Rapid Confirmation of Enzyme Multiplied Immunoassay Technique (EMIT®) Cocaine Positive Urine Samples by Capillary Gas-Liquid Chromatography/Nitrogen Phosphorus Detection (GLC/NPD)

Abstract

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT®) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 µL of pentafluoropropionic anhydride and 25 µL of pentafluoropropropanol at 90°C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 µm of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.

Intrinsic Fluorescence-Based Optical Fiber Sensor for Cocaine Using a Molecularly Imprinted Polymer as the Recognition Element

“…There are several major analytical methods available for the analysis of cocaine and its metabolites including gas chromatography mass spectrometry (GC/MS) [3], [4], high performance liquid chromatography (HPLC) [5], [6], thin layer chromatography [7], voltammetry [8], radioimmunoassay [9] and enzyme-linked immunosorbent assay (ELISA) [10]. These traditional methods, despite having achieved very good results, are generally expensive, time consuming and cumbersome for real-time measurements outside the laboratory, some of which also require sample clean-up and derivatization of cocaine prior to analysis.…”

Derivação de benzoilecgonina urinária com diazometano para verificação da exposição à cocaína por técnicas cromatográficas

“…Métodos analíticos para a identificação de cocaína na urina e seu principal produto de biotransformação (benzoilecgonina) têm sido publicados, utilizando-se de técnicas imunológicas (Ramcharitar et aI., 1995); (Guimarães, 1998), cromatografia em camada delgada (Budd et aI., 1980); (Chasin & Lima, 1998), cromatografia em fase gasosa com detetor de ionização de chama (Von Minden & D'Amato, 1977); (Falk & Harrison, 1985); (Chasin & Lima, 1998) ou nitrogênio-fósforo (Froldi et aI., 1984); (Verebey & Depace, 1989) cromatografia líquida de alta eficiência (Balikova & Vecerkova, 1994); (Clauwaert et aI., 1996) e espectrometria de massa (Mulé & Casella, 1988); (Gerlits, 1993); (Aderjan et aI., 1993); (De Giovanni & Rossi., 1994); (Diamond et aI., 1996).…”

“…A benzoilecgonina é altamente polar e hidrossolúvel e não é facilmente extraída empregando-se solventes orgânicos em extrações líquido-líquido (Hamilton et aI., 1977); (Jain et aI., 1977); (Graas & Watson, 1978). Grandes volumes de solventes (representando cerca de 5-25 vezes o volume de amostra) ou extrações sucessivas são necessárias para produzir razoável índice de recuperação da urina (56-70%) (Wallace et aI, 1976); (Jain et aI., 1977); (Froldi et aI., 1984); (Mulé & Casella, 1988); (Verebey & Depace, 1989); (Gerlits, 1993).…”

“…Apesar da adição de álcoois como etanol ou isopropanol aumentarem a eficiência da extração de benzoilecgonina, esse procedimento também aumenta a extração de substâncias endógenas tipicamente presentes na urina, provocando interferências e possibilitando a contaminação de detetores utilizados em cromatografia em fase gasosa (Verebey & Depace, 1989); (Jennison et aI., 1994).…”

“…Hexafluoroisopropanol (HFIP) ou pentafluoropropanol (PFP), juntamente com anidrido pentafluoropropiônico (PFPA) reagem com o grupo funcional carboxílico gerando os respectivos ésteres halogenados (Mulé & Casella, 1988); (Verebey & Depace, 1989); (Bodor et aI., 1990); (Gonzales et aI., 1995 (Mulé & Casella, 1988). Já o hexafluoroisopropil-derivado de benzoilecgonina é estável por pelo menos quatro dias se mantido a -20°C (Aderjan et aI., 1993).…”