A Quick LC/MS/MS Method for Analysis of Common Benzodiazepines and Opiates

RESULTS AND DISCUSSION

Tania A. Sasaki1, Claire J. Bramwell-German1, Sumandeep Rana2, and Wayne B. Ross2 1Applied Biosystems, Foster City, CA; 2Redwood Toxicology, Santa Rosa, CA

Figure 6. Internal Standard Problem

Figure 6. Metric plots of IS peak areas for α-OH-triazolam-d4 and lorazepam-d4 showed an increase in area as the analyte concentration increased. Lorazepam and α-OH-triazolam both contain two Cl atoms; therefore, at high analyte concentrations,

the double Cl isotope bleeds into the IS MRM channel. Oxazepam-d5 and α-OH-alprazolam-d5 were used as internal standards for lorazepam and α-OH-triazolam, respectively, and yielded linear results.

Table 1. Analytes and Retention Times

Table 1. List of opiates

and benzodiazepines

included in the analytical method and their chromatographic retention times.

Figure 7. Calibration Curve and Statistics

Figure 7. Calibration report for hydrocodone. Other analytes had similar precision and accuracy. The calibration range utilized was 5-5000 ng/mL for all analytes except for fentanyl. A range of 0.5-500 ng/mL was used for fentanyl analysis. CONCLUSIONS

• A fast LC/MS/MS method for analysis of benzodiazepines and opiates in urine was developed.•

Sample preparation was greatly simplified (vs. GC/MS) and consisted of hydrolysis and sample dilution. Total run time for the assay was 6 minutes, including

column equilibration. GC/MS sample analysis used 10x more sample volume and consisted of SPE followed by derivatization and sample concentration.•

The LLOQ for all analytes except fentanyl was 5 ng/mL or better, though a cutoff level of 5 ng/mL was used. For fentanyl, the LLOQ was 0.5 ng/mL. Linearity beyond 5000 ng/mL (500 ng/mL for fentanyl) was not examined.•

Scheduled MRM experiments help optimize dwell times, resulting

in good precision and accuracy in multi-component assays.•

Correlation between LC/MS/MS results and results obtained using a validated GC/MS method was very good with the exception of α-OH-alprazolam. The source of error in α-OH-alprazolam has not yet been determined and is left to future work.•

Fentanyl and norfentanyl were included in this assay and were not included in the GC/MS opiate method. Fentanyl and norfentanyl were confirmed in one sample. •

Benzodiazepines and opiates are analyzed in two different assays using GC/MS. The ability to combine both classes into a single fast analysis can save significant amounts of time and money.

TRADEMARKS/LICENSING

Applied Biosystems and AB (design) are registered trademarks of Applied Biosystems or its subsidiaries in the US and/or certain other countries.QTRAP is a registered trademark of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems and MDS Inc.All other trademarks are the sole property of their respective ownersFor Forensic and Research Use Only. Not for use in diagnostic procedures©2008 Applied Biosystems and MDS Inc. All Rights Reserved.

ABSTRACTBenzodiazepines and opiates are two commonly prescribed drug classes. As a result, it is often necessary to analyze biological samples for the use or abuse of these compounds. A fast LC/MS/MS method was developed to analyze urine for the presence of nine common opiates and ten common benzodiazepines, including metabolites, in six minutes. Sample preparation consisted of enzyme hydrolysis, centrifugation, and dilution. The LLOQ for all analytes except fentanyl was 5 ng/mL or better, though 5 ng/mL was the lowest calibrator used for quantitation. For fentanyl, the lowest calibrator was 0.5 ng/mL. The upper cutoff limit for the assay was set at 5000

ng/mL (500 ng/mL for fentanyl), though linearity for many analytes extended beyond this concentration. LC/MS/MS results were compared with previously obtained data from a validated GC/MS method. Problems were observed with analysis of α-

OH-alprazolam as the LC/MS/MS results were much higher than the GC/MS results. The source of this problem has not yet been identified. Excluding α-OH-alprazolam, a correlation of R2=0.8874 was observed between the two methods.

INTRODUCTIONDrug screening in toxicology is typically performed utilizing immunoassay with GC/MS confirmation. The main advantage of using chromatographic/mass spectral techniques

is their inherent sensitivity and selectivity and GC/MS has long been an indispensable analytical tool in the toxicology laboratory. As easy-to-use benchtop instruments have been developed and become more affordable, the use of LC/MS/MS in the forensic toxicology laboratory has significantly

increased. Several drugs of abuse and their metabolites are relatively polar, semi-volatile, and difficult to analyze using GC/MS, often requiring extensive sample clean-up and derivatization. These compounds are ideal candidates for

LC/MS/MS analysis. LC/MS/MS run times are often much shorter than GC/MS run times resulting in higher throughput and faster sample turnaround time. Furthermore, sample preparation is greatly simplified because derivatization is usually not necessary and it is possible to directly inject aqueous samples, which can eliminate extensive liquid-liquid or solid-phase extraction steps. A simple, quick LC/MS/MS method to detect and quantify nine common opiates and ten common

benzodiazepines and their metabolites in urine has been developed.

MATERIALS AND METHODSStandards: Analytical standards and deuterated internal standards, were all purchased from Cerilliant. A deuterated internal standard for each analyte was used. A list of all analytes and their chromatogrpahic retention times is shown in Table 1. Each analyte used a deuterated analog as an internal standard.Sample preparation: A flow chart of the sample preparation procedure is shown in Figure 1. Synthetic urine was spiked with analytes at known concentrations for use as calibrators. Ion suppression tests were performed using extracted synthetic urine and five different urine samples. No significant ion suppression was observed (data not shown).Instrumentation: The LC/MS/MS system consisted of an Agilent 1200 SL Rapid Resolution LC stack interfaced to an Applied Biosystems/MDS Analytical Technologies 3200 QTRAP®

mass spectrometer. Electrospray positive ionization was used and the mass spectrometer was operated in multi-reaction monitoring mode (MRM). Two transitions per analyte and one transition per internal standard were monitored.

Because of the closely eluting analytes, the Scheduled MRM™ algorithm was used to optimize MRM dwell times. A representative chromatogram is shown in Figure 2

and an explanation of scheduled MRM workflow is shown in Figures 3 and 4. Separation was achieved on a Restek Biphenyl column (2.1 x 50 mm, 1.9 µM) and mobile phases were water and acetonitrile with 0.1% formic acid added to each. A rapid gradient was used and total run time, including column equilibration, was 6 minutes.

Figure 1. Sample Preparation

Figure 1. Sample preparation consisted of a simple hydrolysis step and dilution. Note that a small volume of sample is used and the final sample is diluted >5x prior to injection for LC/MS/MS analysis, which helps minimize potential ion suppression. GC/MS analysis used 5 mL of urine concentrated to 100 µl for analysis.

Figure 4. Dwell time and cycle time can affect data quality. In a multi-component analysis, the ability to schedule MRMs only during a desired retention time window can optimize dwell and cycle times for each analyte, even when there are no “period breaks”

in the chromatogram.

Figure 2. Representative Chromatogram

Figure 2. Representative chromatogram of a 5 ng/mL calibrator.

Figure 8. Sample Summary Report

Figure 5. Summary of a sample. Analytes detected and confirmed by ion ratio are highlighted and the concentrations are shown. The peak review displays integration of analyte peaks.

Figure 9. Correlation ResultsTable 2. Summary of Results

Table 2. Seventeen samples were analyzed and a total of 69 analytes were detected across all samples. The four cases where there was a large discrepancy between LC/MS/MS and GC/MS results were all α-OH-alprazolam. The source of this disagreement has not yet been determined. Of the remaining two instances where LC/MS/MS detected analytes not detected by GC/MS, one was α-OH-alprazolam, which is a known problem; only one significant result was obtained by LC/MS/MS that was missed by GC/MS. The two cases where GC/MS detected analytes missed by LC/MS/MS were near the cutoff levels.

Analyte RT (min)7-Aminoclonazepam 1.70Desalkylflurazepam 2.89a-OH-alprazolam 2.70Diazepam 3.08Temazepam 2.94Lorazepam 2.79Nordiazepam 2.80Oxazepam 2.74Alprazolam 2.84a-OH-triazolam 2.70

Analyte RT (min)Morphine 0.52Codeine 1.39Oxycodone 1.56Hydromorphone 0.68Hydrocodone 1.65Fentanyl 2.57Norfentanyl 1.92Oxymorphone 0.606-MAM 1.62

Figure 3. Separation of Isobaric Compounds

Figure 3. Calibrator (5 ng/mL) showing separation of the isobaric pairs morphine/hydromorphone and codeine/ hydrocodone.

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Figure 4. Illustration of Scheduled MRMFigure 5. Setting up a Scheduled MRM Experiment

Figure 5. To set up a scheduled MRM experiment, the MRM transitions, analyte name, and retention times

are entered into the mass table. The detection window, or duration to monitor the MRM and target scan time

(cycle time) are also entered. The window and scan time are dependent on chromatographic peak width.

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Comparison of GC/MS and LC/MS Results

y = 0.907x - 28.754R2 = 0.8874

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Figure 9. Comparing the GC/MS results to the LC/MS/MS results showed an correlation with an R2

value. An R2

value of 0.8874 shows very good correlation. However, it should also be noted that samples were not split for this study. GC/MS results were prepared and obtained in one laboratory and LC/MS/MS results were obtained at a later date in another laboratory. Data from analysis of α-OH-

alprazolam was not included in this correlation because the results from LC/MS/MS analysis are known to be a problem.

1000 ng/mL

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500 μL urine sample

Add 10 μL IS

Add 250 μL pH 5Β-glucuronidase

Solution (5,000 units/mL)

Centrifuge

Remove 100 µL supernatent, dilute

with 400 µLmobile phase

Inject 20 µLfor LC/MS/MS

Analysis

Vortex and incubate for 180 min at 60°C;