Determination of acyclovir in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

Abstract

Acyclovir {9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV} is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C 18 solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C 8 column (150×2.1 mm, 5 μm). The mobile phase used for the plasma and amniotic fluid was 10 m M acetate/citrate buffer–3.7 m M aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 m M acetate/citrate buffer with 5 m M octanesulfonic acid–acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25–100 μg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R 2>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.