High-Throughput Screening of Drugs of Abuse in Urine by Supported Liquid-Liquid Extraction and UHPLC Coupled to Tandem MS

A qualitative method, involving supported liquid-liquid extraction (SLE) and ultra high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS-MS), was developed for the rapid tentative identification of various drugs of abuse in urine. In this study, 28 drugs and metabolites were covered by the screening procedure. Before analysis, urine samples were extracted by SLE and good extraction recoveries were obtained for most investigated compounds. The UHPLC strategy was then selected for the rapid separation of amphetamines, cocaine, opiates and related compounds in urine. Using columns packed with sub-2µm particles, analysis time was reduced down to 2min, while maintaining acceptable performance. Finally, the detection was by tandem MS operating in the single reaction monitoring (SRM) mode. The most intense transition was selected for the different drugs and SRM dwell times set at 5ms, to maintain sufficient data points across the narrow UHPLC peaks. The tentative identification of the drugs of interest, including amphetamines, opiates and cocaine, was based on both, retention times and mass spectrometry information. With the proposed method, limits of detection were estimated at about 1ngmL−1 and the applicability was assessed by successfully analyzing several samples of drug abusers. Finally, this study demonstrates the potential of UHPLC coupled to tandem MS for the rapid screening of drugs of abuse in urin

Study of radiolysis mechanisms for a better understanding of drugs radiosterilization

In this work, the radiolysis mechanisms in solids as well as in liquid and frozen aqueous solutions have been studied. Liquid chromatography tandem mass spectrometry has evolved so that mass spectral information can now be used to determine the most probable structures of radiolysis products, even those present in traces amounts. Theoretical routes explaining the formation of radiolysis products can be deduced from their structures. The development of strategies to limit the degradation of the active pharmaceutical ingredient during irradiation of the drug requires a better knowledge of the radiolysis mechanisms responsible for the drug degradation. Metoclopramide, an antiemetic drug, has been selected as a model, due to the variety of its chemical bonds. In the solid state, radiation-induced degradation of the drug was very low (<0.1%) and only four radiolysis products were detected in traces. The "major" radiolysis product was formed after the loss of the chlorine by dissociative electron capture. For metoclopramide liquid aqueous solutions, the loss of the drug was important (~30% loss at 25 kGy) and several radiolysis products were detected. The majority of the degradation products were generated following the attacks of hydroxyl radicals and aqueous electrons. The loss of metoclopramide could be lowered up to acceptable levels (<10% loss) provided that radioprotective additives were added and the irradiation dose was limited to 15 kGy, which could be sufficient to reach the required SAL. The selected excipients were mannitol (which reacts mainly with the hydroxyl radical), nicotinamide and pyridoxine that react with both the aqueous electron and the hydroxyl radical. The irradiation of frozen aqueous solutions allowed to minimize the loss of active substance even for a 25 kGy dose. This approach seems to be the most promising method for terminal sterilization of aqueous solutions by ionizing radiations. The major radiolysis product was formed after the attack of the electron. Some of the radiolysis products detected were attributed to the attack of the hydroxyl radical, demonstrating the feasibility of a reaction between the hydroxyl radical from ice radiolysis and the solute. A comparison was performed with irradiated frozen solutions of metoprolol, which has been studied in liquid aqueous solutions (C. Slegers' thesis). Degradation of metoprolol when irradiated in frozen solutions was negligible.(FARM 3)--UCL, 200

Drugs radiosterilization : parameters influencing the selection of the irradiation dose

Radiosterilization is now being recognised as an attractive method for drug sterilization. The irradiation dose must be sufficient to reach sterility, tacking into account the sensistivity of microorganisms contaminating the porduct, but should not be too high in order to avoid excessive degradation of the pharmaceutical compound. This review summarizes the current guidelines concerning the selection of the irradiation dose and the effects of ionizing radiation on microorganisms as well as the mechanisms leading to their radioresistance. The radioresistance of microorganims as a function of the environmental conditions is also critically reviewe

Chemical analysis applied to the radiation sterilization of solid ketoprofen

The aim of this work is to investigate the feasibility of radiation sterilization of ketoprofen from a chemical point of view. Although irradiated ketoprofen has already been studied in the literature [Katusin-Razem et al., Radiat. Phys. Chem. 73 111-116 (2005)], new results, on the basis of electron spin resonance (ESR) measurements and the use of hyphenated techniques (GC-MS and LC-MS), are obtained. The ESR spectra of irradiated ketoprofen consists of four unresolved resonance peaks and the mean G-value of ketoprofen is found to be 4 +/- 0.9 nmoles/J, which is very small. HPLC-UV analyses indicate that no significant loss of ketoprofen is detected after irradiation. LC-MS-MS analyses show that the structures of the non-volatile final products are similar to ketoprofen. Benzaldehyde is detected in the irradiated samples after dynamic-extraction GC-MS. The analyses show that ketoprofen is radioresistant and therefore might be radiosterilized

LC-MS characterization of metoclopramide photolysis products

The aim of this study was to investigate the photodegradation of the antiemetic drug metoclopramide. Metoclopramide aqueous solutions were photoirradiated at 254 nm under argon atmosphere. Irradiated metoclopramide solutions were analyzed by high performance liquid chromatography–ion trap mass spectrometry in order to characterize photolysis products. Rapid decrease in metoclopramide purity, following first-order kinetic, was observed following irradiation. The structures of 18 photolysis products were tentatively identified based on their mass spectra and fragmentation. The main degradation mechanism was scission of the chlorine which could be followed by polymerization of the resulting products since dimeric and trimeric products were observed

Radiosterilization of drugs in aqueous solutions may be achieved by the use of radioprotective excipients

The aim of this study was to assess the feasibility of radiosterilization of drugs aqueous solutions and to evaluate the effects of some additives, such as mannitol, nicotinamide and pyridoxine, which might protect the drug from degradation. Metoclopramide was selected as a model drug. The structures of the degradation products were determined to gain insight on the radiolysis mechanisms in aqueous solution in order to design strategies to lower the drug degradation. Metoclopramide hydrochloride aqueous solutions with and without excipients were irradiated either with gamma rays or high-energy electrons. HPLC-DAD was used to measure the loss of chemical potency and to quantify the degradation products which were also characterized by LC–APCI–MS-MS. Metoclopramide recovery for gamma and electron beam-irradiated solutions containing either mannitol, pyridoxine or nicotinamide meets the pharmacopoeial specifications for metoclopramide content up to a 15 kGy irradiation so that metoclopramide solutions containing these excipients might be radiosterilized at 15 kGy either with gamma rays or high-energy electrons. Structures are proposed for the majority of radiolysis products. Similar radiolysis products were detected for gamma and electron beam irradiations but the chromatographic profiles were different (differences in the distribution of radiolysis products)

Cryo-irradiation as a terminal method for the sterilization of drug aqueous solutions

The aim of this study is to evaluate the specificities of the irradiation of drugs in frozen aqueous solution. The structures of the degradation products were determined to gain insight into the radiolysis mechanisms occurring in frozen aqueous solutions. Metoclopramide hydrochloride and metoprolol tartrate were chosen as models. The frozen solutions were irradiated at dry ice temperature by high energy electrons at various doses. The drug purity (chemical potency) and the radiolysis products were quantified by HPLC-DAD. Characterization of the degradation products was performed by LC-APCI-MS-MS. The structures of the radiolysis products detected in irradiated frozen aqueous solutions were compared to those detected in solid-state and aqueous solutions (previous studies). For both metoclopramide and metoprolol, solute loss upon irradiation of frozen aqueous solutions was negligible. Five radiolysis products present in traces were identified in irradiated metoclopramide frozen solutions. Three of them were previously identified in solid-state irradiated metoclopramide crystals. The two others were formed following reactions with the hydroxyl radical (indirect effect). Only one fragmentation product was observed in irradiated metoprolol frozen solutions. For both drugs, radiosterilization of frozen solutions, even at high doses (25kGy), was found to be possible

Irradiation of human insulin in aqueous solution, first step towards radiosterilization.

The degradation of irradiated human insulin in aqueous solutions was investigated in order to protect the protein against ionizing radiation. The influence of the drug concentration, excipients and irradiation temperature were studied. Aqueous solutions at pH 2 were irradiated by gamma rays or by accelerated electrons. Two different high-performance liquid chromatography (HPLC) methods were used: reverse-phase high-performance liquid chromatography (RP-HPLC)/UV and size exclusion liquid chromatography (SEC/UV) to investigate both the fragmentation and the formation of higher molecular weight proteins. In solution without excipients irradiated at ambient temperature at 10 kGy, the loss of human insulin is almost complete. Addition of radio-protecting excipients (free radicals scavengers) and cryo-irradiation allowed to decrease insulin degradation. The best radio-protector used was ascorbic acid in aqueous solution and oxidized glutathione in the frozen solutions. Only the combination of these two approaches (addition of scavenger and freezing) enables the irradiated human insulin in aqueous solution to meet the European Pharmacopoeia requirements for chemical potency (>or=90%)

Chemical analysis of solid-state irradiated human insulin.

PURPOSE: To study the chemical modifications induced upon irradiation of solid human insulin at radiosterilization doses and investigate the influence of the absorbed dose on radiolysis. MATERIALS AND METHODS: Volatile radiolytic products were monitored by gas chromatography coupled with mass spectrometry (GC-MS) and non-volatile products by two different high performance liquid chromatography (HPLC) methods: the formation of higher molecular weight proteins was assessed by size exclusion liquid chromatography whereas assays for related compounds and chemical potency tests were carried out using reverse-phase HPLC-UV. Conformational changes were investigated by measurements of circular dichroism. RESULTS: After gamma irradiation at 10 kGy, the recovery of insulin was 96.8%; higher molecular weight proteins accounted for 0.35% (relative peak area) and other related compounds (including A21 desamido insulin) represented 1.29%. No major structural changes and no volatile radiolytic compounds were detected. CONCLUSION: Human insulin samples irradiated in the solid-state at 10 kGy (gamma rays) and 14 kGy (electron-beam) meet the European Pharmacopoeia requirements and can be considered as quite stable towards radiation from a chemical analysis viewpoint