Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring

Kim

Journal of Lipid Research

et al. 2011

estrogen defi ciency can cause bone loss in postmenopausal women. Estrogens in women are biosynthesized in the ovaries and metabolized primarily through the NADPH-dependent hydroxylation ( 7 ).Although the biological functions of estrogen metabolism in the target tissues or cells are unclear, its metabolic changes are associated with a range of cancers and in modulating bone density ( 1-6 ). The hydroxylation of estrogens on either the A-ring or D-ring is catalyzed by various cytochrome P450 enzyme isoforms and results in the formation of hydroxy and keto metabolites. Hydroxylation on the A-ring occurs predominately at the C2 position and, to a lesser extent, at the C4 position ( 8 ). The 2-and 4-hydroxy derivatives are further converted to 2-, 3-, and 4-methoxy estrogens by catechol-O -methyltransferase ( 9 ). Hydroxylation at the 16 ␣ position of the D-ring produces 16 ␣ -hydroxyestrone, which can be metabolized further to estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol ( 10 ). According to the carcinogenic effect of breast cancer ( 1,11,12 ), catechol estrogens form quinones that react with DNA and form both stable and depurinating DNA adducts. Other metabolic pathways are also involved for the mitogenic and antiaptotic effects of estrone, estradiol, and some of their hydroxylated metabolites.In contrast to many cancer studies ( 1-4 ), it is diffi cult to measure the concentrations of estrogen metabolites in postmenopausal women with osteoporosis to provide accurate quantifi cation using conventional gas chromatography-mass Abstract Estrogen metabolites play important roles in the development of female-related disorders and homeostasis of the bone. To improve detectability, a validated gas chromatography-mass spectrometry (GC-MS) method was conducted with two-phase extractive ethoxycarbonlyation (EOC) and subsequent pentafl uoropropionyl (PFP) derivatization was introduced. The resulting samples were separated through a high-temperature MXT-1 column within an 8 min run and were detected in the selected ion monitoring (SIM) mode. The optimized analytical conditions led to good separation with a symmetric peak shape for 19 estrogens as their EOC-PFP derivatives. The limit of quantifi cation (LOQ) was from 0.02 to ‫ف‬ 0.1 ng/ml for most estrogens analyzed, except for 2-hydroxyestriol (0.5 ng/ml). The devised method was found to be linear ( r 2 > 0.995) in the range from the LOQ to 40 ng/ml, whereas the precision (% CV) and accuracy (% bias) ranged from Endogenous estrogens play an important role in the development of human cancers, such as breast, endometrial, ovary, thyroid, and prostate cancer, as well as in bone homeostasis ( 1-6 ). In general, higher levels of estrogens are associated with an increased risk of breast cancer, but an

Section: Gc-ms Characteristics Of the Derivativesmentioning

confidence: 99%

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis

Kim

Journal of Lipid Research

et al. 2011

“…The heated capillary temperature was maintained at 250°C. To prepare a calibration curve, we removed T-G and DHT-G from pooled urine by solid-phase extraction (9 ). Urine samples with increasing concentrations of added analyte (0.2-400 g/L T-G and 3-200 g/L DHT-G) and a fixed concentration of added d 3 -T-G (20 L; 1 mg/L) as an IS were then analyzed according to the procedure described above.…”

mentioning

confidence: 99%

Rapid HPLC-Electrospray Tandem Mass Spectrometric Assay for Urinary Testosterone and Dihydrotestosterone Glucuronides from Patients with Benign Prostate Hyperplasia

Choi¹,

Kim

Chung³

2003

Clinical Chemistry

by the locus-specific primer is directly subjected to TaqI digestion and is analyzed on an agarose gel without radioisotopic hybridization. It also appears to be more accurate because all three distinct chimeric CYP21P/ CYP21 genes had 3.2-kb fragments (Fig. 1C, lanes 3 and 4). Simply assuming that the 3.2-kb fragment from conventional Southern analysis (6 ) is a deletion of the CYP21 gene has been shown to be incorrect. The authors of one report have stated that the 3.2-kb fragment analyzed by Southern blotting identified a fused CYP21 gene, but in this study, Southern blotting was performed with radioisotope hybridization and a different endonuclease (8 ).In conclusion, the coexistence of 9.3-and 3.2-kb fragments is consistent with a chimeric CYP21P/CYP21 gene. The method of analysis we have described is a novel tool for identification of such a molecule. These chimeric genes were found to occur frequently in CAH caused by steroid 21-hydroxylase deficiency in a Taiwanese (ethnic Chinese) population. The development of benign prostatic hyperplasia (BPH) is dependent on androgens, primarily dihydrotestosterone (DHT) (1 ). To estimate the activity of 5␣-reductase, which catalyzes the conversion of testosterone to DHT, testosterone and DHT have been quantified in biological samples (2-4 ). Because of their lipophilic properties, they are usually found in the conjugated form, i.e., linked to a hydrophilic sulfuric moiety or ␤-glucuronic acid, which are excreted mainly (Ͼ95%) in human urine. Despite reports of a close association between BPH and testosterone and DHT (1-4 ), to our knowledge, there is no published simultaneous quantitative data for these metabolites as their glucuronides in the urine of patients with BPH.The major problem associated with quantification of total steroids is incomplete hydrolysis, attributable mainly to the matrix effects of urine on the efficiency of enzymatic hydrolysis, which reduces the reproducibility of the assay (5,6 ). Given that it is often necessary to analyze many samples in a short time with good sensitivity and reproducibility, system throughput is a critical issue for many clinical mass spectrometry (MS) groups. In 322 Technical Briefs

“…Afterwards, the coupling rate was calculated according to the Lambert-Beer law and the formulas referred to in Choi et al (2000).…”

Section: Coupling Rate Of Mpa-bsa Formationmentioning

confidence: 99%

“…The relative molecular mass of MPA is only 344.5 and it has no immunogenicity, so it cannot stimulate animals to produce a corresponding antibody directly, though it has immune reactivity and can form a hapten-antibody complex with its specific antibody. Moreover, without coupling with a carrier protein, low-molecular substances are poorly absorbed by solid-phase carriers; through the protein, the hapten can be invested to the solid-phase carrier (Choi et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…The methods currently used for determining anabolic steroid residues in animal muscle tissues are high performance liquid chromatography and tandem mass spectrometry and gas chromatography-mass spectrometry (Daeseleire et al, 1991;Draisci et al, 1998;Philippe et al, 1999;Choi et al, 2000;Stolker et al, 2002;Rambaud et al, 2005), but sample preparation was trivial (Rapp et al, 1989;Yu et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detection of medroxyprogesterone acetate residues in animal muscle tissues by using enzyme-linked immunosorbent assay and liquid chromatography tandem mass spectrometry

Xu¹,

Pen²,

Hao³

et al. 2005

J. Anim. Feed Sci.

Medroxyprogesterone acetate (MPA) has a relative molecular mass of only 344.5 and no immunoreactivity. Methods using carbodiimides and mixed anhydrides were adapted to couple MPA with bovine serum albumin, a carrier protein. The coupling rates of conjugate using the above methods were estimated to be 14 and 20 by ultraviolet spectrophotometry. The coupling was successful according to SDS-PAGE. New Zealand White rabbits were immunized with the conjugate obtained with a coupling rate of 14, and blood was collected after five periods of immunities. Indirect ELISA showed the titer of antiserum to be 2.6×10 5 . Based on the purified antibody, a competitive indirect ELISA was developed. It provided a limit of detection of 0.096 ng/mL, recoveries (edible tissues) between 72 and 91% and a working range of 0.1-8.1 ng/mL. Preliminary evaluation of assay performance in terms of specificity, sensitivity, precision, and accuracy showed that this ELISA can be applied to practical detection of MPA in tissues. Moreover, it was compared with high performance liquid chromatography tandem mass spectrometry. The ion pair for quantification of MPA was 345.2/123.1, and linear equation of MPA was Y = 6.68 × 10 3 X+6.63 × 10 2 . The two analytical methods can be applied to monitor MPA and other anabolic steroid residues in foods.