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).

A Fast and Sensitive Method for the Detection of Leuprolide Acetate: A High-Throughput Approach for the In Vitro Evaluation of Liquid Crystal Formulations.

The suitability of using fluorescence spectroscopy to rapidly assay drug release by quantifying the time-dependent increase in total intrinsic protein fluorescence was assessed. Leuprolide acetate, a synthetic nonapeptide analogue of gonadotropin-releasing hormone (GnRH or LHRH), is the active pharmaceutical ingredient used to treat a wide range of sex hormone-related disorders, including advanced prostatic cancer, endometriosis, and precocious puberty. During the in vitro evaluation of drug delivery technologies for leuprolide acetate, one of the most time-consuming steps is the detection and accurate quantification of leuprolide release from formulation candidates. Thus far, the dominant means for leuprolide detection involves conventional multistep high-performance liquid chromatography (HPLC) methods, requiring sampling, dilutions, sample filtration, and chromatography, which can take up to 40 min for each sample. With the increasing demand for assay adaptation to high-throughput format, here we sought to exploit fluorescence spectroscopy as a tool to develop a novel method to rapidly assay the in vitro release of leuprolide acetate. By utilizing the intrinsic fluorescence of the tryptophan (Trp) and tyrosine (Tyr) amino acid residues present in the leuprolide nonapeptide, the in vitro release from liquid crystal formulations was accurately quantified as a function of fluorescence intensity. Here, we demonstrate that assaying leuprolide release using intrinsic protein fluorescence in a 96-well format requiring volumes as low as 100 µL is a cost-effective, rapid, and highly sensitive alternative to conventional HPLC methods. Furthermore, the high signal-to-noise ratios and robust Z'-factors of >0.8 indicate high sensitivity, precision, and feasibility for miniaturization, high-throughput format adaptation, and automation.

Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human granulocyte-macrophage colony-stimulating factor and its correlation with reversed-phase liquid chromatography method and bioassay.

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75 µm i.d.; effective length, 72 cm) was used at 25 °C; the applied voltage was 12 kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50 mbar for 9s, with detection by photodiode array detector set at 200 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5-200 µg mL(-1) (r(2)=0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79 µg mL(-1) and 2.5 µg mL(-1), respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p>0.05).

Use of collision induced dissociation mass spectrometry as a rapid technique for the identification of pharmacologically active peptides in pharmacopoeial testing.

The applicability of collision induced dissociation mass spectrometry (CID-MS) for the pharmacopoeial identification of pharmacologically active peptides was examined. Two different classes of related peptides were selected, i.e. four synthetic gonadotropin releasing hormone analogues (gonadorelin, goserelin, buserelin and leuprorelin) being either nona- or decapeptides, and human insulin and 2 insulin analogues (insulin lispro and insulin aspart). For all these substances the pharmacopoeial identification currently requires a combination of several partly rather laborious tests using sophisticated equipment. In contrast, CID-MS as a stand alone test can provide increased reassurance about the identity and is rapid and efficient. Moreover, the substance consumption for testing is significantly lower, which is a non-negligible factor for very expensive substances.

Evaluation of acetone as organic modifier in SPE for bioanalytical quantitative LC-MS/MS.

BACKGROUND: To assess the suitability of acetone as an alternative to acetonitrile in SPE under otherwise commonly used conditions, with a focus on selectivity with regard to the most abundant phospholipid class. Two representative analytes were included, leuprolide and tramadol, a peptide and a small molecule, respectively. RESULTS: The use of acetone resulted in analogous elution profiles of all monitored compounds in the majority of conditions. The only significant difference was on silica-based C18, where acetone effected markedly enhanced elution of lysophosphatidylcholines than did acetonitrile. Unmodified silica was shown to operate in per aqueous LC mode in highly aqueous conditions. CONCLUSION: Acetone was established as a selectively similar and slightly more eluotropic alternative to acetonitrile in bioanalytical SPE.

A novel sensitive electrochemical DNA biosensor for assaying of anticancer drug leuprolide and its adsorptive stripping voltammetric determination.

The anticancer drug, leuprolide (LPR) bound to double-stranded fish sperm DNA (dsDNA) which was immobilized onto the surface of an anodically activated pencil graphite electrode (PGE), was employed for designing a sensitive biosensor. The interaction of leuprolide (LPR) with double-stranded DNA (dsDNA) immobilized onto pencil graphite electrode (PGE) have been studied by electrochemical methods. The mechanism of the interaction was investigated and confirmed by differential pulse voltammetry using two different interaction methods; at the PGE surface and in the solution phase. The decrease in the guanine oxidation peak current was used as an indicator for the interaction in acetate buffer at pH 4.80. The response was optimized with respect to accumulation time, potential, drug concentration, and reproducibility for both interaction methods. The linear response was obtained in the range of 0.20-6.00 ppm LPR concentration with a detection limit of 0.06 ppm on DNA modified PGE and between 0.20 and 1.00 ppm concentration range with detection limit of 0.04 ppm for interaction in solution phase method. LPR showed an irreversible oxidation behavior at all investigated pH values on a bare PGE. Differential pulse adsorptive stripping (AdSDPV) voltammetric method was developed for the determination of LPR. Under these conditions, the current showed a linear dependence with concentration within a range of 0.005-0.20 ppm with a detection limit of 0.0014 ppm. Each determination method was fully validated and applied for the analysis of LPR in its pharmaceutical dosage form.

LC determination of leuprolide component amino acids in injectable solution by phanquinone pre-column derivatization labelling procedure.

A sensitive LC method for the determination of leuprolide acetate component amino acids in injectable solution with fluorogenic pre-column derivatization has been developed. The derivatization reaction with phanquinone was optimised by a series of experiments. Histidine, arginine, serine, tryptophan, glutamic acid, tyrosine, methionine, isoleucine, leucine and phenylalanine were separated on a reversed-phase ODS column using as eluent a binary mixture of triethylammonium phosphate buffer-methanol, under gradient elution conditions. The derivatives were eluted in 30 min with good reproducibility. The hydrolysis reaction of the peptide was carried out at reflux with 12 N hydrochloric acid for 2 h 30 min. The intra-day accuracy of the entire procedure (hydrolysis, derivatization, LC separation) ranged from 80.5 to 109.5% of the nominal concentration of leuprolide acetate and the precision (%R.S.D.) was less than 5.8%; the inter-day accuracy was in the range 81.5-107.2% and corresponding R.S.D. values were less than 4.6%. The detection limits (signal-to-noise ratio=3) for the adducts are 30-800 fmol.

Direct determination of the peptide content in microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A quantitative determination of peptides incorporated into poly(d,l-lactide-co-glycolide) microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was accomplished in a single step without pretreatment for extracting the peptide from the microsphere. The conventional extraction methods often underestimate the actual amount of peptide because of incomplete extraction from the microspheres or loss during the procedures. In this study, the microspheres dissolved in acetonitrile containing 0.1% trifluoroacetic acid were mixed with matrix solution containing the internal standard, and the peptide content was directly determined by MALDI-TOF MS. The drug content values determined by MALDI-TOF MS in both the leuprolide- and salmon calcitonin-incorporated microspheres were closer to the theoretical contents than those determined by the conventional extraction method. This method using MALDI-TOF MS could be a good alternative to time-consuming and less-accurate conventional methods.