Hydrolysis step optimisation

The use of beta-glucuronidase is well established in benzodiazepine urinalysis (De Jager and Bailey, 2011; Marin and McMillin, 2010; Bergstrand, Helander and Beck, 2016), the object of this experiment is determine if a lower volume of beta- glucuronidase can be used for this study compared to the usual FMS procedure. The current FMS benzodiazepine urine extraction uses 40 µL of beta- glucuronidase from Helix Pometia but also utilises 1 mL of urine sample and is extracted using SPE. As the method developed for this study would use 0.5 mL of urine sample and use a LLE, a reduced volume of beta-glucuronidase would be advantageous; it would be less expensive and give a cleaner extract. In order to test if the volume of beta-glucuronidase used could be decreased, three real positive urine samples were extracted, once using 20 µL beta-glucuronidase in duplicate and once using 40 µL beta-glucuronidase in duplicate giving a total of 12 sample tubes. The real urine samples were from the SDC cohort; results of the drug court study are detailed in chapter 5. The samples were chosen as the individual had admitted to taking diazepam on their questionnaire so were anticipated to give positive results. Figure 30 shows the process used to carry out this experiment.

89 Figure 30: Hydrolysis step optimisation LLE process

To the 12 sample tubes add 0.5 mL of sodium acetate buffer (pH 5)

Pipette 20 or 40 µL β-glucuronidase solution into each sample in duplicate

Incubate at 60°C for 3 hours

Add 1 mL pH 6 phosphate buffer to all tubes

Vortex mix all tubes

Add 1.75 mL tBME to each tube, mix tubes on mixer for ≥ 5 min

Centrifuge at 3000 rpm for 10 min

Transfer the top layer to clean, labelled 3.5 mL vials

Evaporate to dryness under nitrogen at ≤40°C

Reconstitute in 1000 µL 25:75 MeOH:dH2O

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4.4.2. Method validation experimental

The validation parameters were evaluated for the final LLE method detailed in section 4.5.2 and the final LC-MS/MS method detailed in section 4.3.4. The parameters evaluated were limit of detection, specificity, matrix effects, recovery and process efficiency, carryover and autosampler stability.

4.4.2.1. Limit of detection

The LOD study was carried out in the same way as described in section 3.4.4 of chapter 3. LOD for each analyte was evaluated. Extracted concentrations of 0.002, 0.004, 0.006 and 0.008 mg/L were tested in duplicate. The signal-to-noise ratio should be ≥3 for the analyte to be considered detectable. Retention time for each analyte should be consistent.

4.4.2.2. Specificity

In order to understand if the benzodiazepine drugs would create interference issues within the detection window of each drug within the urine method, a selection of benzodiazepines including all drugs contained in the method plus flunitrazepam, flurazepam, clozapine, midazolam, clobazam, prazepam, 7- aminoflunitrazepam and clonazepam were individually injected, as an unextracted 0.10 mg/L solution. Each drug was injected in triplicate.

4.4.2.3. Matrix effects, recovery and process efficiency

Matrix effects, recovery and process efficiency are described in section 3.4.6 of chapter 3.

For two concentrations investigated, ME, RE and PE were calculated using the post-extraction addition approach detailed by Matuszewski et al. (Matuszewski, Constanzer and Chavez-Eng, 2003)

Blank (drug free) urine from 10 different sources was used for the experiments. The analytes at two different concentrations (ME Low QC 0.015 and ME High QC 0.40 mg/L) were evaluated. The ME, RE and PE experiments were performed as follows:

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 Set A: neat unextracted analyte mix and internal standard in reconstitution solution was prepared ten times.

 Set B: blank urine from 10 different sources were spiked with the analyte mix and internal standard in reconstitution solution after LLE. Set B was prepared in duplicate.

 Set C: blank urine from 10 different sources were spiked with analyte mix and internal standard prior to LLE. Set C was prepared in duplicate.

4.4.2.4. Carryover

Carryover is described in section 3.4.7 of chapter 3.

A solution was prepared to give a concentration of 4 mg/L for all 22 analytes, this was extracted using the LLE method and injected in triplicate followed, by three injections of drug-free reconstitution solution. The concentration 4 mg/L was chosen, as this is ten times the high QC concentration and double the concentration of benzodiazepines used in the FMS in-house method.

4.4.2.5. Autosampler stability

Blank (drug free) urine was spiked using the AS stability solution at a high (0.150 mg/L) and the low (0.015 mg/L) concentration in triplicate and extracted using the LLE procedure detailed in section 4.5.1.3 These extracts were then run on the method detailed in section 4.3.4 to determine a time zero response (t0); the same extracts were then injected at four-hour intervals up to 36 hours. The laboratory was temperature controlled between 16°C and 24°C during the experiment.

4.4.3. Data handling and statistical analysis

Once results had been generated in the Analyst software, they were copied onto a Microsoft® Excel® (version 14.7.3) spreadsheet and batch checked by a second toxicologist before any calculations were performed. The results copied include the peak areas of the analyte and the internal standard, the calculated values of the calibration standards and QCs in mg/L and the datapath information. Microsoft® Excel® was then used to calculate the mean, standard deviation, %CV, %ME, %PE and %RE where necessary. A second toxicologist, as part of the batch checking, also checked the formula used in the spreadsheets for each calculation. Line graphs were generated in the spreadsheet to track autosampler stability. The Statsplus (AnalystSoft™, version 7.7.31) add on for Microsoft® Excel® was used

92 to conduct a paired two sample t-test to determine if there was a statistical difference between the volumes of tBME used. The spreadsheets were saved on a secure drive to protect the data.

4.5. Results and discussion