Simultaneous determination and method validation of clethodim and its metabolites clethodim sulfoxide and clethodim sulfone in tobacco by LC‐MS/MS

A simple method was developed and validated for the simultaneous determination of clethodim, clethodim sulfoxide, and clethodim sulfone in soil and tobacco by liquid chromatography with tandem mass spectrometry. The three target compounds were extracted from tobacco and soil with acetonitrile, and the extracts were purified using octadecyl silane. The proposed method showed satisfactory linearity (R² ≥ 0.9973) for the target compounds. The limits of detection and quantitation of the three analytes in all matrices were 0.024−0.06 and 0.08−0.2 mg/kg, respectively. The recovery was tested in blank soil and tobacco leaf samples and calculated to be 74.8–104.4% with relative standard deviations of 1.9–12.1%. The developed method was successfully applied to the analysis of residues of clethodim, clethodim sulfoxide and clethodim sulfone in real soil and tobacco samples. The results indicated that the developed method can meet the requirements for the analysis of trace amounts of all three analytes in soil and tobacco.     

Measurement of quetiapine and four quetiapine metabolites in human plasma by LC-MS/MS

There is interest in monitoring plasma concentrations of N‐desalkylquetiapine in relation to antidepressant effect. A simple LC‐MS/MS method for quetiapine and four metabolites in human plasma (50 μL) has been developed to measure concentrations of these compounds attained during therapy. Analytes and internal standard (quetiapine‐d8) were extracted into butyl acetate–butanol (10:1, v/v) and a portion of the extract analysed by LC‐MS/MS (100 × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow‐rate 0.5 mL/min; positive ion APCI‐SRM, two transitions per analyte). Assay calibration (human plasma calibrators) was linear across the ranges studied (quetiapine and N‐desalkylquetiapine 5–800, quetiapine sulfoxide 100–15,000, others 2–100 µg/L). Assay validation was as per FDA guidelines. Quetiapine sulfone was found to be unstable and to degrade to quetiapine sulfoxide. In 47 plasma samples from patients prescribed quetiapine (prescribed dose 200–950 mg/day), the (median, range) concentrations found (µg/L) were: quetiapine 83 (7–748), N‐desalkylquetiapine, 127 (7–329), O‐desalkylquetiapine 12 (2–37), 7‐hydroxyquetiapine 3 (<1–48), and quetiapine sulfoxide 3,379 (343–21,704). The analyte concentrations found were comparable to those reported by others except that the concentrations of the sulfoxide were markedly higher. The reason for this discrepancy in unclear. Copyright © 2012 John Wiley & Sons, Ltd.     

Highly sensitive LC-MS/MS-ESI method for simultaneous quantitation of albendazole and ricobendazole in rat plasma and its application to a rat pharmacokinetic study

A highly sensitive and specific LC-MS/MS-ESI method was developed for simultaneous quantification of albenadazole (ABZ) and ricobendazole (RBZ) in rat plasma (50 μL) using phenacetin as an internal standard (IS). Simple protein precipitation was used to extract ABZ and RBZ from rat plasma. The chromatographic resolution of ABZ, RBZ and IS was achieved with a mobile phase consisting of 5 m m ammonium acetate (pH 6) and acetonitrile (20:80, v/v) at a flow rate of 1 mL/min on a Chromolith RP-18e column. The total chromatographic run time was 3.5 min and the elution of ABZ, RBZ and IS occurred at 1.66, 1.50 and 1.59 min, respectively. A linear response function was established for the ranges of concentrations 2.01-2007 and 6.02-6020 ng/mL for ABZ and RBZ, respectively. The intra- and inter-day precision values for ABZ and RBZ met the acceptance as per FDA guidelines. ABZ and RBZ were stable in battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in rats.     

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of ethionamide and ethionamide sulfoxide in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous quantification of ethionamide and ethionamide sulfoxide in human plasma (300 µL) using prothionamide as an internal standard (IS). Solid‐phase extraction was used to extract ethionamide, ethionamide sulfoxide and IS from human plasma. The chromatographic separation of ethionamide, ethionamide sulfoxide and IS was achieved with a mobile phase consisting of 0.1% acetic acid : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Peerless Basic C₁₈ column. The total run time was 3.5 min and the elution of ethionamide, ethionamide sulfoxide and IS occurred at 2.50, 2.18 and 2.68 min, respectively. A linear response function was established for the range of concentrations 25.7–6120 ng/mL (r > 0.998) for ethionamide and 50.5–3030 ng/mL (r > 0.998) for ethionamide sulfoxide. The intra‐ and inter‐day precision values for ethionamide and ethionamide sulfoxide met the acceptance as per FDA guidelines. Ethionamide and ethionamide sulfoxide were stable in battery of stability studies, viz. bench‐top, autosampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of amlodipine in human plasma. Amlodipine was extracted from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.1-10.0 ng/mL with correlation coefficient >or=0.9990. The intrarun and interrun precision and accuracy were within 10.0%. The overall recovery for amlodipine was 63.67%. Total run time was 3.2 min only.     

A validated LC-MS/MS method for the determination of vinflunine in plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive LC‐MS/MS method for the quantification of vinflunine in plasma was developed and validated. The analysis involved a simple liquid–liquid extraction. After making alkaline with NaOH, plasma was extracted with methyl tert‐butyl ether and the organic extract was then evaporated and the residue was reconstituted in mobile phase. The reconstituted solution was injected into an HPLC system and was subjected to reverse‐phase HPLC on a 5 µm ODS‐3 column at a flow‐rate of 0.2 mL/min. The mobile phase consisted of ammonium acetate (0.02 mol/L, pH = 3.0) and acetonitrile (20:80). Vinflunine was detected in the single ion monitoring mode using target ions at m/z 817.4/160.1/142.3 for vinflunine and m/z 447.2/128.3/112.1 for gefitinib (internal standard). Standard curves were linear over the concentration range of 5–1000 ng/mL. The mean predicted concentrations of the quality control samples deviated by less than 2% from the corresponding nominal values; the intra‐assay and inter‐assay precisions of the assay were within 7% relative standard deviation. The extraction recovery of vinflunine was more than 80%. The validated assay was applied to a pharmacokinetic study of vinflunine in plasma following the administration of a single vinflunine injection (2 mg/kg). Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

LC-MS/MS method for the determination of cynandione A in rat plasma and tissues

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the quantitative determination of cynandione A in rat plasma and tissues. The plasma samples were pretreated by liquid-liquid extraction with ethyl acetate after the internal standard (honokiol) had been spiked. The tissue samples were homogenized with physiological saline and treated further like the plasma samples. The separation was performed using a Zorbax SB-C(18) column (3.5 microm, 2.1 x 100 mm) and a C18 guard column (5 microm, 4.0 x 2.0 mm) with an isocratic mobile phase consisting of methanol-0.1% formic acid (78:22, v/v) at a flow rate of 0.2 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode using the electrospray ionization technique in negative mode. The nominal retention times for cynandione A and honokiol were 1.41 and 2.63 min, respectively. The method was validated within the concentration range 0.2-1000 ng/mL in plasma and homogenized tissue for cynandione A, and the calibration curves were linear with correlation coefficients >0.992. The lower limit of quantification of cynandione A was 0.2 ng/mL. The intra-day and inter-day precision and accuracy of the assay in plasma were less than 14.4%, while the intra-day and inter-day precision and accuracy of the assay in tissue homogenate were less than 14.2%. This method proved to be suitable for study of pharmacokinetics and tissue distribution of cynandione A in rat.     

A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.     

Simultaneous densitometric determination of anthelmintic drug albendazole and its metabolite albendazole sulfoxide by HPTLC in human plasma and pharmaceutical formulations

A new, simple, accurate and precise high‐performance thin‐layer chromatographic method has been developed and validated for simultaneous determination of an anthelmintic drug, albendazole, and its active metabolite albendazole, sulfoxide. Planar chromatographic separation was performed on aluminum‐backed layer of silica gel 60G F₂₅₄ using a mixture of toluene–acetonitrile–glacial acetic acid (7.0:2.9:0.1, v /v/v) as the mobile phase. For quantitation, the separated spots were scanned densitometrically at 225 nm. The retention factors (R _f) obtained under the established conditions were 0.76 ± 0.01 and 0.50 ± 0.01 and the regression plots were linear (r ² ≥ 0.9997) in the concentration ranges 50–350 and 100–700 ng/band for albendazole and albendazole sulfoxide, respectively. The method was validated for linearity, specificity, accuracy (recovery) and precision, repeatability, stability and robustness. The limit of detection and limit of quantitation found were 9.84 and 29.81 ng/band for albendazole and 21.60 and 65.45 ng/band for albendazole sulfoxide, respectively. For plasma samples, solid‐phase extraction of analytes yielded mean extraction recoveries of 87.59 and 87.13% for albendazole and albendazole sulfoxide, respectively. The method was successfully applied for the analysis of albendazole in pharmaceutical formulations with accuracy ≥99.32%.     

A rapid LC-MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study

A new, rapid, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous quantification of tenofovir and lamivudine in human plasma using abacavir as an internal standard. An API‐4000 LC‐MS/MS with electrospray ionization was operated in multiple‐reaction monitoring mode for the analysis. The analytes were extracted from plasma by solid‐phase extraction technique using an Oasis HLB cartridge. The reconstituted samples were chromatographed on a Chromolith ROD speed C₁₈ column using a mixture of 0.1% formic acid in water and acetonitrile (90:10 v/v) at a flow‐rate of 1 mL/min. The method was validated as per the FDA guidelines. The calibration curves were found to be linear in the range of 5–600 ng/mL for tenofovir and 25– 4000 ng/mL for lamivudine. The intra‐ and inter‐day precision and accuracy results were well within the acceptable limits. A run time of 2.8 min consumed for each sample made it possible to analyze more samples per day. The proposed assay method was found to be applicable to a pharmacokinetic study in human male volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated LC-MS/MS method for quantification of gabapentin in human plasma: application to pharmacokinetic and bioequivalence studies in Korean volunteers

A sensitive validated liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for gabapentin (GB) in human plasma has been developed and applied to pharmacokinetic (PK) and bioequivalence (BE) studies in human. In a randomized crossover design with a 1-week period, each subject received a 300 mg GB capsule. The procedure involves a simple protein precipitation with acetonitrile and separated by LC with a Gemini C(18) column using acetonitrile-10 mm ammonium acetate (20:80, v/v, pH 3.2) as mobile phase. The GB and internal standard [(S)-(+)-alpha-aminocyclohexanepropionic acid hydrate] were analyzed using an LC-API 2000 MS/MS in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 172.0 --> 154.0 and m/z 172.0 --> 126.0 for GB and IS, respectively. The assay exhibited good linearity over a working range of 20-5000 ng/mL for GB in human plasma with a lower limit of quantitation of 20 ng/mL. No endogenous compounds were found to interfere with the analysis. The accuracy and precision were shown for concentrations over the standard ranges. This method was successfully applied for the PK and BE studies by analysis of blood samples taken up to 36 h after an oral dose of 300 mg of GB in 24 healthy volunteers.     

Development of an LC/MS/MS method in order to determine arctigenin in rat plasma: its application to a pharmacokinetic study

In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2–500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and ?80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half‐life and area under the concentration–time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

An APCI LC‐MS/MS method for routine determination of capecitabine and its metabolites in human plasma

The anticancer drug capecitabine and its metabolites [including the active metabolite 5‐fluorouracil (5‐FU)] display high pharmacokinetic inter‐patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost‐effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid–liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC‐MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry‐over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra‐ and inter‐assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5‐FU was avoided by using a stable isotopic derivative of 5‐FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid and sensitive LC-MS/MS method for quantification of lamotrigine in human plasma: application to a human pharmacokinetic study

A highly sensitive, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lamotrigine (LAM) with 100 μL of human plasma using flucanozole as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using electrospray ionization. A simple liquid–liquid extraction process was used to extract LAM and IS from human plasma. The total run time was 2.0 min and the elution of LAM and IS occurred at 1.25 and 1.45 min; this was achieved with a mobile phase consisting of 0.1% formic acid–methanol (20:40:40, v/v) at a flow rate of 0.50 mL/min on a Discovery CN (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for LAM. A linear response function was established for the range of concentrations 0.1–1500 ng/mL (r > 0.998) for LAM. The intra‐ and inter‐day precision values for LAM met the acceptance as per Food and Drug Administration guidelines. LAM was stable in the set of stability studies, viz. bench‐top, autosampler and freeze–thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of rhein in human plasma: application to a pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of rhein with 100 microL human plasma using celecoxib as an internal standard (IS). The API-4,000 Q-Trap LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of rhein and IS from human plasma with acetonitrile, which yielded consistent recoveries of 36.01 and 65.85% for rhein and IS, respectively. The total chromatographic run time was 5.0 min and the elution of rhein and IS occurred at approximately 1.60 and 3.96 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate (pH 6.0):acetonitrile:methanol (30:58:12, v/v) on an Inertsil ODS-3 column. The method was proved to be accurate and precise at a linearity range of 0.005-5.00 microg/mL with a correlation coefficient (r) of >or=0.995. The lower limit of quantitation was 0.005 microg/mL. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. Rhein was found to be stable in the battery of stability studies. The application of the assay to pre-clinical pharmacokinetic studies confirmed the utility of the assay to derive pharmacokinetic parameters.