For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected pharmacological substances in doping control. In order to improve their performances, professional sportsmen are often tempted to take dietary supplements. However, due to the frequent and widespread occurrence of contaminated supplements, the use of such products is not without risk for the athletes involved. In order to minimize the chances of an unattended positive doping test or serious health problems, fast and reliable screening methods for the detection of anabolic steroids in dietary supplements are needed. A general screening procedure requires the fast and unambiguous detection of a large range of steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the detection of doping substances for the past 40 years. Over time, many laboratories have delivered spectra to be included in standard reference databases, one of which is maintained by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS analysis. In the present study, a new mass spectral library of 88 steroids was developed, along with a fast UPLC-MS method. For the construction of this mass spectral library, three different mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100 V, respectively. This method was then successfully tested on contaminated dietary supplements which had previously been tested by means of a targeted LC-MS/MS method. Overall, the library search was shown to identify the same compounds as the MRM method.
The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.
DHEA (3β-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day during 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated and sulphated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17α-and 17β-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol and 17α-testosterone were observed in the free and sulphated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the ∆ 5 -pathway with 5-androstenediol as the intermediate.Sulphoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulphated form, while glucuronides were negligible.
The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography-mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.
Solvent Front Position Extraction is a novel technique developed for effective sample preparation of biological samples containing coccidiostats prior to LC–MS/MS. In this study the technique was used for isolation and determination of seven coccidiostats, from both main groups being: ionophores and chemical coccidiostats. Its effectiveness was evaluated by comparing with other sample preparation procedures, used in European routine laboratories. Results obtained by Solvent Front Position Extraction were very satisfactory (linearity R2 ≥ 0.971, recovery 90.1–111.1%, RSDr: 8.7–16.6%, RSDR: 9.0–17.7%) and fulfilled requirements described in Commission Regulation (EU) 2021/808 of 22 March 2021, which showed great potential of the technique in sample preparation of coccidiostats in poultry feed.
Fast HPLC offers a definite time advantage for analyses such as content uniformity which requires, in general, a routine analysis of as many as 30 samples and for the analysis of dosage forms of developmental drugs. The columns for Fast HPLC can be used with a minimal amount of modification to conventional HPLC hardware. The resolution obtainable with a Fast HPLC column is poorer than that of a conventional column.(All references to conventional columns in the article imply, unless otherwise indicated, those columns 15-30 cm in length and 3.9-4.6 mm i.d.) improve the resolution of a closely eluting peak resulted in retention times not too different from typical conventional columns. tion and a savings in both solvent and sample consumption, the utility of these columns is limited by the need for specialized hardware. Since the sample volume is not a limiting factor in a typical pharmaceutical analysis and overall cost savings from lesser solvent consumption are not significant in a majority of Attempts to modify a mobile phase to Although microbore columns offered improved resolu-3393 Copyright 0 1987 by Marcel Dekker, Inc. Downloaded by [Duke University Libraries] at 15:44 03 January 2015 3394 PADMANABHAN ET AL.cases as solvent can be recycled, it is concluded that the Fast HPLC columns can play a more important role than the currently available microbore columns in laboratories engaged in pharmaceutical analysis, particularly at the product development stage.Fast high pressure liquid chromatography (1, 2) (Fast HPLC) is a technique in high pressure liquid chromatographic (HPLC) analysis in which the components of analytical interest are eluted in the order of 30 to 100 seconds, which is about 5 to 10 times faster than conventional HPLC.should not be confused with Technicon "Fast-LC"B, which is a sample pretreatment system ( 3 ) . ] Fast HPLC and the columns employed with it have been referred to in the literature by terminologies such as "Fast Analysis by HPLC" ( 4 ) , "Fast LC Separations" (5), "High Speed Chromatography" ( 6 ) , "High Speed, Low Dispersion LC" (7), "Very High Speed Liquid Chromatography" (8, 9, lo), "High Speed LC Using Short Columns" ( 7 ) , "Short High Efficiency HPLC Column" ( 1 1 ) , "Very Short LC Column" ( 1 2 ) , and "Little Champ@" ( 1 3 ) . These columns are typically short [This technique and packed with smaller particles with the following column parameters: size -3 or 5 microns. ferred mainly for the following reasons (14): the gain in chromatographic run time was not significant in terms of total analysis time, which includes sample clean-up and post HPLC length -3 to 5 cm; diameter -4 . 6 mm and particle In the past, Fast HPLC was not predata processing times; columns with 3 micron packings were not available commercially and the 5 micron or larger column Downloaded by [Duke University Libraries] at 15:44 03 January 2015 FAST HPLC IN PHARMACEUTICAL ANALYSIS 3395 packings available at that time had, in general, poor reso tion characteristics; and the hardware available were not s...
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