Testosterone, labelled with

Ring A of cholestenone " as well as of testosterone has been labeled with C by Turner while Heidelberger, Brewer and Dauben have synthesized isotopic 1,2,5,6-dibenzanthracene. 

On the other hand, it is not yet known whether the same effect is found with C-steroids labeled with tritium-containing reagents. Thus values obtained with C-steroids as internal standards and tritiated acetic anhydride for labeling are probably reliable. Similarly the high losses in the S-thiosemicarbazone methods of Riondel et al. (Rl, R2) were attributed largely to losses in the microchemical reactions with progesterone, but poor elution of the thiosemicarbazones from paper and silica gel plates was also regarded as a major factor with testosterone. Since... 

Dehydroepiandrosterone (DHEA), a naturally occurring precursor of endogenous testosterone synthesis, assnmed national attention following its use in professional baseball. Unlike the parenteral administration of testosterone, however, DHEA does not produce a notable elevation in serum testosterone levels or enhance skeletal mnscle hypertrophy.Several adverse effects were reported and the International Olympic Committee considers DHEA an illegal steroid, banning its use. A recent analysis of sixteen commercial DHEA products revealed that only half the products contained the actual amount of DHEA stated on the product label, with actual levels varying between 0% and 150% of the label content. ... 

Generally, methylation of enolate ions with isotopically tagged methyl iodide is a satisfactory labeling procedure. For example, application of this method has given the C-18 labeled steroids, (244) and (245) (see above), 17 -acetoxy-4jS-trideuteriomethyl-4a-methyl-l9-norandrost-5-en-3-one (264) and 19- C-testosterone acetate (268). Methylation of the anion derived from 17jS-acetoxy-4-methyl-l9-norandrost-4-en-3-one (263) with d3-methyl iodide occurs predominantly at C-4, yielding mainly the 4)S-trideuterio-methyl derivative (264) and about 10% of the corresponding C-4 epimer... 

Quest Diagnostics have established a routine system for testosterone, which has now replaced their other methods of analysis (Goldman et al., poster, US Endocrine Society meeting, San Diego, 2005). The analysis is carried out on a Thermofinnigan TSQ Quantum Ultra operated with an APCI source in the positive-ion mode. They use two transitions, 289—>109 and 289—>97, for the analyte and 294—>112 and 294—> 99 for the 2H5-labeled internal standard. On-line extraction and short retention times allow them to assay several thousand samples per month per instrument, making it cost-effective in spite of the high cost of instrumentation. 

Guo and co-workers [24,25] have spearheaded the development of MS/MS serum steroid profiles. Their most recent report describes profiling in 11 min of 12 steroids in 200 pi serum with minimal work-up, comprising acetonitrile protein precipitation. The steroids analyzed were as follows DHEA sulfate, DHEA, aldosterone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, estradiol, testosterone, 17-hy-droxyprogesterone, progesterone, and 25-hydroxyvitamin D3. Stable-isotope-labeled internal standards were incorporated for each steroid. An API-5000 instrument was used with the APPI source in positive-ion mode, with the exception of aldosterone, which had greater sensitivity in negative-ion mode. Separation was carried out on a C8 column, which allowed more rapid separation than the more commonly utilized C18. The MRM transitions utilized are shown in Table 5.3.1. The lower level of sensitivity was between 1.5 and 10 pg/ml, dependent on the steroid. The authors were exhaustive in addressing issues of accuracy, recovery (90-110%) and reproducibility (< 12.2% for same-day and between-day). 

Internal standard Isotope labeled analyte with optimized binding assessment, and verified standard curve IS peak area within 50 % of each analyte 13C6-Estradiol, 16a-Hydroxyestra-diol-rf2, Testosterone-, 13C,-Etheylnylestradiol IS peak area within 50 % of each analyte Testosterone-, Estradiol-rf3 Estron-d4, Estradiol-d3... 

De Ridder, J. J., and Koppens, P. C. (1978). Discrimination between endogenous and exogenous testosterone in human plasma after oral treatment with testosterone undecano-ate, using stable-isotope labeled compounds and mass spectrometric analysis. In Stable Isotopes. Applications in Pharmacology, Toxicology and Clinical Research (T. A. Baillie, ed.), pp. 157-165. Mcmillan, New York. 

The application of the twin ion technique [257] is also of importance in metabolism studies. The doubly labelled steroids [4- C+ 7-l- Ho.44]-androstenedione and [4- C + 7/3- Ho.42]-testosterone, were incubated with human placental microsomes and the resulting metabolites quantitated by counting C and identified by GC-MS [258]. The identified metabolites 17/8,19-dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-en-3,17-dione, 17/8-hydroxy-3-oxo-androst-4-en-3-one, 3,17-dioxoandrost-4-en-19-al, oestradiol-17/3 and oestrone were easily recognisable from the double sets of relevant ions in their spectra due to the mixture of hydrogen and deuterium substitution at C-7. Hence the presence of the aromatizing enzymes in the placental preparation and the intermediates in oestrogen biosynthesis were confirmed. 

The preferred tracer in commercially available RIA kits for labeled testosterone is I. The size of the iodinated group and the site of linkage may greatly affect assay affinity and specificity. Some methods use solvent extraction or chromatography, or both, to remove interferences from other steroids and some drugs. These prefiminaiy steps also permit concentration of the sample when low concentrations of testosterone are expected. Like automated assays, manual RIA assays perform better at higher concentrations, with questionable utility in female and prepubertal subjects. ... 

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