The Development of High-Throughput and High-Precision Analytical Methods for the Screening of Drugs of Abuse in Biological Matrices

Guthery, Bill (2009). The Development of High-Throughput and High-Precision Analytical Methods for the Screening of Drugs of Abuse in Biological Matrices. PhD thesis The Open University.



The qualitative determination of various opiates and benzodiazepines in human serum is described using a two-dimensional gas chromatograph (GC x GC) coupled to a time of flight mass spectrometer (TOFMS). Human serum was 'spiked' with known quantities of benzodiazepines and a 'street heroin' mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N-methyl-N-(tert-butyldimethyl)trifluoracetamide (MTBSTFA) to improve the chromatographic performance.

The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for flunitrazepam and its major metabolite 7-aminoflunitrazepam (7-amino- FN), in the concentration range 5- 1000 ng/ml. The limits of detection (LOD) and limits of quantitation (LOQ), calculated by repeat injections (x10) of the lowest standard, were 1.6 and 5.4 ng/ml (flunitrazepam); 2.5 and 8.5 ng/ml (7-amino-FN), respectively.

A qualitative analysis of hair samples provided by an external collaborator was performed and various drug types detected including opiates, cocaine, methadone and diazepam. The analysis also identified many of the minor components in street drugs which may help forensic scientists to identify the source of the drug.

We propose a method to extract and analyse the pseudo-endogenous drug, gamma-hydroxybutyric acid (GHB), in urine, by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS).

Urine samples (<1.62 μg/ml endogenous GHB) were 'spiked' with 10 μg/ml synthetic GHB, extracted using SPE and derivatised with BSTFA. We obtained δ13C values for 3 x reference standards (#1 mean -31.7 ‰; σn-1 0.15; n=3: #2 mean -34.0 ‰; σn-1 0.85; n=3: #3 mean -42.2 ‰; σn-1. 0.64; n=3).

Selective extraction of GHB by affinity chromatography was investigated as a means to improve the sensitivity. A method was developed to synthesise tetrahydro-5-oxo-3-furanyl acetic acid (furanyl acetic acid) which was identified to contain the GHB sub-structure. It was postulated that the reaction of this compound with the lysine residues of a protein would produce an antigen whereby antibodies, with specific binding sites to GHB, could be created.

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