Vortex-Assisted Liquid-Liquid Microextraction for Steroid …
751
quantification of endogenous steroids related to endocrine disorders has become an
important field of investigation for clinical laboratories such as testosterone replacement therapy (TRT). High sensitivity and selectivity are mandatory in the analytical
determination of steroids, which can be presented at very low concentrations in relatively small and complex biological samples (Regal et al. 2009), veterinary growth
promoter investigation (Noppe et al. 2008), as well as for environmental studies (Peng
et al. 2008), doping control (Hintikka et al. 2008; Kuuranne et al. 2003; Thevis and
Schanzer 2005), Alzheimer disease (Wang 2008) and inherited human disease (Lacey
et al. 2004).
The anabolic androgenic steroids (AAS) are a family of hormones that includes
the natural male hormone testosterone (T), together with numerous closely related
chemical derivatives (Kanayama et al. 2010). All AAS possess both anabolic (musclebuilding) and androgenic (masculine) properties, and they affect a wide range of
physiological systems. The changing trends in steroid abuse have only been preceded
by the rapidly advancing analytical technologies that can cover a wider range of
compounds and achieve lower limits of detection (Scarth et al. 2012).
The method of steroid profiling (T/E ratio) was introduced into routine doping
control by Donike et al. (1993). The ratios of these steroids have been proven to
be very stable (Donike et al. 1993). The administration of steroids such as T, its
precursors are proven to alter one or more parameters of the urinary steroid profile
(Kerkhof et al. 2000). The latest effective date for Endogenous Anabolic Androgenic
Steroids (EAAS) WADA Technical Document (TD) is TDEAAS2016. The purpose
of this TD is to harmonize the approaches to the measurement and reporting of
EAAS in urine, including data in support of the steroidal module of the Athlete
Biological Passport (ABP) or “steroid profile”. Steroid profiling is one of the most
versatile and informative screening tools for the detection of steroid abuse in sports
drug testing. The “steroid profile” in the WADA technical document is composed
of Testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio),
5alpha-androstane-3alpha, 17beta-diol (5αAdiol), 5beta-androstane-3alpha, 17betadiol (5βAdiol) and the ratio of T to E (T/E). These are measured as free steroid
content obtained from the free steroid fraction plus those released from the conjugated
fraction following hydrolysis by glucuronidase enzymes. Other urinary steroids or
ratios of steroid metabolites that could be useful in evaluating a steroid profile are
A/T, A/Etio, 5αAdiol/5βAdiol and 5αAdiol/E) (WADA TDEAAS2016).
5 Microextraction Technique as Sample Preparation
Sample pre-treatment is an important step in any analysis. Prior to the analytical
determination, the analytes are first isolated from the matrix and pre-concentrated.
Also, it is often necessary to clean-up the sample from the complex matrices. Classical
sample pre-treatment techniques [e.g., liquid-liquid extraction (LLE) and solid-phase
extraction (SPE)] are slow, labour intensive (Gyorgy and Karoly 2004) and consume
large amounts of organic solvents. Additionally, in LLE, evaporation is an inevitable
751
quantification of endogenous steroids related to endocrine disorders has become an
important field of investigation for clinical laboratories such as testosterone replacement therapy (TRT). High sensitivity and selectivity are mandatory in the analytical
determination of steroids, which can be presented at very low concentrations in relatively small and complex biological samples (Regal et al. 2009), veterinary growth
promoter investigation (Noppe et al. 2008), as well as for environmental studies (Peng
et al. 2008), doping control (Hintikka et al. 2008; Kuuranne et al. 2003; Thevis and
Schanzer 2005), Alzheimer disease (Wang 2008) and inherited human disease (Lacey
et al. 2004).
The anabolic androgenic steroids (AAS) are a family of hormones that includes
the natural male hormone testosterone (T), together with numerous closely related
chemical derivatives (Kanayama et al. 2010). All AAS possess both anabolic (musclebuilding) and androgenic (masculine) properties, and they affect a wide range of
physiological systems. The changing trends in steroid abuse have only been preceded
by the rapidly advancing analytical technologies that can cover a wider range of
compounds and achieve lower limits of detection (Scarth et al. 2012).
The method of steroid profiling (T/E ratio) was introduced into routine doping
control by Donike et al. (1993). The ratios of these steroids have been proven to
be very stable (Donike et al. 1993). The administration of steroids such as T, its
precursors are proven to alter one or more parameters of the urinary steroid profile
(Kerkhof et al. 2000). The latest effective date for Endogenous Anabolic Androgenic
Steroids (EAAS) WADA Technical Document (TD) is TDEAAS2016. The purpose
of this TD is to harmonize the approaches to the measurement and reporting of
EAAS in urine, including data in support of the steroidal module of the Athlete
Biological Passport (ABP) or “steroid profile”. Steroid profiling is one of the most
versatile and informative screening tools for the detection of steroid abuse in sports
drug testing. The “steroid profile” in the WADA technical document is composed
of Testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio),
5alpha-androstane-3alpha, 17beta-diol (5αAdiol), 5beta-androstane-3alpha, 17betadiol (5βAdiol) and the ratio of T to E (T/E). These are measured as free steroid
content obtained from the free steroid fraction plus those released from the conjugated
fraction following hydrolysis by glucuronidase enzymes. Other urinary steroids or
ratios of steroid metabolites that could be useful in evaluating a steroid profile are
A/T, A/Etio, 5αAdiol/5βAdiol and 5αAdiol/E) (WADA TDEAAS2016).
5 Microextraction Technique as Sample Preparation
Sample pre-treatment is an important step in any analysis. Prior to the analytical
determination, the analytes are first isolated from the matrix and pre-concentrated.
Also, it is often necessary to clean-up the sample from the complex matrices. Classical
sample pre-treatment techniques [e.g., liquid-liquid extraction (LLE) and solid-phase
extraction (SPE)] are slow, labour intensive (Gyorgy and Karoly 2004) and consume
large amounts of organic solvents. Additionally, in LLE, evaporation is an inevitable