Urinary Drug Testing: Small Changes for Big Improvements in Sensitivity

An oversight or mistake in drug testing can have dire consequences—a positive drug test can ruin the possibility of employment, turn an accident into a lawsuit, or cause a physician to discontinue prescribing painkillers for a patient suffering from chronic pain—to cite just a few examples. Tests need to be as sensitive as possible to determine drug-use patterns in individuals, and lab technicians must obtain reliable and statistically significant results; the use of outdated testing techniques can mean less than optimal reporting. Minor adjustments in processes or materials can have a major impact on test accuracy, sensitivity and overall outcome.

High-performance liquid chromatography is commonly used for sample preparation in drug testing because it is sensitive and reliable. When developing an HPLC method, analysts may take steps to improve results. These may include changing the HPLC column, optimizing mobile-phase solvents and altering run conditions. Refining sample preparation improves analysis and can provide greater sensitivity in drug testing, ultimately leading to more accurate, conclusive results.

Blood, urine and oral fluids are widely used in drug testing by LC/MS. The simplest sample matrix is urine, as testing methods are relatively easy and collection is noninvasive.¹ The urine matrix also contains higher concentrations of drug metabolites, allowing more time for analytical runs and increasing the probability of recognizing drug-use patterns.² While urine is a simple matrix, an additional sample preparation step is needed to analyze it.

Prior to excretion, drugs undergo a glucuronidation reaction in the liver, in which they are tagged with a glucuronic acid and their polarity is increased so that they can be absorbed in the kidneys and excreted. Hydrolysis must be performed to cleave the glucuronic acid moiety from the parent drug compound before LC/MS analysis. Two types of hydrolysis are generally performed—acid or enzymatic. The enzymatic method is usually preferred, because it does not introduce harmful solvents into the sample or alter the chromatography. When working with an enzymatic hydrolysis, the remnants of the enzyme used to cleave the bonds, β-glucuronidase, will be left over in the sample matrix. β-glucuronidase weighs about 320 kDa; if left in the sample matrix and injected onto a column, it can precipitate out in the mobile phase during the run, eventually changing the selectivity of the column and increasing backpressure.³ Technicians often perform a “dilute-and-shoot” method, diluting the sample in an effort to minimize the probability of this occurring, but this significantly reduces the test sensitivity.

Finding the right balance in cleaning samples, minimizing solvent usage and concentrating analytes is key to increasing sample prep sensitivity.

Dilute-and-shoot is a simple method in which the sample is diluted 10–30 times; this reduces the concentration of β-glucuronidase enzyme and subsequently the concentration of drug analytes. Although the technique dilutes the analytes and decreases sensitivity, its simplicity and low cost make it a preferred process in clinical and forensic toxicology drug-testing laboratories working with urine.⁴ Techniques that do not eliminate β-glucuronidase completely or that excessively dilute the samples often have signs of ion suppression that decrease analyte detection.⁵ Ion suppression is a problem for those using dilute-and-shoot methods, yet it is still performed in many labs, where its drawbacks are not recognized.

Other types of sample preparation techniques, such as protein precipitation, are alternative methods used to increase sensitivity. Use of protein precipitation plates is an easy way to remove β-glucuronidase without having to take the time to develop a method. This technique still involves a 3:1 or 4:1 dilution, and the test takes about 15 minutes to perform. High-throughput laboratories may not have the time for this technique. Another sample preparation technique, solid-phase extraction (SPE), requires long method development times and numerous, carefully monitored steps. Both methods provide higher sensitivity than dilute-and-shoot; both require additional solvent usage and more time.

β-Gone β-Glucuronidase Removal tubes and 96-well plates (Phenomenex, Torrance, Calif.) provide a simple solution to sensitivity issues that arise when working with urine. They incorporate a specialized sorbent, which is packed in both 96-well plates and 1-mL tubes; this allows easy processing of any sample size at any level of throughput. The sorbent retains β-glucuronidase while releasing analytes of interest in the same amount of time required to perform dilute-and-shoot—less than 1 minute. Hydrolyzed urine is diluted 40% with 0.1% formic acid in methanol and is then passed through the sorbent within seconds with the aid of a vacuum or positive-pressure manifold. This results in a sample that is clean of β-glucuronidase and has high analyte recovery. Figure 1 shows the workflow using dilute-and-shoot and β-Gone. Recovery and sensitivity remain high, and clean samples are ready to be injected onto columns in less than 1 minute. In one representative test, the recovery for 51 drug compounds was over 70% for the entire panel using the non-recombinant form of β-Gone β-Glucuronidase Removal products. Sensitivity increased over three times for THC-COOH using β-Gone when compared to dilute-and-shoot (see Figure 2). Drug compounds such as norbuprenorphine and buprenorphine, which are commonly susceptible to ion suppression when large dilutions occur in the sample matrix, can be detected accurately at low limits using the targeted β-glucuronidase removal approach.⁶

 Figure 1 – Workflow comparison: dilute-and-shoot and β-Gone.

 Figure 2 – Sensitivity increase for THC-COOH using β-Gone.

Laboratories that use the dilute-and-shoot sample preparation method might be well served to evaluate the costs in terms of sensitivity, column lifetime and mass spectrometer maintenance. The β-glucuronidase removal method can increase sensitivity and extend column lifetime.

References

1. Allen, K. Screening for drugs of abuse: which matrix, oral fluid or urine? Ann. Clin. Biochem. 2011, 48(6), 531–41; doi: 10.1258/acb.2011.011116.
2. Moeller, K.E.; Lee, K.C. et al. Urine drug screening: practical guide for clinicians. Mayo Clin. Proceedings 2008, 83(1), 66–76;
3. Trontelj, J. Quantification of glucuronide metabolites in biological matrices by LC-MS/MS, tandem mass spectrometry—applications and principles; Prasain, J., Ed.; ISBN: 978-953-51-0141-3; InTech 2012; www.intechopen.com/books/tandem-mass-spectrometry-applications-andprinciples/quantification-of-glucuronide-metabolites-in-biological-matrices-by-lc-ms-ms
4. Cao, Z.; K, E. et al. Simultaneous quantitation of 78 drugs and metabolites in urine with a dilute-and-shoot LC-MS-MS assay. J. Anal. Toxicol.  June 2015, 39(5), 335–46; doi: 10.1093/jat/bkv024.
5. Grebe, S.K.G. and Singh, R.J. LC-MS/MS in the clinical laboratory—where to from here? Clin. Biochem. Rev 2011, 32, 5–31; www.ncbi.nlm.nih.gov/pmc/articles/PMC3052391/
6. Regina, K.J. and Kharasch, E.D. High-sensitivity analysis of buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide in plasma and urine by liquid chromatography–mass spectrometry. J. Chromatogr. B Anal. Technol. in the Biomed. and Life Sci. 2013, 939, 23–31; doi:10.1016/j.jchromb.2013.09.004.

Jenny Cybulski is product communications manager, Phenomenex, Inc., 411 Madrid Ave., Torrance, Calif. 90501-1430, U.S.A.; tel.: 310-212-0555; e-mail: [email protected]; www.phenomenex.com