Reproducibility assessment for a broad spectrum drug screening method from urine using liquid chromatography time-of-flight mass spectrometry
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ABSTRACT:
SUBMITTER: Metushi I
PROVIDER: S-EPMC11322752 | biostudies-literature | 2018 Feb
REPOSITORIES: biostudies-literature
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Project description:BackgroundLiquid chromatography-mass spectrometry (LC-MS) and tandem LC-MS (LC-MS/MS) are increasingly used in toxicology laboratories as a complementary method to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ultraviolet detection (LC-UV) for comprehensive drug screening (CDS). This study was designed to characterize the sensitivity and specificity of three LC-MS(/MS) vendor-supplied methods for targeted CDS and identify the current limitations associated with the use of these technologies.MethodsFive methods for broad spectrum CDS, including LC-UV (REMEDi), full scan GC-MS, LC-MS (ZQ-Mass Detector with MassLynx-software), LC-QTRAP-MS/MS (3200-QTRAP with Cliquid-software) and LC-LIT-MS/MS (LXQ Linear Ion Trap with ToxID-software) were evaluated based on their ability to detect drugs in 48 patient urine samples.ResultsThe tandem MS methods identified 15% more drugs than the single stage MS or LC-UV methods. Use of two broad spectrum screening methods identified more drugs than any single system alone. False negatives and false positives generated by the LC-MS(/MS) software programs were identified upon manual review of the raw data.ConclusionsThe LC-MS/MS methods detected a broader menu of drugs; however, it is essential to establish manual data review criteria for all LC-MS(/MS) drug screening methods. Use of an EI-GC-MS and ESI-LC-MS/MS combination for targeted CDS may be optimal due to the complementary nature of the chromatographic and ionization techniques.
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Project description:In this study, methasterone urinary metabolic profiles were investigated by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. A healthy male volunteer was asked to take the drug and liquid-liquid extraction was employed to process urine samples. Chromatographic peaks for potential metabolites were hunted out with the theoretical [M - H](-) as a target ion in a full scan experiment and actual deprotonated ions were studied in targeted MS/MS experiment. Fifteen metabolites including two new sulfates (S1 and S2), three glucuronide conjugates (G2, G6 and G7), and three free metabolites (M2, M4 and M6) were detected for methasterone. Three metabolites involving G4, G5 and M5 were obtained for the first time in human urine samples. Owing to the absence of helpful fragments to elucidate the steroid ring structure of methasterone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was employed to obtain structural information of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and the potential structure was inferred using a combined MS method. Metabolite detection times were also analyzed and G2 (18-nor-17β-hydroxymethyl-2α, 17α-dimethyl-androst-13-en-3α-ol-ξ-O-glucuronide) was thought to be new potential biomarker for methasterone misuse which can be detected up to 10 days.
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Project description:The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.
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Project description:BackgroundValid and precise measures of androgen concentrations are needed for etiologic studies of hormonally-related cancers. We developed a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with two sample preparations to measure 11 androgens, including adrenal and gonadal androgenic precursors and their 5α-reduced metabolites.MethodsAndrogen levels were measured in serum from 20 healthy volunteers (5 men, 10 premenopausal women, 5 postmenopausal women). Two blinded, randomized aliquots per individual were assayed in each of three batches. A fourth batch of samples was measured at an external laboratory using comparable methodology to measure 9 of the 11 androgens. Coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated from the individual components of variance. Comparability of 9 androgens across laboratories was assessed using Spearman ranked correlations, Deming regression and bias plots.ResultsThe laboratory CVs were <5% and ICCs were uniformly high (>95%) for all androgens measured across sex/menopausal status groups. Spearman ranked correlations for 9 hormones measured in the comparison laboratory were high (>0.85), suggesting good agreement.ConclusionOur high-performance LC-MS/MS assays of 11 androgens, including adrenal and gonadal androgenic precursors and their 5α-reduced metabolites demonstrated excellent laboratory reproducibility, and good comparability with an established method that measured 9 of the 11 hormones tested. The serum androgen metabolite assays are suitable for use in epidemiologic research.
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Project description:Volatile thiols give a unique flavor to foods and they have been extensively studied due to their effects on sensory properties. The analytical assay of volatile thiols in food is hindered by the complexity of the matrix, and by both their high reactivity and their typically low concentrations. A new ultraperformance liquid chromatography (UPLC) strategy has been developed for the identification and quantification of volatile thiols in Chinese liquor (Baijiu). 4,4'-Dithiodipyridine reacted rapidly with eight known thiols to form derivatives, which provided a diagnostic fragment ion (m/z 143.5) for tandem mass spectrometry (MS/MS). To screen for new thiols, Baijiu samples were analyzed by means of UPLC-MS/MS screening for compounds exhibiting the diagnostic fragment ion (m/z X→143.5). New peaks with precursor ions of m/z 244, 200 and 214 were detected. Using UPLC with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and authentic standards, ethyl 2-mercaptoacetate, 1-butanethiol, and 1-pentanethiol were identified in Baijiu for the first time. Commercial Baijiu samples were analyzed with the new method and the distribution of 11 thiols was revealed in different Baijiu aroma-types. The aroma contribution of these thiols was evaluated by their odoractivity values (OAVs), with the result that 7 of 11 volatile thiols had OAVs > 1. In particular, methanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol had relatively high OAVs, indicating that they contribute significantly to the aroma profile of Baijiu.
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Project description:Aromatic amines are widely used in personal care products and human exposure to this class of chemicals is widespread. Bioanalytical methods to determine trace levels of aromatic amines in human urine are scarce. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine 39 primary aromatic amines (AAs) along with nicotine and cotinine in human urine. Chromatographic separation of the 41 analytes was achieved on an Ultra Biphenyl (100 mm × 2.1 mm, 5 µm) column. Mass spectrometry was operated in electrospray ionization positive ion multiple reaction monitoring (MRM) mode. The method exhibited excellent linear dynamic range (0.1-50 ng/mL) with correlation coefficients (r) > 0.999 for all analytes. Urine samples (2 mL) were hydrolyzed using 10 M NaOH at 95 °C for 15 h and target analytes were extracted using methyl-tert-butyl ether (MTBE). Addition of 15 µL of 0.25 M HCl to the sample extracts improved the recoveries of several target analytes. The method was validated through the analysis of fortified quality control (QC) samples and a certified standard reference material (SRM). Relative recoveries (%) of target analytes fortified in QC samples were in the range of 75-114% for 37 of the 41 analytes while the other analytes exhibited lower recoveries (16-74%). The limits of detection (LOD) and limits of quantification (LOQ) of target analytes were in the range of 0.025-0.20 ng/mL and 0.1-1.0 ng/mL, respectively. Intra-day and inter-day precision of the method assessed through the analysis of fortified urine QC samples at three different concentrations were < 11.7% and < 15.9% (measured as RSD), respectively. The method was applied in the analysis of urine samples from the general population and known smokers; aniline, para-anisidine, para-toluidine, ortho/meta-toluidine, 3-chloroaniline, 4-chloroaniline, 3,4-dichloroaniline, and 4,4'-methylenedianiline were found in all smoker's urine at sum concentrations ranging from 0.04 to 9.16 ng/mL.
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Project description:The cooked meat carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and their principal metabolites produced by cytochrome P450 and/or uridine diphosphate glucuronosyl transferases were simultaneously measured at the parts per trillion level in urine of omnivores, by ultraperformance liquid chromatography (UPLC) with a Michrom Advance CaptiveSpray source and a triple stage quadrupole mass spectrometer. Quantitation was performed in the selected reaction monitoring mode. The UPLC method is much more rapid and sensitive than our earlier capillary HPLC method: the duty cycle of the UPLC method is 19 min compared to 57 min for capillary HPLC. The performance of the UPLC assay was evaluated with urine samples from three subjects over 4 different days. The intraday and interday precisions of the estimates of PhIP, MeIQx, and their metabolites, reported as the coefficients of variation, were ≤10%. The limit of quantification (LOQ) values for PhIP and MeIQx were about 5 pg/mL, whereas the LOQ values of their metabolites ranged from 10 to 40 pg/mL. Furthermore, the identities of the analytes were corroborated by acquisition of full scan product ion spectra, employing between 0.5 and 5 pg of analyte for assay.
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Project description:A untargeted metabolomics approach was proposed in this study based on ultra-high performance liquid chromatography quadrupole time-of-flight (UHPLC-QTOF) and rapid evaporative ionization mass spectrometry (REIMS) to discriminate lamb and mutton meat and investigate their subtle metabolic differences, considering the higher popularity of lamb meat than mutton in the market. Multivariate statistical analysis was performed for data processing in order to distinguish between the two sample types. A total of 42 potential metabolites (20 in positive and 22 in negative ion mode) were defined for UHPLC-QTOF analysis, which provided references for discriminating the two kinds of meat. Furthermore, three potential markers were tentatively identified using LC/MS data against chemical databases. In addition, 14 potential metabolites were putatively identified in negative ion mode using the LipidMaps database. Meanwhile, the data-driven soft independent modeling of class analogy (DD-SIMCA) model was established, which could rapidly differentiate non-pretreated lamb meat and mutton with 92% specificity, rendering REIMS a promising technique for meat identification.
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