Endogenous morphine‐6‐glucuronide (M6G) is present in the plasma of patients: Validation of a specific anti‐M6G antibody for clinical and basic research

Abstract

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers. While endogenous morphine levels are detectable using enzyme-linked immunosorbant assay (ELISA), mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. This study describes the preparation of a specific anti-M6G rabbit polyclonal antibody and its validation. The specificity of this antibody was assessed against 30 morphine-related compounds. Then, a M6G-specific ELISA-assay was tested to quantify M6G in the plasma of healthy donors, morphine-treated, and critically ill patients. The antibody raised against M6G displays a strong affinity for M6G, codeine-6-glucuronide, and morphine-3-6-glucuronide, whereas only weak cross-reactivities were observed for the other compounds. Both M6G-ELISA and LC-MS/MS approaches revealed the absence of M6G in the plasma of healthy donors (controls, n = 8). In all positive donors treated with morphine-patch (n = 5), M6G was detected using both M6G-ELISA and LC-MS/MS analysis. Finally, in a study on critically ill patients with circulating endogenous morphine (n = 26), LC-MS/MS analysis revealed that 73% of the positive-patients (19 of 26), corresponding to high M6G-levels in M6G-ELISA, contained M6G. In conclusion, we show that endogenous M6G can be found at higher levels than morphine in the blood of morphine-naive patients. With respect to the interest of measuring endogenous M6G in pathologies, we provide evidences that our ELISA procedure represents a powerful tool as it can easily and specifically detect endogenous M6G levels.

Revealing the Kinetic Advantage of a Competitive Small-Molecule Immunoassay by Direct Detection

“…Cocaine is a narcotic, while methadone and morphine are both opioids which are known to bind to μ-opioid receptors . These small-molecules are well-known drugs with extensively documented histories of use for both pharmaceutical and recreational purposes. − All three of these drugs act upon the central nervous system and are highly addictive . Immunoassays for the screening of small-molecule drugs are of special interest, as detection and quantification of both prescription and illicit drugs is relevant for toxicological and forensic analyses.…”

Simultaneous analysis of gemfibrozil, morphine, and its two active metabolites in different mouse brain structures using solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry with a deuterated internal standard

“…There are several methods used to detect morphine or its main metabolites M3G and M6G in animal tissues, but not in brain samples [19][20][21][22][23][24]. However, most of them used different molecules as internal standard (IS) such as donepezil, codeine, and naloxone hydrochloride [19][20][21][22].…”

“…However, most of them used different molecules as internal standard (IS) such as donepezil, codeine, and naloxone hydrochloride [19][20][21][22]. There are large differences in the physical and chemical properties among gemfibrozil, morphine, and morphine metabolites.…”