Lcms Analysis Of Synthetic Steroids Or Animal Samples

In veterinary medicine, boldenone, a synthetic anabolic steroid (Figure 2.1), is commercially available, hence it is a concern in the horseracing industry. Pu et al. (2004) used ion-trap LC-MS analysis to detect boldenone conjugates (sulfate and glucuronide) and their 17-epimers in horse urine after intramuscular administration of boldenone undecylenate. Soon afterwards, Ho et al. (2004) reported the occurrence of endogenous boldenone sulfate in the urine of uncastrated male horses, and quantitated it by quadrupole time-of-flight (Q-TOF) LC-MS-MS.

Also known as endogenous in male horses and prohibited as a doping agent is 19-nortestosterone (nandrolone; Figure 2.1). Kim et al. (2000a) validated an LC-MS-MS method for the detection and quantitation of three different commercially available esters, but 19-nortestosterone esters are rapidly hydrolyzed in horse plasma, which limits the usefulness of this method in racehorse doping control.

Another synthetic anabolic steroid available as a veterinary product is stanozolol (Figure 2.1), whose metabolites were investigated by McKinney et al. (2004) by ion-trap LC-MS in horse urine after intramuscular injection.

17a-Methyltestosterone

17a-Methyltestosterone

CH3 CH3

Oxandrolone

Stanozolol

Stanozolol

Trenbolone

Trenbolone

19-Nortestosterone (Nandrolone)

Gestrinone

Gestrinone

Boldenone

Figure 2.1 Examples of synthetic androgen analytes

Boldenone

Tetrahydrogestrinone (THG)

Methenolone

Methenolone

Figure 2.1 Examples of synthetic androgen analytes

Yu et al. (2005) developed an LC-MS-MS screen for deconjugated anabolic steroids in horse urine and characterized the method using horse urine samples spiked with 15 prohibited anabolic steroids. In an excretion study in two horses, methenolone acetate was administered by mouth, methenolone (Figure 2.1) was detected in urine and the 17-epimethenolone metabolite was detected for a longer time.

Racehorse anti-doping work is not the only area that requires screening for prohibited anabolic steroids in animals. The drugs act as growth promoters in cattle and this use is prohibited by European regulations, hence analytical methods have been developed to detect residues in meat for enforcement purposes. Joos and Van Ryckeghem (1999) described a procedure for the LC-MS-MS analysis of 36 anabolic steroids found in animal kidney fat matrices. The method's limits of detection are low enough to meet regulations. In Japan, where maximum residue limits were established in 1995, Horie and Nakazawa (2000) developed a method for the determination of trenbolone (Figure 2.1) and zeranol (an anabolic agent but not a steroid) in bovine muscle and liver tissues by LC-MS with selected-ion monitoring (SIM).

Draisci et al. (2000) used LC-MS-MS to quantitate T (Figure 2.2), 19-nortestosterone and their 17-epimer metabolites in bovine serum and urine, and subsequently stanozolol and its major metabolite (Draisci et al., 2001) and boldenone (Draisci et al., 2003) in bovine urine. Van de Wiele et al. (2000) also worked on stanozolol in cattle urine and feces, with or without derivatization before LC-MS-MS analysis. For trenbolone, Buiarelli et al. (2003) developed and characterized an LC-MS-MS method for bovine urine and serum. Van Poucke and co-workers extended the list of LC-MS-MS target analytes to four anabolic steroids (Van Poucke and Van Peteghem, 2002), then 21 anabolic steroid residues in bovine urine (Van Poucke et al., 2005).

Dehydroepiandrosterone (DHEA)

Dehydroepiandrosterone (DHEA)

Testosterone

Testosterone

Epitestosterone

Epitestosterone

4-Androstene-3,17-dione ^-Dihydrotestosterone Androst-5-ene-3ß,17ß-diol

Androsterone

Androsterone

Etiocholanolone

Etiocholanolone

5a-Androstane-3a,17ß-diol

5a-Androstane-3a,17ß-diol

Figure 2.2 Examples of natural androgen analytes in human samples. Glucuronide or sulfate conjugates of these steroids are formed with the C-3 or C-17 hydroxyl group

Poelmans et al. (2002) reviewed analytical approaches to the detection of stanozolol and its metabolites.

Nielen et al. (2001) focused on a related facet of the illegal use of growth promoters in cattle, namely the detection of anabolic steroids in illegal cocktails. They presented a Q-TOF LC-MS-MS method with the aim of measuring accurate mass and calculating elemental composition for identification purposes.

Turning to the abuse of anabolic steroids by human competitors, Shackleton et al. (1997) conducted LC-MS analyses of pharmaceutical T esters in human plasma because of the potential applications to doping control, especially if sensitivity could be improved.

Leinonen et al. (2002) compared LC-MS-MS with electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoion-ization (APPI) for unconjugated (free) anabolic steroids in human urinary extracts. The selected analytes were synthetic steroids or their metabolites, known to be misused in sports and known to be excreted in urine unconjugated, namely oxandrolone (Figure 2.1), the 3'-hydroxy metabolite of stanozolol and the 6p-hydroxy metabolite of 4-chlorodehydro-methyltestosterone.

Kim et al. (2000b) used GC-MS, LC-MS and LC-MS-MS to study the metabolites of gestrinone (Figure 2.1), a contraceptive and potential sports doping agent, in human urine.

Anti-doping scientists had suspected for years that underground chemists supply performance-enhancing substances to athletes compelled to cheat, including 'designer' drugs made and used only to beat the test. This was proven when the anabolic steroid tetrahydrogestrinone (THG; Figure 2.1), a new chemical entity, was identified by Catlin et al. (2004) in a used syringe anonymously turned in to the US Anti-Doping Agency. LC-MS and LC-MS-MS played key roles in the identification and urine sample screening procedures. Thevis et al. (2005) reasoned that chemical modifications of steroids usually alter their molecular weights, used to detect and identify them in doping control samples, but might not alter their nuclei or some of their characteristic fragments or product ions. When suspicious product ions have been detected, precursor ion scan experiments can help identify unknown steroids.

Another reality that anti-doping scientists must face is that some athletes accused of using banned anabolic steroids offer explanations such as the inadvertent consumption of various alleged sources of the substances. To assess the theory that uncastrated boar meat consumption can result in an adverse finding in a doping control urine test, Le Bizec et al. (2000) tested volunteers and detected 19-norandrosterone and 19-noretiocholanolone for about 24 h after intake. The same team identified and quantitated the boar meat 19-norsteroids responsible for this finding (De Wasch et al., 2001).

To help provide anti-doping scientists with reference standards for LC-MS work, Kuuranne et al. (2002) carried out the enzyme-assisted syntheses of the glucuronides of methyltestosterone and nandrolone (Figure 2.1), and developed a new LC-MS method to control the synthetic product purity.

Watching the world become awash in steroid sex hormones, abused in animals and humans, Lopez de Alda et al. (2003) felt the need to review LC-MS and LC-MS-MS methods for the determination of these and other analytes in the aquatic environment.

In the only publication on the LC-MS analysis of synthetic steroids or animal samples that is unrelated to any steroid misuse, Magnusson and Sandstrom (2004) reported on a quantitative analysis of eight T metabolites in rat intestine mucosa.

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