Detection of Steroid Abuse

 INTRODUCTION TO STEROID HORMONES

Steroid hormones are substances that are essential for the proper functioning of the body. They are derived from cholesterol and are mainly produced by endocrine glands such as the adrenal cortex and gonads- ova or testis. Steroid hormones are grouped into five groups according to the receptors to which  they bind to:

1. ANDROGENS (e.g. testosterone)

  - Found in males. Primarily affect maturation and male sexual determination.

2. ESTROGENS (e.g. estradiol)

  - Found in females. Primarily affect maturation and female sexual determination.

3. GLUCOCORTICOIDS (e.g. cortisol)

  - Mainly affect metabolism such as decreasing inflammation and increasing resistance to stress.

4. MINERALOCORTICOIDS (e.g. aldosterone)

  - Maintain salt and water balance.

5. PROGESTOGENS (e.g. progesterone)

  - Regulate menstrual cycle and maintain pregnancy.

These five types of steroid hormones share similarities in structure which is characterized by three six member rings and one five member ring. Its fat soluble property allows it to enter the cell membrane or target receptor where it will exert its effects.

All hormones have an oxygen at the Carbon-3 and a varied substituent at their Carbon-17. It is this substituent that will vary between different types of hormones or can be Alpha or Beta depending on their positions below the plane of the molecule. 

METHODS OF DETECTION

Since the 1960s, there have been several methods for analyzing steroid hormone drugs in the body- from Thin Layer Chromatography(TLC), followed by Gas Chromatography(GC). However, these methods are limited to the amount of hormone in the analyte especially in pediatric patients or postmenopausal women. Another problem occurs when there is cross-reactivity which would effect the specificity of the result. Therefore, scientists have come up with new methods that are superior in the area regarding specificity and limits of quantification, that will produce a good linearity right down to the lowest concentrations. One such method is the latest version of Gas Chromatography- Mass Spectrometry(GC/MS).

Gas Chromatography- Mass Spectrometry(GC/MS)
Many clinical laboratories use this as the primary analytical method for identification and quantification. A capillary GC column with a thin film (0.25µm or less) of 100% dimethylpolysiloxane is the preferred column for many analysts, because this stationary phase can withstand the highest operating temperature available. Temperatures exceeding 300°C are required to elute the high molecular weight (250-400 Dalton) hormones in a reasonable analysis time while maintaining peak shape and resolution. A phase film thickness of 0.25µm or less maximizes column inertness at these high temperatures. Also, in order to provide reliable quantification, the column must exhibit the inertness necessary to produce symmetric peaks and reproducible results. 

After separating the steroid hormone from the other components in the urine sample, it passes to the mass spectrometer to check for its concentration. Steroid hormones contain three aromatic rings and a phenol group which gives it a distinctive wavelength when absorbing UV radiation (200-278nm).

Some other methods of detecting steroid in the body requires blood sample since the hormone is hardly detectable in the urine of some patients. However, even through this, the result of measurements produced may only reflect just a single time point. This is unlike the GC/MS technology that will reflect an integrated picture of hormone's metabolome over a long period of time.

SAMPLE PREPARATION

Detecting steroid abuse has always been a difficult task for scientists as the preparation procedures are very tedious. Furthermore, a reasonable threshold of steroid concentration on the body to count for steroid abuse is still being debated.
   
Two techniques have been tested and optimized, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5β-androstanedione, 6α-hydroxyestradiol and 7β-hydroxypregnenolone).


Solid Phase Microextraction (SPME)

SPME involves the use of a coated fibres with an extracting phase, that can be a liquid or a solid, which extracts different kinds of analytes (including both volatile and non-volatile) from different kinds of media, that can be in liquid or gas phase. The quantity of analyte extracted by the fibre is proportional to its concentration in the sample as long as equilibrium is reached or, in case of short time pre-equilibrium, with help of convection or agitation. After extraction, the SPME fiber is transferred to the injection port of separating instruments, such as a Gas Chromatograph, where desorption of the analyte takes place and analysis is carried out.

Microextraction by Packed Sorbent(MEPS)


MEPS is suited for sample preparations that involve complex biological compounds- in this case, urine. It reduces the amount of volume and reagent required as compared to other microextraction methods. It is a syringe that involves a Barrel Insert and Needle Assembly(BIN), which contains the stationary phase(packed bed) and is built into the syringe. 

STEROID ABUSE

Steroids are produced naturally by our body in order for our body to continue functioning healthily, however, it is not unknown that drug companies have produces synthetic steroid drug hormones for people who wish to enhance their performance. These drug hormones are known an anabolic steroids. They are related to the male sex hormone and promote male sexual characteristics such as skeletal and muscle growth. They are mainly used by athletes for performance enhancement, and it is for this very reason that steroid drug hormones are susceptible to abuse. Therefore, it is very important for analyst to detect steroid abuse as it may pose a threat tot he body.

 

Steroid abuse for athletes:

Anabolic steroids are natural sex hormone. Athletes will gain strength and muscular mass after steroid intake as steroid could increase muscular endurance, power and speed.

Function:

·         Stimulate receptor within muscle cells and activates specific genes

·         Travel to androgen receptor and mRNA, which signal the brains to construct specific proteins.

·         These proteins travel throughout the body to eliciting the aforementioned anabolic responses.

The effects of steroid on the athletes:

•   Train much harder for longer period of time

•   Accelerate muscle recovery

•   Continue to progress at a rate far beyond normal capacity

The side effects of steroid abuse:

•      Liver damages

•     High blood pressure
•      jaundice
•      Shrinking of the testicles for males
•      Infertility
•      Facial hair for females
•      Menstrual changes
•      gynecomastia