Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry

d ‐Lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased d ‐lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects d ‐lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect d ‐lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of l ‐ and d ‐lactic acid by a two‐step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high‐performance liquid chromatography–tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic? R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2–400 and 0.5–100 µmol/L respectively for l ‐ and d ‐lactic acid. The limit of detection of d ‐lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of raltegravir and raltegravir glucuronide in human plasma by liquid chromatography-tandem mass spectrometric method

Raltegravir is a highly efficacious inhibitor of HIV integrase. Large pharmacokinetic variability has been reported in clinical trials and this could be due to glucuronidation of raltegravir, the only reported metabolism pathway. In order to precisely evaluate and monitor the raltegravir and raltegravir glucuronide simultaneously, a novel, sensitive and robust liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of raltegravir and raltegravir glucuronide in human plasma. A simple protein precipitation with acetonitrile was utilized for plasma sample preparation prior to analysis. Baseline chromatographic separation was achieved on a ZORBAX Eclipse XDB-C8 using gradient elution mode. The run time was 9 min at a constant flow rate of 0.4 ml/min. The mass spectrometer was operated under a positive electrospray ionization condition. Excellent linearity (r(2) ≥ 0.9997) was achieved for raltegravir and raltegravir glucuronide in the range of 2-2000 nmol/l. The average recovery of raltegravir and raltegravir glucuronide was 105.8% and 102.2%, respectively. The precision (coefficient of variation) was 1.6-6.6% for raltegravir and 2.1-6.9 for raltegravir glucuronide, respectively. The accuracy was 98.6-106.1% for raltegravir and 96.3-100.3% for raltegravir glucuronide. The plasma samples were tested to be stable after nine freeze-thaw cycles and exposure to room temperature for 24 h. This well-validated assay was applied for the quantification of raltegravir and raltegravir glucuronide in plasma samples within 24 h after a single oral dose of 400 mg raltegravir in six healthy subjects.     

Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry

A quantitative analysis of polyamines in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The polyamines were carbamylated with isobutyl chloroformate, extracted with diethyl ether under pH 9.0, and analyzed by LC-MS/MS with single reaction monitoring mode. The limit of quantification was 1 ng/mL based on a signal-to-noise ratio>3, and the correlation coefficient (r2) for the calibration curves was >0.99 for both urine and serum samples. The present method was applied to urine and serum samples from 30 breast cancer patients and 30 normal female controls. There was no significant difference in the urinary polyamine levels between breast cancer patients and controls. However, 1,3-diaminopropane, putrescine, spermine and N-acetylspermidine levels in serum increased in breast cancer patients. These four serum polyamines may be a good index to study both production and metabolism of polyamines, and a useful tool in assessment of the polyamine status of breast cancer patients.     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。     

Rapid and simple determination of psychotropic phenylalkylamine derivatives in urine by direct injection liquid chromatography–electrospray ionization–tandem mass spectrometry

A direct injection liquid chromatography–electrospray ionization–tandem mass spectrometric method (LC‐ESI‐MS/MS) was developed and validated for the rapid and simple determination of 13 phenylalkylamine derivatives. Eight deuterium‐labeled compounds were prepared for use as internal standards (ISs) to quantify the analytes. Urine samples mixed with ISs were centrifuged, filtered through 0.22 µm filters and then injected directly into the LC‐ESI‐MS/MS system. The mobile phase was composed of 0.2% formic acid and 2 mM ammonium formate in distilled water and 0.2% formic acid and 2 mM ammonium formate in acetonitrile. The analytical column was a Capcell Pak MG‐II C₁₈ (150 × 2.0 mm i.d., 5 µm, Shiseido). Separation and detection of the analytes were accomplished within 10 min. The linear ranges were 5–750 ng/mL (ephedrine and fenfluramine), 10–750 ng/mL (3,4‐methylenedioxyamphetamine, phendimetrazine, methamphetamine, 3,4‐methylenedioxyethylamphetamine and benzphetamine), 20–750 ng/mL (norephedrine, amphetamine, phentermine and ketamine) and 30–1000 ng/mL (3,4‐methylenedioxymethamphetamine and norketamine), with determination coefficients, R², ≥ 0.9967. The intra‐day and inter‐day precisions were within 19.1%. The intra‐day and inter‐day accuracies ranged from ?16.0 to 18.7%. The lower limits of quantification for all the analytes were lower than 26.5 ng/mL. The applicability of the method was examined by analyzing urine samples from drug abusers (n = 30). Copyright © 2012 John Wiley & Sons, Ltd.     

Highly sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of nifedipine in human plasma and its application to a bioequivalence study

An ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of nifedipine in human plasma using nifedipine‐d6 as the internal standard (IS). The plasma samples were prepared by solid‐phase extraction on Phenomenex Strata‐X cartridges employing 200 μL human plasma. Chromatography was carried out on Waters Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 µm particle size) analytical column under isocratic conditions using a mobile phase consisting of 4.0 mm ammonium acetate‐acetonitrile (15:85, v/v). The precursor → product ion transitions for nifedipine (m/z 347.2 → 315.2) and IS (m/z 353.1 → 318.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive‐ion mode. The method was validated over a wide dynamic concentration range of 0.050–150 ng/mL. Matrix effect was assessed by post‐column analyte infusion and the mean extraction recovery was 95.6% across four quality control levels. The method is rugged and rapid with a total run time of 1.2 min and was applied to a bioequivalence study of 20 mg nifedipine tablet formulation in 30 healthy Indian subjects under fasting condition. Assay reproducibility was confirmed by reanalysis of 116 incurred samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Microwave-assisted extraction and determination of cyanuric acid residue in pet food samples by liquid chromatography-tandem mass spectrometry

Cyanuric acid (CYA) is attracting more attention due to its potential toxicity. In the present work, microwave-assisted extraction method in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was proposed for the determination of CYA in pet food samples. Among different solvents, diethylamine-acetonitrile-water mixture (1:5:4, v/v) was found to be the best one as the extractant due to the strong polarity of CYA in the pet food. An internal standard, (13) C(3) -labeled CYA, was used in the extractions. The separation was performed on a MERCK ZIC HILIC column (150 mm × 2.1 mm id, 5 μm) with gradient elution of 20 mM ammonium acetate solution-acetonitrile. CYA was well retained (Rt = 5.10 min) and eluted with good peak shape. The method could respond linearly with CYA at concentrations from 1.0 to 50 ng/mL with a quantification limit of 0.25 mg/kg. The intra- and inter-day precision was less than 4.0% and the recovery of the assay was in the range of 90.4-108.1%. In the analysis of practical spiked pet food samples, the new method yielded satisfactory results. Due to its simplicity and accuracy the straightforward method is particularly suitable for routine CYA detection.     

Determination of aripiprazole in rat plasma and brain using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Aripiprazole is an important antipsychotic drug. A simple, sensitive and rapid ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC‐ESI‐MS/MS) method was developed and validated for the simultaneous quantification of this compound in rat plasma and brain homogenate. The analyte was extracted from rat plasma and brain homogenate using a weak cation exchange mixed‐mode resin‐based solid phase extraction. The compound was separated on an Agilent Eclipse Plus C₁₈ (2.1 × 50 mm, 1.8 µm) column using a mobile phase of (A) 0.1% formic acid aqueous and (B) acetonitrile with gradient elution. The analyte was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, recoveries and stability were determined. The LOQ was 0.5 ng/mL for aripiprazole in plasma and 1.5 ng/g in brain tissue. The MS response was linear over the concentration range 0.5–100 ng/mL for aripiprazole in plasma and 1.5–300 ng/g in brain tissue. The precision and accuracy for intra‐day and inter‐day were better than 14%. The relative and absolute recoveries were above 72% and the matrix effects were low. This validated method was successfully used to quantify the rat plasma and brain tissue concentrations of the analyte following chronic treatment with aripiprazole. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

反相高效液相色谱-串联质谱法测定尿液中儿茶酚胺和肾上腺素(英文)

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma.The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection.Urine samples were prepared using Oasis weak-cation-exchange cartridges.The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min.Adrenaline,noradrenaline,dopamine,metanephrine,normetanephrine,and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring(MRM).No evidence of ion suppression was observed.The assay was linear up to 5μmol/L for adrenaline,5μmol/L for noradrenaline,6.1μmol/L for dopamine,5.6μmol/L for metanephrine,and 34.6μmol/L for normetanephrine,with lower limits of quantification of 5,5,12,6 and 7nmol/L,respectively.The intra-day and inter-day precisions for all analytes ranged from 0.59%to 4.64%and1.98%to 4.80%,respectively.External quality assurance samples were assayed and showed excellent agreement with the target values.This simple method provides an improved assay for determining urine catecholamines and metanephrines.     

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.     

Determination of lovastatin in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A fast and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of lovastatin in human plasma. With simvastatin as internal standard, sample pretreatment involved one-step extraction with n-hexane-methylene dichloride-isopropanol (20:10:1, v/v/v) of 0.5 mL plasma. Chromatographic separation was carried out on an Acquity UPLC BEH C(18) column with mobile phase consisting of acetonitrile-water (containing 5 mmol/L ammonium acetate; 85:15, v/v) at a flow-rate of 0.35 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization source with positive mode. The analysis time was shorter than 1.7 min per sample. The standard curve was linear (r2>or=0.99) over the concentration range 0.025-50.0 ng/mL with a lower limit of quantification of 0.025 ng/mL. The intra- and inter-day precision values were below 11% and the accuracy (relative error) was within 6.0% at three quality control levels. This is the first method of MS with MRM coupled to UPLC for the determination of lovastatin, which showed great advantages of high sensitivity, selectivity and high sample throughput. It was fully validated and successfully applied to the pharmacokinetic study of lovastatin tablets in healthy Chinese male volunteers after oral administration.     

超高效液相色谱-串联质谱法测定牛尿液中的甲状腺抑制剂(英文)

Five thyreostats(TSs),namely tapazole,thiouracil,methylthiouracil,propylthiouracil,and phenylthiouracil,were determined in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)in positive electrospray ionization mode.Extraction and clean-up were achieved using a ChemElut cartridge with tert-butyl methyl ether,without a derivatization step.Separation was achieved on an Acquity UPLC SS T3 column.The mobile phase was acetonitrile and water containing 0.2%(v/v)formic acid.The mass spectrometer was operated in multiple reaction monitoring mode.Urine samples were spiked with TS solution at levels corresponding to 5,10,15,and 20μg/L.The accuracy(internal standard corrected)ranged from 92%to 107%,with a repeatability precision(relative standard deviation,RSD)less than 15%for all five analytes.The RSDs within-laboratory reproducibility was less than 26%.The decision limits(CCα)and detection capabilities(CCβ)were obtained from a calibration curve and were in the ranges of 3.1-6.1μg/L and 4.0-7.4μg/L,respectively.The CCαand CCβvalues were below the recommended concentration,which was set at 10μg/L.The results show that the described method is suitable for the direct detection of TSs in bovine urine.This method can also be used to determine TSs in porcine urine.     

超高效液相色谱-串联质谱法同时快速确证检测血浆和尿液中的42种精神药物及其代谢产物

针对公共卫生突发事件和临床毒物学检测实践中亟待解决的问题,建立了血浆和尿液中42种精神药物及其代谢产物的超高效液相色谱-串联质谱(UPLC-MS/MS)快速确证分析方法。样品经乙腈沉淀后,以乙酸铵和甲醇-乙腈(1:1, v/v)混合液作为流动相进行梯度洗脱,在Acquity UPLC BEH C18色谱柱上分离后用电喷雾串联质谱法检测,采用正、负离子快速切换多反应监测模式监测,基质标准同位素内标法定量。血浆样品中待测组分的加标回收率除了奋乃静、硫利哒嗪和氯丙嗪的分别为37.6%～57.5%, 36.3%～48.3%和52.4%～67.4%外,尿液样品中待测组分的加标回收率除了曲唑酮和地西泮的分别为100%～142%和108%～177%外,血浆和尿液中其余待测组分的加标回收率分别为60.2%～125%和64.5%～126%,相对标准偏差分别为0.8%～26%和2.6%～18%(n=6);除了巴比妥类药物的检出限为20～100 mg/L外,其余药物的检出限均为0.05～2.0 mg/L。该方法简单、快速、特异性强、灵敏度高。     

Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation‐exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)–tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive‐mode APCI. Responses for all analytes were linear over the calibration ranges. Intra‐assay precision (RSD) was 2–18%, and inter‐assay precision was 2–12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection. Copyright © 2010 John Wiley & Sons, Ltd.     

液相色谱-串联质谱分析组织中基因组脱氧核糖核酸羟甲基胞嘧啶水平

5-羟甲基胞嘧啶通过阻止脱氧核糖核酸(DNA)甲基化转移酶1(DMNT1)甲基化胞嘧啶来影响DNA甲基化的程度。本文建立了液相色谱-串联质谱(LC-MS/MS)测定组织中全基因组5-羟甲基胞嘧啶水平的方法。采用苯酚-氯仿提取组织DNA,提取的DNA用88%甲酸在140 ℃下裂解,DNA裂解液加入同位素胞嘧啶作内标,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用LC-MS/MS检测5-羟甲基胞嘧啶的含量,并计算全基因组中5-羟甲基胞嘧啶的水平。结果表明,5-羟甲基胞嘧啶的线性范围为0.1～30 ng/mL,相关系数为0.9969,检出限(信噪比为3计)和定量限(信噪比为10计)分别为0.057 ng/mL和0.090 ng/mL;日内相对标准偏差和日间相对标准偏差分别为5.13%和6.24%;加标回收率为90.24%～97.53%。用该方法检测了大鼠大脑组织DNA羟甲基化水平,平均结果为0.66%。该方法简便,重现性好,灵敏度较高,能满足全基因组5-羟甲基胞嘧啶定量检测的要求。     

Simultaneous determination of norisoboldine and its major metabolite in rat plasma by ultra-performance liquid chromatography-mass spectrometry and its application in a pharmacokinetic study

Norisoboldine (NIB) is one of the main bioactive isoquinoline alkaloids in Linderae Radix. A rapid, selective and sensitive method using UPLC‐ESI/MS was first developed for simultaneous determination of NIB and norisoboldine‐9‐O‐α‐glucuronide (NIB‐Glu), its major metabolite in rat plasma. A one‐step protein precipitation with methanol was employed as sample preparation technique. Chromatographic separation was carried out on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of acetonitrile and water containing 0.1% formic acid. Detection and quantification were performed using a quadrupole mass spectrometer by selective ion reaction‐monitoring mode. Good linearity was achieved using weighted (1/x²) least squares linear regression over the concentration ranges 0.01–2 µg/mL for NIB and 0.025–25 µg/mL for NIB‐Glu. The lower limit of quantification of NIB and NIB‐Glu was 0.01 and 0.025 µg/mL, respectively. The intra‐ and inter‐day precisions (relative standard deviations) of the assay at all three quality control levels were 4.6–14.1% for NIB, and 5.0–12.2% for NIB‐Glu. The accuracies (relative error) were −13.5–8.1% for NIB and −12.8–7.6% for NIB‐Glu, respectively. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 10 mg/kg NIB. Copyright © 2010 John Wiley & Sons, Ltd.     

固相萃取-高效液相色谱-串联质谱法同时测定尿液中的4种巯基尿酸

建立了固相萃取-高效液相色谱-串联质谱(HPLC-MS/MS)同时检测人体尿液中N-乙酰基-S-(3,4-二羟基丁基)-L-半胱氨酸(DHBMA)、N-乙酰基-S-(3-羟基丙基)半胱氨酸(3-HPMA)、N-乙酰基-S-(2-氰乙基)-L-2-氨基-3-巯基羧酸(CEMA)和苯巯基尿酸(SPMA)的检测方法。冰冻的人体24 h尿液在室温下解冻,混合均匀后离心过滤,经C18固相萃取小柱净化富集后在多反应监测模式下采用HPLC-MS/MS进行定量分析。在3个添加水平下,尿液中DHBMA、3-HPMA、CEMA和SPMA的加标回收率分别为105.6%～124.4%、102.7%～106.5%、103.2%～103.9%和101.7%～104.3%,相对标准偏差为2.6%～7.7%。以不低于3倍的信噪比估算DHBMA、3-HPMA、CEMA和SPMA的检出限分别为0.062、0.031、0.020和0.003 μg/L。应用该方法检测了37例吸烟和非吸烟者的24 h尿液样本,结果发现吸烟者尿液中3-HPMA、SPMA和CEMA的平均含量比非吸烟者高3到6倍。     

Determination of trans,trans‐muconic acid in workers' urine through ultra‐performance liquid chromatography coupled to tandem mass spectrometry

A novel method for the biological monitoring of benzene‐exposed workers has been developed through ultra‐performance liquid chromatography coupled to tandem mass spectrometry. The method uses trans,trans‐muconic acid in urine as the benzene‐exposure biomarker. The method was developed using a triple quadrupole mass spectrometer with enough sensitivity to facilitate diluting and injecting the urine samples directly, rather than performing a solid‐phase extraction procedure as is common in the available protocols. Moreover, compared with a conventional high‐pressure liquid chromatography system, the separation power provided by the ultra‐performance liquid chromatography system allows a 10‐fold reduction in run time. The method was adjusted to a dynamic range of between 198.9 and 4916.7 µg/L to cover the biological exposure index of trans,trans‐muconic acid in urine. Also, the method demonstrated intra‐day and inter‐day precision at 98%, and accuracy within an acceptable range of 101 ± 8%. The method has been used to quantify various types of urine samples, such as workers' urine and inter‐laboratory proficiency tests. Depending on the sample, the quantified levels ranged from less than the limit of quantitation to 3836.7 µg/L. No levels exceeding the calibration range were detected in the urine of workers, and the reported concentrations in urine for the proficiency tests were, as expected, based on known values. Moreover, the new method using sample dilution and faster chromatographic run was more effective, facilitating fast communication of results, as needed, to decision‐makers. Copyright © 2012 John Wiley & Sons, Ltd.