Investigation and Implementation of Analyte Protectants in the Gas Chromatography–Tandem Mass Spectrometry Analysis of Benzodiazepines and Opioids in Whole Blood Extracts

ABSTRACT The determination of benzodiazepines and opioids in whole blood by gas chromatography (GC) remains challenging due to low analyte concentrations and matrix‐related effects that may compromise analytical response. In this study, the use of analyte protectants (APs) was investigated as a means of enhancing detector response in GC–tandem mass spectrometry (GC–MS/MS) analysis of selected benzodiazepines and opioids following Quick, Easy, Cheap, Effective, Rugged, and Safe‐based sample preparation. A series of high‐boiling compounds, including linear alcohols, amines, carboxylic acids, and polyethylene glycols (PEGs), were evaluated as APs capable of modulating the chromatographic response. Among the tested compounds, PEG‐400 and PEG‐600 provided the most consistent signal enhancement, increasing responses of benzodiazepines by 413%–475% and opioids by up to 628% under optimized conditions. Long‐chain amines showed the highest enhancement for selected opioids, reaching up to 1080%. The influence of key experimental parameters on APs efficiency was systematically assessed, demonstrating that acetonitrile as the extraction solvent, elevated injector temperatures (up to 325°C), and low initial oven temperatures favored signal enhancement. The developed GC–MS/MS procedure exhibited satisfactory analytical performance, including high linearity ( R 2 > 0.998), good precision (relative standard deviation <5%), and low limits of quantification. Application of the approach to authentic whole‐blood samples confirmed that signal enhancement can also be achieved in complex biological matrices, although its magnitude was reduced by the presence of residual matrix constituents. In particular, bromazepam exhibited a 593% increase in signal following the addition of PEG‐400. The results demonstrate that the implementation of APs can serve as a simple means of improving analytical response in GC–MS/MS analysis of benzodiazepines and opioids without modification of routine sample preparation procedures. The findings further contribute to the understanding of protectant‐mediated signal enhancement mechanisms in the analysis of biological samples.