For the quantification of pharmaceuticals and their metabolites in biological matrices, mass spectrometric detection using selected reaction monitoring (SRM) mode has become essential for developing sensitive and selective assays. Ultra high pressure liquid chromatographic (UHPLC) separation allows reducing the analysis time while maintaining very good separation efficiency. However, for multiple analytes SRM mode acquisition with short dwell time (≤ 10 msec) is mandatory to maintain good peak shapes and accurate and precise quantitative values.

To allow high sample throughput while maintaining good chromatographic performance we developed a parallel UHPLC system with two low pressure gradient pumps combined to a triple quadrupole mass spectrometry  for the  quantitative analysis of eight HIV protease inhibitors using tube based dried spot sample collection format. This sample collection format has several advantages over card format : 1) sample collection and sample preparation is performed with the same device, 2) larger sample volumes can be collected.  Under ambient conditions the sample drying time is about 2 hours.  Using microwave heating this time could be shortened to only 5 minutes without any analyte degradation. The assay was validated for human plasma and blood spiked with HIV protease inhibitors in the range 25-20,000 ng/mL. The LLOQ was found to be 25 ng/mL and 50 ng/ml for all compounds in plasma and blood respectively. Analysis of clinical samples was also demonstrated. The parallel UHPLC system allowed reducing the analysis time from 6 to 4 minutes and therefore sample throughput was improved significantly without any compromising analytical performance. The assay was applied to analyze clinical plasma samples using a QUAL/QUAN approach with SRM mode for quantification and product ion scan mode with fast scan speed (15000 u/sec) for confirmatory analysis.