Jason S. Wood, Ph.D. Varian, Inc.
Testing for drugs of abuse (DoA) is becoming more prevalent in various environments, including forensics and social justice environments as well as in sporting events. Non-heroin opiates such as morphine, oxycodone, and hydrocodone are appearing increasingly in drug indicator data. For example, in San Francisco, the number of emergency department mentions of oxycodone overdose increased 110 percent in a one year peroid between 2001 and 20021. A US Department of Justice report from the Bureau of Justice Statistics in 2004 showed that the top four drugs utilized by high school seniors (and the percentage of those reporting their use in the last 12 months) were: 1) Alcohol (70.6%), 2) Marijuana (34.3%), 3) Stimulants (10.0%) and 4) Non-heroin Opiates (9.5%)2. Moreover, the US Drug Enforcement Administration (USDEA) reports that since 1990, there has been about a 3-fold increase in morphine products in the United States3.
This application note describes a rapid (5 min), dilute-and-shoot method for the determination of morphine and one of its major metabolites (morphine-glucuronide) in biological fluids (saliva and urine). This method has the advantage that it does not require lengthy derivatization processes (as in GC/MS) and, therefore, can be used in a high-throughput screen environment. It utilizes the advanced features of the Varian 500-MS to divert salts and contaminating proteins away from the API ion source and includes Varians patented OnTrak OraTube as a sampling mechanism for the saliva.
Varian 500-MS LC Ion Trap Mass Spectrometer equipped with an ESI source
Two Varian ProStar 210 Solvent Delivery Modules
CTC Analytics HTS PAL AutoSampler
Materials and Reagents
All chemicals were reagent or HPLC grade from Sigma-Aldrich (St. Louis, MO) with the exception of the morphine and morphine-glucuronide (Cerilliant, Little Rock, TX). Drugs of abuse urine control was obtained from Utak Laboratories, Inc (Valencia, CA). OnTrak OraTube was obtained from the local Varian sales office.
All stock solutions were prepared in 50:50 Methanol:Water at 1 mg/mL. All dilutions were prepared in 5% aqueous methanol + 0.1% acetic acid. Varian OnTrak OraTube (Cat# 6902051) (for obtaining a saliva sample) was used according to its instructions. Utak Laboratories Drugs of Abuse Confirmation Standard (Cat# 98815) was prepared, according to its instructions, by adding 10 mL of HPLC grade water to the dried samples provided.
All saliva and urine samples were diluted 1:10 with 5% aqueous methanol containing 0.1% acetic acid before injection on the 500-MS.
Results and Discussion
The structures of morphine and one of its metabolites are shown in Figure 1. Glucuronidation (catalyzed by uridine-glucuronic transferase or UGTs) is a Phase II metabolism reaction which involves the addition of glucuronic acid from uridine-diphosphoglucuronide to the xenobiotic being metabolized. In general, morphine metabolism is undertaken by UGT2B7.
Typically, simple onsite kits such as the Varian OnTrak TesTcard perform preliminary screening for morphine usage, either on a random basis or as an incident-driven event. However, to eliminate false positives, a second, but more specific chemical method, such as GC/MS, is used as a confirmatory test. However, GC/MS methods for drugs-of-abuse testing are time-consuming, requiring the use of solid-phase extraction and derivatization with pentafluoropropionic anhydride/ pentafluoropropanol. The LC/MS method uses a fast gradient program for complete analysis in only five minutes. Due to the complexity of the final samples injected, a tandem MS technique is employed to increase the method selectivity; the MS/MS conditions are shown in the previous table. Figures 2 and 3 are the calibration curves for morphine and morphine-glucuronide, respectively. Excellent linearity can be demonstrated from 20 to 1000 pg (six points, triplicate runs) for morphine and 40 to 1000 pg (five points, triplicate runs) for morphine-glucuronide. The on column Limits of detection (LOD) and quantification (LOQ) are 10 and 20 pg (respectively) for morphine and 40 and 60 pg (respectively) for morphine-glucuronide. These limits are well within the limits required by the National Institute on Drug Abuse (NIDA) limits for opiates in saliva and urine: 40 ng/mL and 300 ng/mL, respectively.
Figure 4 shows the separation of morphine that was added to a sample of saliva taken with the OraTube sampler. Panel 1 shows the separation of morphine (Arrow marked B) from the salts and matrix interfering peaks (Arrow A). These peaks are now removed when compared to a saliva sample further spiked with morphine (to confirm its identity) but run with the automated 6-port valve of the 500-MS plumbed as a diverter valve.
The diverter valve is an important tool which allows direct injection of urine samples into the ESI source, preventing contamination of the ESI source with urinary salts and extending the maintenance cycle time. To demonstrate this, we have placed a UV detector in-line between the HPLC column and the MS source so that the sample was analyzed by the UV detector prior to the MS. Figure 5 shows the detection of morphine-glucuronide in urine as detected by the 500-MS with the diverter valve, the expanded region on top shows the same sample as detected by UV/Vis at 214 nm.
The bottoming out of the chromatogram (at approximately 0.6 min, UV detection) is the sample void caused (usually) by a high concentration of salt eluting from the LC column.
This application note demonstrates the picogram detection, without derivatization, of morphine and its metabolite morphine-glucuronide in saliva or urine. The limit of quantitation for these analytes is well within the limits set by NIDA and SAMHSA for the detection of opiates in saliva and/or urine. More importantly, this method does not require the lengthy and often tedious derivatization as required in a GC/MS method. All compounds were found to be linear in the calibration range tested and all had very good correlation coefficients (average r2= 0.989).
Finally, this application demonstrates the use of two useful features of the 500-MS, the use of the built-in 6-port injection valve and the syringe pump. Together they can be used as a diverter valve to avoid potential damage to the electrospray interface by diverting salts away from the source and applying a make-up volume of solvent for maintaining optimum spray conditions.
1. NIDA InfoFacts Sept. 2004 (www.drugabuse.gov)
2. US Dept. Of Justice Bureau of Justice Statistics (www.ojp.usdoj.gov/bjs/dcf/du.html)
3. DEA Briefs and Background, Drugs and Drug Abuse www.usdoj.gov/dea/concern/morphine.html