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.     

Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA

Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments. The rapid and accurate analysis of MCs and nodularin (Nod-R) in fish tissue is important for determining occurrence, following trends, and monitoring exposure for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for eight MCs (MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, and MC-LF) and Nod-R in fish, and conduct a validation of the new method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) for analysis and compare the results with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetonitrile/water (3:1) followed by hexane partitioning cleanup was found to be most effective. Thorough validation of the final method was conducted, and 90–115% recoveries were achieved for all analytes except for MC-RR, which gave 130% average recovery (isotopically labeled internal standards were unavailable to correct for possible biases). The use of electrospray ionization in the negative mode gave few interferences and minimal matrix effects in the LC-MS/MS analysis overall. Precision was typically 10–20% RSD among multiple days in experiments, detection limits were <10 ng/g in the fish tissue (catfish, basa, and swai filets), and no false-positives or false-negatives occurred in blind analyses of many spiked samples. The ELISA was unable to distinguish between MCs but was found to correctly assess the presence or absence of MCs and Nod-R in the blind-fortified fish tissues. The capability of these approaches to measure covalently bound MCs was not assessed.     

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

This study aimed to develop and validate a liquid chromatography tandem mass spectrometry method for measuring clematichinenoside AR in rat plasma. Clematichinenoside AR was extracted by solid‐phase extraction and chromatographed on an XTerra MS C₈ column. Pulchinenoside B4 was used as the internal standard. Elution was achieved using an isocratic mobile phase of acetonitrile with 0.1% acetic acid (21:79, v/v) at a flow‐rate of 0.2 mL/min. The detection was performed by multiple reaction monitoring mode via a negative electrospray ionization interface. Standard curves were linear, ranging from 2.5 to 500 ng/mL. The intra‐ and inter‐day precision values were <14.0% and the accuracy was within ±13%. Extraction recovery ranged from 93.2 to 93.9%. This proposed method was successfully applied to a pharmacokinetic study on clematichinenoside AR in rats after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples

Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins.     

Determination of lipoic acid in rat plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a pharamcokinetic study

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.     

Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species

The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.     

Selective LC-MS/MS method for the identification of BMAA from its isomers in biological samples

Algal blooms are well-known sources of acute toxic agents that can be lethal to aquatic organisms. However, one such toxin, β-N-methylamino-L-alanine (BMAA) is also believed to cause amyotrophic lateral sclerosis, also known as Lou Gehrig's disease. The detection and identification of BMAA in natural samples were challenging until the recent introduction of reliable methods. However, the issue of potential interference from unknown isomers of BMAA present in samples has not yet been thoroughly investigated. Based on a systematic database search, we generated a list of all theoretical BMAA structural isomers, which was subsequently narrowed down to seven possible interfering compounds for further consideration. The seven possible candidates satisfied the requirements of chemical stability and also shared important structural domains with BMAA. Two of the candidates, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG) have recently been studied in the context of BMAA. A further isomer, β-amino-N-methyl-alanine (BAMA), has to be considered because it can potentially yield the fragment ion, which is diagnostic for BMAA. Here, we report the synthesis and analysis of BAMA, together with AEG, DAB, and other isomers that are of interest in the separation and detection of BMAA in biological samples by using either high-performance liquid chromatography or ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. We detected for the first time BAMA in blue mussel and oyster samples. This work extends the previously developed liquid chromatography-tandem mass spectrometry platform Spacil et al. (Analyst 135:127, 2010) to allow BMAA isomers to be distinguished, improving the detection and identification of this important amino acid.     

Stabilization of zeylenone in rat plasma by the presence of esterase inhibitors and its LC‐MS/MS assay for pharmacokinetic study

Six esterase inhibitors, namely EDTA·2Na⁺, NaF, phenylmethanesulfonyl fluoride, dichlorvos, bis‐nitrophenyl phosphate (BNPP) and thenoyltrifluoroacetone, and the mixture of NaF and BNPP, were evaluated for the stabilization of labile benzoate containing zeylenone in rat plasma. The mixture appeared to exhibit the most effectively stabilizing effect with the degraded content of zeylenone decreasing from >60% (in the absence of inhibitors) to <6%. Following the stabilization by the addition of NaF (5 mm ) and BNPP (5 mm ), the analytes in rat plasma were acidified by formic acid and extracted into ethyl acetate at 0°C. After chromatographic separation, the detection of zeylenone was performed on a 3200 Q‐Trap with positive ion electrospray mode, monitoring the ion transition m/z 383.2 → 105.0. The method was validated over the range from 2.68 to 1340 ng/mL with inter‐ and intra‐run precision for the quality control samples being less than 6.8%. The assay accuracy was within 100 ± 7.0%. The validated method was successfully applied to a pharmacokinetic study in rats after the intratracheal administration of zeylenone in free drug or polymeric micellar solutions. The results showed that the pulmonary absorption of zeylenone loaded in micelles was significantly retarded compared with that of free drug solutions. Copyright © 2012 John Wiley & Sons, Ltd.     

Utilisation of an enzyme-linked immunosorbent assay (ELISA) for determination of alkylphenols in various environmental matrices. Comparison with LC-MS/MS method

Among the wide range of substances discharged continuously in the environment, alkylphenols became a major focus of environmental research in the last decades, as it was found that they possess endocrine disrupting properties. Knowledge about the occurrence and levels of alkylphenols in environment is critical for the risk assessment of these compounds on both ecosystem and human health. However, the analysis of traces of alkylphenols in environmental matrices is a very difficult task, and the suitable methods involve generally an extraction followed by an extensive sample clean-up before detection, steps often time-consuming and costly.In order to reduce the analysis time, obtain a high throughput of analysis and thus improve work efficiency, the objective of the present study is to investigate the use of immunochemical technique (ELISA) for the determination of nonylphenol and octylphenol in soils and various kinds of water. To our knowledge, this is the first time that the determination of alkylphenols in soil using immunoassay technique is described. A methodology is developed, based on the combination of a single preparation step and the use of a simply ELISA kit. The performances of the method are compared with LC-MS/MS, considered as reference. The developed procedure offers the sensitivity and selectivity necessary for the detection of the target alkylphenols in the ng/g or ng/L range, and is successfully applied to the analysis of several samples. Results indicate that alkylphenols are quantified with concentrations in the same order than LC-MS/MS, meaning that ELISA may be useful not only in screening the samples and get a positive/negative response, but also it allows a good approximation of the concentrations.     

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

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

Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to a patient samples

Warfarin is routinely monitored by assessing its pharmacologic effects on the international normalized ratio. However, having a patient with INR not responding to increasing warfarin dose mandates a direct measurement of warfarin concentrations (total and free) for better patient clinical management of warfarin therapy. Therefore, a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry was developed for the determination of free and total warfarin in human plasma. Free warfarin was measured in plasma filtrate, prepared by ultrafiltration, and sample pretreatment involved protein precipitation with acetonitrile. Linear response (r(2) ≥0.99) was observed over the studied range of free and total warfarin, with the lower limit of detection of 0.25 ng/mL. The intra- and inter-day precision (relative standard deviation) values were <10% and the accuracy (relative error) was ≤6.6 for free and total warfarin. There was no significant difference (p>0.05) between inter- and intra-day studies for the free and total warfarin, which confirmed the reproducibility of the assay method. The mean extraction efficiency was 88.6-107.2% of free and total warfarin. The assay was sensitive to follow warfarin pharmacokinetics (free and total) in a patient with resistance to warfarin up to 24 h after a daily dose of warfarin.     

Development of a fast LC‐MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics

A fast and accurate liquid chromatography/tandem mass spectrometric (LC‐MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 μL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion‐RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor‐to‐parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients. Copyright © 2012 John Wiley & Sons, Ltd.     

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

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

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

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

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

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

Sensitive and selective LC-MS/MS for the determination of tolperisone and etodolac in human plasma and application to a pharmacokinetic study

Tolperisone and etodolac were proven to have synergistic effect for patients of acute low back pain associated with musculoskeletal spasm. In this work, a specific, highly sensitive and reproducible analytical method was developed and validated for the simultaneous determination of tolperisone and etodolac in human plasma using liquid chromatography-tandem mass spectrometric technique. Liquid–liquid extraction was optimized for sample preparation. Zorbax C₈ column (3.5 μm, 50 × 4.6 mm) was used, carrying a mobile phase mixture of 10.0 mM ammonium formate:acetonitrile (40:60, v/v) pH 3.8, running in an isocratic mode. Chlorzoxazone acted as an internal standard. Sample volume of injection was 5.0 μL, and analysis was achieved within 2.5 min. Detection and quantitation were performed by electrospray ionization mass spectrometry using the multiple-reaction monitoring mode. The proposed method could determine the analytes in the range of concentration 0.5–200.0 ng mL⁻¹ for tolperisone and 0.05–20.0 μg mL⁻¹ for etodolac. Findings of inter- and intraday precisions were ≤12.3% with accuracy of ±5.0%. Pharmacokinetics study for the two drugs after oral administration of healthy human volunteers was achieved with the aid of application of the developed study.     

A highly sensitive LC-MS/MS method for the determination of S-raclopride in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and rapid assay method has been developed and validated for the estimation of S‐(−)‐raclopride (S‐RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid–liquid extraction technique for extraction of S‐RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C₁₈ column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S‐RCP and 180.1 → 110.1 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra‐day and inter‐day precisions were 0.23–10.5 and 3.74–7.29%, respectively. This novel method was applied to a pharmacokinetic study of S‐RCP in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

High-throughput LC-MS/MS assay for 6-methoxy-2-naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study

A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.     

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

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

LC-MS/MS methods for determination of unstable pivmecillinam and mecillinam in acidified human plasma: Application to a pharmacokinetic study

Pivmecillinam, the ester of biologically active antibiotic mecillinam, is an effective oral preparation to treat urinary tract infections. To study pharmacokinetics in humans, LC-MS/MS methods were developed to quantify pivmecillinam and mecillinam in human plasma, respectively. Considering cephalexin as internal standard, analytes were separated on UltimateXB-C18 columns after protein precipitation by acetonitrile. The mobile phase was composed of water containing 0.1% formic acid and methanol. The multiple reactions monitoring transitions of m/z 440.2→167.1, 326.1→167.1, and 348.1→158.1 were selected to inspect pivmecillinam, mecillinam, and the internal standard in positive ion mode. No apparent matrix effect was perceived. Linearities were obtained over calibration ranges of 0.0500–12.0 and 10.0–15,000 ng/mL, respectively. The intraday precisions were below 5.5%, the interday precisions were below 6.1%, and accuracies were within –8.1 to 13.0%. Stability tests were conducted and an acidification step was explored to enhance the stability of pivmecillinam and mecillinam. Further stability was validated under various storage and processing conditions. Both methods were applied to a pharmacokinetic study of pivmecillinam and mecillinam after oral administration of 400 mg pivmecillinam hydrochloride tablets in healthy Chinese subjects.