Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

Urinary 19-norandrosterone purification by immunoaffinity chromatography: application to gas chromatography/combustion/isotope ratio mass spectrometric analysis.

The detection of exogenous 19-norandrosterone (19-NA) in urines was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). 19-NA is, for the first time to our knowledge, isolated from urinary matrix by specific immunoaffinity chromatography (IAC) before analysis. The sample preparation consisted of a preliminary purification of urine by solid-phase extraction after hydrolysis by beta-glucuronidase. Unconjugated 19-NA was thus isolated by IAC and directly analysed by GC/C/IRMS. Optimisation of IAC purification was achieved and the reliability of the technique for anti-doping control is discussed.

Long-term detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry.

A method for the detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry (GC-HRMS) is described. The sample preparation involved chemical digestion of the protein structure, which was achieved by incubating the hair with 1 M KOH at 70 degrees C. A single extraction step with tert.-butyl methyl ether provided approximately 90% of the analyte, which was dried and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) to yield clenbuterol N,O-bis-trimethylsilyl (TMS). Hair was collected from four pregnant women who were therapeutically treated with Spiropent (clenbuterol-HCl) and from the infant of one female patient. Hair samples were taken during the application time and two to six months after completion of clenbuterol administration. The detection limit of the method was approximately 4 ng clenbuterol/g hair when 25 mg hair material were processed and 2 ng/g for 50 mg hair samples (corresponds to 4 pg per injection). The method allows clenbuterol to be measured retrospectively for up to at least six months. The levels of clenbuterol determined in hair ranged from 2 to 236 ng/g. No clenbuterol was found in the hair of the infant, which was taken five and a half months after delivery. To improve sample preparation, an additional purification step via immuno affinity chromatography (IAC) was integrated. The IAC purified extracts showed reduced biological background interference and an improved limit of detection (0.8 ng/g).

Comparison of purification procedures for the isolation and detection of anabolic residues in faeces using gas chromatography-mass spectrometry.

Within several regional field laboratories and the national reference laboratory a harmonised methodology for the analysis of anabolic residues in faecal samples was developed. The method consists of a liquid-liquid and a solid-phase extraction step, followed by a high-performance liquid chromatography purification step. Using gas chromatography-mass spectrometry, currently illegally used anabolic steroids can be detected in faeces at the ppb level. Within this context acidification, followed by centrifugation under cooling, allows efficient, practical and rapid defatting of faecal samples. Furthermore, a combination of a silica and an aminopropyl solid-phase extraction column was found to give the best results as regards the sample purification process.

Quantitative determination of several synthetic corticosteroids by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography.

A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.

Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry.

The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3'-hydroxystanozolol (II) and 4 beta-hydroxystanozolol (III)] and metandienone (IV) I17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (V) and 18-nor-17,17-dimethyl-5 beta-androsta-1,13-dien-3 alpha-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation.

By-products of steroid synthesis: a cause of interferences in thin-layer chromatography residue analysis.

Since the late 1980s all of the laboratories involved in high-performance thin-layer chromatography (HPTLC) control of hormonal residues in kidney fat, have occasionally detect a green fluorescent spot with similar RF values and colour to those observed for methyltestosterone (MT). This spot (product) could lead to false positive results for MT and was thus named 'le faux méthyl' (the false methyl) by a french speaking colleague. All of the samples with a false methyl spot also contained a relatively high concentration of progesterone. Differentiation of this product from methyltestosterone can be performed in three ways: firstly, extra HPTLC on reversed-phased plates, secondly, extra purification of the extract with HPLC prior to HPTLC and thirdly, gas chromatography--mass spectrometry. This interference was identified as 20 beta-hydroxyprogesterone, a by-product of progesterone. The problem of the false methyl was not only linked with the TLC characteristics of MT but also to the progesterone used as standard. Some laboratories used an analytical-reagent grade standard and others used commercial progesterone powders as standards (e.g., obtained in crude form from pharmaceutical companies). The commercial-grade progesterones showed two spots in comparison with the analytical standard that showed just one spot. As the false methyl was observed not only in kidney fat and meat samples, but also in illegal hormone cocktails, it was concluded that we had detected a by-product of an illegally used 'natural progesterone'.

Control of the illegal administration of natural steroid hormones in the plasma of bulls and heifers.

In the context of the control of the illegal administration of natural steroid hormones in cattle husbandry, an attempt was made to establish the decision levels for sex steroid hormones in the plasma of adult cattle, taking into account the effect of the treatment. Bulls and heifers were treated with two injections, at a two week interval, of an estradiol-testosterone cocktail. Steroid hormone and biochemical precursor concentrations were measured in plasma samples by using specific radioimmunoassays, before and after the treatment. When the treatment significantly (p < 0.05) modified a hormone concentration, a decision level was established for that hormone concentration. At each decision level, a score was assigned that represented the percentage of treated animals detected when the decision limit was applied. For heifers, 17 beta-estradiol and testosterone concentrations in plasma, which increased after the treatment, are the best criteria to use to detect treated animals, with decision limits of 20 pg ml-1 and 125 pg ml-1, respectively. In the instance of bulls, both testosterone and steroid biochemical precursor concentrations decreased in the plasma after the treatment. We proposed decision limits of 1500 pg ml-1 and 28 pg ml-1 for testosterone and androstenedione concentrations, respectively, the bulls displaying concentrations below these limits being positive. We observed that the repetition of the injection increased the score of the decision limit. The scores for testosterone are 70%, 14d after the first injection and 100% 14 d after the second injection, and for androstenedione, these scores are 60 and 100%, respectively.

Regulation of progesterone during pregnancy in the cat: studies on the roles of corpora lutea, placenta and prolactin secretion.

The regulation of progesterone secretion and release by the corpus luteum and/or by the placenta in cats during pregnancy has been poorly studied and the relative roles of the corpus luteum and the placenta in the release of progesterone into the plasma is not well defined. It is generally assumed that after 40-45 days of pregnancy, circulating progesterone is mainly produced by the placenta and that after 45-50 days the corpus luteum is no longer necessary to maintain pregnancy, even if remnants of corpora lutea are detected in the ovaries during the following interoestrous period. In this study, the relative roles of the placenta and ovary in progesterone production after 30 days of pregnancy have been analysed in an attempt to define the role of progesterone in pregnant cats. Different experiments involved (1) analysis of the effects of an ovariectomy performed at 30 days of pregnancy, and of megestrol acetate supplementation on plasma progesterone during the second half of pregnancy, (2) analysis of the effects of an ovariectomy performed at 45 days on plasma progesterone production and on pregnancy and (3) analysis of the effects of an antiprolactin agent (cabergoline) at days 30 and 40 of pregnancy on progesterone secretion and pregnancy. These results were all compared with the normal plasma progesterone profiles during (a) pseudopregnancy and (b) pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Synthetic anabolic androgens in the fecal material of cattle: radioimmunological determination after purification by high performance liquid chromatography]. / Androgènes anabolisants de synthèse dans les matières fécales des bovins: dosage radio-immunologique après purification par chromatographie liquide à haute performance.

In answer to the mandatory control of the illegal use of anabolizing agents in meat-producing animals imposed by the EEC in farms, a method of analysis of faeces involving high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) has been described. Four HPLC fractions were collected and submitted to corresponding RIA: 17 beta- and 17 alpha-trenbolone, 17 beta-nortestosterone, 17 alpha-nortestosterone and 17 alpha-methyltestosterone. The mean extraction and purification yield was estimated at 44 +/- 7% using tritiated 17 alpha-methyltestosterone as internal standard. Detection limits of the 3 hormones were estimated at 0.2-0.3 ng/g of faeces. About 50 samples can be analysed per week by this method.

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining.

For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HC1 4N: Ethanol 70 degrees (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developing.