24 February / March 2017
A Simplified Mixed-Mode Sample Preparation Strategy for the LC-MS/MS Analysis of Benzodiazepines and Z-Drugs for Forensic Toxicology
by Authors: Jonathan E. Turner, Jonathan P. Danaceau, and Erin E. Chambers
Despite many recent advances in LC-MS/MS for forensic toxicology, the baseline separation of benzodiazepines is often challenging. These challenges can be met by using a high efficiency solid-core column to resolve these chromatographic interferences. Combined with a simplified solid phase extraction (SPE) protocol, accurate and precise quantification of all analytes is achieved.
Introduction:
High-performance liquid chromatography- tandem mass spectrometry (LC-MS/ MS) is a powerful tool for quantitative analysis of drugs of abuse in the field of forensic toxicology. Many laboratories are transitioning to LC-MS/MS as they replace existing immunoassay and GC-MS based screening methods, and consolidate multiple single compound analyses into the analysis of complex compound panels. Benzodiazepines and ‘Z-drugs’ (zolpidem and zopiclone) are frequently prescribed drugs used for their sedative, anxiolytic, and hypnotic properties [1]. They work by potentiating the inhibitory neurotransmitter -amino butyric acid (GABA). Nationally, overdose deaths from benzodiazepines have risen 600% from under 1,600/year in 2001 to 8,000 in 2014, more than any other drug class with the exception of heroin [2].
While many laboratories have increased the use of LC-MS/MS for the analysis of benzodiazepines and other drugs of abuse in recent years, many published techniques still rely on labour- intensive liquid-liquid extraction techniques to prepare the samples .[3-5]. Some of the drawbacks of these techniques include the need to process individual samples one by one, and the use of toxic solvents. Another issue is the need to evaporate and reconstitute samples after extraction, although this can be alleviated by the use of supported liquid exchange (SLE) plates. This manuscript details an abbreviated and modified solid phase extraction (SPE)
0 1.06 1.00 1.12 0 1.00 1.50 1.61 0 1.00 1.50 2.00 3 0 2.00 1.91 0 1.00 1.50 2.00 5 0 1.00 1.50 2.00 6 0 1.00 1.50 2.00 7 0 1.00 1.50 2.00 2.50 2.34 2.50 Time0 2.50 3.00 2.31 2.50 2.35 2.50 4 2.50 100 0 2.50 100 0 2.50 100 0 2.50 3.00 3.00 3.50 13 3.50 14 3.50
4.00 4.00
4.07 4.00 Time 0 4.00 4.50 0 4.00 4.50 5.00 21 5.13 5.00 Time 4.00 3.86 3.00 3.50 12 4.00 3.84 3.85 100 0 4.00 2.50 3.00 11 2.50 1 0 1.50 2 0 2.50 3.00 3.50 3.76
3.47 3.69 3.50
3.77 0 4.00 4.50 4.44 4.50 4.47 20 19 5.00 5.00 4.00 10 4.00 0 4.00 2.00 2.50 2.50 9 0 3.00 2.90 16 4.00 3.50 4.00 2.53 8 0 4.00 4.24 4.50 4.35 17 4.50 4.41 18 5.00 5.00 4.50 4.35 5.00 4.09 15
Figure 1. Chromatograms of benzodiazepines analysed in this application. See Table 1 for compound key. Column: CORTECS UPLC C18
+ 1.6 µm, 2.1 x 100 mm.
method that can rapidly extract this panel of drugs and metabolites from urine samples. It will show the benefits of having sample preparation steps, which include enzymatic hydrolysis, performed within the wells of the µElution plates. It will further show the benefits of having a water-wettable SPE sorbent that can be used without conditioning and equilibration steps. Finally, the chromatographic separation performed using a solid-core charged-surface UHPLC column will be highlighted, which enabled the baseline separation of all target analytes from internal standards with identical nominal masses. The use of a high efficiency solid core column eliminates the risk of
chromatographic interference between the labelled internal standards and the native compounds.
EXPERIMENTAL:
All standards were obtained from Cerilliant (Round Rock, TX). Deuterated internal standards were used for all compounds with the exception of flurazepam. Stock solutions were prepared in methanol (MeOH). Working standards were prepared daily by diluting stock standards in 80:20 (v:v) water:MeOH. Calibrators and quality control (QC) samples were prepared in urine from working standards. High calibration
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