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Abstract Introduction
Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is the ideal solution for the simultaneous analysis of multiple cannabinoids and metabolites due to the high specificity and sensitivity of the instrumentation. A chromatographic method was developed to separate following analytes - cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), tetrahydrocannabinol (THC), nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) - using an Agilent 1290 Infinity II Liquid Chromatograph. Quantitative data was acquired using an Agilent's 6470 triple quadrupole mass spectrometer. Sample throughput was nearly quadrupled (3.7x) by running four simultaneous, staggered chromatographic analyses on a single mass spectrometer using Agilent's MassHunter StreamSelect LC-MS software.
Method
Instrumentation includes an Agilent 6470 Triple Quadrupole LC/MS with Agilent JetStream (AJS), four 1290 Infinity II High Speed Pumps, four 1290 Infinity II Multicolumn Thermostats, and a StreamSelect RSI (PAL3) Autosampler. A 4.4 minute gradient (water:methanol, 0.01% formic acid, 5mM ammonium formate) with a 1.6 minute equilibration was run on an Agilent Poroshell 120 EC-C18 column. Quantitation was performed with optimized MRM transition pairs for each analyte and internal standard in positive mode. Urine was spiked with 6 cannabinoids and serially diluted to get calibration curve, (range 5 - 5000 ng/mL). A batch of 1000 identical samples were made at 100 ng/mL. Calibrators and samples were diluted 1:10 with 70% methanol containing labeled internal standards at 50 ng/mL.
Results
A 1/x weighting factor was applied during linear regression of the calibration curves. The quantitation using chromatographic peak area ratio to a known concentration of the internal standards. Each analyte was quantitated with its own deuterated internal standard, expect for CBDA, which was quantitated with CBD-d3. Samples and calibrators were grouped and quantitated based on the stream on which they were acquired. All calibration curves displayed excellent linearity. Retention time reproducibility between all four chromatographic streams was between 0.43% and 1.54% (n = 1000), depending on the analyte. StreamSelect acquired data for 894 samples over a period of 24-hours, which equates to 97 seconds per analysis. Compared to a 6-minute runtime for the same analysis using traditional LC-MS, this results in a 3.7x increase in sample throughput. Over the course of nearly 27 hours, 1000 identical urine samples containing 100 ng/mL of each of the six analytes and their respective internal standards were run across the four LC streams. Quantitative reproducibility was consistent across all streams. Quantitative CVs ranged from 0.47 to 2.79 %.
Novel Aspect
Full, robust chromatographic separation and LC/MS analysis of 6 cannabinoids at a rate of 97 second per sample.
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= Discovery stage.
= Translation stage.
= Clinically available.
