Liquid chromatography–tandem mass spectrometric assay for the determination of tetrabenazine and its active metabolites in human plasma: a pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α‐dihydrotetrabenazine and β‐dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via solid‐phase extraction using C₁₈ solid‐phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C₁₈ column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API‐4000 LC‐MS/MS in multiple reaction‐monitoring mode was used for detection. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.01–5.03 ng/mL for tetrabenazine and 0.50–100 ng/mL for α‐dihydrotetrabenazine and β‐dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.     

LC–MS/MS method for simultaneous determination of ramelteon and its metabolite M‐II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M‐II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS‐2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol–0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500–30.0 ng/mL for ramelteon and 1.00–250 ng/mL for M‐II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (C_max), the time to the C_max and the elimination half‐life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M‐II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of terbinafine in human plasma using UPLC–MS/MS: Application to a bioequivalence study in healthy subjects

A high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid–liquid extraction of terbinafine and terbinafine‐d7 (used as internal standard) from 100 μL human plasma with ethyl acetate–n‐hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile–8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine‐d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r² ≥ 0.9984). The intra‐batch and inter‐batch precision (CV) was 1.8–3.2 and 2.1–4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.     

Bioanalysis of tolvaptan,a novel AVP‐V2 receptor antagonist in human plasma by a novel LC‐ESI‐MS/MS method: a pharmacokinetic application in healthy South Indian male subjects

A simple, rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) assay method is proposed for the determination of tolvaptan in human plasma samples using tolvaptan d₇ as internal standard (IS). Analyte and the IS were extracted from 100 μL of human plasma via simple liquid–liquid extraction. The chromatographic separation was achieved on a C₁₈ column using a mixture of methanol and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.05–501 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The intra‐day and inter‐day precision (coefficient of variation) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. A run time of 2.0 min for each sample made it possible to analyze more samples in a short time, thus increasing the productivity. The proposed method was successfully applied to a pharmacokinetic study of 15 mg and 60 mg tolvaptan tablet formulation in healthy South Indian male subjects under fasting condition. Copyright © 2013 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometric assay for aliskiren,a novel renin inhibitor in micro‐volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a C₁₈ column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.10–1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.     

A high‐throughput LC–MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses

A high‐throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid–liquid extraction under acidic conditions was used to extract flunarizine and flunarizine‐d8 from 100 μL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C₁₈ (50 × 2.1 mm, 3 μm) column using methanol–10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API‐5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine‐d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1–103.1%), intra‐batch and inter‐batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters C_max and AUC were dose‐proportional.     

Highly sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of nifedipine in human plasma and its application to a bioequivalence study

An ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of nifedipine in human plasma using nifedipine‐d6 as the internal standard (IS). The plasma samples were prepared by solid‐phase extraction on Phenomenex Strata‐X cartridges employing 200 μL human plasma. Chromatography was carried out on Waters Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 µm particle size) analytical column under isocratic conditions using a mobile phase consisting of 4.0 mm ammonium acetate‐acetonitrile (15:85, v/v). The precursor → product ion transitions for nifedipine (m/z 347.2 → 315.2) and IS (m/z 353.1 → 318.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive‐ion mode. The method was validated over a wide dynamic concentration range of 0.050–150 ng/mL. Matrix effect was assessed by post‐column analyte infusion and the mean extraction recovery was 95.6% across four quality control levels. The method is rugged and rapid with a total run time of 1.2 min and was applied to a bioequivalence study of 20 mg nifedipine tablet formulation in 30 healthy Indian subjects under fasting condition. Assay reproducibility was confirmed by reanalysis of 116 incurred samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.     

A sensitive LC–MS/MS‐based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid–liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ column (100 × 4.6 mm, 1.8 μm) with a mobile phase consisting of methanol–water–formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 μL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6–320 ng/mL for salviaflaside and 0.94–188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r²) >0.99. Intra‐ and inter‐day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between −8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.     

Development and validation of a rapid and sensitive UPLC–MS/MS assay for the quantification of tofacitinib in human plasma

A highly sensitive, selective and rapid ultra‐performance liquid chromatography–tandem mass spectrometry method has been developed for the quantification of a Janus kinase (JAK) inhibitor, tofacitinib (TOF). The assay employed liquid–liquid extraction with methyl‐tert butyl ether to extract tofacitinib and tofacitinib‐13C3 15 N (as internal standard) from human plasma. The samples were analyzed on a UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile and 10.0 mm ammonium acetate, pH 4.5 (75:25, v/v) as the mobile phase within 1.4 min. The precursor/product ion transitions were monitored at m/z 313.3/149.2 and 317.4/149.2 for tofacitinib and tofacitinib‐13C3 15 N, respectively, in the positive electrospray ionization mode. The calibration curves were linear (r² ≥ 0.9978) across the concentration range of 0.05–100 ng/mL. The mean extraction recovery of tofacitinib across quality controls was 98.6%. The intra‐ and inter‐batch precision (CV) and accuracy ranged from 2.1–5.1 and 96.2–103.1%, respectively. All validation results complied well with the current guidelines. The method is amenable to high sample throughput and was applied to determine TOF plasma concentration in a pharmacokinetic study with 12 healthy Indian subjects after oral administration of 5 mg tablets.     

A novel and sensitive UPLC–MS/MS method to determine mequitazine in rat plasma and urine: Validation and its application to pharmacokinetic studies

The aim of this study was to develop an analytical method to determine mequitazine in rat plasma and urine. Mequitazine was separated by UPLC–MS/MS equipped with a Kinetex core–shell C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.3 mL/min. Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring positive ion mode. Mass transitions were m/z 323.3 → 83.1 for mequitazine and 281.3 → 86.3 for imipramine as internal standard. Liquid–liquid extraction with ethyl acetate and protein precipitation with methanol were used for sample extraction. Chromatograms showed that the method had high resolution, sensitivity and selectivity without interference from plasma constituents. Calibration curves for mequitazine in rat plasma and urine were 0.02–200 ng/mL, showing excellent linearity with correlation coefficients (r²) >0.99. Both intra‐ and inter‐day precisions (CV%) were within 4.08% for rat plasma and urine. The accuracies were 99.58–102.03%. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of mequitazine after oral and intravenous administration to rats.     

A validated LC–MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay

An accurate and sensitive LC–MS/MS method for determining thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 μL) by liquid–liquid extraction with ethyl acetate and then separated on a BETASIL C₁₈ column (4.6 × 150 mm, 5 μm) with mobile phase composed of methanol–water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor‐to‐product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5‐hydroxy thalidomide, m/z 273.2 → 146.1 for 5′‐hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0–2000.0 ng/mL for thalidomide, 0.2–50.0 ng/mL for 5‐hydroxy thalidomide and 1.0–200.0 ng/mL for 5′‐hydroxy thalidomide. The method was validated with respect to linear, within‐ and between‐batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction to simultaneously determine trans‐resveratrol and its glucuronide and sulfate conjugates in rat plasma and its application to pharmacokinetic assay

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction (SALLE) was applied to simultaneously investigate the pharmacokinetics of trans‐ resveratrol (Res) and its major glucuronide and sulfate conjugates in rat plasma. Acetonitrile–methanol (80:20, v /v) and ammonium acetate (10 mol L⁻¹) were used as extractant and salting‐out reagent to locate the target analytes in the supernatant after the aqueous and organic phase stratification, then the analytes were determined via gradient elution by LC–MS/MS in negative mode in a single run. The analytical method was validated with good selectivity, acceptable accuracy (>85%) and low variation of precision (<15%). SALLE showed better extraction efficiency of target glucuronide and sulfate conjugates (>80%). The method was successfully applied to determine Res and its four conjugated metabolites in rat after Res administration (intragastric, 50 mg kg⁻¹; intravenous, 10 mg kg⁻¹). The systemic exposures to Res conjugates were much higher than those to Res (AUC_0–t , i.v., 7.43 μm h; p.o., 8.31 μm h); Res‐3‐O‐β ‐d ‐glucuronide was the major metabolite (AUC_0–t , i.v., 66.1 μm h; p.o., 333.4 μm h). The bioavailability of Res was estimated to be ~22.4%. The reproducible SALLE method simplified the sample preparation, drastically improved the accuracy of the concomitant assay and gave full consideration of extraction recovery to each target analyte in bio‐samples.     

Development and validation of UPLC‐MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application

A sensitive, rapid and selective ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed for the determination and pharmacokinetic study of domperidone in human plasma. Diphenhydramine was used as the internal standard. Plasma sample pretreatment involved a one‐step liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v). The analysis was carried out on an Acquity UPLC^TM BEH C₁₈ column. The mobile phase consisted of methanol–water containing 10 mmol/L ammonium acetate and 0.5% (v/v) formic acid (60:40, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionizationsource with positive mode. Each plasma sample was chromatographed within 2.1 min. The standard curves for domperidone were linear (r² ≥ 0.99) over the concentration range of 0.030–31.5 ng/mL with a lower limit of quantification of 0.030 ng/mL. The intra‐ and inter‐day precision (relative standard deviation) values were not higher than 13% and accuracy (relative error) was from ?7.6 to 1.2% at three quality control levels. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.