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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine

High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine
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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 1

Bill Yu, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, San Jose, CA Key Words Opiates, benzodiazepines, Prelude SPLC Goal Develop a high-throughput, low solvent consumption, easy-to-run method for quantitative forensic analysis of six opiates and fourteen benzodiazepines in urine. Introduction Analyses of opiate and benzodiazepine panels are some of the highest-volume applications in forensic toxicology labs. In order to meet the need for high throughput, a fast, simple, and cost-effective method was developed, consisting of hydrolysis, simple urine dilution, separation by liquid chromatography (LC), and analysis by mass spectrometry (MS). The method incorporated the Thermo Scientific™ Prelude SPLC™ system (Figure 1), which features two independent channels of sample preparation and liquid chromatography (SPLC). With the Prelude SPLC system, LC methods can be executed in parallel with a different method on each channel (Figure 2) or the same method on both channels (Figure 3) and multiplexed into a mass spectrometer Figure 1. Prelude SPLC system for serial detection. Serial MS detection of multiplexed methods improves mass spectrometer utilization time, increases throughput of forensic toxicology laboratories, and reduces analysis cost. The syringe pumps and high-pressure, low-volume gradient mixing used in Prelude SPLC system provide enhanced HPLC performance: improved peak shape and resolution as well as stable retention times. Conventional LC-MS/MS 14 Benzodiazepines (25 ng/mL each) Prelude SPLC multiplexing Channel 1 14 Benzodiazepines (25 ng/mL each) 6 Opiates (10 ng/mL each) Acquisition of relevent MS data Figure 2. Parallel analysis of 6 opiates (10 ng/mL) and 14 benzodiazepines (25 ng/mL) in multiplexed mode High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urin

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 2

Prelude SPLC Multiplexing - Same Method on Both Channels 6 Opiates (10 ng/mL each) Acquisition of relevent MS data Figure 3. Analysis of six opiates (10 ng/mL) using both channels in multiplexed mode Table 2. SRM transitions for benzodiazepines method Sample Preparation Tables 1 and 2 contain the lists of opiates and benzodiazepines analyzed. Sample preparation consisted of glucuronide hydrolysis followed by dilution. For each sample, a 200 µL aliquot of urine was spiked with 10 µL of internal standards solution and 100 µL of b-glucuronidaze enzyme in an ammonium acetate buffer (pH = 5.0)....

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 3

Liquid Chromatography Chromatographic separations were performed with a Prelude SPLC system by direct injections onto Thermo Scientific™ Accucore™ PFP 50 x 2.1 mm, 2.6 µm analytical columns. The columns were maintained at room temperature. Mobile phases A and B consisted of 10 mM ammonium formate with 0.1% formic acid in water and methanol, respectively. Mobile phase usage was about 3.8 mL per sample. The total gradient run time was 5.3 min for opiates analysis (Figure 4) and 6 min for benzodiazepines analysis (Figure 5). The data acquisition windows were 2 min and 2.8 min for opiates and...

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 4

Results and Discussion Opiates Analysis The limits of quantitation were 10 ng/mL and calibration ranges were 10–6000 ng/mL for all opiates. Figure 6 shows representative calibration curves for selected opiates. Figure 7 shows representative chromatograms at 10 ng/mL for all opiates tested. Intra- and inter-assay quality control statistics shown in Table 3 demonstrate the method to be reproducible across the calibration range for the opiates. Limited matrix effects were seen, and those were largely mediated by deuterated internal standards (Table 4). The data collected with this method...

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 5

3000 Validated Method (ng/mL) Validated Method (ng/mL) Hydromorphone Oxycodone Prelude SPLC-Based Method (ng/mL) 0 Prelude SPLC-Based Method (ng/mL) Figure 8. Data correlation between Prelude SPLC-based opiates method and a previously validated LC/MS method Table 3. Intra- and inter-assay precision for opiates analyses Table 4. Results of matrix effect experiment showing percent recovery of opiates in spiked urine % Recovery Morphine

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 6

Benzodiazepines Analysis The limits of quantitation were 25 ng/mL and calibration ranges were 25–2000 ng/mL for all benzodiazepines. Figure 9 shows representative calibration curves for selected benzodiazepines. Figure 10 shows representative chromatograms at 25 ng/mL for all benzodiazepines tested. Intra- and inter-assay quality control statistics shown in Table 5 demonstrate the method to be reproducible across the calibration range for these benzodiazepines. Use of deuterated internal standard eliminated the small matrix effects we experienced with the method (Table 6). The data...

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 7

Validated Method (ng/mL) a-Hydroxytriazolam Diazepam Lorazepam Nordiazepam Oxazepam Temazepam Prelude SPLC-Based Method (ng/mL) Figure 11. Data correlation between Prelude SPLC-based benzo-diazepines method and a previously validated LC/MS method Table 5. Intra and inter-assay precision for benzodiazepines analyses

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High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine - 8

Table 6. Results of matrix effect experiment showing percent recovery of benzodiazepines in spiked urine % Recovery Desalkyflurazepam Using the Prelude SPLC system, high-throughput, costefficient solutions were developed for forensic analysis of opiates and benzodiazepines in urine. The methods met industry requirements for precision, accuracy, and robustness. Implementation of the method on a Prelude SPLC simplified the work flow and resulted in a 40–60% reduction of solvent usage due to the ability of the system to utilize high efficiency, small diameter columns. The mobile phase volumes...

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