Hemoglobin based blood volume estimation for the (semi-)quantification of methadone, opiates, cocaine and metabolites in Dried Blood Spots (DBS), deposited on non-standardized materials, via GC/MS-MS

The analysis of Dried Blood Spots (DBSs) has been challenging for the forensic toxicological casework in the past years. The broad range of advantages concerning the usage of DBS, including analyte stabilizing effects, reduced infection risks, simplified sampling and storage have led to many successful approaches to establish DBS analytics in routine analysis, but some issues remain. There are only few publications and studies dealing with the quantification of analytes in DBSs on non-standardized materials, which underlines the need for a suitable method. The unknown blood volume of real blood traces is the biggest challenge. Several methods for the blood volume determination have been developed, but have always been limited by the uncertainty of variable hematocrit (hct) concentrations in blood. The following work therefore addresses the analyte quantification of common drugs of abuse and their metabolites in DBSs on non-standardized carrier materials. Since these might be found at a scence of crime, there is a need for a simple determination method for the blood spot volume that allows a parallel analyte measurement and consequently the quantification of analytes.

In the first part of this work, a GC-MS/MS method for the (semi)-quantification of benzoylecgonine (BE), ecgoninemethylester (EME), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine, morphine and dihydrocodeine (DHC) has been validated successfully for DBSs on commonly available printer paper. Since a case-near sample treatment was aspired, DBSs were dried at ambient air without protection against environmental conditions. Validation was performed with respect to the guidelines of the GTFCh (Gesellschaft für Toxikologische und Forensische Chemie) by evaluating linearity, selectivity, accuracy, stability, extraction efficiency and the limits of quantification (LOQ) and detection (LOD). Hereby, long-term stability was evaluated after the samples were exposed to different storage temperatures (-20 °C, 4 °C, 20-22 °C ambient temperature (AT) and 40 °C). The method was also examined with regard to the influences of different extraction buffer amounts (1 mL, 2 mL or 3 mL), different spot volumes (25 µL, 200 µL or 500 µL) and the usage of hct-adjusted blood specimens (hct 34 % or 51 %) on the analyte quantification. The results indicated that the variability of data concerning the stability of analytes and the influences of the amount of blood volume and consequently the amount of extraction buffer were analyte specific. Nevertheless, the method was demonstrated to be suitable for the (semi-)quantification of the analytes in DBSs on printer paper.

In the second part, the applicability of the GC-MS/MS method was tested for DBSs deposited on other non-standardized materials, including cotton, elastane and cotton-elastane composite fabrics, carpet, tile and a wood panel. Selectivity, accuracy and extraction efficiency were investigated at a spot size of 500 µL. Again, the GC-MS/MS method was shown to be sufficient for the (semi-)quantification of most of the analytes on the various carrier materials, with the results indicating that the carrier material has an impact on the analyte quantification. The analytes revealed different reactions to the materials used which resulted in varying degrees of analytes losses.

In part three, a method for the blood volume estimation of a DBS was investigated in parallel to the first and second part. For this purpose the well-known cyanhemiglobin method (HiCN) was used, which enabled a photometric hemoglobin (hb) determination. The hb content was found to correlate with the used amount of blood, so that the initial blood spot volume could be estimated. A standardized correction factor (CF) and specific dilution factors (DF) for each spot could be calculated and applied to the samples of accuracy and extraction efficiency to assess the suitability of the HiCN method in connection with the analyte quantification method. Furthermore, blood samples with adjusted hct levels (hct 34 % and 51 %) were examined to assess whether the HiCN method is suitable to overcome the challenging ´hematocrit effect´. The application of the blood volume estimation to the data of analyte quantification revealed variable results. The different materials produced inconsistent data with respect to blood volume estimation. Nevertheless, the HiCN method was also successfully semi-validated with regard to GTFCh guidelines for blood samples and corresponding DBSs in a physiological hct range (36-50%). Although the suitability of the combination of the GC-MS/MS method and HiCN method was demonstrated for some analytes, the data showed a strong dependency on hct. It was observed that blood samples with highly increased or decreased hct have major impact on the assessment of quantification data.

In the last section of this work, 20 authentic blood samples from post-mortem cases containing relevant analyte concentrations of the investigated substances were used to prepare DBSs on printer paper, cotton-elastane fabric and tile. Analyte concentrations were analyzed in DBSs and liquid samples and compared to each other. HiCN method was also applied. Hereby, highly variable results were reported, indicating that various influences, including the post-mortem interval, putrefaction or post-mortem redistribution of drugs have substance specific impacts. Thus, it was concluded that the assessment of DBSs from post-mortem samples is delicate, as the circumstances of death cannot always be assessed precisely. With respect to the alternative matrix, the results indicated that the use of DBSs was not sufficient to completely cease degradation or other processes affecting the analyte quantification. Nevertheless, correct analyte (semi)-quantification could be performed for most of the selected analytes, including BE, methadone, codeine and morphine.


Citation style:
Could not load citation form.


Use and reproduction:
All rights reserved