LC-MS/MS法定量测定人血浆中的氯氮平

建立了人血浆中氯氮平的固相萃取及LC-MS/MS定量检测方法.血浆中的氯氮平用固相萃取柱(Bond Elut Certify)萃取,采用Waters AtlantisTMd C18色谱柱,电喷雾离子源,正离子检测,多反应监测方式进行定量分析,SKF525a作为内标.对氯氮平的定量下限为100 ng/mL,氯氮平质量浓度...     

Quantification of pseudoephedrine in human plasma by LC-MS/MS using mosapride as internal standard

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of pseudoephedrine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple-reaction monitoring mode using the respective [M + H](+) ions, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 2-1000 ng/mL pseudoephedrine in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 9% for pseudoephedrine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Profiling eicosanoids and phospholipids using LC-MS/MS: principles and recent applications

Eicosanoids are potent lipid mediators involved in numerous physiological and pathophysiological processes. Precursors are polyunsaturated fatty acids liberated from membrane phospholipids. Thus, profiling and quantification of these molecules has gained a lot of attention during last years. Eicosanoids and phospholipids are commonly profiled by LC-MS/MSbecause this technique allows accurate quantification within acceptable run-times. This article therefore focuses on liquid chromatography and the ESI-MS/MS analysis of proinflammatory lipid mediators, particularly arachidonic acid (C20:4) derived eicosanoids and their precursors phospholipids. Recent analytical developments for quantification of these compounds are highlighted and analytical challenges are discussed. Furthermore, applications such as the use of these molecules as biomarkers are presented.     

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid–acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.     

A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum

We report here a novel approach for the extraction and analysis of thyroid hormones (TH) and their metabolites (THM) from human serum samples. Our method features a compact, 96-well micro-titre plate-based pre-analytic extraction/clean-up workflow combined with an isotope dilution LC-MS/MS-MS³ analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 10⁴, with potential to semi-automate the pre-analytics. The method was validated by the assessment of linearity, lower limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves were linear in the concentration range in sample matrix from 0.1–250 nM for T₃, rT₃, T₄ and 3-T₁AM and from 0.005–1 nM for 3,5-T₂ and 3,3′-T₂. Using a 200-μL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T₂) to 0.25 (T₄) nM and LLOD were between 0.002 (3,5-T₂) and 0.052 nM (T₄). We applied the LC-MS/MS-MS³ method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T₄, T₃ and rT₃ concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3′-T₂ concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T₂ concentrations were below the LLOQ and/or LLOD in all but a single sample (0.013 nM). This method expands the analytical spectrum to endogenous thyroid hormone metabolites such as 3,5-T₂ which exert biological actions and rT₃ which may act as surrogate markers for disturbed thyroid hormone metabolism.

Graphical abstract

     

LC-MS/MS法快速测定吡罗昔康制剂中吡罗昔康含量

建立了快速液相色谱-质谱/质谱联用法测定吡罗昔康制剂中吡罗昔康含量的方法。样品以0.1 mol/L盐酸甲醇溶液提取、微孔滤膜过滤、离心后,通过电喷雾离子化(ESI),采用多反应检测(MRM)方式进行正离子检测,用于定量分析的检测离子为m/z 332.2→94.8。采用Shim-pack XR-ODS(3.0 mm×75mm,2.0μm)柱分离,以乙腈-水-甲酸(60:40:0.1,V/V/V)为流动相,流速为0.40 mL/min,在3 min内完成吡罗昔康定量分析。线性范围为2.5～1000.0ng/mL,最低检测限为2.5 ng/mL;日内测定的相对标准偏差小于3.2%,日间测定的相对标准偏差小于3.8%。方法可作为吡罗昔康制剂的质量中吡罗昔康控制方法,并可用于少量血浆样品的测定,也适用于药物代谢动力学研究。     

Simultaneous determination of sulfoxaflor in 14 daily foods using LC-MS/MS

Sulfoxa?or residues in 14 daily foods, including rice, sorghum, chilli, cucumber, white pear, apple, egg, beef brisket, chicken breast, fish, pork liver, milk, pine nut and honey, were simultaneously determined using a modified QuEChERS and LC–MS/MS method. These foods were classified into three categories to be purified. A combination of 25 mg of octadecylsilane (C18) + 25 mg of primary and secondary amine (PSA) + 50 mg of graphitised carbon black (GCB) + 150 mg of MgSO₄ was used to purify the rice, sorghum, honey, apple and white pear. A combination of 25 mg of C18 + 50 mg of PSA + 50 mg of GCB + 150 mg of MgSO₄ was used to purify the chilli and cucumber. A combination of 50 mg of C18 + 25 mg of PSA + 50 mg of GCB + 150 mg of MgSO₄ was used to purify the pine nuts, egg, beef brisket, chicken breast, fish, pork liver and milk. The linearity coefficient values were greater than 0.9975. The limit of detection and limit of quantification were in the ranges of 0.7?1.8 and 2.0?5.0 μg kg^?1, respectively. Average recoveries of the sulfoxa?or at the 14 food matrices at spiking levels of 5.0, 10 and 50 μg kg^?1 ranged from 74.0% to 100.8%, and the relative standard deviation ranged from 2.2% to 11.2%. This is a simple and rapid method for the determination of sulfoxa?or residues in various kinds of daily foods.     

Simultaneous determination of 32 antibiotics in aquaculture products using LC-MS/MS

An analytical multiclass, multi-residue method for the determination of antibiotics in aquaculture products was developed and validated. A fast, cheap, and straightforward extraction procedure followed by liquid chromatography-tandem mass spectrometry analysis was proposed. This method covers 32 antibiotics of different classes, which are frequently used in aquaculture. Three different extraction procedures were compared, and the extraction with acetonitrile (0.1 vol. % formic acid) showed the best results. The selected extraction procedure was validated at four different fortification levels (10 μg kg^?1, 25 μg kg^?1, 50 μg kg^?1, and 100 μg kg^?1). Recoveries of the tested antibiotics ranged from 70 % to 120 %, with the relative standard deviation (RSD) of triplicates lower than 20 %. The limits of quantification (LOQ) ranged from 0.062 μg kg^?1 to 4.6 μg kg^?1, allowing for the analysis of trace levels of these antibiotics in aquaculture products. The method was applied to the analysis of selected antibiotics in fish and shrimp meat available in the Czech market.     

Development and validation of LC-MS/MS method for the quantification of oxcarbazepine in human plasma using an experimental design

A rapid tandem mass spectrometric (MS-MS) method for the quantification of Oxcarbazepine (OXB) in human plasma using imipramine as an internal standard (IS) has been developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 3.0 min, using a mobile phase of acetonitrile-10 mM ammonium formate (90 : 10 v/v) at a flow rate of 0.3 ml/min. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 253>208 and m/z 281>86 for OXB and the IS respectively. Calibration curves were linear over the concentration range of 0.2-16 mug/ml (r>0.999) with a limit of quantification of 0.2 mug/ml. Analytical recoveries of OXB from spiked human plasma were in the range of 74.9 to 76.3%. Plackett-Burman design was applied for screening of chromatographic and mass spectrometric factors; factorial design was applied for optimization of essential factors for the robustness study. A linear model was postulated and a 2(3) full factorial design was employed to estimate the model coefficients for intermediate precision. More specifically, experimental design helps the researcher to verify if changes in factor values produce a statistically significant variation of the observed response. The strategy is most effective if statistical design is used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic and bioequivalence studies.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.     

Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS

Analytical and Bioanalytical Chemistry - DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS...     

Rapid analysis of the nitrification inhibitor 3,4-dimethylpyrazole phosphate in soil using LC-MS/MS

Nitrate from the biological nitrification of ammonium fertilisers causes environmental damage via groundwater contamination and nitrous oxide emission. To limit nitrate formation, nitrification inhibitors (NIs) are used in conjunction with ammonium-based fertilisers in agricultural land management. The NI 3,4-dimethyl-1H-pyrazole phosphate (DMPP), with an active constituent 3,4-dimethyl-1H-pyrazole (3,4-DMP), is commercially available and its effectiveness and behaviour in soils have been studied. However, only one method for the analysis of 3,4-DMP in soil has been reported and relies on extensive sample preparation to remove matrix interferences prior to HPLC analysis. A new method was developed to allow monitoring of 3,4-DMP residues in soil after appliaction, which utilises the greater selectivity and sensitivity of LC-MS/MS. A 3,4-DMP limit of quantitation of 0.5 ng/g was achieved, which is 10 times more sensitive than the published method, and was achieved using 10,000 times less 3,4-DMP injected on-column, with an injection volume 100 times smaller. Four internal standards were evaluated to improve the accuracy of the extraction method. The isotope-substituted structural isomer 3,5-dimethyl pyrazole-¹⁵N₂ provided the best and most consistent recoveries over the 300-fold concentration range tested. The new method was employed to investigate the persistence and mobility of 3,4-DMP in an agricultural soil. 3,4-DMP had a half-life of 5 days in the top 0.5 cm of soil at normal and double recommended application rates, while half-lives in the 2.5 cm soil profile were 28 and 21 days, respectively. 3,4-DMP mobility in the clay loam soil tested was low, with only 15–25% of applied 3,4-DMP detected below the top 0.5 cm, suggesting the loss of 3,4-DMP was either due to volatilisation or degradation, rather than leaching into the soil profile.     

液相色谱-串联质谱法测定食品中甲基磺草酮

食品用乙腈-水(3+1)溶液进行提取,经凝胶色谱、固相萃取柱净化后,用液相色谱-串联质谱法进行测定和确证,外标法定量。色谱分离用甲醇和甲酸-水(0.1+99.9)溶液以不同体积比混合为流动相梯度洗脱,采用负离子模式电喷雾离子源在多反应监测模式下进行检测。甲基磺草酮的质量浓度在0.01~0.2 mg·L-1范围内与其峰面积呈线性关系。以4种食品样品为基体,加入3种浓度水平的甲基磺草酮标准做回收试验,测得回收率在73.2%~100.6%之间;测定值的相对标准偏差(n=10)在4.1%~11%之间。     

An integrated strategy for identification and relative quantification of site-specific protein phosphorylation using liquid chromatography coupled to MS2/MS3

Reversible and differential multisite protein phosphorylation is an important mechanism controlling the activity of cellular proteins. Here we describe a robust and highly selective approach for the identification and relative quantification of site-specific phosphorylation events. This integrated strategy has three major parts: visualisation of phosphorylated proteins using fluorescently stained polyacrylamide gels, determination of the phosphorylation site(s) using automatic MS3 triggered by the loss of phosphoric acid, and relative quantification of phosphorylation by integrating MS2- and MS3-extracted ion traces using a fast-scanning, linear ion trap mass spectrometer. As a test case, recombinant sucrose-phosphate synthase (SPS) from Arabidopsis thaliana (At5g1110) was used for identification and quantification of site-specific phosphorylation. The identified phosphorylation site of the actively expressed protein coincides with the major regulatory in vivo phosphorylation site in spinach SPS. Site-specific differential in vitro phosphorylation of native protein was demonstrated after incubation of the recombinant protein with cold-adapted plant leaf extracts from A. thaliana, suggesting regulatory phosphorylation events of this key enzyme under stress response.     

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.     

Measurement of quetiapine and four quetiapine metabolites in human plasma by LC-MS/MS

There is interest in monitoring plasma concentrations of N‐desalkylquetiapine in relation to antidepressant effect. A simple LC‐MS/MS method for quetiapine and four metabolites in human plasma (50 μL) has been developed to measure concentrations of these compounds attained during therapy. Analytes and internal standard (quetiapine‐d8) were extracted into butyl acetate–butanol (10:1, v/v) and a portion of the extract analysed by LC‐MS/MS (100 × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow‐rate 0.5 mL/min; positive ion APCI‐SRM, two transitions per analyte). Assay calibration (human plasma calibrators) was linear across the ranges studied (quetiapine and N‐desalkylquetiapine 5–800, quetiapine sulfoxide 100–15,000, others 2–100 µg/L). Assay validation was as per FDA guidelines. Quetiapine sulfone was found to be unstable and to degrade to quetiapine sulfoxide. In 47 plasma samples from patients prescribed quetiapine (prescribed dose 200–950 mg/day), the (median, range) concentrations found (µg/L) were: quetiapine 83 (7–748), N‐desalkylquetiapine, 127 (7–329), O‐desalkylquetiapine 12 (2–37), 7‐hydroxyquetiapine 3 (<1–48), and quetiapine sulfoxide 3,379 (343–21,704). The analyte concentrations found were comparable to those reported by others except that the concentrations of the sulfoxide were markedly higher. The reason for this discrepancy in unclear. 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.