Hair Steroid Research Articles

Simultaneous determination of hair cortisol, cortisone and DHEAS with liquid chromatography–electrospray ionization-tandem mass spectrometry in negative mode

The present study aimed to develop a novel method for simultaneous assay of cortisol, cortisone and dehydroepiandrosterone sulfate (DHEAS) in human hair. The method was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) in negative mode. Analytes were extracted from 20-mg hair incubated in 1ml of methanol for 5 days. 100μl non-SPE supernatant of the incubation solution was utilized in LC-MS/MS analysis. The liquid chromatography separation was performed on a reversed-phase C18 column with a mobile phase of 80:20 (v:v) methanol and deionized water containing 0.1% formic acid. The use of ESI in negative mode and the use of a small volume of the incubation solution significantly improved assay sensitivity. The linear range was 5-250pg/mg for cortisol and cortisone, and 5-500pg/mg for DHEAS. The limit of detection was 2pg/mg for the three analytes in hair. The coefficients of variation for intra-day and inter-day assay were less than 10%. The method was applied to determine the three analytes mentioned above of hair samples from 103 participants. The results indicated that there was no significant effect of age and education level on the hair cortisol, cortisone and DHEAS levels. The simple treatment procedure developed in the present study may facilitate simultaneous measurement of more steroids in hair.

Quantitative analysis of steroid hormones in human hair using a column-switching LC–APCI–MS/MS assay

The analysis of steroid hormones in hair is increasingly used in the field of stress-related research to obtain a retrospective index of integrated long-term hormone secretion. Here, most laboratories have so far relied on immunochemical assays originally developed for salivary analyses. Although these assays are fast and easy to perform, they have a reduced reliability and specificity due to cross-reactivity with other substances and are limited to the detection of one hormone at a time. Here, we report the development of a LC-MS/MS-based method for simultaneous identification of endogenous concentrations of seven steroid hormones (cortisol, cortisone, testosterone, progesterone, corticosterone, dehydroepiandrosterone (DHEA) and androstenedione) in human hair. Hair samples were washed with isopropanol and steroid hormones were extracted from 10mg whole, nonpulverized hair by methanol incubation. A column switching strategy for on-line solid phase extraction (SPE) was applied, followed by analyte detection on an AB Sciex API 5000 QTrap mass spectrometer. Results indicated linearity of the method for all steroids over ranges of 0.09-90pg/mg (0.9-900pg/mg for DHEA) with correlation coefficients ranging between 0.9995 and 0.9999. Intra- and inter-assay coefficients of variation were between 3.7 and 9.1%. The limits of quantification (LOQ) were below (or equal to) 0.1pg/mg for all steroids, except of DHEA for which the LOQ was 0.9pg/mg. An analysis of 30 natural hair samples (15 men/15 women) using this method confirmed that all steroid hormones could be quantified at endogenous levels in each individual. In addition, the use of whole hair samples and on-line SPE resulted in a significant reduction in sample throughput times, increasing the applicability of this method for research questions where a larger number of samples needs to be processed.

Gas chromatography/mass spectrometry based hair steroid profiling may reveal pathogenesis in hair follicles of the scalp

A method of steroid profiling, including androgens, progestins, corticoids and sterols, was developed to evaluate the concentrations of steroids as well as the activities of the enzymes responsible for steroidogenesis in hair by gas chromatography/mass spectrometry. The extraction efficiencies of steroids from the hair matrix were improved by ultrasonication for 1 h at 50 °C. The overall recoveries ranged from 71 to 132%, with a limit of quantification for all analytes ranging from 1 to 50 ng/g. The devised method was used to identify the metabolic changes for both male-pattern baldness (MPB) and the drug efficiency of dutasteride, which inhibits 5α-reductase. Increased dihydrotestosterone levels and the dihydrotestosterone/testosterone (DHT/T) ratio, which is responsible for the 5α-reductase activity, were observed in the MPB patients. A dutasteride treatment resulted in decreases in the DHT and 5α-androstanedione concentrations and DHT/T ratio in the hair samples. Hair steroid profiling reflects the sebaceous status in the scalp and may be useful for monitoring the metabolic responses to both the disease and drug actions.

Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS n spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2–4 h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS 3 data from hair samples incurred with high levels (300–800 μg kg −1) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.

Analysis of anabolic steroids in human hair using LC–MS/MS

New highly sensitive, specific, reliable, reproducible and robust LC–MS/MS methods were developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for nandrolone. Positive samples were confirmed on LC–MS/MS in selective reaction monitoring (SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range 1–400 pg/mg and 5–400 pg/mg, respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5 pg stanozolol and 3.0 pg nandrolone per mg of hair, when approximately 20 mg of hair were processed. Analysis using LC–MS/MS confirmed 11 athletes’ positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and 1 for nandrolone (14.0 pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non-invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing.

Analysis of anabolic steroids in hair: Time courses in guinea pigs

Sensitive, specific, and reproducible methods for the quantitative determination of eight anabolic steroids in guinea pig hair have been developed using LC/MS/MS and GC/MS/MS. Methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone, methenolone and DHEA were administered intraperitoneally in guinea pigs. After the first injection, black hair segments were collected on shaved areas of skin. The analysis of these segments revealed the distribution of anabolic steroids in the guinea pig hair. The major components in hair are the parent anabolic steroids. The time courses of the concentrations of the steroids in hair (except methenolone, which does not deposit in hair) demonstrated that the peak concentrations were reached on days 2–4, except stanozolol, which peaked on day 10 after administration. The concentrations in hair appeared to be related to the physicochemical properties of the drug compound and to the dosage. These studies on the distribution of drugs in the hair shaft and on the time course of their concentration changes provide information relevant to the optimal time and method of collecting hair samples. Such studies also provide basic data that will be useful in the application of hair analysis in the control of doping and in the interpretation of results.

Physiological concentrations of anabolic steroids in human hair

Doping with endogenous anabolic steroids is one of the most serious issues in sports today. The measurement of anabolic steroid levels in human hair is necessary in order to distinguish between pharmaceutical steroids and natural steroids. This is the first investigation into the physiological concentrations of anabolic steroids in human hair in Chinese subjects. A gas chromatography–tandem mass spectrometry (GC/MS/MS) method was developed for the simultaneous identification and quantitation of five endogenous anabolic steroids (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) in hair. After basic hydrolysis, hair samples were extracted with diethyl ether, derivatized and then detected using GC/MS/MS in the multiple-reaction monitoring mode (MRM). The one precursor/two product ion transitions for each anabolic steroid were monitored. The limits of detection for the five endogenous anabolic steroids were in the 0.1–0.2 pg/mg range. All analytes showed good linearity and the extraction recoveries were 74.6–104.5%. Within-day and between-day precisions were less than 20%. This method was applied to the analysis of testosterone, epitestosterone, androsterone, etiocholanolone, and dehydroepiandrosterone in human hair. Full-length hair samples were taken at the skin surface from the vertex of 39 males, 30 females and 11 children from China. None of the subjects were professional athletes. Testosterone and dehydroepiandrosterone were detected in all the hair segments. The physiological concentrations of testosterone were in the range 0.8–24.2 pg/mg, 0.1–16.8 pg/mg and 0.2–11.5 pg/mg in males, females and children, respectively, however, the mean values of dehydroepiandrosterone were much higher than the concentrations of testosterone. These data are suitable reference values and are the basis for the interpretation of results from investigations into the abuse of endogenous anabolic steroids.

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.

Monitoring Anabolic Steroids in Meat-Producing Animals. Review of Current Hyphenated Mass Spectrometric Techniques

Monitoring anabolic steroids in meat-producing animals is a challenging task. It implies very specific and sensitive analytical methods able to detect and identify sub-μg kg−1 residue levels in complex biological matrices such as meat, urine, or hair. Gas and liquid chromatography coupled to mass spectrometry are the most efficient means of achieving these objectives. In this paper we review how developments in mass spectrometry have been rapidly applied to this problem, how efficient analysis of anabolic steroids in urine, edible tissue, and hair has been achieved, and, later, how measurement of conjugate steroids and determination of the origin of natural steroid hormones has been achieved. The performance characteristics of different mass spectrometers (quadrupole, ion-trap, electromagnetic, isotope-ratio, tandem, and hybrid instruments), the efficiency of different acquisition techniques (LR-SIM, HR-SIM, MRM), and, finally, sample introduction (gas chromatography and liquid chromatography, with discussion of alternative interfaces) are discussed, with numerous applications.

Multiresidue analysis of anabolic compounds in bovine hair by gas chromatography–tandem mass spectrometry

A rapid and specific multiresidue method was developed to perform screening and confirmation of 15 anabolics steroids in bovine hair, namely: dienestrol, DES, hexestrol, 17α- and 17β-estradiol, 17α- and 17β-19-nortestosterone, 17α- and 17β-boldenone, testosterone (β) and epitestosterone (α), ethynylestradiol, α-methyltestosterone, and α- and β-Zearalanol. After methanolic and solid-phase extraction, trimethylsilyl derivatization (TMS) was performed and the derivatized extracts submitted to GC–MS–MS analysis. TMS derivatives of the studied hormones yielded specific MS–MS fragmentation with a high abundance for ions selected in the screening procedure. Validation was performed regarding qualitative parameters: decision limit (CCα), detection capability (CCβ) and specificity. For confirmation, European union criteria 2002/657/EC were considered. Detection capability ranged between 1.0 and 10.0 ng g −1 depending on the compound.

Identification of anabolic steroids in serum, urine, sweat and hair: Comparison of metabolic patterns

Anabolic steroids usually undergo a complex metabolism which is mainly dependent on structural variation and application pathway. In contrast to doping analysis, where the presence of a prohibited substance constitutes a doping offence without regarding the origin or application, in forensic cases any information related to anabolic steroids may be required to evaluate the cause of death, liability of defendants, or self-consumption. 19-Norsteroids proved to be appropriate model compounds for the investigation, because of their prevalence in recent doping cases, analytical and biochemical similarity to endogenous steroids, well explored biochemistry, and the availability of numerous reference standards. Typical urinary metabolites are conjugates of terminal biotransformation products like norandrosterone–glucuronide, which are relatively unspecific and do not reveal details about administration time, pathway, dosages or structural details. Therefore, metabolic patterns in blood, hair and sweat were examined to study metabolism, distribution and incorporation behaviour of the steroids and hence the traceability in the respective matrices. Concentration profiles of the steroids in blood and the resulting bioavailability show distinct particularities after oral or transdermal application of steroids. Oral application of steroid precursors is followed by extensive first pass biotransformation of the steroid to inactive metabolites, while transdermal administration appears to be an efficient pathway, leading to effective and durable concentrations of active steroids in blood. Due to the mutual similarity of metabolic patterns in hair and sweat corresponding to the regional presence of sweat and sebum glands, steroid incorporation appears to proceed mainly via sweat absorption.

Analytical strategy for detecting doping agents in hair

Lists of banned classes of doping agents are released by the International Olympic Committee, adopted by other sports authorities and updated regularly, including the substance classes stimulants, narcotics, diuretics, anabolic agents, peptide hormones, beta-blockers etc. There are different classes of restriction:•anabolic and masking agents (anabolic steroids, diuretics etc.) are always banned for athletes regardless of their topical activity (training or competition)•several substances are permitted with certain restrictions (caffeine below a cut-off value, or inhalation of some β2 agonists)•β-blockers are prohibited in competitions of certain sports disciplines•the majority of the substances (stimulants, narcotics etc.) is prohibited during competitions, so that they do not have to be analysed in out-of-competition samples.A differentiation between training and competition period is impossible by means of hair analysis due to the uncertainty of (especially short-term) kinetic considerations related to hair growth. Therefore, the analytical identification of doping relevant substances in hair is not always a sufficient criterion for a doping offence and the identification of stimulants, beta-blockers etc. in hair would be entirely irrelevant. The most interesting target substances are certainly the anabolic agents, because their desired action (enhanced muscle strength) lasts longer than the excretion, leading to sophisticated procedures to circumvent positive analytical results in competition control. Besides the analysis of out-of-competition control samples, the long term detection of steroids in hair could provide complementary information. An analytical approach to the identification of exogenous steroids in hair requires consideration of the presence of many other steroids in the hair matrix interfering the analysis at trace levels, and of a limited chemical stability. The analysis of endogenous steroids in hair appears to be even more complicated, because the possibility of many biotransformation reactions from (into) other precursors (metabolites) has to be taken into account. Precursor substances of anabolic steroids (especially esters as application forms) are very promising analytical targets of hair analysis, because they can only be detected after an exogenous intake. The quantitative evaluation of active parent compounds like testosterone (which is actively involved in physiological processes of hair growth) in hair is still controversial. Clinical applications under reproducible conditions can be useful, but the biovariability of these parameters will probably prevent the definition of acceptable cut-off levels as a criterion of abuse.

Gas chromatographic–tandem mass spectrometric determination of anabolic steroids and their esters in hair: Application in doping control and meat quality control

We have developed a powerful and simple sensitive method for testing hair for anabolic steroids and their esters. A 100-mg amount of powdered hair was treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid-phase extraction on amino and silica cartridges. The residue was derivatized with N-methyl- N(trimethylsilyl)-trifluoracetamide (MSTFA) prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. The generally chosen parent ion was the molecular ion while two daughter ions were selected for each compound with collision energies ranging from −16 to −21 eV. Internal standards were nandrolone d 3 for non-esterified drugs and testosterone phenyl propionate for esters. The limits of detection calculated from an analysis of the blanks ( n=30) were 0.08 pg/mg for nandrolone, 6.20 pg/mg for boldenone, 0.07 pg/mg for methyl testosterone, 0.15 pg/mg for ethinyl estradiol, 2.10 pg/mg for metandienone, 0.86 pg/mg for testosterone propionate, 0.95 pg/mg for testosterone cypionate, 1.90 pg/mg for nandrolone decanoate, 3.10 pg/mg for testosterone decanoate and 4.80 pg/mg for testosterone undecanoate. Application to doping control has been demonstrated. In a series of 18 sportsmen, two tested positive for anabolic steroids in hair whereas urinalysis was negative for both of them. The first positive case was nandrolone and the second case concerned the identification of testosterone undecanoate. Measured in 10 white males aged between 22 and 31 years, the testosterone concentration was in the range 1.7–9.2 pg/mg (mean=5.0 pg/mg). The method was also applied in meat quality control. Of the 187 analyses realized based upon hair and urine sampling in slaughter houses, 23 were positive for anabolic steroids in hair: one case for boldenone, one case for metandienone, two cases for testosterone propionate, three cases for nandrolone, five cases for testosterone decanoate and 11 cases for methyl testosterone. In the meantime, urinalysis was always negative for these drugs or their metabolites.

Testing for anabolic steroids in hair from two bodybuilders

Two male bodybuilders were recently arrested by the French customs in Strasbourg (France) in possession of 2050 tablets and 251 ampoules of various anabolic steroids. It was claimed that the steroids were for personal use and not for trafficing as suggested by the police. Urine and hair specimens were collected from both suspects to clarify the claims. Nandrolone, stanozolol, testosterone and their corresponding metabolites were identified in the urine of both subjects. After decontamination, the hair was hydrolyzed by sodium hydroxide in presence of deuterated internal standards. After extraction with ethyl acetate and silylation, the drugs were identified by GC–MS in the electron impact mode. Hair from both males were positive for nandrolone (196 and 260 pg/mg), testosterone (46 and 71 pg/mg) and stanozolol (135 and 156 pg/mg), clearly indicating steroids abuse. Although not yet recognized by the International Olympic Committee, hair analysis may be a useful adjunct to conventional drug testing in urine from athletes.

Detection of Anabolic Steroids in Head Hair

We developed a gas chromatography/mass spectrometry method for detection and quantitation of anabolic steroids in head hair. Following alkaline digestion and solid-phase extraction, the MO-TMS derivatives gave a specific fragmentation pattern with EI ionization. For stanozolol, the TMS-HFBA derivative showed several diagnostic ions. For androstanolone, mestanolone (methylandrostanolone), and oxymetholone two chromatographic peaks for cis and trans isomers of derivatives were seen. Recoveries were 35 to 45% for androstanolone, oxymetholone, chlorotestosterone-acetate, dehydromethyltestosterone, dehydrotestosterone, fluoxymesterone, mestanolone, methyltestosterone, and nandrolone; 52% for mesterolone, trenbolone; 65% for bolasterone; 24% for methenolone and 17% for stanozolol. Limits of detection were 0.002 to 0.05 ng/mg and of quantitation were 0.02 to 0.1 ng/mg. Seven white male steroid abusers provided head hair samples (10 to 63 mg) and urine. In the hair samples, methyltestosterone was detected in two (confirmed in urine); nandrolone in two (also confirmed in urine); dehydromethyltestosterone in four (but not found in urine); and clenbuterol in one (but not in urine). Oxymethalone was found in urine in one, but not in the hair. One abuser had high levels of testosterone: 0.15 ng/mg hair, and 1190 ng/mL urine. We conclude that head hair analysis has considerable potential for the detection and monitoring of steroid abuse.

A preliminary study of steroid reproductive hormones in human hair

Nowadays research and clinical studies of human reproductive endocrinology are generally carried out using human blood reproductive hormone assays. However the acquisition of human blood samples has some shortcomings. In search of new approaches, we paid attention to the fact that progesterone can be detected in cow's hair. Consequently we investigated whether or not steroid hormones are measurable in human hair. The results showed that the levels of steroid hormones in hair are not affected by shampoo and do not significantly vary between different segments of hair (i.e. top, middle and basal segments). The menstrual estradiol and progesterone rhythm of female hair is similar to that of female serum. The ratio of hair estradiol to serum estradiol in the female is 41.2% and that of hair progesterone to serum progesterone is 59.0%; the ratio of hair testosterone to serum testosterone in male is 116%. There are significant correlations between hair and serum steroid hormones of healthy human adult: γ (estradiol)=0.395 ( n=20), p<0.05; γ (progesterone)=0.440 ( n=22), p<0.025 and γ (testosterone)=0.395 ( n=25), p<0.05.

Detection of estradiol and testosterone in hair of cattle by HPL/EIA

This study concerns the detection of natural steroid hormones in hair of cattle. Estradiol (E2) and testosterone (T) were chosen as representatives of estrogens and androgens. In particular, the influence of age, sex and hair pigmentation on the steroid concentrations was investigated. Samples were obtained from numerous steers, cows, bulls, and female and male calves. The extraction procedure for E2 and T from hair comprised liquid-liquid and solid-phase extraction and was followed by an essential high-performance liquid chromatography (HPLC) step for further purification of the extracts. Final quantification was performed with specific enzyme immunoassays (EIA). Lower E2-concentrations were detected in the hair of some steers, cows, and bulls (approximately 1 ng/g), in several of these hair samples the concentrations of E2 were below the limit of detection. Testosterone was measured in the hair of steers (approximately 3 ng/g), cows (approximately 6 ng/g), and bulls (in average 15 ng/g). There was a significant difference in the testosterone concentrations of white (approximately 8 ng/g) and of black hair (approximately 33 ng/g) of bulls. In hair from all male and female calves, E2 and T were measured. The concentrations amounted approximately to 9 ng E2/g and 3 ng T/g for female calves and to 5 ng E2/g and 7 ng T/g for male calves. There was no significant influence of sex or hair colour on the steroid concentrations in hair of calves. The results suggest that the method is a powerful means to detect natural steroid hormones in hair of animal origin.

Simultaneous quantification of five odorous steroids (16-androstenes) in the axillary hair of men

Five 16-androstenes have been simultaneously quantified in extracts of the axillary hair of men (age range 18–40 years) using combined capillary gas chromatography-mass spectrometry, with specific ion monitoring. Quantities found (pmol/mg.hair, with approximate 24-h totals in parentheses) were: 5α-androst-16-en-3-one, 0–15 (0–433); 4, 16-androstadien-3-one, 0–143 (0–4103); 5,16-androstadien-3β-ol, 0–3.5 (0–728); 5α-androst-16-en-3α-ol, 0–17 (0–1752) and 5α-androst-16-en-3β-ol, 0–4 (0–416). There were no significant relationships with age of the subjects for any of the steroids measured but significant relationships were found between the amounts of the two ketones and between 5α-androst-16-en-3α- and 3β-ols. These findings may indicate the existence of a pathway of metabolism in axillary bacteria in which 4,16-androstadien-3-one is reduced to 5α-androst-16-en-3-one and thence to the 3α- and 3β -alcohols. The data are discussed in the context of axillary odour because of the low olfactory thresholds of several of the 16-androstenes measured and because of the relatively large quantities found in some subjects.