Development of a method for determining xenobiotics in the form of dried blood spots on cotton material and Whatman® 903 cards.

The aim of the study: The aim of this study was to develop and validate a quantitative analysis procedure for four benzodiazepines (alprazolam, diazepam, flunitrazepam, clonazepam) and one Z-drug (zolpidem), applied to cotton material and Whatman® 903 cards in the form of blood spots.

Material and methods: The extraction was conducted using a mixture of phosphate-carbonate buffer pH=6, acetonitrile, and methanol. The blood spots were analyzed using high-performance liquid chromatography coupled with triple quadrupole mass spectrometry.

Results: All calibration curves maintained linearity (R²>0.995) in the concentration ranges of 1-50 ng/ml on cotton material and Whatman ® 903 cards. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) for the analyzed substances were in the ranges of 0.04- 0.40 ng/ml (LOD) and 0.12-1.08 ng/ml (LOQ) isolated from Whatman® 903 cards, and 0.06-1.15 ng/ml (LOD) and 0.17-2.04 ng/ml (LOQ) isolated from cotton. Accuracy for concentrations of 5 ng/ml, 10 ng/ml, and 50 ng/ml was comparable in both groups, ranging from 76-101% for Whatman® 903 cards and 73-117% for cotton. No significant matrix effect was observed on the quantitative results of the analyzed benzodiazepines.

Conclusions: In the presented study, a qualitative and quantitative analysis procedure for four xenobiotics from the benzodiazepine group and zolpidem, applied as dried blood spots on cotton material and Whatman® 903 cards, was developed and validated. The extraction method presented focused on isolating xenobiotics from blood spots of known volume. Sample extraction using a simple liquid-liquid extraction (LLE) technique creates opportunities for applying the described method to real samples secured on cotton material or stored on Whatman® 903 cards.