Methadone group

Overweight group

Control group

Figure 5. GC/C/IRMS chromatograms for 5αAdiol and 5βAdiol - methadone , overweight and control groups.

Drugs of abuse and other influences on steroid profile using new validated methods Radostina Popova1, Assia Petrova1 and prof. d-r Maria Orbetzova2 1Bulgarian Doping Control Laboratory, Bulgarian Anti-doping Centre, Sofia, Bugaria 2Medical University of Plovdiv, Plovdiv, Bulgaria

Abstract We compared urinary levels of androgen steroids and their ratios such as T/E, A/Etio, A/T, DHT/E, 5⍶/5β-diols, making part of the steroid profile. The ratios are calculated according the TD and WADA’s International standard for laboratories. The comparison is between three groups - control, people using drugs and overweight people. The Testosterone/ Epitestosterone ratio was significantly decreased in the narcotics group, the urinary testosterone concentrations were decreased. The group of overweight people shows much higher levels of steroids compared to control group. The abnormal steroid metabolism has been investigated. T/E ratios is a parameter used to indicate the suppression of male gonadal function after usage of narcotics. After optimization of sample preparation with new SPE columns we have validated a confirmative mass spectrometric method with GC/C/IRMS. This technique provides the capability to measure the carbon isotopic ratio in order to determine the origin of these steroids. The 13C/12C ratio is different whether the steroid is endogenously produced or has its origin from a pharmaceutical preparation. The nature and change in urinary metabolites ratios in humans is of great interest in doping control because it is affected by various drugs, therapeutical medicines and nutritional supplements.

Introduction Determination of steroid profile parameters is a major concern of doping control specialists for detection and confirmation of endogenous anabolic steroids. The urinary steroid profile is composed of concentrations and ratios of various endogenous hormones such as Testosterone, Androsterone, Etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, 5α-androstane-3α,11β-diol-17-one, DHEA, DHT and others. Biosynthesis of endogenous steroid is shown on Figure 1. Sex hormones are often studied as biological basis for sex differences due to their role in the central nervous system regulation, implicating the endocrine system in the pathophysiology of substance use disorders and addictive behaviour. Addictive behaviour is an opioid dependence. The purpose of this study is to examine endocrinal damages at a chronic treatment with opioid agonist (methadone therapy). The increase in body weight and fat tissue is associated with several abnormalities of steroid balance. Androgens have an important impact on glucose and lipid metabolism and fat homeostasis. It is likely that an androgen imbalance in obesity may play a role in the pathophysiology of the metabolic syndrome and increase the risk for cardiovascular diseases.

Materials and Methods All reference standards were obtained from AGNMI (Australia). The GC-IRMS STANDARD C15/C20/C25 was obtained from Chiron (Norway). The C18 cartridge (200 mg/3 ml; 500mg/10ml) were from Thermo Scientific (Bremen, Germany), Silicycle (Canada). All organic solvents were of HPLC grade. All samples were measured on an Agilent 7890A gas chromatograph and G3170A mass spectrometer. The GC system was equipped with a DB-1MS UI, 20m x 0.180mm x 0.18µm film thickness column. The injection volume was 2 µl; the injections were performed split 1:5 at 280°С. A constant flow of 1,0 ml/min of helium carrier gas was used. The initial oven temperature was 190°С, followed by a ramp at 2°С/min to 218°С held for 3 min, followed by a ramp at 2°С/min to 230 °С and 20 °С/min to 300 °С and held for 6 min. After hydrolysis with β-glucuronidase (E. coli) and liquid-liquid extraction with TBME at pH 9-10 trimethylsilyl derivatives of the steroids were analysed by GC/MS. Table 1 shows the screening method validation data.

Compound Correlation coefficient,(r²)

Extraction recovery,

[%]

Range, [ng/ml]

Androsterone 0,996 91 240-4445 Etiocholanolone 0.999 92 250-4664 d4-Androsterone 1,000 91 50-810 Epitestosterone 0,996 97 2.4-289 Testosterone 0,999 92 2.5-312 5-Androsterone-3,17-diol

0,999 84 5-368

Androstendione 0.995 91 5-375 5-Androstane-3,17-diol

1,000 81 5-384

Dehydroepiandrosterone 0,997 97 2.4-374 5-Dihydrotestosterone 0,998 97 2.4-310 5-Androstane-3,11-diol-17-one

0,999 93 100-1900

5-Androstane-3,11-diol-17-one

0,999 85 100-1907

Table 1. Screening method: validation data – correlation coefficient, extract recovery, range

Results and Discussion Using the two validated methods we analyzed three groups: control group, overweight group and methadone group. The ratio between different steroids was compared. Results are presented in figures 3 and 4. The period of methadone treatment is from 6 months to 60 months. The concentration of methadone is between 0.94 ng/ml and 15.35 ng/ml and depends on the course of treatment. Methadone treatment group had suppressed levels of steroids (A=2.80 ng/ml, E=2.0 ng/ml, T=0.03 ng/ml, 5αAdiol=0.07 ng/ml, 5βAdiol=0.11ng/ml) compared to controls (A=3.60 ng/ml, E=2.03 ng/ml, T=0.05 ng/ml, 5αAdiol=0.08 ng/ml, 5βAdiol=0.13ng/ml). The levels of steroids in overweight group were A=6.34 ng/ml, E=3.81 ng/ml, T=0.07 ng/ml, 5αAdiol=0.25 ng/ml, 5βAdiol=0.24ng/ml compared to controls A=3.60 ng/ml, E=2.03 ng/ml, T=0.05 ng/ml, 5αAdiol=0.08 ng/ml, 5βAdiol=0.13ng/ml. The control group has universal steroid profile genotype ins/ins, del/ins, while the other two groups show a sudden increase in the ratio of 5αAdiol/5βAdiol. Comparing the control group and the methadone group shows that in a long term methadone use had lower levels of testosterone. Samples were tested by isotope mass spectrometer for determining the origin of steroids and showed that they have an endogenous character. Compound Reference

standards [‰]

Overweight

group [‰]

Methadone group [‰]

Control group, [‰]

Androsterone -32,1 -21,83 -19,78 -18,36

Etiocholanolone -24,5 -22,85 -22,31 -20,10

5-Androsterone-3,17-diol

-34,7 -24,37 -22,79 -20,47

5-Androstane-3,17-diol

-30,4 -23,72 -22,70 -18,61

5-Androstane-3,11-diol-17-one

-23.6 -22,22 -20,12 -19,57

Table 2. Confirmation method: δ13C values

Conclusions From the above results, the following conclusions can be drawn: It can be concluded that the chronic use of methadone leads to a direct toxic effect on the sex hormone testosterone. Testing of samples in isotopic mass spectrometry shows that their decrease in the concentration is not due to additional intake of prohibited substances. The results have confirmed the suppressive effect of methadone on testosterone undergoing methadone treatment. The average Testosterone level in men receiving methadone treatment was significantly lower than controls. When comparing the two groups, the control and overweight once, the conclusion can be made that slow metabolism leads to increased amounts of endogenous steroids. Testing with the isotope mass spectrometer has confirmed that the higher levels are due to abnormalities in the metabolic pathway. References

WADA Technical Document TD2016EAAS Version Number 1.0; TD2016IRMS Version Number 1.0 E. Nolteernsting, W. Schaenzer, M. Donike,Standartisation of the T/E-Determination by Deuterated internal standards, Recent advances in doping analysis (2), 113-120. 1995 A.Maitre, Ch.Saudan,P.Mangin, M.Saugy, Urinary Analysis of four Testosterone metabolites and pregnanediol by gas chromatography-combustion-isotope ratio mass spectrometry after oral administration of testosterone, Journal of Anal. Toxicology, vol.28, Sept. 2014 R. Pasquali,V. Vicennati, Steroids and the metabolic syndrome, Journal of Steroid Biochemistry&Molecular Biology 109 (2008) 258-265

All samples were measured on an Agilent 7890A gas chromatograph coupled through GC Isolink and CONFLO IV to a DELTA PLUS isotope ratio mass spectrometer (Thermo Scientific). The GC system was equipped with a DB-35MS UI, 20m x 0.180mm x 0.18µm film thickness column. The injection volume was 2 µl; the injections were performed splitless at 300 °С. A constant flow of 1,2 ml/min of helium carrier gas was used. The initial oven temperature of 120°С was held for 1,5 min, increased at 40 °С/min to 240°С, followed by a ramp at 2°С/min to 256°С held for 1 min., followed by a ramp at 2°С/min to 280°С and 20°С/min to 300°С and held for 4 min. The oxidation reactor was operated at 940°С and oxidized for 60 min after each sequence of 60-70 injections. The water removal was done by a Nafion membrane. The δ13С value of the CO2 reference gas was calibrated towards the GC-IRMS STANDARD C15/C20/C25.

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Figure 4. Comparison between control group and overweight group

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Figure 3. Comparison between control group and methadone treatment group

Figure 2. Sample preparation for confirmatory analysis

Figure 1. Biosynthesis of endogenous steroid

The overweight group set higher hormone levels. However, the resulting steroid ratios did not show such large deviations from the control group. Samples were placed on the isotopic mass spectrometer and showed that steroids were of an endogenous nature. In both groups it was observed that when comparing the ratios of 5αAdiol/5βAdiol, the ratio of control group was lower than the one of the others. It can be concluded that this ratio is not affected vastly by the drug’s concentration and abnormalities in the metabolic pathway. Due to the observed lower levels of testosterone the ratio of Androsterone/Testosterone in the methadone group was in higher ratios than in the overweight group. The δ13C values of the following androgenic endogenous steroids were measured: Androsterone, Etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, 5α-androstane-3α,11β-diol-17-one. The results are shown in Table 2. In Figure 5 are shown GC/C/IRMS chormatograms for 5αAdiol and 5βAdiol.

The method developed for confirmatory analysis consists of: solid phase extraction (SPE), liquid-liquid extraction (LLE) and preparative – HPLC purification. A 10-25 ml urine sample, depending on the previously measured steroid profile, was prepared by a standard operating procedure for steroids – figure 2. The clean-up was performed on a Dionex Softron GmbH and analytical column BDS Hypersil C18, dim. (mm) 250x 4.6, particle size 5 µ, with guard column Hypersil gold 3um 10x4mm.The injection volume was 100 µl, the flow rate 1 ml/min and the DAD wavelength 196 nm. For HPLC clean-up 1, linear gradient was used increasing from 30:70 acetonitrile/water to 100 % acetonitrile in 25 min; after 3 min at 100% acetonitrile, the column was re-equilibrated for 6 min. Four fractions were collected, two of them (F1 and F4) were dried, reconstituted in acetone and injected in GC/C/IRMS; the other fractions were dried and acetylated (with 50 µl acetic anhydride и 50 µl pyridine, at 80 °С, 2 hours). Then after evaporation, they were reconstituted in methanol: water 60:40 and submitted to a second HPLC clean-up. For HPLC clean-up 2, a linear gradient was used increasing from 70:30 acetonitrile/water to 100 % acetonitrile in 33 min; after 3 min at 100% acetonitrile, the column was re-equilibrated for 6 min.