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 for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility 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 specific 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 error in prediction to be less than 10%, the working range was established between 2 x 10(10) and 7 x 10(10) VP/ml with correlation coefficient of 0.99975, 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 lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.

Determination of the inorganic ion composition of rat airway surface fluid by capillary electrophoresis: direct sample injection to allow multiple analyses from nanoliter volumes.

The pulmonary airways are covered by a layer of airway surface fluid (ASF) which is typically < 30 microm in thickness. ASF composition is an important factor in the pathogenesis of several lung diseases including cystic fibrosis. Because of the very small volume of ASF, it is difficult to determine ASF composition, Particularly for inorganic ions, since sampling by lavage is not suitable. With nanoliter injected volumes, capillary electrophoresis (CE) is ideally suited to ASF analysis. We have developed a novel technique using separate sampling and injection capillaries whereby submicroliter volumes of ASF (typically approximately 100 nL) can be collected from airways and then analyzed by CE. Cations (Na+, K+, Ca2+, Mg2+) and anions (including Cl-) are quantitated (RSD < 10%) using indirect UV detection. In healthy rat lungs, ASF was found to be hypotonic, consistent with observations made in human airways. This technique has been developed using rats, which have not previously been studied because their small size prohibits the use of other sampling techniques.