Positive Electrospray Tandem Mass Spectrometry Research Articles

Characterization of isomeric lipid A species from Pseudomonas aeruginosa PAO1 by non-aqueous capillary electrophoresis with positive and negative ion electrospray tandem mass spectrometry

Pseudomonas aeruginosa is a priority target pathogen for antibiotic research and development because of its high resistance to a wide range of antibiotics. Acquisition of resistance is sometimes associated with modifications of the lipid A component present in the outer membrane of most Gram-negative bacteria. For a deeper understanding of subtle chemical variations of lipid A in relation to biological properties, the structural elucidation of lipid A species within a bacterial strain is of crucial importance. However, the simultaneous presence of monophosphorylated positional isomers in naturally heterogeneous lipid A samples cannot easily be recognized with direct MS measurements or with conventional LC-MS strategies. Herein, we present the application of a non-aqueous capillary electrophoresis (NACE) method coupled to positive and negative ion electrospray tandem mass spectrometry with collision-induced dissociation (CID) activation technique for the in-depth analysis of the lipid A isolate of P. aeruginosa PAO1. The main advantage of our NACE strategy is its separation power regarding the site of phosphorylation of the lipid A structures, which makes it a good orthogonal technique to chromatographic characterizations of the bacterial lipid A composition, where the separation is mainly based on acylation differences of the species. Overall, the results of this electrophoretic approach revealed hitherto unreported isomeric monophosphorylated PAO1 lipid A constituents, including both phosphate and acyl chain positional isomers. The parallel fragmentation in the complementary positive and negative ion modes enabled the unequivocal assignment of the phosphorylation site and position of acyl chains in lipid A compounds of three acylation families ranging from tetra- to hexa-acylation. Moreover, C1-monophosphorylated lipid A species have been identified in P. aeruginosa for the first time.

A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue

Despite its critical role in neurodevelopment and brain function, vitamin D (vit-D) homeostasis, metabolism, and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive, and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely 1,25(OH)2D3, 3-epi-1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 24,25(OH)2D2. Chromatographic separation was achieved on a pentaflurophenyl column with 3 mM ammonium formate water/methanol [A] and 3 mM ammonium formate methanol/isopropanol [B] mobile phase components. Detection was by positive ion electrospray tandem mass spectrometry with the EVOQ elite triple quadrupole mass spectrometer with an Advance ultra-high-performance liquid chromatograph and online extraction system. Calibration standards of each metabolite prepared in brain matrices were used to validate the detection range, precision, accuracy, and recovery. Isotopically labelled analogues, 1,25(OH)2D3-d3, 25(OH)D3-c5, and 24,25(OH)2D3-d6, served as the internal standards for the closest molecular-related metabolite in all measurements. Standards between 1 fg/mL and 10 ng/mL were injected with a resulting linear range between 0.001 and 1 ng, with an LLOD and LLOQ of 1 pg/mL and 12.5 pg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12–11.53% and 0.28–9.11%, respectively. Recovery in acetonitrile ranged between 99.09 and 106.92% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D2, 1,25(OH)D2, and 24,25(OH)2D2, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological and neuropsychiatric disorders, and neurodegeneration.Graphical

Kynurenine to tryptophan ratio as a biomarker of acute stress in fish

The aim of this study was to determine the kynurenine (KYN) to tryptophan (TRP) ratio (KTR) in fish tissue to assess its usefulness as a biomarker of acute stress. Laboratory held rainbow trout (Oncorhynchus mykiss) were subjected to an acute stressor and KYN, TRP and cortisol were measured in liver and brain tissues at 4- and 48-h post-stress. The analytical method used to determine our analytes was based on lyophilization, and liquid-solid extraction followed by isotope dilution high-performance liquid chromatography positive ion electrospray tandem mass spectrometry. The [KYN]/[TRP] ratio (KTR) was greater in fish liver and brain in the 48-h post-stress exposure group (n = 8) relative to controls (n = 8, p < 0.05); a similar increase was not observed in fish in the 4-h post-stress exposure group. Hepatic and brain cortisol levels were also elevated in fish from both stress-induced groups relative to their respective controls implying that cortisol responded more quickly to the stressful stimulus than KYN and TRP. Our results suggest that the KTR is a promising acute stress diagnostic biomarker in fish. Efforts are ongoing to assess whether the KTR can be used as a biomarker for chronic stress in fish exposed to aquatic contaminants and other environmental stressors and if similar assessments can be made on tissues collected via non-lethal approaches.

An ultra-sensitive UHPLC-MS/MS assay for the quantification of orally administered vancomycin in plasma

Though marketed for over half a century, little is known about the pharmacokinetics of oral vancomycin except that its bioavailability is low, thus making accurate determination of plasma concentrations difficult. To quantify plasma concentrations of vancomycin after oral administration, we developed an ultra-sensitive UHPLC-MS/MS assay and validated it according to FDA´s and EMA´s pertinent guidelines. A fast and simple protein precipitation method followed by short UHPLC chromatography was developed for extraction and separation of vancomycin from plasma. Quantification was performed via heated positive electrospray tandem mass spectrometry with multiple reaction monitoring using deuterated internal standard. The assay was linear in the calibrated concentration range of 0.05–100 ng/mL showing correlation coefficients >0.997. Intraday and interday accuracy showed coefficients of variation <12% at the lower limit of quantification (LLOQ) of 0.05 ng/mL and <6% in the calibrated range while corresponding values for precision were <13% and <8%, respectively. With its high sensitivity, the assay allows for the accurate quantification of therapeutic plasma concentrations in the therapeutic range (up to 100 μg/mL) in 1000-fold diluted samples with a sample volume decreased down to 1 μL. The UHPLC-MS/MS assay was successfully used for the determination of trough plasma concentrations of two patients with Clostridium difficile infection receiving oral vancomycin therapy and its performance was compared to a commercial immunoassay for concentrations close to its LLOQ.

A diethylpyrocarbonate-based derivatization method for the LC-MS/MS measurement of plasma arginine and its chemically related metabolites and analogs

BackgroundChanges in NO metabolism correlate with cardiovascular risk factors and are associated with endothelial dysfunction. NO availability is regulated by nitric oxide synthase (NOS) and arginine and some chemically related metabolites and analogs have the capacity to alter NOS activity. Hence the need for analytical methods for the simultaneous assessment of these analytes. MethodsAnalytes (L-arginine (Arg), NG-monomethyl-L-arginine (MMA), L-homoarginine (hArg), asymmetric dimethyl-L-arginine (ADMA), symmetric dimethyl-L-arginine (SDMA), and L-citrulline (CIT)) were isolated from human plasma by thermal coagulation of plasma followed by a derivatization with diethylpyrocarbonate. Carbetoxy derivatives were separated on a C18 reversed-phase column in <10 min using an aqueous solution of 0.4% v/v formic acid and acetonitrile (95:5, v/v) mixture as a mobile phase. Positive electrospray ionization and tandem mass spectrometry in combination with specific multiple reaction monitoring transitions were used for detection of analytes and three deuterated forms of the analytes used as internal standards. ResultsIntra- and inter-day precision %RSD values ranged between 3 and 5.5% and percentage recoveries were close to 100% for all analytes. Plasma concentrations in 20 healthy male volunteers were 58.62 ± 8.81 μmol/L for Arg, 105.08 ± 21.66 nmol/L for MMA, 1.88 ± 0.57 μmol/L for hArg, 0.612 ± 0.140 μmol/L for ADMA, 0.581 ± 0.172 μmol/L for SDMA, and 28.62 ± 11.60 μmol/L for Cit, respectively. ConclusionThis LC–MS/MS method provides the capacity to quantify the plasma concentrations of arginine and some of its chemically related metabolites. Sample preparation was simple, inexpensive and effortless. Overall, given the short sample preparation and chromatographic run time, the method may be suitable for the fast and reproducible quantitative determination of the analytes in large clinical trials and routine analysis.

Overcoming the stability, solubility and extraction challenges in reversed-phase UHPLC–MS/MS bioanalysis of a phosphate drug and its prodrug in blood lysate

BMS-986104 is a S1P1R modulator drug candidate under development and has been evaluated in Phase I clinical trials. BMS-986104 functions as a prodrug and undergoes enzymatic transformations in vivo to form the pharmacologically active phosphate drug, BMS-986104-P. Here, we report approaches to overcome the stability, solubility and extraction challenges in developing a sensitive, accurate and rugged LC–MS/MS method for the simultaneous quantification of the phosphate drug and its prodrug in blood lysate. An effective stabilization strategy using a cocktail of phosphatase and kinase inhibitors was developed to ensure the stability of both analytes during sample collection, storage, and processing. A combination of surfactant (CHAPS) and weak base (Tris) was found to be able to effectively improve the solubilization of the phosphate drug. The blood lysate samples were extracted by protein precipitation followed by solid-phase extraction using an Oasis HLB 96-well SPE plate. The method achieved acceptable matrix effect and recovery for the two analytes that have very different chemical properties. Stable-isotope labeled D6-BMS-986104 and D13-BMS-986104-P were utilized as the internal standards. Chromatographic separation was achieved using isocratic conditions on an Acquity UPLC BEH C18 analytical column. The two analytes and their internal standards were detected by positive ion electrospray tandem mass spectrometry. The calibration curves, which ranged from 2.00 to 2000 ng/mL for BMS-986104 and 4.00 to 4000 ng/mL for BMS-986104-P, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision was within ±5.0% CV, inter-assay precision was within ±4.9% CV, and the assay accuracy was within ±5.8% of the nominal values for both analytes in rat blood lysate. The method was validated and successfully applied to support multiple pre-clinical toxicity studies.

Development of a Novel, Sensitive, Selective, and Fast Methodology to Determine Malondialdehyde in Leaves of Melon Plants by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Early production of melon plant (Cucumis melo) is carried out using tunnels structures, where extreme temperatures lead to high reactive oxygen species production and, hence, oxidative stress. Malondialdehyde (MDA) is a recognized biomarker of the advanced oxidative status in a biological system. Thus a reliable, sensitive, simple, selective, and rapid separative strategy based on ultra-high-performance liquid chromatography coupled to positive electrospray-tandem mass spectrometry (UPLC-(+)ESI-MS/MS) was developed for the first time to measure MDA, without derivatization, in leaves of melon plants exposed to stress conditions. The detection and quantitation limits were 0.02 μg·L−1 and 0.08 μg·L−1, respectively, which was demonstrated to be better than the methodologies currently reported in the literature. The accuracy values were between 96% and 104%. The precision intraday and interday values were 2.7% and 3.8%, respectively. The optimized methodology was applied to monitoring of changes in MDA levels between control and exposed to thermal stress conditions melon leaves samples. Important preliminary conclusions were obtained. Besides, a comparison between MDA levels in melon leaves quantified by the proposed method and the traditional thiobarbituric acid reactive species (TBARS) approach was undertaken. The MDA determination by TBARS could lead to unrealistic conclusions regarding the oxidative stress status in plants.

Quantitative analysis of N-acylphosphatidylethanolamine molecular species in rat brain using solid-phase extraction combined with reversed-phase chromatography and tandem mass spectrometry.

A novel method for the sensitive and selective identification and quantification of N‐acylphosphatidylethanolamine molecular species was developed. Samples were prepared using a combination of liquid–liquid and solid‐phase extraction, and intact N‐acylphosphatidylethanolamine species were determined by reversed‐phase high‐performance liquid chromatography coupled to positive electrospray tandem mass spectrometry. As a result of their biological functions as precursors for N‐acylethanolamines and as signaling molecules, tissue concentrations of N‐acylphosphatidylethanolamines are very low, and their analysis is additionally hindered by the vast excess of other sample components. Our sample preparation methods are able to selectively separate the analytes of interest from any expected biological interferences. Finally, the highest selectivity is achieved by coupling chromatographic separation and two N‐acyl chain specific selected reaction monitoring scans per analyte, enabling identification of both the N‐acyl chain and the phosphatidylethanolamine moiety. The validated method is suitable for the reliable quantification of N‐acylphosphatidylethanolamine species from rat brain with a lower limit of quantification of 10 pmol/g and a linear range up to 2300 pmol/g. In total, 41 N‐acylphosphatidylethanolamine molecular species with six different N‐acyl chains, amounting to a total concentration of 3 nmol/g, were quantified.

Development, validation and application of a comprehensive stereoselective LC/MS–MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine

A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion β-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by β-glucuronidase. Hydroxybupropion β-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete β-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R) were 10, 50, 100, and 100ng/mL, respectively. The limits of quantification for (1R,2R)-threohydrobupropion β-d-glucuronide, (1S,2S)-threohydrobupropion β-d-glucuronide, and (1R,2R)-erythrohydrobupropion β-d-glucuronide were each 50ng/mL. Due to the abundance of bupropion and metabolites in human urine, no efforts were made to optimize sensitivity. All analytes were stable following freeze thaw cycles at -80°C. This assay was applicable to clinical pharmacokinetic investigations of bupropion in patients and to in vitro metabolism of the primary bupropion metabolites to their glucuronides.

A validated enantioselective LC-MS/MS assay for quantification of a major chiral metabolite of an achiral 11-β-hydroxysteroid-dehydrogenase 1 inhibitor in human plasma: Application to a clinical pharmacokinetic study.

BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid-liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC-MS/MS assay was validated over a concentration range of 0.200-200ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days at -20°C and -70°C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.

Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-min run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1ng/mL, respectively. All analytes were stable following freeze thaw cycles at -80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM01183 (lurbinectedin), a novel antineoplastic agent, in mouse, rat, dog, Cynomolgus monkey and mini-pig plasma

Lurbinectedin (PM01183) is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected sequences in the minor groove of DNA, inducing PM01183-DNA adducts that stall replication, DNA repair and transcription and gives rise to double-strand breaks and finally, caspase-dependent apoptotic cell death. PM01183 has demonstrated clinical antitumor activity in platinum resistant/refractory ovarian cancer patients.A rapid and sensitive liquid chromatography/tandem mass spectrometry assay was developed and validated to quantify PM01183 in plasma from nonclinical species. The bioanalysis consisted of a supported liquid extraction, followed by a gradient phase chromatography and, detection by positive ion electrospray tandem mass spectrometry. The calibration range for PM01183 was established using PM01183 standards from 0.1 to 100ng/mL in blank plasma. The multiple reaction monitoring, based on the transition m/z 767.3→273.0, was specific for PM01183, and that based on the transition m/z 771.4→277.0 was specific for the internal standard (deuterated PM01183).No endogenous material interfered with the analysis of PM01183 and the internal standard from blank plasma. The limit of detection (LOD) of the assay was calculated as 0.025ng/mL. The correlation coefficients for the calibration curves ranged from 0.9937 to 0.9987. The mean inter-day accuracies for all calibration standards ranged from 92 to 108% (≤8% bias), and the mean inter-day precision for calibration standards was always less than 12%. The mean intra and inter-day assay accuracy for all quality control replicates remained between 91 and 109%. The mean intra and inter-day assay precision was less than 10% for all QC levels.The method was validated to demonstrate the specificity, recovery, limit of quantification, accuracy and precision of measurements. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies of PM01183 in nonclinical species. The main PK parameters in dogs (3 male and 3 female, respectively) were calculated as follows: maximum concentration (Cmax, 12.9±0.6 and 10.2±3.0ng/mL) and the area under the plasma concentration-time curve (AUC, 24.9±0.7 and 22.6±6.1ngh/mL). The results showed that plasma samples could be monitored for PM01183 for long enough to accurately estimate pharmacokinetics information.

Chemical derivatization of neurosteroids for their trace determination in sea lamprey by UPLC-MS/MS

This article describes the development and validation of a sensitive and robust method for the determination of neurosteroids in sea lamprey, an ancestral animal in vertebrate evolution. Chemical derivatization was used to enhance the detection of neurosteroids containing ketone function by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Iminooxy derivatives of 12 oxosteroids and three internal standards were monitored by positive electrospray tandem mass spectrometry using the neutral loss of sulfate. Limit of quantification, extraction recovery and matrix effect were first evaluated and SPE using C18 sorbent was selected after comparison with liquid/liquid extraction and protein precipitation. Matrix effect ranged from 89.6% to 113.1% in plasma and from 79.8% to 100.0% in the brain. Recovery values ranged from 80.0% to 103.8% in plasma and from 86.3% to 107.9% in the brain. Chromatographic separation was achieved by reverse phase chromatography (2.1mm×100mm, 1.7µm particle size, C18) with a binary gradient between methanol and 0.1% formic acid in water. Limit of quantification ranged from 5 to 10pg/mL and was up to 80 times lower than those for non-derivatized steroids. Accuracy and precision parameters were determined and inter- and intra-day at three concentrations: 50, 500 and 5000pg/mL. This method was applied to analyze real samples. progesterone (P), pregnenolone (P5), 7-hydroxy-pregnenolpne (7P5), 17-hydroxy-pregnenolpne (17P5)dehydroepiandrosterone (DHEA), androstenedienone (A4), testosterone (T), dihydrotestosterone (DHT), allopregnanolone (THP), 11-hydroxy-androstenedienone (11A4) and 11-Deoxycortisol (S) were measured in sea lamprey brain and plasma matrixes.

A UHPLC-MS/MS bioanalytical assay for the determination of BMS-911543, a JAK2 inhibitor, in human plasma.

Herein we report a rapid, accurate and robust UHPLC-MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled (13)C4-BMS-911543 as the internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved within 2min on a Zorbax Extend-C18 column with an isocratic elution. BMS-911543 and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The assay range was from 1 to 500ng/mL, and the standard curve was fitted with 1/x(2) weighted linear regression. The intra-assay precision was within 5.0% CV and the inter-assay precision was within 2.6% CV. The inter-assay mean accuracy, expressed as percents of theoretical, was between 99.8% and 102.3%. The assay has high recovery (∼80%) and minimal matrix effect (0.95-1.00). BMS-911543 was stable in human plasma for at least 24h at room temperature, 90 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in clinical studies.

Determination of Pinaverium Bromide in Human Plasma by a Sensitive and Robust UPLC-MS-MS Method and Application to a Pharmacokinetic Study in Mexican Subjects.

A high-throughput ultra-performance liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS-MS) method was developed for the determination of pinaverium bromide in human plasma. Protein precipitation with acetonitrile was used to extract pinaverium and itraconazole (as internal standard) from 500 µL plasma samples. The chromatographic separation was achieved with an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm) using a mixture of acetonitrile-5 mM ammonium formate (80:20, v/v) as mobile phase. Isocratic elution at 0.3 mL/min was used. Detection was performed by positive ion electrospray tandem mass spectrometry on a XEVO TQ-S by multiple reaction monitoring mode. The mass transitions monitorized were as follows: m/z 511.2 → 230 for pinaverium bromide, and m/z 705.29 → 392.18 for the itraconazole. The method was validated over a concentration range of 12-12,000 pg/mL. The chromatographic method runtime is 2.5 min and was applied to characterize the pharmacokinetics of pinaverium bromide after the oral administration of 100 mg to healthy Mexican subjects.

Sewage-based epidemiology in monitoring the use of new psychoactive substances: Validation and application of an analytical method using LC-MS/MS.

Sewage-based epidemiology (SBE) employs the analysis of sewage to detect and quantify drug use within a community. While SBE has been applied repeatedly for the estimation of classical illicit drugs, only few studies investigated new psychoactive substances (NPS). These compounds mimic effects of illicit drugs by introducing slight modifications to chemical structures of controlled illicit drugs. We describe the optimization, validation, and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine (MPA), 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA). Sample preparation was performed using solid-phase extraction (SPE) with Oasis MCX cartridges. The LC separation was done with a HILIC (150 x 3 mm, 5 µm) column which ensured good resolution of the analytes with a total run time of 19 min. The lower limit of quantification (LLOQ) was between 0.5 and 5 ng/L for all compounds. The method was validated by evaluating the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recoveries and matrix effects. The method was applied on sewage samples collected from sewage treatment plants in Belgium and Switzerland in which all investigated compounds were detected, except MPA and PMA. Furthermore, a consistent presence of MXE has been observed in most of the sewage samples at levels higher than LLOQ.

A novel approach for concurrent quantitation of glutathione, glutathione disulfide, and 2‐hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide, using liquid chromatography–positive electrospray tandem mass spectrometry

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.

Collision-Induced Dissociation Analysis of Brevianamide A and C in Electrospray Ionization Mass Spectrometry

Brevianamides A and C are isomeric cyclic peptides with several reported biological activities, isolated from diverse microorganisms. Currently, there has been no previous investigation of brevianamide fragmentation utilizing electrospray ionization mass spectrometry (ESI-MS). In this work experiments were carried out in the positive mode using two different spectrometers (low and high resolution) with an ESI source. Computational chemistry studies helped identify the protonation sites based upon atomic charges, proton affinities and molecular orbitals, computed using the B3LYP/6-31++G (d,p) model. The data suggests that the presence of the allylic position of the lactamic N in brevianamides C governs its fragmentation pathways. Distinguishing between brevianamides A and C using positive ion electrospray tandem mass spectrometry (ESI(+)MS/MS) is made possible by the spectral difference of each isomer and offers an alternative to other spectroscopic techniques.

A validated LC-MS/MS method for the simultaneous determination of BMS-791325, a hepatitis C virus NS5B RNA polymerase inhibitor, and its metabolite in plasma.

BMS-791325 is a hepatitis C virus (HCV) non-structural protein 5B (NS5B) RNA polymerase inhibitor that is being developed for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of BMS-791325 and its metabolite, BMS-794712, in rat and dog plasma. This method utilized stable-isotope labeled [D6]-BMS-791325 and [13CD3]-BMS-794712 as internal standards. The samples were extracted using liquid-liquid extraction with n-butyl-chloride. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column (2.1mm×50mm, 3.0μm). Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curves, which ranged from 5.00 to 2000ng/mL for BMS-791325 and from 1.00 to 400ng/mL for BMS-794712, were fitted to a 1/x2 weighted linear regression model. For both species, the intra-assay precision was within ±4.3% CV, the inter-assay precision was within ±6.2% CV, and the assay accuracy was within ±10.8% of the nominal values for BMS-791325 and BMS-794712. The validated method was successfully applied to support pre-clinical toxicokinetic studies.

Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC-MS/MS assay.

A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (∼90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.