ABSTRACT
A new procedure using high-performance liquid chromatography (HPLC) with ultraviolet detection to assay hydroxyurea (HU) levels in plasma has been developed. The drug was isolated from plasma by a direct deproteinization process with sulfosalicylic acid. Following neutralization of the acidic supernatant, an aliquot was loaded onto an Aminex HPX-72S column (300x7.8 mm). Chromatography was performed at 55 degrees C using a mobile phase consisting of acetonitrile-0.025 M ammonium sulfate buffer (pH 8.5) including 0.1% triethylamine, 0.01 M sodium sulfate, and 5 mM sodium heptane sulfonate. The UV absorbance of effluent was monitored at 214 nm. A flow-rate of 0.8 ml/min was used for analyzing HU in both human and mouse plasma. Under these conditions, the drug eluted at 12.6 min. The assay possessed linearity up to 425 microg/ml, with a lower limit of quantitation of 3.32+/-0.0004 microg/ml (mean+/-S.D., n=10). Intra-day and inter-day coefficients of variation were less than 8.5% and 8.7% respectively. Absolute differences were less than 7.4%. The method has been employed in clinical studies and the sensitivity of the assay was shown to be adequate for characterizing the plasma pharmacokinetics of HU in mice. In conclusion, the procedure described herein could be ideally suited for therapeutic monitoring of hydroxyurea.
Subject(s)
Antisickling Agents/blood , Chromatography, High Pressure Liquid/methods , Hydroxyurea/blood , Animals , Antisickling Agents/pharmacokinetics , Humans , Hydroxyurea/pharmacokinetics , Mice , Mice, Inbred ICR , Reproducibility of Results , Sensitivity and Specificity , Ultraviolet RaysABSTRACT
Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.
Subject(s)
Anemia, Sickle Cell , Disease Models, Animal , Mice, Transgenic , Animals , Hemoglobin, Sickle/genetics , Humans , MiceABSTRACT
The convenience of dried blood filter paper specimens for genetic screening programs has prompted us to test the stability of these specimens for hemoglobin identification by cation exchange high performance liquid chromatography. This report shows that identification of Hb AA, Hb AF, Hb AS, Hb FAS, Hb AJ, Hb FJ, Hb EF, and Hb SS can be achieved by high performance liquid chromatography even after six weeks of storage at room temperature. Also, accurate hemoglobin quantitation can be obtained from the same samples within three weeks of storage at room temperature. The combination of dried blood samples and high performance liquid chromatography provides an accurate system to screen for hemoglobinopathies, even after long periods of sample storage at ambient conditions.
