Fast Sample Preparation with Liquid Chromatography Tandem Mass Spectrometry for Illegal Opioid Analysis

Article

The Column

ColumnThe Column-03-12-2018
Volume 14
Issue 3
Pages: 19–22

With the growing use of illegal opioids, analysts should be prepared for a large influx of samples in their laboratories. While the workload may be increasing, the number of analysts and equipment may not, so the need for faster and better liquid chromatography–mass spectrometry (LC–MS) methods is important. By implementing an efficient sample preparation technique for matrix cleanup for some of the most common and traditional opioid matrices-blood and urine-coupled with a rapid and accurate LC method, laboratories can address the analytical needs for this growing problem.

Photo Credit: Iryna Imago/Shutterstock.com

Daniel Spurgin and Jenny Cybulski, Phenomenex, Torrance, California, USA

With the growing use of illegal opioids, analysts should be prepared for a large influx of samples in their laboratories. While the workload may be increasing, the number of analysts and equipment may not, so the need for faster and better liquid chromatography–mass spectrometry (LC–MS) methods is important. By implementing an efficient sample preparation technique for matrix cleanup for some of the most common and traditional opioid matrices-blood and urine-coupled with a rapid and accurate LC method, laboratories can address the analytical needs for this growing problem.

The abuse of opioids has become a global crisis. In 2015, opioids led to the death of over 33,000 Americans, and for one of the most potent and deadly opioids, fentanyl, the death rate from 2015–2016 more than doubled (1,2). According to the October 2016 issue of Medical Care, opioid abuse costs the USA about $78.5 billion annually as a result of the increased use of public and private healthcare, lost productivity, addiction treatment, and criminal justice involvement (3,4). Given the size of this problem, the demand for laboratories to process samples is massive.

Common sample matrices for opiates testing are urine and whole blood. These matrices require proper sample preparation before liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis to ensure that results are accurate, but often this can add a significant amount of time. For laboratories that are trying to keep up with the increase in samples without additional resources or head count, faster and more accurate results help to ensure that they can keep up with demands. By implementing proper sample preparation and a quick, yet reliable LC–MS/MS method, forensic toxicology laboratories can keep up with the heightened demand from the growing opioid epidemic.

 

Urine Sample Preparation

Urine drug testing can be complicated by the metabolic process that occurs after drug ingestion and there are several recommended approaches for preparing urine samples before analysis. During metabolism, drugs are tagged with a glucuronic acid to help aid in adsorption into the kidneys, but this glucuronide form must be cleaved, which is primarily performed with a β-glucuronidase enzyme, prior to analysis, for accurate results regardless of the sample preparation technique performed. The sample preparation techniques range from the quick and dirty “dilute-and-shoot” method to solid-phase extraction (SPE), which requires a deeper understanding of the analytes of interest and method development, but leaves a cleaner, more concentrated extract. While dilution is a simple sample preparation method, it leads to lower analyte response and poor signal‑to‑noise ratio, and the large β-glucuronidase enzyme is still present in the sample, which will lead to premature LC column death from buildup on the column’s stationary phase. The simplest approach to removing β-glucuronidase without losing sensitivity is utilizing a chemical filter that selectively captures the enzyme, passing cleaner sample through the cartridge or plate. Figure 1 shows the resulting increase of sensitivity in comparison to dilute‑and‑shoot. If greater analyte concentration is necessary, another quick and simple sample preparation technique would be supported liquid extraction (SLE). Either a synthetic or a traditional diatomaceous earth sorbent can be used; the type of SLE sorbent is dependent on sample volume, specific analytes, and extraction solvents. Table 1 displays good recoveries for a panel of analytes using a traditional diatomaceous earth product and an extraction solvent of 95:5 dichloromethane–IPA.

The most extensive approach to preparing an opioid panel for LC–MS/MS testing is SPE. SPE leverages a selective sorbent that targets analytes of interest while allowing contaminants and other interferences to be washed away, resulting in the cleanest extract possible. The two main drawbacks are sample processing time and method development. The proper sample preparation methods for urine depend on the needs of laboratories, paired with the costs that can accompany sample preparation products. While products do come at a price, they can reduce system maintenance and avoid inaccurate results.

Sample Preparation from Whole Blood

Whole blood is a more complex matrix for extraction of opioid analytes because of the many components present. In addition to sample preparation, it also requires a pretreatment step to release drug compounds from the blood cells. Pretreatment can include a hemolysis, sonication, or osmotic breakdown before sample preparation and the technique should be selected based on application or method needs. To eliminate matrix interferences from whole blood, a sample preparation step is essential. Depending on the analysis, the methods to clean up the matrix can vary. Viable, efficient, and quick sample preparation options include protein precipitation, phospholipid removal, SLE, or SPE.

Protein precipitation and phospholipid removal products are not analyte-specific and work as a type of chemical filter, making them excellent candidates for unknown drug screening. However, more targeted techniques are available for cleanup of known analytes such as opioids. SLE is a reliable option for proper sample preparation and having two diverse SLE sorbent options, synthetic and a traditional diatomaceous earth, ensures that the SLE product used yields the best results for specific opioid analyses. The most reliable, clean, and selective sample preparation option is SPE. With so many diverse sorbent options, SPE allows analysts an opportunity to optimize their method by retaining the opioid analytes of interest while removing specific matrix interferences. In all four sample preparation techniques, cleaner samples, higher throughput, and longer column lifetimes can be achieved while reducing MS maintenance or cleaning.

Regardless of the sample preparation method used, it is essential to analyze samples quickly and accurately. Forensic toxicologists need an LC method with a short run time that still resolves critical isobars.

Experimental

LC conditions were as follows: Column: 50 × 3.0 mm, 2.6-µm Kinetex Pheny‑Hexyl (Phenomenex); guard: SecurityGuard ULTRA Biphenyl (Phenomenex); cartridges: AJ0‑9208 (Phenomenex); SecurityGuard ULTRA Holder: AJ0-9000; mobile phase: A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol; gradient: 0 min 10% B, 0.2 min 10% B, 1.5 min 40% B, 2 min 95% B, 2.5 min 95% B, 2.51 min 10% B, 4 min 10% B; flow rate: 0.7 mL/min; injection volume: 10 µL; column temperature: ambient; detector: MS/MS (Sciex Triple Quad 4500), ESI +.

 

Results and Discussion

In this example (Figure 2) critical isobars morphine–hydromorphone and codeine–hydrocodone were resolved without sacrificing run time. The phenyl properties of the phenyl-hexyl phase utilized pi-pi interactions to retain hydrophilic compounds like morphine, while the alkyl properties helped to resolve isomeric species like morphine–hydromorphone. The 2.6-µm core–shell particles produced sharper peaks and faster elution times compared to fully porous particles. All analytes in this 19-drug panel eluted in less than 2.8 min, with a total run time of ~4 min.

Conclusion

Depending on analytical needs and system performance criteria, there are many different sample preparation options to choose from, ranging from simple and fast to detailed and lengthy as well as generic to assay-specific. Coupling a well-prepared sample with good chromatographic separation is crucial to improve the understanding of this growing problem.

References

  1. US Department of Health and Human Service (2017). HHS announces over $70 million in grants to address the opioid crisis [press release]. Published 31 May 2017. Accessed 05/01/2018 https://www.hhs.gov/about/news/2017/05/31/hhs-announces-over-70-million-in-grants-to-address-the-opioid-crisis.html.
  2. Centers for Disease Control and Prevention. Annual Surveillance Report of Drug-Related Risks and Outcomes - United States, 2017. Surveillance Special Report 1. Centers for Disease Control and Prevention, U.S. Department of Health and Human Services. Published 31 August 2017. Accessed 05/01/2018 from https://www.cdc.gov/drugoverdose/pdf/pubs/2017­ cdc-drug-surveillance-report.pdf.
  3. H.A. Pollack, Medical Care54(10), 899–900 (2016). DOI: 10.1097/MLR.0000000000000652
  4. Pain News Network, CDC: Prescription Opioid Abuse Costs $78.5 Billion. Published 15 September 2016. Accessed 15/01/2018. https://www.painnewsnetwork.org/stories/2016/9/15/cdc-prescription-opioid-abuse-costs-785-billion-annually.

Daniel Spurgin holds a B.S in combined sciences and minor in biotechnology from Santa Clara University (California, USA) and a Masters of Business and Science from the Keck Graduate Institute for Applied Life Sciences at the Claremont Colleges (California, USA). Prior to joining Phenomenex as the Global Marketing Manager-Clinical, Forensics, and Toxicology, he worked in management consulting and at startup biotech companies.

Jenny Cybulski graduated from Vanguard University (California, USA) with a B.S. in biology and a minor in chemistry and now works as a product marketing manager at Phenomenex located in Torrance, California, USA.

E-mail:DanielS@phenomenex.comWebsite:www.phenomenex.com

 

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