Fast, Definitive LC/MS/MS Analysis of THC and THC Metabolites in Urine Using an Ultra II® Biphenyl Column
By Amanda Rigdon a, Clinical/Forensic Innovations Chemist, Becky Wittrig a, Ph.D., Global HPLC Specialist, and Takeo Sakuma b, Ph.D., Principal Application Scientist
a Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823
b AB SCIEX, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8
- Good retention and peak shape allow direct quantification of THC-COOH-glucuronide.
- Faster sample throughput than GC/MS methods--short analysis time and no derivatization.
- Indisputable identification using two or more MRM transitions.
Analyzing THC samples by LC/MS/MS cuts analysis time in half.
As marijuana, hashish and other cannabinoid formulations are consumed, the main psychoactive component, ?9-tetrahydrocannabinol (?9-THC), is quickly absorbed and metabolized to several compounds, including 11-nor-9-carboxy-?9-tetrahydrocannabinol (THC-COOH). Since ?9-THC is metabolized so rapidly, testing programs are generally based on THC-COOH, which can be found in urine, blood, hair, and tissues. To facilitate excretion in the urine, THC-COOH undergoes a conjugation reaction, in which a sugar-like group is added to form THC-COOH-glucuronide, a more hydrophilic compound (Figure 1). Testing often is accomplished using GC/MS; however, this requires time-consuming steps, including derivatization, to obtain acceptable chromatography. Even with derivatization, THC-COOH-glucuronide cannot be determined directly; instead, the glucuronide must be removed by either chemical or enzymatic means prior to analysis as THC-COOH. The yield of this conversion back to the acid can vary, thus potentially adding to inaccuracy. LC/MS/MS analysis can be a better alternative than GC/MS, but column choice is critical as adequate retention of hydrophilic compounds is difficult to achieve with a standard C18 column.
By using LC/MS/MS, fast, conclusive results can be obtained since THC-COOH-glucuronide can be determined directly and because derivatization can be eliminated. As shown in Figure 2, samples can be prepared for LC/MS/MS in 75% less time and analyzed in approximately half the time required for GC/MS. The key to successful LC/MS/MS analysis is using a column that can retain THC-COOH-glucuronide. For this analysis of cannabinoids in urine, an Ultra II® Biphenyl column was chosen because it provides better hydrophilic retention than a C18 column and exhibits excellent peak shape which simplifies quantification. Additionally, since retention is increased, higher amounts of methanol can be used in the mobile phase, which improves desolvation efficiency and increases sensitivity in the electrospray interface.
As shown in Figure 3, THC and its metabolites were fully resolved in just under 4 minutes on an Ultra II® Biphenyl column. Chromatographic resolution is important because THC-COOH and THC-COOH-glucuronide share the same transitions, as a result of in-source fragmentation of the fragile THC-COOH-glucuronide analyte. Fragmentation may be avoided by further optimization of ionization conditions, but in this case it is beneficial, as matrix components interfered with the 521/345 transition of THC-COOH-glucuronide. Although the MRM transitions monitored for THC-COOH and THC-COOH-glucuronide are identical, the two compounds are chromatographically resolved, allowing for accurate determination of both analytes.
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Analyzing cannabinoids in urine by LC/MS/MS instead of GC/MS saves time without sacrificing sensitivity. The Ultra II® Biphenyl column provides good retention of all compounds with a total run time of 6 minutes—including baseline resolution of compounds with the same transitions. By using MS/MS detection, with two +MRM transitions per analyte, compound identity could be reliably determined. Using the unique selectivity of the Ultra II™ Biphenyl column coupled with the detecting power of LC/MS/MS, THC and its metabolites can be directly analyzed and reported much more efficiently than with conventional GC/MS methods.
