LC-MS/MS-based multibiomarker approaches for the assessment of human exposure to mycotoxins

Mycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide, and hence represent a major hazard for food and feed safety. To estimate human exposure arising from contaminated food, so-called biomarker approaches have been developed as a complementary biomonitoring tool besides traditional food analysis. The first methods based on radioimmunoassays and enzyme-linked immunosorbent assays as well as on liquid chromatography were developed in the late 1980s and early 1990s for the carcinogenic aflatoxins and in the last two decades further tailor-made methods for some major mycotoxins have been published. Since 2010, there has been a clear trend towards the development and application of multianalyte methods based on liquid chromatography–electrospray ionization tandem mass spectrometry for assessment of mycotoxin exposure made possible by the increased sensitivity and selectivity of modern mass spectrometry instrumentation and sophisticated sample cleanup approaches. With use of these advanced methods, traces of mycotoxins and relevant breakdown and conjugation products can be quantified simultaneously in human urine as so-called biomarkers and can be used to precisely describe the real exposure, toxicokinetics, and bioavailability of the toxins present. In this article, a short overview and comparison of published multibiomarker methods focusing on the determination of mycotoxins and relevant excretion products in human urine is presented. Special attention is paid to the main challenges when analyzing these toxic food contaminants in urine, i.e., very low analyte concentrations, appropriate sample preparation, matrix effects, and a lack of authentic, NMR-confirmed calibrants and reference materials. Finally, the progress in human exposure assessment studies facilitated by these analytical methods is described and an outlook on probable developments and possibilities is presented.

Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing

A method based on the direct injection of diluted urine for the identification and quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide, ethylmorphine, ethylmorphine-6-glucuronide and 6-acetylmorphine (6AM) in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Four deuterium labelled analogues were used as internal standards: morphine-3-glucuronide-D3, morphine-D3, codeine-D3 and 6AM-D3. Twenty microlitre aliquots of urine were mixed with 80 mul of the internal standard solution in autosampler vials and 10 mul was injected. The chromatographic system consisted of a 2.0 x 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/l formic acid. Two product ions produced from the protonated molecular ions were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was below 10% at higher levels for all analytes, but at the reporting limits the variation was above 20% for 6AM, morphine-3-glucuronide and codeine-6-glucuronide. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using authentic urine samples. The two methods agreed almost completely (99%) regarding the identified analytes, but for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by gas chromatography-mass spectrometry.We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for opiates     

A novel approach for LC-MS/MS-based chiral metabolomics fingerprinting and chiral metabolomics extraction using a pair of enantiomers of chiral derivatization reagents

Chiral metabolites are found in a wide variety of living organisms and some of them are understood to be physiologically active compounds and biomarkers. However, the overall analysis of chiral metabolomics is quite difficult due to the high number of metabolites, the significant diversity in their physicochemical properties, and concentration range from metabolite-to-metabolite. To solve this difficulty, we developed a novel approach for chiral metabolomics fingerprinting and chiral metabolomics extraction, which is based on the labeling of a pair of enantiomers of chiral derivatization reagents (i.e., DMT-(S,R)-Pro-OSu and DMT-3(S,R)-Apy) and precursor ion scan chromatography of the derivatives. The multivariate statistics is also required for this strategy. The proposed procedures were evaluated by the detection of a diagnostic marker (i.e., d-lactic acid) using the saliva of diabetic patients. This method was used for the determination of biomarker candidates of chiral amines and carboxyls in Alzheimer's disease (AD) brain homogenates. As the results, l-phenylalanine (L-Phe) and l-lactic acid (L-LA) were identified as the decreased and increased biomarker candidates in the AD brain, respectively. Therefore, the proposed approach seems to be helpful for the determination of non-target chiral metabolomics possessing amines and carboxyls.     

Fully automated standard addition method for the quantification of 29 polar pesticide metabolites in different water bodies using LC-MS/MS

A reliable quantification by LC-ESI-MS/MS as the most suitable analytical method for polar substances in the aquatic environment is usually hampered by matrix effects from co-eluting compounds, which are unavoidably present in environmental samples. The standard addition method (SAM) is the most appropriate method to compensate matrix effects. However, when performed manually, this method is too labour- and time-intensive for routine analysis. In the present work, a fully automated SAM using a multi-purpose sample manager “Open Architecture UPLC®-MS/MS” (ultra-performance liquid chromatography tandem mass spectrometry) was developed for the sensitive and reliable determination of 29 polar pesticide metabolites in environmental samples. A four-point SAM was conducted parallel to direct-injection UPLC-ESI-MS/MS determination that was followed by a work flow to calculate the analyte concentrations including monitoring of required quality criteria. Several parameters regarding the SAM, chromatography and mass spectrometry conditions were optimised in order to obtain a fast as well as reliable analytical method. The matrix effects were examined by comparison of the SAM with an external calibration method. The accuracy of the SAM was investigated by recovery tests in samples of different catchment areas. The method detection limit was estimated to be between 1 and 10 ng/L for all metabolites by direct injection of a 10-μL sample. The relative standard deviation values were between 2 and 10 % at the end of calibration range (30 ng/L). About 200 samples from different water bodies were examined with this method in the Rhine and Ruhr region of North Rhine-Westphalia (Germany). Approximately 94 % of the analysed samples contained measurable amounts of metabolites. For most metabolites, low concentrations ≤0.10 μg/L were determined. Only for three metabolites were the concentrations in ground water significantly higher (up to 20 μg/L). In none of the examined drinking water samples were the health-related indication values (between 1 and 3 μg/L) for non-relevant metabolites exceeded.     

Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam,meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes

Among the new psychoactive substances (NPS), so-called designer benzodiazepines have become of particular importance over the last 2 years, due to their increasing availability on the internet drug market. Therapeutically used nitrobenzodiazepines such as flunitrazepam are known to be extensively metabolized via N-dealkylation to active metabolites and via nitro reduction to the 7-amino compounds. The aim of the present work was to tentatively identify phase I and II metabolites of the latest members of this class appearing on the NPS market, clonazolam, meclonazepam, and nifoxipam, in human urine samples. Nano-liquid chromatography-high-resolution mass spectrometry was used to provide data about their detectability in urine. Data revealed that clonazolam and meclonazepam were extensively metabolized and mainly excreted as their amino and acetamino metabolites. Nifoxipam was also extensively metabolized, but instead mainly excreted as the acetamino metabolite and a glucuronic acid conjugate of the parent. Based on analysis of human urine samples collected in cases of acute intoxication within the Swedish STRIDA project, and samples submitted for routine drug testing, the most abundant metabolites and good targets for urine drug testing were 7-aminoclonazolam for clonazolam, 7-acetaminomeclonazepam for meclonazepam, and 7-acetaminonifoxipam for nifoxipam.     

Optimization of LC-MS/MS using triple quadrupole mass analyzer for the simultaneous analysis of carbosulfan and its main metabolites in oranges

This paper describes an analytical method involving a simple solvent extraction for the simultaneous liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) determination of carbosulfan, its most toxic metabolite -carbofuran -, and its other main metabolites - 3-hydroxycarbofuran, 3-ketocarbofuran, 3-hydroxy-7-phenolcarbofuran, 3-keto-7-phenolcarbofuran, 7-phenolcarbofuran and dibutylamine - in oranges. Chromatography was performed on a Zorbax Bonus-RP (150 mm × 2.1 mm, 5 μm). The mobile phase was a ternary gradient water-methanol-acetonitrile with 1.0 mM ammonium acetate at flow rate of 0.2 ml min⁻¹. The LC separation and MS/MS optimization were studied to select the most appropriate operating conditions. The method developed has also been validated. The limits of quantification (LOQs) were from 1 μg kg⁻¹ for carbofuran to 10 μg kg⁻¹ for 3-keto-7-phenolcarbofuran. Extracts spiked with carbosulfan and its metabolites, at LOQ level, yielded average recoveries in the range 60-94%, with relative standard deviations (R.S.D.s) less than 15%. Calibration curves for carbosulfan and its metabolites (range LOQ-1000LOQ) were linear, with coefficients of correlations better than 0.990. The method was successfully applied to establish the primary degradation products in oranges treated with carbosulfan. The LC-MS/MS method developed is simple, rapid, and suitable for the quantification and confirmation of carbosulfan and seven of its main metabolites in orange at levels lower than 10 μg kg⁻¹.     

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n?=?100), in hospitalized patients receiving blood transfusion (n?=?10), and in athletes (n?=?468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.     

水产品中孔雀石绿、结晶紫及其代谢产物残留量的检测

孔雀石绿（MG）和结晶紫（CV）具有高毒素、高残留和致癌、致畸、致突变等特点,当其进人生物体内,就会产生具有更强危害的隐性孔雀石绿（LMG）和隐性结晶紫（LCV）。鉴于孔雀石绿和结晶紫的危害性,包括我国在内的许多国家都将它们列为水产养殖中的禁用药物。     

Principles for the characterisation and the value assignment of the candidate reference material in the new ISO Guide 35:2017

Accreditation and Quality Assurance - The production of reference materials (RMs) is a key activity for the improvement and maintenance of a worldwide coherent measurement system. General...     

Development of a certified reference material for the content of nitroimidazole parent drugs and hydroxy metabolites in pork meat

Nitroimidazoles have been applied in the past to poultry and pigs to treat protozoan diseases and to combat bacterial infections, but due to adverse health effects their use in food-producing animals has meanwhile been banned in the EU. The request for a certified reference material in a representative matrix was stipulated by the responsible Community Reference Laboratory and is underpinned by the need to improve the accuracy and comparability of measurement data and to establish metrological traceability of analytical results. The Institute for Reference Materials and Measurements (IRMM) has responded to this demand by developing and producing a new certified matrix reference material, ERM-BB124. This incurred lyophilised pork meat material was certified according to ISO guides 34 and 35 for the mass fractions of six nitroimidazole compounds. Processing of the frozen muscle tissue to the final material was accomplished by application of cutting, freeze-drying, mixing and milling techniques. Homogeneity and stability measurements were performed using liquid chromatography tandem mass spectrometry. The relative standard uncertainty due to possible heterogeneity showed to be below 1.8% for all analytes. Potential degradation during transport and storage was assessed by isochronous stability studies. No significant instability was detected at a storage temperature of −20 °C for a shelf-life of 2 years. The certified mass fraction values were assigned upon evaluation of the data acquired in an international laboratory inter-comparison involving 12 expert laboratories using different sample preparation procedures, but exclusively LC-MS/MS methods. Relative standard uncertainty contributions for the characterisation (between-lab variation of mean values) were found to be between 1.6 and 4.8%. Certified values for five analytes were in the range of 0.7 to 6.2 μg kg⁻¹, with expanded relative uncertainties ranging between 7 and 14%. Dimetridazole could be certified as “<0.25 μg kg⁻¹ with a probability of 95%”. All values are traceable to the International System of Units (SI). The material is intended to be used for method validation purposes (including trueness estimation) and for method performance assessment.     

Identification of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the peripheral cannabinoid receptor (CB2) by HPLC-MS/MS

The in vitro microsomal metabolism of JWH-015, a ligand that exhibits a high binding affinity at the peripheral cannabinoid receptor CB₂, has been studied. A total of 22 metabolites were identified and structurally characterized. The metabolites are products of: 1) monohydroxylation on the naphthalene ring (m/z 344, M20 and M21), indole ring (m/z 344, M17 and M18), or the N-alkyl group (m/z 344, M14); 2) arene oxidation leading to dihydrodiols (m/z 362, M12 and M15); 3) dihydroxylation on the naphthalene ring (m/z 360, M7) or indole ring (m/z 360, M13), resulting from a combination of monohydroxylations on both the naphthalene and indole rings (m/z 360, M16), or a combination of monohydroxylations on the naphthalene ring and on the N-propyl group (m/z 360, M9); 4) trihydroxylation (m/z 378, M1, M3, M4, M6, and M10); 5) N-dealkylation (m/z 286, M19); 6) N-dealkylation and monohydroxylation on the naphthalene ring (m/z 302, M11); 7) N-dealkylation and dihydrodiol formation from arene oxidation (m/z 320, M2 and M5); 8) dehydrogenation after monohydroxylation on the N-alkyl group (m/z 326, M22); 9) dehydrogenation and monohydroxylation on the indole ring (m/z 342, M8).     

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT

The iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MSⁿ analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N ³-ethyl-N ¹-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent.     

GC/MS-based metabolomic approach to validate the role of urinary sarcosine and target biomarkers for human prostate cancer by microwave-assisted derivatization

A recent study showed that sarcosine may be potentially useful for the diagnosis and prognosis of prostate cancer (PCa). The aim of this study was to validate diagnostic value of sarcosine for PCa, to evaluate urine metabolomic profiles in patients with PCa in comparison of non-cancerous control, and to further explore the other potential metabolic biomarkers for PCa. Isotope dilution gas chromatography/mass spectrometry (ID GC/MS) metabolomic approach was applied to evaluate sarcosine using [methyl-D₃]-sarcosine as an internal standard. Microwave-assisted derivatization (MAD) together with GC/MS was utilized to obtain the urinary metabolomic information in 20 PCa patients compared with eight patients with benign prostate hypertrophy and 20 healthy men. Acquired metabolomic data were analyzed using a two-sample t test. Diagnostic models for PCa were constructed using principal component analysis and were assessed with receiver–operating characteristic curves. Results showed that the urinary sarcosine level has no statistical difference between the PCa group and the control group. In addition, nine metabolomic markers between the PCa group and the healthy male group were selected, which constructed a diagnostic model with a high area under the curve value of 0.9425. We conclude that although urinary sarcosine value has limited potential in the diagnostic algorithm of PCa, urinary metabolomic panel based on GC/MS assay following MAD may potentially become a diagnostic tool for PCa.     

A CBNM isotopic reference material set for testing linearity of isotope mass spectrometers (CBNM IRM 072/1-15)

Summary Any mass spectrometric measurement system has inherent inaccuracies caused by isotope fractionation in the ion source and non-linearity of the detector and or measurement system. A set of well known triple isotope mixtures is an excellent means to verify and correct non-linearity of the mass spectrometer detector and measurement system after the isotope fractionation has been corrected for. CBNM IRM 072/1-15 is a set of such mixtures. Its preparation and characterization is described. It is available for interested users.

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CBNM Isotopen-Referenzmaterial zur Prüfung der Linearität von Isotopen-Massenspektrometern (CBNM IRM 072/1-15)

     

LC-MS/MS method for the simultaneous quantification of artesunate and its metabolites dihydroartemisinin and dihydroartemisinin glucuronide in human plasma

Artesunate (AS), a hemisuccinate derivative of artemisinin, is readily soluble in water and can easily be used in formulations for parenteral treatment of severe malaria. AS is rapidly hydrolyzed to the active metabolite dihydroartemisinin (DHA) and primarily eliminated by biliary excretion after glucuronidation. To investigate systematically the AS metabolism and pharmacokinetics, a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of AS and its metabolites DHA and DHA glucuronide (DHAG) in human plasma samples was developed. Compared to previous methods, our method includes for the first time the quantification of the glucuronide metabolite using a newly synthesized stable isotope-labeled analogue as internal standard. Sample preparation was performed with only 50 μL plasma by high-throughput solid-phase extraction in the 96-well plate format. Separation of the analytes was achieved on a Poroshell 120 EC-C₁₈ column (50*2.1 mm, 2.7 μm, Agilent Technologies, Waldbronn, Germany). The method was validated according to FDA guidelines. Calibration curves were linear over the entire range from 1 to 2,500 nM (0.4–961.1 ng/mL), 165 to 16,500 nM (46.9–4,691.8 ng/mL), and 4 to 10,000 nM (1.8–4,604.7 ng/mL) for AS, DHA, and DHAG, respectively. Intra- and interbatch accuracy, determined as a deviation between nominal and measured values, ranged from ?5.7 to 3.5 % and from 2.7 to 5.8 %, respectively. The assay variability ranged from 1.5 to 10.9 % for intra- and interbatch approaches. All analytes showed extraction recoveries above 85 %. The method was successfully applied to plasma samples from patients under AS treatment.

A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry

Discovery stage studies that address issues of absorption, distribution, metabolism and excretion (ADME) are vital for lead optimization resulting in new drug candidates. Often pharmacokinetics (PK) is assessed in these experiments without regard for the metabolism of the compound or the potential for metabolites to circulate in vivo. This work presents a strategy for drug level determination and detection of metabolites using dried blood spots for sample collection. Initially, metabolites are detected from microsomal incubations and characterized using tandem mass spectrometry. Data dependent enhanced MS and enhanced product ion (EMS-EPI) scanning with dynamic background subtraction was used on a hybrid quadruple linear ion trap mass spectrometer. On-the-fly background subtraction greatly improved the detection of metabolites. These data were used to build a multiple reaction monitoring (MRM) method for the parent and metabolites. MRM-EPI scanning was used to analyze the extracted dried blood spots from the PK study. Circulating metabolites were detected using MRM and their identities confirmed on the basis of fragment ion spectra collected simultaneously. The use of dried blood spots provides a means for re-analysis of PK samples for metabolite identification without the need for complex sample storage and preparation. Both parent compound and metabolite information can be collected in these studies, resulting in a savings of time and resources.     

酒类分析专用柱气相色谱仪校准用标准物质的选择

对酒类分析专用柱气相色谱仪校准用标准物质进行了研究.分别用乙醇中甲醇、乙醇中叔戊醇和甲苯中苯标准溶液校准配有酒类分析专用聚乙二醇石英毛细管柱的气相色谱仪,色谱峰面积相对标准偏差、检出限均满足检定校准技术要求.乙醇中甲醇标准溶液的色谱峰对称性差,甲苯中苯标准溶液的色谱响应值最低.乙醇中叔戊醇标准溶液是理想的聚乙二醇酒类分...     

LC-MS/MS method for the quantification of myo- and chiro-inositol as the urinary biomarkers of insulin resistance in human urine

A simple LC‐MS/MS method has been developed and validated for the quantification of endogenous myo‐ and chiro‐inositol in human urine. myo‐ and chiro‐Inositol were completely resolved from other carbohydrates and there were no interference peaks in human urine. The correlation coefficient (n = 3) was greater than 0.9991 over the range 0.05–25.0 µg/mL with the weighted (1/C²) least square method. Precision (%RSD) and accuracy (%RE) were 0–10.0% and 0–6.0% for the intra‐day assay (n = 5) and 0–14.3% and 0–10.0% for the inter‐day assay (n = 5). myo‐ and chiro‐Inositol have been shown to be stable in human urine stored at room temperature and for three freeze–thaw cycles. Copyright © 2011 John Wiley & Sons, Ltd.