Postmortem drug screening by non-targeted and targeted ultra-performance liquid chromatography-mass spectrometry technology

In the medical examiner setting, comprehensive drug screening is an essential analytical tool in the investigation of cause and manner of death.We have validated non-targeted and targeted screening assays for drugs and metabolites using ultra-performance liquid chromatography (UPLC) interfaced with mass spectrometry (MS) in single and tandem stages. For non-targeted screening by UPLC-MS electrospray interface, in-source fragmentation was used along with MS scanning (m/z 80-650) and library search for over 700 drug and metabolite analytes. Targeted detection of over 200 analytes by UPLC-MS-MS was performed with dual transition ion monitoring. Validation studies confirmed reproducibility of both mass spectra produced by in-source fragmentation and transition ion ratios by collision-cell dissociation. Lower limit of detection by UPLC-MS (10-150 ng/mL) and UPLC-MS-MS (1-50 ng/mL) was determined for a subset of drugs and correlated with extraction recovery and matrix effect. Drug findings by UPLC-MS and UPLC- MS-MS were compared with gas chromatography-mass spectrometry (GC-MS) screening in postmortem blood from 410 medical examiner cases with 1121 positive drug findings by all methods. Accuracy, based on results of confirmation testing, was high (98-99%) across all screening assays and detection sensitivity by GC-MS (71%), UPLC-MS (73%), and UPLC-MS-MS (76%) was determined. UPLC-MS plus UPLC-MS-MS screening resulted in the highest drug detection rate (95%) and provided optimal dual-screening for the postmortem casework.     

Evaluation of the use of UPLC-TOFMS with simultaneous [14C]-radioflow detection for drug metabolite profiling: application to propranolol metabolites in rat urine

The non-selective beta-adrenergic receptor antagonist propranolol [1-(isopropylamino)-3-(1-naphthoxy)-2-propanol] is metabolised extensively in vivo. Enumerating and identifying the many metabolites that result from multiple biotransformations provides a considerable analytical challenge, greatly aided by efficient chromatography coupled to sensitive mass spectrometric detection. Here the use of the newly introduced high-resolution technique of "ultra performance liquid chromatography" (UPLC) linked to quadrupole time-of-flight mass spectrometry (TOFMS) with simultaneous [(14)C]-radioflow detection was applied to rapid metabolite profiling. [(14)C]-propranolol, dosed intraperitoneally to rat at 25mgkg(-1) and 200muCikg(-1) was used as a model compound for this evaluation. Some 14 metabolites were detected in the urine by this technique including a number of conjugated metabolites such as sulphates, several isobaric glucuronides and two novel di-glucuronides.     

Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS

A UPLC/Q-TOF-MS/MS method for analyzing the constituents in rat plasma after oral administration of Yin Chen Hao Tang (YCHT), a traditional Chinese medical formula, has been established. The UPLC/MS fingerprints of the samples were established first in vitro and in vivo, with 45 compounds in YCHT and 21 compounds in rat plasma after oral administration of YCHT were detected. Of the 45 detected compounds in vitro, 30 were identified, and all of the 21 compounds detected in rat plasma were identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by mass spectrometry analysis and retrieving the reference literatures. Of the identified 21 compounds in rat plasma, 19 were the original form of compounds absorbed from the 45 detected compounds in vitro, 2 were the metabolites of the compounds existed in YCHT. It is concluded that a rapid and validated method has been developed based on UPLC-MS/MS, which shows high sensitivity and resolution that is more suitable for identifying the bioactive constituents in plasma after oral administration of Chinese herbal medicines, and provides helpful chemical information for further pharmacology and active mechanism research on the Chinese medical formula.     

Development and validation of a rapid UPLC/MS method for the simultaneous determination of I3C,DIM, and related metabolites and its application to pharmacokinetics studies

Indole-3-carbinol (I3C) and diindolylmethane (DIM) are naturally derived dietary phytochemicals with promising anti-cancer properties that have been demonstrated both in vitro and in vivo. Using reversed-phase ultra-performance liquid chromatography(UPLC) coupled with mass spectrometry (MS), a rapid, specific, and high throughput method was developed and validated for the quantification and identification of I3C, DIM, and other I3C metabolites in plasma. Samples containing I3C or DIM and the internal standard 4-methoxy indole (IS) were extracted using a liquid-liquid extraction technique. The mean recovery was 96.21% for I3C and 108.5% for DIM. Separation was achieved using a Waters Acquity UPLC HSS T3, 1.8 µm, 2.1 mm×150 mm column and acetonitrile–water gradient elution. The flow rate was 0.3 mL/min and the run time was 9 min. The limits of detection and quantification for I3C and DIM were 15 ng/mL and 25 ng/mL, respectively. Calibration curves for I3C and DIM were linear (r²>0.99) over a concentration range of 0.025–20 µg/mL. Precision, accuracy, and stability analysis fulfilled the CDER guidelines criteria. The method was successfully applied to the determination of the pharmacokinetic parameters of I3C or DIM after oral, intravenous, or intraperitoneal administration to Sprague Dawley rats. The method described here is superior over existing analytical methods for I3C and its metabolites in terms of sensitivity, speed, and separation.     

Determination of febuxostat in human plasma using ultra‐performance liquid chromatography tandem mass spectrometry

A rapid and sensitive liquid chromatography‐tandem mass spectrometry method has been developed and validated for the determination of febuxostat in human plasma. The liquid‐liquid extraction technique was used for the extraction of febuxostat from human plasma using trandolapril as the internal standard (IS). Chromatography was performed on a ultra‐performance liquid chromatography (UPLC) BEH C18, 50 mm X 2.1 mm, 1.7 µm particle size column, with the mobile phase consisting of 0.1% formic acid and acetonitrile (in a 25:75 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub‐microgram levels. The method was validated and the lower limit of quantification for febuxostat was found to be 0.075 µg/ml. The mean recovery for febuxostat ranged from 100.9 to 106.5%. This method increased the sensitivity and selectivity; resulting in high‐throughput analysis of febuxostat using commercially available IS for pharmacokinetic, bioavailability, and bioequivalence studies, with a chromatographic run time of 1.5 min only. Copyright © 2012 John Wiley & Sons, Ltd.     

Gas chromatography-mass spectrometry in the investigation of on-column dehydration of steroid hormones during gas-liquid chromatography.

Some underivatized steroids when injected onto conventional packed columns for gas-liquid chromatography underwent varying degrees of dehydration. This problem was traced to the presence of small pieces of broken glass on the top of the column at the point of injection. This observation provoked an examination of the effect of pre-column dehydration on a number of different types of steroids. Powdered aluminium was placed in the injection liner of a Hewlett-Packard gas chromatograph fitted with an HP1 capillary column connected to a mass selective detector, and injections were made using a new high temperature septumless injection system at temperatures between 200 and 400 degrees C. 5 alpha-androstan-3 alpha-ol, a simple monofunctional C19 steroid chosen as a model to establish optimum conditions, underwent dehydration at injection temperatures greater than 250 degrees C and the product reached a maximum at 400 degrees C when no unchanged steroid was present. Monohydroxylated androgens and oestrogens underwent dehydration at 400 degrees C producing products whose mass spectra indicated they were monenes, although the position of the double bond could not be assigned. Polyfunctional androgens and oestrogens and corticosteroids underwent complex changes producing a number of products some of whose structures could not be determined. The dehydration products had the advantage that they had relatively intense high mass ions and for suitable steroids this might provide enhanced sensitivity of detection during mass fragmentography. In such cases dehydration was reproducible and straight line standard curves were obtained. C27 and C28 secosteroids (vitamins D2 and D3) and some of their metabolites (e.g. 25-hydroxyvitamin D) underwent efficient dehydration, again producing products with intense molecular ions. In the case of 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3, dehydration produced different products which were easily resolved in the chromatographic system used. Dehydration of vitamin D metabolites eliminates the need for derivatization and gives enhanced sensitivity of measurement by gas chromatography-mass spectrometry.     

Determination of urinary 13C-caffeine metabolites by liquid chromatography-mass spectrometry: the use of metabolic ratios to assess CYP1A2 activity

A method using liquid chromatography coupled with mass spectrometry with an atmospheric pressure electrospray source was developed for analysis of labelled caffeine and fourteen of its metabolites in urine. Caffeine metabolic ratios were determined after an oral bolus of labelled caffeine in 20 healthy subjects with different characteristic CYP1A2 activity, relative to smoking habit and oral contraceptive intake. The use of labelled caffeine for the calculation of metabolic ratios avoided taking into account the important background of endogenous caffeine metabolites, very difficult to eliminate even after a specific diet. The selectivity and high sensitivity of mass spectrometry detection allowed urine collections for only a 3h period. Comparison between characteristic groups showed that labelled caffeine metabolic ratios were sensitive markers of changes in CYP1A2 activity.     

Identification and characterization of amiodarone metabolites in rats using UPLC–ESI-QTOFMS-based untargeted metabolomics approach

Amiodarone is a class III anti-arrhythmic benzofuran derivative extensively utilized in treatment of life-threatening ventricular and supraventricular arrhythmias. However, amiodarone also produces adverse side effects including liver injury due to its metabolites rather than parent drug. The purpose of the present study was to identify metabolites of amiodarone in the plasma and urine of rats administered the drug by using an untargeted metabolomics approach. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS) and results subjected to multivariate data analysis. A total of 49 amiodarone metabolites were identified and their structures were characterized by tandem mass spectrometry. Amiodarone metabolites are presumed to be generated via five major types of metabolic reactions including N-desethylation, hydroxylation, carboxylation (oxo/hydroxylation), de-iodination, and glucuronidation. Data demonstrated that an untargeted metabolomics approach appeared to be a reliable tool for identifying unknown metabolites in a complex biological matrix.     

基于UPLC/Q-TOF-MS/MS对雷公藤次碱在小鼠体内代谢的分析

目的:研究雷公藤次碱在小鼠体内的生物转化规律。方法:以昆明山海棠根皮提取物中雷公藤次碱为研究对象,借助UPLC/Q-TOF-MS/MS高分辨质谱系统,基于多重质量亏损技术,结合MetabolitePilot代谢物分析鉴定软件,系统分析雷公藤次碱在小鼠体内的代谢产物。结果:雷公藤次碱在小鼠体内经一系列生物转化后,生成包括氧化、N-乙酰化、脱C₂H₂O基团等在内的多种代谢产物。结论:本方法能鉴定雷公藤次碱在小鼠体内的生物转化规律,为中药昆明山海棠的临床使用提供重要参考。     

液相色谱-串联质谱法在药物代谢研究中应用最新进展

液相色谱-质谱联用技术因其具有高分离能力、高灵敏度、应用范围广和较强的专属性等特点,已成为一种重要的现代分离分析技术。串联质谱法比单极质谱具有更高的灵敏度和选择性,可以得到更多的结构信息。本文首先介绍了液相色谱-串联质谱法中串联质谱基本原理、常见质谱质量分析器及其特点、串联质谱的连接方式和数据采集方法等。然后将其在药物代谢研究中的主要应用作简要介绍,主要包括利用串联质谱测定药物的代谢动力学参数,利用几种串联质谱应用方式对药物代谢物的结构进行鉴定,概括了每种方式的优劣,总结了串联质谱在药物代谢物结构鉴定中的应用策略,并对其在药物代谢未来的应用进行了展望。     

用液相色谱-离子阱质谱联用法检测兔体液中阿普唑仑及其主要代谢产物

建立鉴别体液中阿普唑仑及其主要代谢物的高效液相色谱 电喷雾离子阱质谱联用(LC/MSn)法 .采用LC/MSn联用技术 ,检测家兔灌胃给药后血样和尿样中阿普唑仑原形药及其羟基化代谢物、葡萄糖苷酸型结合物 ,得到了它们的色谱、质谱以及特征碎片离子信息 .阿普唑仑的最低检测限小于 1ng .本法专属性强 ,可推广至其他生物检材的测定 ,尤其适用于需快速响应的临床和法医毒物分析     

Semi‐quantitative analysis of tramadol,dextromethorphan, and metabolites in decomposed skeletal tissues by ultra performance liquid chromatography quadrupole time of flight mass spectrometry

The use of filtration/pass‐through extraction (FPTE) and ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC–qTOF–MS) to detect tramadol (TRAM), dextromethorphan (DXM), and metabolites from skeletal remains is described. Rats (n=5) received 50 mg/kg tramadol and were euthanized by CO₂ asphyxiation approximately 30 minutes post‐dose. Rats (n=4) received 75 mg/kg dextromethorphan and were euthanized by CO₂ asphyxiation approximately 45 minutes post‐dose. Remains decomposed to skeleton outdoors and vertebral bones were collected. Bones were cleaned, dried, and pulverized to a fine powder. Bones underwent dynamic methanolic extraction followed by FPTE before analysis using UPLC–qTOF–MS. Recovery was at least 90% of maximal value within the first 10 minutes of methanolic extraction for all samples assayed. Analytical response was measured over the concentration range of 1–500 ng/mL, with precision and bias <20% in triplicate analyses of all calibrators, and a limit of detection of 1 ng/mL for TRAM, DXM, and all metabolites. The vertebral bone analyzed using this method detected TRAM, DXM, and their respective metabolites in all samples analyzed.     

A UPLC–MS/MS application for profiling of intermediary energy metabolites in microdialysis samples—A method for high-throughput

Research within the field of metabolite profiling has already illuminated our understanding of a variety of physiological and pathological processes. Microdialysis has added further refinement to previous models and has allowed the testing of new hypotheses. In the present study, a new ultra-performance liquid chromatography/electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the simultaneous detection and quantification of intermediary energy metabolites in microdialysates was developed. The targeted metabolites were mainly from the citric acid cycle in combination with pyruvic acid, lactic acid, and the ATP (adenosine triphosphate) hydrolysis product adenosine along with metabolites of adenosine. This method was successfully applied to analyze the microdialysates obtained from an experimental animal study giving insight into the hitherto unknown concentration of many interstitial energy metabolites, such as succinic acid and malic acid. With a total cycle time of 3 min, injection to injection, this method permits analysis of a much larger number of samples in comparison with conventional high performance liquid chromatography/tandem mass spectrometry HPLC–MS/MS strategies. With this novel combination where microdialysis and high sensitivity UPLC–MS/MS technique is combined within cardiologic research, new insights into the intermediary energy metabolism during ischemia–reperfusion is now feasible.     

Atrazine metabolite screening in human microsomes: detection of novel reactive metabolites and glutathione adducts by LC-MS

Atrazine (ATZ), one of the most widely used herbicides worldwide, has been the subject of several scientific studies associated with its human and ecological risks. In order to study atrazine's toxicity, the formation of its metabolites and the result of their exposure must be assessed. This relies on our ability to detect and identify all of atrazine's metabolites; however, no previous untargeted screening method has reported the detection of all known metabolites and glutathione conjugates at once. In this study, a compound-specific, postacquisition metabolic screening method was employed following a generic HPLC separation coupled with high resolution time-of-flight mass spectrometry (TOF-MS) to detect Phase I metabolites and glutathione conjugates generated by in vitro human liver microsomal incubations. Our method was designed to be unbiased and applicable to a wide variety of compounds since methods that can detect a broad range of metabolites with high sensitivity are of great importance for many types of experiments requiring thorough metabolite screening. On the basis of incubations with atrazine and three closely related analogues (simazine, propazine, and cyanazine), we have proposed a new Phase I metabolism scheme. All known Phase I transformations of atrazine were successfully detected, as well as a new N-oxidation product. Novel reactive metabolites were also detected as well as their glutathione conjugates. These newly detected species were produced via imine formation on the N-ethyl group, a biotransformation not previously observed for atrazine or its analogues.     

Early human ADME using microdoses and microtracers: bioanalytical considerations

Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time. High radiological doses and the associated costs have been a major obstacle to the routine (and early) use of (14)C despite the recognized advantages of a (14)C-tracer for quantifying drug metabolism and disposition. Accelerator mass spectrometry eliminates this long-standing problem by reducing radioactivity levels while delivering matrix-independent quantitation to attomole levels of sensitivity in small samples or fractionated isolates. Accelerator mass spectrometry and trace (14)C-labeled drugs are now used to obtain early insights into the human metabolism of a drug candidate in ways that were not previously practical. With this article we describe some of our empirically based approaches for regualted bioanalysis and offer perspectives on current applications and opportunities for the future.     

Detection of the designer benzodiazepine flunitrazolam in urine and preliminary data on its metabolism

Designer benzodiazepines have emerged as recreational drugs. They are available via the Internet without control and are found in the form of falsified (fake) medicines. For some of them, limited information concerning their effects, their toxicity, and their detection in bio fluids is available in the literature. For others, nothing has been published, as in the case of flunitrazolam (FNTZ). To gain preliminary data on its elimination parameters in urine and to investigate its metabolism, one of the authors ingested one pink tablet bought on the Internet, after confirming the absence of other compounds and agreement with the labeled dosage (0.25 mg) by nuclear magnetic resonance (NMR). A software algorithm (MetaboLynx, Waters, Milford, MA, USA) was used to predict FNTZ biotransformation and four potential metabolites were proposed: 4‐hydroxy‐FNTZ, desnitro‐FNTZ, 7‐amino‐FNTZ, and 7‐acetamido‐FNTZ. Urine samples were collected over 72 hours following oral administration of one tablet. After liquid/liquid extraction at pH 9.5, FNTZ concentrations were determined using ultra performance liquid chromatography–triple quadrupole–mass spectrometry (UPLC–QqQ–MS/MS). FNTZ remained detectable in hydrolyzed urine for 21 hours after ingestion, with concentrations ranging between 1 and 18 ng/mL. About 3% of the initial dose was excreted in urine as total unchanged FNTZ during this period. In vitro experiments (HLM incubations) were performed using ultra performance liquid chromatography–quadrupole time of flight‐mass spectrometry (UPLC–QTOF–MS) in order to investigate the potential CYP‐ and UGT‐dependent metabolites where only 7‐amino‐FNTZ was detected as the only metabolite. However, in the urine specimens, desnitro‐FNTZ, 7‐acetamido‐FNTZ and 7‐amino‐FNTZ were the main detected compounds. The identification of FNTZ metabolites dramatically improves the detection windows of the drug up to 37 hours.     

超高效液相色谱法检测西红花中非法添加的染料金橙Ⅱ

目的建立检测中药西红花中非法添加的金橙Ⅱ的液质联用鉴别法和超高效液相色谱含量测定法。方法用超高效液相色谱法分离中药西红花中非法添加的金橙Ⅱ,用离子阱进行定性鉴别,流动相为乙腈-0.01 mol/L醋酸铵溶液(30∶70),离子化模式为ESI(+),碰撞能量为30 V;用超高效液相色谱法测定金橙Ⅱ含量,流动相为乙腈-0.01 mol/L醋酸铵溶液(30∶70),检测波长484 nm。结果在拟订的质谱条件下,金橙Ⅱ产生[M+H-Na]+分子离子峰,二级质谱的特征碎片离子明显;定量检测方面,进样量与峰面积的线性范围为5.435～745.0 ng,r=0.999 0,平均加样回收率为98.29%,RSD为1.25%(n=5)。结论液质联用法定性准确、可靠,液相色谱含量测定法简便、快速、准确。     

Characterization of ornidazole metabolites in human bile after intraveneous doses by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry

Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to characterize ornidazole metabolites in human bile after intravenous doses. A liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of the bile level of ornidazole. Bile samples, collected from four patients with T-tube drainage after biliary tract surgery, were prepared by protein precipitation with acetonitrile before analysis. A total of 12 metabolites, including 10 novel metabolites, were detected and characterized. The metabolites of ornidazole in human bile were the products of hydrochloride (HCl) elimination, oxidative dechlorination, hydroxylation, sulfation, diastereoisomeric glucuronation, and substitution of NO₂ or Cl atom by cysteine or N-acetylcysteine, and oxidative dechlorination followed by further carboxylation. The bile levels of ornidazole at 12 h after multiple intravenous infusions were well above its minimal inhibitory concentration for common strains of anaerobic bacteria.     

Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data.

An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.     

Determination of two COX-2 inhibitors in serum and synovial fluid of patients with inflammatory arthritis by ultra performance liquid chromatography–inductively coupled plasma mass spectroscopy and quadrupole time-of-flight mass spectrometry

The determination of two sulphur-containing drugs, the COX-2 inhibitors celecoxib and etoricoxib, in the serum and synovial fluid of inflammatory arthritis patients, is described using a sensitive ultra performance liquid chromatography–inductively coupled plasma mass spectroscopy (UPLC/ICPMS) method. Confirmation of the identity of the analytes in the samples was also performed by electrospray quadruple time-of-flight mass spectrometry in positive electrospray ionisation mode. The two COX-2 inhibitors were extracted from serum and synovial fluid following dilution with acetate buffer (pH 5) and liquid–liquid extraction (LLE) into ethyl acetate. Extracted samples were then analysed using UPLC/ICPMS with sulphur-specific detection. The limit of detection by UPLC/ICPMS was 0.45 ng/ml of sulphur in both serum and synovial fluid. The UPLC/ICPMS method was applied to the analysis of samples from patients receiving either 200 mg/day of celecoxib (2× 100 mg), 90 mg/day etoricoxib or placebo. The range of concentrations detected in the samples for the two drugs was from 0.3 to 3.3 μg/ml.