Classification and detection of testosterone propionate and nandrolone residues in duck meat using surface-enhanced Raman spectroscopy coupled with multivariate analysis.

There is a critical need for a rapid and simple method of qualitative and quantitative analysis of testosterone propionate (TP) and nandrolone (NT) residues in duck meat. In this study, we applied surface-enhanced Raman spectroscopy (SERS) coupled multivariate analysis for the classification and detection of TP and NT residues in duck meat. A total of 294 duck meat extract samples were obtained from duck breast meats based on a LC-MS/MS sample preparation method with slight modification including 102 duck meat extract samples without TP and NT, 43 duck meat samples containing TP, 47 duck meat extract samples containing NT, and 102 duck meat extract samples containing TP and NT. Raw Raman spectra were pretreated by using adaptive iteratively reweighted penalized least squares (airPLS), normalization and first derivative, and then the score values of first 10 principal components were selected as the inputs of the developed models. A particle swarm optimization-support vector classification (PSO-SVC) model was created to classify all the duck meat samples into the 4 groups (i.e., control group, TP group, NT group, and TP combined with NT group) with the classification accuracies of 99.49 and 100% for training set and test set, respectively. Furthermore, 2 least squares support vector regression (LS-SVR) models were developed to predict the TP values in samples with a determination coefficient (R2) value of 0.9316, root mean square error (RMSE) value of 2.1739, and ratio of prediction to deviation (RPD) value of 3.2189 for the test set, and NT values in samples with an R2 value of 0.9038, RMSE value of 2.2914, and RPD value of 2.9701 for the test set. Surface-enhanced Raman spectroscopy technology, in combination with multivariate analysis, has the potential to become the qualitative and quantitative analysis tool for TP and NT residues in duck meat extract.

Determination of steroids in bovine hair: Validation of a microwave-assisted chemical derivatization method using liquid chromatography-tandem mass spectrometry and in vivo studies.

Hair analysis has attracted great attention in the regulatory analysis of food-producing animals, particularly due to the wider detection window of veterinary drugs in this matrix and also the possibility of confirming parent drugs with minimum metabolization. This work involved the development and validation of a quantitative liquid chromatography-tandem mass spectrometry method to determine 25 steroids and steroid esters in bovine hair. Sensitivity was improved using a fast and effective microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was conducted in accordance with the Decision 657/2002/EC guidelines. An animal experimentation procedure was performed on 12 bovine animals in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections on the neck. The samples were collected for 78 days in which the detection of the administrated analytes was only observed near the application sites. For some of the monitored days, no analyte was detected on the neck area. Since the migration of the analytes was not observed in areas other than the application site, false-negative results should be carefully considered when monitoring animal hair samples.

Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry.

Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS2 mode was developed and validated to improve the coverage to 65 AAS and/or their esters in a single injection. In addition, a GC/MS/MS method in selected reaction monitoring (SRM) mode was developed to screen for another seven AAS and/or their esters not adequately covered by the UHPLC/HRMS method using the same sample extract after derivatisation with pentafluoropropionic anhydride. The UHPLC/HRMS and GC/MS/MS methods in combination allowed the detection of 72 AAS and/or their esters with estimated limits of detection down to sub to low ppb levels with good interday precision. Method applicability was demonstrated by the detection of boldione and 4-androstenedione in two out-of-competition hair samples and testosterone propionate in a referee hair sample.

Development and validation of rapid and simultaneous method for determination of 12 hair-growth compounds in adulterated products by UHPLC-MS/MS.

Synthetic hair-growth compounds have been illegally used in diverse products to enhance the short-term efficacy of these products. In this study, a rapid and simultaneous method for the determination of hair-growth compounds in adulterated products based on ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The limit of detection (LOD) and limit of quantitation (LOQs) of the method were 0.08-43.6ng/mL and 0.27-145ng/mL for the solid-, liquid-, and cream-type samples, respectively. Good calibration linearity for all compounds was demonstrated with a correlation coefficient (r2) higher than 0.997. The intra- and inter-assay precisions were within 11%. The corresponding accuracies were 86-117% and 81-113%, respectively. The mean recoveries obtained for the solid-, liquid, and cream-type samples ranged from 87 to 114%, with a relative standard deviation (RSD) within 6%. The RSD of the stability evaluated at 4°C for 48h was less than 6%. The established method was used to screen 76 samples advertised as hair-growth treatments, from online and offline markets, over the course of two years. In 10% of the samples, four compounds, including triaminodil, minoxidil, finasteride, methyltestosterone, and testosterone-propionate were detected. The concentrations were in the range of 0.5-16.4mg/g. This technique provides a reliable platform for technical analysis for continuous monitoring of adulterated products to protect public health.

Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry.

Anabolic and androgenic steroids (AASs) are a class of prohibited substances banned in horseracing at all times. The common approach for controlling the misuse of AASs in equine sports is by detecting the presence of AASs and/or their metabolites in urine and blood samples using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This approach, however, often falls short as the duration of effect for many AASs are longer than their detection time in both urine and blood. As a result, there is a high risk that such AASs could escape detection in their official race-day samples although they may have been used during the long period of training. Hair analysis, on the other hand, can afford significantly longer detection windows. In addition, the identification of synthetic ester derivatives of AASs in hair, particularly for the endogenous ones, can provide unequivocal proof of their exogenous origin. This paper describes the development of a sensitive method (at sub to low parts-per-billion or ppb levels) for detecting 48 AASs and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Decontaminated horse hair was pulverised and subjected to in-situ liquid-liquid extraction in a mixture of hexane - ethyl acetate (7:3, v/v) and phosphate buffer (0.1M, pH 9.5), followed by additional clean-up using mixed-mode solid-phase extraction. The final extract was analysed using UHPLC-HRMS in the positive electrospray ionisation (ESI) mode with both full scan and parallel reaction monitoring (PRM). This method was validated for qualitative identification purposes. Validation data, including method specificity, method sensitivity, extraction recovery, method precision and matrix effect are presented. Method applicability was demonstrated by the successful detection and confirmation of testosterone propionate in a referee hair sample. To our knowledge, this was the first report of a comprehensive screening method for detecting as many as 48 AASs and/or their esters in horse hair. Moreover, retrospective analysis of non-targeted AASs and/or their esters was made feasible by re-examining the full scan UHPLC-HRMS data acquired.

Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry.

A sensitive method for the identification and quantification of anabolic steroids and clenbuterol at trace levels in dietary supplements by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in atmospheric pressure ionisation (APCI) mode using a single-stage Orbitrap analyser operating at a resolution power of 100 000 full width at half maximum (FWHM) was developed and validated. A total of 1 g of dietary supplement was added with testosterone-d3 as internal standard, dissolved in methanol, evaporated to dryness, diluted in sodium hydroxide solution and extracted with a mixture of pentane/ethyl ether 9:1. The extract was directly injected into the LC-HRMS system. The method was fully validated. Limits of detection (LODs) obtained for anabolic androgenic steroids (AASs) varied from 1 to 25 ng g(-1) and the limit of quantitation (LOQ) was 50 ng g(-1) for all analytes. The calibration was linear for all compounds in the range from the LOQ to 2000 ng g(-1), with correlation coefficients always higher than 0.99. Accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Good values of matrix effect and recovery were achieved. The ease of the sample preparation together with a fast run time of only 16 min permitted rapid identification of the analytes. The method was applied to the analysis of 30 dietary supplements in order to check for the presence of anabolic agents not labelled as being present in these supplements. Many AASs were often detected in the same sample: indeed, androstenedione was detected in nine supplements, 5-androsten-3ß-ol-17-one (DHEA) in 12, methandienone in three, stanozolol in one, testosterone in seven and testosterone esters in four of them. A retrospective analysis of suspected compounds not included at the beginning of the method development was also possible by means of the full acquisition spectra obtained with the HRMS technique.

[Study on simultaneous determination method for banded synthesis hormones residues in egg products].

OBJECTIVE: To develop method of 7 banded synthesis sex hormones residues in egg products determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). METHODS: The sample were enzymolied and target compounds were extracted with methanol. ZnCl2 was added to the extract solution to remove lipid and then analytes were purified by LC-C18 and LC-NH2 solid phase extraction cartridge and further determined by UPLC-MS/MS under positive ionization and multiple reaction monitoring (MRM) modes. RESULTS: The limits of detection (LOD) of UPLC-MS/MS method used for testing Chlormadione acetate (CDA), Medroxypogesterone acetate (MPA), Megestrol Acetate (MA), Testosterone propionate (TSP), Norgestrel (NG), Methyltestosterone (MTS) and Nandrolone (NT) in egg products ranged from 0.012 to 0.23 microg/kg, and the limits of quantification (LOQ) were from 0.04 to 0.76 microg/kg. Experiments on spiked samples of egg products showed that at addition level of 2.0 microg/kg, the average recoveries of the sex hormones ranged from 80.2% to 114%, and coefficients of variation from 6.7% to 14.3%; while at addition level of 4.0 microg/kg, the average recoveries ranged from 75% to 119%, and coefficient of variation from 2.9% to 7.3%. CONCLUSION: The method could be able to identify and quantify banded synthesis hormones residues in eggs and egg products. It could be simple and sensitive, suitable for statutory residue testing.

Hair analysis of anabolic steroids in connection with doping control-results from horse samples.

Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).

Establishment of an HPLC identification system for detection of counterfeit steroidal drugs.

A set of simple HPLC methods employing UV detection were developed for detection of counterfeit drugs by the qualitative and quantitative analysis of nine steroidal drugs, ethinylestradiol, diethylstilbestrol, norethisterone, norgestrel, methyltestosterone, medroxyprogesterone acetate, progesterone, testosterone propionate and nilestriol. The methods were based on studies of the relationships between the retention factors (k) of the nine compounds and the percentages of water to methanol in the mobile phases on a reverse phase Alltima C(18) column giving reliable separation of the compounds under three sets of chromatographic conditions. The methods were validated using statistical tests and were used on nine commercial samples for detection of possible counterfeit drugs.

Detection of testosterone propionate administration in horse hair samples.

A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incubating for 15 min at 60 degrees C. After evaporation, 30 microL of hexane was added and 2.5 microL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m x 0.25 mm i.d. x 0.25 microm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS(2) full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg(-1). The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg(-1)) showed the presence of testosterone in the range of 1-6 pg mg(-1) in hair collected during 5 months after administration.

HPLC method development for testosterone propionate and cipionate in oil-based injectables.

Two isocratic liquid chromatographic methods for the determination of testosterone propionate (TP) and cipionate (TC) in oil-based injectables using methyltestosterone and bolasterone as internal standards, respectively, have been developed and validated. Mobile phases 57% water:acetonitrile 43% (v:v) and 54% water:acetonitrile 46% (v:v) were used for TP and TC, respectively. For both methods, a bonded-silica Luna CN (250 mm x 4.6 mm i.d., 5 microm) (25 degrees C) column, a flow-rate 1 ml min(-1) and UV absorbance detection at 245 nm were used and two separations up to base line were achieved. Prior to HPLC analysis, sample preparation was required, including extraction of TP and TC from oil-based injectables using the surfactant sodium dodecyl sulphate.