Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer.

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.

New database on hormone and veterinary drug residue determination in animal products.

A new database was created which provides a carefully judged inventory of analytical methods available for the determination of residues of growth promoters (steroidal anabolic hormones, beta-agonists and glucocorticoids) and veterinary drugs (antibiotics and growth inhibitors), which are or will be regulated by EU legal acts. This database is available on the Internet at http:/(/)cemu10.fmv.ulg.ac.be/OSTC.

Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg.

A new and efficient procedure for the clean-up of tetracycline residues in animal tissues and egg prior to reversed-phase high-performance liquid chromatography is described. The principal steps involve homogenization of the tissues in sodium succinate buffer and methanol, followed by centrifugation and clean-up with metal chelate affinity chromatography (MCAC). After further concentration on an Empore extraction membrane with cation-exchange properties, the sample is analysed by HPLC with fluorescence detection. The method was tested on porcine kidney and muscle, bovine liver and whole chicken's egg. The recoveries were determined from spiked tissues for oxytetracycline, tetracycline, chlortetracycline and doxycycline and ranged from 40 to 70%, with repeatabilities below 10% R.S.D.. The analytical responses were linear in the range up to at least 1000 ng/g. The detection limits of the method were estimated at 0.42 ng/g of oxytetracycline, 0.49 ng/g of tetracycline, 0.66 ng/g of chlortetracycline and 1.38 ng/g of doxycycline in porcine muscle, using signal-to-noise ratios of 4:1. Similar detection limits were estimated for kidney, liver and egg. The measured limits of quantification were 2 ng/g for oxytetracycline, 3 ng/g for tetracycline, 4 ng/g for chlortetracycline and 5 ng/g for doxycycline in porcine kidney. The advantage of this method over existing methods is its increased limit of detection.

Multiresidue analysis of anabolic agents in muscle tissues and urines of cattle by GC-MS.

The illegal use of anabolic steroids in livestock breeding has taken enormous proportions the last few decades. To protect the consumer against possible harmful effects due to the consumption of contaminated meat or meat products, a multiresidue analysis of anabolic steroids has been developed for muscle tissues and urine. The pretreatment of the meat and urine samples consists of an enzymatic digestion, liquid or solid-phase extraction, and finally high-performance liquid chromatography (HPLC) fractionation. Five fractions or windows are collected, each containing a number of analytes. The residues are derivatized prior to the detection by gas chromatography-mass spectrometry (GC-MS). Both gas chromatographic retention data and mass spectral data are used for identification of nortestosterone, testosterone, estradiol, ethynylestradiol, trenbolone, zeranol, diethylstilbestrol, boldenone, methandienone, methyltestosterone, megestrol acetate, chlormadinone acetate, medroxyprogesterone acetate, chlorotestosterone, progesterone, and chlorotestosterone acetate. The limit of detection varies from matrix to matrix and from analyte to analyte but is, in the most favorable case, on the order of 0.3 ppb (micrograms/kg).

A quantitative method for the detection of sulfonamide residues in meat and milk samples with a high-performance thin-layer chromatographic method.

Sulfonamides are widely used in veterinary medicine for prophylactic purposes and for the treatment of various infections of food-producing animals. This means that residues of these drugs and their possible metabolites may occur in food of animal origin. In Belgium, a zero tolerance level for sulfonamides in edible animal tissues has been set. In order to check this zero level on a routine basis, a rapid and sensitive method has to be available. For this purpose, a quantitative high-performance thin-layer chromatographic (HPTLC) method for the detection of sulfonamide residues in animal tissue and milk samples has been developed. The sample preparation consists of a liquid extraction followed by a solid phase extraction (SPE) on disposable columns for the meat samples and a matrix solid phase dispersion (MSPD) for the milk samples. A three-multiple development chromatographic system is used for the separation and a derivatization with fluorescamine decreases the minimal detectable quantity per spot from 1.42 to 0.32 ng. The limit of quantification is 4 micrograms/kg for milk and meat samples.

Development of a competitive enzyme immunoassay for 17 alpha-19-nortestosterone.

Because 17 beta-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17 alpha-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17 alpha-19-nortestosterone-3-carboxy-methyloxime-bovine serum albumin (17 alpha-19-NT-3-CMO-BSA), the competitive incubation of 17 alpha-19-NT and the 17 alpha-19-nortestosterone-3-CMO-horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17 alpha-19-nortestosterone was used to produce an antibody with selective affinity for the 17 alpha-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B0 50% of +/- 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.

Peroxidative in vitro metabolism of diethylstilbestrol induces formation of 8-hydroxy-2'-deoxyguanosine.

The generation of superoxide and hydroxyl radicals is known to be implicated in the hydroxylation of 2'-deoxyguanosine (dG) at the C-8 position and of guanine base residues in DNA. It was also shown previously that in the presence of horseradish peroxidase, hydrogen peroxide and Fe3+ - EDTA complex, diethylstilbestrol (DES) induces single strand breaks in DNA, caused by the production of superoxide anion (O2-) and hydroxyl (OH.) radicals. By means of high-pressure liquid chromatography and electrochemical detection a strong indication is adduced that dG is oxidized to 8-hydroxy-2'-deoxyguanosine during peroxidative in vitro metabolism of DES, which might be at the basis of DES induced cell transformation.

Fluorimetric determination of aflatoxin M1 in cheese.

A simple and sensitive method is proposed for the determination of aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water (3 + 1). Acetone is evaporated under vacuum, and the aqueous phase is passed through a C18 disposable cartridge. After the cartridge is washed with acetonitrile-water (1 + 9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a silica cartridge. Final analysis is performed by 2-dimensional thin layer chromatography (TLC) combined with fluorodensitometry or by liquid chromatography on a reverse phase C18 column with fluorescence detection. Recovery is greater than 90%, and the coefficient of variation is 6% or less. The detection limit is in the range of 10 ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1 derivative and rechromatography.

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay.

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.

Radioimmunoassay of ochratoxin A in barley.

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.

Systematic identification of antioxidants in lards, shortenings, and vegetable oils by thin layer chromatography.

A simple and reliable method is described for rapid identification of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin, gallates (lauryl, octyl, propyl), nordihydroguaiaretic acid, 3,3'-thiodipropionic acid, tocopherol, t-butylhydroquinone, and 2,4,5-trihydroxybutyrophenone in lards, shortenings, and vegetable oils. The antioxidants are extracted with 95% methanol, concentrated under vacuum at less than or equal to 45 degrees C, and analyzed by thin layer chromatography. Three elution solvents, 2 adsorbent types, 2 visualization sprays, and UV viewing at 254 and 36 nm are used. Sunflower and corn oil samples, fortified with 100 ppm antioxidant, were analyzed to establish to validity of the method.

Spectroscopic properties of the methyl esters of chlorophenoxy acid herbicides.

The mass and infrared spectra of the methyl esters of 9 chlorophenoxy acid herbicides are presented. Ultraviolet data are discussed and proton magnetic resonance spectra are tabulated. Because of the sensitivity of the technique, the mass spectra are most useful for the identification of those compounds in residues, especially by combined gas chromatography-mass spectrometry. The pure herbicides used for the recording of the spectra were obtained by synthesis and recrystallization.