Unique potentiometric detection systems for HPLC determination of some steroids in human urine (original) (raw)
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Journal of Chromatography A, 2008
Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C 18 column (150 mm × 4.6 mm I.D., 5 m) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.
Rapid Communications in Mass Spectrometry, 2000
A gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method is described and validated for measurement of d 13 C values of the acetate derivatives of urinary etiocholanolone and androsterone. The analysis was performed with only 2 mL of urine. The sample preparation consisted of deconjugation with b-glucuronidase, solid phase extraction, and derivatization with acetic anhydride and pyridine. The within-assay precision of two quality control (QC) urine samples ranged from 0.5 to 2.1 CV%. The between-assay precision in the same QC urines ranged from 1.7 to 3.4 CV%. Administration of testosterone enanthate to a subject resulted in a 6% decrease in d 13 C values from À25% (baseline) to À31%. Two weeks after testosterone administration was discontinued, the d 13 C values remained abnormally low while the urine testosterone/epitestosterone (T/E) ratio returned to less than 6. This relatively simple method is useful for rapidly screening a large number of urine samples, including those with T/E`6.
Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography
Journal of Microcolumn Separations, 2000
A capillary-liquid chromatography LC method was developed for the quantification of the endogenous steroids testosterone and epitestosterone in human urine. One milliliter of urine was used for the overall method. Free testosterone was first separated by liquid᎐liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically hydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary Ž . column switching system with a low back pressure precolumn PC was used for Ž . fast loading of large sample volumes 20 L . Chromatographic separation was Ž . carried out on a 15 cm = 300 m inner diameter i.d. column, packed with 3 m Hypersil BDS-C at a flow rate of 4 Lrmin with isocratic elution and UV 18 Ž . absorbance detection 240 nm . Limit of detection for free testosterone was established at 0.5 ngrmL. Limits of detection were established at 1.5 and 3.2 ngrmL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed Ž . through the entire calibration range up to 250 ngrmL . ᮊ
Analytica Chimica Acta, 2010
Voltammetric investigation of two corticoid isomers-testosterone and epitestosterone has been carried out at bare and single-wall carbon nanotubes (SWNT)-modified edge plane pyrolytic graphite electrode (EPPGE). Square wave voltammetry (OSWV) has been used for the simultaneous determination of isomeric steroids. The reduction of the two isomers occurred in a pH dependent, 2e, 2H + process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5-1000 nM for both the steroids with the limit of detection 2.8 × 10 −9 and 4.1 × 10 −9 M for testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results obtained from the proposed voltammetric method were also compared with HPLC analysis and found to be similar.
Talanta, 2011
A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination of testosterone (T) and epitestosterone (E) in human urine samples has been developed and elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis using 36% hydrochloric acid for determination of total level of T has been applied. Unconjugated (free) form of the both androgens were determined without hydrolysis steps, what makes novelty of the method, because simplifies the proposed procedure. In turn, the measurements of urinary free T and E provided the diagnostic information for excess adrenal production of steroids. The proposed LC assay was evaluated by analyzing a series of urine samples containing T, E and methyltestosterone (MT) as internal standard at the range of concentration 2-300 ng −1 mL of both analyzed hormones. The proposed method was fully validated for specificity, linearity, limits of detection and quantitation, precision and trueness according to the current requirements concerning analytical methods. Interestingly, the developed LC method allows to obtain a sensitive enhancement with respect to UV detection with the quantitation limit for T and E equaled 2 ng mL −1 . The method was selective and reliable for identity and enable to detect changes of endogenous levels of T and E in urine independently of fluctuations characteristic for both analyzed endogenous hormone level in plasma. .pl (L. Konieczna). androgen deficiency in clinical conditions . The normal amounts of total endogenous T and epitestosterone (E) practically measured in healthy male in urine are in the range 30-60 ng mL −1 [11]. T and E and their ratio T/E is stable in males, what was well established . Since 1983, T was forbidden in sports by the International Olympic Committee (IOC). The detection of illicit use of T is currently carried out measuring the ratio between the concentration of T and its isomer E. A ratio of their concentrations (T/E ratio) higher than 4 is considered as potentially indicative of T administration. On the other hand, because the T/E ratio can be artificially modified by the administration of E, a urinary concentration of epitestosterone above 200 ng mL −1 has been established as indicative of its misuse as a masking agent . The World Anti-Doping Agency (WADA) indicated that if the T/E ratio was equal or above 4, or concentration of E higher than 200 ng mL −1 , a confirmation procedure to prove doping would be necessary .
Reactive Arrays of Colorimetric Sensors for Metabolite and Steroid Identification
Journal of sensor technology, 2014
The work described herein examines a rapid mix-and-measure method called DETECHIP suitable for screening of steroids and metabolites. The addition of steroids and metabolites to reactive arrays of colorimetric sensors generated characteristic color "fingerprints" that were used to identify the analyte. A color analysis tool was used to identify the analyte pool that now includes biologically relevant analytes. The mix-and-measure arrays allowed the detection of disease metabolites, orotic acid and argininosuccinic acid; and the steroids androsterone, 1,4-androstadiene, testosterone, stanozolol, and estrone. The steroid 1,4-androstadiene was also detected by this method while dissolved in synthetic urine. Some of the steroids, such as androstadiene, stanozolol, and androsterone were co-dissolved with (2-hydroxypropyl)-β-cyclodextrin in order to increase solubility in aqueous buffered solutions. The colorimetric arrays do not intend to eliminate ELISA or mass spectroscopy ba...
Journal of Chromatography A
An isotope dilution mass spectrometry (IDMS) method for the determination of selected endogenous anabolic androgenic steroids (EAAS) in urine by UHPLC-MS/MS has been developed using the isotope pattern deconvolution (IPD) mathematical tool. The method has been successfully validated for testosterone, epitestosterone, androsterone and etiocholanolone, employing their respective deuterated analogs using two certified reference materials (CRM). Accuracy was evaluated as recovery of the certified values and ranged from 75% to 108%. Precision was assessed in intraday (n=5) and interday (n=4) experiments, with RSDs below 5% and 10% respectively. The method was also found suitable for real urine samples, with limits of detection (LOD) and quantification (LOQ) below the normal urinary levels. The developed method meets the requirements established by the World Anti-Doping Agency for the selected steroids for Athlete Biological Passport (ABP) measurements, except in the case of androsterone, which is currently under study.
Molecules, 2022
Solid-phase analytical derivatization (SPAD) is a promising hybrid sample preparation technique combining the clean-up and preconcentration of the sample in a single step. In this work, a novel SPAD method based on the preparation of trimethylsilyl (TMS) derivatives of steroid hormones (testosterone, estrone, DHT, estriol, estradiol, and progesterone) in Phenomenex Strata C18-E (100 mg, 1 mL) cartridges has been developed and applied for their GC-MS/MS determination in human urine samples. The proposed procedure allows the detection and quantification of steroids with limits of 1.0-2.5 and 2.5-5 ng/mL, respectively. These characteristics are comparable with those obtained with a conventional liquid-liquid extraction, while the recovery of analytes in the proposed SPAD procedure is higher. The major advantages of SPAD are a short derivatization time, high efficiency, and the possibility to automatize the procedure. However, its cost-effectiveness in routine practice is still questionable.
2021
Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO T...