MSACL 2019 EU Abstract

Confirmation Method for the Determination of Drugs of Abuse in Saliva: Validation of a RUO Kit by LC-MS/MS, Ready for Use

Ugo de Grazia (1), Giuseppe Maria Merone (2), Elisa Bassotti (3), Annachiara D'Urso (1), Fabio Savini (4)
(1) Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy, (2) University of Chieti–Pescara “G. d’Annunzio”, Chieti, Italy, (3) R&D Department Eureka Lab Division, Chiaravalle, Italy, (4) Hospital “Santo Spirito”, Pescara, Italy

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Ugo de Grazia (Presenter) Fondazione IRCCS Istituto Neurologico Carlo Besta >> POSTER (PDF)

Presenter Bio: My career starts in 1991 with a Degree in Biol Sci at the University of Rome. Then I get Ph.D. in Experimental Medicine and then specialty in Clinical pathology. At that time my focus was on molecular biology in neuroimmunology and neuroncology. In the late 90s my interest switched on molecular biology of rare diseases. In 2001 I reach Milano to work in Clinical Pathology laboratory of Fondazione Carlo Besta. During this period I began to work with chromatography and antiepileptic drugs and I find this field particularly stimulating. At present I’m still in the same lab and I’m responsible for Clinical pharmacology service dealing either with diagnostic routine and research on epilepsy management and therapeutic drug monitoring. My present interest are bioanalytical method development and use of microsampling in TDM/DOA.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Road checks carried out by the police contemplate the collection of a salivary sample, which is analyzed with first level screening methods. These semi-quantitative results must be confirmed with confirmatory techniques, such as liquid chromatography combined with mass spectrometry. The aim of this study was is to validate a method for the quantitative analysis of the major substances of abuse on a salivary sample with LC-MS / MS systems.

METHODS: The developed and validated method allows the quantification of 17 drugs of abuse (DOA): Cocaine, Benzoylecgonine, Cocaethylene, THC, Buprenorphine, 6-monoacetylmorphine, Morphine, Codeine, Methadone, EDDP, Amphetamine, Methamphetamine, MDMA, MDA, MDE, Ketamine, MBDB. The analytical procedure consists of a dilution of saliva sample with 15 internal standards (drug of abuse labelled with stable isotopes). After the centrifugation, the supernatant is directly injected in LC-MS/MS system. Multiparametric and multi levels calibrators (six levels) and controls (two levels) are supplied with the kit to ensure a stable calibration curve in the suitable range of interest for each molecule.

RESULTS: An LC–MS/MS method for determination of 17 different drugs of abuse, in saliva samples, was successfully developed and validated according to FDA guidelines for bioanalytical method validation. The method was considered to be selective: analyzing 6 blank samples there were no unexpected endogenous interferences >20% of LLOQ for all drugs, nor >5% of the IS signals. Recovery from collection devices was comprised between 91 and 110%. No matrix effect was found. The method was linear for all drugs with R2 ≥0.996 in all cases. Intra-assay (CVs variable from 5.67 to 6.85%) and inter-assay (CVs variable from 5.84 to 7.25%) reproducibility analyses demonstrated accuracy and precision within acceptance criteria. Carry-over and interferences were negligible. Lower limits of quantitation are below the limits defined by international guidelines (European Guidelines for Workplace in Oral Fluid, Version 2.0, by European Workplace Drug Testing Society, 2015) and Italian national laws. Samples were tested for stability and resulted stable at least six month at -20°C and one week at +4°C.

DISCUSSION: The research use only (RUO) version of this new kit (from Eureka Lab Division) for the determination of drugs of abuse in saliva has been successfully validated according to FDA guidelines in a collaborative study and is now becoming applied to real samples. This kit, thanks to its easy of use and its performances in terms of accuracy and precision could become a reference standard for the laboratories involved in toxicology and confirmation tests for drug of abuse.