Liquid chromatographic-tandem mass spectrometric urine assay for a highly metabolized cyclic ureidobenzenesulfonamide: issues concerning assay specificity and quality control preparation

An LC–MS–MS method was validated for the quantitation of a β₃ agonist (A) in human urine to support Phase I studies. A was designed to accelerate metabolism for weight reduction. During assay development a significant loss of A was apparent from frozen urine quality control samples. The addition of 0.75% bovine serum albumin (BSA) in urine (v/v) was required to maximize the recovery of A from urine. Urine samples were basified and extracted into methyl t-butyl ether–isopropyl alcohol (90:10, v/v). The organic layer was washed, evaporated, reconstituted, and injected onto a 5 cm, C8 HPLC column prior to MS–MS analysis. The standard curve was linear from 5 to 500 ng/ml. Intraday precision for peak area ratios from BSA urine samples at seven separate concentrations over a range of 5–500 ng/ml (n=5) was <4.0% and calculated concentrations were within 91–115% of nominal concentrations. Interday precision for BSA urine quality control (QC) samples at four separate concentrations (n=10 of each) was <5.0% and individual calculated concentrations were within 90–111% of nominal concentrations. This work emphasizes that potential metabolites and quality control standards should be prepared and assayed as early as possible in method development, especially before the sample collection section of the clinical protocol is prepared. The methods described here have wide utility to other compounds containing basic benzene sulfonamides and to β₃ agonist candidates.     

Specific and sensitive analysis of nefopam and its main metabolite desmethyl-nefopam in human plasma by liquid chromatography-ion trap tandem mass spectrometry

A specific and sensitive liquid chromatography–tandem mass spectrometric (LC–MS–MS) method using an ion trap spectrometer was developed for quantitation of nefopam and desmethyl-nefopam in human plasma. Nefopam, desmethyl-nefopam and the internal standard (ethyl loflazepate) were extracted in a single step with diethyl ether from 1 mL of alkalinized plasma. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v:v). It was delivered at a flow-rate of 0.3 mL/min. The effluent was monitored by MS–MS in positive-ion mode. Ionisation was performed using an electrospray ion source operating at 200 °C. Nefopam and desmethyl-nefopam were identified and quantified in full scan MS–MS mode using a homemade MS–MS library. Calibration curves were linear over the concentration range of 0.78–100 ng/mL with determination coefficients >0.996. This method was fast (total run time < 6 min), accurate (bias < 12.5%), and reproducible (intra- and inter-assay precision < 17.5%) with a quantitation limit of 0.78 ng/mL. The high specificity and sensitivity achieved by this method allowed the determination of nefopam and desmethyl-nefopam plasma levels in patients following either intermittent or continuous intravenous administration of nefopam.     

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug-protein binding in human plasma

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid–liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent (S)-(−)--(1-naphthyl)ethylamine (S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C₁₈ (4.6 mm × 250 mm, 5 μm) column with a mixture of acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal–0.01 mol·L⁻¹ potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL·min⁻¹ and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 μg·mL⁻¹ for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 μg·mL⁻¹ (R.S.D. < 10%, n = 5). The method developed was used to study the drug–protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.     

Simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation by liquid chromatography

A rapid and accurate liquid chromatographic method is described for the simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation. Chromatographic separation of the two drugs was achieved on a Diamonsil™ C₁₈ column (200 mm×4.6 mm, 5 μm). The mobile phase consisting of a mixture of acetonitrile, methanol and 0.5% ammonium acetate (39:11:50 (v/v), pH 5.5) was delivered at a flow rate of 1.0 ml/min. Detection was performed at 220 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 201.2–2012.0 μg/ml for roxithromycin and 42.7–427.0 μg/ml for ambroxol hydrochloride, respectively. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

Development and validation of a bioanalytical method using automated solid-phase extraction and LC-UV for the simultaneous determination of lumefantrine and its desbutyl metabolite in plasma

A bioanalytical method for the determination of lumefantrine (LF) and its metabolite desbutyl-lumefantrine (DLF) in plasma by solid-phase extraction (SPE) and liquid chromatography has been developed. Plasma proteins were precipitated with acetonitrile:acetic acid (99:1, v/v) containing a DLF analogue internal standard before being loaded onto a octylsilica (3 M Empore) SPE column. Two different DLF analogues were evaluated as internal standards. The compounds were analysed by liquid chromatography UV detection on a SB-CN (250 mm × 4.6 mm) column with a mobile phase containing acetonitrile–sodium phosphate buffer pH (2.0; 0.1 M) (55:45, v/v) and sodium perchlorate 0.05 M. Different SPE columns were evaluated during method development to optimise reproducibility and recovery for LF, DLF and the two different DLF analogues. The within-day precisions for LF were 6.6 and 2.1% at 0.042 and 8.02 μg/mL, respectively, and for DLF 4.5 and 1.5% at 0.039 and 0.777 μg/mL, respectively. The between-day precisions for LF were 12.0 and 2.9% at 0.042 and 8.02 μg/mL, respectively, while for DLF 0.7 and 1.2% at 0.039 and 0.777 μg/mL, respectively. The limit of quantification was 0.024 and 0.021 μg/mL for LF and DLF, respectively. Different amounts of lipids in plasma did not affect the absolute recovery of LF or DLF.     

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC–MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020–10.0 and 0.100–50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102–153 ng/ml for norfentanyl in urine.

Liquid–liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d₅ and norfentanyl-d₅ from the matrixes was applied and separation was performed on a reversed phase YMC Pro C₁₈-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine.

The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied.

The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 μg fentanyl.     

A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine

The purpose of this research project was to investigate potential matrix effects of anticoagulant and lipemia on the response of olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈ in an LC/MS/MS assay. Blank human serum and sodium heparin, sodium citrate, and K₃EDTA plasma with various degrees of lipemia were fortified with olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈. Six replicates of each sample were extracted using Waters Oasis^® MCX cartridges and analyzed using electrospray LC/MS/MS. The analytes were separated on a Phenomenex LUNA phenyl hexyl, 2 mm×50 mm, 5 μm, analytical column and a gradient rising from 2 to 85% mobile phase B. Mobile phase A consisted of acetonitrile–ammonium acetate (20 mM) (52:48 v/v) and mobile phase B was formic acid–acetonitrile (0.1:100 v/v). Ion suppression was investigated through post column infusion experiments. The degree of lipemia of each sample, indicated by turbidity, was ranked into categories from least to greatest and used for statistical analyses. The results from analysis of variance testing indicated that lipemia, anticoagulant and their interaction significantly influenced mass spectral matrix effects and extraction matrix effects. Differential behavior between the analytes and labeled internal standards contributed to variability. The most significant source of variability however, was ion suppression due to co-eluting matrix components.     

Simultaneous determination of cinnarizine and domepiridone maleate from tablet dosage form by reverse phase ion pair high performance liquid chromatography

A new simple, precise, rapid and selective reverse phase ion pair high performance liquid chromatography (HPLC-RP) method has been developed for the simultaneous determination of cinnarizine (CINN) and domepiridone maleate (DOME) from tablets using acetonitrile–methanol–water–0.1 N sulfuric acid (37:10:48:5 v/v/v/v) containing sodium lauryl sulfate (0.01 M), as a mobile phase and a Machery Nagel nitrile column (10 μ, 25 cm×4.0 mm i.d.) as the stationary phase. The flow of mobile phase through the column was kept at 1.0 ml min⁻¹ through out the analysis. Detection was carried out using a UV detector at 225 nm. The retention times for CINN and DOME were 4.73 and 9.41 min, respectively. The linearity range and percentage recoveries for CINN and DOME were 4–1000 and 60–750 μg ml⁻¹ and 99.90 and 99.60%, respectively.     

Determination of caderofloxacin lactate in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) method for the quantification of a newly active quinolone carboxylic acid caderofloxacin lactate in rat plasma was developed and validated after precipitation method with methanol. Chromatographic separation was achieved on a reversed-phase Shimadzu 2.0 μm C18 column (150 mm × 2.00 mm) with the mobile phase of methanol–0.02% formic acid and step gradient elution resulted in a total run time of about 10.0 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (5–2000 ng/mL) (r = 0.9998). The lowest limit of quantification (LLOQ) was 5 ng/mL and the lowest limit of detection (LLOD) was 2 ng/mL. Average recoveries ranged from 88.80 to 93.05% in plasma at the concentrations of 10, 100 and 1000 ng/mL. Intra- and inter-day relative standard deviations were 4.01–7.30 and 4.15–7.51%, respectively. This method was successfully applied in the pharmacokinetic studies in rats.     

Evaluation of impurities level of perindopril tert-butylamine in tablets

Perindopril tert-butylamine is a new member of angiotensin-converting enzyme inhibitors group used in the treatment of hypertension and heart failure. In this paper, the evaluation of reversed-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of impurities level of perindopril tert-butylamine in tablets was done. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system with DAD detector. Separations were performed on a YMC-Pack C8 column (250 mm × 4.6 mm; 5 μm particle size) at 50 °C column temperature. Mobile phase was a mixture of acetonitrile–potassium phosphate buffer (0.05 M) (37:63, v/v) (pH 2.5). pH of the mobile phase was adjusted with ortophosphoric acid. Mixture of acetonitrile–water (40:60, v/v) was used as a solvent. Injection volume was 50 μl, flow rate 1.7 ml min⁻¹ and UV-detection was performed at 215 nm. The developed method subjected to method validation and parameters in terms of selectivity, linearity, precision, accuracy, limit of detection, limit of quantitation and robustness were defined. The validated method is suitable for the simultaneous determination of perindopril tert-butylamine as well as its impurities in pharmaceuticals.     

Method development and validation of repaglinide in human plasma by HPLC and its application in pharmacokinetic studies

In this study, the development and validation of a high-performance liquid chromatography (HPLC) assay for determination of repaglinide concentration in human plasma for pharmacokinetic studies is described. Plasma samples containing repaglinide and an internal standard, indomethacin were extracted with ethylacetate at pH 7.4. The recovery of repaglinide was 92% ± 55.31. Chromatographic separations were performed on Purospher^® STAR C-18 analytical column (4.8 mm × 150 mm; 5 μm particle size). The mobile phase composed of acetonitrile–ammonium formate (pH 2.7; 0.01 M) (60:40, v/v). The flow rate was 1 ml/min. The retention time for repaglinide and indomethacin were approximately 6.2 and 5.3 min, respectively. Calibration curves of repaglinide were linear in the concentration range of 20–200 ng/ml in plasma. The limits of detection and quantification were 10 ng/ml and 20 ng/ml, respectively. The inter-day precision was from 5.21 to 11.84% and the intra-day precision ranged from 3.90 to 6.67%. The inter-day accuracy ranged 89.95 to 105.75% and intra-day accuracy ranged from 92.37 to 104.66%. This method was applied to determine repaglinide concentration in human plasma samples for a pharmacokinetic study.     

Pharmacokinetics and tissue distribution study of orientin in rat by liquid chromatography

A simple HPLC–UV method was established for the determination of orientin in plasma and different tissues of rat (heart, liver, spleen, lung, kidney, brain, stomach and small intestine). The separation was achieved by HPLC on a C₁₈ column with a mobile phase composed of acetonitrile–0.1% acetic acid (20:80, v/v), UV detection was used at 348 nm. Good linearity was found between 0.250–50.0 μg/ml (r² = 0.9966) for plasma samples and 0.050–50.0 μg/ml (r² ≥ 0.9937) for the tissue samples, respectively. Within- and between-day precisions expressed as the relative standard deviation (R.S.D.) for the method were 2.3–9.6% and 3.0–7.4%, respectively. The relative recoveries of orientin ranged from 95.4 to 100.6% for plasma and 93.1 to 107.9% for tissue homogenates. The developed method was successfully applied to the pharmacokinetics and tissue distribution research after intravenous administration of a 20 mg/kg dose of orientin to healthy Sprague–Dawley rats. The main pharmacokinetics parameters obtained presented that orientin was quickly distributed and eliminated within 90 min after intravenous administration. The tissue distribution results showed that liver, lung and kidney were the major distribution tissues of orientin in rats, and that orientin had difficulty in crossing the blood–brain barrier. It was also found that there was no long-term accumulation of orientin in rat tissues.     

Development of a HPLC method for the determination of cyclosporin-A in rat blood and plasma using naproxen as an internal standard

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 205 nm has been developed for the determination of cyclosporin-A (CyA) in rat blood and plasma. Naproxen was successfully used as an internal standard. Blood or plasma samples were pretreated by liquid–liquid extraction with diethyl ether. The ether extract was evaporated and the residue was reconstituted in acetonitrile–0.04 M monobasic potassium phosphate buffer (pH 2.5) solvent mixture. After washing with n-hexane, 30 μl of the reconstituted solution was injected into HPLC system. Good chromatographic separation between CyA and internal standard peaks was achieved by using a stainless steel analytical column packed with 4 μm Nova-Pak Phenyl material. The system was operated at 75 °C using a mobile phase consisting of acetonitrile–0.04 M monobasic potassium phosphate (pH 2.5) (65:35 v/v) at a flow rate of 1 ml/min. The calibration curve for CyA in rat blood was linear over the tested concentration range of 0.0033–0.0166 M with a correlation coefficient of 0.989. For rat plasma, the range of the concentrations tested were between 0.002 and 0.0166 M and showed linearity with a correlation coefficient of 0.953. The intra- and inter-run precision and accuracy results were 1.24–21.87 and 3.1–12.23%, respectively. The low volume of blood or plasma needed (200 μl), simplicity of the extraction process, short run time (5 min) and low injection volume (30 μl) make this method suitable for quick and routine analysis.     

Simultaneous determination of 10 active components in traditional Chinese medicine "YIGONG" capsule by RP-HPLC-DAD

A simple, reliable and accurate method for the simultaneous separation and determination of 10 active components (psoralen, isopsoralen, emodin, oleanolic acid, stachydrine hydrochloride, ammonium glycyrrhizinate, glycyrrhizinate, schizandrol, imperatorin and isoimperatorin) in traditional Chinese medicine “YIGONG” capsule was developed using reverse phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection. The chromatographic separation was performed on a Eurospher C₁₈ column (250 mm × 4.6 mm i.d. with 5.0 μm particle size) with a acetonitrile–water gradient containing 0.5% (v/v) aqueous phosphoric acid as mobile phase. Two detection wavelengths (210 and 250 nm) were utilized for the quantitative analysis due to the different UV spectra of these components. Good linear behaviors over the investigated concentration ranges were observed with the values of R² higher than 0.999 for all the analytes. The recoveries, measured at three concentration levels, varied from 95.0 to 105.3%. The validated method was successfully applied to the simultaneously determination of these active components in “YIGONG” capsule from different production batches.     

Liquid chromatographic analysis of physostigmine salicylate and its degradation products

A simple stability-indicating HPLC assay has been developed for physostigmine salicylate, capable of following its degradation. A 250×5 mm i.d. column packed with 10 μm Bondapak C₁₈ was used, with a mobile phase of acetonitrile–ammonium acetate (pH 6.0; 0.1 M) (50:50, v/v) and flow rate 1.2 ml min⁻¹. All peaks are eluted in <10 min and the method has good precision. The optimum wavelength for detection of degradation products is 305 nm. Application of the assay for a commercial preparation of physostigmine salicylate for injection is presented.     

LC determination of glimepiride and its related impurities

Five impurities in glimepiride drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. For the identification and characterization purpose these impurities were isolated from a crude reaction mixture of glimepiride using a normal phase HPLC system. Based on the spectroscopic data like NMR, FTIR, UV and MS these impurities were characterized and used as impurity standards for determining the relative response factor during the validation of the proposed isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Phenomenex Luna C8 (2) 100 Å, 5 μm, 250 mm × 4.6 mm using a mobile phase consisting of phosphate buffer (pH 7.0)–acetonitrile–tetrahydrofuran (73:18:09, v/v/v) with UV detection at 228 nm and a flow rate of 1 ml/min. The column temperature was maintained at 35 °C through out the analysis. The method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of glimepiride as active pharmaceutical ingredient (API).     

HPLC method for enantioselective analysis of cloprostenol

A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile–sodium dihydrogenphosphate (pH 3.0; 20 mM) (33:67, v/v) as the mobile phase. Baseline resolution of (±)-cloprostenol (R = 2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of μmol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.     

A simplified analytical method for a phenotyping cocktail of major CYP450 biotransformation routes

An efficient, fast and reliable analytical method was developed for the simultaneous evaluation of the activities of five major human drug metabolising cytochrome P450 (1A2, 2C9, 2C19, 2D6 and 3A4) with a cocktail approach including five probe substances, namely caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam. All substances were administered simultaneously and a single plasma sample was obtained 2 h after the administration. Plasma samples were handled by liquid-liquid extraction and analysed by gradient high performance liquid chromatography (HPLC) coupled to UV and fluorescence detectors. The chromatographic separation was achieved using a Discovery semi-micro HS C18 HPLC column (5 μm particle size, 150 mm×2.1 mm i.d.) protected by a guard column (5 μm particle size, 20 mm×2.1 mm i.d.) The mobile phase was constituted of a methanol, acetonitrile and 20 mM ammonium acetate (pH 4.5) with 0.1% triethylamine mixture and was delivered at a flow rate of 0.3 mL min⁻¹. All substances were separated simultaneously in a single run lasting less than 22 min. The HPLC method was formally validated and showed good performances in terms of linearity, sensitivity, precision and accuracy. Finally, the method was found suitable for the screening of these compounds in plasma samples.     

Simultaneous determination of berberine and palmatine in rat plasma by HPLC-ESI-MS after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction and the pharmacokinetic application of the method

A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method has been developed and validated for the identification and quantification of berberine and palmatine in rat plasma. After the addition of the internal standard (IS) and alkalization with 0.5 M sodium hydroxide solution, plasma samples were extracted by ethyl ether and separated by HPLC on a Shim-pack ODS (4.6 μm, 150 mm × 2.0 mm i.d.) column using a mobile phase composed of A (0.08% formic acid and 2 mmol/l ammonium acetate) and B (acetonitrile) with linear gradient elution. Analysis was performed on a Shimadzu LC/MS-2010A in selected ion monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. [M]⁺ = 336 for berberine; 352 for palmatine and [M + H]⁺ = 340 for IS were selected as detecting ions, respectively. The method was validated over the concentration range of 0.31–20 ng/ml for berberine and palmatine. Inter- and intra-CV precision (R.S.D.%) were all within 15% and accuracy (%bias) ranged from −5 to 5%. The lower limits of quantification were 0.31 ng/ml for both analytes. The extraction recovery was on average 68.6% for berberine, 64.2% for palmatine. The validated method was used to study the pharmacokinetic profile of berberine and palmatine in rat plasma after oral administration of Huang-Lian-Jie-Du decoction.