Unique Structural/Stereo-Isomer and Isobar Analysis of Novel Fentanyl Analogues in Postmortem and DUID Whole Blood by UHPLC–MS-MS

The presented analytical method enabled the Toxicology Department at the Cuyahoga County Medical Examiner’s Office to identify 26 and quantitatively report 24 compounds in 500 μL of whole blood, including fentanyl analogues (fentalogues) such as methoxyacetyl fentanyl (MeOAF) and cyclopropyl fentanyl (CPF). This second-generation method (FG2) was developed with the objective to improve the existing analysis (FG1) by decreasing sample size, lowering limits of detection (LOD) and lower limit of quantitation, minimizing ion suppression and resolving chromatographic interferences. Interferences may occur in the analysis of fentanyl, MeOAF, CPF, 3-methylfentanyl (3MF), butyryl fentanyl and isobutyryl fentanyl due to isobars and structural or geometric isomerism with another analogue or metabolite. The isomeric and isobaric fentalogues were grouped into three sets. The LOD established for Set 1 [MeOAF, para-methoxyacetyl fentanyl, para-fluoro acryl fentanyl (isobar), fentanyl carbamate], 2-furanyl fentanyl, Set 2 [CPF, (E)-crotonyl fentanyl] and carfentanil was 0.0125 ng/mL. The LOD established for N-methyl norfentanyl, norfentanyl, norcarfentanil, despropionyl fentanyl (4-ANPP), acetyl fentanyl, β-hydroxy fentanyl, benzyl fentanyl, acryl fentanyl, alfentanil, fentanyl, para-fluoro fentanyl, Set 3 [(±)-trans-3MF, (±)-cis-3MF, isobutyryl and butyryl fentanyl], para-fluoroisobutyryl fentanyl, sufentanil, phenyl fentanyl and cyclopentenyl fentanyl was 0.0625 ng/mL. Seven-point linear calibration curves were established between 0.025 and 4.0 ng/mL for the 8 analytes with the lower LOD and 0.125 and 20 ng/mL for the 18 analytes with the higher LOD. 4-ANPP and cyclopentenyl fentanyl met qualitative reporting criteria only. The results for five postmortem and two driving under the influence of drugs authentic case samples are presented. To the authors’ knowledge, FG2 is the first published method that achieved baseline resolution of the nine structural/stereo isomers and one isobar by ultra-high performance liquid chromatography–MS-MS and provided quantitative validation data for nine compounds. FG2 may be used as the new baseline for future isomers that need to be chromatographically separated.

Evaluation of a Method for Determining Concentrations of Isoeugenol, an AQUI-S Residue, in Fillet Tissue from Freshwater Fish Species

AQUI-S is a fish anesthetic/sedative that is approved for use in a number of countries throughout the world and has the potential for use in the United States. The active ingredient in AQUI-S is isoeugenol. A method for determining isoeugenol concentrations in edible fillet tissue is needed for regulatory purposes, including surveillance and potential use in studies fulfilling human food safety data requirements if U.S. Food and Drug Administration approval is pursued. A method was developed and evaluated for determining isoeugenol concentrations in fillet tissue using relatively common procedures and equipment. The method produced accurate and precise results with fillet tissue from 10 freshwater fish species. The percentage of isoeugenol recovered from samples fortified with isoeugenol at nominal concentrations of 1, 50, and 100 g/g for all species was always >80 and <97. Within-day precision for samples fortified at those same concentrations was 10, and day-to-day precision was 4.0. Method precision with fillet tissue containing biologically incurred isoeugenol was 8.1. There were no or minimal chromatographic interferences in control fillet tissue extracts from 9 of the 10 species. The method detection limits for all but one species ranged from 0.004 to 0.014 g/g, and the quantitation limits ranged from 0.012 to 0.048 g/g.

Validation of HPLC-amperometric detection to measure serotonin in plasma, platelets, whole blood, and urine

We describe an isocratic liquid-chromatographic method with amperometric detection for determination of serotonin by rapid sample preparation. Platelet-poor plasma and platelets were injected after a single deproteinization step with perchloric acid. Addition of sodium borohydride to whole blood avoids oxidation of serotonin during the deproteinization step without any chromatographic interferences. We purified urinary serotonin by two successive cationic and anionic extraction steps. After urine dilution, urinary 5-hydroxyindoleacetic acid (5-HIAA) was measured under the same chromatographic conditions. Platelet serotonin concentrations correlated more closely with whole-blood serotonin concentrations after correction for platelet number than with concentrations expressed in nmol/L. This suggests that whole-blood serotonin measurements should be corrected for platelet count to eliminate the variability of circulating platelets. Combined determination of serotonin in whole blood and urine and of 5-HIAA in urine provides a useful tool for detecting and monitoring carcinoid tumors.