Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Determination of metformin and other biguanides in forensic whole blood samples by hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry

Metformin is an anti-diabetic drug in the biguanide class which also includes phenformin and buformin. Because of the potential adverse effects of the biguanides, a reliable liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionization was developed for the quantification of the drugs in both live and post-mortem human whole blood. The blood proteins were precipitated by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by cation-exchange solid-phase extraction to eliminate ion suppression effects. The separation was performed by hydrophilic interaction liquid chromatography. Matrix-matched calibrants combined with isotope dilution of metformin were used for calibration. The detection limits were 0.01 mg/L for metformin and phenformin and the relative intra-laboratory reproducibility standard deviations were less than 6% at concentrations of 1-10 mg/L. The mean true recoveries were greater than 86%.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry

The interest in therapeutic drug monitoring has increased over the last few years. Inter‐ and intra‐patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield^TM C₁₈ column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of seven metabolites of oxyresveratrol in rat urine and bile using liquid chromatography/tandem mass spectrometry

Oxyresveratrol (trans‐2,4,3′,5′‐tetrahydroxystilbene) is a major compound isolated from Smilax china, a Chinese herbal medicine. The rat urine and bile samples were pretreated by solid‐phase extraction method after oral administration at a dose of 100 mg/kg of oxyresveratrol. Seven metabolites were identified by LC‐MS/MS method with electrospray ionization in negative ion mode. The results indicated that main metabolites of oxyresveratrol were monoglucuronided and monosulfated oxyresveratrol. Based on the results, the metabolic pathway of oxyresveratrol in rat urine and bile was proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry

N-acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a 'dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d(3)-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C(8) minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d(3)-NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 micromol/L with limit of quantification at 1 micromol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9-102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases.     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸

建立了液相色谱-电喷雾离子阱串联质谱(LC-ESI-IT-MS/MS)同时分析烟草中20种游离氨基酸的方法。烟草样品经萃取后过滤直接进样,无需进行衍生和固相萃取等其他前处理步骤。液相色谱采用HyPURITY C18反相色谱柱(200 mm×2.1 mm, 5 μm),采用1%(体积分数,下同)乙腈水溶液(含0.1%九氟戊酸)和90%乙腈水溶液(含0.1%九氟戊酸)为流动相进行梯度洗脱。结果表明,20种氨基酸的检出限(LOD)为0.01～0.05 μmol/L (S/N=3),线性相关系数均大于0.9977,峰面积测定的相对标准偏差(RSD)为0.78%～4.93%。该方法分析效率、灵敏度和选择性高,已成功应用于多种烟草样品中氨基酸的分析测定。     

Determination of urinary S-sulphocysteine, xanthine and hypoxanthine by liquid chromatography-electrospray tandem mass spectrometry

Molybdenum cofactor and isolated sulphite oxidase deficiencies are two related rare autosomal recessive diseases characterized by severe neurological abnormalities, dislocated lens and mental retardation. Determination of three biochemical markers S-sulphocysteine (SSC), xanthine (XAN) and hypoxanthine (HXAN) in urine is essential for a definitive diagnosis and identification of the exact defect. We developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of SSC, XAN and HXAN in urine. The analysis was carried out in the negative-ion selected-reaction monitoring mode. The turnaround time for the assay was 7 min. Linear calibration curves for the three biomarkers were obtained in the range of 12-480 micromol/L. The intra- and inter-day assay variations were <2.5%. Mean recoveries of SSC, XAN and HXAN added to urine at two significantly different concentrations were in the range 94.3-107.3%. At a normal SSC urine excretion value of 3.2 micromol/mmol creatinine, the signal-to-noise ratio was 337:1. This stable isotope dilution LC-MS/MS method is specific, rapid and simple, and provides definitive diagnosis for molybdenum cofactor and isolated sulphite oxidase deficiencies in very small volumes of urine. We have identified seven new cases of isolated sulphite oxidase deficiency from four Saudi families and one Sudanese family.     

Rapid and sensitive determination of acetylsalicylic acid and salicylic acid in plasma using liquid chromatography–tandem mass spectrometry: application to pharmacokinetic study

A simple and sensitive analytical method using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of acetylsalicylic acid (aspirin, ASA) and its major metabolite, salicylic acid (SA), in animal plasma has been developed and validated. Both ASA and SA in plasma samples containing potassium fluoride were extracted using acetonitrile (protein precipitation) with 0.1% formic acid in it. 6‐Methoxysalicylic acid was used as the internal standard (IS). The compounds were separated on a reversed‐phase column. The multiple reaction monitoring mode was used with ion transitions of m/z 178.9 → 136.8, 137.0 → 93.0 and 167.0 → 123.0 for ASA, SA and IS, respectively. The lower limits of quantification for ASA and SA were 3 and 30 ng/mL, respectively. The developed method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after p.o. and i.v. administration of 1 mg/kg to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of sialic acids in infant formula by liquid chromatography tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionization (LC-ESI-MS/MS) was developed for determination of N-acetylneuramic acid and N-glycolylneuramic acid in infant formula. Reconstituted samples were hydrolysed in dilute sulfuric acid and deproteinized with acetonitrile. The extract was analysed directly without further clean-up by hydrophilic interaction chromatography. The substances were detected in negative ion mode and matrix matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviation was better than 6% for both substances. An R2 of 0.985 was obtained by comparison with a classical colorimetric assay based on reaction with resorcinol. The developed method is expected to be applied for accurate routine analysis of infant formulas.     

Determination of penciclovir in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the determination of penciclovir in human plasma. The method involved simple, one‐step SPE procedure coupled with a C₁₈, 75 × 4.mm, 3µm column with a flow‐rate of 0.5 mL/min, and acyclovir was used as the internal standard. The Quattro Micro mass spectrometry was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Using 250 µL plasma, the methods were validated over the concentration range 52.555–6626.181 ng/mL, with a lower limit of quantification of 52.55 ng/mL. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid simultaneous determination of 14 sulfonamides in wastewater by liquid chromatography tandem mass spectrometry

This paper describes a rapid method for the determination of 14 kinds of sulfonamides (SAs) in wastewater using SPE, and LC-MS/MS with positive ESI (ESI(+)) and selected reaction monitoring (SRM) mode. The SPE was performed on an Oasis hydrophilic-lipophilic-balanced (HLB) cartridge. Chromatographic separation on a C18 column was achieved using a binary eluent containing methanol and water with 0.2% formic acid. Typical recoveries of the analytes ranged from 22.3 to 87.0% at a fortification level of 100 ng/L. The LODs in wastewater except sulfathiazole (3 ng/L) could be detected and quantified at levels as low as 1 ng/L. Finally, the method was applied to water from the municipal outlet and the aquaculture wastewater effluent. Sulfamethazine (SM(2)), sulfamethoxypyridazine (SMP), and sulfamethoxazole (SMZ) were most frequently found in wastewater in a concentration range between 1.2 and 31.7 ng/L.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.     

Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study

A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C₁₈ reversed‐phase column, eluted with mobile phase of acetonitrile–ammonium acetate (5 m m ; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi‐reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.