A Simple and Accurate Method for the Rapid Quantitation of Drugs of Abuse in Urine Using Liquid Chromatography

Transcription

1 Application Note LCMS-109 A Simple and Accurate Method for the Rapid Quantitation of Drugs of Abuse in Urine Using Liquid Chromatography Time of Flight (LC-TOF) Mass Spectrometry Introduction Many clinical research methods for drug quantitation involve labor-intensive, time-consuming sample preparation protocols. Methods with minimal preparation that are accurate and reproducible are therefore highly desirable for high throughput laboratories. In this study, we describe a sensitive, selective and reproducible LC-QTOF mass spectrometry method employing a simple dilute and shoot approach. The results demonstrate the suitability of LC-QTOF technology to quantitate wide panels of drugs of abuse in urine with minimal sample preparation and at low cost. Methods Sample Preparation Fifty microliters of urine, 25 µl β-glucuronidase (IMCSzyme, Columbia, S.C., USA), 50 µl acetate buffer (ph = 7) and 50 µl of working internal standard solution (400 ng/ml) were added to a 96 well plate which was vortexed and incubated (hydrolyzed) in a heating unit for 1 60 o C. 50 µl of mobile phase A (0.1% formic acid in water) was then added and centrifuged at 3000 RPM for 5 min to mix the sample and remove any particulates. Authors E. Howard Taylor, Shannon Johnson Addiction Labs of America, Brentwood, TN, USA Tony Drury, Carsten Baessmann Bruker Daltonik GmbH, Bremen, Germany Matt Willetts, Adi Kulkarni, Cory Lytle Bruker Daltonics Inc., Billerica, MA, USA Keywords Accurate mass Instrumentation and Software Bruker compact QTOF Quantitation Shimadzu LC-20 AD XR UHPLC TOF Drugs of abuse Toxicology research TASQ 1.0 software

2 Two hundred microliters of the supernatant were then transferred into a 96 well analytical plate for analysis (see figure 1). Table 1 shows the list of drugs and metabolites along with the deuterated internal standard (I.S) that were analyzed. Table 1: Drugs and metabolites analyzed along with respective deuterated internal standards denoted in brackets. Opiates/Opioids: morphine (D6), codeine (D6), hydrocodone (D6), hydromorphone (D3), oxycodone (D3), oxymorphone (D3), meperidine (D4), buprenorphine (D4), norbuprenorphine (D3), methadone (D3), methadone metabolite EDDP (D3), mono-acetylmorphine (D3), fentanyl (D5), norfentanyl (D5) Benzodiazepines: alprazolam (D5), alpha-hydroxyalprazolam (D5), diazepam (D5), nordiazepam (D5), oxazepam (D5), temazepam (D5), lorazepam (D4), clonazepam (D4), 7-aminoclonazepam (D4) Stimulants: cocaine metabolite benzoylecgonine (D3), amphetamine (D5), methamphetamine (D5), MDMA (D5), MDA (D5); methylphenidate (D9) Tricyclic antidepressants: amitriptyline (D3), nortriptyline (D3), imipramine (D3), desipramine (D3), doxepin (D3) desmethylydoxepin (D3), and Others: tapentadol (D3), tramadol (13C D3), carisoprodol (D7), meprobamate (D7), zolpidem (D7), PCP (D5) Instrumentation Mass Spectrometry Instrument: Bruker compact QTOF Software: Bruker Compass 1.9 (data acquisition) TASQ 1.0 (data processing, review and reporting) Chromatography: Shimadzu LC-20 AD XR UHPLC Column: Phenomenex analytical column (Kinetex 2.6 µm: C18: 2.1 x 100mm) Chromatographic separation of the analytes was performed using a linear gradient with 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile Phase B) at 40 o C and a flow rate of 0.4 ml/min (see figure 2). Injection volume was 5 µl. Initial conditions were 95% A / 5% B, and then a linear gradient to 65% A / 35% B from 1 to 5 min. with a second linear gradient to 10% A / 90% B from 5 to 8 min returning to the initial conditions for 3 min. Chromatographic elution profile Sample preparation Figure 2: Gradient Programming Add 50 μl mobile phase A and centrifuge - Transfer 200 μl Figure 1: Sample preparation for urine samples in 96 well plates. The compact TM QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) operating in positive electrospray mode ( 2,500 V ) was calibrated with sodium formate clusters at the beginning of every injection delivering a mass accuracy of < 2 ppm. Data were acquired using a TOF MS acquisition rate of 3 Hz over the mass range of m/z. The concentrations of drugs / metabolites chosen for the calibration range were designed to represent the expected concentration values from actual donor samples. For drugs and metabolites that were expected at lower concentrations such as fentanyl and norfentanyl the calibration range was ng/ml and ng/ml respectively. Calibrators for drugs expected to be present at mid-range concentrations were prepared from ng/ml. For drugs and metabolites expected at higher concentrations, urine calibrators were prepared from ng/ml.

3 Results All data were processed with Bruker TASQ 1.0 software. Quantitation was based on high resolution extracted ion chromatograms using a narrow ion extraction window of +/- 5 mda. Figure 3 shows the elution profile of the analytes measured with good chromatographic peak shape throughout. Sample chromatogram Figure 3: High-resolution extracted ion chromatogram for all compounds. All compounds eluted within 8 minutes showing good separation (either by mass or retention time or both) and peak shape. Examples of the elution profiles for all compounds and the benzodiazepines in isolation are shown in figures 3 and 4 respectively. Figure 5 shows the efficient hydrolysis of morphine glucoronide by using the purified form of β-glucuronidase enzyme. Benzodiazepine chromatogram For quantitation purposes, the accurate mass of the precursor ion (M+H) and deuterated internal standard was used. As a further step to ensure the correct ions were used for quantitation, the measured isotopic pattern of the precursor ion was fitted against the theoretical pattern in the TASQ results viewer as illustrated in figure 6. Note the use of the narrow extraction windows throughout the experiment resulted in interference free chromatograms. Figure 7 shows an example of the excellent linearity and back calculated accuracies for the oxymorphone calibrants. The chromatogram, calibration curve and associated results table are interactively linked and can be viewed simultaneously in TASQ software. Figure 4: High resolution extracted ion chromatogram for benzodiazepines Alprazolam, Clonazepam, and alpha-hydroxyalprazolam present at 50 ng/ml and Diazepam, Nordiazepam, Temazepam, Lorazepam, and Oxazepam at 500 ng/ml

4 Chromatogram showing hydrolysis a b Morphine Glucuronide Morphine Morphine Figure 5: Chromatogram from a specimen containing 1875 ng/ml of Morphine Glucuronide and 625 ng/ml of Morphine: a) Before β-glucuronidase hydrloysis and b) after hydrolysis showing that the enzymatic hydrolysis is complete. Sample chromatogram Quantifier ion Excellent fit to True Isotopic Pattern Internal Standard (IS) Figure 6: Quantitation of Oxymorphone (left) and confirmation using true isotopic pattern matching (right). Figure 7: TASQ quantitation pane for Oxymorphone showing good accuracy and linearity R 2 =

5 Results table Compound Retention Time (min) m/z Lowest Level (ng/ml) LOQ (ng/ml) Highest Level (ng/ml) %CV at cutoff 6-Acetylmorphine Acetylmorphine D Aminoclonazepam Aminoclonazepam D alpha-hydroxyalprazolam alpha-hydroxyalprazolam D Alprazolam Alprazolam D Amitriptyline Amitriptyline D Amphetamine Amphetamine D Benzoylecgonine Benzoylecgonine D Buprenorphine Buprenorphine-D Carisoprodol Carisoprodol D Clonazepam Clonazepam D Codeine Codeine D Desipramine Desipramine D Desmethyldoxepin Desmethyldoxepin D Diazepam Diazepam D Doxepin Doxepin D EDDP EDDP D Fentanyl Fentanyl D Hydrocodone Hydrocodone D Hydromorphone Hydromorphone D Imipramine Imipramine D Lorazepam Lorazepam D

6 Compound Retention Time (min) m/z Lowest Level (ng/ml) LOQ (ng/ml) Highest Level (ng/ml) %CV at cutoff Meprobamate Meprobamate D MDA MDA D MDMA MDMA D Meperidine Meperidine-D Methadone Methadone D Methamphetamine Methamphetamine D Methylphenidate Methylphenidate D Morphine Morphine D Norbuprenorphine Norbuprenorphine D Nordiazepam Nordiazepam D Norfentanyl Norfentanyl D Nortriptyline Nortriptyline D Oxazepam Oxazepam D Oxycodone Oxycodone D Oxymorphone Oxymorphone D PCP PCP D Tapentadol Tapentadol-HCL D Temazepam Temazepam D Tramadol Tramadol-13C,D3-HCl Zolpidem Zolpidem D

7 Discussion The use of a purified form of β-glucuronidase produced a significantly visibly cleaner sample for LC-MS analysis and no interferences were detected. A calibration curve (performed daily) of peak area ratio with each drug or metabolite s respective deuterated internal standard vs target concentration was linear with a correlation coefficient greater than 0.99 for all analytes. The CV% for benzodiazepines at the LOQ and cutoff averaged 3.9% and 2.8%, respectively. The opiates/opioids CV% at LOQ and cutoff averaged 4.5% and 4.8% respectively and all other drugs averaged a CV of 3.2% and 3.8% at the LOQ and cutoff. Conclusion This dilute and shoot method using a purified form of β-glucuronidase, combined with high resolution, QTOF mass spectrometry produced excellent quantitative results for all drug types considered in this experiment. The method is rapid, robust, low cost and relatively simple to implement. An advantage of using QTOF full scan accurate mass measurement over typical triple quadrupole methods is that it allows the possibility to considerably increase the number of drugs quantitated per run without the need to change the chromatographic or data acquisition parameters. This approach therefore lends itself to many high throughput research applications without significantly adding to the cost or complexity of analysis.

8 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , LCMS-109, For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bruker Daltonics Inc. Bremen Germany Phone +49 (0) Fax +49 (0) Billerica, MA USA Phone +1 (978) Fax +1 (978)