Quantitative analysis of ES-285, an investigational marine anticancer drug,in human,mouse, rat,and dog plasma using coupled liquid chromatography and tandem mass spectrometry

A method was developed for the quantitative analysis of the novel anticancer agent ES-285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre-clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d(3)) ES-285 as internal standard. Aliquots of 10 micro l of the supernatant were injected directly on to an Inertsil ODS-3 column (50 x 2.0 mm i.d., 5 micro m). Elution was carried out using methanol-10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow-rate of 0.2 ml min(-1) with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple-quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml(-1). A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES-285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre-clinical trials, and will be implemented in the planned clinical phase I studies.     

Rapid Quantification of Astilbin in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry and Its Application to Pharmacokinetic Study

Astilbin is a potential immunosuppressive agent with minor cytotoxicity. Its oral bioavailability is supposed to be rather low and therefore a sensitive analytical method is required for its pharmacokinetic study after oral administration. A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed and validated for the determination of astilbin in rat plasma. Plasma samples were subjected to liquid-liquid extraction with ethyl acetate and separated by reversed phase high performance liquid chromatography（HPLC） with methanol-0.01%（volume fraction） formic acid（50：50, volume ratio） as mobile phase. Quantitive determination was achieved on negative LC-MS/MS by a multiple reaction moitoring method with transitions m/z 449.1→150.9（quantifier） and m/z 449.1→284.9（qualifier） for astilbin and m/z 128.9→42.0 for internal standard（IS）. A lower limit of quantification（LLOQ） of ng/mL was achieved within a short cycle time of 3.4 min. The method was successfully applied to a pharmacokinetic study involving oral and intravenous administrations of 6 mg/kg astilbin to six rats.     

Determination of the lipophilic antipsychotic drug ziprasidone in rat plasma and brain tissue using liquid chromatography-tandem mass spectrometry

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LCESI-MS/MS) method with low matrix effects was developed and validated for the quantification of the lipophilic antipsychotic ziprasidone from rat plasma and brain tissue. Ziprasidone was extracted from rat plasma and brain homogenate using a single-step liquid-liquid extraction. Ziprasidone was separated on an Agilent Eclipse XDB C8 column (150 x 2.1 mm i.d., 5 microm) column using a mobile phase of acetonitrile-0.02% ammonia in water (pH 7.20 adjusted with formic acid) using gradient elution. Ziprasidone was detected in the positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recovery, matrix effects and stability were determined. The LLOQ was 0.2 ng/mL for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/mL for plasma and 0.833-833.3 ng/g for brain tissue. The correlation coefficient (R2) values were more than 0.996 for both plasma and brain homogenate. The precision and accuracy intra-day and inter-day were better than 8.13%. The relative and absolute recovery was above 81.0% and matrix effects were lower than 5.2%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of ziprasidone after chronic treatment.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for simultaneous quantification of olanzapine, clozapine, ziprasidone, haloperidol, risperidone, and its active metabolite 9-hydroxyrisperidone, in rat plasma using midazolam as internal standard (IS). The analytes were extracted from rat plasma using a single step liquid-liquid extraction technique. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 0.1 ng/mL and correlation coefficient (R(2)) values for the linear range of 0.1-100 ng/mL were 0.997 or greater for all the analytes. The intra-day and inter-day precision and accuracy were better than 8.05%. The relative and absolute recovery was above 77% and matrix effects were low for all the analytes except for ziprasidone. This validated method has been successfully used to quantify the plasma concentration of the analytes after chronic treatment with antipsychotic drugs.     

Quantitative determination of ragaglitazar in rat plasma by HPLC: validation and application in pharmacokinetic study

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of ragaglitazar [(-) DRF 2725, NNC 61-0029], a novel anti-diabetic agent, in rat plasma. The assay procedure involved simple liquid/liquid extraction of ragaglitazar and internal standard (IS, troglitazone) from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100 - 5C(18) column (4.6 x 250 mm, 5 micro m). Mobile phase consisting of 0.01 M potassium dihydorgen ortho phosphate (pH 3.2) and acetonitrile (30:70, v/v) was used at a flow rate of 1.0 mL/min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of IS and ragaglitazar were 6.9 and 12.2 min, respectively. The standard curve for ragaglitazar was linear (r(2) > 0.999) in the concentration range 0.2-100 micro g/mL. Absolute recovery was >87% from rat plasma for both analyte and IS. The lower limit of quantification (LLOQ) of ragaglitazar was 0.2 micro g/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples, 0.2, 1.0, 5.0 and 50 micro g/mL, were in the range 1.32-3.70% relative standard deviation (RSD) and 1.19-9.39% RSD, respectively. Accuracy in the measurement of QC samples was in the range 94.28-107.45%. Analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze/thaw cycles. Stability of ragaglitazar was established for 1 month at -20 degrees C. The application of the assay to a pharmacokinetic study in rats is described.     

A simple RP-HPLC method for the simultaneous determination of curcumin and its prodrug, curcumin didecanoate, in rat plasma and the application to pharmacokinetic study

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of curcumin and its prodrug, curcumin didecanoate (CurDD), in rat plasma. The analytes were extracted by ethyl acetate following the addition of sodium dodecyl sulfate, and separated on a reverse-phase C(18) column using a gradient mobile phase system of acetonitrile-tetrahydrofuran-water containing 0.1% formic acid. Detection by UV absorption at 425 nm gave a lower limit of quantitation (LLOQ) of 5 and 10 ng/mL for curcumin and CurDD in 50 μL of plasma, respectively. Intra- and inter-day precisions of quality control samples except those at LLOQ were within 15% for curcumin and CurDD, respectively, and the accuracies for both compounds were between 93.9 and 108%. The method was successfully applied to determine plasma concentration-time curves of curcumin and CurDD in rats following intravenous (i.v.) administration of curcumin or CurDD at doses of 1 mg/kg (calculated as curcumin). The results suggested that i.v. dosed CurDD provided sustained plasma levels of curcumin.     

Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of benzyl butyl phthalate and its metabolites,monobenzyl phthalate and monobutyl phthalate,in rat plasma,urine, and various tissues collected from a toxicokinetic study

This study describes the development of a sensitive liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of benzyl butyl phthalate (BBP) and its major metabolites, monobenzyl phthalate (MBzP) and monobutyl phthalate (MBuP), in rat plasma, urine, and 10 different tissues. The method was validated with regard to the specificity, linearity, precision, accuracy, lower limit of quantification (LLOQ), recovery, and stability by using the matrix-matched quality control samples. The assay achieved LLOQ of 1 ng/ml of BBP for plasma and urine, 4 ng/g for kidney and liver, 10 ng/g for fat, and 20 ng/g for all other tissues. For MBzP and MBuP, the assay achieved LLOQ of 5 ng/ml for plasma and urine, 10 ng/g for fat, and 20 ng/g for all other tissues. The disposition of BBP was characterized by a large volume of distribution (71.1–82.9 l/kg) and a high clearance (838.7–871.0 ml/min/kg). It was extensively metabolized to MBzP and MBuP, with their levels consistently exceeding the BBP levels. The distribution of BBP, MBzP, and MBuP to tissues of kidney, liver, stomach, small intestine, large intestine, spleen, brain, testis, thyroid, and fat was determined under steady-state conditions. For BBP, the steady-state tissue-to-plasma partition coefficient (K _p) was the highest for fat (25.0) followed by small intestine (2.6), thyroid (2.0), and stomach (1.1). In contrast, for MBzP and MBuP, it was the highest for kidney (2.0 and 4.3, respectively) and liver (4.3 and 2.1, respectively) but was less than unity for all other tissues. The developed assay method and findings of this study may be useful to evaluate the exposure and toxic potential of BBP and its metabolites in risk assessment.     

Quantitative determination of the polypeptide motilin in rat plasma by externally calibrated liquid chromatography/electrospray ionization mass spectrometry

We present a method for the quantitation of motilin from rat plasma by protein precipitation and liquid chromatography/mass spectrometry (LC/MS). Using external calibration, the method was linear over the concentration range 10-1000 ng/mL with an initial sample volume of 150 microL. The LC system included a C(18) column with a 300 A pore size. A linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Motilin was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 675.5. The approximated limit of detection was less than 1 ng/mL and the lower limit of quantitation (LLOQ) was 10 ng/mL. The method showed a high degree of precision and accuracy both within and between runs at five validation points, including the LLOQ.     

Development and validation of a sensitive ELISA for quantitation of Grastim (rhG-CSF) in rat plasma: Application to a pharmacokinetic study

Grastim is bacterially produced recombinant counterpart of human granulocyte colony stimulating factor (G-CSF). It has biological activity similar to that of endogenous G-CSF. In the present work a sensitive, accurate, precise and enzyme-linked immunosorbent assay (ELISA) for the quantitation of G-CSF in rat plasma was developed and validated. The ELISA method employed a technique in which anti-human-G-CSF was adsorbed onto 96-well maxisorp plates and used to capture the G-CSF in rat plasma samples. The captured G-CSF was then detected using streptavidin-HRP amplification system. Absolute recovery was >90% from rat plasma. The validation includes assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLOQ), storage stability (30 days) in rat plasma and assay specificity. The standard curve for G-CSF was linear (R2 > 0.996) in the concentration range 4.88-625 pg/mL. The LLOQ was established at 4.88 pg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 15, 250 and 500 pg/mL, were in the range 3.00-8.66% relative standard deviation (RSD) and 1.03-4.69% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.28-110.79% of the nominal values. The assay shows dilutional linearity and specificity. Stability of G-CSF was established for 30 days at -80 degrees C and through three freeze-thaw cycles. The validated assay was successfully employed for the assessment of pharmacokinetic disposition of G-CSF in rats.     

Development and validation of a high‐performance liquid chromatography–tandem mass spectrometry method for the determination of the novel proteasome inhibitor CEP‐18770 in human plasma and its application in a clinical pharmacokinetic study

CEP‐18770, [(1R)‐1‐{[(2S,3R)‐3‐hydroxy‐2‐{[(6‐phenyl‐2‐pyridinyl)carbonyl]amino}butanoyl]amino}‐3‐methylbutyl]boronic acid, is a novel proteasome inhibitor, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to measure the drug in human plasma, based on simple protein precipitation with acetonitrile after the addition of irbesartan as internal standard. The method requires a small volume of sample (100 µl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise and accurate, with overall precision, expressed as coefficient of variation (CV%), always < 10.0%, accuracy in the range 93.8–107.7% and high recovery, close to 100%. The limit of detection is 0.01 ng/ml and the lower limit of quantitation (LLOQ) is 0.20 ng/ml. The assay was validated in the range from the LLOQ up to 50.00 ng/ml. This is the first method developed and validated for analyzing a proteasome inhibitor with a boronic‐acid‐based structure in human plasma. The method was successfully applied to study the pharmacokinetics of CEP‐18770 in cancer patients with solid tumors or multiple myeloma who had received the drug as a short intravenous bolus during the initial Phase I trial. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry pharmacokinetic study of DL-praeruptorin A in rat plasma

dl ‐Praeruptorin A is a novel drug with valuable apoptosis and inflammation inhibitory effects in cardiac muscle. Previous pharmacokinetic studies of dl ‐praeruptorin A have had limited success due to its very low plasma concentrations. In this study, we developed and validated a new rapid, sensitive and specific high‐performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS) method for quantitative analysis of dl ‐praeruptorin A in rat plasma. dl ‐Praeruptorin A and diazepam (internal standard) extracted from rat plasma samples with chloroform and analyzed on an XTerra? RP₁₈ column (150 mm × 4.6 mm i.d., 5 µm) were chromatographically separated within 5.5 min using methanol–water (75:25, v/v; flow rate 1 mL/min) as the mobile phase. dl ‐Praeruptorin A was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ, 2.5 ng/mL), precision (intra‐ and inter‐day <11.0%), accuracy (90.2–96.3%), recovery (>79.2%) and stability were determined. The correlation coefficient (r²) for the linear range of 2.5–2500.0 ng/mL was >0.999. No matrix effects were observed. The validated method was successfully applied to pharmacokinetic studies of dl ‐praeruptorin A after intravenous administration to rats. The LLOQ obtained with this method was lower than in previous studies and could be valuable for determination of dl ‐praeruptorin A in therapeutic drug monitoring and preclinical studies to establish appropriate dose and frequency. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and Validation of an Improved LC Method for the Simultaneous Determination of Pirfenidone and Its Carboxylic Acid Metabolite in Human Plasma

A sensitive and specific assay based on liquid chromatography with ultraviolet detection was developed for the simultaneous determination of pirfenidone (PFD), a novel antifibrotic agent, and its carboxylic acid metabolite in human plasma. The carboxylic acid metabolite was further identified by mass spectrometric analysis. PFD, its carboxylic acid metabolite and the internal standard methyl-p-aminobenzoate were extracted from plasma by a simple one-step liquid-liquid extraction with ethyl acetate and subsequently separated on a Zorbax SB-C18 column with a mobile phase of trifluoroacetic acid–triethylamine–acetonitrile–water (0.1:0.15:28:71.75, v/v/v/v) and monitored at 314 nm. Extraction recovery was over 70% in plasma. The calibration curves were linear over the concentration range of 0.05–25 μg mL^?1. The limit of detection (LOD) and lower limit of quantitation (LLOQ) in human plasma were 10 and 50 ng mL^?1, respectively. Intra- and inter-assay precision of the method were within 8.6%. The accuracy as expressed by the bias ranged between ?4.5 and 4.0%. The method was successfully applied to determine pharmacokinetic parameters of PFD and its carboxylic acid metabolite after a single oral dose of 200 mg of PFD in healthy volunteers.     

Determination of levetiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to bioequivalence studies

The first liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of levetiracetam, an antiepileptic drug, in human plasma is described. The plasma filtrate obtained after solid-phase extraction (SPE), using a polymer-based, hydrophilic-lipophilic balanced (HLB) cartridge, was submitted directly to a short column LC/MS/MS assay. There was no significant matrix effect on the analysis. For validation of the method, the recovery of the free analytes was compared to that from an optimized extraction method, and the analyte stability was examined under conditions mimicking sample storage, handling, and analytical procedures. The extraction procedure yielded extremely clean extracts with a recovery of 79.95% and 89.02% for levetiracetam and the internal standard (IS), respectively. The intra-assay and inter-assay precision for the samples at the lower limit of quantitation (LLOQ) were 6.33 and 6.82%, respectively. The calibration curves were linear for the dynamic range of 0.5 to 50 microg/mL with a correlation coefficient r >/= 0.9971. The intra-assay accuracy at LLOQ, LQC, MQC, and HQC levels ranged from 81.60 to 95.40, 93.00 to 103.47, 95.97 to 104.09, and 91.15 to 95.18%, respectively, while the inter-assay accuracy at LLOQ, LQC, MQC and HQC levels varied from 80.20 to 95.40, 88.53 to 107.53, 95.97 to 108.45, and 91.15 to 112.70%, respectively. The method is rugged and fast with a total instrumental run time of 2 min. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 1000 mg immediate release (IR) formulations.     

Determination of rosuvastatin in rat plasma by HPLC: Validation and its application to pharmacokinetic studies

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of rosuvastatin (RST), a novel, synthetic and potent HMG-CoA inhibitor in rat plasma. The assay procedure involved simple liquid-liquid extraction of RST and internal standard (IS, ketoprofen) from a small plasma volume directly into acetonitrile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). Mobile phase consisting of 0.05 m formic acid and acetonitrile (55:45, v/v) was used at a flow rate of 1.0 mL/min for the effective separation of RST and IS. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 240 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of RST and IS were 8.6 and 12.5 min, respectively. The standard curve for RST was linear (r2 > 0.999) in the concentration range 0.02-10 microg/mL. Absolute recoveries of RST and IS were 85-110 and >100%, respectively, from rat plasma. The lower limit of quantification (LLOQ) of RST was 0.02 microg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.02, 0.06, 1.6 and 8.0 microg/mL, were in the range 7.24-12.43% relative standard deviation (RSD) and 2.28-10.23% RSD, respectively. Accuracy in the measurement of QC samples was in the range 93.05-112.17% of the spiked nominal values. Both analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. RST was found to be stable for a period of 30 days on storage at -80 degrees C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.     

Validation of a simple HPLC method for DRF-4848, a novel COX-2 inhibitor suitable for pharmacokinetic application in rats

For pharmacokinetic and toxicokinetic purpose a simple HPLC-UV method has been developed and validated for the estimation of DRF-4848, a novel COX-2 inhibitor in rat plasma. A liquid-liquid extraction was used to extract DRF-4848 and internal standard (IS, DRF-4367) from rat plasma. The analysis was performed on a C(18) column with UV detection at 285 nm. The isocratic mobile phase, 0.01 M potassium dihydrogen ortho phosphate (pH 3.2) and acetonitrile (50:50, v/v) was run at a flow rate of 1 mL/min. The retention times of DRF-4848 and IS were 6.8 and 11.2 min, respectively. Absolute recovery for analyte and IS was >80% from rat plasma. A linear response was observed over a concentration range 0.1-20 microg/mL. The lower limit of quantification (LLOQ) of DRF-4848 was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.1, 0.3, 8.0 and 15.0 microg/mL, were in the range 1.74-8.70% relative standard deviation (RSD) and 0.75-8.43% RSD, respectively. Accuracy in the measurement of QC samples was in the range 93.29-116.51% of the nominal values. Analyte and IS were stable in the battery of stability studies viz., benchtop, autosampler, long-term and freeze/thaw cycles.     

Development and validation of a generic liquid chromatographic method for the simultaneous determination of five commonly used antimalarial drugs: Application to pharmaceutical formulations and human plasma

A simple, sensitive, and rapid liquid chromatographic method was developed and validated using diode array detection for the determination of five commonly used antimalarial drugs in pharmaceutical formulations and in human plasma. Chromatographic separation of antimalarial drugs and internal standard (ibuprofen) was achieved on a C₁₈ column with a mobile phase composed of 10 mM dipotassium orthophosphate at pH 3.0, methanol, and acetonitrile in a ratio of 20:38:42 v/v, at a flow rate of 1 mL/min. The analytes were monitored at 220 nm and separated in ?10 min. The method was validated for linearity, accuracy, precision, limit of quantification, and robustness. Both intra‐ and interday precisions (in terms of %RSD) were lower than 3% and accuracy ranged from 98.1 to 104.5%. Extraction recoveries were ≥96% in plasma. The limits of quantitation for artemether, lumefantrine, pyrimethamine, sulfadoxine, and mefloquine were 0.3, 0.03, 0.06, 0.15, and 0.15 μg/mL in human plasma. Stability under various conditions was also investigated. The method was successfully applied for quantification of antimalarial drugs in marketed formulations and in spiked human plasma. The method can be employed for routine QC purposes and in pharmacokinetic investigations.     

Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantification of three iridoids in rat plasma after oral administration of Zhi-zi-chi decoction using LC-MS

A sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rat plasma. After the addition of internal standard (I.S.) salidroside and acidification (formic acid, 0.1%), plasma samples were carried out by protein precipitation with acetonitrile and separated on a Kromasil C(18) column (200 mm × 4.6 mm, 5 μm) within a runtime of 15.0 min. The linear ranges were 2-250 ng/mL for both 6α-hydroxygeniposide and genipin gentiobioside and 2-2000 ng/mL for geniposide, respectively. The lower limit of quantification (LLOQ) was 2 ng/mL for all the analytes. The validated method was successfully applied to the pharmacokinetics study of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rats after oral administration of Zhi-zi-chi decoction.     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Quantification of leonurine, a novel potential cardiovascular agent, in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in rats

Leonurine (SCM‐198), an alkaloid from Herba Leonuri, has been suggested as a novel cardiovascular agent by pharmacology studies in preclinical stage. In present study, we report a simple, rapid and sensitive high‐performance liquid chromatography–tandem mass spectrometry method (HPLC‐MS/MS) for determination of leonurine in rat plasma. Leonurine and its internal standard (IS) n‐benzoyl‐l ‐arginine ethyl ester (BAEE) were extracted from plasma samples by one‐step protein precipitation with perchloric acid. Chromatographic separation was performed on an Agilent Zorbax SB‐C₁₈ column (150 × 2.1 mm, 5 µm) using an isocratic elution with acetonitrile–ammonium acetate buffer (10 mm , pH 4.0; 25:75, v/v) as mobile phase at a flow rate of 0.2 mL/min. Analytes were detected by tandem mass spectrometry in positive electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) with the transitions of m/z 312.3 → 181.1 for leonurine and m/z 307.2 → 104.6 for IS. The calibration curves were linear over the range of 4–256 ng/mL with a lower limit of quantitation (LLOQ) of 4 ng/mL. The intra‐ and inter‐day assay precision (as relative standard deviation) were <15%, except which at LLOQ were <20%, with accuracy in the range 98.73‐105.42%. The validated HPLC‐MS/MS method was successfully applied to the pharmacokinetic study in rats following oral administration of leonurine. Copyright © 2011 John Wiley & Sons, Ltd.