Sample preparation is one of the most important steps in the majority of bioanalytical procedures to determine trace psychoactive substances in biological samples with complex matrices. An ideal biological sample preparation technique should be simple, inexpensive, efficient, selective, and compatible with various bioanalytical techniques [1]. It should give a recovery as high as possible, use the minimum amount of solvent, and be environmentally friendly. Therefore, researchers are making great efforts to develop rapid, accurate, precise, and sensitive bioanalytical methods for the determination of psychoactive substances and their metabolites in real biological samples [1-3].
Therefore, the main aim of my study was the development of a new sensitive and specific bioanalytical method based on fast gas chromatography with negative-ion chemical ionization mass spectrometry (GC/NICI-MS) using a mixed-mode solid phase extraction for the identification and quantification of psychoactive substances in biological samples. Moreover, the speed of the analytical separation was emphasized by modifying various GC/NICI-MS parameters. The fully validated bioanalytical method was applied for the identification and quantification of several psychoactive substances in real whole blood and urine samples.
The proposed GC/NICI-MS method coupled with a mixed-mode solid phase extraction, and derivatization by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide:acetonitrile:ethyl acetate mixture (20:40:40 (v/v/v)) was shown to be useful for the analysis of psychoactive substances in biological samples. For that purpose, the derivatization step using different silylation reagents, duration, and temperature was investigated. This method is the fastest among the others reported up to now [2,3]. Under the optimized GC conditions derivatives of analytes were completely separated within 3.9 min. Sensitive and specific NICI-MS detection combined with fast GC resulted in a sharp and symmetric peak shape of the target analyte while maintaining sufficient resolution.
One of the greatest challenges with a residue analysis of psychoactive substances is the selection of a sorbent suitable to achieve an acceptable sensitivity to all compounds characterized by different physicochemical properties. In this research, a mixed-mode polymeric sorbent with ion-exchange properties was applied. This sorbent is capable of hydrophilic-lipophilic and ion-exchange interactions [2,4,5], and therefore it is suitable for achieving an efficient extraction of all herein-investigated analytes by a single extraction step. For that reason, biological sample preparation conditions including a selection of the solvent for washing and elution steps, pH values were optimized. To the best of my knowledge, this method has been used for the first time for the optimization of sample preparation at pH 1.0.
The developed bioanalytical method for the determination of psychoactive substances in biological samples was validated following the recommendations for new methods [2,6,7]. The linear relationships with the correlation coefficients (r2) better than 0.996 were evaluated. It was determined that extraction efficiency ranged from 82.9 (±6.2) % to 94.6 (±3.4) %. The precision (RSD) for psychoactive substances was 4.08 - 9.52 %, while the accuracy was in the range of 93.0 - 106.3 %. The developed method provides significant advantages in comparison with other previously published methods [7-9]. It shows higher sensitivity (the limit of detection ≤ 0.62 ng mL-1) in biological samples. Moreover, this bioanalytical method has several advantages: elimination of interferences, low volume of samples (0.2 mL), a multi-residue analysis, and very fast chromatographic separation of analytes (3.9 min). According to the results, the developed bioanalytical method is accurate, precise, effective, selective, sensitive, and specific enough to detect analytes after a long time of use of a single oral administration of some psychoactive substances. Furthermore, this method enables reaching the highest specificity for major analytes and meets the requirements of good laboratory practice, especially when applied to pharmacodynamic investigations. Ultimately, the developed bioanalytical method has been applied in routine toxicological analysis during the investigation of both clinical and forensic cases.