Detection of Cannabinoids by LC–MS-MS and ELISA in Breast Milk
Cristina Sempio, Erica Wymore, Claire Palmer, Maya Bunik, Thomas K. Henthorn, Uwe Christians, Jost Klawitter
Abstract
Cannabis is the most commonly used drug of abuse in pregnancy and after delivery. However, little is known regarding the disposition of cannabinoids in breast milk, although delta-9-tetrahydrocannabinol (THC), the main psychoactive component, is highly lipophilic. Quantification of cannabinoids in breastmilk is essential for clinical monitoring and research studies and breastmilk banks mainly rely on enzyme-linked immunosorbent assay (ELISA) in terms of screening for cannabinoids. To support clinical studies on disposition of cannabinoids in breastmilk, we validated a high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay for the simultaneous quantification of 12 cannabinoids and their metabolites in human breast milk. Said assay was based upon a simple one-step protein precipitation, online column extraction and detection in the positive multiple reaction monitoring mode. After successful validation, the assay was used to analyze 30 samples from a clinical research study that had tested negative using an ELISA kit that is commonly used by breastmilk banks. In human breast milk, depending on the analyte, the lower limits of quantification of the LC-MS-MS assay ranged from 0.39 to 7.81 ng/mL. Acceptance criteria for intra- and inter-batch accuracy (85-115%) and imprecision (<15%) were met for all compounds. Mean extraction efficiencies were above 60% for all analytes. Mean matrix effect ranged from -12.5% to 44.5% except of THC-glucuronide for which significant matrix effects were noted. No carry-over was detected. Although cannabinoid-negative based on the ELISA, all 30 samples tested positive for THC using LC-MS-MS (0.8-130 ng/mL) and several also for 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD) and cannabigerol (CBG). We validated a sensitive and specific assay for the quantification of 12 cannabinoids in human breastmilk that outperformed an ELISA commonly used by breastmilk banks.