MSACL 2022 Abstract

Interfering Compound in Urine Cannabinoid Analysis Agnes Cua (1), Amadeo Pesce (2) Precision Diagnostics Agnes Cua, PhD (Presenter) Precision Diagnostics

Presenter Bio: Currently, I am an Associate Lab Director at Precision Diagnostics. As part of the Research and Development team, I develop analytical methods for urine and oral fluid drug analysis using liquid chromatography tandem mass spectrometry. Also, I analyze and help interpret patients’ drug test results. I earned my PhD in Chemistry, ABCC board certified in Toxicological Chemistry and NRCC certified as Toxicological Chemist.

PROBLEM

An interfering compound coelutes with delta-9-THC-COOH (11-Nor-9-carboxy-delta-9-tetrahydrocannabinol) in urine drug analysis, with a LC-MS/MS method that uses Phenyl Hexyl column and a 4.2 min gradient program. This causes a small shift in the retention time (RT) of delta-9-THC-COOH preventing its identification, and subsequently is reported as negative or with interference comment, even when delta-9-THC-COOH is present in the urine sample. There is also a possibility that with the RT shift being very small, it becomes not significant, and peak is identified as delta-9-THC-COOH. This becomes an issue when delta-9-THC-COOH is absent in the urine sample and is reported as positive in the presence of the interfering compound.

We also sought to identify the identity of the interfering compound.

METHOD INFORMATION

50 µL urine processed with ‘Dilute and Shoot’ method
Shimadzu LC – MPX (2 streams)

A. SCIEX 6500(+) Mass Spectrometer
MP-A: 0.1% Formic Acid in H2O
MP-B: Methanol
4.2 min gradient @ 0.8-1.2 mL/min flow rate
Phenyl Hexyl column, 50 x 4.6 mm, 2.6 µm, with guard cartridge
Column oven @ 50oC
5 µL injection volume
Quantitative MRM acquisition:
delta-9-THC-COOH 345: 193, 119
delta-9-THC-COOH-d3 348: 302


TROUBLESHOOTING STEPS

To resolve the issue, a LC-MS/MS method that uses a C18 column was developed, where an initial isocratic phase with 60% B was followed by an increase to 95% B and subsequent re-equilibration.

B. SCIEX 5500 Mass Spectrometer
MP-A: 2mM Ammonium Acetate in H2O
MP-B: Methanol
3.5 min isocratic/gradient, 0.8 mL/min flow rate
C18 column, 50 x 2.1 mm, 5 µm, with guard cartridge
Column Oven @ 40oC
5 µL injection volume
Quantitative MRM acquisition:
delta-9-THC-COOH 345: 193, 119
delta-8-THC-COOH 345.2: 299.1, 165.1
delta-9-THC-COOH-d3 348: 302

For identification of interfering compound, a delta-8-THC-COOH (11-Nor-9-carboxy-delta-8-Tetrahydrocannabinol) Standard was used to confirm its identity.

OUTCOME

The method results in the separation of the interfering compound from delta-9-THC-COOH, allowing definite quantitation of delta-9-THC-COOH. Used of the Standard also allowed identification of the interfering compound to be delta-8-THC-COOH.