A novel approach for in-process monitoring and managing cross-contamination in a high-throughput high-performance liquid chromatography assay with tandem mass spectrometric detection

Abstract

Cross-contamination among wells of a high-throughput, high-density assay is a risk that cannot be detected or controlled by the performance of calibration standards and quality control samples. In the current practice, carryover and cross-contamination is detected only when analytes are detected in blank, zero, placebo, pre-dose samples, in a low standard or low quality control sample. There is no mechanism that allows bioanalytical scientists to determine if cross-contamination has occurred among other samples. As a result, erroneous results can be released to clients even though a batch meets the acceptance criteria. We tested a new approach that quantifies the cross-contamination of each sample and allows the scientist to make quality decisions with documentation. The approach will also detect carryover in over 90% of the wells. Briefly, two additional analytes were added as contamination markers. The markers were added to a multi-well plate alternatively creating a pattern of a checkerboard. The spiked multi-well plate was then used to perform the assay. If both markers were detected in a well, the sample was considered contaminated. The amount of the unexpected marker detected in a well measures the degree of contamination and may be used to make deactivation decisions. Depending on the relative impact of the contamination, a scientist can choose to tolerate the bias, reject the sample, reject the batch or raise the lower limit of quantitation for the batch. A guideline for rejection decisions is presented for discussion.