Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity and inhibition by LC-MS/MS method.

In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-hydroxykynurenine is removed by a specific transaminase (3-hydroxykynurenine transaminase, 3-HKT) which converts the compound into the more stable xanthurenic acid. Interfering with 3-hydroxykynurenine metabolism in A. gambiae is a potential approach for the development of transmission-blocking drugs and insecticides. Hence, the aims of this work were to develop and validate a new LC-MS/MS method for the evaluation of A. gambiae 3-hydroxykynurenine transaminase (Ag-HKT) activity and the determination of the potency of inhibitors of the enzyme. We set up a LC-MS/MS based enzymatic assay for the determination of kinetic constants values of the recombinant Ag-HKT enzyme and for the evaluation of Ag-HKT inhibition by a known protein inhibitor used as reference and a newly synthesized compound. The chromatographic separation was performed in a gradient mode on a Phenomenex Synergi Polar-RP (150 mm × 2.0, 4 µm) with methanol and water containing both 0.2% formic acid. Mass spectrometric detection was achieved with an ion trap equipped with an ESI source, in positive ionization scan, operating in SRM mode. The LC-MS/MS method was validated in terms of selectivity, linearity, precision and accuracy.

Troxerutin, a mixture of O-hydroxyethyl derivatives of the natural flavonoid rutin: Chemical stability and analytical aspects.

Troxerutin (TRX) is a mixture of semisynthetic hydroxyethylrutosides (Hers) arising from hydroxyethylation of rutin, a natural occurring flavonoid. TRX is commonly used for its anti-oxidant and anti-inflammatory properties in chronic venous insufficiency and other vascular disorders. In recent studies, the protective effects of TRX in Alzheimer's disease, colon carcinogenesis and hepatocellular carcinoma are emerged. However, the chemical stability of TRX has never been studied. Hence, the aims of the work were to study the TRX chemical stability through a forced degradation study and to develop and validate a new stability indicating LC-UV method for determination of TRX. In order to perform the study, TRX stability was tested in various stress conditions analysing the degradation samples by LC-MS. Three degradation products (DPs; D1, D2 and D3, 3',4',7-Tri-O-(ß-hydroxyethyl)quercetin, 3',4',5,7-Tetra-O-(ß-hydroxyethyl)quercetin and 3',4'-Di-O-(ß-hydroxyethyl)quercetin respectively) arising from degradation in acidic conditions were identified and synthesized: among them, D1 resulted the stability indicator for hydrolytic degradation. Furthermore, a stability-indicating LC-UV method for simultaneous determination of triHer (3',4',7-Tri-O-(ß-hydroxyethyl)rutin, the principal component of the mixture) and D1 was developed and validated. The LC-UV method consisted in a gradient elution on a Phenomenex Kinetex EVO C18 (150 × 3 mm, 5 µm) with acetonitrile and ammonium bicarbonate buffer (10 mM, pH 9.2). The method was linear for triHer (20-60 µg mL-1) and D1 (5.1-35 µg mL-1). The intraday and interday precision were determined and expressed as RSDs: all the values were ≤ 2% for both triHer and D1. The method demonstrated also to be accurate and robust and the average recoveries were 98.8 and 97.9% for triHer and D1, respectively. Moreover, the method resulted selective and specific for all of the components present in the degradation pattern of TRX (diHer (3',4'-Di-O-(ß-hydroxyethyl)rutin), triHer, tetraHer (3',4',5,7-Tetra-O-(ß-hydroxyethyl)rutin), D3, D1 and D2) and it was successfully applied for the stability studies of both drug substances and drug products.

New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone.

1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography-tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M1-3) demonstrating its lack of antimuscarinic activity. 5. Despite the presence of the α,ß-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Development and validation of a stability-indicating HPLC-UV method for the determination of Thiocolchicoside and its degradation products.

A stability indicating high performance liquid chromatography method has been developed for the determination of thiocolchicoside (TCC) and its main degradation products thiocolchicoside S-oxide (D1SO) and 3-O-demethylthiocolchicine (D3) in liquid and solid formulations. The method was developed based on a previous forced degradation study showing that TCC underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation being the main degradation products D3 and D1SO respectively. The analytes separation and quantification were achieved on a Synergi™ 4µm Polar-RP 80Å, column 150×4.6mm (Phenomenex) using the mobile phase constituted (flow rate 1mLmin-1) of eluant A: 20mM sodium acetate buffer (pH 5.0) and eluant B: MeOH:CH3CN (20:80); the elution was performed in gradient mode detecting the analytes at 254nm. The method showed linearity for TCC assay in the 5-15µgmL-1, range and for unknown (TCCfu) and known (D1SO and D3) degradation products assay, in the 0.5-10µgmL-1 range: all the square of the correlation coefficients were greater than 0.999. The precision, determined in terms of intra-day and inter-day were expressed as RSDs and resulted to be 1.19, 1.10, 1.37 and 1.04% and 0.95, 0.83, 1.30 and 0.72 for TCC, TCCfu, D1SO and D3, respectively. The method demonstrated also to be accurate; indeed, the average recoveries were 102.1/102.0% for TCC (ampoules and hard capsules respectively), 101.3/100.3% for TCCfu, 101.7/100.2% for D1SO, and 101.4/101.4% for D3. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated by analysis of commercial liquid and solid dosage forms.

New insights in oxybutynin chemical stability: Identification in transdermal patches of a new impurity arising from oxybutynin N-oxide rearrangement.

Oxybutynin hydrochloride (Oxy), the first choice drug used for the management of urinary incontinence, is available in different types of formulations. However, due to its better lipophylicity and permeability, Oxyfree base was used in the new topical formulations such as transdermal patch and gel. The presence of an unprecedented impurity (Oxy-EK) in transdermal patches led to reinvestigate the chemical stability of Oxyfree base in oxidative conditions assigning, to Oxy-EK, the structure of (3E)-4-(N,N-diethylamino)-2-oxo-3-buten-1-yl 1-cyclohexyl-1-phenylglycolate. Oxy-EK arises from the prototropic rearrangement of oxybutynin N-oxides leading to the formation of an enamino ketone function which shows a long-wavelength UV-absorption. The total synthesis of Oxy-EK was performed, allowing to propose it as the indicator of stability for oxidative degradation of Oxy free base in transdermal formulations. The presence in the structure of Oxy-EK of an α,ß-unsaturated carbonyl function, a potential Michael acceptor, suggested the need of evaluating its possible mutagenic power. Accordingly, the Ames test was performed: at nontoxic concentrations, Oxy-EK did not increase the number of revertant colonies in all strains tested both in the absence and presence of the exogenous metabolic activator S9.

Development and validation of a stability-indicating LC-UV method for the determination of pantethine and its degradation product based on a forced degradation study.

Pantethine (d-bis-(N-pantothenyl-ß-aminoethyl)-disulfide, PAN), the stable disulfide form of pantetheine, has beneficial effects in vascular diseases being able to decrease the hyperlipidaemia, moderate the platelet function and prevent the lipid peroxidation. Furthermore, recent studies suggested that PAN may be an effective therapeutic agent for cerebral malaria and, possibly, for neurodegenerative processes. Interestingly, in the literature, there were no data dealing with the chemical stability and the analytical aspects of PAN. Hence, in the present work the chemical stability of PAN was for the first time established through a forced degradation study followed by liquid chromatography tandem mass spectrometry investigation showing the formation of three degradation products of PAN (PD1, PD2 and POx) arising from hydrolytic, thermal and oxidative stresses. Based on these data a stability-indicating LC-UV method for simultaneous estimation of PAN, and its most relevant degradation product (PD1) was developed and validated; moreover the method allowed also the separation and the quantification of the preservative system, constituted by a paraben mixture. The method showed linearity for PAN (0.4-1.2mgmL(-1)), MHB, PHB (0.4-1.2µgmL(-1)) and PD1 (2.5-100µgmL(-1)); the precision, determined in terms of intra-day and inter-day precision, expressed as RSDs, were in the ranges 0.4-1.2 and 0.7-1.4, respectively. The method demonstrated to be accurate and robust; indeed the average recoveries were 100.2, 99.9, and 100.0% for PAN, MHB and PHB, respectively, and 99.9% for PD1. By applying small variations of the mobile phase composition, counter-ion concentration and pH the separation of analytes was not affected. Finally, the applicability of this method was evaluated analyzing the available commercial forms at release as well as during stability studies.