Comparative LC/MS Analysis of the Extraction of Cocaine and its Metabolites Using QuEChERS and Column Extraction Methods

Abstract

<b>Abstract ID 56031</b> <b>Poster Board 97</b> Cocaine abuse is a major public health problem worldwide and monoclonal antibodies are at the forefront as a possible treatment for its abuse. Our laboratory has developed a recombinant humanized anti-cocaine monoclonal antibody, known as h2E2, and a Fab fragment that has shown promising results in animal studies. In order to study the efficacy of this antibody against cocaine, we have also developed methods to measure cocaine and its metabolites in rodent tissue and fluids such as brain, blood, or urine. We compared two cocaine extraction methods, the Quick Easy Cheap Effective Rugged Safe (QuEChERS) salt mix of NaCl, MgSO4, sodium citrate (dihydrate + sesquihydrate); and United Chemical Technologies (UCT) Clean Screen DAU columns. Cocaine extracted from urine by both methods was quantified via a Shimadzu LCMS 8060. Our objective was to find a quick and reproducible extraction method. We used 3D printing to develop custom holding stands for the columns. QuEChERS is a dispersive solid-phase extraction method for sample preparation. For this comparison, we used urine from a study examining the effect of the Fab fragment of h2E2 on the distribution of cocaine and its metabolites in Sprague-Dawley rats per IUACUC regulations. We selected a subset of urine samples (n=9) that would have high (&gt;1000ng/mL), mid (≤100ng/mL), and low (≤10ng/mL) levels of cocaine. We compared a 10-fold dilution of the extracts obtained by the two methods. The urine samples were extracted with 50mg of preweighed QuEChERS and 200 uL acetonitrile. The organic supernatant (150uL) of cocaine and its metabolites was used for the analysis. For the initial estimates, the standards used ranged from 3.2-7000ng/mL. Six months later, a 10-fold dilution of the samples, with a similarly diluted standard set, was analyzed (0.3-700ng/mL). The columns utilize both a reverse (C8) phase and an ion exchange (benzenesulfonic acid) phase bonded to silica gel. The extraction of cocaine and its metabolites was carried out as recommended by the manufacturer’s instructions. Fused deposition modeling (FDM), commonly known as 3D printing, was used to make custom holders for the extraction columns through design iteration. The final eluate was collected with the 3D printed stands and then evaporated with a Reacti-Therm III Heating Module(using air). This was reconstituted in 150uL of acetonitrile and stored in vials for analysis. For both extraction methods, 0.5uL of each sample was injected into the instrument. Samples (n=48) in duplicate were analyzed twice over 41 days between experiments using the QuEChERS method. Each sample concentration was within 25% over the two days. The three sample levels were reanalyzed at a 10x dilution using the two methods. All but one (20.7%) of the high samples were within 20% of the first run for both methods. For the mid to low concentrations, none of the values fell within 25% of the earliest run for either method which needs further investigation. This will be done with naive urine spiked with similar concentrations of cocaine and its metabolites. For the QuEChERS method, the most tedious step was weighing the salts. Comparatively, the other process needed conditioning, application, washing, and elution of the column before drying and reconstituting the sample. Overall, we found the QuEChERS method of extracting cocaine and its metabolites to be easier to implement with fewer required steps. Thanks to United Chemical Technologies for providing the columns used Supported by NIDA grant U01DA050330