Development and validation of the simultaneous determination of artemisone, clofazimine and decoquinate with HPLC.

The aim of this study was to develop and validate a novel HPLC method for the simultaneous analysis of artemisone, clofazimine and decoquinate. Detection was obtained at two wavelengths; 284 nm (clofazimine) and 210 nm (artemisone and decoquinate). Gradient elution was used with mobile phase A (A) consisting of 0.005 M sodium octanesulphonic-acid (pH 3.5) and mobile phase B (B) of HPLC grade acetonitrile. The flow rate was set to 1.0 ml/min with (A) at 35% and (B) at 65% for 2 min, followed by a gradient shift of 10/90% ((A)/(B)) over a duration of 4 min. After 10 min, the initial gradient conditions were readjusted to 35/65% ((A)/(B)). Distinctive peaks were identified for clofazimine, artemisone and decoquinate, respectively. The proposed HPLC assay method was validated and found to be reliable, reproducible and accurate for simultaneous analysis of the three compounds.

Age and sex differences in brain gene expression in neonatal rats.

Gene expression in the central nervous system is highly region-specific. We tested the hypothesis that certain developmental biomarkers could be detected in the whole brain or in cortical, subcortical or cerebellar structures. Brain gene expressions of male and female rats at birth, 3 days, and 10 days of age were measured by microarray technique ( approximately 10 K genes; n=9/category). We found 53 significantly up-regulated and 8 down-regulated genes at 10 days of age, relative to birth and 3 days of age. The whole brain, however, showed no significant sex differences in gene expression patterns up to 10 days of age. Ten genes with the highest up-regulation, and 5 down-regulated genes were further confirmed by quantitative real-time PCR (Q-PCR), using the whole brain, cortices, subcortical structures, and cerebellum. The Q-PCR confirmed genes are known to be involved in neuronal differentiation, axonal myelination and growth, neurotransmission and glycolytic pathways. With a few exceptions, the expression levels of Q-PCR confirmed genes were significantly different in the whole brain, compared to other regions. In a separate study, we tested the potential utility of the Q-PCR confirmed genes, as whole brain biomarkers, after a six-hour exposure to hyperoxia (>98% oxygen breathing) in 10 days old rats. This relatively mild oxidative challenge created a 3.5-fold increase in the expression of T-cell receptor beta Variable 8.3b, known to have regulatory function during development. We suggest that genes displaying significant expression in the whole brain, regardless of their origin, could be used to screen normal brain development in neonatal rat models of experimental neurology.