It is estimated that the United States consumes approximately 75 percent of the world’s prescription drugs, and a staggering 52 million people over the age of 12 have used prescription drugs for nonmedical purposes in their lifetime.1 As more and more people become addicted to prescription pills, it is no surprise that an increase in illicit drug abuse has also been observed. For example, recent data indicate that there is a link between prescription opiate addiction and heroin abuse because the two substances target the same receptors. The link is so strong that approximately 75 percent of patients addicted to prescription opioids switch to heroin—because it is less expensive.2This has resulted in a 63 percent increase in heroin use since 2002.3 The large usage and addiction rates for both substances has created increased demand for drug screening.
“Dilute and shoot”
The growing demand for opioid screening and the increasing number of abused drugs in general puts pressure on labs to develop new techniques and increase throughput. This can pose both qualitative and quantitative challenges for analysts. Many labs are turning to a “dilute and shoot” LC/MS/MS approach when analyzing urine samples, because this technique ensures that drug compounds are not lost during the preparation procedure. But it can also cause many negative downstream effects, which can lead to increased cost per sample due to maintenance and downtime. The root cause of these issues can often be attributed to the hydrolysis procedure, which is required when performing urine analysis.
In a urine sample matrix, drugs are often present as a glucuronidated metabolite. It is standard practice to remove the glucuronide moiety prior to LC/MS/MS analysis using a hydrolysis procedure to ensure that the drugs are accurately confirmed and quantified. Hydrolysis can be performed in several ways; enzymatic hydrolysis using ß-glucuronidase is often preferred because it does not require the use of harsh acids and is a rather simple procedure. While simple, however, the procedure does introduce a new component to the sample: an enzyme. Enzymes are protein molecules that, when injected onto an HPLC column or into a mass spectrometer, can reduce sensitivity, clog columns, and build up on the mass spec source. This will eventually lead to required maintenance and subsequent downtime.
There are several methods to remove the enzyme prior to analysis, but most of these methods include more targeted sample preparation procedures that require method development such as solid phase extraction (SPE). So, despite its limitations, many labs still prefer simple sample preparation methods such as “dilute and shoot.” An optional centrifugation procedure can be performed to remove the ß-glucuronidase enzyme, but removal is often not complete. Incomplete removal of the enzyme will eventually increase backpressure (Figure 1), reduce sensitivity, and cause problems with the mass spec instrument.
ß-glucuronidase: an “out of the box” approach
Historically, protein precipitation has been favored as a go-to preparation technique for samples that are protein-rich, such as plasma. Urine does not typically contain significant amounts of protein and would not usually benefit from a protein precipitation procedure. However, urine that has been subjected to enzymatic hydrolysis with ß-glucuronidase contains a significant amount of enzyme, which by definition is a protein.
Recently, screening labs have been applying a protein precipitation technique to remove the enzyme and ensure minimum wear and tear on the column and instrument. The technique is both rapid and simple, requiring about five minutes plus dry-down time and virtually no method development. Protein precipitation is also effective, resulting in >33x increase in HPLC column lifetime and a significant reduction in backpressure (Figure 2) and mass spec downtime for maintenance. Because the technique is non-selective, it removes only proteins (ß-glucuronidase) from the sample without risking the loss of any drug compounds that may be present.
As the link between prescription and illicit drugs increases, labs must ensure that their testing procedures are able to accurately detect a wide variety of unknown compounds. These methods need to be fast and simple while not adding to downtime and maintenance, particularly in high-throughput settings. To address this challenge, labs can look outside of their traditional tool box and incorporate a cleanup technique that is not typically performed in drug screening labs. By implementing a rapid protein precipitation technique that has historically been used to clean up plasma samples in pharmaceutical and clinical labs, analysts can easily remove the ß-glucuronidase from their samples without risking the loss of drug compounds. The technique also significantly increases HPLC column lifetime and reduces mass spec maintenance requirements without the need to perform additional method development.
References
- National Institute on Drug Abuse (NIDA). Popping pills: prescription drug abuse in America. http://www.drugabuse.gov/related-topics/trends-statistics/infographics/popping-pills-prescription-drug-abuse-in-america. Accessed September 2, 2015.
- American Society of Addiction Medicine, Opioid Addiction Disease 2015 Facts & Figures. http://www.asam.org/docs/default-source/advocacy/opioid-addiction-disease-facts-figures.pdf?sfvrsn=2#search=%22opioid%20addiction%20facts%20and%20figures%22. Accessed September 2, 2015.
- Popovich, Nadja. Rapid rise of heroin use in US tied to prescription opioid abuse, CDC suggests. The Guardian, July 8, 2015. http://www.theguardian.com/us-news/2015/jul/08/heroin-use-overdose-deaths-us-prescription-opioids. Accessed September 2, 2015.
