Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange-high-performance liquid chromatography (AE-HPLC) method f or the quantification of adenovirus type 5 (Ad5) total particles was valida ted according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accu racy and recovery. The viral particles were detected by absorbance at 260 n m using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad 5 and lysate samples were used for the validation of the method. Relative s tandard deviations (RSDs) for the inter-day, intra-day precision and reprod ucibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specifi c for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the err or in prediction to be less than 10%, the working range was established bet ween 2 X 10(10) and 7 X 10(11) VP/ml with correlation coefficient of 0.9997 5, standard deviation of 6.14 X 10(9) VP/ml and a slope of 3.04 X 10(5) VP/ ml. The recovery of the method varied between 88 and 106% in all of the lys ate samples investigated which is statistically similar to 100% recovery at 95% confidence interval. (C) 2001 Elsevier Science B.V. All rights reserve d.