Evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on N-nitrosamine formation when heating cured meat.

N-nitrosamines are meant to be probable or possible carcinogenic components, possibly formed out of a reaction between nitrite and N-containing substances such as amino acids and secondary amines. Nitrite is often used for processing meat products because of its colouring and antimicrobial properties. During this experimental setup, the influence of proline, hydroxyproline or pyrrolidine on N-nitrosamine formation in meat samples was evaluated. The N-nitrosamines concentrations were measured with gas chromatography-thermal energy analyzer. Only the concentrations of N-nitrosodimethylamine and N-nitrosopyrrolidine were found above the limit of detection in a number of tested experimental conditions. The concentration of these two N-nitrosamines was modelled as a function of temperature and nitrite concentration for different situations (presence or absence of added natural N-containing meat components). It could be concluded that proline and pyrrolidine promoted the formation of N-nitrosopyrrolidine. It could also be confirmed that the higher the temperature of the meat processing procedure and the higher the sodium nitrite amounts added, the higher were the yields of the respective N-nitrosamines.

Optimization and validation of a liquid chromatography tandem mass spectrometry (LC/MSn) method for analysis of corticosteroids in bovine liver: evaluation of Keyhole Limpet beta-glucuronidase/sulfatase enzyme extract.

A liquid chromatography tandem mass spectrometry (LC/MS(n)) method for the determination of 12 corticosteroids in bovine liver has been optimized and validated in accordance with the European Commission Decision 2002/657/EC. A bovine liver sample was deconjugated with beta-glucuronidase/sulfatase enzyme, extracted with diethyl ether and further cleaned up with Solid Phase Extraction (SPE) before analysis with LC/MS(n). Two different enzyme extracts (originating from Helix Pomatia and Keyhole Limpet) and three SPE elution solvents (ethyl acetate, acetonitrile and methanol) were compared during the optimization. Helix Pomatia is generally known as the enzyme most being used for enzymatic hydrolysis purposes. Nevertheless, when detecting corticosteroids in the low microg kg(-1) concentration range, the Helix Pomatia extract may lead to interferences in the final LC/MS(n) chromatogram. When using the Keyhole Limpet enzyme extract, no interferences were observed and therefore, this extract was the best choice for enzymatic hydrolysis tested in this case. Ethyl acetate was used as elution solvent during the validation procedure since SPE elution with acetonitrile resulted in higher chromatographic backgrounds, while elution with methanol showed less reproducible results. Validation of the optimized method was carried out for 10 of the 12 corticosteroids, giving mean recoveries between 91 and 109%, and repeatability and reproducibility coefficients of respectively maximum 13.7 and 18.0%. The working ranges for the linear calibration curves were 5-20 microg kg(-1) for prednisolone, methylprednisolone and prednisone and 0.5-4 microg kg(-1) for the other compounds (coefficients of determination R(2)> or =0.97). Specificity, decision limit (CCalpha) and detection capability (CCbeta) were for all compounds within the EC specified limits.

Calculation of the decision limit (CCalpha) and the detection capability (CCbeta) for banned substances: the imperfect marriage between the quantitative and the qualitative criteria.

Initially in the Decision 2002/657/EC the criteria for the calculation of the decision limit (CCalpha) and the detection capability (CCbeta) have been estimated as purely quantitative (alpha-error is 1% and beta-error is 5%). In 2004, the European Commission has issued a document to provide guidance for the interpretation of the 2002/657/EC. In this document it is mentioned that also qualitative criteria should be fulfilled. Therefore, the calculated CCalpha and CCbeta must be verified by using fortified samples. The method should be able to detect/identify the target component in 50% of the cases at CCalpha and in 95% of the cases at CCbeta. Analytical methods for the analysis of nitroimidazoles, nitrofurans and corticosteroids with LC-MS/MS have been validated by fortifying blank samples below and above the MRPL. CCalpha and CCbeta were calculated using the ISO 11843 approach. In addition, the frequency of methodical compliance for the qualitative criteria was determined at each concentration level. It was observed that at the calculated CCalpha and CCbeta levels the qualitative criteria were not fulfilled. It was concluded that the detection capability of the analytical method should be calculated by using decreasing fortification levels at and below the MRPL. A protocol validating methods for banned substances by limiting the number of samples is presented and the qualitative criteria for the assessment of CCalpha and CCbeta were verified based on the same set of data without the need of performing additional validation experiments.

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3).

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.

Determination of anabolic steroids with gas chromatography-ion trap mass spectrometry using hydrogen as carrier gas.

Helium is considered to be the ideal carrier gas for gas chromatography/mass spectrometry (GC/MS) in general, and for use with an ion trap in particular. Helium is an inert gas, can be used without special precautions for security and, moreover, it is needed as a damping gas in the trap. A disadvantage of helium is the high viscosity resulting in long GC run times. In this work hydrogen was tested as an alternative carrier gas for GC in performing GC/MS analyses. A hydrogen generator was used as a safe source of hydrogen gas. It is demonstrated that hydrogen can be used as a carrier gas for the gas chromatograph in combination with helium as make-up gas for the trap. The analysis time was thus shortened and the chromatographic performance was optimized. Although hydrogen has proven useful as a carrier gas in gas chromatography coupled to standard detectors such as ECD or FID, its use is not mentioned extensively in the literature concerning gas chromatography-ion trap mass spectrometry. However, it is worth considering as a possibility because of its chromatographic advantages and its advantageous price when using a hydrogen generator.

Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry.

The objective of this study was to provide LC and GC-multiple mass spectrometry (MSn) data in positive and negative ion modes to prove the distinction between dexamethasone and betamethasone in a mixture of both components. Using GC-MS, the differentiation was based on a difference in the ratio of the ion traces of the two chromatographic peaks of the alpha and beta epimer with m/z 310 and 330. A minimum of 15% dexamethasone should be present in a mixture of both to detect it as present with a probability of 95%. In the same way betamethasone can be detected from 15% on. Because of the very similar structures of the dexamethasone and betamethasone epimers, no reversed-phase (RP) separations have been reported. Normal-phase separations have been reported in other studies. However because of the compatibility of RP mobile phases in the coupling with MS, the latter was the method of choice. In LC-MSn positive ion mode the product ion 355 was plotted against the sum of 337 and 319. With this combination dexamethasone and betamethasone could be discriminated in a mixture of 20 to 80% of each combination of analytes. In negative ion mode only two product ions were formed from the fragmentation of the acetate adduct, [M-H]- and [M-H-CH2O]-. The intensity of the fragment 391 ([M-H]-) was determined in the discrimination of the two epimers.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine.

In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated.

Determination of mercaptobenzimidazol and other thyreostat residues in thyroid tissue and meat using high-performance liquid chromatography-mass spectrometry.

This paper describes a method for extraction of tapazol, thiouracil, methylthiouracil, propylthiouracil and mercaptobenzimidazol (MBI) from thyroid tissue. The solid-phase extraction procedure is optimized to obtain the maximum results for the main thyreostats including MBI. Different combinations of sample application, column conditioning and wash steps were tested. The analytes were extracted from the matrix with methanol. After solid-phase extraction they were derivatised with 7-chloro-4-nitrobenzo-2-furazan. Determination is carried out using liquid chromatography-electrospray mass spectrometry. The identification of the analytes was performed according to the final revision of the EU criteria (93/256/EC decision). The detection capability was 20 microg kg(-1) for all mentioned thyreostats.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry.

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.