Supported liquid extraction combined with liquid chromatography tandem mass spectrometry for the quantitative analysis of a TLR7 agonist imiquimod LFX453 in plasma at low picograms per milliliter: Method validation and its application to a pharmacokinetic study in minipig.

Sample preparation is essential for low-level compound determination. In the present work, supported liquid extraction (SLE) was used as sample preparation for the low-level determination of a new TLR7 agonist imiquimod compound, LFX453. Samples were extracted on ISOLUTE® SLE 96-well plates using tert-butyl-methyl ether followed by evaporation and dry residue reconstitution with 150 µl of a mixture of 0.1% formic acid in acetonitrile-water (50/50, v/v). Samples were eluted using a flow rate of 0.750 ml/min on a C18 column (50 × 2.1 mm, 2.7 µm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Tandem mass spectrometry was used to analyze the samples in positive mode. The method run time was 6.5 min, and the low limit of quantification was 1.00 pg/ml with 0.100 ml of minipig plasma. Intra-run and inter-run precision and accuracy were within the acceptance criteria at four concentration levels over a concentration ranging from 1.00 to 200 pg/ml. There was no matrix effect and recovery, three freeze-thaw cycles and incurred samples reanalysis were validated. The method was successfully applied for measuring LFX453 in minipig plasma after application on minipig skin.

Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of eight Xiakucao Oral liquid-related compounds in rat plasma and its application in pharmacokinetic study.

Xiakucao Oral Liquid (XKCOL) has been widely used for treating mammary gland hyperplasia and goiter in China. However, its pharmacokinetic data have been missing to date. To conduct its pharmacokinetic study, we established an LC-tandem mass spectrometry method for the simultaneous determination of eight XKCOL-related compounds in rat plasma. Liquid-liquid extraction was used for the sampling process. Chromatographic separation was performed on a Phenomenon Luna C18 column with a mobile phase of methanol and 2 mM ammonium acetate, using gradient elution at a flow rate of 0.8 mL/min. Detection was performed in the multiple reaction monitoring mode using negative electrospray ionization (ESI-) with optimized MS parameters. Endogenous substances and carryover did not interfere in the detection of analytes. The calibration curves showed a good linear relationship within the linear ranges. The intra- and inter-batch accuracy and precision were 94.8%-110.0% and ≤11.2%, respectively. There was no significant matrix effect and the recovery was reproducible. The dilution of samples did not affect the accuracy and precision. The solution and plasma samples were stable under the various test conditions. The major components of XKCOL absorbed into the blood were salvianic acid A and rosmarinic acid. They demonstrated linear kinetics over the dose range used in this study.

Pharmacokinetics of Novel Furoxan/Coumarin Hybrids in Rats Using LC-MS/MS Method and Physiologically Based Pharmacokinetic Model.

Novel furoxan/coumarin hybrids were synthesized, and pharmacologic studies showed that the compounds displayed potent antiproliferation activities via downregulating both the phosphatidylinositide 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway. To investigate the preclinical pharmacokinetic (PK) properties of three candidate compounds (CY-14S-4A83, CY-16S-4A43, and CY-16S-4A93), liquid chromatography, in tandem with the mass spectrometry LC-MS/MS method, was developed and validated for the simultaneous determination of these compounds. The absorption, distribution, metabolism, and excretion (ADME) properties were investigated in in vitro studies and in rats. Meanwhile, physiologically based pharmacokinetic (PBPK) models were constructed using only in vitro data to obtain detailed PK information. Good linearity was observed over the concentration range of 0.01−1.0 µg/mL. The free drug fraction (fu) values of the compounds were less than 3%, and the clearance (CL) values were 414.5 ± 145.7 mL/h/kg, 2624.6 ± 648.4 mL/h/kg, and 500.6 ± 195.2 mL/h/kg, respectively. The predicted peak plasma concentration (Cmax) and the area under the concentration-time curve (AUC) were overestimated for the CY-16S-4A43 PBPK model compared with the experimental ones (fold error > 2), suggesting that tissue accumulation and additional elimination pathways may exist. In conclusion, the LC-MS/MS method was successively applied in the preclinical PK studies, and the detailed information from PBPK modeling may improve decision-making in subsequent new drug development.

Development and validation of LC-MS/MS method for amlexanox in rat plasma and its application in preclinical pharmacokinetics.

Amlexanox, an anti-inflammatory and anti-allergic agent, has been widely used clinically for the treatment of canker sores, asthma, and allergic rhinitis. Recently, amlexanox has received considerable attention in curing nonalcoholic fatty liver diseases and hepatitis virus infection. Herein, we first established a sensitive high-performance liquid chromatography-tandem mass spectrum (LC-MS/MS) method for the determination of amlexanox in rat plasma. Propranolol was used as the internal standard (IS). Using a simple protein precipitation method, the amlexanox and IS were separated with Capcell Pak C18 column (2.0 × 50 mm, 5 µm) and eluted with water and acetonitrile each containing 0.1% formic acid using gradient elution condition at a flow rate of 0.4 mL·min-1 . Amlexanox and IS were detected by a triple quadrupole mass in multiple reactive monitoring (MRM) under the transitions of m/z 299.2 → 281.2 and m/z 259.9 → 116.1 with positive electrospray ionization, respectively. The calibration curves of amlexanox were established with the range of 50 to 2000 ng·mL-1 (r2 > 0.99). The validation method consisted of selectivity, accuracy, precision, carryover effect, matrix effect, recovery, dilution effect, and stability. The fully validated method was successfully applied to the pharmacokinetic study of amlexanox in Wistar rats.

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.

In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0−250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.

A High-Throughput LC-MS/MS Method for the Simultaneous Quantification of Twenty-Seven Drug Molecules in Ocular Tissues.

The purpose of this study was to develop a validated LC-MS/MS analytical method for the simultaneous analysis of a large cassette containing a wide range of drug substances with positive, negative, or neutral charge and further apply the method to assess octanol partition coefficient and eye tissue recovery of the drug cassette. A twenty-seven-drug cassette (N-in-one) including beta blockers, NSAIDs, and corticosteroids that range from extremely hydrophilic (sotalol) to very hydrophobic (triamcinolone hexacetanide) was used to develop an LC-MS/MS assay using QTrap 4500. An LC-MS/MS method based on gradient elution, with an eighteen-minute run time including equilibration time, was developed and validated for the rapid and simultaneous quantitation of drugs with a wide range of lipophilicities. Scheduled multiple reaction monitoring was used to maximize the scan time for each peak, ensuring sufficient scans. Method validation included lower limit of quantitation (LLOQ) and intra- and inter-day reproducibility. The LLOQ ranged from 0.5 (sotalol) to 40 fmols (dexamethasone) on column with a %RSD < 20%. The method was tested by measuring octanol:water and octanol:buffer (PBS, pH 7.4) partition coefficients and by quantitation of the drug cassette extracted from rabbit aqueous humor and cornea. Measured partition coefficients correlated positively with predicted values (r2=0.5-0.7). Drug recovery was ≥ 79% from aqueous humor and between 61 and 67% on average from cornea. A rapid, sensitive LC-MS/MS method suitable for N-in-one drug delivery screening was developed for simultaneous quantification of twenty-seven drugs in aqueous solutions and eye tissues.

Simultaneous determination of sacubitrilat and fimasartan in rat plasma by a triple quad liquid chromatography-tandem mass spectrometry method utilizing electrospray ionization in positive mode.

An LC-tandem mass spectrometry method was developed and validated for the simultaneous quantitation of fimasartan and sacubitrilat using positive ion mode. The protein precipitation method was employed for the extraction of fimasartan, sacubitrilat and alprazolam (internal standard) from rat heparinized plasma. Baseline separation of the analytes was accomplished using an ACE-5, C18 (4.6 × 50 mm) column and gradient elution of mobile phase A (5 mm ammonium formate and 0.1% formic acid in purified water) and B (acetonitrile:methanol, 80:20; v/v). All peaks of interest were eluted within a 5-min runtime. The quantitation was achieved in the selected reaction monitoring mode. The developed method was validated as per US Food and Drug Administration guidelines and met the pre-defined acceptance criteria. The method showed linearity from 5 to 10,000 ng/mL. The accuracy/precision of intra- and inter-batch assays was 96.64%/2.05% to 109.17%/13.70% and 100.74%/3.76% to 106.39%/9.75% for fimasartan and 100.02%/1.49% to 113.80%/9.38% and 100.75%/2.31% to 108.40%/7.74% for sacubitrilat, respectively, in rat plasma. Fimasartan and sacubitrilat remained stable in rat plasma at different experimental conditions up to 21 days. The developed method was sensitive, selective and applied successfully to monitor plasma concentrations of fimasartan and sacubitrilat in an oral rat pharmacokinetic study.

A sensitive liquid chromatography-tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: Application to pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 µl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 µm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5-2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.

Simultaneous Determination of Acrylamide and Hydroxymethylfurfural in Extruded Products by LC-MS/MS Method.

The aim of this study was to develop a liquid chromatography/tandem mass spectrometry LC-MS/MS method for the simultaneous determination of acrylamide and hydroxymethylfurfural (HMF) in corn snack products enriched with food industry by-products: brewer's spent grain (BSG), sugar beet pulp (SBP) and apple pomace (AP). Development of the method included the study of different sources for ionization, different mobile phases, different extraction conditions as well as different methods of sample preparation. Finally, the single LC-MS/MS method was developed for the analysis of both analytes in one step with a duration of 20 min using a simple single-step extraction. The method with apparent recoveries of 91.4 and 90.4 for acrylamide and HMF, respectively, was applied for the analysis of non-extruded and extruded samples. The obtained results shown that the acrylamide content was

Cocaine-Induced Reinstatement of Cocaine Seeking Provokes Changes in the Endocannabinoid and N-Acylethanolamine Levels in Rat Brain Structures.

There is strong support for the role of the endocannabinoid system and the noncannabinoid lipid signaling molecules, N-acylethanolamines (NAEs), in cocaine reward and withdrawal. In the latest study, we investigated the changes in the levels of the above molecules and expression of cannabinoid receptors (CB1 and CB2) in several brain regions during cocaine-induced reinstatement in rats. By using intravenous cocaine self-administration and extinction procedures linked with yoked triad controls, we found that a priming dose of cocaine (10 mg/kg, i.p.) evoked an increase of the anadamide (AEA) level in the hippocampus and prefrontal cortex only in animals that had previously self-administered cocaine. In the same animals, the level of 2-arachidonoylglycerol (2-AG) increased in the hippocampus and nucleus accumbens. Moreover, the drug-induced relapse resulted in a potent increase in NAEs levels in the cortical areas and striatum and, at the same time, a decrease in the tissue levels of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) was noted in the nucleus accumbens, cerebellum, and/or hippocampus. At the level of cannabinoid receptors, a priming dose of cocaine evoked either upregulation of the CB1 and CB2 receptors in the prefrontal cortex and lateral septal nuclei or downregulation of the CB1 receptors in the ventral tegmental area. In the medial globus pallidus we observed the upregulation of the CB2 receptor only after yoked chronic cocaine treatment. Our findings support that in the rat brain, the endocannabinoid system and NAEs are involved in cocaine induced-reinstatement where these molecules changed in a region-specific manner and may represent brain molecular signatures for the development of new treatments for cocaine addiction.

A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.

Comprehensive and temporal analysis of secreted proteins in the medium from IL-6 exposed human hepatocyte.

We have previously identified intracellular secretory acute phase response (sAPR) proteins in human hepatocytes following interleukin-6 (IL-6) induction by fluorogenic derivatization (FD)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS). In this report, we utilized this method, which uses 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as the FD reagent, to comprehensively and time-dependently analyze secreted proteins in the medium, including sAPR proteins. Since DAABD-Cl selectively reacts with thiol moieties of cysteinyl residues, direct derivatization, high-resolution LC separation and identification of the secreted proteins in the culture medium were successfully achieved without a pretreatment step. As a result, 14 sAPR proteins were identified simultaneously during a 72 h induction by IL-6. The secretion levels of 11 proteins increased, whereas the secretion levels of three important transport proteins decreased (albumin, retinol-binding protein 4 and transthyretin). In addition, the secretion level of a haptoglobin was found to increase significantly between 0 and 6 h by 1.88-fold compared with the control sample. The secretion levels of four cytoplasmic proteins increased: LDH, a known marker for cell damage, and GSTA1, FABP1 and ADH1B, which are marker proteins for hepatocellular damage. The secretion levels of the other two newly identified cytoplasmic proteins, profilin-1 and SOD2, were also found to increase, suggesting that these two proteins represent novel markers for cell damage. These results suggest that the FD-LC-MS/MS proteomics method can be used to analyze comprehensively and time-dependently the secreted proteins and thereby can offer information that aids our understanding of the dynamics of protein secretion affected by the exposure of cytokines such as IL-6.

A sensitive and specific LC-MS/MS method for determination of a novel antihypertensive peptide FR-6 in rat plasma and pharmacokinetic study.

The investigation of peptide drugs has become essential in the development of innovative medications for hypertension. In this study, a sensitive high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to determine the plasma concentration and stability of the antihypertensive peptide FR-6 in rats. An isotopically labeled peptide (with an unchanged sequence) was utilized as an internal standard (IS) for validation purposes. Subsequently, this assay was employed to examine the pharmacokinetics of different administration methods (tail vein and gavage) in Sprague Dawley (SD) rats. Extracted plasma samples underwent sample preparation through methanol protein precipitation, followed by elution of FR-6 on Wondasil C18 Superb column (4.6 × 150 mm, 5 µm), using a mobile phase consisting of formic acid (0.1%) in water (A) and formic acid (0.125%)-ammonium formate (2 mM) in methanol (B). Ion pairs corresponding to FR-6 and IS were monitored via multiple reaction monitoring (MRM) under positive ion mode: m/z 400.7 â†’ 285.1 for FR-6 and m/z 406.1 â†’ 295.1 for IS detection respectively. The method exhibited excellent linearity with respect to FR-6 concentrations. In addition, the inter-day and intra-day precision were 0.61-6.85% and 1.76-11.75%; the inter-day and intra-day accuracy were -7.28-0.13% and -7.20-2.28%, respectively. In conclusion, the matrix effect, extraction recovery, and stability data were validated according to FDA recommended acceptance criteria for bioanalytical methods. This validated method serves as a reliable tool for determining the concentration of antihypertensive peptide FR-6, and has been successfully applied in pharmacokinetic studies involving rats.

Development and validation of an LC-MS/MS method for simultaneous quantification of eight drugs in plasma and brain: Application in a pharmacokinetic study in mice.

A selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantitation of a cassette of 8 drugs, including docetaxel, erlotinib, loperamide, riluzole, vemurafenib, verapamil, elacridar and tariquidar. Stable isotopically labeled compounds were available for use as internal standards for all compounds, except for tariquidar for which we used elacridar-d4. Sample pre-treatment involved liquid-liquid extraction using tert-butyl-methyl ether as this resulted in good recovery and low ion suppression. Chromatographic separation was achieved using a Zorbax Extend C18 analytical column and a linear gradient from 20 % to 95 % methanol in 0.1 % (v/v) formic acid in water. MS/MS detection using multiple reaction monitoring was done in positive ionization mode. We validated this assay for human and mouse plasma and mouse brain homogenates. The calibration curves were linear over a range 1-200 nM for each drug in the mix, except for tariquidar probably due to the lack of a stable isotope labeled analog. The intra-day and inter-day accuracies were within the 85-115 % range for all compounds at low, medium and high concentrations in the three different matrices. Similarly, the precision for all compounds at three different concentration levels ranged below 15 %, with the exception of tariquidar in mouse plasma and brain homogenate and riluzole in brain homogenate. Pilot studies have confirmed that the method is suitable for the analysis of mouse plasma samples and brain homogenates following cassette dosing of this mixture in mice.

Trace analysis of benzophenone-type UV filters in water and their effects on human estrogen and androgen receptors.

To carry out risk assessments of benzophenone-type UV filters (BPs), fast and accurate analytical methods are crucial to determine and monitor levels in the environment. This study presents an LC-MS/MS method that requires minimal sample preparation and yet can identify 10 different BPs in environmental samples such as surface or wastewater resulting in a LOQ range from 2 to 1060 ng/L. The method suitability was tested through environmental monitoring, which showed that, BP-4 is the most abundant derivative found in the surface waters of Germany, India, South Africa and Vietnam. BP-4 levels correlate with the WWTP effluent fraction of the respective river for selected samples in Germany. Peak values of 171 ng/L for 4-hydroxybenzophenone (4-OH-BP), as measured in Vietnamese surface water, already exceed the PNEC value of 80 ng/L, elevating 4-OH-BP to the status of a new pollutant that needs more frequent monitoring. Moreover, this study reveals that during biodegradation of benzophenone in river water, the transformation product 4-OH-BP is formed which contain structural alerts for estrogenic activity. By using yeast-based reporter gene assays, this study provides bio-equivalents of 9 BPs, 4-OH-BP, 2,3,4-tri-OH-BP, 4-cresol and benzoate and complements the existing structure-activities relationships of BPs and their degradation products.

Hair Sample Analysis as a Method of Monitoring Exposure to Bisphenol A in Dogs.

Bisphenol A (BPA) is an organic substance widely used in the plastics industry. It penetrates food and environment and, as an endocrine disruptor, has detrimental effects on human organisms. Pet animals, which live in the immediate vicinity of humans, are also exposed to BPA; however, knowledge regarding the exposure of dogs to this substance is extremely scarce. This is the first study in which hair analysis has been used to biomonitor BPA in 30 dogs using liquid chromatography and tandem mass spectrometry techniques. The presence of BPA in concentration levels above the method detection limit (1.25 ng/g) was noted in 93.33% of samples. BPA concentration levels were found to range from 7.05 ng/g to 436 ng/g (mean 81.30 ng/g). Statistically significant differences in BPA concentration levels were found between animals with physiological weight and animals with abnormal weight (skinny and obese). In turn, differences between males and females, as well as between young, middle-aged and old dogs, were not statistically significant. The obtained results have clearly shown that hair analysis is a useful method to evaluate the exposure of dogs to BPA. This study also confirmed that dogs are exposed to BPA to a large extent, and this substance may play a role as a pathological factor in this animal species. However, many aspects connected to the influence of BPA on canine health status are unclear and need further study.

Biomonitoring parabens in dogs using fur sample analysis - Preliminary studies.

Parabens are widely used in the food, cosmetics and pharmaceutical industry and are widespread in the environment. As endocrine disruptors, parabens have adverse effects on living organisms. However, knowledge of the exposure of domestic animals to parabens is extremely scarce. Therefore, this study assessed the exposure level of dogs to three parabens commonly used in industry (i.e. methylparaben - MeP, ethylparaben - EtP and propylparaben - PrP) using fur sample analysis in liquid chromatography-tandem mass spectrometry. The presence of parabens has been noted in the samples collected from all dogs included in the study (n = 30). Mean concentrations of MeP, EtP and PrP in the fur of dogs were 176 (relative standard deviation - RSD = 127.48%) ng/g dry weight (dw), 48.4 (RSD = 163.64%) ng/g dw and 79.8 ng/g dw (RSD = 151.89%), respectively. The highest concentrations were found for MeP (up to 1023 ng/g dw). Concentrations of MeP and EtP in males were statistically higher than those in females (p < 0.05). Statistically significantly higher concentration levels of PrP in young animals (up to three years old) were also found. This is the first study concerning the use of fur samples to evaluate the exposure of domestic animals to parabens. The results indicate that an analysis of the fur may be a useful tool of paraben biomonitoring in dogs. The presence of parabens in the canine fur also suggests that these substances may play a role in veterinary toxicology. However, many aspects connected with this issue are not clear and require further study.

Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities.

The present manuscript reports on monitoring data of 12 ergot alkaloids (EAs) in cereal and cereal-derived products, collected in Italy over the period 2017-2020, for official control purposes under the edge of the Commission Recommendation 2012/154/EU on the monitoring of the presence of EAs in feed and food. To these purposes, an LC-MS/MS method was set up and applied, after in-house verification of its analytical performance. Besides satisfactory recoveries and precision, the method's quantification limits proved suitable to assess the compliance of cereals and cereal-based foods with the recently issued EU maximum permitted levels (Commission Regulation 2021/1399/EU). The validity of the generated data was also evaluated through the adoption of four proficiency tests, from which acceptable z-score values (-2 ≤ z ≤ 2) were obtained. The method was then applied to analyse a total of 67 samples, collected in Italy over the period 2017-2020. The samples consisted of 18 cereal grains, 16 flours (14 of wheat and 2 of spelt) and 31 other types of cereals derivatives (including 9 for infants). Overall, the EAs analysis returned a high percentage of left-censored data (>86%). Among the positive samples, the highest contamination levels, up to 94.2 µg/kg, were found for ergocristine (12% incidence), followed by ergocristinine (7% incidence) with levels of up to 48.3 µg/kg.

Mycotoxin Biomarkers in Pigs-Current State of Knowledge and Analytics.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig' exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique-tandem mass spectrometry-is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper.

Development of a multi-mycotoxin LC-MS/MS method for the determination of biomarkers in pig urine.

An LC-MS/MS method has been developed for the sensitive and selective determination of 35 mycotoxins (biomarkers of exposure) in pig urine samples. Sample preparation includes creatinine adjustment (with the developed LC-UV method) with enzymatic hydrolysis of pig urine samples followed by liquid-liquid (LLE) extraction. The LLE protocol, as well as enzymatic hydrolysis for indirect mycotoxin glucuronides determination, was optimized in this study. Additionally, two other sample preparation protocols were compared with the developed LLE method: immunoaffinity columns and solid-phase extraction cartridges (Oasis HLB). The detection and quantification of the biomarkers were performed using triple quadrupole mass spectrometry.The method was validated with regard to the guidelines specified by the EMEA (European Medicines Agency). The extraction recoveries were higher than 60% for 77% of the analytes studied, with the intra- and inter-day relative standard deviation being lower than 20% for most of the compounds at four different concentration levels. The limits of quantification ranged from 0.1 ng/mL for zearalenone and sterigmatocystin to 8 ng/mL for nivalenol. To the best knowledge of the authors, the matrix effect was evaluated for the first time in this study for six different urine samples, and the coefficient of variation was found to be lower than 15% for most analytes studied. Finally, the developed method was applied to analyse 56 pig urine samples. Deoxynivalenol (1-20 ng/mL), zearalenone (0.1-1.5 ng/mL) and ochratoxin A (1.5-15 ng/mL) were the main analytes detected in these samples. Moreover, the co-occurrence of alternariol monomethyl ether and alternariol in pig urine is reported herein for the first time.