Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated beta-CD

CE with multiple isomer sulfated beta-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of norketamine in liquid/liquid extracts of the two body fluids. Ketamine, norketamine, and dehydronorketamine could be unambiguously identified via HPLC fractionation of urinary extracts and using LC-MS and LC-MS/MS with 1 mmu mass discrimination. The CE assay was used to characterize the stereoselectivity of the compounds' enantiomers in the samples of five ponies anesthetized with isoflurane in oxygen and treated with intravenous continuous infusion of racemic ketamine. The concentrations of the ketamine enantiomers in plasma are equal, whereas the urinary amount of R-ketamine is larger than that of S-ketamine. Plasma and urine contain higher S- than R-norketamine levels and the mean S-/R-enantiomer ratios of dehydronorketamine in plasma and urine are lower than unity and similar.     

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.     

Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in echinococcosis patients

An assay for the simultaneous determination of the enantiomers of hydroxymebendazole (OH-MBZ) and hydroxyaminomebendazole (OH-AMBZ) together with aminomebendazole (AMBZ) in human plasma is described for the first time. It is based upon liquid-liquid extraction at alkaline pH from 0.5 mL plasma followed by analysis of the reconstituted extract by CE with reversed polarity in the presence of a 50 mM, pH 4.2 acetate buffer containing 15 mg/mL sulfated beta-CD as chiral selector. For all compounds, detection limits are between 0.01 and 0.04 microg/mL, and intraday and interday precisions evaluated from peak area ratios are <6.9 and <8.5%, respectively. Analysis of 39 samples of echinoccocosis patients undergoing pharmacotherapy with mebendazole (MBZ) revealed that the ketoreduction of MBZ and AMBZ is highly stereoselective. One enantiomer of each metabolite (firstly detected peak in both cases) could only be detected. The CE data revealed that OH-MBZ (mean: 0.715 microg/mL) is the major metabolite followed by AMBZ (mean: 0.165 microg/mL) and OH-AMBZ (mean: 0.055 microg/mL) whereas the MBZ plasma levels (mean: 0.096 microg/mL, levels determined by HPLC) were between those of AMBZ and OH-AMBZ.     

Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma.

An enantioselective high-performance liquid chromatographic assay for the quantitation of the enantiomers of ketamine and its major metabolite norketamine in human plasma is described (assay I). The procedure involved extraction of the compounds from alkalized plasma into cyclohexane. Stereoselective separation was achieved with a prepacked alpha 1-acid glycoprotein column without any derivatization procedure. A second assay using a conventional reversed-phase column to determine total (racemic) ketamine and norketamine is also described. Because of interfering plasma peaks (assay II) the cyclohexane solution was reextracted into 1 M hydrochloric acid. The detection wavelength was 215 nm for all substances. The limit of quantification of the method was ca. 40 ng/ml in plasma. The assays were sensitive and reproducible. The method was demonstrated to be sensitive for stereoselective pharmacokinetic studies of ketamine after clinical doses.     

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics

A method of capillary electrophoresis with contactless conductivity detection has been developed for non‐enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 μm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 μmol/L) for ketamine and 75 ng/mL (0.3 μmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 μg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 μL of plasma with 30 μL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.     

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.     

Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro

Glucuronidation catalyzed by uridine‐5′‐diphospho‐glucuronosyl‐transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O‐glucuronidation is more common than N‐glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β‐glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Concentrations of HNK diastereomers and norketamine were significantly higher in comparison to untreated urine and an increase of ketamine and DHNK levels was found in selected but not all samples. This suggests that O‐glucuronides of HNK and N‐glucuronides of the other compounds are formed in equines. N‐glucuronidation of norketamine was studied in vitro with liver microsomes of different species and the single human enzyme UGT1A4. With equine liver microsomes (ELM) a stereoselective N‐glucuronidation of norketamine was found that compares well to the results obtained with urines collected after ketamine administration. No reaction was observed with canine liver microsomes, human liver microsomes and UGT1A4. Incubation of ketamine and DHNK with ELM did not reveal any glucuronidation. Enantioselective CE is suitable to provide insight into the phase II metabolism of ketamine and its metabolites.     

Analysis of ketamine and norketamine in hair samples using molecularly imprinted solid-phase extraction (MISPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS)

An anti-ketamine molecularly imprinted polymer (MIP) was synthesized and used as the sorbent in a solid-phase extraction protocol to isolate ketamine and norketamine from human hair extracts prior to LC-MS/MS analysis. Under optimised conditions, the MIP was capable of selectively rebinding ketamine, a licensed anaesthetic that is widely misused as a recreational drug, with an apparent binding capacity of 0.13 μg ketamine per mg polymer. The limit of detection (LOD) and lower limit of quantification (LLOQ) for both ketamine and norketamine were 0.1 ng/mg hair and 0.2 ng/mg hair, respectively, when 10 mg hair were analysed. The method was linear from 0.1 to 10 ng/mg hair, with correlation coefficients (R ²) of better than 0.99 for both ketamine and norketamine. Recoveries from hair samples spiked with ketamine and norketamine at a concentration of 50 ng/mg were 86% and 88%, respectively. The method showed good intra- and interday precisions (<5%) for both analytes. Minimal matrix effects were observed during the LC-MS/MS analysis of ketamine (ion suppression −6.8%) and norketamine (ion enhancement +0.2%). Results for forensic case samples demonstrated that the method successfully detected ketamine and norketamine concentrations in hair samples with analyte concentrations ranging from 0.2 to 5.7 ng/mg and from 0.1 to 1.2 ng/mg, respectively.     

Simultaneous Measurement of Urinary Ketamine,Norketamine, and Dehydronorketamine by Liquid Chromatography‐Atmospheric Pressure Chemical Ionization Mass Spectrometry

A liquid chromatography/atmospheric pressure ionization mass spectrometry has been developed for the determination of ketamine, norketamine, and dehydronorketamine in human urine. A separation of these analytes in urine samples without tedious pretreatment procedures has been achieved within 10 min. Linear calibration curves of these analytes with coefficients better than 0.998 have been obtained over a wide range from 12.5 to 200 ng/mL. The accuracy was between 2.1% and 7.2% with detection limits at levels of 0.02 ng/mL, 0.02 ng/mL and 0.93 ng/mL for ketamine, norketamine and dehydronorketamine, respectively. The results demonstrate the suitability of the liquid chromatography/atmospheric pressure ionization mass spectrometry approach to analyze trace ketamine, norketamine and dehydronorketamine in urine. Urinary ketamine and norketamine levels were relatively low at 4–24 h intervals and were difficult to assay in a normal laboratory. In the present study, the determination of urinary dehydronorketamine levels at 2–24 hours appears to have a great potential for use in Schedule III controlled drugs management.     

Development and validation of a stereoselective HPLC method for the determination of the in vitro transport of nateglinide enantiomers in rat intestine

A simple stereoselective high performance liquid chromatographic method was developed for the determination of the in vitro transport of the enantiomers of nateglinide (N-(trans-4-isopropylcyclohexyl-carbonyl)-phenylalanine) in the rat intestine using a Chiralcel OJ-RH column (150 x 4.0 mm, 5 microm). The effects of the mobile phase composition, pH, the flow rate, and the temperature on the chromatographic separation were investigated. The enantioseparation was achieved at 33 degrees C using a mobile phase containing 100 mM potassium dihydrogen phosphate, pH 2.5, and ACN (32:68 v/v) delivered at a flow rate of 1 mL/min. The analytes were monitored at 210 nm and linearity (r >0.99) was obtained for a concentration range of 0.5-50 microg/mL. The LOD and LOQ were 0.2 and 0.5 microg/mL for the R-enantiomer and 0.2 and 0.8 microg/mL for the S-enantiomer, respectively. Both, the intra- and interday accuracy and precision of the calibration curves were determined. The method was successfully applied to estimate the in vitro passage of the enantiomers and the racemate of nateglinide in duodenum, jejunum, and ileum of rats. Generally, higher concentrations of nateglinide and the S-enantiomer were observed when the racemate was administered compared to administration of the individual enantiomers of nateglinide.     

GC—MS定量测定大鼠血,脑中的氯胺酮和二甲苯胺噻嗪

描述了大鼠血、脑中氯胺酮和二甲苯胺噻嗪同时测定的定量分析方法。该法用溶剂同时提取血中或脑中的氯胺酮和二甲苯胺噻嗪,并在气相色谱上与杂质有效分离。方法简单、可靠、灵敏度高。已用于给药后大鼠血、脑中氯胺酮和二甲苯胺噻嗪的定量测定,得到了有意义的结果。     

Simultaneous separation of eight beta-adrenergic drugs using titanium dioxide nanoparticles as additive in capillary electrophoresis

The analysis is described for separating seven beta-adrenergic blocking agents (atenolol, celiprolol, clorprenaline, fenoterol, metoprolol, propranolol, terbutaline) and clenbuterol (sympathomimetic beta-2 receptor stimulating agonist, decongestant and bronchodilator, illicit anabolic used in athletics) by CE with UV detection. In order to simultaneously separate all analytes, Tris-H3PO4 solution was applied containing titanium dioxide nanoparticles (TiO2 NPs) as BGEs. The effects of important factors, such as concentration of TiO2 NPs, optimum pH, run buffer concentration, and separation voltage, were investigated so as to achieve best CE separation. The eight analytes could be well separated applying a separation voltage of 15 kV in 75 mM Tris-H3PO4 buffer at a pH of 2.40, containing 6.0 x 10(-6) g/mL TiO2 NPs. Under these optimal conditions, the RSDs for peak areas and for migration times were less than 2.7 and 2.3%, respectively. The detection limits were 0.1 microg/mL for celiprolol, 0.1 microg/mL for propranolol, 0.2 microg/mL for fenoterol, 1.0 microg/mL for atenolol, 1.0 microg/mL for clenbuterol, 1.0 microg/mL for clorprenaline, 1.0 microg/mL for metoprolol, and 1.0 microg/mL for terbutaline. The proposed method was successfully applied for the rapid CE determination of the frequently applied antihypertensive beta-blocking compounds atenolol, metoprolol, terbutaline, and propranolol in pharmaceutical tablets.     

固相萃取-毛细管电泳法测定兔血清中的山莨菪碱对映体

建立一种可用于定量的毛细管电泳法分离山莨菪碱对映体. 系统研究了三种手性选择剂: 羟丙基-β-环糊精 (HP-β-CD), 甲基-β-环糊精 (Me-β-CD), 羧甲基-β-环糊精(CM-β-CD) 及其浓度、缓冲溶液浓度和 pH 对山莨菪碱拆分的影响. 在110 mmol/L Tris-H3PO4缓冲液中加入20.0 mg/mL HP-β-CD和5.0 mg/mL CM-β-CD (pH 4.0)条件下, 山莨菪碱的4个对映体达到基线分离. 血清样品通过固相萃取预处理和浓缩, 对映体的固相萃取回收率在82.9%～90.7%, 相对标准偏差RSD%均小于7 %. 山莨菪碱的4个对映体血标准溶液浓度与电泳峰面积在77.86～0.39 μg/mL范围内呈良好的线性, r≥0.999, 检出限(S/N＝3)为0.08 μg/mL. 平均日间和日内精密度(RSD% )分别小于6.1% 和4.8%, 方法回收率为97.4% 和105.4%. 建立的方法准确、可靠, 应用于监测兔连续3 d口服75 mg 山莨菪碱后血清中山莨菪碱的血药浓度, 结果满意.     

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.     

Optimization and validation of a new CE method for the determination of pantoprazole enantiomers

A new CE method using sulfobutylether-beta-cyclodextrin (SBE-beta-CD) as chiral additive was developed and validated for the determination of pantoprazole enantiomers. The primary factors affecting its separation efficiency, which include chiral selector, buffer pH, organic additive, and applied voltage, were optimized. The best results were obtained using a buffer consisting of 50 mM borax-150 mM phosphate adjusted to pH 6.5, 20 mg/mL SBE-beta-CD, and a 10 kV applied voltage. The optimized method was validated for linearity, precision, accuracy, and proved to be robust. The LOD and LOQ for R-(+)-pantoprazole were 0.9 and 2.5 μg/mL, respectively. The method is capable of determining a minimum limit of 0.1% (w/w) of R-enantiomer in S-(-)-pantoprazole bulk samples.     

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.     

Enantioselective CE method for pharmacokinetic studies on ibuprofen and its chiral metabolites with reference to genetic polymorphism

A stereospecific CE method was elaborated for the quantification of ibuprofen enantiomers and their major phase I metabolites: 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen in plasma and urine. Optimal temperature and pH of BGE were established to obtain complete separation of eight ibuprofen chiral compounds and (+)-S indobufen, applied as an internal standard, during one analytical run. After isolation from biological matrices using SPE on an octadecyl stationary phase, the analytes were separated and resolved up to 10 min in a silica capillary filled with BGE, consisting of heptakis 2,3,6-tri-O-methyl-beta-CD in triethanolamine-phosphate buffer, pH 5.0. Complete enantioseparation of the all analytes confirmed specificity of the method. The calibration curves were linear in the range of 0.1-25.0 mg/L for IBP enantiomers and their chiral metabolites in 0.5 mL of plasma and 1.0-200.0 mg/L in 0.05 mL of urine. Following SPE procedure, recovery of the chiral analytes from the two media was in the ranges of 82-87%, 90-95% and 70-76% for ibuprofen, 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen enantiomers, respectively. The validated method was successfully applied in pharmacokinetic investigations of IBP enantiomers as well as free chiral metabolites in reference to the genetic polymorphism of CYP450 2C isoenzymes.     

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane

An enantioselective assay for the determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ‐cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ‐cyclodextrin. The developed assay is precise (intra‐ and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.     

Determination of the enantiomeric impurity in S-(-)pantoprazole using high performance liquid chromatography with sulfobutylether-beta-cyclodextrin as chiral additive

A new and accurate HPLC method using sulfobutylether-beta-cyclodextrin (SBE-beta-CD) as chiral mobile phase additive (CMPA) was developed and validated for the determination of R-(+)pantoprazole in S-(-)pantoprazole. The influences of type and concentration of CD, ACN content and buffer pH of mobile phase on the resolution and retention of enantiomers were investigated. A baseline resolution of pantoprazole enantiomers was achieved on a Spherigel C18 column (150 mm x 4.6 mm, 5 microm) using ACN and 10 mM phosphate buffer (pH 2.5) containing 10 mM SBE-beta-CD (15:85 v/v) as mobile phase with a flow rate of 0.9 mL/min at 20 degrees C. The detection wavelength was set at 290 nm. The method was extensively validated in terms of accuracy, precision and linearity according to the International Conference on Harmonisation (ICH) guidelines and proved to be robust. The LOD and LOQ for R-(+)pantoprazole were 0.2 and 0.5 microg/mL, respectively, with 5 microL injection volume. A good linear relationship was obtained in the concentration range of 0.5-6.0 microg/mL with r(2) >0.999 for R-(+)pantoprazole. The percentage recovery of the R-(+)pantoprazole ranged from 92.1 to 101.2 in bulk drug of S-(-)pantoprazole. The method is capable of determining a minimum limit of 0.05% w/w of R-enantiomer in S-(-)pantoprazole bulk samples.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.