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).

Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC-MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers.

Gonadotropin-releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti-Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti-doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow-release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute-and-shoot and solid-phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non-physiological levels. Thus, for doping-control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data.

Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances.

Identification of gonadotropin-releasing hormone metabolites in greyhound urine.

Gonadotropin-releasing hormone (GnRH) is a 10-residue peptide hormone that induces secretion of luteinizing hormone (LH) and follicle-stimulating hormone into the blood from the pituitary gland. In males, LH acts on the testes to produce testosterone. The performance-enhancing potential of testosterone makes administration of exogenous GnRH a concern in sports doping control. Detection of GnRH abuse is challenging owing to its rapid clearance from the body and its degradation in urine. Following recent investigations of GnRH abuse in racing greyhounds in New Zealand, we carried out a GnRH administration study in greyhounds in an attempt to identify GnRH metabolites that might provide more facile detection of GnRH abuse; little information is available on in vivo metabolites of exogenous GnRH in any species and none in dogs. We identified three C-terminal GnRH metabolites in urine: GnRH 5-10, GnRH 6-10, and GnRH 7-10. These metabolites and intact GnRH, which was also detected in urine, were all excreted over a 1-3 h period after GnRH administration. Two of the GnRH metabolites - GnRH 5-10 and GnRH 6-10 - were more stable in urine than intact GnRH offering improved potential to detect GnRH administration. Copyright © 2017 John Wiley & Sons, Ltd.

Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

Outcome from consecutive ICSI cycles in patients treated with recombinant human LH and those supplemented with urinary hCG-based LH activity during controlled ovarian stimulation in the long GnRH-agonist protocol.

Clinical results were compared in a well-established, assisted reproduction program during the cross-over from highly purified (HP)-human menopausal gonadotropin (hMG) to rhFSH/rhLH. We included the last 33 patients treated with HP-hMG and the first 33 patients receiving rhFSH/rhLH for ovarian stimulation in their first intracytoplasmic sperm injection cycle. Patient baseline characteristics were almost identical in the two groups. Ovarian stimulation characteristics (days of stimulation, total amount of FSH administered using a modest initial loading dose of 150 IU/d, patients with oocyte retrieval) were similar for the two groups. However, the number of total and leading follicles and E2 serum levels on the human chorionic gonadotropin injection day were significantly higher in the rhFSH/rhLH group. The oocyte yield was significantly higher in the rhFSH/rhLH group as well as the number of metaphase II oocytes, difference almost reaching the statistical significance. The number of oocytes fertilized was also higher in patients receiving rhFSH/rhLH treatment. Implantation and clinical pregnancy rates were similar in both the study groups. It is concluded that in women undergoing controlled ovarian hyperstimulation under pituitary suppression for ART, the recombinant combined product containing FSH and LH in a fixed 2:1 ratio is more effective than HP-hMG in terms of follicle development, oocyte yield and quality, and fertilization rates.

Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS.

The decapeptide gonadotrophin-releasing hormone (GnRH) is endogenously produced in the hypothalamus and secreted into the microcirculation between hypothalamus and pituitary gland. Here, the bioactive hormone is responsible for the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. Because an intermittent application of exogenous GnRH in young males increases the testosterone plasma level by stimulation of the Leydig cells, the potential misuse of the administered substance offers a reasonable relevancy for doping controls and is prohibited in accordance to the list of banned substances of the World Anti-Doping Agency (WADA). The presented method provides a mass spectrometric approach to determine the nondegraded hormone in regular doping control samples by utilizing a sample preparation procedure with solid phase extraction, immunoaffinity purification and a subsequent separation by liquid chromatography with ESI-MS/MS detection. For liquid chromatography/mass spectrometry two alternative instrumental equipments were tested: the first consisted of an Agilent 1100 liquid chromatograph coupled to an Applied Biosystem Q Trap 4000 mass spectrometer, the second equipment was assembled by a Waters Aquity nano-UPLC coupled to a Thermo LTQ Orbitrap high resolution/high accuracy mass spectrometer. In urine specimens provided from healthy volunteers GnRH was not detected in accordance to the recent literature, but in postadministration samples urinary concentrations between 20 to 100 pg/ml of the intact peptide were determined. The method offered good validation results considering the parameter specificity, linearity (5-300 pg/ml), limit of detection (LOD, approx. 5 pg/ml), precision (inter/intraday, < 20%) and accuracy (105%) using Des-pGlu(1)-GnRH as internal standard to control each sample preparation step.

Symptoms of premenstrual syndrome and their association with migraine headache.

OBJECTIVES: To determine the association between the severity of premenstrual (PMS) symptoms and headache outcome measures during natural menstrual cycles and after medical oophorectomy. BACKGROUND: Premenstrual syndrome may occur in 64% of those with pure menstrual migraine and 33% of those with menstrually related migraine. Few past studies have examined the relationship between the severity of PMS symptoms and migraine headache. METHODS: Data were obtained from a 6.5-month randomized-controlled trial examining the role of medical oophorectomy in the prevention of migraine headache and later divided into two data sets for analysis purposes. The menstrual cycle data set was composed of data from three natural menstrual cycles obtained from 21 participants during lead-in and placebo run-in phases. Each menstrual cycle was subdivided into seven 3-day intervals based on urine hormone metabolites. The medical oophorectomy data set included data from a 2-month treatment period in which a medical oophorectomy was induced by gonadotropin-releasing hormone agonists (GnRHa) and participants were randomized to transdermal estradiol or a matching placebo (GnRHa/estradiol and GnRHa/placebo groups, respectively). All participants completed a daily diary recording the severity of PMS symptoms and headache outcome measures. The primary outcome measures were the PMS index (mean of the daily PMS severity scores) and the headache index (mean of the headache severity scores). Pearson correlation coefficients were used to assess the degree of association between the outcome measures. RESULTS: Menstrual Cycle Data Set.-The PMS index was significantly correlated with the headache index during native menstrual cycles (correlation coefficient of 0.47; P < .05) and during all seven intervals of the menstrual cycle (correlation coefficients of 0.39 to 0.65; all P values < .05). Medical Oophorectomy Data Set.-Correlation coefficients between the PMS and headache indices were 0.58 and 0.47 for the GnRHa/estradiol (n = 9) and GnRHa/placebo groups, respectively (P-values of <.05). CONCLUSIONS: Moderate correlations exist within female migraineurs between the severity of PMS symptoms and headache outcome measures throughout natural menstrual cycles as well as after medical oophorectomy. Our data would suggest that the presence and severity of headache might modulate PMS symptoms in female migraineurs.

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry.

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE-MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.

Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography-electrospray ionization mass spectrometry.

A sensitive LC-MS quantitation method of cetrorelix, a novel gonadotropin releasing hormone (GnRH) antagonist, was developed. Plasma and urine samples to which brominated cetrorelix was added as an internal standard (I.S.) were purified by solid-phase extraction with C8 cartridges. The chromatographic separation was achieved on a C18 reversed-phase column using acetonitrile-water-trifluoroacetic acid (35:65:0.1, v/v/v) as mobile phase. The mass spectrometric analysis was performed by electrospray ionization mode with negative ion detection, and the adduct ions of cetrorelix and I.S. with trifluoroacetic acid were monitored in extremely high mass region of m/z 1543 and 1700, respectively. The lower limit of quantitation was 1.00 ng per 1 ml of plasma and 2.09 ng per 2 ml of urine, and the present method was applied to the analysis of pharmacokinetics of cetrorelix in human during phase 1 clinical trial.

Enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone releasing hormone (LH-RH) using a heterobifunctional cross-linking agent, N-[beta-(4-diazophenyl)ethyl]maleimide.

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and beta-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and LH-RH4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.

A monthly cycle in the plasma concentration of GnRH and the glomerular filtered load of GnRH during second trimester pregnancy.

Six healthy women were examined three times a week during the second trimester. The aim of the study was to test the existence of a monthly cycle in plasma GnRH. Because the kidney plays a dominant part in the elimination of small peptides from the circulation by filtration, the filtered load (P-GnRH x GFR) of GnRH was estimated and tested for a cycle. A mathematical model (parabolas overlaid with a cosine curve and containing parameters for cycle length, cycle amplitude and phase) was used in a multivariate analysis of changes in plasma GnRH, creatinine clearance and GnRH filtered load. We found significant monthly cycles in plasma GnRH (p = 0.003) and in GnRH filtered load (p = 0.004), but no significance for a cycle in creatinine clearance. As an intermediate result we demonstrated that the tubular reabsorption of GnRH was unsaturated for all values of plasma GnRH and GnRH filtered load in question. The increased pregnancy level of plasma GnRH originates in part in the placenta, but we assume that the cycle generator is contained in the maternal neuroendocrine system.

High-performance liquid chromatography followed by radioimmunoassay for the determination of a luteinizing hormone-releasing hormone analogue, leuprorelin, and its metabolite.

A sensitive method for the determination of leuprorelin (TAP-144), a luteinizing hormone-releasing hormone analogue, and its C-terminal metabolite, M-I, in serum and urine has been developed. Leuprorelin and M-I were extracted from serum or urine samples with Sep-Pak C18 cartridges, and separated completely by high-performance liquid chromatography and determined by radioimmunoassay using [125I]leuprorelin as the labelled antigen. The detection limit of the method was 0.05 ng/ml for leuprorelin and M-I, and the recovery of the compounds added to serum and urine was over 88% with a coefficient of variation (within-assay) of less than 5%. The method was applied to the determination of leuprorelin and M-I-like immunoreactivity in serum or urine after administration of once-a-month injectable microspheres of leuprorelin acetate (TAP-144-SR) to patients with prostate cancer.

Time-related neuroendocrine manifestations of puberty: a combined clinical and experimental approach extracted from the 4th Belgian Endocrine Society lecture.

The neuroendocrine manifestations of puberty converge on changes in GnRH secretion. Their appraisal through the assay of GnRH-like material in 24-hour urine extracts shows an increased excretion of this material in the late prepubertal period. The most striking pubertal changes in GnRH secretion occur on a circadian and ultradian basis. In man, they can be evaluated only indirectly. The circadian variations in LH and FSH secretion characteristic of puberty may be observed in timed fractions of 24-hour urine with some delay when compared to the variations of plasma levels. Studies on the frequency of pulsatile LH secretion and during chronic intermittent administration of GnRH support the existence of an increased frequency of GnRH secretory episodes at puberty. LH response to synthetic GnRH is directly related to the frequency of stimulation by endogenous GnRH pulses and provides a very useful index of neuroendocrine maturation in patients with delayed or precocious puberty. A direct evaluation of pulsatile GnRH secretion is possible using the rat hypothalamus in vitro. In these experimental conditions, the frequency of pulsatile GnRH release increases during very early stages of sexual maturation in the male rat. GnRH itself and beta-endorphin are inhibitory regulators of GnRH secretion in vitro and may participate in the mechanisms restraining the pulse-generating machinery in the hypothalamus before puberty.

Urinary excretion of immunoreactive luteinizing hormone-releasing hormone-like material in children correlation with pubertal development.

Immunoreactive LRH (iLRH)-like material has been measured in extracts of urine from normal children and adolescents, adult men and women, and postmenopausal women. The urinary excretion of iLRH-like material was significantly greater in pubertal than in prepubertal subjects and in boys than girls at both stages of sexual maturation [prepubertal males, 3.26 +/- 0.49 ng/24 h (SE; n = 24); pubertal males, 5.94 +/- 1.36 (n = 12); prepubertal females, 1.14 +/- 0.21 (n = 19); pubertal females, 2.85 +/- 0.56 (n = 13)]. In adult males (n = 5) the urinary excretion of iLRH-like material was 7.8 +/- 1.3 ng/24 h, and in adult women in the follicular phase of the menstrual cycle (n = 8) it was 2.9 +/- 0.3. In five postmenopausal women the urinary iLRH-like content was 7.32 +/- 0.92 ng/24 h (P less than 0.01 relative to normal pubertal and adult women). In children the 24-h urinary excretion of iLRH-like material was positively correlated with chronological and bone ages, Tanner stage of genital (male) and breast (female) development, and the urinary excretion of LH and FSH in males. It did not correlate with the urinary excretion of either LH or FSH in females. Carboxymethylcellulose chromatography of extracts of urine from pubertal boys and girls, adult men and women, and postmenopausal women suggested that the iLRH-like material may be the 2-10 fragment of LRH rather than the intact decapeptide.

[Urinary LHRH: radioimmunological assay and physiological significance]. / LHRH urinaire: dosage radioimmunologique et signification physiologique.

A LHRH-like substance is detectable in the urine. It is extractable by glass powder, which suggests that it is a peptide, and it can be concentrated by anti-LHRH affinity chromatography, strongly suggesting that it is not a contaminant in the RIA. It is composed of very little intact LHRH, and the great majority appears as multiple smaller fractions, although the possibility of "urinary LHRH" being a slightly larger peptide containing a sequence similar or identical to that of LHRH has not been entirely eliminated. Urinary measurements show no correlation with the clinical state, and the widely differing values obtained using different antisera in the present study emphasize the need for in-depth antiserum binding studies prior to clinical use in the LHRH radioimmunoassay.

Urinary gonadotrophins and luteinizing hormone-releasing hormone (LH-RH) like immunoreactivity during three normal reproductive cycles.

Highly specific and sensitive radioimmunological methods were applied to determine the levels of LH-RH like immunoreactivity in urine previously extracted by spherosil and methanol, and to assay the gonadotrophins, after extraction with acetone. The endogenous urinary LH-RH like immunoreactivity material was identified by chromatography on Sephadex G25, as having physicochemical properties similar to those of the hormone found in unextracted urine after iv injection of synthetic LH-RH, but different from those of the synthetic decapeptide. The LH-RH like immunoreactivity and the gonadotrophins were assayed in daily collected urine during the reproductive cycle of 3 normal women. A midcycle peak of both FSH and LH was found in each subject. No increases of LH-RH like immunoreactivity were found before or concomittant with the gonadotrophins surge. But peaks of urinary LH-RH like immunoreactivity were observed during the luteal phase, without subsequent increase of gonadotrophin secretion.