Combined Enzyme Immunoassay-LCEC Method for the Identification, Confirmation, and Quantitation of Opiates in Biological Fluids

Abstract

An enzyme multiplied immunoassay technique (EMIT) was used to test for opiates (morphine, hydromorphone, and codeine) in extracts of blood, bile, and tissue homogenates. All immunoassay opiate positive specimens were then tested by a reversed-phase liquid chromatographic procedure using electrochemical detection (LCEC). Blood specimens were then quantitated by LCEC. The sensitivity of the immunoassay (as morphine) was 0.020 mg/L, 0.200 mg/L, and 0.100 mg/kg for blood, bile, and tissue homogenates, respectively, with 2% intrarun and 7% interrun precision. The LCEC method was linear from 0.005 to 0.300 mg/L for morphine, hydromorphone, and codeine (nalorphine internal standard) with detection limits of 0.005 mg/L for each analyte. Intrarun and interrun precision varied from 1 to 2% and 6 to 11%, respectively. Recoveries, using a double extraction technique, ranged from 70 to 95%. These two methods, applied to 495 post mortem cases, demonstrated a 6% incidence of opiates, with no false positives.

Forensic science

“…It involves hydrolysis with two-dimensional TLC analysis using ferric chloride/ferricyanide visualization (73). Analysis and identification of heroin and related opiates in biological fluids has been accomplished by GLC (74,75), HPLC (76)(77)(78)(79)(80)(81), GC/MS (82), thin-layer immunoassay (83), and a combined enzyme immunoassay-LCEC method (84). Pholcodeine, a common component in cough medicines, has been reported as a possible interfering compound in the immunoassay for opiates in urine (85).…”

Bile Analysis in Heroin Overdose

“…The EHC may prolong the pharmacological effect of the drug or drug metabolites (3–5). Furthermore, many of these drugs accumulate in bile, and the gall bladder serves as a collection and storage depot for xenobiotics (6–8). In fact, it has been shown that these drugs have significantly higher concentrations in bile than in blood (7,8).…”

“…Furthermore, many of these drugs accumulate in bile, and the gall bladder serves as a collection and storage depot for xenobiotics (6–8). In fact, it has been shown that these drugs have significantly higher concentrations in bile than in blood (7,8). Among the xenobiotics excreted in the bile, there are some drugs of abuse such as opioids (7,9).…”

“…Among the xenobiotics excreted in the bile, there are some drugs of abuse such as opioids (7,9). Bile concentrations of morphine, the main metabolite of heroin, have been shown to be often several‐fold higher than in blood (7,8,10). Morphine is mainly metabolized in the liver by conjugation with glucuronic acid to form morphine‐6‐glucuronide (M6G) and morphine‐3‐glucuronide (M3G).…”