Quantitative determination of pimozide in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A simple, sensitive and specific LC–ESI/MS method was developed for the determination of pimozide in human plasma. Pimozide and cinnarizine (internal standard) were isolated from plasma samples by liquid–liquid extraction. The chromatographic separation was accomplished on a Thermo Hypersil-HyPURITY C18 reversed-phase column (150 mm × 2.1 mm, i.d., 5 μm) with the mobile phase consisting of 5 mM ammonium acetate (pH 3.5, adjusted with acetic acid)–methanol–acetonitrile (39:5:56, v/v/v). The lower limit of quantification was 0.02 ng/mL, and the assay exhibited a linear range of 0.025–12.800 ng/mL. The established method has been successfully applied to a bioequivalence study of 2 pimozide formulations in 32 healthy male Chinese volunteers.     

Simultaneous quantification of losartan and active metabolite in human plasma by liquid chromatography–tandem mass spectrometry using irbesartan as internal standard

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing electronspray ionization was developed and validated for quantification of losartan and its carboxylic acid metabolite in human plasma using irbesartan as internal standard (IS). Following a simple pretreatment procedure, the analytes were separated using a gradient mobile phase on reverse phase C₁₈ column. Selected reaction monitoring was specific for losartan, losartan acid and irbesartan. The method validation demonstrated the specificity, lower limit of quantification, accuracy and precision of measurements. The assay exhibited a linear dynamic range of 2.0–400 ng/mL for losartan and 1.85–370 ng/mL for losartan acid. A run time of 3.5 min for each sample made it possible to analyze more than 200 samples per day. The validated method has been successfully used to analyze human plasma samples for application in bioavailability/bioequivalence studies.     

Simultaneous quantitation of hydrochlorothiazide and metoprolol in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method for the simultaneous quantitation of hydrochlorothiazide (HCT) and metoprolol (MET) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated. MS/MS detection involved switching the electrospray ionization (ESI) mode during chromatography from negative to detect HCT and its internal standard (I.S.) 5-bromouracil to positive to detect MET and its I.S. tramadol. Sample preparation by liquid–liquid extraction with diethyl ether–dichloromethane (60:40, v/v) was followed by chromatography on a Venusil MP-C18 column using methanol–ammonium acetate (10 mM)–formic acid (pH 3.4) (50:50:0.05, v/v/v) at a flow rate of 0.8 mL/min. The method was linear in the concentration range 3–1000 ng/mL for both HCT and MET using 100 μL human plasma. Intra- and inter-day precisions (as relative standard deviation) for HCT were 2.9–3.9% and 3.9–4.7%, respectively and for MET were 2.4–4.1% and 4.7–6.2%, respectively. Accuracies (as relative error) were ±3.8% and ±2.6% for HCT and MET, respectively. The assay was successfully applied to a pharmacokinetic study involving a single oral dose of a combination tablet (25 mg HCT, 50 mg MET) in healthy volunteers.     

Determination of a peroxisome proliferator-activated receptor γ agonist, 1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy-3-phenyl-1H-indene-2-carboxylic acid ethyl ester (KR-62980) in rat plasma by liquid chromatography–tandem mass spectrometry

A novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, KR-62980, was determined by liquid–liquid extraction with ethyl acetate and liquid chromatography–tandem mass spectrometry (LC/MS/MS) in rat plasma. In order to evaluate the pharmacokinetics of KR-62980, a reliable, selective and sensitive high-performance liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantification of KR-62980 in rat plasma. KR-62980 and imipramine (IS) were separated on Hypersil GOLD C18 column with a mixture of acetonitrile–ammonium formate (10 mM) (80:20, v/v) as mobile phase. The ion transitions monitored were m/z 437.2 → 114.2 for KR-62980, m/z 281.3 → 86.1 for imipramine in multiple reaction monitoring (MRM) mode. The percent recoveries of KR-62980 and imipramine were 90.1 and 98.4% from rat plasma, respectively. The linear dynamic range extended from 0.01 to 10 μg/ml with a correlation coefficient (R²) greater than 0.99 and the lower limit of quantification was 0.01 μg/ml. The mean of intra- and inter-assay precisions was 2.1 and 9.3%. The method was validated and successfully applied to the pharmacokinetic study of KR-62980 in rat.     

Simultaneous determination of magnolin and epimagnolin A in rat plasma by liquid chromatography with tandem mass spectrometry: Application to pharmacokinetic study of a purified extract of the dried flower buds of Magnolia fargesii, NDC-052 in rats

A purified extract isolated from the dried flower buds of Magnolia fargesii (NDC-052) is currently being evaluated for phase III clinical trials as a new anti-asthma drug. A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of magnolin and epimagnolin A, the major bioactive components of NDC-052, in rat plasma was developed. After liquid–liquid extraction with tolterodine as an internal standard, magnolin and epimagnolin A were separated on a Luna phenyl-hexyl column with the mobile phase of 70% methanol in 10 mM ammonium formate. The analytes were detected by an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curves were linear over the concentration range of 50–2500 ng/mL for magnolin and epimagnolin A in rat plasma. The intra- and inter-day coefficients of variation and relative errors for magnolin and epimagnolin A at four QC concentrations were 1.5–11.4% and 5.9–12.5%, respectively. The lower limits of quantification for magnolin and epimagnolin A were 50.0 ng/mL using 50 μL of plasma. This method was successfully applied to the pharmacokinetic study of magnolin and epimagnolin A after an oral administration of NDC-052 in male Sprague–Dawley rats.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.     

Liquid chromatography-tandem mass spectrometry for the determination of jaceosidin in rat plasma

Jaceosidin (4′,5,7-trihydroxy–3′,6-dimethoxyflavone), isolated from Artemisia species as well as Eupatorium species, has antiallergic, anticancer, anti-inflammatory and antioxidant activity. A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the quantification of jaceosidin in rat plasma was developed to characterize the pharmacokinetics of jaceosidin. Jaceosidin and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at acidic pH and analyzed on a Luna phenyl-hexyl column using the mixture of acetonitrile and 0.1% formic acid (45:55, v/v) as a mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The calibration curve was linear (r² = 0.9973) over the concentration range of 2.00–500 ng/ml. The lower limit of quantification for jaceosidin was 2.0 ng/ml using 50 μl of plasma sample. The coefficients of variation of intra- and inter-assay at four QC levels were 2.4–9.6% and the relative errors were −9.1 to 10.0%. The matrix effects for jaceosidin and linezolid were practically absent. The recoveries of jaceosidin and linezolid were 87.0 and 87.7%, respectively. This method was successfully applied to the pharmacokinetic study of jaceosidin in rats.     

Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography–tandem mass spectrometry

A chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) simultaneous stereoselective analysis of bambuterol and its active metabolite terbutaline enantiomers in Wistar rat plasma has been developed and validated. All analytes and the internal standard were extracted from rat plasma samples by liquid–liquid extraction, separated on macrocyclic glycopeptide teicoplanin column with mobile phase constituted of 20 mM ammonium acetate solution–methanol (10:90, v/v) at a flow-rate of 0.4 mL/min. Detection was performed on an API 3000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves in the range 1–800 ng/mL were linear and the accuracy for each analyte was within 8.0%. The intra- and inter-day precision as determined from quality control samples was less than 10.1%. The validated assay was successfully used to determine the enantiomers of bambuterol and terbutaline in rat plasma samples in the pharmacokinetic studies of rac-bambuterol.     

Determination of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry

A sensitive and rapid method was developed for quantification of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS). An aliquot of 1 mL plasma sample was extracted with ethyl acetate–dichloromethane. Separation of olprinone and the milrinone (internal standard, IS) from the interferences was achieved on a C₁₈ column followed by MS/MS detection. The analytes were monitored in the positive ionization mode. Multiple reaction monitoring using the transition of m/z 251 → m/z 155 and m/z 212 → m/z 140 was performed to quantify olprinone and IS, respectively. The method had a total chromatographic run time of 3 min and linear calibration curves over the concentration range of 0.5–60 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-day precisions were less than 16.3% for low QC level, and 7.1% for other QC levels, respectively. The intra- and inter-day relative errors were ranged between −12.2% and 3.7% for three QC concentration levels. The validated method was successfully applied to the quantification of olprinone concentration in human plasma after intravenous (i.v.) administration of olprinone at a constant rate of infusion of 2 μg/(kg min) for 5 min in order to evaluate the pharmacokinetics.     

Application of a rapid and selective method for the simultaneous determination of protease inhibitors,lopinavir and ritonavir in human plasma by UPLC–ESI-MS/MS for bioequivalence study in Indian subjects

A high throughput and rugged ultra performance liquid chromatography tandem mass spectrometry (UPLC–ESI-MS/MS) method is developed and validated for the selective determination of protease inhibitors – lopinavir (LPV) and ritonavir (RTV) in human plasma. Plasma samples were prepared by solid phase extraction of the analytes and their deuterated analogs as internal standard (IS) using Waters Oasis HLB cartridges. The chromatographic separation was achieved in a run time of 1.2 min on Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) under isocratic conditions. The mobile phase consisted of 10 mM ammonium formate, pH 4.0 adjusted with formic acid and methanol (10:90, v/v). The protonated precursor → product ion transitions for lopinavir, ritonavir, d₈-lopinavir and d₆-ritonavir were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. A linear dynamic range of 2.9–1452 ng/mL and 29.6–14379 ng/mL was established for ritonavir and lopinavir respectively using 0.1 mL human plasma. The mean relative recovery of lopinavir (96.6%), ritonavir (97.5%), d₈-lopinavir (85.5%) and d₆-ritonavir (86.3%) from spiked plasma samples was consistent and reproducible. The method was successfully applied to a bioequivalence study of [200(lopinavir) + 50(ritonavir)] mg tablet formulation in 36 healthy human subjects under fasting conditions.     

Validated LC–MS/MS method for the determination of sarpogrelate in human plasma: Application to a pharmacokinetic and bioequivalence study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C₁₈ column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3 → 135.3 and of I.S. at m/z 264.1 → 58.0. The assay was validated over the concentration range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 μL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were ≤6.4% and ≤5.4%, respectively, with accuracy (relative error, R.E.) in the range 0.5–3.6%. The method was successfully applied to a pharmacokinetic and bioequivalence study enrolling 22 Chinese volunteers administered sarpogrelate tablets.     

Simultaneous quantification of enalapril and enalaprilat in human plasma by high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to simultaneously determine enalapril and enalaprilat in human plasma. With benazepril as internal standard, sample pretreatment involved in a one-step protein precipitation (PPT) with methanol of 0.2 ml plasma. Analysis was performed on an Ultimate™ XB-C₁₈ column (50 mm × 2.1 mm, i.d., 3 μm) with mobile phase consisting of methanol–water–formic acid (62:38:0.2, v/v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction-monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for enalapril and enalaprilat were both obtained in the concentration range of 0.638–255 ng/ml (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.638 ng/ml. The intra-day precision (R.S.D.) was below 7.2% and inter-day R.S.D. was less than 14%, while accuracy (relative error R.E.) was within ±8.7 and ±5.5%, determined from QC samples for enalapril and enalaprilat which corresponded to requirement of the guidance of FDA. The HPLC–MS/MS method herein described was fully validated and successfully applied to the pharmacokinetic study of enalapril maleate capsules in 20 healthy male volunteers after oral administration.     

Determination of tropisetron in human plasma by liquid chromatography–tandem mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry method (LC–MS/MS) for the determination of tropisetron in human plasma was developed and validated over the concentration range of 0.100–100 ng/mL. Diphenhydramine was used as the internal standard (IS). The tropisetron and the IS were extracted from alkalized plasma samples into diethyl ether–dichloromethane (2:1, v/v) and the LC separation was performed by a Diamonsil C₁₈ column (150 mm × 4.6 mm, i.d., 5 μm). The mobile phase was methanol:water (80:20, v/v) containing 0.2% formic acid delivered at a flow rate of 0.5 mL/min. The total chromatographic run time was 4.5 min. The MS data acquisition was accomplished by selected reaction monitoring (SRM) mode with positive atmospheric pressure chemical ionization (APCI) interface. The lower limit of quantification (LLOQ) achieved was 0.100 ng/mL with precision (RSD) of 3.1% and accuracy (RE) of −0.7%. For both inter-batch and intra-batch tests, the precision (RSD) for the entire validation was less than 6.0%, and the accuracy (RE) was within the −0.5% to 0.2% range. This validated LC–MS/MS method was later used to characterize the pharmacokinetics as well as the bioequivalence of tropisetron formulations.     

A liquid chromatography–tandem mass spectrometry method for the quantification of PAC-1 in rat plasma

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on an ACQUITY UPLC™ BEH C₁₈ column, with acetonitrile and water (39:61 (v/v) both containing 0.1% formic acid) as mobile phase at a flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 10–1500 ng/mL (r > 0.99). The LOQ was evaluated to be 0.3 ng/mL. The method described herein is sensitive, selective and faster than other existing method, and was successfully applied to the pharmacokinetic study and gender difference investigation of PAC-1 after oral administration in rats.     

Simultaneous quantitation of paracetamol,caffeine, pseudoephedrine,chlorpheniramine and cloperastine in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantitation of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma has been developed and validated. After sample preparation by liquid–liquid extraction, the analytes and internal standard (diphenhydramine) were analyzed by reversed-phase HPLC on a Venusil Mp-C₁₈ column (50 mm × 4.6 mm, 5 μm) using formic acid:10 mM ammonium acetate:methanol (1:40:60, v/v/v) as mobile phase in a run time of 2.6 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode. The method was linear for all analytes over the following concentration (ng/ml) ranges: paracetamol 5.0–2000; caffeine 10–4000; pseudoephedrine 0.25–100; chlorpheniramine 0.05–20; cloperastine 0.10–40. Intra- and inter-day precisions (as relative standard deviation) were all ≤11.3% with accuracy (as relative error) of ±5.0%. The method was successfully applied to a study of the pharmacokinetics of the five analytes after administration of a combination oral dose to healthy Chinese volunteers.     

Determination of mangiferin in rat plasma by liquid–liquid extraction with UPLC–MS/MS

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for the determination of mangiferin in rat plasma using diphenhydramine as the internal standard (IS). Liquid–liquid extraction (LLE) was used for sample preparation and the analysis was achieved with gradient elution on C₁₈ reversed phase column. The method was validated over the concentration range 0.02–5.0 μg/mL for oral administration and 0.4–100 μg/mL for intravenous administration. The intra-day and inter-day precision of mangiferin expressed as RSD < 15% and the accuracy (RE) did not exceed 15%. This validated method is a novel technique for sample preparation and quantitation, which was successfully applied to estimate the bioavailability of mangiferin.     

An LC–MS/MS method for the simultaneous determination of chlorogenic acid,forsythiaside A and baicalin in rat plasma and its application to pharmacokinetic study of Shuang-huang-lian in rats

An LC–MS/MS method was developed for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin, three major ingredients in Shuang-huang-lian preparations, in rat plasma. Following extraction by methanol–ethyl acetate–trifluoroacetic acid (49:49:2, v/v/v), the extracted analytes were separated on a reverse phase C12 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The limits of quantification were between 1.0 and 2.1 ng/mL, the precision was <7% and the accuracy was between 94% and 107%. The validated method was applied to a comparative pharmacokinetic study in rats after administration of Shuang-huang-lian solutions via intravenous, peroral or intratracheal routes. The results showed that the three chemical markers were more rapidly and thoroughly absorbed following pulmonary delivery as compared with peroral administration.     

PK and tissue distribution of docetaxel in rabbits after i.v. administration of liposomal and injectable formulations

A simple and sensitive HPLC method was established and validated for the determination of docetaxel (DTX) in rabbit plasma and tissue samples. Biosamples were spiked with paclitaxel as an internal standard and pre-treated by solid phase extraction (SPE). Sample separation was performed on a reverse-phase HPLC column at 30 °C by using a mobile phase of acetonitrile–methanol–0.02 M ammonium acetate buffer (pH 5.0) (20:47.5:32.5, v/v/v) at flow rate of 1.0 mL/min The UV absorbance of the samples was measured at the wavelength of 230 nm. The standard curves were linear over the ranges of 0.02525–2.525 μg/mL for plasma, 1.010–202.00 μg/g for lung, 0.202–20.20 μg/g for spleen, liver and kidney, 0.202–10.10 μg/g for heart and stomach, 0.0505–2.02 μg/g for brain, respectively. The limits of quantification (LOQ) were 10.0 ng/mL in the plasma samples and 20.0 ng/g in the tissue samples, respectively. The analysis method was successfully applied to pharmacokinetics and tissue distribution studies of DTX liposomes and DTX injection after i.v. administration to the rabbits. The results showed that the liposome carrier led to a significant difference in pharmacokinetics and tissue distribution profile compared to the conventional DTX injection.     

Liquid chromatography/tandem mass spectrometry for the determination of changrolin in rat plasma: Application to a bioavailability study

A sensitive and specific LC–CMS/MS method was developed for the quantification of changrolin, an anti-arrhythmic drug, in rat plasma using tiapride as internal standard. Liquid–liquid extraction was employed for sample preparation and analyzed using a multiple reaction monitoring mode with electrospray positive ionization source. The calibration curve for changrolin was linear over the range 5–1600 ng/mL with coefficients of correlation >0.99. The intra- and inter-batch precision was less than 8.6%, and accuracy ranged from 94.6% to 104.5%. This validated LC–MS/MS method was successfully applied to a bioavailability study of oral and intravenous administration of changrolin with 20 mg/kg dosage in SD rats.