Exploring the potential of using ion mobility-mass spectrometry to separate matrix interferences from analytes in food control.

During residue analysis in complex matrices for food safety purposes, interfering signals can sometimes overlap with those of the analyte of interest. Access to an additional separation dimension besides chromatographic and mass separation, such as ion mobility, can aid in removing interfering signals, allowing for correct analyte identification in these cases. In our laboratory, during routine LC-MS/MS analysis of liver samples for growth promoter residues, an interfering signal was found that matches the retention time and m/z values for stanozolol, a synthetic anabolic steroid. In the present work, the performance of a liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS) method has been evaluated to study whether this LC-MS/MS false positive in liver samples could be eliminated by LC-IM-MS analysis. A cyclic ion mobility system already allowed the separation of stanozolol from the interfering peak after only one pass, showing a significant improvement compared to the conventional LC-MS/MS method. Additionally, collisional cross section (CCS) values were calculated and successfully compared with those from literature for identification purposes, eventually allowing both the identification and quantification of stanozolol in this complex matrix.

Comparison of Compound Identification Tools Using Data Dependent and Data Independent High-Resolution Mass Spectrometry Spectra.

Liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) is a frequently applied technique for suspect screening (SS) and non-target screening (NTS) in metabolomics and environmental toxicology. However, correctly identifying compounds based on SS or NTS approaches remains challenging, especially when using data-independent acquisition (DIA). This study assessed the performance of four HRMS-spectra identification tools to annotate in-house generated data-dependent acquisition (DDA) and DIA HRMS spectra of 32 pesticides, veterinary drugs, and their metabolites. The identification tools were challenged with a diversity of compounds, including isomeric compounds. The identification power was evaluated in solvent standards and spiked feed extract. In DDA spectra, the mass spectral library mzCloud provided the highest success rate, with 84% and 88% of the compounds correctly identified in the top three in solvent standard and spiked feed extract, respectively. The in silico tools MSfinder, CFM-ID, and Chemdistiller also performed well in DDA data, with identification success rates above 75% for both solvent standard and spiked feed extract. MSfinder provided the highest identification success rates using DIA spectra with 72% and 75% (solvent standard and spiked feed extract, respectively), and CFM-ID performed almost similarly in solvent standard and slightly less in spiked feed extract (72% and 63%). The identification success rates for Chemdistiller (66% and 38%) and mzCloud (66% and 31%) were lower, especially in spiked feed extract. The difference in success rates between DDA and DIA is most likely caused by the higher complexity of the DIA spectra, making direct spectral matching more complex. However, this study demonstrates that DIA spectra can be used for compound annotation in certain software tools, although the success rate is lower than for DDA spectra.

Occurrence of resorcyclic acid lactones in porcine urine: discrimination between illegal use and contamination.

Zeranol (α-zearalanol, α-ZAL), is a resorcyclic acid lactone (RAL). Its administration to farm animals to improve meat production has been prohibited in the European Union due to the potential risk to human health. However, it has been demonstrated that α-ZAL may be present in livestock animals due to Fusarium fungi that produce fusarium acid lactones contamination in feed. The fungi produce a small amount of zearalenone (ZEN), which is metabolized to zeranol. The potential endogenous origin of α-ZAL makes it difficult to correlate positive samples to a potential illicit treatment with α-ZAL. We present two experimental studies that investigated the origin of natural and synthetic RALs in porcine urine. Urine samples from pigs that were either fed with ZEN-contaminated feed or administered α-ZAL by injection were analyzed by liquid chromatography coupled to tandem mass spectrometry, with the method validated according to Commission Implementing Regulation (EU) 2021/808. The data show that although the concentration of α-ZAL in the ZEN feed-contaminated samples is significantly lower than in the illicit administration samples, α-ZAL can occur in porcine urine via natural metabolism. Additionally, the feasibility of using the ratio of forbidden/fusarium RALs in porcine urine as a reliable biomarker for illicit treatment with α-ZAL administration was evaluated for the first time. This study demonstrated that the obtained ratio in the contaminated ZEN feed study was close to 1, while in the illegally administered α-ZAL samples the ratio is always higher than 1 (up to 135). Therefore, this study proves that the ratio criteria (already used when a forbidden RAL is detected in bovine urine) may also be used for porcine urine.

Immunoaffinity Plastic Blade Spray Mass Spectrometry for Rapid Confirmatory Analysis of Food Contaminants.

The lack of chromatographic separation in ambient and direct mass spectrometry (MS) ionization techniques jeopardizes the overall selectivity of the developed methods. Incorporating a biosensing element at the ionization source could compensate for that inherent lack of selectivity. Thus, a simplified immunoaffinity-direct MS technique was developed, immunoaffinity blade spray (iBS), featuring a conductive polystyrene blade material. In iBS, the generic coating used in conventional coated blade spray is replaced with a layer of highly specific monoclonal antibodies (mAbs), while the stainless steel is replaced with conductive polystyrene to allow for simple ELISA platelike hydrophobic immobilization of mAbs. Because of its high relevance for climate change-induced food safety issues, the mycotoxin deoxynivalenol (DON) was chosen as a model substance. Following a rapid extraction from wheat flour, DON is immuno-captured, and the blade is positioned in front of the MS for direct iBS-MS/MS analysis. The method's applicability was demonstrated by analyzing spiked and incurred wheat flour samples, omitting the need for time-consuming chromatographic separation. Apart from DON, cross-reacting DON conjugates could be successfully analyzed as well. The direct iBS-MS/MS method is generic and adaptable to detecting any analyte in sample extracts, provided that specific mAbs are available.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

Insights of ion mobility spectrometry and its application on food safety and authenticity: A review.

Ion mobility spectrometry (IMS) is gaining importance in the field of food safety and authenticity in recent years due to its main potential to overcome the challenges that arise from the complexity of food matrices. For many years, IMS has been used as a stand-alone analytical detector due to its quick response, high sensitivity, and portability, and stand-alone applications in food analysis have been explored in recent years. At the same time, IMS hyphenation to mass spectrometry (MS) techniques, usually combined with liquid or gas chromatography (LC/GC), provides an additional dimension to separate isobaric compounds and thus improves method selectivity. Besides, with such ion mobility - mass spectrometry (IM-MS) methods, background noise decreases, increasing method sensitivity, and it provides complementary information to mass spectra and retention time with the collision cross section (CCS). The development of CCS databases within the food safety field would even permit the identification of compounds in non-targeted approaches. Furthermore, it would increase the confidence of control laboratories when determining a sample as non-compliant. Therefore, the number of applications by IMS on food safety and authenticity has increased remarkably in recent years. This review provides the general insights of IMS with the current state and recent approaches for its performance improvement and a general outlook of its applicability in food safety and authenticity.

Simplified screening approach of anabolic steroid esters using a compact atmospheric solid analysis probe mass spectrometric system.

Due to the absence of chromatographic separation, ambient ionization mass spectrometry had the potential to improve the throughput of control laboratories in the last decades and will soon be an excellent approach for on-site use as well. In this study, an atmospheric solids analysis probe (ASAP) with a single quadrupole mass analyzer has been evaluated to identify anabolic steroid esters rapidly. Sample introduction, applied scan time, and probe temperature were optimized for sensitivity. The in-source fragmentations of seventeen selected steroid esters, commonly found in illicit samples, were determined by applying different cone voltages (12, 20, 30, and 40 V). A spectral library was created for these steroid esters based on the four stages of in-source fragmentation spectra. The applicability of this method was demonstrated for the rapid identification of steroid esters in oily injection solutions, providing test results in less than 2 min.

Immuno-Enriched Microspheres - Magnetic Blade Spray-Tandem Mass Spectrometry for Domoic Acid in Mussels.

Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

Discrimination between the exogenous and endogenous origin of thiouracil in farm animals, the final chapter?

Thiouracil (2-thiouracil) is a thyreostatic compound that can be used as an illegal growth promoter. In bovine, porcine and other farm animals, low concentrations of thiouracil are detected in urine. There is much debate on which concentrations can be considered to originate from feed ('natural') and which concentrations are caused by the illegal administration of thiouracil for growth-promoting purposes. Currently, a threshold value of 10 µg/L in urine is applied. The threshold value is based on epidemiological data. Data on thiouracil from animals treated with thiouracil is scarce. We conducted a study whereby animals were fed with rapeseed, rapeseed with thiouracil, or regular feed with thiouracil (low and high concentration). It was determined that administration of thiouracil leads to concentrations higher than the current 10 µg/L threshold of thiouracil and its metabolites in urine during treatment. Animals fed with rapeseed showed higher thiouracil concentrations than the control group, mostly above 10 µg/L and in some cases above 30 µg/L. In the discovery study, several biomarkers for thiouracil treatment were tentatively identified and confirmed with reference standards. One metabolite was identified as indicative for thiouracil abuse, namely 6-methyl-thiouracil. Another metabolite, 4-thiouracil, was indicative for endogenous formation and did not increase during 2-thiouracil treatment. 6-Methyl-thiouracil was not found in urine samples from the Dutch routine control programmes that contained (endogenous) 2-thiouracil above the threshold value. However, 4-thiouracil was found at high concentrations in the same samples when 2-thiouracil was present. This study's overall conclusion is that the threshold value for thiouracil in bovine urine samples should be set at 10 µg/L and for porcine urine samples at 30 µg/L. Also, confirmation of 6-methyl-thiouracil and 4-thiouracil should be used as indicators for exogenous or endogenous origin in routine control monitoring programmes.

Hand-Held Diode Laser for On-Site Analysis Using Transportable Mass Spectrometry.

A hand-held laser diode thermal desorption electrospray ionization (LDTD-ESI) mass spectrometry (MS) method was developed for rapid screening of illegal substances in solid samples. To achieve that, a simple, inexpensive, battery-powered surgical laser diode at 940 nm was employed to ablate the solid samples. The potential of using a black polytetrafluoroethylene substrate to enhance the analytes' desorption to the gas phase was investigated and demonstrated. Among the optimized ESI parameters, the solvent (methanol/water, 50:50, v/v) and the flow rate (50 µL h-1) were critical to obtain the best sensitivity. The applicability was demonstrated for the rapid identification of selective androgen receptor modulators (SARMs) in pills and powders based on accurate mass measurements by time-of-flight MS. Also, the hand-held LDTD-ESI was combined with a transportable single quadrupole MS. The same SARMs samples were analyzed, and identifications were based on in-source cone voltage fragmentation patterns observed. These initial results demonstrate the applicability of the developed simplified LDTD-ESI MS method for future on-site testing of organic compounds in solid samples.

Detection of methionine- and alanine-recombinant bovine somatotropins and their induced antibodies in serum and milk of cows suggests blood-milk barrier specificity for these compounds.

The elimination of recombinant bovine somatotropin (rbST) and its induced antibodies through milk of 2 formulations is studied to propose a control strategy for its use or abuse. Two dairy cows were treated with alanine-rbST (Ala-rbST), which is identical to endogenous bovine somatotropin, and ten dairy cows were treated with methionine-rbST (Met-rbST), which differs by 1 amino acid from endogenous bovine somatotropin. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to measure rbST at a decision limit (CCα) of 0.8 ng/mL and 2.3 ng/mL for serum and milk, respectively. The results show that the administered Ala-rbST is transferred from blood to milk but that this is not the case for Met-rbST. This suggests a blood-milk barrier-related specificity for these compounds. In addition, rbST-induced antibodies were formed in animals treated with Ala-rbST and those treated with Met-rbST. In both treatments, the rbST-induced antibodies were transferred from blood to milk, showing no blood-milk barrier specificity for these antibodies. These elimination patterns show that, for enforcement purposes, the detection of rbST-induced antibodies in tank milk can serve to screen for rbST administration, and subsequent confirmatory serum analysis by LC-MS/MS is needed to identify whether Ala-rbST or Met-rbST has been used.

USB-Powered Coated Blade Spray Ion Source for On-Site Testing Using Transportable Mass Spectrometry.

On-site testing in food analysis using mass spectrometry (MS) requires miniaturization of vacuum systems, mass analyzers, sample cleanup, and ionization sources. In this study, a simple coated blade spray (CBS) ion source was developed that enables high voltage generation on the blade by ubiquitous certified (micro-)USB On-The-Go devices like smartphones, tablets, and power banks. CBS is capable of performing both analyte enrichment by solid-phase microextraction (SPME) material coated on the metal substrate and direct-spray ionization. The USB-CBS device was used on two different MS systems, a transportable single-quadrupole and a benchtop triple-quadrupole tandem MS. Various characteristics of the USB-CBS device, including high voltage generation and angular positioning, were studied. The potential of the newly developed device for food safety applications is demonstrated by banned and regulated veterinary drugs such as ß-agonists and sulfonamide antibiotics, covering a wide range of molecular weights and polarities. The results highlight the potential of the developed, simplified, inexpensive (less than 10 USD), and universal vendor-independent USB-powered CBS ion source coupled with MS(/MS) systems for semiquantitative applications, in laboratories, and in future on-site food quality and safety testing. Apart from that, most likely on-site environmental, biomedical, and forensic testing will also benefit from this USB-CBS instrumental development that is compatible with any atmospheric inlet MS system.

Evaluation of attenuation of pharmaceuticals, toxic potency, and antibiotic resistance genes in constructed wetlands treating wastewater effluents.

The performance of constructed wetlands (CWs) in the removal of pharmaceutically active compounds (PhACs) is generally evaluated on the basis of chemical analysis. In this work, we used a combination of chemical, toxicological, and molecular analyses to assess the attenuation of PhACs, toxic potency and antibiotic resistance genes (ARGs) in a field study of three CWs serving as tertiary treatment of wastewater treatment plants. First, 17 PhACs were analysed chemically, of which 14 were detected and seven at concentrations >0.1µg/l. Even though some of the individual PhACs were moderately or highly removed in the CWs investigated, median removal of overall PhACs was approximately 50% in the vertical subsurface flow CW (VSF-CW) with a lower hydraulic loading rate while the removal in the other two free water surface flow CWs (SF-CWs) was negligible. Second, toxic potency of wastewater extracts was assessed in a range of bioassays. Estrogenicity was overall attenuated in CWs, while the neurotoxic potency of wastewater extracts did not decrease after passage through the two CWs investigated. Third, the VSF-CW and one of the SF-CW showed a positive removal of an integrase gene and three ARGs tested. The increased concentrations of ARGs in the other SF-CW, as well as the increase of total bacteria in all CWs, may relate to regrowth of resistance-carrying bacteria. Finally, multivariate analysis shows that most PhACs are positively correlated to the observed toxic potency. Additionally, low removal of organics and nutrients seems to parallel with low removal of PhACs. ARGs positively correlated with organics, nutrients and some PhACs, and the integrase gene but not to the respective antibiotics. The insufficient removal of PhACs, toxic potency, and ARGs indicates the need of an optimal design of CWs as tertiary treatment facilities.

Enhancing detectability of anabolic-steroid residues in bovine urine by actively modulated online comprehensive two-dimensional liquid chromatography - high-resolution mass spectrometry.

In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices. Compared to one-dimensional liquid chromatography (1D-LC), LC × LC methods offer higher separation power, thanks to the combined effect of two different selectivities and a higher peak capacity. However, when using state-of-the-art LC × LC instrumentation, the price paid for the increase in separation power is a decrease in sensitivity and detectability of trace-level analytes. This can be ascribed to the sample dilution that takes place during each of the two chromatographic steps. The way in which fractions are collected and transferred from the first to the second column is also of paramount importance, especially the volume and the solvent composition of the fractions injected in the second column. To overcome the detection limitation, we present an active-modulation strategy, based on concentrating the fractions of the first-dimension effluent using a modulation interface that employs trap columns. We obtained a signal enhancement for anabolic-steroid compounds in a bovine-urine sample by a factor of 2.4-7.6 and an increase in the signal-to-noise ratio up to a factor of 7 in comparison with a standard loop-based modulation interface. In addition, thanks to the increased sensitivity of our method, a substantially larger number of peaks were detected (76 vs. 36). Moreover, we could reduce the solvent consumption by a factor of three (160 mL vs. 500 mL per run).

Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and their and the corresponding N-oxides (PAs-ox) are genotoxic plant metabolites which can be present as unwanted contaminants in food products of herbal origin like tea and food supplements. PAs and PAs-ox come in a wide variety of molecular structures including many structural isomers. For toxicity assessment it is important to determine the composition of a sample and to resolve all isomeric PAs and PAs-ox, which is currently not possible in one liquid or gas chromatographic (LC or GC) run. In this study an online two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC QToF-MS) method was developed to resolve isomeric PAs and PAs-ox. After comprehensive column and mobile phase selection a polar endcapped C18 column was used at pH 3 in the first dimension, and a cross-linked C18 column at pH 10 in the second dimension. Injection solvents, column IDs, flow rates and temperatures were carefully optimized. The method with column selection valve switching described in this study was able to resolve and visualize 20 individual PAs/PAs-ox (6 sets of isomers) in one 2D-LC QToF-MS run. Moreover, it was shown that all isomeric PAs/PAs-ox could be unambiguously annotated. The method was shown to be applicable for the determination and quantification of isomeric PAs/PAs-ox in plant extracts and could be easily extended to include other PAs and PAs-ox.

Metabolism of Ibuprofen by Phragmites australis: Uptake and Phytodegradation.

This study explores ibuprofen (IBP) uptake and transformation in the wetland plant species Phragmites australis and the underlying mechanisms. We grew P. australis in perlite under greenhouse conditions and treated plants with 60 µg/L of IBP. Roots and rhizomes (RR), stems and leaves (SL), and liquid samples were collected during 21 days of exposure. Results show that P. australis can take up, translocate, and degrade IBP. IBP was completely removed from the liquid medium after 21 days with a half-life of 2.1 days. IBP accumulated in RR and was partly translocated to SL. Meanwhile, four intermediates were detected in the plant tissues: hydroxy-IBP, 1,2-dihydroxy-IBP, carboxy-IBP and glucopyranosyloxy-hydroxy-IBP. Cytochrome P450 monooxygenase was involved in the production of the two hydroxy intermediates. We hypothesize that transformation of IBP was first catalyzed by P450, and then by glycosyltransferase, followed by further storage or metabolism in vacuoles or cell walls. No significant phytotoxicity was observed based on relative growth of plants and stress enzyme activities. In conclusion, we demonstrated for the first time that P. australis degrades IBP from water and is therefore a suitable species for application in constructed wetlands to clean wastewater effluents containing IBP and possibly also other micropollutants.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial - the potential of enantiomeric separation for doping control analysis.

The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes' claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.

Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

Evaluation of the discriminative potential of a novel biomarker for estradiol treatments in bovine animals.

The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry (GC-C/IRMS). A serum dipeptide composed of pyroglutamic acid and phenylalanine was identified as a potential biomarker of estradiol treatments in adult cows. To evaluate its potential to pinpoint suspicious samples, samples from prepubertal females under different estrogenic treatments have been analyzed. The results confirmed the up-regulation of the dipeptide in adult bovines. The 2-week-old females exhibited short-lasting responses only in a few animals. The 6-month-old female showed a delayed but clear increase on the biomarker level. The composition of the anabolic preparations, the dose, and/or the administration route are possible additional reasons for the reduced response in young animals. A comparison to previous results reported by various researchers is included.

Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach.

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17ß-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.