Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase
Patent
1998-09-24
2000-10-31
Ceperley, Mary E.
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
435 79, 435 793, 435 794, 435188, 436815, 5303889, 5303898, 530403, 530405, G01N 33536, C07K 1644, C12N 996
Patent
active
061401374
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates generally to the field of the detection of drug metabolites in biological samples. More specifically, it provides a system for developing hapten conjugates and specific antibodies for use in assay systems for detection or quantitation of the title methadone metabolite.
BACKGROUND ART
Methadone has been widely used as an aid in withdrawal from heroin addiction. Compliance with methadone therapy is frequently monitored by analysis of urine samples for the presence of methadone, which can be performed using one of several commercially available immunoassays for methodone.
However, there are several occasions when a simple assay for methadone provides in incorrect or incomplete diagnostic information. For example, the methadone may be so extensively metabolized that the concentration excreted falls below that of the assay being used. To the extent that the assay distinguishes between methadone and excreted metabolites, the test can be negative even for a patient in full compliance with therapy (Nicar et al., Clin. Chem. 42:543, 1996). In another example, a patient may add methadone to their sample to disguise the fact that they are not adhering to the treatment protocol. Samples that have been tampered with can in principle be distinguished in that they will not contain filterable metabolites of methadone that are also present in urine when the methadone treatment protocol is being properly adhered to.
Both these types of situations could be easily recognized if it were possible to independently measure the presence of the excreted metabolite. In practical terms, detecting methadone metabolites is difficult because of the chemical and immunological similarity with methadone itself. Accordingly, there is a need for reagents and techniques that would permit routine monitoring of methadone metabolites in a clinical setting.
A major pathway of metabolism of methadone is demethylation to the presumed intermediate N-desmethylmethadone, leading to urinary excretion of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine herein abbreviated as EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), and their ring-hydroxylated analogs (Sullivan et al., J. Medicinal Chem. 16:909, 1973). These structures are depicted in FIG. 1. Lesser amounts of 4-dimethylamino-2,2-diphenylvaleric acid (formed by side-chain oxidation); 1 ,5-dimethyl-3,3-diphenyl-2-pyrrolidone (resulting from subsequent N-demethylation and cyclization), ring-hydroxylated methadone, and normethadol are also found. Methadone N-oxide is formed by storage of samples at 30.degree. C. In human volunteers receiving.about.100 mg of dl-methadone orally, EDDP was found by Sullivan et al. to be the most prominent metabolite in urine, present at a level roughly comparable with that of methadone itself.
In order to detect methadone metabolites, Sullivan et al. extracted urine with methylene chloride, hydrolyzed the reconstituted extract using .beta.-glucuronidase and aryl sulfatase, optionally acetylated or methylated the product, and then characterized it by gas chromatography or combined gas chromatography-mass spectroscopy.
The synthesis and spectral properties of optically active EDDP were described by Brine et al. (J. Heterocyclic Chem., 23:369, 1986). The .sup.1 H--NMR analysis suggested that the free base exists predominantly in an enamine form. CD and ORD studies provided data consistent with a fairly rigid enamine structure.
Simultaneous gas chromatography mass spectrometry (GC/MS) assay for EDDP in urine was described by Baugh et al. (J. Forensic Sci. 36:548, 1991). Urine was extracted with 1-chlorobutane at pH.about.9, the organic phase was back-extracted into acetate buffer, adjusted to pH.about.9, and re-extracted with I -chlorobutane. Area corresponding to ions at m/z 277, 262, and 276 was measured. Quantitation was enhanced by using deuterated methadone as the internal standard. Although the assay is quantitative, it relies on a multi-step extraction procedure and the availability of equipment to perform GC/MS.
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Sung et al.,
Coty William A.
Powell Michael J.
Sanchez Anthony J.
Sigler Gerald F.
Ceperley Mary E.
Microgenics Corporation
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