Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.     

Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds.     

An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert-hydroxyl groups.

An improved method for a suitable derivatization of polyhydroxylated steroids having one or two tert-hydroxyl groups at the 5beta-, 14alpha-, 17alpha-, 24-, and/or 25-positions by capillary gas chromatography (CGC) is described. By using trimethylsilyl triflate as a silylating reagent and 2,6-lutidine as a catalyst, each of 5beta-cholane and 5alpha-cholestane series of steroids was successfully transformed into trimethylsilyl (TMS) ether derivatives to give a single CGC peak under mild conditions. More bulky triethylsilyl (TES) etherification of 14alpha- and 17alpha-hydroxy compounds provided multiple CGC peaks arising from completely- and/or incompletely-derivatized TES ethers accompanied by their thermal elimination products.     

Thermal analysis of polyesters containing oxirane groups

The preliminary studies of the thermal behaviour of polyester obtained in polycondensation process of cyclohex-4-ene-1,2-dicarboxylic anhydride and ethylene glycol and its new epoxidized form have been performed. The thermal characterization of initial polyester and its completely oxidized form was done by using differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). The non-isothermal DSC was applied to determine the influence of time and the temperature on the chemical modification of initial polyester using 38-40% solution of peracetic acid. On the basis of DSC profiles it has been found that the endothermic transition, due to the degradation process of initial polyester was characteristic feature under controlled heating program. The two characteristic transitions for the new epoxidized polyester, the exothermic peak corresponded to the thermal crosslinking of epoxidized polyester (322.8–336.4°C) and the endothermic decomposition peak of the cured material (363.8–388.9°C) were observed. The peak maximum temperatures (Tmax) and the heat of cross-linking reaction (ΔH_c) for epoxypolyester prepared at 20–60°C under 1–4 h were evaluated. The Tmax1 were almost independent from epoxidation conditions, while, the values of ΔH_c were dependent from conditions of synthesis. The ΔH_c values of this process decreased when time of oxidation increased. The highest values of ΔH_c at 40°C were obtained. Additionally, TG experiments confirmed two separated degradation steps of the new epoxidized polyester indicating the ester (370–380°C) and ether (450–460°C) bond breakdown.     

Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.     

Multivariate optimization of a derivatisation procedure for the simultaneous determination of nine anabolic steroids by gas chromatography coupled with mass spectrometry

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and alpha-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction "temperature-reaction time" is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30 microg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.     

Evaluation of equine urine reactivity towards phase II metabolites of 17‐hydroxy steroids by liquid chromatography/tandem mass spectrometry

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (?18°C, 4°C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17β‐ and 17α‐glucuronide steroids were exposed to hydrolysis leading to non‐conjugated steroids. Only 17β‐hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17α‐hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4°C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at ?18°C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination. Copyright © 2008 John Wiley & Sons, Ltd.     

Case Study: Doping Substances in Equestrian Food Supplements

In the course of investigations on equestrian supplemental products for the presence of doping substances, two products were found to contain forbidden substances. As reported earlier a plant extract (Mexican cactus extract) named “Energy 5” contained the anabolic androgenic steroids (AAS) stanozolol, 17β-hydroxy-17α-methyl-5α-androstane-3β-ol (3β,5α-THMT) as well as mestanolone not declared on the label. In the present study, a product called “Super Kalm Paste” was tested. Analysis by gas chromatography - mass spectrometry (GC-MS) revealed that the preparation contained the class I anti-arrhythmics quinine (trade names Kinidin^TM, Durules) and cinchonine. The samples were prepared according to a sample preparation procedure established for anabolic steroids in nutritional supplements for humans. The sample treatment comprised the extraction and purification of the analytes as well as the chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the administration of such products could lead to positive doping tests, a pilot excretion study on “Energy 5” was conducted with two geldings, and urine samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS) after solid phase extraction and mixed derivatisation has demonstrated the presence of the stanozolol metabolite 16β-hydroxy-stanozolol in urine samples after “Energy 5” application.     

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3)

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.     

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.     

The role of self-protonation under direct GC-MS determination of amphetamine hydrochloride

Summary We studied the possibility of direct GC-MS determination of amphetamine hydrochloride. In order to avoid the double peak we determined the optimal injector temperature (260°C) and heating rate (40°C min⁻¹). In the direct analysis of amphetamine hydrochloride containing samples, calibration for fragmentm/z=59 (originating from amphetaminium ion fragmentation; the calibrating agent is amphetamine hydrochloride) gave better results than calibration form/z=44 (derived from both free amphetamine and amphetaminium ion; calibration agents were amphetamine or amphetamine hydrochloride). We also compared the mass spectra of amphetamine and amphetamine hydrochloride taken by ion-trap GC-MS. The fragmentation path was cleavage of the C-C bond at benzyl position for the free base or between the phenyl group and the aliphatic moiety, in the case of free base and amphetaminium ion, respectively.     

Improved ultrasonic‐based sample treatment for the screening of anabolic steroids by gas chromatography/mass spectrometry

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with β‐glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t‐test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA)/NH₄I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t‐test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1‐testosterone, danazol and etiocholanolone‐D5, the same results were obtained using a sonication time of 5 min. The overall applicability of the ultrasonic‐based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput. Copyright © 2010 John Wiley & Sons, Ltd.     

Some Physico-chemical Properties of Doxazosin Mesylate Polymorphic Forms and its Amorphous State

Seven polymorphic modifications of doxazosin mesylate, designed as forms A, D, E, F, G, H, I, and the amorphous state were studied by thermal methods (TG and DSC), temperature resolved X-ray powder diffractometry, hot stage and scanning electron microscopy and by FT-IR spectroscopy. Amorphous form was obtained either by fast evaporation of the solvent or by fast cooling of the melt in the DSC. Polymorphs A and F were found to be stable in the temperature range from room temperature to their melting points at 277.9 and 276.5°C, respectively. Form G, which melts at 270.8°C, was found to be hygroscopic. Polymorph D undergoes irreversible solid–liquid–solid phase transition at 235.5°C to polymorph I which melts at 274.9°C. Form H, which melts at 258.0°C, was found to be unstable at high temperatures. DSC examinations revealed that form H is irreversibly transformed to polymorph F during heating above the temperature of about 240°C. The amorphous state was found to be stable at room temperature but when heating above the glass transition (T _g=144.1°C) it crystallizes at 221.6°C, what leads into a mixture of polymorphic forms. The new polymorphic form designed as E was identified in the mixture. The polymorph E is converted by heating to the more stable form F. The solubilities at 25°C for forms A, and F in methanol are 3.5 and 7.7 mg mL⁻¹and in water they are 3.8 and 6.2 mg mL⁻¹, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Determination of residual anabolic steroid in meat by gas chromatography-ion trap-mass spectrometer

The use of natural and synthetic anabolic steroids in animal fattening has been prohibited in Taiwan and many countries because of their potential toxic effect on public health. This paper describes a newly developed gas chromatography-ion trap-mass spectrometry (GC-IT-MS) method for the quantitative determination of various residual anabolic steroids in meat. Anabolic steroid was derivatized with N-methyl-N-trimethylsilytrifluoroacetamide prior to GC-IT-MS analysis. MS² was employed for quantitative measurement. In addition, 2d-estradiol was used as an internal standard. Quantitative determination was based on the ratio of peak area of steroid derivative to peak area of internal standard derivative. Good linearity of each compound, 0.03-1.0 μg/ml, was determined. Solvent extraction was used to extract residual anabolic compounds in meat samples and a solid phase extraction (SPE) procedure was utilized for sample cleanup and pre-concentration. The limits of detection of anabolic compounds approximately ranged from 0.1 to 0.4 μg/kg. The detection limit was comparable with or better than reported methods and was below the minimum required performance limits (MRPLs) established by the European Community (EC). The application of this newly developed method was demonstrated by analyzing various beef, pork, chicken and several animal internal organ samples from local markets.     

Changes of mechanical properties in cold-crystallized syndiotactic polypropylene during aging

Many semicrystalline polymers undergo a process of aging when they are stored at temperatures higher than their glass-transition temperature (T _g). Syndiotactic polypropylene was quenched from the melt to −40 °C, crystallized from the glassy state at 20 or 40 °C and stored at the respective temperature for different aging times up to 7200 h. A significant increase in the tensile modulus and stress at yield and a decrease in strain at yield were observed for both aging temperatures. Differential scanning calorimetry (DSC) scans of aged material showed an endothermic annealing peak 15–30 °C above the previous aging temperature, the maximum temperature and enthalpic content of which increased with aging time. The position and the shape of the melting peak were not affected by aging. Scans of the storage modulus obtained from dynamic mechanical analyser measurements indicated a softening process starting at about 20 °C above the aging temperature and correlating with the annealing peak detected by DSC. Density measurements and wide-angle X-ray scattering investigations revealed that neither the crystallinity increased significantly nor did the crystal structure change. So the observed property changes induced by aging are attributed to microstructural changes within the amorphous phase. Furthermore, it could be shown by annealing experiments carried out at 60 °C, that aging above T _g is, analogous to aging below T _g (physical aging), a thermoreversible process. Received: 18 September 2000 Accepted: 2 January 2001     

Characterization of thermally-damaged LX-17

Thermal damage was applied to LX-17 at 190°C for several hours. The damaged LX-17 samples, after cooled down to room temperature, were characterized for their material properties, safety and performance. Mass losses upon thermal exposure were insignificant (<0.1 mass%). The damaged LX-17 samples expanded, resulting in a bulk density reduction of 4.3%. Subsequent detonation measurements (cylinder tests) were conducted on the thermally-damaged LX-17 samples. The results showed that the fractions of damaged LX-17 reacted were slightly lower than those of pristine LX-17. The thermally damaged LX-17 samples had average detonation velocity of 7.341 mm μs⁻¹, lower than that (7.638 mm μs⁻¹) of pristine LX-17. The average detonation energy density for the damaged LX-17 was 5.18 kJ cm⁻³, about 6.0% lower than the detonation energy density of 5.51 kJ cm⁻³ for the pristine LX-17. The break-out curves showed reaction zone lengths for pristine LX-17 and damaged LX-17 were similar but the damaged samples had ragged detonation fronts. DSC curves showed no significant difference between pristine LX-17 and damaged LX-17 with a peak temperature of 381°C observed.     

Thermal Measurements on Poly[2,2'-(m-phenylene)-5,5'-bibenzimidazole] Fibers

Fibers drawn form poly[2,2'-(m-phenylene)-5,5'-bibenzimidazole] (PBI) were studied by DSC and DMA. PBI is a high temperature polymer T _g is between 387 and450°C depending on the measurement technique used. The as-spun fiber is free of orientation. The oriented fiber exhibits considerable dependence on whether the DSC measurements were carried out in free-to-shrink or fixed-length modes. The β-relaxation is at 290°C, and was associated with loss of water. The γ-transition at 20°C was not identified, while theδ-transition at –90°C seems to correspond to rotation of the m-phenylene ring. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Improvement in steroid screening for doping control with special emphasis on stanozolol

The Medical Commission of the International Olympic Committee forbids the use of anabolic androgenic steroids and beta2-agonists to improve athletic performance. In this work we have selected examples of anabolic androgenic compounds and their metabolites to evaluate the GC-MS analysis of some trimethylsilyl derivatives. The aim is to set the best GC conditions to improve the detection within the whole range of analyte elution temperatures. The initial column temperature was changed to 105 or 140 degrees C followed by 40 degrees C min(-1) to 200 degrees C and then 15 degrees C min(-1) to 300 degrees C. Using 140 degrees C as the initial oven temperature it was possible to obtain narrower initial analyte distributions for the compounds that elutes at the beginning of the chromatogram as clenbuterol, mabuterol, epimethylenediol and norandrosterone, without loss of derivatized metabolites signal. Later. eluting analytes, such as the stanozolol metabolites, furazabol and oxandrolone were not affected. Temperatures below 140 degrees C. resulted in partial derivatization for some analytes mainly stanozolol related structures. Therefore evaluation of derivatization conditions as occurring in three steps, the vial, vaporization chamber and capillary column, was thoroughly assessed. The new program temperature improves the signal-to-noise ratio for some compounds and shows adequate resolution for endogenous compounds. Some of the difficult key separations necessary for doping control enforcement were also obtained with the proposed method.