Deuterium-labeled Analogs Research Articles

Biological Evaluation of d-[18F]Fluoroalanine and d-[18F]Fluoroalanine-d3 as Positron Emission Tomography Imaging Tracers for Bacterial Infection.

d-Amino acids such as d-alanine are substrates for bacterial peptidoglycan biosynthesis and are selectively taken up by bacteria and not mammalian cells. Consequently, d-amino acid metabolism is an attractive target for antibiotic discovery and the development of bacteria-specific imaging agents. d-Fluoroalanine and the deuterium-labeled analogue fludalanine (MK641) were originally explored as antibiotics by Merck but failed in clinical trials due to unaccepted toxicity. Herein, we synthesized a fluorine-18 labeled d-fluoroalanine, d-3-[18F]fluoroalanine (d-[18F]FAla), and its deuterated analogue, d-3-[18F]fluoroalanine-d3 (d-[18F]FAla-d3), and evaluated their capability to image bacterial infection. Both d-[18F]FAla and d-[18F]FAla-d3 can accumulate up to 0.64-0.78% ID/cc in the infectious area at 15 min postinjection. Despite the reduction of in vivo defluorination not being observed for deuterated 18F-labeled d-fluoroalanine, these radiolabeled d-alanine analogues were able to differentiate bacterial infection from sterile inflammation in a soft-tissue model of S. aureus infection.

Synthetic and analytical routes to the L-amino acid conjugates of cis-OPDA and their identification and quantification in plants

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Transition-metal-free silylboronate-mediated cross-couplings of organic fluorides with amines

C–N bond cross-couplings are fundamental in the field of organic chemistry. Herein, silylboronate-mediated selective defluorinative cross-coupling of organic fluorides with secondary amines via a transition-metal-free strategy is disclosed. The cooperation of silylboronate and potassium tert-butoxide enables the room-temperature cross-coupling of C–F and N–H bonds, effectively avoiding the high barriers associated with thermally induced SN2 or SN1 amination. The significant advantage of this transformation is the selective activation of the C–F bond of the organic fluoride by silylboronate without affecting potentially cleavable C–O, C–Cl, heteroaryl C–H, or C–N bonds and CF3 groups. Tertiary amines with aromatic, heteroaromatic, and/or aliphatic groups were efficiently synthesized in a single step using electronically and sterically varying organic fluorides and N-alkylanilines or secondary amines. The protocol is extended to the late-stage syntheses of drug candidates, including their deuterium-labeled analogs.

Liquid chromatography-tandem mass spectrometry analysis of delamanid and its metabolite in human cerebrospinal fluid using protein precipitation and on-line solid-phase extraction

The penetration of the antituberculosis drug delamanid into the central nervous system is not established. The distribution of delamanid and its major metabolite, DM-6705, into the cerebrospinal fluid requires investigation. A liquid chromatography-tandem mass spectrometry method for the quantification of delamanid and DM-6705 in human cerebrospinal fluid was developed and validated. The calibration range for both analytes was 0.300 – 30.0 ng/mL. The deuterium-labelled analogue of delamanid (delamanid-d4) and OPC-14714 were used as internal standards for delamanid and DM-6705, respectively. Samples were processed by protein precipitation followed by on-line solid-phase extraction and high-performance liquid chromatography on an Agilent 1260 HPLC system. A Phenomenex Gemini-NX C18 (5.0 µm, 50 mm × 2.0 mm) analytical column was used for on-line solid-phase extraction, and a Waters Xterra MS C18 (5.0 µm, 100 mm × 2.1 mm) analytical column for chromatographic separation using gradient elution, at a flow rate of 300 µL/min. The total run time was 7.5 min. Analytes were detected by multiple reaction monitoring on an AB Sciex 5500 triple quadrupole mass spectrometer at unit mass resolution, with electrospray ionization in the positive mode. Accuracy and precision were assessed over three independent validation batches. Extraction recoveries were more than 98% and were consistent across the analytical range. Both analytes in CSF exhibited non-specific adsorption to polypropylene tubes. The method was used to analyse cerebrospinal fluid samples from patients with pulmonary tuberculosis in an exploratory pharmacokinetic study.

Use of a liquid chromatography-tandem mass spectrometry method to assess the concentration of epinephrine, norepinephrine, and phenylephrine stored in plastic syringes

Abstract Objectives There are concerns about the potency of epinephrine (EPI), norepinephrine (NE), and phenylephrine (PE) stored in syringes for later infusions in clinical care. The objective of our study was to optimize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentrations EPI, NE, and PE dissolved in normal saline and stored in 50 mL 3-part Becton Dickinson syringes. Methods Medications were diluted in normal saline to 80 μg/mL for EPI and NE, and 100 μg/mL for PE. The solutions were stored in syringes for 0 (fresh), 3, and 7 days in a medical refrigerator. United States Pharmacopeia grade EPI, NE, and PE and their deuterium-labeled analogs were used as calibration standards. Stored samples and standards were diluted and analyzed by LC-MS/MS operated in selected reaction monitoring mode. Results The calculated limit of quantification for EPI, NE and PE were well below the concentrations used in clinical practice. The coefficient of variation remained below 12 % for all samples. The standard linear calibration regressions for EPI, NE, and PE had r 2 values of between 0.96 and 0.98 (p < 0.001). EPI and NE stored in the refrigerator remained within 10 % of the of their initial concentrations at all time points. The concentration of PE in syringe decreased by 19.85 % at 3 days, with no further decrease at 7 days, compared to fresh PE. Conclusions The sample preparation steps and optimized LC-MS/MS method allowed simple and reliable measurements of EPI, NE, and PE.

Identification, characterization, synthesis of major metabolites biotransformed from vonoprazan fumarate

A first synthetic research concerning four observed metabolites and their deuterium-labeled analogues of reflux esophagitis drug vonoprazan fumarate is reported. Among which the synthetic methods of three metabolites M − I, M-III, M-IV-Sul, and four stable isotop labeled analogues M-I-d4, M-II-d4, M-III-d4, M-IV-Sul-d4 have not yet been reported before. The structures of these compounds were elucidated on the basis of MS and NMR spectroscopy.

Development and validation of a simple, selective, and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma

Remdesivir, formerly GS-5734, has recently become the first antiviral drug approved by the U.S. Food and Drug Administration (FDA) to treat COVID-19, the disease caused by SARS-CoV-2. Therapeutic dosing and pharmacokinetic studies require a simple, sensitive, and selective validated assay to quantify drug concentrations in clinical samples. Therefore, we developed a rapid and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma with its deuterium-labeled analog, remdesivir-2H5, as the internal standard. Chromatographic separation was achieved on a Phenomenex® Synergi™ HPLC Fusion-RP (100 × 2 mm, 4 μm) column by gradient elution. Excellent accuracy and precision (<5.2% within-run variations and.<9.8% between-run variations) were obtained over the range of 0.5–5000 ng/mL. The assay met the FDA Bioanalytical Guidelines for selectivity and specificity, and low inter-matrix lot variability (<2.7%) was observed for extraction efficiency (77%) and matrix effect (123%) studies. Further, stability tests showed that the analyte does not degrade under working conditions, nor during freezing and thawing processes.

Creatine transport and pathological changes in creatine transporter deficient mice.

The severe impact on brain function and lack of effective therapy for patients with creatine (Cr) transporter deficiency motivated the generation of three ubiquitous Slc6a8 deficient mice (-/y). While each mouse knock-out line has similar behavioral effects at 2 to 3 months of age, other features critical to the efficient use of these mice in drug discovery are unclear or lacking: the concentration of Cr in brain and heart differ widely between mouse lines, there are limited data on histopathologic changes, and no data on Cr uptake. Here, we determined survival, measured endogenous Cr and uptake of its deuterium-labeled analogue Cr-d3 using a liquid chromatography coupled with tandem mass spectrometry assay, and performed comprehensive histopathologic examination on the Slc6a8-/y mouse developed by Skelton et al. Our results show that Slc6a8-/y mice have widely varying organ-specific uptake of Cr-d3, significantly diminished growth with the exception of brain, progressive vacuolar myopathy, and markedly shortened lifespan.

Differential adsorption of analyte and deuterated analogue onto the rotor seal of the injector in high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Samples were injected onto the HPLC system via either a Valco C14W.1 internal sample injector or a Rheodyne 7125 injector, both with dissimilar Vespel rotor seals, and linked to a quadrupole time-of-flight (QTOF) mass spectrometer. Alternatively, samples were infused directly into the mass spectrometer via a syringe-driver. Electrospray ionisation in positive mode was used in both cases. Experiments demonstrated significant differential adsorption of the analyte (RAC inhibitor, NSC23766) and its tri-deuterated internal standard onto the Vespel rotor seal, with the latter seeming to be preferentially adsorbed. The deuterated analogue also showed lower electrospray sensitivity, as demonstrated by syringe infusion of a mixture of the compounds, compared with their separate infusion at the same concentration. This study has demonstrated the problem of differential adsorption onto HPLC Vespel rotor seals, and that the use of a stable-isotope-labelled analogue as internal standard does not guarantee the constancy of the analyte/internal response ratio in quantitative methods. The partial solution was to work at much higher concentration where adsorption, whilst still apparent, was relatively insignificant.

Flash vacuum pyrolysis of oxo-stabilised phosphonium ylides containing methoxythiophene and methylthiophene groups

Ten new oxo-stabilised phosphonium ylides containing substituted thiophene groups, as well as two deuterium-labelled analogues, are prepared and fully characterised. Upon flash vacuum pyrolysis at 800 °C, the simpler examples undergo extrusion of Ph3PO coupled with domino cyclisation to give thieno[3,2-b]furan and thieno[3,4-b]furan products but the corresponding approach to a thieno[2,3-b]furan fails. One ylide designed to give a thieno[3,4-b]furan instead gives phenylbutadiyne and 2-phenyl-3-vinylthiophene at 725 °C and the mechanism of this unusual process is elucidated by deuterium labelling. Attempts to access more complex heterocyclic products from extended methoxythienyl ylides failed. Two ylides bearing a 3-methyl-2-thienylacryloyl group gave 5-benzylbenzothiophene, 5-(α-methylbenzyl)benzothiophene and fluoreno[3,4-b]thiophene upon FVP at 800 °C and the mechanism was again elucidated by deuterium labelling.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort.A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8–250 ng mL−1 for free hydroxycholesterols, 0.2–10 ng mL−1 for dihydroxycholesterols, 0.2–500 ng mL−1 for bile acids and 16–2000 μg mL−1 for cholesterol with acceptable accuracy and precision.In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained.In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.

Synthetic Access to Noncanonical Strigolactones: Syntheses of Carlactonic Acid and Methyl Carlactonoate

Strigolactones are plant hormones regulating essential stages of a plant's development. Their low natural abundance combined with a low chemical stability significantly hampered the detailed investigation of their biological activity. Noncanonical strigolactones lack the fused tricyclic ABC-ring system commonly present in canonical-type strigolactones but feature an open-chain unit linking structurally diverse A-ring moieties to the butenolide D-ring. We herein present an efficient synthetic access to enantiomerically pure noncanonical strigolactones by a Stille cross-coupling approach to forge the central diene moiety and demonstrate this strategy by syntheses of natural products methyl carlactonoate and carlactonic acid. Furthermore, a synthetic access to deuterium-labeled analogues of these natural products has been developed.

A Book-Type Dried Plasma Spot Card for Automated Flow-Through Elution Coupled with Online SPE-LC-MS/MS Bioanalysis of Opioids and Stimulants in blood

Despite many benefits including simple point-of-care sample collection, reduced costs, and simplified shipping and storage, dried blood spot (DBS) techniques have faced adoption resistance due to factors such as the hematocrit (Hct) effects and the established preference for bioanalysis of plasma rather than whole blood. One way to potentially circumvent these challenges is to adopt the concept of dried plasma spot (DPS) techniques. One approach to accomplishing this is through an on-card red blood cell (RBC) filtration to generate plasma from whole blood without the need for centrifugation. In this report, a book-type DPS card has been developed and validated by employing fully automated flow-through elution coupled with online SPE-LC-ESI-MS/MS for the quantitative determination of four representative opioids (morphine, codeine, oxycodone, hydrocodone) and five stimulants (amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), phentermine, and mephedrone) in one method using their corresponding deuterium-labeled analogues as internal standards. Method validation results showed good linearity (R2 ≥ 0.9963) ranging from 5 to 1000 ng/mL. Intraday and interday precision and accuracy were within the acceptable limits at four quality control (QC) levels. Extraction recovery was ≥87.9% at both the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ) along with acceptable selectivity and sensitivity. DPS on-card short-term stability was compound-dependent and storage-dependent. The additional benefits of the validated book-type DPS card include a wider applicability range of Hct (30% to 60%), automated online analysis compatibility, and a higher plasma volume yield.

Microquantification of phospholipid classes by stable isotope dilution and nanoESI mass spectrometry.

A new method for microquantification of phospholipid classes by nanoelectrospray mass spectrometry and stable isotope dilution is presented. The method covers the sum of phosphatidylcholine and sphingomyelin and in addition selectively quantifies phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. A phospholipid class is quantified together with its corresponding lyso-species due to the presence of a common head group. Phospholipids are extracted from tissue lysates, hydrolysed by hydrofluoric acid, and the liberated polar head groups choline, ethanolamine, serine, and inositol are quantified by nanoelectrospray mass spectrometry using deuterium-labeled analogs of the head groups as internal standards. The method is applied to tissue samples of a gastrointestinal tumor and of corresponding non-affected control tissue. In the tumor sample, the abovementioned phospholipids were found at roughly threefold elevated concentrations with a virtually unaltered relative abundance profile.

Quantification of Synthetic Cathinones in Rat Brain Using HILIC-ESI-MS/MS.

The abuse of synthetic cathinones, formerly marketed as "bath salts", has emerged over the last decade. Three common drugs in this class include 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). An LC-MS/MS method has been developed and validated for the simultaneous quantification of MDPV, mephedrone, and methylone in brain tissue. Briefly, MDPV, mephedrone, methylone, and their deuterium-labeled analogs were subjected to solid phase extraction (SPE) and separated using an HILIC Silica Column. The HPLC was coupled to a Shimadzu IT-TOF (ion trap-time of flight) system with the electrospray source running in positive mode (+ESI). The method was validated for precision, accuracy, and extraction efficiency. All inter-day and intra-day % RSD (percent relative standard deviation) and % error values were less than 15% and extraction efficiency exceeded 80%. These conditions allowed for limits of detection of 1ng/mL for MDPV, and 5 ng/mL for both mephedrone and methylone. The limits of quantification were determined to be 5ng/mL for MDPV and 10 ng/mL for mephedrone and methylone. The method was utilized to evaluate the pharmacokinetics of these drugs in adult male rats following administration of a drug cocktail including MDPV, mephedrone, and methylone. All three compounds reached peak concentrations in the brain within 15 min. Although methylone and mephedrone were administered at the same dose, the peak concentration (Cmax) of mephedrone in the brain was significantly higher than that for methylone, as was the area under the curve (AUC). In summary, this quick and sensitive method for measuring synthetic cathinones may be used for future pharmacokinetic investigations of these drugs in target tissue.

Continuous-Flow Synthesis of Deuterium-Labeled Antidiabetic Chalcones: Studies towards the Selective Deuteration of the Alkynone Core.

Flow chemistry-based syntheses of deuterium-labeled analogs of important antidiabetic chalcones were achieved via highly controlled partial C≡C bond deuteration of the corresponding 1,3-diphenylalkynones. The benefits of a scalable continuous process in combination with on-demand electrolytic D2 gas generation were exploited to suppress undesired over-reactions and to maximize reaction rates simultaneously. The novel deuterium-containing chalcone derivatives may have interesting biological effects and improved metabolic properties as compared with the parent compounds.

Insight into the mechanism of the thermal reduction of graphite oxide: deuterium-labeled graphite oxide is the key.

For the past decade, researchers have been trying to understand the mechanism of the thermal reduction of graphite oxide. Because deuterium is widely used as a marker in various organic reactions, we wondered if deuterium-labeled graphite oxide could be the key to fully understand this mechanism. Graphite oxides were prepared by the Hofmann, Hummers, Staudenmaier, and Brodie methods, and a deuterium-labeled analogue was synthesized by the Hofmann method. All graphite oxides were analyzed not only using the traditional techniques but also by gas chromatography-mass spectrometry (GC-MS) during exfoliation in hydrogen and nitrogen atmospheres. GC-MS enabled us to compare differences between the chemical compositions of the organic exfoliation products formed during the thermal reduction of these graphite oxides. Nuclear analytical methods (Rutherford backscattering spectroscopy, elastic recoil detection analysis) were used to calculate the concentrations of light elements, including the ratio of hydrogen to deuterium. Combining all of these results we were able to determine graphite oxide's thermal reduction mechanism. Carbon dioxide, carbon monoxide, and water are formed from the thermal reduction of graphite oxide. This process is also accompanied by various radical reactions that lead to the formation of a large amount of carcinogenic volatile organic compounds, and this will have major safety implications for the mass production of graphene.

Mass spectral studies on 1-n-pentyl-3-(1-naphthoyl)indole (JWH-018), three deuterium-labeled analogues and the inverse isomer 1-naphthoyl-3-n-pentylindole.

A number of synthetic cannabinoids such as the 1-alkyl-3-acylindoles are the target of significant designer drug activity. One of the first waves of these compounds identified in clandestine samples was 1-n-pentyl-3-(1-naphthoyl)indole, JWH-018. These totally synthetic molecules can be prepared in a number of regioisomeric forms. The electron ionization mass spectrometric (EI-MS) fragmentation of the 1-n-pentyl-3-(1-naphthoyl)indole is compared to its inverse isomer 1-naphthoyl-3-n-pentylindole. These two substances are directly available from indole using identical precursor reagents and similar reaction conditions. Stable isotope deuterium labeling of the three major regions of the JWH-018 molecule allows confirmation of the structures of the major fragment ions. The spectra for the 1-n-pentyl-3-(1-naphthoyl)-d(5) -indole, 1-n-pentyl-3-(1-d(7) -naphthoyl)indole and 1-d(11) -n-pentyl-3-(1-naphthoyl)indole provide significant assistance in elucidating the structures for the major fragment ions in JWH-018. The EI mass spectra for these isomers show a number of unique ions which allow for the differentiation of the 1-alkyl-3-acylindole compounds from the inverse regioisomeric 1-acyl-3-alkylindoles. The fragment ion [M-17](+) at m/z 324 for JWH-018 was formed by the elimination of a hydroxyl radical and the spectra of the three deuterium-labeled derivatives indicated the loss of hydrogen from the naphthalene ring. Further structural analogues suggest the hydrogen to come from the 8-position of the naphthalene ring. The three deuterium-labeled analogues provide significant assistance in confirming the structures for the major fragment ions in the mass spectrum of the traditional synthetic cannabinoid compound, 1-n-pentyl-3-(1-naphthoyl)indole, JWH-018. The 1-naphthoyl-3-n-pentylindole inverse regioisomer can be easily differentiated from the traditional synthetic cannabinoid compound.

Hydrogen-deuterium exchange of α-carbon protons and fragmentation pathways in N-methylated glycine and alanine-containing peptides derivatized by quaternary ammonium salts.

Recently, we developed a selective and efficient method of hydrogen-deuterium exchange (HDX) at the α-carbon (α-C) of sarcosine residue (N-methylglycine) in model peptides [Bąchor et al. J. Mass Spectrom. 2014, 49, 43]. Here, we report the influence of quaternary ammonium (QA) group on HDX at the α-C of sarcosine and N-methylalanine in peptides. The obtained results suggest a significant acceleration of the HDX in sarcosine residue caused by the presence of QA. The effect depends on the distance between the sarcosine residue and QA moiety. The deuterons, introduced at α-C, are resistant to the back-exchange in acidic aqueous solution. The collision induced dissociation of the deuterium-labeled analogs of QA-tagged oligosarcosine peptides without mobile hydrogen revealed the mobilization of the hydrogens localized at α-C of sarcosine residue.

Simultaneous determination of glimepiride and pioglitazone in human plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study.

The rapid, sensitive, and selective liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) for the simultaneous estimation and pharmacokinetic investigation of glimepiride and pioglitazone in human plasma has been developed and fully validated. Glimepiride and pioglitazone, compounds which exert synergistic effects on blood glucose control, were investigated in human plasma using deuterium-labeled analogs as internal standards (IS). Liquid-liquid extraction was carried out on 0.2 mL of human plasma using ethyl acetate, and chromatographic separation was performed on an Agilent Eclipse plus C18 column (4.6 mm × 100 mm, 3.5 μm) using a mobile phase consisting of methanol-water-formic acid (95:5:0.1, v/v/v, plus 5mM ammonium acetate) at a flow rate of 0.8 mL/min. To quantify glimepiride, pioglitazone and their IS, multiple reaction monitoring (MRM) transitions of m/z 491.2→352.2, m/z 496.2→357.2, m/z 357.2→134.2 and m/z 361.2→138.2 were performed in positive mode. The total run time was 3.0 min and the elution time was about 2.4 min. The method exhibited good separation of analytes, without interference from endogenous substances. The linear calibration curves were 0.2-250 ng/mL for glimepiride and 0.2-1,250 ng/mL for pioglitazone; the lower limit of quantification (LLOQ) was 0.2 ng/mL for both analytes. Intra- and inter-day reproducibility was less than 10% for glimepiride and less than 5% for pioglitazone, with relative errors ranging from -8.00% to 2.80% at the three concentrations of analytes used for quality control (QC). The matrix effect was negligible and recoveries were similar for each analyte and its IS. Glimepiride and pioglitazone were found to be stable under the assay conditions and the method was successfully applied to the evaluation of pharmacokinetic studies of glimepiride and pioglitazone, following oral doses of 2mg glimepiride tablets and 15 mg pioglitazone tablets to 16 healthy volunteers.