Determination of sterigmatocystin in cheese by high-performance liquid chromatography-tandem mass spectrometry

Different cheese samples produced in Latvia (eight) and Belgium (13) were analysed for their sterigmatocystin (STC) content. Only two (9.5%) of the samples were positive for STC with concentration levels of 1.23 and 0.52 µg kg^?1, respectively. Five (24%) samples contained STC above the limit of detection (0.03 µg kg^?1) but below the limit of quantification (0.1 µg kg^?1), A sensitive liquid chromatography-electrospray positive ionisation-tandem mass spectrometry (LC-MS/MS) method, which was previously developed for the analysis of STC in grains, was modified and applied to the analysis of STC in cheese. This method involved sample extraction with acetonitrile–water (90 : 10, v/v), defatting with n-hexane, solid-phase extraction, separation on a reversed-phase C₁₈ column, and STC detection by LC-MS/MS.     

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides,trimethoprim and dapsone in muscle,egg, milk and honey

A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO₃H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCβ for sulfonamides ranged between 5.6 and 8.2 µg kg^?1 for eggs, between 11.1 and 69.9 µg kg^?1 for milk, between 64.7 and 87.9 µg kg^?1 for muscle, and between 2.7 and 5.3 µg kg^?1 for honey. CCβ values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg^?1 and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg^?1 calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.     

Moniliformin analysis in maize samples from North-West Italy using multifunctional clean-up columns and the LC-MS/MS detection method

A fast clean-up method has been developed to purify maize extracts and to detect moniliformin (MON) in maize samples from North-West Italy over a four-year period (2008–2011). The method is based on the use of MycoSep® 240 Mon clean-up columns (Romer Labs^®). Samples were extracted using acetonitrile/water (84:16, v/v), and the extracts were purified with previously described clean-up columns. The liquid chromatography–tandem mass spectrography (LC-MS/MS) analysis has been carried out by means of hydrophilic interaction chromatography (HILIC), combined with negative electrospray mass spectrometry. The method has a recovery of 76–91% (relative standard deviation, RSD%: 6–14%), a limit of detection (LOD) of 1 µg kg^–1 and a limit of quantification (LOQ) of 4 µg kg^?1. Naturally contaminated maize (108 samples) was analysed for MON content. The average percentages of positive samples was 93% with the following ranges (µg kg^?1): 33–2606 (2008); <LOD–527 (2009); <LOD–920 (2010); <LOD–409 (2011).     

Occurrence of chloramphenicol in cereal straw in north-western Europe

Two surveys are presented of straw analysed for naturally occurring chloramphenicol (CAP), a drug banned for use in food-producing animals. In the first study, CAP was analysed by LC-MS/MS and detected in 37 out of 105 straw samples originating from the Netherlands, France, the UK, Germany and Denmark. The highest level found was 6.3 µg kg^?1, the average 0.6 µg kg^?1 and the median 0.2 µg kg^?1. The second study included a method comparison between ELISA and LC-MS/MS and a survey of CAP in cereal straw sampled at farms in all areas of Sweden. A total of 215 samples were screened by ELISA and a subset of 26 samples was also analysed by LC-MS/MS. Fifty-four of the samples contained more than 1 µg kg^?1 CAP and the highest level found was 32 µg kg^?1 (confirmed by LC-MS/MS). The highest contents of CAP in this study were allocated to the Baltic sea coast in the south-eastern part of Sweden (the county of Skåne and the Baltic Sea isle of Gotland). These results indicate a high incidence of CAP in straw in north-west Europe and have a severe impact on the enforcement of European Union legislation.     

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS)

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS³) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol–water mixture and clean-up by immunoaffinity columns and detection using the MS³ scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r² > 0.998), excellent precision (RSD < 2.9%) and recovery (94%). The limit of quantification (LOQ) for coffee beans and espresso beverages was 0.010 and 0.003 µg kg^?1, respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018–1.80 µg kg^?1 and 0.020–0.440 µg l^?1, respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg^?1).     

Oxytetracycline,tetracycline, chlortetracycline and doxycycline in pasteurised cow’s milk commercialised in Brazil

The aim of this study was to investigate the presence of tetracycline residues in pasteurised cow’s milk using high-performance liquid chromatography coupled with UV/VIS detection to determine the exposure of Brazilian’s population to antibiotic residues. One hundred samples collected from the State of Paraná, Brazil, were analysed. Three of these samples were contaminated at the following concentrations: 121.8 µg·kg^?1 for oxytetracycline, 93.5 µg·kg^?1 for tetracycline and 134.6 µg·kg^?1 for chlortetracycline (61.6 µg·kg^?1) and doxycycline (73.0 µg·kg^?1). The median tetracycline residue concentration found in the samples was 42.3 µg·kg^?1, and the estimated daily intake (EDI) was 0.05 µg Kg^?1 bw day^?1 in Brazil. These results demonstrate that the occurrence of tetracycline in Brazilian milk was low (3%) and only for 2% above the maximum residue limit, so the risk to the population from the presence of these residues in milk was low (<1% of the acceptable daily intake).     

Aflatoxins in animal feed in Iran

One hundred and forty-six samples of animal feed (barley, n = 60; wheat bran, n = 22; wheat dry pulp, n = 29; and canola meal, n = 35) were collected in 2011 from Mashhad (Khorasan, Iran). Aflatoxins (AFs) were determined in these samples after immunoaffinity column clean-up by high-performance liquid chromatography (HPLC) with fluorescence detection. Aflatoxin B₁ (AFB₁) contamination was found in 28 samples: in five of the barley samples (8.3%) at a mean level of 0.48 µg·kg^?1, in two wheat bran samples (9.0%) at a mean level of 0.88 µg·kg^?1, in 10 wheat dry pulp samples (34.5%) at a mean level of 0.30 µg·kg^?1 and in 11 canola meal samples (31.4%) at a mean level of 0.92 µg·kg^?1. AFB₁ levels were below the maximum levels of Iran regulations (5 µg·kg^?1) and the EU maximum limit (5 µg·kg^?1).     

Rapid and sensitive method for the determination of four EU marker polycyclic aromatic hydrocarbons in cereal-based foods using isotope-dilution GC/MS

A rapid and sensitive method has been developed for the determination of the four European Union marker polycyclic aromatic hydrocarbons (PAHs; benz[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene) in some cereal-based foods. The method is based on pressurised liquid extraction (PLE), solid-phase extraction clean-up (SPE) and isotope-dilution gas chromatography with mass-spectrometric detection (GC/MS). The developed method was calibrated for the content range of 0.05–12.5 µg kg^?1 (expressed on a product basis). Recoveries of PAH were monitored in each sample via the recovery of ¹³C-labelled PAHs. Recovery values were in the range between 86% and 91%, with relative standard deviations (RSDs) between 5% and 9%. The achieved limits of detection for all analytes were below 0.05 µg kg^?1. The applicability of the method for the analysis of routine samples was studied by the analysis of a set of commercial bread and breakfast cereal samples. In all analysed samples, benzo[a]pyrene (BAP) was the most prevalent PAH with the content between 0.09 and 0.30 µg kg^?1. On average, samples showed low levels of the sum of the four EU marker PAHs (ΣPAH4) that ranged between 0.11 and 0.22 µg kg^?1 for bread samples and between 0.23 and 0.87 µg kg^?1 for breakfast cereal samples. The developed method was found suitable for the determination of PAHs in cereal-based foods like cornflakes and breads with total relative fat contents below 3.5%.     

Validation data for HPLC/FLD determinations of ochratoxin A in red paprika and black pepper adopting a one-step clean-up procedure

Validation data for the determination of ochratoxin A (OTA) in two spice matrices, red paprika and black pepper, were obtained for samples prepared with a simplified single-step clean-up column. Extracts of finely ground samples of red paprika and black pepper were prepared and applied to a Mycosep® 229 Ochra clean-up column. The purified extract was then subjected to HPLC/FLD analysis. The relative standard deviation for repeatability (RSDr) of the method was 11.8% for red paprika and 9.9% for black pepper. The limit of detection (LOD) value (three times the noise) was estimated as corresponding to the response of an extract derived from a blank matrix (previously washed) and spiked at 1.0 µg kg^?1. The limit of quantitation (LOQ) (three times LOD) was 3.0 µg kg^?1. The performance of the one-step column clean-up procedure appears to be a suitable alternative to commonly used clean-up techniques and allows the precise determination of OTA in two complex matrices.     

Preparation and application of an immunoaffinity column based on an antibody with strong affinity and packing material with good stability

This article describes the production of an anti-citrinin antibody that showed a high affinity constant (K_a) of 6.28 × 10⁹ and good tolerance to organic solvent and low pH, the synthesis of a Cu (II)-embedded polymer that showed strong binding with this antibody and the preparation of packing material for an immunoaffinity column (IAC) that show good stability. Most of the IACs reported either use harsh elution conditions and are used only once or use gentle elution conditions and are reused many times. Here, through the combined use of a strong-affinity antibody and packing material with good stability, high recoveries during clean-up and yet simultaneously good stability of the IAC were successfully achieved. Under optimised conditions of 80% methanol (pH 3), the IACs were used to clean-up the extracts of Monascus colour and red yeast rice samples, followed by HPLC detection. The recoveries of citrinin from spiked samples at levels of 50–200 μg kg^?1 were in the range of 84–97%.     

Polycyclic aromatic hydrocarbons in milk powders marketed in Argentina and Brazil

The aim of this study was to quantify polycyclic aromatic hydrocarbon (PAH) levels in milk powder samples commercialised in Argentina and Brazil during 2012. Thirty-one samples were available from the retail market. An HPLC method for the determination of PAHs was applied involving a clean-up step with silica cartridges. Recoveries were greater than 79% for all PAHs analysed. Reproducible determination with adequate detection and quantification limits (LOD and LOQ) were attained by HPLC with fluorescence detection for 14 PAHs. Acenaphthylene was determined with a UV–VIS detector. There is no significant difference in any PAHs or in the sum of them between the Argentinean and Brazilian samples. Therefore, the samples were evaluated together. The highest concentration of benzo(a)pyrene (BaP) detected was 0.57 µg kg^?1 in milk powder. Contamination of samples expressed as the sum of 15 analysed PAHs varied between 11.8 and 78.4 µg kg^?1 and as PAH4 (BaP, chrysene, benzo(a)anthracene and benzo(b)fluoranthene) was between 0.02 and 10.16 µg kg^?1. The correlation coefficient for PAH2 (BaP and chrysene) and PAH4 groups was 0.95, for PAH2 and PAH8 it was 0.71, and for PAH4 and PAH8 it was 0.83. All the samples were below the regulatory limit for BaP, but 65% of commercial milk powders do not comply with the European Union limit for PAH4. This is the first report of PAH contamination in powder milk from Argentina and Brazil.     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography–tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC–APCI–MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5–4.0 µg kg^?1 for NIV, 2.8–5.3 µg kg^?1 for DON, 0.4–1.7 µg kg^?1 for HT-2 and 0.4–1.0 µg kg^?1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^?1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.     

Distribution of semduramicin in hen eggs and tissues after administration of cross-contaminated feed

Semduramicin is an ionophore coccidiostat used in the poultry industry as a feed additive. Cross-contamination of feeds for non-target animals with semduramicin is unavoidable. However, it is not known whether undesirable residues of semduramicin may occur in food after cross-contaminated feed is administered to animals. The aim of the work was to determine the levels of semduramicin in hen eggs (yolks and albumen) and tissues (liver, muscle, spleen, gizzard, ovarian yolks and ovaries) after administration of feed contaminated with 0.27 mg kg^?1 of this coccidiostat. The residues were determined using LC-MS/MS. The distribution pattern confirmed the high lipophilicity of semduramicin. Residues were found mainly in egg yolks (28.8 µg kg^?1), ovarian yolks (19.5 µg kg^?1) and liver (2.57 µg kg^?1), while hens’ muscle was free from semduramicin (LOD = 0.1 µg kg^?1). Among edible tissues, the maximum level (2 µg kg^?1) was exceeded only in the liver.     

Optimization and Validation of a Method Without Alkaline Clean-Up for Patulin Analysis on Apple Puree Agar Medium (APAM) and Apple Products

A sensitive high-performance liquid chromatography-UV (HPLC-UV) method, based on the Association of Analytical Communities (AOAC) Official method 2000.02, was developed and validated for the high-throughput analysis of patulin in in vitro experiments on apple puree agar medium (APAM). The importance of repeating the ethyl acetate extraction step at liquid-liquid extraction (LLE) was examined, as well as the extent of patulin degradation during the sodium carbonate clean-up. In addition to this alkaline clean-up, the efficiency of using an Oasis HLB or C₁₈ cartridge as solid-phase extraction (SPE) clean-up was compared. This resulted in a two-step ethyl acetate LLE, followed by an Oasis HLB SPE clean-up, without alkaline clean-up conditions. The method was fully validated for APAM, cloudy apple juice, and apple puree. Average patulin recoveries at levels of 100, 500, and 1000 μg kg^?1 of APAM varied between 95 and 113 % over 3 independent days, with an interday precision (RSD_R) of 5 to 10 %. Recovery experiments carried out with the spiked apple juice (at 50 μg kg^?1) and apple puree (10 μg kg^?1) showed average recovery rates laying between 80–101 % (RSD_R?=?12 %) and 77–100 % (RSD_R?=?9 %), respectively. This method offered a detection limit of 3–4 μg kg^?1 and a quantification limit of 5–8 μg kg^?1 for APAM, apple juice, and puree.     

Tropane and ergot alkaloids in grain-based products for infants and young children in the Netherlands in 2011–2014

An LC-MS/MS multi-method was developed to simultaneously quantify ergot alkaloids (EAs) and tropane alkaloids (TAs) in 113 cereal-based food for infants and young children. To assess yearly variation, samples were collected in 2011, 2012 and 2014. EAs were detected in 54% and TAs in 22% of the samples. Mean EA levels in the three sampling years were 10.6, 6.2 and 8.6 µg kg^?1, respectively (maximum: 115.4 µg kg^?1), indicating that exposure to EAs would not have exceeded the health-based guidance values set by EFSA in 2012. Mean TA levels were 3.9, 2.4 and 0.4 µg kg^?1, respectively (maximum: 80.8 µg kg^?1). The acute reference dose for TAs, derived by EFSA in 2013, would have been exceeded by young children when consuming some of the products sampled in 2011–2012. TA levels had decreased drastically in 2014, possibly due to measures taken by producers as response to the EFSA Opinion.     

Development and validation of an UHPLC-MS/MS method for the determination of mycotoxins in grass silages

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) multi-mycotoxin analytical method was developed to simultaneously identify and quantify 20 mycotoxins in grass silages, inclusive of mycotoxins that are currently regulated in European Union feeds. Extraction of mycotoxins from dried grass silages was performed using of a modified QuEChERS extraction employing an acidified aqueous extraction (0.1 N HCl) with no further clean-up. Following chromatographic separation, analytes were detected using a fast polarity-switching MS/MS method that allowed both positive and negative ions to be analysed from a single injection, thus the reducing time and cost of analysis. The limits of detection and quantification ranged between 3 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A and A₁) and 200 µg kg^–1 DM (deoxynivalenol), and between 10 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A₁) and 500 µg kg^–1 DM (deoxynivalenol), respectively. Inter-assay accuracy and precision ranged between 90% and 107% and between 3.9% and 15.0% CV, respectively. The accuracy of the method was assessed through the application to a range of incurred samples in an inter-laboratory study.     

Occurrence of bound 3-monochloropropan-1,2-diol content in commonly consumed foods in Hong Kong analysed by enzymatic hydrolysis and GC-MS detection

The aim of this study was to determine the level of bound 3-monochloropropan-1,2-diol in foodstuffs commonly consumed in Hong Kong, China, by an enzymatic hydrolysis indirect method which proved to be free from interferences. A total of 290 samples were picked up randomly from the local market and analysed. About 73% of these samples were found to contain detectable amounts of bound 3-MCPD. Amongst the 73 food items, bound 3-MCPD was not detected in 13 food items, including extra virgin olive oil, beef ball/salami, beef flank, ham/Chinese ham, nuts, seeds, soy sauce, oyster sauce, butter, yoghurt, cream, cheese and milk. For those found to contain detectable bound 3-MCPD, the content ranged up to 2500 µg kg^?1. The highest mean bound 3-MCPD content among the 14 food groups was in biscuits (440 [50–860] µg kg^?1), followed by fats and oils (390 [n.d.–2500] µg kg^?1), snacks (270 [9–1000] µg kg^?1), and Chinese pastry (270 [n.d.–1200] µg kg^?1). Among the samples, the highest bound 3-MCPD content was in a grape seed oil (2500 µg kg^?1), followed by a walnut flaky pastry (1200 µg kg^?1) and a grilled corn (1000 µg kg^?1). Basically, the results of this study agreed well with other published results in peer-reviewed journals, except for cheese, cream, ham, nuts and seeds.     

Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Screening Phenylethanolamine A Residues in Pork Samples

Phenylethanolamine A (PEA), a new alternative β-agonist, has been illegally used in farming to promote the muscle growth in food-producing animals. In this study, a sensitive and convenient competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for determination of PEA residues in pork samples. The produced antibody was highly specific to PEA and exhibited a negligible cross-reactivity toward some other β-agonists. The developed technique was characterized by the limit of detection below 0.08 μg kg^?1 and the IC₅₀ value of 0.93 pmol mL^?1 (0.32 ng mL^?1). Validation of the technique was done using artificially spiked and naturally contaminated pork samples. The recoveries ranged from 79.6 to 112.6 % for the samples spiked at levels of 0.1–5 μg kg^?1 with the variation coefficients below 15 %. The analysis of naturally contaminated samples showed that the obtained data corresponded with the data obtained by the LC-MS/MS. The developed ciELISA was shown to be a feasible highly sensitive and specific screening tool for PEA residue analysis.     

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine,cattle, sheep,and chicken

A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C₁₈ column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5–16 µg kg^?1 ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5–2.0 µg kg^?1 in muscle and 1–4 µg kg^?1 in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.     

High-performance liquid chromatography with fluorescence detection for the determination of nitrofuran metabolites in pork muscle

A simple and sensitive HPLC method with fluorescence detection (HPLC-FLD) is reported for the simultaneous determination of metabolites of four nitrofuran drugs (furazolidone, furaltadone, nitrofurantoin and nitrofurazone) in pork muscle. The method involves acid hydrolysis of the protein-bound drug metabolites and the conjugation of the released side-chains with a novel fluorescence agent 2-hydroxy-1-naphthaldehyde. After liquid–liquid extraction and effective separation of the derivatives on a YMC-Pack Polymer C18 column at 40°C under alkaline conditions, the high fluorescence intensity of these derivatives at emission wavelength λ_em = 463 nm enables their simultaneous determination in pork muscle at concentrations as low as 1 µg kg^?1. The method was validated using blank pork muscle fortified with all four metabolites at 0.5, 1.0 and 2.0 µg kg^?1. Recoveries were > 92.3% with RSDs < 8.5% for all four metabolites. The results obtained with HPLC-FLD and LC-MS/MS methods showed very good agreement for pork muscle samples.