Bromazepam determination in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry: a highly sensitive and specific tool for bioequivalence studies

A rapid, sensitive and specific method to quantify bromazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using diethyl ether-hexane (80 : 20, v/v). The extracts were analyzed by high-performance liquid chromatography (HPLC) coupled to electrospray tandem mass spectrometry (MS/MS). Chromatography was performed isocratically on a Genesis C(18) analytical column (100 x 2.1 mm i.d., film thickness 4 microm). The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 5.0-150 ng ml(-1) (r(2) > 0.9952). The limit of quantification was 5 ng ml(-1). This HPLC/MS/MS procedure was used to assess the bioequivalence of two bromazepam 6 mg tablet formulations (bromazepam from Medley SA Indústria Farmacêutica as the test formulation and Lexotan from Produtos Roche Químico e Farmacêutico SA as the reference formulation). A single 6 mg dose of each formulation was administered to 24 healthy volunteers (12 males and 12 females). The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. Since the 90% CI for C(max), AUC(last), AUC(0-240 h) (linear) and AUC((0- infinity )) ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the bromazepam formulation from Medley is bioequivalent to the Lexotan formulation for both the rate and the extent of absorption.     

Determination of levonorgestrel in human plasma by liquid chromatography-tandem mass spectrometry method: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine levonorgestrel in human plasma was developed and fully validated. After hexane-ethyl acetate (70:30, v/v) induced extraction from the plasma samples, levonorgestrel was subjected to LC/MS/MS analysis using electro-spray ionization. The MS system was operated in the selected reaction monitoring mode. Chromatographic separation was performed on a Hypersil BDS C18 column (i.d. 2.1x50 mm, particle size 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.25-90 ng/mL for levonorgestrel. The lower limit of quantification of the method was 0.25 ng/mL for levonorgestrel. The intra- and inter-batch precision was 3.7-10.2 and 5.1-12.9%, respectively, for all quality control samples at concentrations of 0.5, 6.0 and 45.0 ng/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for levonorgestrel compared with those methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method was successfully used for a bioequivalence study of two tablet formulations of levonorgestrel in healthy volunteers.     

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for topotecan determination in beagle dog plasma and its application in a bioequivalence study

Topotecan (TPT) is an important anti‐cancer drug that inhibits topoisomerase I. A sensitive and robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method that potentially determines TPT in beagle dog plasma is needed for a bioequivalence study of TPT formulations. We developed and validated LC‐MS/MS to evaluate TPT in beagle dog plasma in terms of specificity, linearity, precision, accuracy, stability, extraction recovery and matrix effect. Plasma samples were treated with an Ostro^TM sorbent plate (a robust and effective tool) to eliminate phospholipids and proteins before analysis. TPT and camptothecin (internal standard) were separated on an Acquity UPLC BEH C₁₈ column (1.7 µm, 2.1 × 50 mm) with 0.1% formic acid and methanol as the mobile phase at a flow rate of 0.25 mL/min. TPT was analyzed using positive ion electrospray ionization in multiple‐reaction monitoring mode. The obtained lower limit of quantitation was 1 ng/mL (signal‐to‐noise ratio > 10). The standard calibration curve for TPT was linear (correlation coefficient > 0.99) at the concentration range of 1–400 ng/mL. The intra‐day and inter‐day precision, accuracy, stability, extraction recovery and matrix effect of TPT were within the acceptable limits. The validated method was successfully applied in a bioequivalence study of TPT in healthy beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of chlordesmethyldiazepam by liquid chromatography–tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC‐MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid‐liquid extraction (diethyl‐ether/hexane, 80/20, v/v) procedure. The LC‐MS/MS method on a RP‐C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5–50 ng/mL (R > 0.999). The between‐run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification—LLOQ (0.500 ng/mL). The between‐run accuracies were 0.1, –1.5, –2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam—test, Eurofarma Lab. Ltda and Olcadil®— reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0‐inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of metformin and gliclazide in human plasma by liquid chromatography-tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine gliclazide and metformin in human plasma using huperzine A as the internal standard (IS). After acetonitrile-induced protein precipitation of the plasma samples, gliclazide, metformin and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 10-10,000 ng ml(-1) for gliclazide and 7.8-4678.9 ng ml(-1) for metformin. The recoveries of the method were found to be 71-104%. The lower limits of quantification (LOQ) of the method were 10.0 and 7.8 ng ml(-1) for gliclazide and metformin, respectively. The intra- and interday precision was less than 15% for all quality control samples at concentrations of 100, 500, and 2000 ng ml(-1). The validated LC/MS/MS method has been used to study bioequivalence in healthy volunteers. These results indicate that the method was efficient with a very short running time (2.0 min) for metformin and gliclazide compared to the methods reported in the literature. The presented method had acceptable accuracy, precision and sensitivity and was used in clinical bioequivalence study.     

Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers

A simple, sensitive and selective LC-MS-MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C(18) column with a mobile phase consisting of 0.2% formic acid-methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508-5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC(0-24), AUC(0-infinity), C(max), T(max) and t(1/2) of huperzine A were 16.35 +/- 3.42 vs 16.38 +/- 3.61 ng h/mL, 17.53 +/- 3.80 vs 17.70 +/- 3.97 ng h/mL, 2.47 +/- 0.49 vs 2.51 +/- 0.51 ng/mL, 1.3 +/- 0.4 vs 1.2 +/- 0.3 h and 5.92 +/- 0.75 vs 6.18 +/- 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent.     

Ticlopidine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify ticlopidine in human plasma using clopidogrel as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid-liquid extraction using diethyl ether-hexane (80 : 20, v/v). The extracts were analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC/MS/MS). Chromatography was performed isocratically on a Jones Genesis C(8) 4 microm analytical column (150 x 4.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 1.0-1000 ng ml(-1) (r(2) > 0.999427). The limit of quantification was 1.0 ng ml(-1). This HPLC/MS/MS procedure was used to assess the bioequivalence of two ticlopidine 250 mg tablet formulations (ticlopidine test formulation from Apotex do Brasil, Brazil, and Ticlid from Sanofi-Synthelabo, standard reference formulation). A single 250 mg dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and area under the curve ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that ticlopidine formulation from Apotex do Brasil is bioequivalent to Ticlid formulation with respect to both the rate and the extent of absorption.     

Nevirapine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify nevirapine in human plasma using dibenzepine as the internal standard (IS) was developed and validated. The method employed a liquid-liquid extraction. The analyte and the IS were chromatographed on a C(18) analytical column, (150 x 4.6 mm i.d. 4 microm) and analyzed by tandem mass spectrometry in the multiple reaction monitoring mode. The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 10-5000 ng ml(-1) (r(2) > 0.9970). The between-run precision, based on the relative standard deviation for replicate quality controls was 1.3% (30 ng ml(-1)), 2.8% (300 ng ml(-1)) and 3.6% (3000 ng ml(-1)). The between-run accuracy was 4.0, 7.0 and 6.2% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two nevirapine tablet formulations (Nevirapina from Far-Manguinhos, Brazil, as a test formulation, and Viramune from Boehringer Ingelheim do Brasil Química e Farmacêutica, as a reference formulation) in 25 healthy volunteers of both sexes who received a single 200 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Nevirapina/Viramune was 96.4-104.5% for AUC((0-last)), 91.4-105.1% for AUC((0-infinity)) and 95.3-111.6% for C(max) (AUC = area under the curve; C(max) = peak plasma concentration). Since both 90% CI for AUC((0-last)) and AUC((0-infinity)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Nevirapina was considered bioequivalent to Viramune according to both the rate and extent of absorption.     

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5 > 166.1 for itopride and m/z 342.3 > 111.6 for IS, respectively. Analytes were chromatographed on an YMC C₁₈ reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r² = 0.9999) over the studied range (0.5-1000 ng mL⁻¹) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.     

Determination of lafutidine in human plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry: application to a bioequivalence study

A rapid, sensitive and specific high-performance liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the first time to determine the concentration of lafutidine in human plasma. After the addition of diazepam (the internal standard, IS) and 1 M sodium hydroxide solution to 0.5-ml plasma sample, lafutidine was extracted from plasma with n-hexane : isopropanol (95 : 5, v/v). The organic layer was evaporated and the residue was redissolved in 200-microl mobile phase. The analyte was chromatographically separated on a prepacked Shimadzu Shim-pack VP-ODS C(18) column (250 x 2.0 mm i.d.) using a mixture of methanol-water (20 mM CH(3)COONH(4)) = 80 : 20 (v/v) as mobile phase. Detection was performed on a single quadrupole mass spectrometer using an electrospray ionization interface and the selected-ion monitoring (SIM) mode. The method showed excellent linearity (r = 0.9993) over the concentration range of 5-400 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection was 1 ng/ml. The validated LC/ESI-MS method has been successfully applied to the bioequivalence study of lafutidine in 24 healthy male Chinese volunteers.     

Validated ultra-performance liquid chromatography tandem mass spectrometry method for the determination of pramipexole in human plasma

A sensitive and high throughput ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method has been developed for the determination of pramipexole, a dopamine agonist, in human plasma. Sample preparation involved liquid-liquid extraction of pramipexole and ranitidine as the internal standard (IS) in ethyl acetate from 100 μL human plasma. The chromatographic separation is achieved on a Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) analytical column using an isocratic mobile phase, consisting of 10 mM ammonium formate (pH 7.50)-acetonitrile (15:85, v/v), at a flow-rate of 0.5 mL/min. The precursor → product ion transition for pramipexole (m/z 212.1 → 153.0) and IS (m/z 315.0 → 176.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over a wide dynamic concentration range of 20-4020 pg/mL. Matrix effect is assessed by post-column infusion experiment and the process efficiency were 91.9% and 85.7% for pramipexole and IS, respectively. The method is rugged and rapid with a total run time of 1.5 min and is applied to a bioequivalence study of 0.25 mg PPX tablet formulation in 30 healthy Indian male subjects under fasting condition.     

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.     

A validated liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma: application to a pharmacokinetic study

A new liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma has been developed and fully validated. The article describes in detail the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using liquid-liquid extraction with a mixture of 1-chlorobutane/isopropanol/ethyl acetate (88:2:10, (v/v/v)). The chromatographic separation was performed on a reversed-phase XTerrra MS C₈ column ( i.d.; 3.5 μm particle size) using a mobile phase consisting of 0.010 M ammonium formate (pH 7.8) and acetonitrile (35:65, (v/v)), pumped at a flow rate of 0.80 ml min⁻¹. The analytes were detected using a Finnigan LCQ advantage ion-trap mass spectrometer with positive electrospray ionization in selected reaction monitoring (SRM) mode. Tandem mass spectrometric detection enabled the quantitation of both compounds down to 0.10 ng ml⁻¹. Calibration graphs were linear (r better than 0.990, n=11), in concentration ranges 0.10 to 200 ng ml⁻¹ for mirtazapine demethylmirtazapine. The intra- and inter-day R.S.D. values were less than 14.8 and 16.6% for mirtazapine and demethylmirtazapine, respectively. The method was successfully applied to a kinetic study in order to assess the main pharmacokinetic parameters of mirtazapine and demethylmirtazapine.     

Sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of metoclopramide in human plasma: application to a bioequivalence study

A simple, sensitive and rapid method has been developed and validated for determination of the metoclopramide (MCP) in 100 μL human plasma. The analytical procedure involves a liquid–liquid extraction method using tramadol as an internal standard (IS). Chromatographic separation was carried out on a HyPURITY ADVANCE column using a mobile phase consisting of acetonitrile and 10 mm ammonium acetate buffer in the ratio of 80:20 (v/v) at a flow rate of 0.3 mL/min. The total run time of analysis was 2.5 min and elution of MCP and IS occurred at 0.9 and 1.3 min, respectively. A linear response function was established for the range of concentrations 0.53–42.07 ng/mL (r > 0.99). The intra‐ and inter‐day precision values for MCP met the acceptance as per FDA guidelines. MCP was stable in a battery of stability studies viz., bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay method was successfully applied to an oral bioequivalence study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry method for the quantitation of mycophenolate mofetil in human plasma: Application to a bioequivalence study and metabolite identification

We established a sensitive, selective, and rapid analytical method for the quantitation and pharmacokinetic investigation of mycophenolate mofetil in human plasma. To our knowledge, this is the first method that characterizes presence of mycophenolate mofetil glucuronide in clinical samples through tandem mass spectrometry detection and resolves mycophenolate mofetil from its glucuronide metabolite. Liquid chromatography coupled to tandem mass spectrometry detection in positive ion mode was selected to provide optimal selectivity and sensitivity. Due to the ionizable characteristics of the mycophenolate mofetil, a mixed‐mode cation‐exchange disposable extraction cartridge was prudently chosen. The chromatographic separation was achieved on Luna^® C18(2) (100×4.60 mm) column using mobile phase consisting of a mixture of 1±0.05 mM ammonium formate in water, titrated to pH 3.1±0.1 with formic acid, and methanol (20:80, v/v), at a flow rate of 0.7 mL/min. The detection was led at m/z ratios of 434.4→ 114.2 and 438.4→ 118.3, for mycophenolate mofetil and mycophenolate mofetil‐D4, respectively. The developed method was linear between 40.2–4986.0 pg/mL. All validation parameters were within the defined limits. The validated method was then successfully applied for the evaluation of bioequivalence parameters of mycophenolate mofetil after an oral administration of 500 mg mycophenolate mofetil tablet to healthy male Indian volunteers.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of febuxostat in human plasma

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d₇‐febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C₁₈ column (4.6 × 100 mm, 5 µm) using acetonitrile–5 mm ammonium acetate–formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor‐to‐product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d₇‐febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0–5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra‐ and inter‐day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at ?20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of sparfloxacin in rat plasma by liquid chromatography/tandem mass spectrometry

Sparfloxacin, a fluoroquinolone antibiotic, is used for the treatment of bacterial infection. A quantification method using mass spectrometry was developed for the determination of sparfloxacin in rat plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed‐phase C₁₈ column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma sparfloxacin concentrations after a single oral administration of sparfloxacin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

High-throughput quantification of perindopril in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of perindopril in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 369/172 for perindopril and m/z 417/234 for the internal standard. The method exhibited a linear dynamic range of 0.1-100 ng/mL for perindopril in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6.1%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 450 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

超高效液相色谱-串联质谱法测定人血浆中盐酸氨溴索及其制剂的生物等效性评价

采用超高效液相色谱-串联质谱（UHPLC-MS/MS）技术,建立了快速、简单、灵敏的测定人血浆中盐酸氨溴索含量的方法,并用于盐酸氨溴索人体生物等效性预试验研究。取50 μL血浆样品,采用蛋白沉淀法处理,以盐酸氨溴索-d5为内标。采用Waters XBridge BEH C18色谱柱（50 mm×2.1 mm,2.5 μm）,以0.1%（v/v）甲酸水-含0.1%（v/v）甲酸的甲醇为流动相,在0.4 mL/min流速下进行梯度洗脱。采用电喷雾电离（ESI）源以正离子模式进行MRM检测。结果显示,盐酸氨溴索在2~400 ng/mL范围内线性关系良好,相关系数（r）为0.998,准确度为97.1%~108.7%,精密度为1.0%~5.6%。将该方法用于6名健康受试者口服盐酸氨溴索受试制剂和参比制剂30 mg后血药浓度的测定,结果显示二者相对生物利用度为（102.3±14.8）%,血药浓度-时间曲线下面积（AUC_0-t、AUC_0-∞）和最大血药浓度（C_max）的90%置信区间均在80.0%~125.0%范围内,两种制剂生物等效。