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Abstract Introduction: The Multicenter AIDS Cohort Study/ Women’s Interagency HIV Study Combined Cohort Study (MACS/WIHS) is a collaboration researching the effects of chronic health conditions of people living with HIV/AIDS. The main focus of the collaboration is to research heart, lung, blood, and sleep disorders. Tobacco and cannabis use potentially can add additional risk to cardiologic and pulmonary health in these immunocompromised individuals. The analytes targeted in this method are nicotine, cotinine, 3-OH-cotinine, THC-COOH, and creatinine. Nicotine presence in the urine is an indicator of active or passive use dependent on the concentration. Cotinine and 3-OH-cotinine are the primary metabolites of nicotine and are good indicators of secondhand smoke exposure (SHSE) at certain concentrations. THC-COOH is the primary metabolite of delta-9-THC and is the best indicator of cannabis use. Quantitation of the metabolites of nicotine and THC, respectively, can help to provide a clearer understanding of the effects of their use on lung and heart health. MACS/WIHS has a biorepository of over 8000 specimens ready to be analyzed with this method. Although various methods exist for the quantitation of nicotine and THC metabolites respectively, there are none yet published that quantify both simultaneously.
Objectives: To facilitate the quantitation of THC and nicotine metabolites in urine by providing a targeted method for the selected analytes.
Methods: Urine samples are thawed and vortexed, then centrifuged. 200µL of urine are aliquoted to a well in a glass coated plate. 30µL of IMCSzyme glucuronidase mix is added to each sample for the reaction to begin. After 15 minutes, 20µL of deuterium labeled IS mix is added to each sample. As the reaction is taking place, prep of a µElution HLB SPE takes place. 200 µL of MeOH is pushed through to waste with a positive pressure N2 manifold. Next, 200µL LCMS grade H2O is pushed through to prime and equilibrate the sorbent. Next, 200uL of the urine mixed with glucuronidase and IS is added to the sorbent and pushed through. Two washes of 10mM Ammonium Acetate (aqueous) are pushed through. The waste plate is switched out to a collection plate, a new glass coated plate. Two steps of 25µL 50:50 ACN: Ethyl Acetate are used to elute the desired analytes off of the sorbent. The elute is dried down under a stream of N2 for 5 minutes. The dried sample is then resuspended in 50µL of 80:20 MPA:MPB (MPA = 10mM Ammonium Acetate, MPB = 80:20 ACN:MeOH). The plate is sealed and ready for injection. The calibration curve is made by spiking stripped human urine from a range of 1ng/mL to 10,000 ng/mL, the only variation being creatinine which is measured from 0.4-400 mg/dL. The LC/MSMS method is 6.1 minutes beginning at 10%B and gradually reaching a point of 99%B. Chromatographic separation was achieved using a Phenomenex 100mm Kinetex EVO C18 2.6um column. The injection volume is 10µL The detector used is a SciEx 6500+ Qtrap TripleQuadrupole Mass Spectrometer. The MS was run in MRM-ESI mode. The transitions for the chosen analytes and their internal standard counterparts are as follows. The following are done in positive mode, Creatinine 113.9/44.1, Creatinine-d3 116.9/47.1, Nicotine 163.1/130.0, Nicotine-d3 166.0/130.0, Cotinine 177.1/80.0, Cotinine-d3 180.1/80.0, 3-OH-Cotinine 193.0/80.1. The following are done in negative mode, THC-COOH 343.2/299.2, THC-COOH-d9 352.1/308.2. Each analyte also has a qualifier transition used solely for confirmation of the analyte's presence in the sample.
Results: The resulting method’s reproducibility was tested by inter-day injections of calibration curves (1-10000ng/mL)(n=5), and the injection of QCs at 10ng/mL (n=5). The R2 values for all curves are over 0.987, however most averaged over 0.996. All curves were weighted with 1/x, and linear when possible, otherwise quadratic regressions. Inter-day imprecision was less than 0.035% for all curves. The inter-day imprecision for the QCs were 2.0% or less. No carryover was observed for any of the analytes after injections at the high end of the calibration curve (10,000ng/mL). There is no crosstalk with the current set of deuterium-labeled standards (THC-COOH-d3 had some crosstalk with THC-COOH but was replaced with THC-COOH-d9 to resolve the issue). There is some matrix suppression dependent on the saturation of the urine, but the assay is sensitive enough and the concentration of the target analytes is high enough that it does not make a significant difference. A test to determine recovery has not yet been performed but will be done. The LLOQ for nicotine, cotinine, 3-OH-cotinine, and THC-COOH were below 1ng/mL (the lowest calibration concentration), as well as below 0.4 mg/dL for creatinine. The lowest calibrator concentration being much lower than the accepted concentration of what would be considered “positive” in a clinical setting. So far 1702 patient specimens from MACS/WIHS have been run with this method. Within these specimens 527 (31%) have tested positive for THC-COOH (>15ng/mL), 635 (37%) tested positive for nicotine (>15ng/mL), 251 (15%) were positive for 3-OH-Cotinine (>50ng/mL), 280 (16%) were positive for cotinine, with 110 (6%) of the cotinine positives being within the SHSE range.
Conclusion: Although many studies have focused on individual metabolites of nicotine or THC, there has not been a published method that encompasses various metabolites of both compounds. It seems pertinent to run one method that quantifies all the aforementioned metabolites within one method. The developed method is very sensitive with respect to what is considered a positive value within human urine. The method is efficient and more cost-effective and faster than running multiple methods to analyze the various metabolites. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
