HPLC-grade formic acid was purchased from Waters (Milford, MA, USA). Deionized water was used throughout the study. Other reagents were of analytical grade. Instrumentation and chromatography Liquid chromatography For fast, selective and sensitive analyses of analytes, a Waters UPLC–MS-MS system (Waters) consisting of a Waters ACQUITYTM UPLC and Xevo TQ mass spectrometer with a triple quadruple mass analyzer and electrospray ionization (ESI) interface in a negative ion mode were used in this assay. UPLC–MS-MS and data analysis were performed with the MassLynxTM NT4.1 workstation (Waters). The separation of three analytes and the IS from endogenous substances was performed on a Capcell core C18 column (2.1 mm × 100 mm, 2.6 μm; Shiseido, Japan). The column and autosampler tray temperatures were kept constant at 40 and 10°C, respectively. The mobile phase consisting of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) was run at 10–90% A over 0–3.0 min, 90% A over 3.0–3.2 min, 90–10% A over 3.2–3.5 min and 10%A over 3.5–4.0 min in a gradient elution mode at a flow rate of 0.2 mL/min. MS conditions The optimal MS parameters were as follows: capillary voltage set at 2.0 kV, source temperature set at 150°C, desolvation gas (nitrogen) at 400°C at a flow rate of 400 L/h, and cone gas flow rate at 50 L/h. Multiple reaction monitoring (MRM) was used to quantitate the three analytes and the IS. The MRM mode was conducted by monitoring the transitions of the precursor ion and product ion. The dwell time was set at 0.2 s to observe a good peak shape. Argon was used as the collision gas. The cone voltage, collision energy and MRM transitions of three analytes and the IS are listed in Table I. Table I. Mass Parameters for Liquiritin (1), Protocatechualdehyde (2), Rosmarinic Acid (3) and Bendroflumethiazide (IS) Compound  MRM (m/z)  Cone (V)  Collision (kV)  Dwell (s)  1  417.2 → 255.1  40  25  0.2  2  137.0 → 108.1  40  25  0.2  3  359.0 → 161.2  30  20  0.2  IS  420.1 → 289.0  40  30  0.2  Compound  MRM (m/z)  Cone (V)  Collision (kV)  Dwell (s)  1  417.2 → 255.1  40  25  0.2  2  137.0 → 108.1  40  25  0.2  3  359.0 → 161.2  30  20  0.2  IS  420.1 → 289.0  40  30  0.2  Preparation of standard samples and quality control samples Stock solutions of liquiritin, PAL and rosmarinic acid (200 μg/mL) and the IS (40 ng/mL) were prepared in methanol. A set of standard solutions and quality control (QC) solutions for the three bioactive constituents were prepared by diluting the stock solutions (50 ng/mL) successively with methanol. All of the solutions were stored at −20°C. Plasma standards were prepared as follows: 20 μL of standard solutions containing liquiritin, PAL and rosmarinic acid and 20 μL IS were added to Eppendorf microtubes, dried under air flow at 37°C, and mixed with 100 μL blank plasma and 10 μL hydrochloric acid followed by vortexing for 30 s. The final concentrations of the plasma standards were 5.00, 10.0, 25.0, 50.0, 100, 200 and 400 ng/mL for liquiritin and rosmarinic acid; and 5.00, 10.0, 25.0, 50.0, 100 and 200 ng/mL for PAL. The QC samples were prepared at concentrations of 10.0, 50.0, 320.0 ng/mL for liquiritin and rosmarinic acid, and at 10.0, 40.0, 160.0 ng/mL for PAL. The samples were processed with liquid–liquid extraction by adding 2 mL ethyl acetate. The extraction procedure included vortexing the mixture for 3 min, centrifuging at 13,000 rpm (4°C) for 5 min, blowing to dry the transferred supernatant, and reconstituting with the initial mobile phase. After vortexing for 1 min and centrifuging at 13,000 rpm (4°C) for 5 min, an aliquot of 5 μL supernatant was injected into the UPLC–MS-MS system. Method validation The assay was validated using plasma from healthy Sprague Dawley rats, following the guidelines for bioanalytical method validation issued by the US Food and Drug Administration Center for Drug Evaluation and Research (23). Specificity and selectivity Interference by the endogenous matrix was evaluated using six Sprague Dawley rats by comparing the chromatograms of blank plasma, plasma spiked with the three analytes and the IS, and plasma after oral administration of ZBPYR. Sensitivity The lower limit of quantification (LLOQ) was defined as the lowest concentration measurable with precision <20%, and accuracy between 80 and 120% of the theoretical value. The signal-to-noise ratio for the LLOQ must be ≥10 (23). Linearity The linearity of the assay was assessed using three analyte plasma standards over a concentration range of 5–400 ng/mL for liquiritin and rosmarinic acid and 5–200 ng/mL for PAL in three consecutive patches. Calibration curves were constructed by the weighted (1/x2) linear regression of three analytes/IS peak area ratios versus the spiked concentrations. Two calibration curves were determined and fitted to one curve with a coefficient that not <0.990 in one patch. Precision and accuracy The intra- and inter-day precision and accuracy were assessed by quantitating three concentration levels of QC samples (six samples for each concentration level) on the same day and on three consecutive validation days. The precision was evaluated by relative standard deviation (RSD, %) and accuracy by the relative error (RE, %) of measured concentration versus the spiked concentration. The extraction recovery and matrix effect The recoveries of three analytes and the IS were determined at three QC levels with six replicates for each level by comparing the peak areas from extracted samples with those from post-extracted blank plasma samples spiked with the three analytes and IS. The matrix effect was measured at three QC levels by comparing the peak area of post-extracted blank plasma samples spiked with the three analytes and IS (A) with that of pure standard solution containing equivalent amounts of the compounds (B). The ratio (A/B × 100)% was used to evaluate the matrix effect. Stability Freeze–thaw stability, post-preparative stability, short-term stability and long-term stability were evaluated at three QC levels to assess the stability of three analytes and the IS in rat plasma. QC samples at concentrations of 10.0, 50.0 and 320 ng/mL for liquiritin and rosmarinic acid, and at concentrations of 10.0, 40.0 and 160 for PAL were frozen at −80°C, and then thawed at room temperature for each cycle. The three freeze–thaw cycles were evaluated. The post-preparative stability of the processed samples in an autosampler at 10°C was assessed to determine whether a delay in analysis could lead to instability of the analytes over 8 h. The short-term stability was assessed by putting the QC samples on the benchtop for 4 h before analysis. The long-term stability of the analytes in rat plasma at −80°C was evaluated by analyzing the QC samples over 4 weeks. The analyte was considered stable if the response of the measured analyte concentration to its corresponding theoretical value differed by <15%. Pharmacokinetic study Animals Male Sprague Dawley Rats weighing 220–250 g were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China). Before this assay, the animals were raised with free access to normal standard rodent chow and water in specific-pathogen free (SPF) conditions for 1 week, and were then fasted with access to only water for 12 h prior to the experiment. The animal study was performed in accordance with the guidelines for the use of experimental animals of Dalian Medical University and was approved by the Institutional Review Committee on Animal Care. The protocol was approved by the Animal Ethics Committee of the institution. Preparation of ZBPYR, dosing and sampling ZBPYR consists of 12 crude drugs: Panax ginseng C.A. Mey. (30.0 g), Dioscorea opposita Thunb. (15.0 g), Poriacocos (Schw.) Wolf (15.0 g), Paeonia lactiflora Pall.(15.0 g), S. miltiorrhiza (12.0 g), Dolichos lablab L. (15.0 g), Nelumbo nucifera Gaertn (20.0 g), Acoru statarinowii Schott (10.0 g), Polygala tenuifolia Willd (10.0 g), Santalum album L. (4.5 g), Citrus reticulate Blanco (9.0 g) and G.uralensis Fisch. (9.0 g) (8). According to the constituents of ZBPYR, 12 herbs (164.5 g) were subjected to reflux extraction with water for 2 h. The water decoction was evaporated under vacuum to obtain a final concentration of 34.2 g extract. The extract was accurately weighed and then dissolved in water to prepare the 0.3591 g/mL prescription solution. The concentrations of liquiritin, PAL and rosmarinic acid in ZBPYR were 1.74, 0.05 and 0.03 mg/g, respectively. The pharmacokinetic study in the Sprague Dawley rats was a single-dose, randomized study. The rats were orally administered ZBPYR extract at a dose of 3.591 g/kg (equivalent to 17.2725 g/kg crude drug). About 0.3 mL blood samples were collected from the orbital venous plexus in heparinized centrifuge tubes at 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 12, 20, 24, 30 h after oral administration of 1 mL/100 g ZBPYR extract (the concentrations of liquiritin, PAL and rosmarinic acid in ZBPYR were 1.74, 0.05 and 0.03 mg/g, respectively). Plasma samples were obtained following centrifugation at 12,000 rpm for 5 min, and stored at −80°C until subsequent use. Data analysis The pharmacokinetic parameters (AUC0−∞, AUC0−t, T1/2) of the three analytes rats were calculated with DAS2.1 software supplied by the Pharmacological Society of China (Beijing, China). The maximum concentration (Cmax) and time to reach the maximum concentration (Tmax) were directly obtained from the experimental data. All of the data are expressed as mean ± SD. Results Method development Optimization of sample extraction method We selected acetonitrile and methanol as the protein precipitation (PPT) solvents, and ethyl acetate, methyl tert-butyl ether, and diethyl ether as the liquid–liquid extraction solvents (data not shown). The optimization extraction method was evaluated by the recovery and matrix effect using QC samples. According to the data, the PPT method showed severe endogenous compound interference, which caused a large matrix effect. In addition, the protein residue can contaminate the LC–MS-MS system. Meanwhile, ethyl acetate yielded excellent results for PAL and liquiritin. On the other hand, the recovery was low (<50%) for rosmarinic acid, possibly due to its acidity; thus, an additive (hydrochloric acid, formic acid, acetic acid) was added to increase its yield. Finally, hydrochloric acid was used for optimization in peak shape, recovery and matrix effects of the analytes. Optimization of LC–MS-MS conditions MRM mode was selected to monitor the analytes. The presence of the proton adduction [M − H]− was more abundant than that of [M + H]+. The collision energy and fragment voltage were adjusted to produce the maximum response and optimal MRM transition (417.2 → 255.1 for liquiritin, 137.0 → 108.1 for PAL, 359.0 → 161.2 for rosmarinic acid and 420.1 → 289.0 for IS). The full-scan product ion spectra of three analytes and the IS are shown in Figure 1. The concentration of IS was chosen based on the response of the IS, which should be equal to the response intensity of three analytes with middle concentration. The constitution of the mobile phase was investigated to ensure the stability of the analytes. A Capcellcore C18 column (2.1 mm × 100 mm, 2.6 μm; Shiseido, Japan) was achieved better separation and shorter analytical time by comparing with Kinetex C18 column (100 mm × 4.6 mm, 2.6 um; Phenomenex, American) and ZORBAX SB-C18 column (150 mm × 4.6 mm, 3.5 um; Agilent, American). In the binary gradient system, acetonitrile–water led a higher response than methanol-water, as well as peak tailing. Compared with ammonium acetate, 0.1% formic acid promoted symmetry of the chromatographic peak and led to a robust response, so it was eventually chosen as the mobile phase additive. Figure 1. View largeDownload slide Full scan product ions of [M − H]− of liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS). Figure 1. View largeDownload slide Full scan product ions of [M − H]− of liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS). Method validation Specificity and selectivity Typical chromatograms obtained from rat plasma (Figure 2A), rat plasma spiked with the three analytes and IS (Figure 2B), and rat plasma sample obtained after oral administration of ZBPYR (Figure 2C) are shown in Figure 2. There were no endogenous component interference at the retention time of liquiritin (1.14 min), PAL (1.37 min), rosmarinic acid (1.84 min) and the IS (1.58 min) at their transitions. The optimized extraction method and LC–MS-MS conditions allowed good separation of the analytes from interference components. Thus, the specificity of this method was adequate for studying these three analytes in rat plasma. Figure 2. View largeDownload slide MRM chromatograms of blank rat plasma sample (A); blank rat plasma spiked with analytes (LLOQ) and IS (B); and rat plasma (spiked with IS) after administration of Zibupiyin Recipe at 2 h (C) for liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS, 4). Figure 2. View largeDownload slide MRM chromatograms of blank rat plasma sample (A); blank rat plasma spiked with analytes (LLOQ) and IS (B); and rat plasma (spiked with IS) after administration of Zibupiyin Recipe at 2 h (C) for liquiritin (1), protocatechuic aldehyde (2), rosmarinic acid (3) and bendroflumethiazide (IS, 4). Linearity and LLOQ The calibration curves showed good linearity over the concentration range of 5–400 ng/mL for liquiritin and rosmarinic acid, and 5–200 ng/mL for PAL. Typical linear regression equations for the calibration curves (weighted factor 1/x2) were Y = 3.24E−3X + 4.18E−4 (r = 0.9928) for liquiritin, Y = 2.06E−2X + 1.80E−4 (r = 0.9946) for PAL, and Y = 4.82E−3X + 2.11E−5 (r = 0.9941) for rosmarinic acid, where X represents the concentration of three analytes in plasma and Y represents the peak area ratio. These results indicate that the LC–MS-MS response was proportional to the plasma concentration of the analytes, and that the assay was linear. The LLOQs for the three analytes were all set at 5 ng/mL with accuracy and intra- and inter-day precisions within 12.5%, which was stable and sufficient for this assay. Accuracy, precision, recovery and matrix effects Intra- and inter-day precision, accuracy, extraction recovery, and matrix effects for the three analytes are summarized in Table II. All of the intra- and inter-day, precision and accuracy results were within the acceptable criteria of ±15%, which indicated that this preprocessing method was consistent, precise and reproducible for the analytes at different concentrations. The extraction recovery and matrix effects ranged from 83.4–89.5% to 76.4–81.2% for liquiritin, 87.4–93.1% to 81.7–84.5% for PAL, and 95.7–99.4% to 99.9–107.4% for rosmarinic acid (Table II). The mean recovery and matrix effects of the IS were 92.3 and 102.9%, respectively, at a concentration of 40 ng/mL. These results indicate that the recovery and matrix effect for the three analytes were within the recommended values for assay validation, as stated in the “Guidance for Industry: Bioanalytical Method Validation” (23). Table II. Precision, Accuracy, Extraction Recovery and Matrix Effect of Liquiritin (1), Protocatechualdehyde (2), and Rosmarinic Acid (3) and IS in Rat Plasma Analytes  Spike concentration (ng mL−1)  Accuracy, RE (%)  Intra-day  Inter-day  Recovery (%, Mean ± SD)  Matrix effect (%, Mean ± SD)  RSD (%)  RSD (%)  1  10  −3.0  12.8  12.9  83.4 ± 7.2  78.9 ± 4.3  50  −4.4  11.8  12.6  89.5 ± 5.1  76.4 ± 3.7  320  3.2  9.4  9.2  88.1 ± 4.3  81.2 ± 4.9  2  10  −4.4  4.1  5.7  89.8 ± 3.6  82.7 ± 1.8  40  8.8  8.8  4.9  87.4 ± 2.8  84.5 ± 4.9  160  −5.2  5.3  6.4  93.1 ± 5.0  81.7 ± 3.8  3  10  −4.3  9.9  11.0  95.7 ± 8.4  102.1 ± 2.1  50  2.6  12.2  10.3  99.4 ± 6.7  99.9 ± 9.1  320  −3.0  8.6  9.2  96.1 ± 7.8  107.4 ± 3.8  IS  40  –  –  –  92.3 ± 3.7  102.9 ± 4.1  Analytes  Spike concentration (ng mL−1)  Accuracy, RE (%)  Intra-day  Inter-day  Recovery (%, Mean ± SD)  Matrix effect (%, Mean ± SD)  RSD (%)  RSD (%)  1  10  −3.0  12.8  12.9  83.4 ± 7.2  78.9 ± 4.3  50  −4.4  11.8  12.6  89.5 ± 5.1  76.4 ± 3.7  320  3.2  9.4  9.2  88.1 ± 4.3  81.2 ± 4.9  2  10  −4.4  4.1  5.7  89.8 ± 3.6  82.7 ± 1.8  40  8.8  8.8  4.9  87.4 ± 2.8  84.5 ± 4.9  160  −5.2  5.3  6.4  93.1 ± 5.0  81.7 ± 3.8  3  10  −4.3  9.9  11.0  95.7 ± 8.4  102.1 ± 2.1  50  2.6  12.2  10.3  99.4 ± 6.7  99.9 ± 9.1  320  −3.0  8.6  9.2  96.1 ± 7.8  107.4 ± 3.8  IS  40  –  –  –  92.3 ± 3.7  102.9 ± 4.1  Stability The stability of liquiritin, PAL and rosmarinic acid in rat plasma under different conditions is summarized in Table III, in which the precision and accuracy were within the acceptable criteria of ±15%. These results indicate that liquiritin, PAL and rosmarinic acid were stable in plasma at room temperature for 4 h, at −80°C for at least 30 days, after three freeze–thaw cycles, and at 10°C in an autosampler for 8 h in processed samples. Table III. Stability of Liquiritin (1), Protocatechualdehyde (2), and Rosmarinic Acid (3) in Rat Plasma At Different Conditions Determined by UPLC–MS-MS Analytes  Spike concentration (ng mL−1)  30 d, −80°C  4 h, room temperature  Three freeze–thaw circles  8 h,10°C  RE (%)  RSD (%)  RE(%)  RSD (%)  RE (%)  RSD (%)  RE (%)  RSD (%)  1  10  9.4  6.1  8.4  0.6  7.8  6.3  8.3  0.8  50  1.2  0.4  3.8  6.8  −3.8  3.4  0.3  0.6  320  −8.1  0.7  8.0  7.3  −4.4  10.2  6.3  6.1  2  10  −2.5  7.9  −0.7  4.7  1.5  7.5  7.2  5.5  40  12.9  1.5  −9.9  3.9  −8.6  10.0  7.8  −9.9  160  9.1  1.9  9.5  4.2  10.0  4.0  2.7  6.6  3  10  12.5  5.7  2.1  5.4  2.7  7.8  3.6  4.3  50  10.9  −3.4  8.7  4.4  9.9  2.9  12.6  5.9  320  6.7  3.2  −10.5  7.8  11.6  9.0  8.8  11.2  Analytes  Spike concentration (ng mL−1)  30 d, −80°C  4 h, room temperature  Three freeze–thaw circles  8 h,10°C  RE (%)  RSD (%)  RE(%)  RSD (%)  RE (%)  RSD (%)  RE (%)  RSD (%)  1  10  9.4  6.1  8.4  0.6  7.8  6.3  8.3  0.8  50  1.2  0.4  3.8  6.8  −3.8  3.4  0.3  0.6  320  −8.1  0.7  8.0  7.3  −4.4  10.2  6.3  6.1  2  10  −2.5  7.9  −0.7  4.7  1.5  7.5  7.2  5.5  40  12.9  1.5  −9.9  3.9  −8.6  10.0  7.8  −9.9  160  9.1  1.9  9.5  4.2  10.0  4.0  2.7  6.6  3  10  12.5  5.7  2.1  5.4  2.7  7.8  3.6  4.3  50  10.9  −3.4  8.7  4.4  9.9  2.9  12.6  5.9  320  6.7  3.2  −10.5  7.8  11.6  9.0  8.8  11.2  Pharmacokinetic study The developed method was successfully applied to the pharmacokinetic study of ZBPYR in rat plasma after oral administration of 0.3591 g/mL ZBPYR. The concentration–time curves (mean ± SD) are presented in Figure 3 and the corresponding pharmacokinetic parameters are listed in Table IV. The concentration of the drug was readily measurable up to 30 h. By observing the pharmacokinetic time profile curves, the mean Cmax of liquiritin, PAL and rosmarinic acid in male rats were 216, 124 and 223 ng/mL, respectively, with analogous concentration trends. Table IV. Pharmacokinetic Parameters of Liquiritin, Protocatechualdehyde and Rosmarinic Acid in Rat Plasma After Oral Administration of ZibuPiyin Recipe (ZBPYR) Parameters  Units  Liquiritin  Protocatechualdehyde  Rosmarinic acid  AUC0−∞  ug/L h  1447.4 ± 440.4  1425.5 ± 396.5  3099.4 ± 782.7  AUC0−t  ug/L h  1569.4 ± 494.4  1640.4 ± 325.6  3640.6 ± 1189.4  Cmax  ug/L  215.9 ± 24.9  123.7 ± 36.9  222.7 ± 86.7  Tmax  h  0.6 ± 0.2  1.2 ± 0.4  1.7 ± 0.7  T1/2  h  7.8 ± 0.8  9.7 ± 4.4  10.0 ± 2.8  Parameters  Units  Liquiritin  Protocatechualdehyde  Rosmarinic acid  AUC0−∞  ug/L h  1447.4 ± 440.4  1425.5 ± 396.5  3099.4 ± 782.7  AUC0−t  ug/L h  1569.4 ± 494.4  1640.4 ± 325.6  3640.6 ± 1189.4  Cmax  ug/L  215.9 ± 24.9  123.7 ± 36.9  222.7 ± 86.7  Tmax  h  0.6 ± 0.2  1.2 ± 0.4  1.7 ± 0.7  T1/2  h  7.8 ± 0.8  9.7 ± 4.4  10.0 ± 2.8  Figure 3. View largeDownload slide Rat plasma concentration (μg/L)–time (h) curves of liquiritin (1), protocatechuic aldehyde (2) and rosmarinic acid (3) after administration of ZBPYR. Figure 3. View largeDownload slide Rat plasma concentration (μg/L)–time (h) curves of liquiritin (1), protocatechuic aldehyde (2) and rosmarinic acid (3) after administration of ZBPYR. Discussion In a previous report, the pharmacokinetic profiles of PAL in rat serum after oral administration of Radix Salviae miltiorrhizae extract showed a double peak (18), which was different from our results, possibly because of the different pharmacokinetic profiles in different herbs. The mean Tmax of male rats for liquiritin, PAL and rosmarinic acid were ~0.6, 1.2 and 1.7 h, respectively, showing that the absorption of liquiritin was more rapid than that of PAL and rosmarinic acid, which is in accordance with previous studies (17–19). The data from our pharmacokinetic study demonstrated that liquiritin, PAL and rosmarinic acid in rat plasma had a rapid absorption and short terminal half-life, which may illustrate a short dosing interval and reveal the pharmacokinetic features of these constituents of ZBPYR in rats. The pharmacokinetic profile of these three analytes in rats after administration of ZBPYR was characterized for the first time in this study. These data may provide a foundation for future pharmacological studies on ZBPYR. Conclusions A rapid and specific UPLC–ESI–MS-MS method was established for the simultaneous determination of liquiritin, PAL and rosmarinic acid in rat plasma, which was highly sensitive and accurate. The method was successfully applied to the pharmacokinetic study of liquiritin, PAL and rosmarinic acid in rats after oral administration of ZBPYR extract, and the pharmacokinetic parameters may provide a suitable reference for clinical application. Funding This research was supported by National Natural Science Foundation of China (No. 81230084 and 81403306) and the Specialized Research Fund for the Doctoral Program of Higher Education of China from Ministry of Education, Science and Technology Development Center (20132105130001). References 1 Liu, D., Jia, X.B.; Analysis and discussion about current development of relevant studies on” traditional Chinese medicine components; China Journal of Chinese Material Medica , ( 2014); 39: 171– 174. 2 Sun, E., Xu, F.J., Zhang, Z.H.; Discussion about research progress and ideas on processing mechanism of traditional Chinese medicine; China Journal of Chinese Material Medica , ( 2014); 39: 363– 369. 3 Lao, Y., Wang, X., Xu, N.; Application of proteomics to determine the mechanism of action of traditional Chinese medicine remedies; Journal of Ethnopharmacology , ( 2014); 155: 1– 8. Google Scholar CrossRef Search ADS PubMed  4 Zhang, J., Gao, W., Hu, X.; The influence of compatibility of traditional Chinese medicine on the pharmacokinetic of main components in Fructusaurantii; Journal of Ethnopharmacology , ( 2012); 144: 277– 283. Google Scholar CrossRef Search ADS PubMed  5 Sun, H., Dong, T., Zhang, A.; Pharmacokinetics of hesperetin and naringenin in the Zhi Zhu Wan, a traditional Chinese medicinal formulae, and its pharmacodynamics study; Phytotherapy Research , ( 2013); 27: 1345– 1351. Google Scholar CrossRef Search ADS PubMed  6 Ma, W., Peng, Y., Wang, W., Bian, Q., Wang, N., Lee, D.Y.-W., et al.  .; Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC-MS/MS; Biomedical Chromatography , ( 2016); 30( 10): 1573– 1581. Google Scholar CrossRef Search ADS PubMed  7 He, S.M., Chan, E., Zhou, S.F.; ADME properties of herbal medicines in humans: evidence, challenges and strategies; Current Pharmaceutical Design , ( 2011); 17: 357– 407. Google Scholar CrossRef Search ADS PubMed  8 Shi, X., Lu, X.G., Zhan, L.B.; The effects of the Chinese medicine ZiBuPiYin recipe on the hippocampus in a rat model of diabetes-associated cognitive decline: a proteomic analysis; Diabetologia , ( 2011); 54: 1888– 1899. Google Scholar CrossRef Search ADS PubMed  9 Zhan, L., Jiang, W., Lu, X.; Mechanism of protective effect of spleen yin nourishing recipe on dendritic spines in rats; Journal—Beijing University of Traditional Chinese Medicine , ( 2007); 30: 597. 10 Zhan, L.B., Lin, H.Y., Gong, X.Y.; Effects of recipe on endoplasmic reticulum stress in brain tissue of spleen-yin deficiency Alzheimer’s Disease rats; World Science and Technology (Modernization of Traditional Chinese Medicine and Materia Medica) , ( 2011); 13: 993– 998. 11 Jia, S.L., Wu, X.L., Li, X.X., Dai, X.L., Gao, Z.L., Lu, Z., et al.  .; Neuroprotective effects of liquiritin on cognitive deficits induced by soluble amyloid-β1-42 oligomers injected into the hippocampus; Journal of Asian Natural Products Research , ( 2016); 18( 12): 1186– 1199. Google Scholar CrossRef Search ADS PubMed  12 Huang, X., Wang, Y., Ren, K.; Protective effects of liquiritin on the brain of rats with Alzheimer’s disease; The West Indian Medical Journal , ( 2016). 10.7727/wimj.2016.058. 13 Zhao, X., Zhai, S., An, M.S., Wang, Y.H., Yang, Y.F., Ge, H.Q., et al.  .; Neuroprotective effects of protocatechuic aldehyde against neurotoxin-induced cellular and animal models of Parkinson’s disease; PLoS One , ( 2013); 8( 10): e78220. Google Scholar CrossRef Search ADS PubMed  14 Hasanein, P., Seifi, R., Hajinezhad, M.R., Emamjomeh, A.; Rosmarinic acid protects against chronic ethanol-induced learning and memory deficits in rats; Nutritional Neuroscience , ( 2016). 10.1080/1028415X.2016.1203125. 15 Sotnikova, R., Okruhlicova, L., Vlkovicova, J., Navarova, J., Gajdacova, B., Pivackova, L., et al.  .; Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta; The Journal of Pharmacy and Pharmacology , ( 2013); 65( 5): 713– 723. Google Scholar CrossRef Search ADS PubMed  16 Sotnikova, R., Kaprinay, B., Navarova, J.; Rosmarinic acid mitigates signs of systemic oxidative stress in streptozotocin-induced diabetes in rats; General Physiology and Biophysics , ( 2015); 34( 4): 449– 452. Google Scholar PubMed  17 Yan, Y., Chai, C.Z., Wang, D.W.; Simultaneous determination of puerarin, daidzin, daidzein, paeoniflorin, albiflorin, liquiritin and liquiritigenin in rat plasma and its application to a pharmacokinetic study of Ge-Gen Decoction by a liquid chromatography–electrospray ionization-tandem mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis , ( 2014); 95: 76– 84. Google Scholar CrossRef Search ADS PubMed  18 Ye, G., Wang, C.S., Li, Y.Y.; Simultaneous determination and pharmacokinetic studies on (3, 4-dihydroxyphenyl)-lactic acid and protocatechuic aldehyde in rat serum after oral administration of Radix Salviae miltiorrhizae extract; Journal of Chromatographic Science , ( 2003); 41: 327– 330. Google Scholar CrossRef Search ADS PubMed  19 Ma, B., Wang, Y., Zhang, Q.; Simultaneous determination of oridonin, ponicidin and rosmarinic acid from herbaisodi rubescentis extract by LC–MS-MS in rat plasma; Journal of Chromatographic Science , ( 2013); 51: 910– 918. Google Scholar CrossRef Search ADS PubMed  20 Bi, X., Gong, M., Di, L.; Review on prescription compatibility of shaoyaogancao decoction and reflection on pharmacokinetic compatibility mechanism of traditional chinese medicine prescription based on in vivo drug interaction of main efficacious components; Evidence-Based Complementary and Alternative Medicine , ( 2014); 2014: 208129. Google Scholar CrossRef Search ADS PubMed  21 Chen, Y., Ouyang, D.S., Kang, Z.; Effect of a traditional Chinese medicine Liu Wei Di Huang Wan on the activities of CYP2C19, CYP2D6 and CYP3A4 in healthy volunteers; Xenobiotica; the Fate of Foreign Compounds in Biological Systems , ( 2012); 42: 596– 602. Google Scholar CrossRef Search ADS PubMed  22 Tang, J., Song, X., Zhu, M.; Study on the pharmacokinetics drug–drug interaction potential of Glycyrrhiza uralensis, a traditional Chinese medicine, with lidocaine in rats; Phytotherapy Research , ( 2009); 23: 603– 607. Google Scholar CrossRef Search ADS PubMed  23 U.S. Food and Drug Administration. http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm64964.htm ( 2013). © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Journal

Journal of Chromatographic ScienceOxford University Press

Published: Feb 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve Freelancer

DeepDyve Pro

Price
FREE
$49/month

$360/year
Save searches from
Google Scholar,
PubMed
Create lists to
organize your research
Export lists, citations
Read DeepDyve articles
Abstract access only
Unlimited access to over
18 million full-text articles
Print
20 pages/month
PDF Discount
20% off