Overview of accessibility and quality of antiepileptic drugs in Madagascar.

PURPOSE: To determine the accessibility of treatment and the quality of antiepileptic drugs (AEDs) in the Haute Matsiatra district of Madagascar. METHODS: Cross-sectional descriptive study and interviews. Samples of 10 units of each available AED were collected, and the active ingredient was quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode-array UV detection. The quality of an AED was considered satisfactory if the quantity of active ingredient in each tablet was in the range ±15% of the average value according to the European Pharmacopeia (6th edition, 2008). RESULTS: The area was well served with health infrastructure but rescue facilities were poorly distributed. Available AEDs were all first-generation, and 73% were generic formulations. People with epilepsy (PWE) surveyed consulted traditional healers and most were treated with plants. PWE did not consider themselves sick but believed they were "possessed"; they consulted a doctor only immediately after a seizure, following the advice of traditional healers. The most prescribed AED was phenobarbital, costing between 0.03 and 0.12 US Dollar (US$) per 100mg. The purchase of full treatment was difficult for 77% of PWE and as a result, 39% took nothing. The quality of AEDs were considered unsatisfactory in 2.8% of cases. CONCLUSION: The AEDs collected in Haute Matsiatra were globally of good quality. The main limiting elements were a lack of knowledge among PWE that epilepsy is a disease, and the cost of traditional treatments.

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: a review.

Endogenous ribonucleotides and deoxyribonucleotides play a crucial role in cell function. The determination of their levels is of fundamental interest in numerous applications such as energy metabolism, biochemical processes, or in understanding the mechanism of nucleoside analog compounds. Nucleoside analogs are widely used in anticancer therapy. Their mechanisms of action are related to their structural similarity with natural nucleotides. Numerous assays have been described for the determination of endogenous nucleotides or anticancer nucleotide analogs in different matrices such as cellular cultures, tissue or peripheral blood mononuclear cells. The determination of these compounds is challenging due to the large difference of concentrations between ribonucleotides and deoxyribonucleotides, the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules due to the phosphate moiety. The extraction was generally performed at low temperature and was based on protein precipitation using acid or solvent mixture. This first phase could be coupled with extraction or cleaning step of the supernatant. Liquid chromatography coupled with UV detection and based on ion-exchange chromatography using non-volatile high salt concentrations was largely described for the quantification of nucleotides. However, the development of LC-MS and LC-MS/MS during the last ten years has constituted a sensitive and specific tool. In this case, analytical column was mostly constituted by graphite or C18 stationary phase. Mobile phase was usually based on a mixture of ammonium buffer and acetonitrile and in several assays included a volatile ion-pairing agent. Mass spectrometry detection was performed either with positive or negative electrospray mode according to compounds and mobile phase components. The purpose of the current review is to provide an overview of the most recent chromatographic assays (over the past ten years) developed for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy. We focused on sample preparation, chromatographic separation and quantitative considerations.

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry.

In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography-electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10pmol injected according to deoxyribonucleotides and from 12.5 to 3000pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3-107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS.

To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.

Polysaccharides as a marker for detection of corn sugar syrup addition in honey.

Honey is a natural product of high quality. However, because of its limited production and of its relatively high price, some beekeepers or unscrupulous traders do not hesitate to modify and falsify this natural product in order to try to increase its market value. Then, these involved falsification practices, for example intentional addition of cheap sugar syrup to honeys, are sometimes difficult to detect. An effective and simple analytical method is proposed in order to detect adulteration in honey by analysis of polysaccharide profiles. Samples were previously treated with reversed-phase solid phase extraction first to remove monosaccharides and small oligosaccharides and second to concentrate simultaneously traces of polysaccharides. A chromatographic separation using anion exchange stationary phase and pulse amperometric detection was further performed. Polysaccharide fingerprints (degree of polymerization from 11 to 17) were shown to be present in laboratory doped samples, and not detectable or present at very low concentrations in the authentic honey samples. Application to acacia, mountain polyfloral and polyfloral honeys enabled readily the detection of fraud resulting from deliberate addition of 1% of corn syrup.