Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation.

Non-steroidal drugs that increase endogenous testosterone (T) may be used to exploit ergogenic effects of androgens in power sports. While superactive GnRH analog use is suspected, neither screening nor detection tests are developed. This study aimed to determine if (a) stimulation for 5 days by leuprolide (a superactive GnRH analog) of serum and urine steroids and urine LH is reproducible at a 2 week interval, (b) nandrolone decanoate (ND) co-administration masks responses to leuprolide administration, (c) performance of urine measurement of leuprolide and M1, its major metabolite, as a detection test. Healthy men were randomized into a 4 week parallel group, open label clinical study in which all men had daily sc injections of leuprolide (1mg) for 4 days in the 1st and 3rd weeks with hormone-free 2nd and 4th weeks. In the 3rd week, men were randomized to either ND injections or no extra treatment. Serum steroids were determined by liquid chromatography, tandem mass spectrometry (LC-MS), urine steroids by gas chromatography, mass spectrometry (GC-MS), urine leuprolide and M1 by high resolution LC-MS and urine LH by immunoassay. Leuprolide stimulated striking, reproducible increases in serum and urine LH and steroids (serum T, dihydroT (DHT), 3α diol; urine T, epitestosterone (E) and androsterone (A). ND suppressed basal serum T, E2, 3α diol, and urinary E but did not mask or change the magnitude of responses to leuprolide. Urine leuprolide and M1 measurement had 100% sensitivity and specificity in detecting leuprolide administration up to one day after cessation of injections with the detection window between 1 and 3 days after last dose. Screening using urine steroid and LH measurements, optimally by urinary log10(LHxT), correctly classified 82% of urine samples. It is concluded that leuprolide stimulation of endogenous testosterone is reproducible after a 10-day interval, is not masked by ND and is reliably detected by urine leuprolide or M1 measurement for at least 1 day after administration.

Quantitative LC-MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration.

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and -20 °C as well as after three freeze-thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.