Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3α-hydroxy-5α-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 °C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N = 3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.     

Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid–liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at ?20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r?≥?0.990) over a range of 50–1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n?=?3) and inter-day (n?=?5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N?=?5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.     

Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.     

Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle.

The use of anabolic steroids is prohibited in the European Community by Directive 96/22/EC and the control of compliance is regulated by Directive 96/23/EC. Multi-residue methods are necessary for screening for the use of forbidden substances. Because accreditation is gaining more and more importance, validation of the methods used and of the results obtained has become indispensable. The developed GC-MS methods for the detection of anabolic steroids in urine and muscle tissue were validated with regard to the following parameters: specificity, recovery at the 2 micrograms kg-1 level and limit of detection. For urine the recoveries ranged from 17 to 81% and for muscle tissue from 26 to 65%. The limit of detection ranged from 0.1 to 2.6 micrograms kg-1 for urine and from 0.3 to 4.6 micrograms kg-1 for muscle tissue. Specificity was guaranteed in both matrices by the selection of four specific ions. Blank samples were evaluated for interferences and it could be concluded that in no case did the four selected ions appear simultaneously at the correct retention time. The practicability of the criteria for low resolution mass spectrometry set in Decision 93/256 in the low micrograms kg-1 range is also briefly discussed.     

Effect of extended use of single anabolic steroids on urinary steroid excretion and metabolism

Long-term use of single anabolic steroids by weightlifters and body builders at dosages greater than or equal to 25 mg per 24 h resulted in reduced excretion of urinary androgen metabolites, androsterone and etiocholanolone, compared to values prior to anabolic use. The excretion of major urinary metabolites of glucocorticoids was not affected by anabolic use. Urinary excretion of anabolic steroids or anabolic metabolites averaged 20-25% of total anabolic steroid administered. The major excreted metabolites of methandrostenolone, nandrolone, oxandrolone and oxymetholone were identified by gas chromatography-mass spectrometry based on the major mass spectral ion peaks.     

Metabolites in feces can be important markers for the abuse of anabolic steroids in cattle.

In Belgium, to control the abuse of anabolic steroids in cattle, urine samples have been gradually replaced by feces samples, because the latter can be obtained more easily from living animals. Urine and feces samples were collected from heifers after administration of boldenone, norethandrolone or ethylestrenol. Metabolites present in feces or urine were determined by GC-MS. Large qualitative and quantitative differences in the metabolic profiles were observed. In feces, in contrast to urine, the parent compounds or their major metabolites were detectable only shortly after administration. On the other hand, metabolites resulting from the reduction of the 3-oxo group and the unsaturated carbon-carbon bonds, present on the A-ring, allow for long-term detection in feces. A-ring reduced metabolites have been identified in samples found positive for norgestrel, boldenone, methylboldenone and methyltestosterone, respectively. These results are in agreement with concomitant in vivo experiments.     

Direct analysis of anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption-dielectric barrier discharge ionization mass spectrometry

Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the consumption of performance enhancing anabolic steroids by athletes. The present article describes a novel strategy for identifying the trace anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are enriched within a liquid droplet during the thermal desorption process and desorbed all-together at the last moment of droplet evaporation in a short time domain. The desorbed molecules were ionized using a dielectric barrier discharge ion-source in front of the mass spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement with several orders of magnitude compared to the thermal desorption at a lower temperature. The limits of detection (LODs) of various steroid molecules were found to be in the range of 0.05–0.1 ng mL⁻¹ for standard solutions and around two orders of magnitude higher for synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by using a simple and rapid dichloromethane extraction technique. The analytical figures of merit of this technique were evaluated at open atmosphere using suitable internal standards. The technique is simple and rapid for high sensitivity and high throughput screening of anabolic steroids in urine.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

The external review committee on pure reference materials at the National Analytical Reference Laboratory

The preceding paper described the implementation of a quality assurance system suitable for accreditation to ISO Guide 34 by the Pure Substance Reference Material (PSRM) team of the National Analytical Reference Laboratory (NARL). One of the key components of this system has been the establishment of an external advisory committee that scrutinises each candidate material that is to be offered as a reference material. At the time of writing 190 reference materials were available from NARL, including a range of illicit drugs and agricultural chemicals in addition to anabolic steroids and their metabolites, and all have been reviewed by the external committee prior to final approval by the accredited production signatory.     

Determination of anabolic steroids in dietary supplements by liquid chromatography-tandem mass spectrometry

Nineteen different dietary supplements, ordered through the internet and intercepted by the Belgian pharmaceutical inspection at the post office, were analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the presence of anabolic steroids. After a methanolic extraction the samples were screened for the presence of 49 compounds. This resulted in almost 60% of the samples being suspected of containing one of these 49 anabolic compounds and being subjected to a confirmatory product ion scan. In all of these suspected samples we were able to confirm at least one anabolic steroid with concentrations between 0.01 and 2.5 mg unit(-1) (unit: one capsule or tablet or for liquids: the prescribed dose). The anabolic steroids that was mostly encountered was testosterone (50%) followed by beta-boldenone (25%). These results once more confirm the dubious reputation of over-the-counter dietary supplements.     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

Nutritional supplements cross-contaminated and faked with doping substances

Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15% of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of 'classic' anabolic steroids such as metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new 'designer' steroids such as prostanozol, methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the near future also cross-contaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the beta2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk. For the detection of new 'designer' steroids in nutritional supplements, mass spectrometric strategies (GC/MS, LC/MS/MS) are presented.     

Investigations into the feasibility of routine ultra high performance liquid chromatography–tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids,anabolic steroid esters and related compounds

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored.     

Detection of Anabolic Steroids By Gc/Sim/Ms With Trifluoroacetylation in Equine Plasma and Urine

ABSTRACT

A method to detect three anabolic steroids (boldenone, nandrolone and mesterolone) is presented. The anabolic steroids are isolated from equine plasma and urine by extraction with diethyl ether and C₁₈ Sep-Pak cartridge adsorption, respectively. The extracts obtained were derivatized with trifluoroacetyl anhydride and analyzed by GC/SIM/MS. The selected ion monitoring (SIM) mode was applied to increase the sensitivity and, when possible, the higher m/z ions were selected to improve identification. Stability of derivatives was good and compounds having hydroxy and conjugated ketone groups produced trifluoroacetyl ester derivatives that were also stable. Repeatability of the chromatographic analysis was evaluated on the basis of area repeatability, and the coefficient of variation obtained was lower than 4.4. The detection limit was 1 and 5 ng/ml for all the anabolic steroids studied in equine plasma and urine, respectively.     

Selecting effective siRNA target sequences by using Bayes’ theorem

Short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells but varies markedly in its gene silencing efficacy. Although many design rules/guidelines for effective siRNAs based on various criteria have been reported recently, there are few consistencies among them. This makes it difficult to select effective siRNA sequences in mammalian genes. Another shortcoming of most previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. The analytical prediction method proposed in the present study uses Bayes’ theorem to select effective siRNA target sequences from many possible candidate sequences. It is quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The results of evaluating it by applying it to recently reported effective and ineffective siRNA sequences for various genes indicate that it would be useful for many other genes. It should therefore be useful for selecting siRNA sequences effective for mammalian genes.     

一种预测药物活性的神经元计算新方法

提出一种基于全变异算子遗传算法(MGA)的神经元计算新方法,用于辨识复杂药物构效关系。在MGA中,表达变量的各基因使用不同的变异概率,以便提高局部搜索效率。通过将随机初始化技术与局部搜索策略相结合,该算法能在有限时间内得到满意解。使用74个抑制还原酶的嘧啶类化合物所组成的数据集作为构效关系神经元计算的典型对象,用来考核MGA法在预测药效活性计算中的有效性。交叉验证及活性预测试验表明,用MGA法建立的构效关系模型的预测能力优于其他方法。     

Simultaneous Doping Analysis of Main Urinary Metabolites of Anabolic Steroids in Horse by Ion-trap Gas Chromatography-Tandem Mass Spectrometry

The use of anabolic steroids in racehorses is strictly regulated. We have developed a method for the simultaneous analysis of 11 anabolic steroids: fluoxymesterone, 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, oxymetholone, boldenone, furazabol, methenolone, nandrolone, and stanozolol, for possible application to a doping test in racehorses. We selected 15 kinds of target substances for a doping test from the main metabolites of these anabolic steroids, and established a method for simultaneous analysis. Urine was hydrolyzed and subjected to solid-phase extraction. Then, the residue from the extracts was derivatized by trimethylsilylation. The derivatized samples were subjected to ion-trap gas chromatography-tandem mass spectrometry, and their mass chromatograms and product ion spectra were obtained. The limit of detection of the target substances was 5 - 50 ng/mL, and the mean recovery and coefficient of variation were 71.3 - 104.8% and 1.1 - 9.5%, respectively.