Low Extraction Efficiency in Liquid-Liquid Extraction and Quantification of Nicotine, Opioids and Cannabinoids from Plasma, Brain, and Placental Tissue Dominique Figueroa (Presenter) University of Maryland

Authors: Dominique B. Figueroa, Maureen A. Kane
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD

Methods for extraction and quantification of nicotine and its metabolites, opioids, and cannabinoids in plasma, brain, and placenta using liquid-liquid or solid phase extraction have been published, however due to the differing chemistries of these compounds, simultaneous extraction and subsequent detection has yet to be achieved. This assay aims to apply liquid-liquid extraction of nicotine, opioids, and cannabinoids in plasma, brain and placental tissue toward simultaneous detection of these chemically diverse compounds. We explore different methods of liquid-liquid extraction, aiming for the highest extraction efficiency.

Problem

We aim to extract and quantify nicotine and its metabolites, opioids, and cannabinoids in plasma, brain, and placental tissue using liquid-liquid extraction, however due to the differing chemistries of these compounds, we are unable to achieve consistent extraction efficiency across all compounds greater than 50%.

Method Information

Compounds included in assay development were caffeine, nicotine, cotinine, trans-hydroxycotinine, morphine, 6-acetylmorphine (6AM), ecgonine methyl ester, benzoylecgonine, codeine, cocaine, cannabidiol (CBD), tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol (11-nor-9-TCH-COOH). To examine extraction from tissue, brain and placental tissue were suspended at 20 mg/mL in 50% methanol, 50% water and homogenized using a Precellys 24 bead beater. Neat drug standards were then spiked into the homogenate. The resulting spiked homogenate was exposed to 1 mL of methanol, acetonitrile, and 0.1% formic acid in acetonitrile. Extraction of compounds from plasma used 50 µL of drug-spiked plasma exposed to the same extraction solvents. LC-MS/MS was applied using a WatersTM I-Class UPLC and a Xevo TQ-S mass spectrometer operating in positive ion mode.

Troubleshooting Steps

This method aims to examine extraction of nicotine, opioids, and cannabinoids in human plasma, brain, and placental tissue. Differing solubilities of the nicotine and cotinine alkaloids, polar opioids, and less polar cannabinoids yield inconsistent recoveries across compounds. Extraction of nicotine and opioids using methanol was > 50% while extraction of cannabinoids yielded a <50% recovery. Using acetonitrile as an extraction solvent generated similar results while addition of 0.1% formic acid to acetonitrile extraction resulted in a 50% of greater recovery of all compounds including cannabinoids.

Outcome

Current studies are exploring further optimization of extraction methods, examining homogenate concentration and other solvents such as chloroform-methanol extraction. Quantification of nicotine, opioids, and cannabinoids was performed using targeted quantitation on a tandem quadrupole mass spectrometer according to a unique precursor to product ion transition for each compound. The quantitative method was validated in accordance with the Guidance for Industry: Bioanalytical Method Validation by the US Food and Drug Administration for selectivity, sensitivity, linearity, carryover, dilution integrity, accuracy, precision, recovery, matrix effect, and stability (freeze-thaw, bench-top, and autosampler/post-preparative stability). The assay will be applied to clinical samples of plasma, fetal brain, and placenta to investigate the effect of in utero exposure to nicotine, opioids and cannabinoids.