Quantification of mephenytoin and its metabolites 4'-hydroxymephenytoin and nirvanol in human urine using a simple sample processing method

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method without the use of sample extraction was developed for the simultaneous quantification of urinary concentrations of mephenytoin, a standard phenotyping substrate for the cytochrome P450 enzyme CYP2C19, and its phase I metabolites 4'-hydroxymephenytoin and nirvanol. Fifty microL of urine were diluted with a buffered beta-glucuronidase solution and incubated at 37 degrees C for 6 h followed by addition of methanol, containing the internal standard 4'-methoxymephenytoin. The chromatographic separation was achieved using a 100 x 3 mm, 5 micro Thermo Electron Aquasil C18 column with a gradient flow, increasing the organic fraction (acetonitrile/methanol 50:50) of the mobile phase from 10 to 90%. Quantification by triple-stage mass spectrometry (TSQ Quantum, Thermo Electron) was accomplished by negative electrospray ionization in the selected reaction monitoring mode. Linearity was observed for all substances in the concentration range 15-10 000 ng/mL. The lower limit of quantification (LLOQ) was 20 ng/mL for 4'-hydroxymephenytoin and 30 ng/mL for nirvanol and mephenytoin, respectively. Intra- and inter-day inaccuracy did not exceed 9.5% for all substances from LLOQ to 10 000 ng/mL. Intra- and inter-day precision were in the range of 0.8-10.5%. The method was validated according to international ICH and FDA guidelines and successfully applied for phenotyping of Caucasian male volunteers who received an oral dose of 50 mg mephenytoin.     

液相色谱-电喷雾串联四极杆质谱法测定鸡肉中17种磺胺类药物的残留

建立了鸡肉中17种磺胺类药物残留量的液相色谱-电喷雾串联四极杆质谱测定方法。以稳定同位素13C6-磺胺二甲基嘧啶作为内标,采用多反应监测定量。样品经过匀浆、乙腈提取、正己烷脱脂、硅胶柱净化后进行液相色谱-质谱分析。采用Capcell Pak C8DD色谱柱,以均含0.2%甲酸的水和甲醇为流动相进行梯度洗脱。方法检出限为0.02~1 μg/kg,17种磺胺类药物的加标回收率为52.3%~124.9%（添加水平为1,5,10　μg/kg),相对标准偏差为1.0%~17.6%。     

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.     

A Rapid Sample Preparation Procedure Using MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine Using LC–MS/MS Analysis

Clinical diagnosis of tetrodotoxin (TTX) poisoning can be difficult because of the lack of characteristic morphological findings and a screening test, such as an immunoassay. Here, we present a fully validated method for the analysis of TTX in serum and urine. In this method, serum and urine samples were extracted using MonoSpin CBA or amide columns, followed by LC–MS/MS analysis. The TTX was eluted from the column by 0.1 mL of 10 % acetic acid solution, and was directly injected into LC–MS/MS. An Agilent 1200 HPLC system equipped with a HILIC separation column (Zorbax HILIC Plus 2.1 × 100 mm, 3.5 μm) was used for isocratic elution, with a mobile phase of 10 mM ammonium formate with formic acid (95:5, v/v), along with 5 mM trifluoroacetic acid and 2 % acetonitrile. TTX was detected with an Agilent 6410 mass spectrometer utilizing positive electrospray ionization and multiple reaction monitoring. Limits of quantification for serum and urine were established to be 1 and 0.5 ng mL^?1, respectively. Limits of detection for serum and urine were 0.5 and 0.25 ng mL^?1, respectively. Intra-day and inter-day precision varied from 1.5 to 8.5 %. The recovery was >86.5 % for both matrices. In this method, the sample preparation process prior to injection into the LC–MS/MS takes approximately 10–15 min, which reduces the extraction time to one-tenth of that of previous methods. The application of this method was further verified by analysis of biological materials from a patient suffering from puffer fish poisoning.     

Quantitative analysis of clindamycin in human plasma by liquid chromatography/electrospray ionisation tandem mass spectrometry using d1-N-ethylclindamycin as internal standard

A new method for the quantitative analysis of clindamycin in human plasma by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) is presented. Recently published methods possess a disadvantage because of their use of internal standards with extraction and ionisation properties different from those of clindamycin. To avoid these problems, d(1)-N-ethylclindamycin was synthesised for use as internal standard by N-demethylation and subsequent d(1)-N-ethylation. Plasma sample preparation was done by an easy and rapid liquid-liquid extraction using ethyl acetate. The method was validated in the expected concentration range for a pharmacokinetic study. Calibration graphs were linear within the range 0.05-3.2 microg/mL plasma. Intra-day precision was between 0.90% (2.8 microg/mL) and 3.25% (0.05 microg/mL), inter-day variability was found to be between 1.33% (0.7 microg/mL) and 2.60% (0.05 microg/mL). Inter-day accuracy showed deviations between 0.4% (0.05 microg/mL) and -4.8% (0.2 microg/mL). The method is simple and robust, and has been applied to the batch analysis of clindamycin during a pharmacokinetic study.     

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.     

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。     

Determination of cefuroxime in human serum or plasma by liquid chromatography with electrospray tandem mass spectrometry

Cefuroxime is a second-generation cephalosporin used against different kinds of bacterial infections. To be able to optimize the dosing it is necessary to characterize the pharmacokinetics of cefuroxime which requires a selective and sensitive analytical method for cefuroxime in plasma or serum. A new rapid liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using cefotaxime as internal standard, was developed for analysis of cefuroxime in human serum. The work-up procedure consisted of protein precipitation with acetonitrile/cefotaxime, and after centrifugation the supernatant was dissolved in mobile phase. The sample was injected on a SB-CN column and the detection was performed using tandem mass spectrometry (MS/MS). The limit of quantification was determined to 0.025 microg/mL. The method was linear in the range 0.025-50 microg/mL with a coefficient of correlation >0.999. The limit of quantification and intra-day variability were found to be the same for plasma samples, which indicates that the method is valid for serum as well as plasma samples.     

High-throughput determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine carbocysteine in human plasma was developed and fully validated. After methanol-induced protein precipitation of the plasma samples, carbocysteine was subjected to LC/MS/MS analysis using electrospray ionization (ESI). The MS system was operated in the selected ion monitoring (SRM) mode. Chromatographic separation was performed on a Hypurity C18 column (i.d. 2.1 mm x 50 mm, particle size 5 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.1-20 microg/mL for carbocysteine. The lower limit of quantification (LLOQ) of the method was 0.1 microg/mL for carbocysteine. The intra- and inter-day precision was less than 7% for all quality control samples at concentrations of 0.5, 2.0, and 10.0 microg/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for carbocysteine compared with methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used to a bioequivalence study of two tablet formulations of carbocysteine in healthy volunteers.     

LC–ESI–MS Determination of Flupentixol in Human Plasma

Flupentixol and an internal standard, loperamide were extracted from human plasma by liquid–liquid extraction and analyzed on a Thermo Hypersil HyPURITY C18 column, with 10 mM ammonium acetate–acetonitrile–methanol (26:62:12, v/v/v) as mobile phase, coupled with electrospray ionization mass spectrometry (ESI–MS). The protonated analyte was quantified by selected-ion monitoring (SIM) with a quadrupole mass spectrometer in a positive-ion mode. The calibration curve was linear (r = 0.9990) over the concentration range: 0.039–2.5 ng mL^?1. Intra-day and inter-day precision (RSD%) were less than 13.05%. The established method was successfully applied for the determination of pharmacokinetics of flupentixol in human plasma.     

Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies

A liquid chromatography/mass spectrometry method for simultaneous determination of paracetamol and dextropropoxyphene in human plasma is described. Paracetamol and dextropropoxyphene, together with their internal standards (tolbutamide and pyrroliphene), were extracted from 0.5 mL of plasma using solid-phase extraction. The chromatography was performed using a Thermo Hypersil APS-2 Amino column (250 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile and 0.4% glacial acetic acid in water (20:80). The total run time was 6 min for each sample. The triple-quadrupole mass spectrometer was operated in both positive (for detection of dextropropoxyphene and its IS pyrroliphene) and negative (for detection of paracetamol and its IS tolbutamide) modes using a polarity-switching technique. Multiple reaction monitoring was used for quantification. The method was linear over the concentration range of 0.1-20 microg/mL for paracetamol and 0.5-80 ng/mL for dextropropoxyphene. The intra- and inter-day precision were less than 10%, and the accuracy ranged from 92.2-110.9%. The lower limits of quantification were 0.1 microg/mL for paracetamol and 0.5 ng/mL for dextropropoxyphene. The present method provides a robust, fast and sensitive analytical tool for both paracetamol and dextropropoxyphene, and has been successfully applied to a clinical bioequivalence study in 14 subjects.     

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.     

Determination of zaltoprofen in human plasma by liquid chromatography with electrospray tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, we developed a method coupling liquid-liquid extraction (LLE) to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) to determine zaltoprofen levels in human plasma, using enalapril as internal standard (IS). The high sensitivity and specificity of MS/MS detection enabled the use of small plasma volumes (250 microL) and a simple LLE procedure. Furthermore, the short run-times (2 min) involved are compatible with the requirements of large-scale clinical studies. Ion acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions m/z 299.3 > 225.0 for zaltoprofen and m/z 377.4 > 234.2 for the IS enalapril. The limit of detection (LOD) was 0.01 microg/mL and the lower limit of quantitation (LLOQ) was 0.05 microg/mL. The devised method was linear over the studied range (0.05-20 microg/mL), with r2 > 0.99 and a run-time of 2 min. Intra-day precisions fell in the range 2.0-13.8%, inter-day precisions in the range 2.1-3.9%, and intra- and inter-day accuracies in the range 102.8-114.1%. The described method provides a fast and sensitive analytical tool for zaltoprofen and was successfully applied to a 24-subject pharmacokinetic study.     

Determination of four nitroimidazoles in poultry and swine muscle and eggs by liquid chromatography/tandem mass spectrometry

A rapid method is presented for the determination of 4 nitroimidazoles in poultry and swine muscle and eggs by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). Samples were extracted with acetonitrile, then evaporated the organic phase. After filtration, the extract was directly injected into the LC/ESI-MS/MS system. The LC separation was made on a C8 column by applying a gradient composed of water and acetonitrile. Overall average recoveries ranged from 50-86% for egg, 66-115% for poultry muscle, and 79-111% for swine muscle. The decision limits ranged from 0.05-0.25 microg/kg for egg, 0.07-0.27 microg/kg for poultry muscle, and 0.07-0.26 microg/kg for swine muscle; and detection capabilities ranged from 0.51-0.68 microg/kg for egg, 0.41-0.75 microg/kg for poultry muscle, and 0.53-0.78 microg/kg for swine muscle.     

Determination of glufosfamide in rat plasma by liquid chromatography/tandem mass spectrometry

A sensitive and selective high-performance analytical method based on liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed for the quantification of glufosfamide in rat plasma. Zidovudine was employed as internal standard. Glufosfamide was determined after methanol-mediated plasma protein precipitation using LC/MS/MS with an electrospray ionization interface in negative ion mode. Two sets of standard curves were developed, from 0.005 to 1.0 microg/mL and from 1.0 to 50.0 microg/mL. The assay was accurate (% deviations from nominal concentrations < 5%), precise and reproducible (intra- and inter-day coefficients of variation < 10%). Glufosfamide in rat plasma was stable over three freeze/thaw cycles, and at ambient temperatures, for at least 2 h. The validated method was successfully applied to the determination of glufosfamide plasma concentrations in rats for 24 h following an intravenous administration of 25 mg/kg.     

Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionisation

A new method for simultaneous determination of amiloride and hydrochlorothiazide by liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) operated in positive and negative ionization switching mode was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation. The analytes were separated on a Phenomenex Curosil-PFP (250x4.6 mm, 5 microm) column by a gradient elution with a mobile phase consisting of 0.15% formic acid solution containing 0.23% ammonium acetate and methanol pumped at a flow rate of 1.0 mL.min(-1). Rizatriptan was used as the internal standard (IS) for quantification. The determination was carried out on a Waters Quattro-micro triple-quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode using the following transitions monitored simultaneously: positive m/z 230-->171 for amiloride, m/z 270-->158 for rizatriptan, and negative m/z 296-->205 for hydrochlorothiazide. The lower limits of quantification (LLOQs) were 0.1 and 1.0 ng.mL(-1) for amiloride and hydrochlorothiazide, respectively, which were lower than other published methods by using ultraviolet (UV), fluorimetric or mass spectrometric detection. The intra- and inter-day precision and accuracy were studied at three different concentration levels and were always better than 15% (n=5). This simple and robust LC/MS/MS method was successfully applied to the pharmacokinetic study of compound amiloride and hydrochlorothiazide tablets in healthy male Chinese volunteers.     

Application of 1-alkylamines to a liquid chromatographic/turbo ionspray tandem mass spectrometric method for quantifying metabolites of a new bone anabolic agent,TAK-778, in human serum

We investigated the application of alkylamines, as additives to the mobile phase, to a quantification method for the metabolites, M-III and M-IV, of TAK-778, which is a new bone anabolic agent, in human serum using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Prior to setting up the analytical method, we found that 1-alkylamines co-existing with M-III and M-IV in the turbo ionsprayed solution formed 1-alkylammonium adduct molecules of these metabolites during the ionization process, and the abundance of the adduct ions was considerably higher than that of protonated molecules ([M + H](+)s) of these metabolites. Based on these findings, we investigated a variety of 1-alkylamines and their spiked concentrations in the mobile phase for LC/MS/MS analysis to obtain higher sensitivities for the quantification of these metabolites. After these examinations, we found that 1-hexylamine at a final concentration of 0.05 mmol l(-1) was the most suitable additive for the mobile phase, and set the selected reaction monitoring (SRM) ions for the 1-hexylammonium adduct molecule and [M + H](+), allowing about a fivefold gain in the SRM chromatographic peak compared with that without 1-hexylamine. The adduct ion was considered to be formed by interaction between the amino group of 1-hexylamine and the phosphoryl group of M-III and M-IV. The internal standard (I.S.) used was deuterated M-III for each metabolite. The analytes and I.S. were extracted with diethyl ether from serum samples at neutral pH and injected into the LC/MS/MS system with a turbo ionspray interface. The limit of quantification for both analytes was 0.5 ng ml(-1) when 0.1 ml of serum was used, and the calibration curves were linear in the range 0.5-100 ng ml(-1). The method was precise; the intra- and inter-day precisions of the method were not more than 5.6%. The accuracy of the method was good, with deviations between added and calculated concentrations of M-III and M-IV being typically within 16.6%. This method provided reliable pharmacokinetic data for M-III and M-IV after the intramuscular administration of TAK-778 sustained-release formulation in humans.     

Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of residues of penicillins and cephalosporins in milk has been developed. After a simple extraction with acetonitrile, the extract was directly injected into the LC/MS/MS system on a C(18) column. A gradient consisting of acetonitrile and water, each containing 0.1% formic acid, was applied. The abundant parent ions [M + H](+) produced by positive electrospray ionisation were selected for fragmentation with argon. For each compound at least one fragment was recorded with multiple reaction monitoring. The limits of detection ranged from 1.5 to 25 microg/kg and the limits of quantification ranged from 4 to 50 microg/kg. Recoveries were examined at three levels (MRL, 0.5 x MRL, 2 x MRL) and ranged from 57 to 88%. The coefficients of variation obtained for the repeatability experiments were in agreement with those specified by the Horwitz equation. Linearity was checked by injecting extracts of samples spiked with increasing amounts of the different standards ranging from 0 to 150 microg/kg. The advantage of this method over existing methods is the very simple sample pre-treatment which makes the method very suitable for routine analysis.     

Development of a liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of apicidin, a novel histone deacetylase inhibitor, in rat serum: application to a pharmacokinetic study

Apicidin, a fungal metabolite isolated from Fusarium pallidoroseum, is a cyclic tetrapeptide that exhibits potent anti-protozoal and anti-angiogenic activities. Although extensive studies have been recently conducted to examine the biological and pharmacological action, no information is available on the quantitative analysis of apicidin. To our knowledge, this study is the first to describe a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay method for the quantification of apicidin in rat serum. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, and precision. The multiple reaction monitoring was based on the transitions m/z 624.7 --> 84.3 and 372.1 --> 176.1 for apicidin and trazodone, respectively. The assay utilized a single liquid-liquid extraction and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL of rat serum. The assay was linear over a range from 0.5-1000 ng/mL, with correlation coefficients >0.9994. The mean intra- and inter-day assay accuracy ranged from 99.9-101.5% and 94.8-102.1%, respectively, and the mean intra- and inter-day precision was between 2.7-5.9% and 1.6-11.5%, respectively. The developed assay method was applied to a pharmacokinetic study after intravenous injection of apicidin in rats at a dose of 1 mg/kg.