Ruth Verplaetse

The evaluation of the applicability of a high pH mobile phase in ultrahigh performance liquid chromatography tandem mass spectrometry analysis of benzodiazepines and benzodiazepine-like hypnotics in urine and blood

A sensitive liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous detection of benzodiazepines, benzodiazepine-like hypnotics and some metabolites (7-aminoflunitrazepam, alprazolam, bromazepam, brotizolam, chlordiazepoxide, chlornordiazepam, clobazam, clonazepam, clotiazepam, cloxazolam, diazepam, ethylloflazepate, flunitrazepam, flurazepam, loprazolam, lorazepam, lormetazepam, midazolam, N-desmethylflunitrazepam, nitrazepam, N-methylclonazepam (internal standard), nordiazepam, oxazepam, prazepam, temazepam, tetrazepam, triazolam, zaleplon, zolpidem, zopiclone) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge. Electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher retention and higher electrospray ionization signals than the conventional low pH mobile phases. Considering the benefits of a high pH mobile phase on both chromatography and mass spectrometry, its use should be encouraged. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). The optimized method was fully validated.

Development and validation of a sensitive UPLC-MS/MS method for the analysis of narcotic analgesics in urine and whole blood in forensic context.

Narcotic analgesics are widely (ab) used and sometimes only occur in low concentrations in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry method was developed for simultaneous analysis of 9 narcotic analgesics and metabolites (buprenorphine, O-desmethyltramadol, fentanyl, norbuprenorphine, norfentanyl, pethidine, piritramide, tilidine and tramadol) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization with electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher electrospray ionization signals than the conventional low pH mobile phases. In the final method, gradient elution with 10mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). Selectivity, matrix effects, recovery, linearity, sensitivity, precision, accuracy and stability were validated in urine and whole blood. All parameters were successfully evaluated and the method showed very high sensitivity, which was the major aim of this study. The applicability of the method was demonstrated by analysis of several forensic cases involving narcotic analgesics.

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 microm, 2.1 mm x 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.

Development and validation of a new TD-GC/MS method and its applicability in the search for human and animal decomposition products.

Differentiation between human and animal remains by means of analysis of volatile compounds released during decomposition is impossible since no volatile marker(s) specific for human decomposition has been established today. Hence, the identification of such a marker for human decomposition would represent great progression for the discovery of buried cadavers by analytical techniques. Cadaver dogs can be trained more efficiently, the understanding of forensic entomology can be enhanced, and the development of a portable detection device may be within reach. This study describes the development and validation of a new analytical method that can be applied in the search of such (a) specific marker(s). Sampling of the volatile compounds released by decomposing animal and human remains was performed both in a laboratory environment and outdoors by adsorption on sorbent tubes. Different coatings and several sampling parameters were investigated. Next, the volatile compounds were analyzed and identified by a thermal desorber combined with gas chromatography coupled to mass spectrometry (TD-GC/MS). Different GC columns were tested. Finally, the analytical method was validated using a standard mixture of nine representative compounds.

Screening of Urine and Blood Using Limited Sample Preparation and Information Dependent Acquisition with LC-MS/MS as Alternative for Immunoassays in Forensic Toxicology

Screening of Urine and Blood Using Limited Sample Preparation and Information Dependent Acquisition with LC-MS/MS as Alternative for Immunoassays in Forensic Toxicology Immunoassays are widely used to perform an initial toxicological screening of biological samples. However, LC-MS/MS is described as a promising technique that can overcome the limitations of immunoassays (such as their lack of selectivity). The objective of this project was to implement a LC-MS/MS method for screening of forensic ante- and post-mortem urine and whole blood samples that can replace the immunoassays. Easy and rapid sample preparation techniqueswere evaluated. Protein precipitation with acetonitrile combined with aqueous dilution (dilution factor 5 for urine and 10 for blood) proved to be an effective procedure. On the LC-MS/MS, 1 scheduled multiple reaction monitoring transition for each of 414 compounds was analyzed in positive mode, followed by an enhanced product ion scan if the peak height exceeded a specified threshold.