Development of a Novel LC-MS/MS Multi-Method for the Determination of Regulated and Emerging Food Contaminants Including Tenuazonic Acid, a Chromatographically Challenging Alternaria Toxin.

The regulation of food contaminants in the European Union (EU) is comprehensive, and there are several compounds in the register or being added to the recommendation list. Recently, European standard methods for analysis have also been issued. The quick analysis of different groups of analytes in one sample requires a number of methods and the simultaneous use of various instruments. The aim of the present study was to develop a method that could analyze several groups of food contaminants: in this case, 266 pesticides, 12 mycotoxins, 14 alkaloid toxins, and 3 Alternaria toxins. The main advantage of the herein described approach over other methods is the simultaneous analysis of tenuazonic acid (TEA) and other relevant food contaminants. The developed method unites the newly published standard methods such as EN 15662:2018, EN 17194:2019, EN 17256:2019, EN 17425:2021, EN 17521:2021, which describes the analysis of both regulated and emerging contaminants. The developed method is based on a QuEChERS sample preparation, followed by LC-MS/MS analysis under alkaline mobile phase conditions. The pH of the aqueous eluent was set to 8.3, which resulted in baseline separation among ergot alkaloids and their corresponding epimers, a symmetric chromatographic peak shape for analyzing TEA and fit-for-purpose sensitivity for MS/MS detection in both positive and negative ionization modes. Those compounds, which possess the corresponding isotopically labeled internal standards (ISTD), allowed for direct quantification by the developed method and no further confirmation was necessary. This was proven by satisfactory analyses of a number of quality control (QC), proficiency test (PT), and validation samples.

An Alternative Strategy for Screening and Confirmation of 330 Pesticides in Ground- and Surface Water Using Liquid Chromatography Tandem Mass Spectrometry.

The presence of pesticide residues in water is a huge worldwide concern. In this paper we described the development and validation of a new liquid chromatography tandem mass spectrometric (LC-MS/MS) method for both screening and quantification of pesticides in water samples. In the sample preparation stage, the samples were buffered to pH 7.0 and pre-concentrated on polymeric-based cartridges via solid-phase extraction (SPE). Highly sensitive detection was carried out with mobile phases containing only 5 mM ammonium formate (pH of 6.8) as an eluent additive and using only positive ionization mode in MS/MS instrument. Hence, only 200-fold sample enrichment was required to set a screening detection limit (SDL) and reporting limit (RL) of 10 ng/L. The confirmatory method was validated at 10 and 100 ng/L spiking levels. The apparent recoveries obtained from the matrix-matched calibration (5-500 ng/L) were within the acceptable range (60-120%), also the precision (relative standard deviation, RSD) was not higher than 20%. During the development, 480 pesticides were tested and 330 compounds fulfilled the requirements of validation. The method was successfully applied to proficiency test samples to evaluate its accuracy. Moreover, the method robustness test was carried out using higher sample volume (500 mL) followed by automated SPE enrichment. Finally, the method was used to analyze 20 real samples, in which some compounds were detected around 10 ng/L, but never exceeded the assay maximum level.

Automation in quantifying phenoxy herbicides and bentazon in surface water and groundwater using novel solid phase extraction and liquid chromatography tandem mass spectrometry.

The occurrence of phenoxy herbicides is a financial and regulatory concern for drinking water treatment plants. This paper presents a new method of quantification for nine phenoxy-acids and bentazon in different water samples using liquid chromatography tandem mass spectrometry (LC-MS/MS). The method is based on an automated solid phase extraction (SPE) process that applied hydrophilic modified polystyrene and divinylbenzene cartridges at low pH (<2.0). Main advantages of the presented method include the reduced consumption of organic solvent in extraction and the fully automated sample pre-concentration. The method is thus more environmentally-friendly. In the quantification step, five stable isotopically labelled analogues were used as internal standards to account for the losses during sample preparation and to calibrate the ion source response under the mass spectrometric detection. The method was optimized in terms of sample preparation and subsequent LC-MS/MS separation to obtain reliable measurement of the analyte concentration during real sample analysis. The method quantification limit was between 1.5 and 10.0 ng/L for target compounds in surface water and groundwater samples. The method was validated at three fortification levels between 10.0 and 1000 ng/L, and the results showed fit-for-purpose recovery with appropriate precision at low concentration levels. The method was also utilized to analyse thirty-two actual water samples from different sources. Forty percent of the analysed samples contained detectable level of herbicides, ranging from 1.91 to 40.5 ng/L. The concentrations of targeted herbicides in our study were comparable to those found in water samples in other regions of world.

Determination of Alternaria Toxins in Tomato, Wheat, and Sunflower Seeds by SPE and LC-MS/MS-A Method Validation Through a Collaborative Trial.

BACKGROUND: Alternaria toxins are ubiquitous contaminants in highly consumed food products. Therefore, they are candidates to be regulated by EU legislation. In this context, the availability of reliable analytical methods is a keystone both for protecting the health of citizens and smooth functioning of the European market. OBJECTIVE: This paper describes an advanced LC-MS/MS method based on isotope dilution quantification suitable for the determination of altenuene, alternariol, alternariol monomethyl ether, tenuazonic acid, and tentoxin in tomato puree, wheat, and sunflower seeds. METHODS: The method has been validated in an interlaboratory study that included the analysis of both spiked and naturally contaminated food commodities. Twenty-three participants contributed with analytical data. RESULTS: The average recoveries and relative standard deviations for repeatability and reproducibility obtained across the tested matrixes were: 97, 8.0, and 23%, for altenuene, respectively; 95, 9.2, and 17% for alternariol, respectively; 98, 6.4, and 13% for alternariol monomethyl ether, respectively; 97, 4.2, and 9.3% for tenuazonic acid, respectively; and 102, 5.6, and 15% for tentoxin, respectively. The method enabled the determination of all tested Alternaria toxins close to or below 1 µg/kg. CONCLUSION: Overall, the method showed a satisfactory trueness and precision, complying with the requirements for the monitoring of mycotoxins in food in the EU. It is currently under evaluation by the European Committee for Standardization for adoption as a standard method. HIGHLIGHTS: Isotope dilution mass spectrometry method for the determination of Alternaria toxins in food.

Separation of fosetyl and phosphonic acid in food matrices with mixed-mode HPLC column coupled with tandem mass spectrometric detection and method application to other highly polar pesticides.

The aluminum salt of fosetyl (tris(ethyl phosphonate)) is an antifungal agrochemical. This paper presents a novel high performance liquid chromatography (HPLC) method for the simultaneous determination of fosetyl and the phosphonic acid, its main metabolite, in food samples. The method is based on an ion-displacement separation performed on the recently released Luna Omega PS C18 mixed-mode HPLC column. Baseline separation of fosetyl and phosphonic acid was feasible. This was achieved by optimizing the mobile phase composition and by introducing ethylenediaminetetraacetate for all matrices in the generally used extraction medium for polar pesticides and the injection solution. The binary mobile phase consisted of 10% (v/v) methanol in water and aqueous formate buffer (pH = 3.5) in gradient elution mode. The main advantages of the method over previous method include the stable retention time and peak resolution without the need for long column priming, conditioning or regeneration. Moreover, the approach was tested with other polar pesticides including glyphosate, glufosinate, and perchlorate and showed fit-for-purpose separation. The method was validated for spinach, cherry, and wheat flour samples, and was successfully applied on oat flour and arugula quality control samples. The results obtained for the five analytes met the requirements set by EU. The limit of quantifications was much lower than the maximum residue limits and ranged from 0.02 to 0.20 mg/kg.

A Dilute and Shoot Strategy for Determining Alternaria Toxins in Tomato-Based Samples and in Different Flours Using LC-IDMS Separation.

Alternaria toxins are emerging mycotoxins whose regulation and standardization are in progress by the European Commission and the European Committee for Standardization. This paper describes a dilute and shoot approach to determine five Alternaria toxins in selected food samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strategy involves sample extraction with acidified aqueous methanol, followed by a solvent change accomplished via sample evaporation and reconstitution. The quantification is based on isotope dilution, applying all corresponding isotopically labeled internal standards to compensate possible matrix effects of the analysis. The main advantages of the present method over other existing methods includes simple and effective sample preparation, as well as detection with high sensitivity. The five-fold sample dilution can decrease matrix effects, which were evaluated with both external and internal standard methods. The results demonstrated a limit of quantification lower than 1.0 µg/kg for all five analytes for the first time. The newly presented method showed acceptable accuracy (52.7-111%) when analyzing naturally contaminated and spiked standard samples at the described levels. The method was validated for tomato-based and flour samples (wheat, rye, and maize). The absolute recovery ranged from 66.7% to 91.6% (RSD < 10%). The developed method could be an alternative approach for those laboratories that exclude sample cleanup and pre-concentration of state-of-the-art instruments with enhanced sensitivity.

Determination of Alternaria Toxins in Sunflower Oil by Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry.

Alternaria toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

Quantification of aromatic amines derived from azo colorants in textile by ion-pairing liquid chromatography tandem mass spectrometry.

Azo dyes can metabolize back to precursor aromatic amines (arylamines), which are potentially carcinogenic. The ISO 14362-1:2017 standard requires the determination of the arylamines, produced from textile samples, preferably by using the gas chromatography mass spectrometric (GC-MS) method. This paper presents an ion-pairing high performance liquid chromatography tandem mass spectrometric (LC-MS/MS) method for determining aromatic amines, derived from azo colorants, in both natural and synthetic textiles. The separation enables adequate apparent retention factor (k' > 2.1 for the most hydrophilic compounds) and has appropriate sensitivity without sample clean-up procedure. The background matrix constituents influence the quantification in even a 100-fold diluted sample, therefore, calibration requires background compensation. The method allows fast confirmation and quantification of twenty-five arylamines in complex matrices of textiles. The method was validated with success and used to both natural and synthetic textile proficiency test (PT) samples.

Determination of acrylamide in gingerbread and other food samples by HILIC-MS/MS: A dilute-and-shoot method.

This paper describes a hydrophilic interaction liquid chromatography tandem mass spectrometric (HILIC-MS/MS) method for determining acrylamide in food. The method primary optimised for gingerbread samples that have high sugar contents. A new sample preparation process was optimized using an experiment design based on a central composition design (CCD). A mixture of acidified aqueous acetonitrile was established as a suitable extraction medium. The extracts were further diluted and separation of acrylamide on TSKgel Amide-80 HILIC column was carried out within 8 min. The method was validated using naturally contaminated quality check (QC) as well as spiked samples. The developed method showed acceptable accuracy (101% - 105%) and precision (2.9% - 7.6%). The limit of quantification was 20 µg/kg. The method was also tested by analysing acrylamide in other food samples (bread, roasted coffee, instant coffee, cappuccino powder and fried potato). The acrylamide concentrations found in samples were between 20 µg/kg and 667 µg/kg, which were lower than the benchmark levels set by the European Union (EU). The main advantage of the newly developed method over the standard methods included the easier sample preparation and faster analysis with reduced ion suppression.

Determination of Thyreostats in Urine Using Supported Liquid Extraction and Mixed-Mode Cation-Exchange Solid-Phase Extraction: Screening and Confirmatory Methods.

The application of thyreostats in livestock has been banned in the European Union since 1981, but these drugs are currently in the focus due to the natural occurrence of thiouracil (TU). Studies have been published on TU contamination in urine samples of animal and human origins without any drug administration of it. This paper presents new analytical methods to analyze thyreostats to support the legislation on the recommended concentration (RC) levels of these drugs. Both screening and confirmatory methods are developed for analyzing thyreostats in porcine and bovine urines using a liquid chromatography-tandem mass spectrometry technique. The new methods include a chemical derivatization with 3-iodobenzyl bromide, followed by novel purification approaches using supported liquid extraction and mixed-mode cation-exchange solid-phase extraction (SPE) for screening and confirmatory purposes, respectively. The optimized derivatization in combination with the cation-exchange SPE gives high sensitivity and reducing matrix effect of the analysis. The methods are validated in accordance with the guidelines for the validation of screening methods and European Commission Decision 2002/657/EC. The confirmatory method is used in the national monitoring plan. The detected levels of TU in urine samples are below the currently applicable RC level (10 µg L-1).

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56µg/L-4.95µg/L and 0.17µg/kg-2.14µg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples.

Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimised method using liquid chromatography-tandem mass spectrometry.

Alternaria toxins and citrinin are mycotoxins produced by fungi growing on different raw materials and agricultural commodities. Maximum levels of these toxins in foods are currently under consideration by the European Commission as a risk management measure. In this study, a new quantitative method is described for the determination of five Alternaria toxins and citrinin in tomato and tomato juice samples based on LC-MS/MS detection. Samples were extracted with pure methanol, followed by a derivatisation step with 2,4-dinitrophenylhydrazine to improve the determination of tenuazonic acid and to decrease the wide polarity difference between the compounds of interest. Samples were purified on hydrophilic-modified styrene polymer solid-phase extraction cartridges. High-performance liquid chromatographic columns packed with different core-shell materials were tested for the separation of toxins and a C-18 phase was in the final method applied to achieve sufficient separation of all relevant analytes. A key element of this approach was to prove successful transferability of the method to three different triple quadrupole mass spectrometers. A full single laboratory method validation was performed on two LC-MS/MS systems and performance characteristics met the predefined requirements. Moreover, the method was used in an international proficiency test and the satisfactory z-scores obtained (-0.1 to 0.8 in tomato juice samples) demonstrated the reliability of the approach described. The method will be validated in an inter-laboratory collaborative study and if the criteria for method precision are met, the method will be proposed as a new Work Item to the European Committee for Standardisation.

Determination of tetracyclines in pig and other meat samples using liquid chromatography coupled with diode array and tandem mass spectrometric detectors.

Two high performance liquid chromatographic methods (HPLC-DAD and LC-MS/MS) were developed to analyze tetracycline (TC) residues in pig meat (pork) samples. The method involved a sample preparation using a solid-liquid extraction (SLE) by McIlvaine buffer, followed by a solid-phase extraction (SPE) clean-up using Strata-XL cartridges. The developed sample clean-up resulted in a selective chromatogram in the HPLC-DAD separation and a reduced matrix effect (ME) in LC-MS/MS analysis. Moreover, HPLC columns packed with core-shell particles were tested for separation, which further enhanced the sensitivity and the selectivity of determinations. The validation of the methods for pig samples was carried out according to European Union 2002/657/EC decision. In addition, validation was also performed for bovine, chicken, and turkey meat samples using HPLC-DAD method. The performance characteristics of determinations were evaluated with both spiked and incurred samples, and were systematically compared. LC-MS/MS technique was found to be more accurate for spiked samples; however, HPLC-DAD method resulted in more reliable concentrations for incurred samples.

Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography-tandem mass spectrometry.

This paper describes a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the analysis of three stanozolol metabolites (16ß-hydroxystanozolol, 3'-hydroxystanozolol, and 4ß-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC-MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core-shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15µg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.

Simultaneous determination of eight corticosteroids in bovine tissues using liquid chromatography-tandem mass spectrometry.

This paper describes a newly developed method for the simultaneous determination of eight corticosteroid residues in bovine muscle, liver and kidney samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The determination of methylprednisone, the main metabolite of methylprednisolone, in bovine tissues using LC-MS/MS is carried out for the first time. The method development demonstrates that the pH is important in optimizing the sample preparation. Tests performed using different solid-phase extraction (SPE) cartridges were enabled to produce conditions for reducing the matrix effects (ion suppression and enhancement) of analysis. Acidic condition and mixed-mode cation exchange SPE columns resulted in the most suitable clean-up for muscle and liver, and also yielded acceptable results for kidney. The enhanced sample clean-up resulted in excellent clear baselines of ion transitions, and therefore, a higher delta electron multiplier voltage (ΔEMV) could be set in the MS/MS detector. The application of 500 V of ΔEMV improved the signal responses, however, the noise level did not change, and consequently, the overall sensitivity and analytical limits (limit of detection, limit of quantification) could be enhanced. In the HPLC separation, the recently introduced Kinetex phenyl-hexyl core-shell type column was used that enabled baseline separation for dexamethasone and its ß-epimer, betamethasone. Dexamethasone and betamethasone were eluted within 12 min and such reduced retention, obtained with core-shell HPLC type column, further enhanced the sensitivity. The method was validated according to the European Union (EU) 2002/657/EC Decision; the studied parameters met the EU standards. The decision limits and limit of detections were calculated in each matrix for all corticosteroids and varied from 0.01 to 13.3µg/kg and from 0.01 to 0. 1 µg/kg, respectively.

Development of a rapid method for the determination and confirmation of nitroimidazoles in six matrices by fast liquid chromatography-tandem mass spectrometry.

A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to identify and to quantify nitroimidazoles, metronidazole (MNZ), ronidazole (RNZ) and dimetridazole (DMZ) and their corresponding hydroxy metabolites, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMNNI) in plasma, milk, muscle, egg, honey and feed samples. The same sample clean-up procedure including a novel solid-phase extraction (SPE) on polymeric Strata-SDB cartridges was used for each matrix. The analytes were separated on Kinetex XB C-18 core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water/methanol (88/12, v/v, pH 2.6) at a flow rate of 0.7 ml/min. The main advantage of the developed method is that the analysis time of only 3 min, which is about three to ten times shorter than in other reported HPLC methods. The developed method was validated using a matrix-comprehensive in-house validation strategy. The matrix effect of LC-MS/MS analysis was also investigated. Results are presented from the successful application of the developed method to an incurred pork meat certified reference material and to incur porcine plasmas in a proficiency test in year 2011.

Analysis of sub µg/kg lincomycin in honey, muscle, milk, and eggs using fast liquid chromatography-tandem mass spectrometry.

A fast liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is developed to determine lincomycin (LM) in honey, muscle, milk, and egg. Samples are cleaned-up at pH 4.7 using Strata-X-C mixed-mode polymeric strong cation exchange solid-phase extraction (SPE) cartridges, which could selectively adsorb the lincomycin from matrices under the acidic condition. LM is separated on the recently introduced Kinetex XB core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water/acetonitrile (93/7, v/v, pH 2.6) at a flow rate of 0.7 mL/min. The subsequent MS/MS detection has decreased ion effect, which allows the limit of detection (LOD) of LM for honey to be 0.05 µg/kg for honey and 0.5 µg/kg for muscle, milk, and egg. These LODs are much lower than those reported previously. The other main advantage of the developed method is the analysis time of only 3.5 min, which is about three times shorter than other reported LC-MS-MS methods. Recoveries varies between 94.2% and 125.2% and in-house reproducibility ranges from 3.7% to 28.7%. The developed method is validated according to European Union (EU) Commission Decision 2002/657/EC using a matrix-comprehensive validation strategy. All studied analytical parameters fulfills the EU guidelines.

Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography-tandem mass spectrometry.

A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub µg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 µg/kg for MRL compounds and varied from 0.1 to 0.2 µg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3 µg/kg.

Quantitative determination of corticosteroids in bovine milk using mixed-mode polymeric strong cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A new method was developed to identify and quantify corticosteroids (prednisolone, methylprednisone, flumetasone, dexamethasone, and methylprednisolone) in raw bovine milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing mixed-mode polymeric strong cation exchange and reversed-phase (MCX) solid-phase extraction (SPE) to reduce ion effects in a multimode ion (MMI) source. The main advantage of this method over other commonly used methods includes the use of a single SPE cartridge with a low volume for sample preparation and fast separation on the HPLC system with reduced ion suppression. This study is the first to report the determination of methylprednisone, a metabolite of methylprednisolone, in bovine milk. This method was validated in accordance with the European Union (EU) Commission Decision 2002/657/EC. The recoveries vary between 90% and 105%. The within-laboratory reproducibility (precision) is less than 30%. The decision limits and detection capabilities were calculated along with LODs, which ranged from 0.02 to 0.07 microg/kg. The method was further enhanced by its successful adaptation to other LC-MS/MS systems equipped with the newly developed ion source, Agilent Jet Stream (AJS). After optimization of the AJS ion source and MS parameters, even lower LOD values were achieved (0.001-0.006 microg/kg) for the corticosteroids. Analytical results obtained with the AJS were characterized by an enhanced area response and similar noise level comparable to those obtained with conventional orthogonal atmospheric ionization (API).

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry.

This paper presents the development of a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0-9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC-MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCalpha) for the first time to 0.1 microg/L (1 and 0.2 microg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 microg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1-1.0 microg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 microg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 microg/L.