B-319 Drugs of Abuse: Correlation of Immunoassay Absorbance With Concentration By LC-MS/MS and False Negative Rates on the Roche Analyzer
Authors: Militello L , Gqamana P P , Zhang V 
Published: September 2023

Abstract
A common practice for drug abuse testing is to first screen using immunoassay (IA) then confirm using tandem mass spectrometry (LC-MS/MS) if the screening result is indeed positive. Immunoassays are qualitative and a positive result is based on an absorbance reading over a cutoff value specified by the manufacturer. Despite that, one method of investigating unexpected results is to check for an absorbance close to the cutoff value, as that may indicate an analyte concentration close to the limit of detection and the need for further investigation. Here we investigate the validity of this approach regarding potential false positives/false negatives by using quantitative LC-MS/MS assays. These explicitly model instrument response as a function of concentration and are expected to be more reliable than absorbance. This abstract aims to answer two questions: Is there a relationship between absorbance by IA and concentration of analyte as measured by LC-MS/MS that can be used in clinical interpretations? What is the false negative rate (FNR) by IA screening on the Roche automated analyzer? We collected discarded patient urine samples that were screened for amphetamines (AMP), cocaine (COC) and THC to get a total of 10 negative and 10 positive samples of each analyte with a range of absorbance values, as well as preliminary data for benzodiazepines, opiates, and oxycodone. All samples were tested again 5 times in one day using a Roche Cobas c501 chemistry analyzer. The cutoff concentrations for the immunoassays are 500 ng/mL for AMP, 300 ng/mL for COC and 50 ng/mL for THC. In-house LC-MS/MS assays gave quantitative measurements with a limit of quantitation of 30 ng/mL for AMP and COC and 10 ng/mL for THC. The results obtained from LC-MS/MS were considered as reference values to determine the FNR of the IAs. The linear regression between absorbance and concentration was calculated in Excel using the average absorbance of each sample. The amphetamine IA results showed a concordance with LC-MS/MS of 80%, a FN rate of 20%, and an R2 of 0.80 between absorbance and concentration. The cocaine IA results showed a concordance with LC-MS/MS of 65%, a FN rate of 41%, and an R2 of 0.24. The THC IA results showed a concordance with LC-MS/MS of 95%, a FN rate of 9%, and an R2 of 0.94. The amphetamine IA shows moderate agreement with LC-MS/MS and accurately detected 80% of the positive specimens. The absorbance and concentration show good fit to the regression model. The cocaine IA shows fair agreement with LC-MS/MS and has an LOD too high to detect the presence of drug in most specimens that were found positive by LC-MS/MS. The lack of fit to the regression model is due to specimens with very high concentrations of COC that do not cause a proportional increase in absorbance. The THC IA shows excellent agreement with LC-MS/MS and excellent fit to the regression model. These findings indicate the use of absorbance may be able to assist in test interpretation for specimens near the cutoff value.