Determination of anabolic steroids in bovine serum by liquid chromatography-tandem mass spectrometry.

In the present paper we report the LC-MS/MS determination of residues of 12 anabolic steroids in bovine serum, as an expansion of our work protocols for steroids determination in biological matrices. Steroids analyzed included α-zearalanol, ß-zearalanol, α-trenbolone, ß-trenbolone, methyltestosterone, α-estradiol, ß-estradiol, ethynylestradiol, α-boldenone, ß-boldenone, α-nortestosterone and ß-nortestosterone. Following protein precipitation, serum samples were cleaned up by solid-phase extraction using Oasis HLB and Amino cartridges. Atmospheric pressure chemical ionization (APCI) in both positive and negative ionization modes was used and mass spectrometry detection was carried out in multiple reaction monitoring mode following two or (in most cases) three product ions per precursor ion. The method was validated in accordance with the Commission Decision 2002/657/EC. The decision limit (CCα) values obtained, ranged from 0.01 to 0.07 ng/ml and the detection capability (CCß) values obtained ranged from 0.02 to 0.12 ng/ml. The recoveries ranged from 70.2% to 118.2%. The developed method is suitable for routine and confirmatory purposes such as control of illegal use in livestock production.

Gel permeation chromatography clean-up for the determination of gestagens in kidney fat by liquid chromatography-tandem mass spectrometry and validation according to 2002/657/EC.

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of gestagens in kidney fat (medroxyprogesterone acetate, megestrol acetate and melengestrol acetate). The procedure involved a clean-up procedure with gel permeation chromatography (GPC). The analytes were analyzed by reversed-phase LC-MS/MS, in positive multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from the chosen precursor for the unambiguous confirmation of the gestagens. The method was validated at the validation level of 1.0 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCalpha ranged from 0.20 to 0.22 ng/g while the detection capabilities CCbeta ranged from 0.33 to 0.38 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate mean for residue analysis studies.

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry.

A specific and sensitive multi-method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha/beta-nortestosterone, methyltestosterone, beta-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, alpha/beta-zearalenol, alpha/beta-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCalpha ranged from 0.03 to 0.14ng/g while the detection capabilities CCbeta ranged from 0.05 to 0.24ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.

Determination of anabolic steroids in bovine urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) multi-method has been developed for the determination of 15 anabolic steroids in bovine urine (diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha-nortestosterone, alpha/beta-zearalenol, alpha/beta-zaeralanol, zearalenone, stanozolol and 16beta-OH-stanozolol). The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, a washing step with hexane and final clean-up with SPE with Oasis HLB and Amino cartridges. The analytes were quantified by liquid chromatography coupled to a tandem mass spectrometer (LC-TSQ Quantum AM) operating in both positive and negative atmospheric pressure chemical ionisation (APCI). Data acquisition was performed in multiple reaction monitoring (MRM) mode quantifying two diagnostic product ions from a chosen precursor. The method was validated according to the Commission Decision 2002/657/EC, for the detection and confirmation of residues in products of animal origin. The method specificity, sensitivity, accuracy and precision were evaluated. The decision limits CCalpha ranged from 0.06 to 0.26 ng/ml and the detection capabilities CCbeta ranged from 0.11 to 0.49 ng/ml. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in bovine urine and can be used for residue control programs.

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.