Development and validation of a method to analyse the prodrug hydroxymethylnitrofurazone (NFOH) in human plasma by high-performa

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Serafim, Eliana Ometto Pavan (1*); Peccinini, Rosangela Gonçalves (2); Ribeiro, Maria Lúcia(3); Chung, Man Chin (1).

1  Lapdesf - Department of Drugs and Medicines - School of Pharmaceutical Sciences – University of São Paulo State – UNESP, Araraquara, SP, Brazil.

Chagas disease is still a serious Public Health problem and considered extremely neglected because it afflicts exclusively populations in developing countries. Benznidazol (Rochagan^q, Roche) is the only drug available in the Brazilian market. Among the potentially anti-Chagas compounds under study, the prodrug hydroxymethylnitrofural (NFOH) has shown lower toxicity and higher tripanomicide activity than nitrofural (NF, active compound) in all stages of development of the parasite. In 2006, preclinical toxicity studies showed that NFOH presented an LD₅₀ higher than 2000 mg in rats. This value is considered safe by the World Health Organization (WHO) and promising for continued testing. In this context, the purpose of this work was to investigate the in vitro stability of NFOH. To this end, an analytical methodology, not yet described in literature, was developed and validated for the determination of NF and NFOH in human plasma by LLE-HPLC-UV.  NF and NFOH were extracted from samples of human plasma at pH 11 with ethyl acetate.  The organic layer was filtered with anhydrous sodium sulfate and dried under nitrogen. The extracts were re-dissolved in sodium carbonate : acetonitrile (79:21) and analysed by HPLC, using: a C18 (250 mm x 4.6 mm I.D, 5µm) column, sodium acetate/acetic acid buffer at pH 4.6: acetonitrile (79:21) as mobile phase and detection at 365 nm. The response of the LLE-HPLC-UV method for NF and NFOH was linear over a dynamic range from 0.20-10.0 µg/mL, with correlation coefficients of NF: 0.9978, NFOH: 0.9942. The accuracy and precision of the extraction procedure was evaluated by means of the recovery of five replicates (inter-day) and ten replicates (intra-day) of fortified plasma samples. Mean recoveries for NF ranged from 86 to 98% on the same day and 84 to 98% intra days and coefficients of variation of ≤ 3.0% on the same day and  ≤ 4.0% inter- days.  For NFOH, the mean recoveries were 88 to 96% on the same day and 90 to 96% intra – days with coefficients of variation being ≤ 11.0% on the same day and ≤ 13.0% between days.The quantification limits of the method were: 0.19 and 0.12 µg/mL  for NF and  NFOH, respectively. The stability assays in frozen plasma indicated that NF remains stable for up to 22 days (limit of the test) and NFOH begins to convert on the second day. These results indicate that plasma samples with NFOH should be prepared on the day of the experiment.The efficiency of the methodology was demonstrated by the adequate validation of the parameters obtained for the quantification of NF and the prodrug NFOH, thus  permitting its application in pharmacokinetic studies.