Development and validation of a highly sensitive GC/MS method for the determination of buprenorphine and nor-buprenorphine in blood

A sensitive and specific GC/MS method for the determination of buprenorphine (BPN) and its main metabolite nor-buprenorphine (nor-BPN) in blood has been developed, optimized and validated. Sample preparation includes solid-phase extraction of both analytes and their derivatization with acetic anhydride in pyridine. BPN-d4 was used as internal standard for the determination of both analytes. Limits of detection and quantification for BPN and nor-BPN were 0.02 and 0.05 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (0.05-30.0 μg/L) with a correlation coefficient higher than 0.996. Absolute recovery ranged from 90.2 to 97.6% for both analytes and their internal standard. Intra- and inter-day accuracy was found to be between -5.40 to 1.73% and -2.45 to 2.80%, respectively, while intra- and inter-day precision were less than 5.8 and 4.7%, for both analytes. The method was applied to real blood samples obtained from patients that follow BPN maintenance program. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic drug level monitoring in order to adjust BPN dosage of BPN maintained patients or for the investigation of forensic cases.     

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

LC-MS/MS法测定人血浆中甘草次酸及其临床药代动力学研究

目的建立人血浆中甘草次酸的LC-MS/MS测定方法,研究男性健康志愿者单剂量服用甘草酸二铵胶囊,其代谢产物甘草次酸体内药代动力学行为。方法健康男性志愿者单剂量口服甘草酸二铵胶囊150 mg,血浆样品经乙酸乙酯提取,进行LC-MS/MS分析。色谱柱为Agilent ZORBAXSB C18(3.0×100 mm,5μm),流动相为甲醇∶乙腈∶醋酸铵缓冲液(5 mmol.L-1醋酸铵,0.2%冰醋酸)(15∶60∶25,V/V/V),检测离子为m/z469.4/355.2(甘草次酸)、m/z358.9/279.9(内标泼尼松龙)。测定甘草次酸血药浓度,计算其药代动力学参数。结果在1.5～192μg.L-1内,甘草次酸与内标的峰面积比值与浓度的线性关系良好,定量限为1.5μg.L-1,提取回收率为77.14%～83.64%。人体中甘草次酸药代动力学参数:Cmax为(73.85±25.25)μg.L-1,Tmax为(11.50±3.07)h,T21β为(11.82±3.56)h,AUC0-60为(1252.49±489.06)μg.h.L-1。结论建立的LC-MS/MS分析方法准确灵敏,适于临床药代动力学研究。口服甘草酸二铵胶囊,其代谢产物甘草次酸在体内的药代动力学特点是达峰时间长,约占受试者总人数50%的药时曲线有双峰现象。     

LC-MS/MS法测定人血清中亮丙瑞林的浓度

目的:建立人血清中亮丙瑞林浓度的LC-MS/MS测定方法。方法:采用乙腈沉淀蛋白提取血清中目标成分。选用Agela Venusil ASB C18色谱柱(150 mm×4.6 mm,5μm),以乙腈-5 mmol.L-1醋酸铵-甲酸(30∶70∶0.1)为流动相,采用多反应离子监测(MRM)模式进行正离子检测,选择监测离子反应为m/z605.6→248.9[M+2H]2+(亮丙瑞林)和151.8→110.1[M+H]+(对乙酰氨基酚)。结果:血清中内源性物质不干扰亮丙瑞林的测定;亮丙瑞林的线性范围为0.1～10.0 ng.mL-1,最低定量限为0.1 ng.mL-1。方法准确度为88.5%～111.5%,日内和日间精密度均<15%。结论:本文建立的LC-MS/MS法,简单快速,专属性强,灵敏度高,可用于亮丙瑞林缓释制剂在人体内的药代动力学研究。     

A simple extraction and LC‐MS/MS approach for the screening and identification of over 100 analytes in eight different matrices

In the present study, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach using one single work‐up approach in whole blood, plasma, serum, post‐mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post‐mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether‐ethyl acetate mixture (1:1). Separation and identification were performed using LC‐QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi‐reaction‐mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z‐drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in‐house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work‐up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of colistin A and colistin B in plasma and culture medium using a simple precipitation step followed by LC/MS/MS

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm × 250 mm, 5 μm particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor–product ion pairs were m/z 1167.7 → 1079.6 for colistin A and m/z 1153.7 → 1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 μL plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV <6.2% and accuracy < ±12.6%. For culture medium (50 μL + 50 μL plasma), LLOQ was 24.2 and 13.2 ng/mL for colistin A and B, respectively, with CV <11.4% and accuracy < ±8.1%. The quick sample work-up method allows for determination of colistin A and B in clinical samples without causing hydrolysis of the prodrug colistin methanesulfonate (CMS).     

Validation of a GC/MS method for the determination of tramadol in human plasma after intravenous bolus

Tramadol quantitative determination by gas chromatography-mass spectrometry (GC/MS) using nefopam hydrochloride as internal standard (IS) and two calibration curves because of the large range of concentration attended in the plasmatic samples is described. Plasma samples drawn from subjects in postoperative period treated with two different initial intravenous (iv) bolus of tramadol (50 and 100 mg) followed by tramadol at the same infusion rate (12 mg h(-1)) are analysed. We operated for the qualitative analysis in Scan mode while for the quantitative analysis in SIM mode, selecting the ion m/z 58 for tramadol and m/z 179 for IS. The limit of detection (LOD) was 0.01 microg ml(-1) and the limit of quantification was 0.04 microg ml(-1).     

Oral and i.v. pharmacokinetics of isosteviol in rats as assessed by a new sensitive LC-MS/MS method

A sensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed to investigate isosteviol pharmacokinetics in vivo. Isosteviol was extracted from plasma with hexane and 4% formic acid. A Phenomenex Synergi 2mu Fusion reversed phase analytical HPLC column (50 mm x 2.0 mm) equipped with a Synergi 2micro Fusion guard column was employed for chromatographic separations. The gradient mobile phase consisted of acetonitrile (ACN) and 20mM ammonium acetate at pH 6.5, starting at 20% ACN and ramping to 80% at 7 min, followed by 80% ACN for 1 min, then 20% ACN for 5 min. Negative SRM was used to monitor the m/z 317.1/317.1 and 317.3/317.3 transitions for isosteviol and 395.0/395.0 and 397.0/397.0 transitions for internal standard. The retention time of isosteviol was 9.2 min. The assay was linear over the range of 50-2,000 ng/mL. The accuracy of the method was in the range of 97-105%. Intra- and inter-day precisions were in the range of 1.5-4.6%. Isosteviol (4 mg/kg) was dosed intravenously and orally to Sprague-Dawley rats (n=6). Plasma samples were collected and analysed. Intravenous isosteviol has a distribution half-life of 35.7+/-9.0 min with the initial distribution volume of 68.1+/-9.4 mL. The total clearance, terminal half-life and steady-state volume of distribution were 1.25+/-0.12 mL/min, 150.6+/-50.5 min and 272.6+/-95.9 mL, respectively. The oral bioavailability of isosteviol was found to be 60.4+/-15.5%.     

Use of GC × GC/TOF‐MS and LC/TOF‐MS for metabolomic analysis of Hyalella azteca chronically exposed to atrazine and its primary metabolite,desethylatrazine

Atrazine is one of the most commonly detected contaminants in the U.S. Little information is available on one of atrazine's metabolites, desethylatrazine (DEA). Two‐dimensional gas chromatography and liquid chromatography coupled with time of flight‐ mass spectrometry were used to examine metabolite profiles of Hyalella azteca chronically exposed to 30 µg/L atrazine and DEA. The majority of identified metabolites were by‐products of β‐oxidation of fatty acids suggesting possible disruption in energy metabolism. Eicosanoids increased in exposed females suggesting possible perturbations in neuropeptide hormonal systems. Overall, this research demonstrates the feasibility of utilizing metabolomic profiling of invertebrate species exposed to environmental contaminants as a way to determine mechanisms of toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid and sensitive LC-MS/MS method for determination of Pravastatin in human plasma

快速灵敏的LC-MS/MS方法检测人血浆中普伐他汀的浓度@张敏$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @周宏灏$Ins     

快速灵敏的LC-MS/MS法测定人血浆中缬沙坦浓度

目的：建立一种快速、灵敏的高效液相色谱-串联质谱法（LC-MS/MS）测定人血浆缬沙坦浓度。方法：200μL血浆样品经乙腈一步沉淀蛋白后,在Inertsil ODS-色谱柱（2.1mm×150mm,5μm）上分离,流动相由乙腈和1‰甲酸水溶液（70∶30）组成。采用电喷雾离子源（ESI源）正离子多反应监测（MRM）扫描分析,缬沙坦和厄贝沙坦的离子选择通道分别为：m/z436.3→235.2和429.4→207.2。结果：缬沙坦的线性范围为24.2～3100.0μg/L,日内和日间相对标准差均小于15%。结论：本法操作快速、灵敏,适用于缬沙坦的临床药动学研究。     

A simple and rapid ESI-LC-MS/MS method for simultaneous screening of doping agents in urine samples

Objective:

The use of performance enhancing substances is banned in sports by the World Anti-Doping Agency (WADA). Though most prohibited substances can be detected by GC/MS, inclusion of corticosteroids and designer drugs has made it essential to detect these critical doping agents on LC/MS/MS due to their better separation and detection.

Materials and Methods:

A common extraction procedure for the isolation of acidic, basic and neutral drugs from urine samples was developed. A total of 28 doping drugs were analyzed on API 3200 Triple quadrupole mass spectrometer using C18 column in atmospheric pressure electrospray ionization. The mobile phase composition was a mixture of 1% formic acid and acetonitrile with gradient time period.

Results:

The method developed was very sensitive for detection of 28 doping agents. The linearity was performed for each drug and the total recovery percentage ranged from 57 to 114. Limit of detection is found to be 0.5 ng/ml for carboxy finasteride and 1-5 ng/ml for other drugs. The method was successfully used to detect positive urine samples of 3-OH-stanozolol, methyl phenidate, mesocarb, clomiphene metabolite and carboxy finasteride.

Conclusion:

The method developed based on controlled pH extraction method and HPLC-mass spectrometry analysis allowed better identification and confirmation of glucocorticosteroids and a few other drugs in different categories. The validated method has been used successfully for testing of 1000 In-competition samples. The method helped in detection of chemically and pharmacologically different banned drugs in urine in a single short run at a minimum required performance limit set by WADA.     

Development and validation of a GC/MS method for the determination of tadalafil in whole blood

Tadalafil is a phosphodiesterase type 5 (PDE-5) inhibitor and it is used in the treatment of pulmonary arterial hypertension and erectile dysfunction. A sensitive and specific method is described for the determination of tadalafil in whole blood. Tadalafil and its internal standard (protriptyline) were isolated from the matrix by solid phase extraction, and were analyzed by gas chromatography/mass spectrometry (GC/MS) after derivatization by N,O-bis(trimethylsilyl)-trifluoracetamide (BSTFA) with 1% trimethylchlorsilane (TMCS). Limits of detection and quantification for tadalafil were 0.70 and 2.00 μg/L, respectively. The calibration curve was linear between 2.00 and 500.0 μg/L, with a correlation coefficient higher than 0.991. The values obtained for intra- and inter-day accuracy was found to be between −10.5 to 8.5% and −4.2 to 4.5%, respectively, while intra- and inter-day precision were less than 8.4 and 11.2%, correspondingly. Absolute recovery was determined at three concentration levels and ranged from 92.1 to 98.9%. The proposed method is the first fully validated GC/MS method for the determination of tadalafil in whole blood and it can be routinely applied by toxicological laboratories, for pharmacokinetic studies, for therapeutic drug level monitoring or for the investigation of related forensic cases.     

UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Zolpidem and zopiclone (Z‐compounds) are non‐benzodiazepine hypnotics of new generation that can be used in drug‐facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z‐compounds and their metabolites by ultra‐high‐pressure liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC‐HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy‐ and three hydroxy‐ metabolites, that could be also detected by gas chromatography/mass spectrometry (GC‐MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N‐demethyl and N‐oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC‐MS/MS method for the determination of Z‐compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC‐MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive method for determination of veliparib (ABT-888), in human plasma,bone marrow cells and supernatant by using LC/MS/MS

A rapid and sensitive method was developed and validated using a liquid chromatographic method with tandem mass spectrometry detection (LC/MS/MS) for determination of veliparib (ABT-888) in plasma, bone marrow supernatant, and bone marrow cells. Sample preparation involved a single protein precipitation step by the addition of the sample with acetonitrile. Separation of veliparib and the internal standard, A620223.69, was achieved on a Atlantis™ dC₁₈ column (100 mm × 2.1 mm, 3 μm) column using a mobile phase consisting of acetonitrile–ammonium acetate (2 mM) containing formic acid (0.1%, v/v) using isocratic flow at 0.2 mL/min for 3 min. The analyte and internal standard were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5–1000 nM. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods. This method was subsequently used to measure concentrations of veliparib in cancer patients receiving an oral daily dose of 10 mg with demonstration of drug accumulation in the marrow compartment and in the target leukemia bone marrow cells.     

UPLC-MS/MS法测定Beagle犬血浆中S-奥拉西坦浓度

目的:建立灵敏、准确的Beagle犬血浆中S-奥拉西坦浓度的UPLC-MS/MS检测方法,并用于该药在Beagle犬体内的药动学研究.方法:血浆样品经甲醇沉淀蛋白后,以甲醇-水溶液(85∶15,V/V,内加10 mmoL乙酸铵和0.1％甲酸)为流动相,用HP Amide LC-MS/MS Column(100 mm×3 mm ID,5μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 159.0/114.1(S-奥拉西坦)和m/z 143.0/126.1(内标,吡拉西坦).结果:S-奥拉西坦线性范围为0.05～50 μg/mL,定量下限为0.05μg/mL,日内、日间精密度(RSD)均小于15％.S-奥拉西坦大鼠血浆样品在储存、预处理、分析期间均显示良好的稳定性.应用此法研究了6只Beagle犬单剂量口服S-奥拉西坦50 mg/kg后的药动学特点.结论:该方法快速、专属、灵敏、适用性强,可应用于5-奥拉西坦的临床前药动学研究.     

Validation of rapid and simple LC-MS/MS method for determination of voriconazole in rat plasma

A rapid, simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of voriconazole (VRC) in rat plasma, using ketoconazole as internal standard (IS). Analysis was performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water-formic acid (60:40:0.05, v/v/v), at a flow of 1.0 mL/min (split ratio 1:5), and a mass spectrometer Micromass, equipped with a double quadrupole and an electrospray ionization interface, operated in a positive mode. Plasma samples were deproteinized with methanol (1:2) and 30 microL of the supernatant was injected into the system. The retention times of VRC and IS were approximately 3.3 and 2.7 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 50-2500 ng/mL with determination coefficient >0.98. The lower limit of quantification was 50 ng/mL. The accuracy of the method was within 5%. Intra- and inter-day relative standard deviations were less or equal to 12.5 and 7.7%, respectively. The applicability of the LC-MS-MS method for pharmacokinetic studies was tested using plasma samples obtained after intravenous administration of VRC to male Wistar rats. The reported method provided the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of VRC in pre-clinical pharmacokinetic studies.     

Phenolic compounds from Byrsonima crassifolia L. bark: phytochemical investigation and quantitative analysis by LC-ESI MS/MS

Phytochemical investigation of the methanolic extract of Byrsonima crassifolia's bark led to the isolation of 8 known phenolic compounds 5-O-galloylquinic acid, 3-O-galloylquinic acid, 3,4-di-O-galloylquinic acid, 3,5-di-O-galloylquinic acid, 3,4,5-tri-O-galloylquinic acid, (+)-epicatechin-3-gallate along with (+)-catechin and (+)-epicatechin.Due to their biological value, in the present study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, working in multiple reaction monitoring (MRM) mode, has been developed to quantify these compounds. B. crassifolia bark resulted in a rich source of phenolic compounds and particularly of galloyl derivates. The proposed analytical method is promising to be applied to other galloyl derivatives to quantify these bioactive compounds in raw material and final products.     

Hair testing for cocaine and metabolites by GC/MS: criteria to quantitatively assess cocaine use

A simple, rapid and sensitive method has been developed and validated for the determination of cocaine and its main metabolites (benzoylecgonine and cocaethylene) in human hair. The method involved solid‐phase extraction with an Oasis HLB extraction cartridge and subsequent analysis by GC/MS. The limit of detection was 0.01 ng mg^?1 for cocaine, 0.04 for benzoylecgonine and 0.03 for cocaethylene. The method validation included linearity (with a correlation coefficient >0.99 over the range 0.2–50 ng mg^?1), intra‐ and inter‐day precision (always lower than 12%) and accuracy (mean relative error always below 17%) to meet the bioanalytical acceptance criteria. The procedure was further applied to 40 hair samples from self‐reported cocaine users arrested by the police who provided a positive urine‐analysis for cocaine, and was demonstrated to be suitable for its application in forensic toxicology. New approaches were raised to detect false‐negative results that allow a better interpretation of hair testing results. Copyright © 2012 John Wiley & Sons, Ltd.