Full-scan High-resolution Mass Spectrometry Research Articles

Nontarget Insights into the Fate of Cl-/Br-Containing DOM in Leachate during Membrane Treatment.

Halogenated organic compounds in wastewater are persistent and bioaccumulative contaminants of great concern, but few are known at the molecular level. Herein, we focus on nontarget screening of halogenated dissolved organic matter (DOM) in highly concentrated organic matrices of waste leachates and their concentrates. Solid-phase extraction (SPE) was optimized before capturing halogenated signatures via HaloSeeker 2.0 software on mining full-scan high-resolution mass spectrometry (HRMS) fingerprints. This study identified 438 Cl-/Br-containing DOM formulas in 21 leachates and membrane concentrates. Among them, 334 formulas were achieved via SPE with mixed-sorbent cartridges (mixed-SPE), surpassing the 164 formulas achieved through Bond Elut PPL cartridges (PPL-SPE). Herein, only four samples identified via PPL-SPE exhibited a resolution of >50% for extracted Cl-/Br-containing DOM by either SPE. The halogenated DOM constituted 6.87% of the total DOM mass features. Nevertheless, more abundant adsorbable organic halogens deciphered waste leachates and highly concentrated waste streams as reservoirs for halogenated contaminants. Remarkably, 75.7-98.1% of Cl-/Br-containing DOM in primary membrane concentrates remained stable through the secondary membrane treatment, indicating the persistence of these unknown contaminants even post-treatment.

Evidence for solanidine as a dietary CYP2D6 biomarker: Significant correlation with risperidone metabolism.

The extensive variability in cytochrome P450 2D6 (CYP2D6) metabolism is mainly caused by genetic polymorphisms. However, there is large, unexplained variability in CYP2D6 metabolism within CYP2D6 genotype subgroups. Solanidine, a dietary compound found in potatoes, is a promising phenotype biomarker predicting individual CYP2D6 metabolism. The aim of this study was to investigate the correlation between solanidine metabolism and the CYP2D6-mediated metabolism of risperidone in patients with known CYP2D6 genotypes. The study included therapeutic drug monitoring (TDM) data from CYP2D6-genotyped patients treated with risperidone. Risperidone and 9-hydroxyrisperidone levels were determined during TDM, and reprocessing of the respective TDM full-scan high-resolution mass spectrometry files was applied for semi-quantitative measurements of solanidine and five metabolites (M402, M414, M416, M440 and M444). Spearman's tests determined the correlations between solanidine metabolic ratios (MRs) and the 9-hydroxyrisperidone-to-risperidone ratio. A total of 229 patients were included. Highly significant, positive correlationswere observed between all solanidine MRs and the 9-hydroxyrisperidone-to-risperidone ratio (ρ>0.6, P < .0001). The strongest correlation was observed for the M444-to-solanidine MR in patients with functional CYP2D6 metabolism, i.e., genotype activity scores of 1 and 1.5 (ρ 0.72-0.77, P< .0001). The present study shows strong, positive correlations between solanidine metabolism and CYP2D6-mediated risperidone metabolism. The strong correlation within patients carrying CYP2D6 genotypes encoding functional CYP2D6 metabolism suggests that solanidine metabolism may predict individual CYP2D6 metabolism, and hence potentially improve personalized dosing of drugs metabolized by CYP2D6.

Pseudotargeted Metabolomics Approach Enabling the Classification-Induced Ginsenoside Characterization and Differentiation of Ginseng and Its Compound Formulation Products.

The use of diversified ginseng extracts in health-promoting foods is difficult to differentiate, as they share bioactive ginsenosides among different Panax species (e.g., P. ginseng, P. quinquefolius, P. notoginseng, and P. japonicus) and different parts (e.g., root, leaf, and flower). This work was designed to develop a pseudo-targeted metabolomics approach to discover ginsenoside markers facilitating the precise authentication of ginseng and its use in compound formulation products (CFPs). Versatile mass spectrometry experiments on the QTrap mass spectrometer achieved classified characterization of the neutral, malonyl, and oleanolic acid-type ginsenosides, with 567 components characterized. A pseudo-targeted metabolomics approach by multiple reaction monitoring (MRM) of 262 ion pairs could assist to establish key identification points for 12 ginseng species. The simultaneous detection of 14 markers enabled the identification of ginseng from 15 ginseng-containing CFPs. The pseudo-targeted metabolomics strategy enabled better performance in differentiating among multiple ginseng, compared with the full-scan high-resolution mass spectrometry approach.

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.

Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control.

High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 reversed-phase column using mobile phases of 5 mM ammonium formate and acetonitrile. A 7.5 min LC gradient was employed to elute substances and results indicated that the LC method generated sharp and symmetric chromatographic peaks. An in-house equine doping compound database and a spectral library were built to increase method specificity for substances of interest. Five criteria, i.e. accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions (compound database) and HRMS/MS spectra (spectral library), were employed for targeted screening. We utilized these criteria to validate targeted detection of 451 substances within our in-house equine doping compound database. By using all five criteria in screening, the false screening positive rate is significantly reduced. A screening strategy and a Microsoft Excel macro were developed to facilitate interpretation and reporting of results. As the simultaneous acquisition of the full scan HRMS data provides the opportunity for retrospective non-targeted analysis, our findings highlight the use of this novel methodology as a simple, rapid, and reliably reproducible strategy to meet the challenge of identifying an increasing number of doping substances that could potentially impact the integrity of the horse racing community.

Metabolomics by UHPLC-MS: benefits provided by complementary use of Q-TOF and QQQ for pathway profiling.

Non-targeted metabolic profiling using high-resolution mass spectrometry (HRMS) is a standard approach for pathway identification despite technical limitations. To assess the performance of combining targeted quadrupole (QQQ) analysis with HRMS for in-depth pathway profiling. Serum of exercising patients with type 1 diabetes (T1D) was profiled using targeted and non-targeted assays. Non-targeted analysis yielded a broad unbiased metabolic profile, targeted analysis increased coverage of purine metabolism (twofold) and TCA cycle (three metabolites). Our screening strategy combined the benefits of the unbiased full-scan HRMS acquisition with the deeper insight into specific pathways by large-scale QQQ analysis.

HaloSeeker 1.0: A User-Friendly Software to Highlight Halogenated Chemicals in Nontargeted High-Resolution Mass Spectrometry Data Sets.

In the present work, we address the issue of nontargeted screening of organohalogenated chemicals in complex matrixes. A global strategy aiming to seek halogenated signatures in full-scan high-resolution mass spectrometry (HRMS) fingerprints was developed. The resulting all-in-one user-friendly application, HaloSeeker 1.0, was developed to promote the accessibility of associated in-house bioinformatics tools to a large audience. The ergonomic web user interface avoids any interactions with the coding component while allowing interactions with the data, including peak detection (features), deconvolution, and comprehensive accompanying manual review for chemical formula assignment. HaloSeeker 1.0 was successfully applied to a marine sediment HRMS data set acquired on a liquid chromatography-heated electrospray ionization [LC-HESI(-)] Orbitrap instrument ( R = 140 000 at m/z 200). Among the 4532 detected features, 827 were paired and filtered in 165 polyhalogenated clusters. HaloSeeker was also compared to three similar tools and showed the best performances. HaloSeeker's ability to filter and investigate halogenated signals was demonstrated and illustrated by a potential homologue series with C12H xBr yCl zO2 as a putative general formula.

Ultra-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Direct Infusion-High-Resolution Mass Spectrometry for Combined Exploratory and Targeted Metabolic Profiling of Human Urine.

The application of metabolic phenotyping to epidemiological studies involving thousands of biofluid samples presents a challenge for the selection of analytical platforms that meet the requirements of high-throughput precision analysis and cost-effectiveness. Here direct infusion–nanoelectrospray (DI–nESI) was compared with an ultra-performance liquid chromatography (UPLC)–high-resolution mass spectrometry (HRMS) method for metabolic profiling of an exemplary set of 132 human urine samples from a large epidemiological cohort. Both methods were developed and optimized to allow the simultaneous collection of high-resolution urinary metabolic profiles and quantitative data for a selected panel of 35 metabolites. The total run time for measuring the sample set in both polarities by UPLC–HRMS was 5 days compared with 9 h by DI–nESI–HRMS. To compare the classification ability of the two MS methods, we performed exploratory analysis of the full-scan HRMS profiles to detect sex-related differences in biochemical composition. Although metabolite identification is less specific in DI–nESI–HRMS, the significant features responsible for discrimination between sexes were mostly the same in both MS-based platforms. Using the quantitative data, we showed that 10 metabolites have strong correlation (Pearson’s r > 0.9 and Passing–Bablok regression slope of 0.8–1.3) and good agreement assessed by Bland–Altman plots between UPLC–HRMS and DI–nESI–HRMS and thus can be measured using a cheaper and less sample- and time-consuming method. A further twenty metabolites showed acceptable correlation between the two methods with only five metabolites showing weak correlation (Pearson’s r < 0.4) and poor agreement due to the overestimation of the results by DI–nESI–HRMS.

A Case Study Involving U-47700, Diclazepam and Flubromazepam-Application of Retrospective Analysis of HRMS Data.

The number of new psychoactive substances (NPS) available is constantly increasing, making it difficult for toxicology laboratories to keep screening methods up to date. Full scan high-resolution mass spectrometry (HRMS) is a versatile technique which allows for progressive updating of spectral databases to increase the scope of screening. It also allows for retrospective screening of data-specifically, reprocessing of data files using an updated spectral database without the need for re-extraction or reanalysis.The coronial case reported here illustrates the application of retrospective processing of HRMS data in the detection of emerging NPS. A 28-year-old male with a history of illicit drug use was found deceased at home. Initial routine screening of the post-mortem peripheral blood identified only methylamphetamine, amphetamine and trace amounts of lorazepam. A compound with an accurate mass and isotope ratio consistent with the opioid AH-7921 was also detected in the liquid chromatography (LC)-HRMS screen; however; the retention time and mass spectrum did not match the library. Further investigation confirmed the compound to be U-47700, another opioid and structural isomer of AH-7921. Several months later, after additional NPS had been added to the in-house HRMS database, retrospective screening of the HRMS data was performed, revealing the presence of designer benzodiazepines, diclazepam and flubromazepam as well as the psychedelic drug 2,5-dimethoxy-4-chloroamphetamine (DOC). Quantitative analysis gave the following results in peripheral post-mortem blood: U-47700 (330 μg/L), diclazepam (70 μg/L), flubromazepam (10 μg/L), methylamphetamine (290 μg/L) and amphetamine (150 μg/L) (DOC not quantitated). These substances, along with lorazepam and etizolam, were also confirmed in the post-mortem urine and an investigation into blood and urinary metabolites was carried out. All analyses were performed using the same LC-quadrupole-time of flight method. The cause of death was aspiration (of gastric content into airways and lungs) due to mixed drug toxicity.

Trace analysis of multi-class phytohormones in Oryza sativa using different scan modes in high-resolution Orbitrap mass spectrometry: method validation, concentration levels, and screening in multiple accessions.

Phytohormones are signaling and regulating metabolites involved in numerous plant processes, including growth, development, and responses to stress. Currently, the focus is on the analysis of multiple phytohormones in order to characterize crosstalk and hormone signaling networks. In this paper, representative phytohormones of the major classes are simultaneously determined in rice tissues by a generic solid-liquid extraction, followed by liquid chromatography and electrospray ionization high-resolution tandem mass spectrometry using a Q-Exactive™ instrument. After a thorough optimization of the sample preparation, the analytical method was fully validated toward the ultra-trace quantification of six a priori selected plant hormones using three scan modes of the quadrupole-Orbitrap instrument: full-scan high-resolution mass spectrometry, targeted single ion monitoring (t-SIM), and t-SIM followed by data-dependent tandem mass spectrometry. Overall, a similar quantitative performance was noticed for the different scan modes. The analytical method was successfully applied to measure basal phytohormone levels in six different rice accessions, comprising Oryza sativa ssp. japonica, indica, and Oryza glaberrima. Hormone concentrations were higher in shoots than in roots or at least similar. Except for a lower level of salicylic acid in shoots of O. glaberrima versus O. sativa, no other differences in hormone levels could be noticed that were dependent of the (sub)species assignment of the analyzed accessions. Making use of the benefits of full-scan high-resolution mass spectrometry, a first post-run suspect screening was performed, suggesting - based on accurate mass measurements and isotopic patterns - the possible presence of about 50 additional plant hormones in the rice tissues. Graphical abstract ᅟ.

Matrix-effect free quantitative liquid chromatography mass spectrometry analysis in complex matrices using nanoflow liquid chromatography with integrated emitter tip and high dilution factors

Matrix effects are probably the Achilles heel of most quantitative liquid chromatography mass spectrometry (LC–MS) methods based on electrospray ionization. This work reports the evaluation of matrix effects in challenging matrices such as food extracts, human urine or wastewater at different dilution factors using nanoflow liquid chromatography-high resolution mass spectrometry (LC–MS). For this purpose, a suite of representative low-molecular weight compounds such as pesticides, drugs of abuse, sport drugs or environmental contaminants were selected. The approach is based on the use of reversed-phase C18 nano columns furnished with an integrated emitter tip. The nanoflow LC system was combined with full-scan high resolution mass spectrometry using a HRMS (orbitrap) instrument operated at a resolution of 70000. The sensitivity achieved with this configuration enables the implementation of high dilution factors (e.g. 1:20, 1:50 or beyond). When combining nanoflow LC–MS analysis with such high dilution factors (e.g. 1:50), signal suppression was negligible in most cases, so that matrix-matched standards may eventually be skipped, simplifying laboratory workflows by using external calibration in demanding applications such as drug analysis in urine, environmental contaminants in wastewater or pesticide testing in food, thus, eliminating the need for standard addition, matrix-matched calibration or isotopically-labelled standards.

Evaluation of nanoflow liquid chromatography high resolution mass spectrometry for pesticide residue analysis in food

This article reports on the evaluation of nanoflow liquid chromatography-mass spectrometry (LC–MS) for pesticide residue analysis in food. The approach is based on the use of reversed-phase C18nano columns with an integrated emitter, so that separation, ionization and detection are performed minimizing dead volumes. The use of nanoflow not only increases ionization efficiency and minimizes ionization suppression but also boost sensitivity compared to analytical-scale LC–MS methods. The nanoflow LC system was combined with full-scan high resolution mass spectrometry using a Q-Exactive Orbitrap instrument. The analytical performance was assessed for over 60 representative pesticides in five representative commodities (tomato, baby food, orange, fruit-based jam and olive oil). The sensitivity achieved with this configuration enables the implementation of high dilution factors (eg. 1:20, 1:50 or beyond) in pesticide residue workflows without compromising sensitivity, featuring limits of quantitation in the low ng kg−1 range. Using this dilution factors, signal suppression was found negligible in most cases (<10% in most cases, especially with 1:50 dilution), so that matrix-matched standards may be skipped, thus simplifying laboratory workflows. The robustness of the nanoflow LC system and its capability to withstand long analytical runs was also evaluated. Appropriate precision in terms of peak area and retention time was obtained at different concentration levels for over 125 injections without any instrument servicing. The main benefits of the nanoflow liquid chromatography approach are the high sensitivity gain and the outstanding reduction in matrix effects thanks to the high sample dilution factors that can be implemented, along with the substantial reduction in solvent usage.

Shifting the paradigm in gas chromatography mass spectrometry pesticide analysis using high resolution accurate mass spectrometry

In the last 10 years, the majority of the pesticide food control laboratories moved from GC–MS to GC–MS/MS as the preferred analytical technique to address GC amenable compounds. The main reason for this change was the negative impact on the single stage GC–MS analysis caused by the interference from coeluting matrix compounds. The new development of gas chromatography coupled to high resolution accurate mass spectrometry (GC-HRAMS) provides opportunities to overcome such matrix interferences as a result of its much higher selectivity; allowing laboratories to carry out full-scan analysis covering the most exigent analytical performance criteria.The aim of this work is the evaluation of the main analytical performance parameters for the multiresidue screening of pesticide residues by a gas chromatography-Orbitrap™ mass spectrometer in fruits and vegetables. Electron ionization and full-scan high resolution mass spectrometry was selected for this evaluation. Mass resolving power, selectivity, sensitivity, analyte identification and database generation are discussed in light of the analysis of various commodities and a large scope of pesticides. The selected operational conditions provided optimal mass accuracy, within ±1mDa and achieved LOQs of 10μg/kg or lower for practically all the selected pesticides and commodities. Applicability for quantitative residue analysis was verified by the validation of the 210 pesticides in four matrices (tomato, apple, leek and orange). The performance of the GC-EI-full scan Orbitrap HRAMS developed method was confirmed with the analysis of 102 real samples providing good results.

A global inter-laboratory study to assess acquisition modes for multi-compound confirmatory analysis of veterinary drugs using liquid chromatography coupled to triple quadrupole, time of flight and orbitrap mass spectrometry

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of “identification points”. A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors.In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points.

Algal toxin profiles in Nigerian coastal waters (Gulf of Guinea) using passive sampling and liquid chromatography coupled to mass spectrometry

Algal toxins may accumulate in fish and shellfish and thus cause poisoning in consumers of seafood. Such toxins and the algae producing them are regularly surveyed in many countries, including Europe, North America, Japan and others. However, very little is known regards the occurrence of such algae and their toxins in most African countries. This paper reports on a survey of phytoplankton and algal toxins in Nigerian coastal waters.Seawater samples were obtained from four sites for phytoplankton identification, on three occasions between the middle of October 2014 and the end of February 2015 (Bar Beach and Lekki in Lagos State, Port Harcourt in Rivers State and Uyo in Akwa Ibom State). The phytoplankton community was generally dominated by diatoms and cyanobacteria; however several species of dinoflagellates were also identified: Dinophysis caudata, Lingulodinium polyedrum and two benthic species of Prorocentrum.Passive samplers (containing Diaion® HP-20 resin) were deployed for several 1-week periods on the same four sites to obtain profiles of algal toxins present in the seawater. Quantifiable amounts of okadaic acid (OA) and pectenotoxin 2 (PTX2), as well as traces of dinophysistoxin 1 (DTX1) were detected at several sites. Highest concentrations (60 ng OA g−1 HP-20 resin) were found at Lekki and Bar Beach stations, which also had the highest salinities. Non-targeted analysis using full-scan high resolution mass spectrometry showed that algal metabolites differed from site to site and for different sampling occasions. Screening against a marine natural products database indicated the potential presence of cyanobacterial compounds in the water column, which was also consistent with phytoplankton analysis.During this study, the occurrence of the marine dinoflagellate toxins OA and PTX2 has been demonstrated in coastal waters of Nigeria, despite unfavourable environmental conditions, with regards to the low salinities measured. Hence shellfish samples should be monitored in future to assess the risk for public health through accumulation of such toxins in seafood.

Liquid chromatography–full scan-high resolution mass spectrometry-based method towards the comprehensive analysis of migration of primary aromatic amines from food packaging

European Union legislation has established that plastic food contact materials shall not release primary aromatic amines (PAAs), which are toxic compounds and suspected human carcinogens. As valid alternative to existing methods for PAA determination, which are based on spectrophotometric test or targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) approaches, in this study a LC-Orbitrap–full scan-high resolution mass spectrometry (HRMS) method was devised and validated for the determination of migration levels of 22 PAAs from food contact materials, thus exploiting the specificity of accurate mass measurement. Direct injection of the simulant (acetic acid 3%, w/v) into the LC–MS system after migration, without any pre-treatment step, makes the developed method of great value for rapid screening analysis of a large number of amines. A very fast and efficient separation (<11min) of PAAs was achieved. Detection limits in the 0.06–0.7μgkg−1 range were calculated for 17 out of 22 of the investigated PAAs, however obtaining values within 5.3μgkg−1 for the other 5 amines. Good dynamic linear ranges from two to four orders of magnitude (r2≥0.990) were obtained and satisfying results were achieved in terms of intra-day (RSDs<10%) and inter-day repeatability (RSDs<17%). Trueness values in the 70±1–131±5% range proved reliability of the developed method for PAAs quantification also at very low concentration levels. Finally, the method was successfully applied to a range of different real plastic multilayer food packaging materials, noticing in all cases levels below the established limits of detection.

Overcoming matrix effects in electrospray: Quantitation of β-agonists in complex matrices by isotope dilution liquid chromatography–mass spectrometry using singly 13C-labeled analogues

In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of β2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different β2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC–ESI-MS in complex samples. In this procedure singly 13C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of β2-agonists in human urine and bovine liver, even at the sub ngg−1 and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97–114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of 13C1-labeled standards and IPD is a reliable approach for accurate LC–MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry.

Combined data mining strategy for the systematic identification of sport drug metabolites in urine by liquid chromatography time-of-flight mass spectrometry

The development of comprehensive methods able to tackle with the systematic identification of drug metabolites in an automated fashion is of great interest. In this article, a strategy based on the combined use of two complementary data mining tools is proposed for the screening and systematic detection and identification of urinary drug metabolites by liquid chromatography full-scan high resolution mass spectrometry. The proposed methodology is based on the use of accurate mass extraction of diagnostic ions (compound-dependent information) from in-source CID fragmentation without precursor ion isolation along with the use of automated mass extraction of accurate-mass shifts corresponding to typical biotransformations (non compound-dependent information) that xenobiotics usually undergo when metabolized. The combined strategy was evaluated using LC–TOFMS with a suite of nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol, ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline), after single doses administered to rats. The metabolite identification coverage rate obtained with the systematic method (compared to existing literature) was satisfactory, and provided the identification of several non-previously reported metabolites. In addition, the combined information obtained helps to minimize the number of false positives. As an example, the systematic identification of urinary metabolites of propranolol enabled the identification of up to 24 metabolites, 15 of them non previously described in literature, which is a valuable indicator of the usefulness of the proposed systematic procedure.