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A LC–MS method was proposed for the analysis of 28 drugs of abuse in exhaled breath.Development of an analytical method which is sensitive, fully validated and applied on authentic sample analysis from drug abusers.Authentic sample analysis from the patients revealed the presence of drugs of abuse in exhaled breath.This study will also help to understand the prevalence of drugs of abuse in blood, oral fluid and exhaled breath.Exhaled breath carries aerosol micro-particles containing nonvolatile organic substances. Recently, the analysis of drugs of abuse (DOA) have become of interest in exhaled breath particles (EBP). In this study, a liquid chromatography – tandem mass spectrometry (LC–MS/MS) method was developed and validated to analyze 28 DOA in 30 L of EBP collected on a permeable polymer filter. After extraction, the chromatographic separation was achieved on a UPLC BEH phenyl column using a mobile phase consisting of methanol and water both containing 4 mmol/L ammonium formate and 0.05% ammonia. The column temperature was set at 50 °C and mobile phase flow rate 0.5 mL/min in gradient mode with a total run time of 5 min. The mass spectrometer was operated in positive electrospray ionization and selected reaction monitoring mode. Acquired limits of quantification were in the range of 1–66 pg/filter for all substances except DM-tramadol. Excellent linearity over the concentration range from LLOQs – 15 ng/filter with r2 values >0.99 and satisfactory recoveries (70–116% at 100 pg/filter) were achieved. During method application a total 26 samples were analyzed of which 24 were found to be positive for 13 analytes. The highest amount was found for methadone (56 ng/filter) and the lowest amount was found for the methadone metabolite EDDP (2 pg/filter) in two different samples.