Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Quantitative analysis of two opioid peptides in plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

Quantitative analysis of two opioid peptides, DSLET [(d-Ser²)Leu-enkephalin-Thr⁶] and Met-enkephalin-Arg-Gly-Leu, was performed using microbore liquid chromatography interfaced to electrospray ionization tandem mass spectrometry. Validation of the methodology was demonstrated for each peptide in plasma. Quantitative analyses were performed through the use of a deuterium labelled peptide analog as an internal standard. Linearity was observed for the analysis of DSLET (5–1000 ng/ml) and Met-enkephalin-Arg-Gly-Leu (1–1000 ng/ml) in plasma with a limit of detection of 0.25 ng/ml for Met-enkephalin-Arg-Gly-Leu and 1.0 ng/ml for DSLET. In general, the observed concentrations showed good reproducibility with coefficients of variation of within 15%. In the concentration range studied, only 0.5 ml of plasma was required for optimal detection of Met-enkephalin-Arg-Gly-Leu and 0.25 ml for DSLET. Application of this method was demonstrated by studying the disposition of DSLET in a rat. DSLET administered to a rat exhibited a short half-life and a high clearance value.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.     

Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry

A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C₁₈ column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H]⁺ m/z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation (r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Determination of melatonin in Acyrthosiphon pisum aphids by liquid chromatography–tandem mass spectrometry

Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75–110%), and good repeatability, expressed as relative standard deviation (<10%). Limits of detection (LOD) and quantitation (LOQ) were 1 pg/mg and 5 pg/mg, respectively. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 200 times LOQ. The validated method was successfully applied to 26 aphid samples demonstrating its usefulness for melatonin determination in insects. This is -to our knowledge- the first identification of melatonin in aphids by LC–MS/MS.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μM range for GSH and 0.05–20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r² > 0.997, n = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n = 5) and inter-day (n = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography–electrospray ionization tandem mass spectrometry

Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53–7.09% and 0.40–2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Simultaneous quantification of venlafaxine and O-desmethylvenlafaxine in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine venlafaxine (VEN) and O-desmethylvenlafaxine (ODV) in human plasma. Sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The separation was carried out on an ACQUITY UPLC? BEH C₁₈ column with 10 mmol/L ammonium acetate and methanol as the mobile phase at a flow rate of 0.30 mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for VEN and ODV were both obtained in the concentration range of 0.200–200 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.200 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were less than 13% and the accuracy (relative error, R.E.) was within ±5.3% and ±3.6% for VEN and ODV. The method herein described was superior to previous methods in sensitivity and sample throughput and successfully applied to clinical pharmacokinetic study of venlafaxine sustained-release capsule in healthy male volunteers after oral administration.     

Investigations on the biodegradation of alkylpolyglucosides by means of liquid chromatography–electrospray mass spectrometry

The biodegradation of alkylpolyglucosides (APGs) was studied under the conditions of the OECD Screening Test with activated sludge as an inoculum. An influence of alkyl and sugar chain length on the biodegradation rate and a central scission pathway of the biodegradation were investigated. The liquid chromatography-electrospray mass spectrometry technique was used for alkylpolyglucoside analysis and for identification and semiquantitative determination of metabolites. It was found that APGs with a longer alkyl chain were biodegraded faster than those with a shorter one. However, a longer sugar chain caused slower biodegradation of APGs. The central scission pathway of biodegradation was also confirmed.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

Simultaneous determination of clarithromycin,rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to ?10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.     

Determination of methocarbamol concentration in human plasma by high performance liquid chromatography–tandem mass spectrometry

A simple and reproducible high performance liquid chromatography–tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150–12,000 ng/mL with good intraday assay and interday assay precision (CV% < 10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.     

Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC–MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.