Quantification of 31 illicit and medicinal drugs and metabolites in whole blood by fully automated solid-phase extraction and ultra-performance liquid chromatography–tandem mass spectrometry

Abstract

An efficient method for analyzing illegal and medicinal drugs in whole blood using fully automated sample preparation and short ultra-high-performance liquid chromatography-tandem mass spectrometry (MS/MS) run time is presented. A selection of 31 drugs, including amphetamines, cocaine, opioids, and benzodiazepines, was used. In order to increase the efficiency of routine analysis, a robotic system based on automated liquid handling and capable of handling all unit operation for sample preparation was built on a Freedom Evo 200 platform with several add-ons from Tecan and third-party vendors. Solid-phase extraction was performed using Strata X-C plates. Extraction time for 96 samples was less than 3 h. Chromatography was performed using an ACQUITY UPLC system (Waters Corporation, Milford, USA). Analytes were separated on a 100 mm × 2.1 mm, 1.7 μm Acquity UPLC CSH C(18) column using a 6.5 min 0.1 % ammonia (25 %) in water/0.1 % ammonia (25 %) in methanol gradient and quantified by MS/MS (Waters Quattro Premier XE) in multiple-reaction monitoring mode. Full validation, including linearity, precision and trueness, matrix effect, ion suppression/enhancement of co-eluting analytes, recovery, and specificity, was performed. The method was employed successfully in the laboratory and used for routine analysis of forensic material. In combination with tetrahydrocannabinol analysis, the method covered 96 % of cases involving driving under the influence of drugs. The manual labor involved in preparing blood samples, solvents, etc., was reduced to a half an hour per batch. The automated sample preparation setup also minimized human exposure to hazardous materials, provided highly improved ergonomics, and eliminated manual pipetting.

Nitrobenzodiazepines: Postmortem brain and blood reference concentrations

“…The pre-analytical variation was lower in brain than blood, corroborating the notion that brain tissue is a useful postmortem specimen and a suitable alternative to blood. Table 5: Validation data for brain tissue together with lower and upper limit of quantification (LLOQ; ULOQ) in both brain and blood, previously reported for blood in [38]. Figure 1: Correlation between brain and blood concentrations (mg/kg) of the sum of clonazepam and 7-aminoclonazepam (N=37) with a Spearman's rank correlation coefficient (r s ) of 0.96.…”

“…The published methods were validated in blood including limit of quantification, extraction recovery etc. for illicit and medicinal drugs including the drugs and metabolites investigated in the present article [37,38].…”

“…Later, blood samples (N=10) were applied to the sample preparation used for brain tissue, which was performed by a fully automated solid phase extraction setup with 96-well plates according to a previously published method developed for blood [38]. Brain homogenates (100 mg) were initially diluted manually with water 1:1.…”

“…Validation data are listed in table 5 together with lower and upper limits of quantification (LLOQ, ULOQ) previously reported for each compound in femoral blood [38]. For a subset of samples, matrix effect and extraction recovery were separately determined (N=4).…”

Comparison of conventional liquid chromatography–tandem mass spectrometry versus microflow liquid chromatography–tandem mass spectrometry within the framework of full method validation for simultaneous quantification of 40 antidepressants and neuroleptics in whole blood

“…These procedures allow faster and more effective analyses e.g. if only a limited amount of sample is available and the number of different analytes is unknown in the beginning of a typical analysis in clinical and forensic toxicology [6][7][8][9][10][11][12][13][14]. Antidepressant and neuroleptic drugs are widely used in the treatment of psychiatric diseases such as depression, mood and anxiety disorders, and schizophrenia.…”