A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum (original) (raw)
Related papers
… of Chromatography B, 2009
A simple and novel HPLC-MS method for the simultaneous quantification of testosterone, 11ketotestosterone, and 11-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water-acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2-50 ng/ml), limits of detection (0.1-0.2 ng/ml) and quantification (0.2-0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).
Steroid determination in fish plasma using capillary electrophoresis
Environmental Toxicology and Chemistry, 2010
A capillary separation method that incorporates pH-mediated stacking is employed for the simultaneous determination of circulating steroid hormones in plasma from Perca flavescens (yellow perch) collected from natural aquatic environments. The method can be applied to separate eight steroid standards: progesterone, 17a,20b-dihydroxypregn-4-en-3-one, 17a-hydroxyprogesterone, testosterone, estrone, 11-ketotestosterone, ethynyl estradiol, and 17b-estradiol. Based on screening of plasma, the performance of the analytical method was determined for 17a,20b-dihydroxypregn-4-en-3-one, testosterone, 11-ketotestosterone, and 17b-estradiol. The within-day reproducibility in migration time for these four steroids in aqueous samples was 2%. Steroid quantification was accomplished using a calibration curve obtained with external standards. Plasma samples from fish collected from the Choptank and Severn Rivers, Maryland, USA, stored for up to one year were extracted with ethyl acetate and then further processed with anion exchange and hydrophobic solid phase extraction cartridges. The recovery of testosterone and 17b-estradiol from yellow perch plasma was 84 and 85%, respectively. Endogenous levels of testosterone ranged from 0.9 to 44 ng/ml, and when detected 17a,20bdihydroxypregn-4-en-3-one ranged from 5 to 34 ng/ml. The reported values for testosterone correlated well with the immunoassay technique. Endogenous concentrations of 17b-estradiol were 1.7 ng/ml. 11-Ketotestosterone was not quantified because of a suspected interferant. Higher levels of 17a,20b-dihydroxypregn-4-en-3-one were found in male and female fish in which 17b-estradiol was not detected. Monitoring multiple steroids can provide insight into hormonal fluctuations in fish. Environ. Toxicol. Chem. 2010;29:1950-1956. # 2010 SETAC Keywords-Steroidal compounds pH-Mediated stacking Capillary electrophoresis Perca flavescens Micelle electrokinetic chromatography 1954 Environ. Toxicol. Chem. 29, 2010 L. Bykova et al.
Aquatic Toxicology, 2006
The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 g MDHT l −1 for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 g l −1 was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 g l −1 . Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 g/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 g l −1 showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 g l −1 up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had matured.
American Journal of Analytical Chemistry, 2013
17α-methyltestosterone is used to induce the sex reversal of Tilapia sp. to obtain cultures mono-sex to an economically viable. This practice may lead to environmental contamination and problems in human health. Therefore methods need to be developed to detect residues of 17α-methyltestosterone in aqueous matrices. A simple high-performance liquid chromatographic method using ultraviolet detection (245 nm) and testosterone as internal standard has been developed for the monitoring 17α-methyltestosterone in freshwater samples of tilapia aquaculture. The method described involves limited sample preparation as it includes a filtration followed by a single solid-phase extraction step using C18 cartridge. Validation data indicated that the HPLC-UV method for 17α-methyltestosterone determination in the concentration range of 50-2000 μg/L provided good linearity, sensitivity, accuracy and precision. Method performance was efficiently applied to monitoring the freshwater samples of fish ponds and the surrounding aquatic channels.
Analysis of hormonal steroids in fish plasma and bile by coupling solid-phase extraction to GC/MS
Analytical and Bioanalytical Chemistry, 2006
An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, dihydrotestosterone, 11α-ketotestosterone, 17α-hydroxyprogesterone, 17α-hydroxypregnenolone, 17α,20β-dihydroxy-4-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC-MS) has been developed. After enzymatic hydrolysis for bile samples only, samples were concentrated and purified using two successive SPE (C 18 and NH 2 ) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) / mercaptoethanol / ammonium iodide (NH 4 I) and determined by GC-MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range 90-120% in plasma and in the range 60-70% in bile, and the reproducibility was below 10% for the complete procedure. Limits of detection obtained ranged from 0.1 to 0.4 ng/g in fish plasma and from 1.6 to 14 ng/g in fish bile. The developed method was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries.
Jurnal Akuakultur Indonesia
Tilapia fish farming using monosex male population has been reported to have 10% higher productivity compared to mix-sex culture. This study aimed to determine immersion dose of 17α-metiltestosterone (MT) that allowed higher male percentage, growth, survival, and measure testosterone level in fish body. The experiment was designed as three immersion doses, namely: 0; 1.8; and 5.4 mg/L MT, each with three replications. Immersion was conducted to 14-day-old larvae for four hours, with density of 100 fish/L of water. Testosterone levels in fish was measured using ELISA method, and sex identification was performed by histology method. The result showed that percentage of male fish was the same in the treatment of 1.8 mg/L and 5.4 mg/L, and 53–65% higher than the control without MT treatment (54% male). Growth and survival of fish until day 56 post immersion were the same. By ELISA analysis, the levels of testosterone in larvae just after immersion was similar in 1.8 mg/L and 5.4 mg/L ...