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.     

Analysis of enantiomers of sibutramine and its metabolites in rat plasma by liquid chromatography–mass spectrometry using a chiral stationary-phase column

Sibutramine, a monoamine reuptake inhibitor, is used as a racemate, for the treatment of obesity. It is converted in vivo mainly to two desmethyl active metabolites, mono-desmethylsibutramine (MDS) and di-desmethylsibutramine (DDS). In the present study, we introduced a rapid and simple chromatographic method for separating the R(+)- and S(−)-isomers of sibutramine, MDS, and DDS, respectively. The stereoisomers of the three compounds were extracted from rat plasma using diethyl ether and n-hexane under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (10 mM ammonium acetate buffer adjusted to pH 4.03 with acetic acid:acetonitrile, 94:6, v/v). The enantiomers in the extract were separated on a Chiral-AGP stationary-phase column and were quantified in a tandem mass spectrometry. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of the enantiomers of sibutramine, MDS, and DDS in plasma after a single oral dose of 10 mg/kg racemic sibutramine in rats.     

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 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.     

Stereoselective pharmacokinetic study of rhynchophylline and isorhynchophylline epimers in rat plasma by liquid chromatography–tandem mass spectrometry

1.?In this study, the stereoselective pharmacokinetics of rhynchophylline (RIN) and isorhynchophylline (IRN) in rat plasma were investigated using liquid chromatography–tandem mass spectrometry (LC–MS/MS).

2.?A rapid, robust and sensitive LC–MS/MS method for simultaneous quantification of RIN and IRN in rat plasma was established and validated. Chromatographic separation was performed on a Poroshell 120 EC-C₁₈ column under a gradient elution with methanol and water containing 0.01% ammonia as mobile phase. Calibration curve was linear over a concentration range of 1–2000?ng/mL for both epimers.

3.?After intravenous administration, there was no apparent difference in pharmacokinetic parameters between two epimers. However, after oral administration, RIN showed remarkable higher plasma exposure than IRN. The bioavailability, C_max and AUC_0–t of RIN were about 9.2-fold, 6.4-fold and 9.1-fold higher than those of IRN at 10?mg/kg, and 7.8-fold, 4.3-fold and 7.7-fold at 20?mg/kg, respectively. Additionally, with dosage enhanced from 10?mg/kg to 20?mg/kg, the plasma concentrations of RIN or IRN increased significantly and the bioavailability enhanced about three times.

4.?In conclusion, the results of this work demonstrated for the first time that the pharmacokinetics of RIN and IRN have stereoselectivity.     

Screening and confirmation methods of the major urinary metabolite of finasteride–carboxy-finasteride by liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

Screening and confirmation methods of the major urinary metabolite of finasteride–carboxy-finasteride for doping control purpose were developed. Liquid–liquid extraction was adopted for the sample preparation. Analytes were detected by positive electrospray ionization in single quadrupole and triple quadrupole mass spectrometer. In the screening method, selected ion monitoring (SIM) mode was used to monitor m/z 403 for carboxy-finasteride. In the confirmation method, product ion mode was used to monitor the precursor ion m/z 403. The limit of detection was below 2 ng/mL for the screening method. Good linearity was obtained in the range 10.0–500.0 ng/mL. The intra-run and inter-run precision calculated from quality control (QC) samples was less than 5.3%. The accuracy as determined from QC samples was within ±6.6%. The screening method was applied for the analysis of excretion samples, allowing the detection of carboxy-finasteride for up to 49 h in urine specimen collected after an oral administration of 5 mg of finasteride.     

Determination of repaglinide in human plasma by high-performance liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of repaglinide in human plasma. The analyte and internal standard (I.S.), diazepam, were extracted from plasma (25 μL) by liquid–liquid extraction with diethyl ether–dichloromethane (60:40, v/v) and separated on a XDB-C₁₈ column using acetonitrile–ammonium acetate buffer (pH 6.8, 0.01 mol/L) as mobile phase. The retention times of repaglinide and I.S. were 1.95 and 2.35 min, respectively. Detection was carried out using API 4000 mass spectrometer with an ESI interface operating in the multiple reaction monitoring (MRM) mode. The assay was linear over the concentration range 0.050–50 ng/mL with a limit of detection (LOD) of 0.010 ng/mL. Intra- and inter-day precisions (as relative standard deviation, R.S.D.) were ≤5.07% and ≤11.2%, respectively, and accuracy (as relative error, R.E.) was from ?0.593% to ?1.26%. The assay was successfully applied to a pharmacokinetic study involving a single oral administration of a tablet containing 2 mg repaglinide to each of 10 healthy volunteers.     

Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography–tandem mass spectrometry

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu_n, n = 2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu_n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 × 10⁶ cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H]⁺ ions of MTX and MTXGlu_n to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3 → 294.2. Calibration curves for all analytes were linear in the range 2–250 nM (r² > 0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60 ± 4 to 108 ± 13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.     

Long-term detection of clodronate in equine plasma by liquid chromatography–tandem mass spectrometry

Clodronate is a non-nitrogen-containing bisphosphonate drug approved in equine veterinary medicine. Clodronate is prohibited for use in competition horses; therefore, to set up an appropriate control, detection times and screening limits are required. The quantitative method in plasma consisted of addition of chloromethylene diphosphonic acid as internal standard. Automated sample preparation comprised a solid phase extraction with weak anion exchange properties on microplate. After methylation of the residue with trimethyl orthoacetate, analysis was conducted by high-performance liquid chromatography–tandem mass spectrometry. Using a weighting factor of 1/(concentration)², good linearity was observed in the range of 1 to 500 ng/ml, with low limits of detection and quantification of 0.5 and 1 ng/ml, respectively. Precision and accuracy determined at four concentrations were satisfactory, with an error percentage less than 15%. Absence of carry-over and good stability of clodronic acid in plasma after a long-term storage at −20°C were verified. The method was successfully applied to the quantification of clodronic acid in plasma samples from horses administered with a single intramuscular administration of Osphos® at a mean dose of 1.43 ± 0.07 mg/kg. The observed detection time will be verified in a clinical population study conducted in diseased horses.     

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.     

Bioanalysis of an oligonucleotide and its metabolites by liquid chromatography–tandem mass spectrometry

An ion-pair reversed phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed for the quantification of a phosphorothioate oligonucleotide (PS-OGN) PF-ODN and its metabolites 5′N-1/3′N-1, 5′N-2 and 5′N-3 in rat plasma. Plasma samples were prepared with an initial phenol/dichloromethane liquid–liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a Phenomenex Gemini C₁₈ column using hexafluoro-2-propanol/triethylamine buffer and methanol as the mobile phase at a flow rate of 0.5 mL/min. Except for 5′N-1 and 3′N-1, which were coeluted and could not be differentiated by mass spectrometer, the other analytes were separated chromatographically and mass spectrometrically. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 4.0 ng/mL for PF-ODN and its four metabolites with acceptable precision and accuracy. Inter-day and intra-day RSD for three quality control (QC) levels across validation runs were less than 12.0% and the accuracy ranged from −9.6% to 6.0% for the analytes. This validated LC–MS/MS method was applied to a preliminary pharmacokinetic study of PF-ODN in rats.     

Simultaneous quantification of antibiotic dyes in aquatic products and feeds by liquid chromatography–tandem mass spectrometry

A confirmatory and quantitative method based on liquid chromatography–tandem mass spectrometry (LC/MS/MS) has been developed for the determination of low-level residues of three antibiotic dyes and two metabolites in fish muscle and feed. The target compounds include methylene blue (MB), crystal violet (CV), leucocrystal violet (LCV), malachite green (MG), and leucomalachite green (LMG). The procedures involve solvent extraction by 50% McIlvaine’s buffer with acetonitrile, followed by solid phase extraction (SPE) with an MCX cartridge. High performance liquid chromatography (HPLC) and positive electrospray ionization (ESI) MS with multiple reaction monitoring of two transition reactions was applied for each compound. The detected ion ratios of MB, CV, LCV, MG, and LMG were 11.8, 34.9, 88.4, 25.6, and 42.0, respectively. The average fortification recoveries of the MB, CV, LCV, MG, and LMG of the level of 0.8 μg/kg tested in fish muscle and feed samples were 99.68, 98.93, 100.49, 100.01, and 100.00%, respectively. The precision of analysis of analytes in fish muscle and feed ranged from 4% to 14% and from 7% to 14%, respectively. The decision limits (CCα) were 0.28–0.54 μg/kg, and the detection capabilities (CCβ) were 0.35–0.67 μg/kg (n = 99).     

Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography–tandem mass spectrometry assay

A stable isotope dilution liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) assay to measure amiodarone, the most frequently used agent for maintaining sinus rhythm in patients with atrial fibrillation, and its major metabolite desethylamiodarone in human plasma and serum was developed. Measurement of amiodarone and desethylamiodarone was performed during a 4.0-min run-time using amiodarone-D₄ and desethylamiodarone-D₄ as internal standards. Calibration curves covering 12 calibrators measured in four replicates each for the analysis of both amiodarone and desethylamiodarone were linear and reproducible in the range of 0.01–40.0 mg/L (r > 0.999). Limits of detection in plasma matrix were 2.7 μg/L for amiodarone and 1.9 μg/L for desethylamiodarone, and lower limits of quantification in plasma matrix were 7.5 μg/L for amiodarone and 2.5 μg/L for desethylamiodarone. Interassay imprecision and inaccuracy were <8% and <9% for both substances. Mean extraction yield was 99.6% (range 92.6–107.7%) for amiodarone and 90.2% (range 80.0–94.7%) for desethylamiodarone. Agreement was moderate for amiodarone (n = 162) and desethylamiodarone (n = 117), respectively, between the present method and a HPLC method with UV detection using a commercially available reagent set for the HPLC analysis of these drugs. The Passing–Bablok regression line was HPLC = 0.98 (LC–MS/MS) + 0.10 [mg/L]; r = 0.94 for amiodarone and HPLC = 1.05 (LC–MS/MS) + 0.02 [mg/L]; r = 0.90 for desethylamiodarone. This sensitive and interference-free LC–MS/MS assay permits rapid and accurate determination of amiodarone and desethylamiodarone in human plasma and other body fluids.     

Quantitation of the tacrine analogue octahydroaminoacridine in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of octahydroaminoacridine in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5 μm, 100 mm × 4.6 mm) using acetonitrile:10 mM ammonium acetate:formic acid (30:70:1, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1 → 175.1 and of the I.S. at m/z 264.1 → 58.0. The method was linear in the range 0.01–10 ng/ml with a lower limit of quantitation of 0.01 ng/ml. Intra- and inter-day precisions measured as relative standard deviation were <3.15% and <5.01%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of a tablet containing 4 mg octahydroaminoacridine succinate to healthy volunteers. Pharmacokinetic parameters for octahydroaminoacridine include C_max 1.19 ± 0.53 ng/ml, T_max 0.77 ± 0.17 h, AUC_0−t 3.42 ± 1.01 ng h/ml and t_1/2 2.89 ± 0.56 h.     

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 alprostadil in rat plasma by ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry after intravenous administration

A rapid, highly selective ultra performance liquid chromatography–electrospray ionisation–tandem mass spectrometry method (UPLC–ESI–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of alprostadil in rat plasma. After a simple sample preparation procedure involving a one-step liquid–liquid extraction, alprostadil and the internal standard, diphenhydramine, were chromatographed on an ACQUITY UPLC™ BEH C₁₈ column with gradient elution using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.25 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r² = 0.99) over the concentration range 0.4–250.0 ng mL⁻¹, with a lower limit of quantification of 0.4 ng mL⁻¹ for alprostadil. The inter- and intra-day precision (%R.S.D.) was less than 8.5% and 2.4%, respectively, and the accuracy (RE%) was between 9.3% and 1.0% (n = 6). Alprostadil in rat plasma was stable when stored at room temperature for 0.5 h and at −20 °C for two weeks. The method was very rapid, simple and reliable, and was employed for the first time for the pharmacokinetic studies of alprostadil in rats after a single intravenous administration of 50 μg kg⁻¹.     

Liquid chromatography–tandem mass spectrometry for quantification of cefaclor in rat plasma and its application to pharmacokinetic studies

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the positive electrospray ionization mode was developed and validated in order to analyze cefaclor, a second generation cephalosporin antibiotic, in rat plasma. The plasma was pre-treated by a single step of protein precipitation with methanol, and then injected directly into the LC/MS/MS system for quantification. Drugs were separated on a Synergi 4μ Polar-RP 80 A column (150 × 2.0 mm) with a mixture of 0.1 % formic acid and methanol (65 : 35, v/v) as the mobile phase at 0.2 mL/min. Detection was performed with multiple reaction-monitoring modes at m/z 368.1 → 174.2 (for cefaclor) and m/z 396.0 → 227.1 (for cefdinir, the internal standard). The limit of quantification (LOQ) was determined to be 10 ng/mL with acceptable linearity ranging from 10 to 10,000 ng/mL. Validation parameters for cefaclor, including accuracy, precision, absolute matrix effect, and stability in rat plasma, were acceptable according to the assay validation guidelines of the FDA (U.S. Department of Health & Human Services, Food and Drug Administration, Guidance for Industry, Bioanalytical Method Validation, 2001). The developed analytical method was successfully applied to the pharmacokinetic studies of cefaclor in rats. These observations suggest, therefore, that the validated assay can be used in routine pharmacokinetic studies of cefaclor in rats.     

Simultaneous quantitation of lamivudine,zidovudine and nevirapine in human plasma by liquid chromatography–tandem mass spectrometry and application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the simultaneous quantitation of lamivudine, zidovudine and nevirapine in human plasma using abacavir as internal standard has been developed and validated. The analytes and IS were extracted from plasma by solid phase extraction using Oasis HLB cartridges and separated on a Hypurity Advance C18 column using a mixture of acetonitrile:0.1% formic acid (76:24, v/v) at a flow rate of 0.8 mL/min. Detection involved an API-4000 LC–MS/MS with electrospray ionization in the positive ion mode and multiple-reaction monitoring for analysis. The method was validated according to FDA guidelines and shown to provide intra- and inter-day precision and accuracy within acceptable limits in a run time of only 3.5 min. The method was successfully applied to a pharmacokinetic study involving a single oral administration of a combination tablet to human male volunteers.