Morphine assay

K. E. Rasmussen, Quantitative morphine assay by means of gas-liquid chromatography and on-column silylation, J. Chromatogr., 120 491 (1976). 

These site heterology experiments with the morphine assay show that substitution at the R position with linking groups yielded the best selectivity for morphine over codeine. The reason is that the R group is the same for morphine and codeine, and the R group is far from the R group, which is where morphine and codeine are structurally different. The antibodies raised to this conjugate thus have a paratope... 

Figure 6.13. Selectivity of morphine assay in the presence of codeine.

Exogenously administered substances are not the only agents which might interfere with a receptor binding assay. For example, opiate receptor binding as a radioreceptor assay for morphine would appear to be a relatively specific procedure since only opiates can interfere with the attachment to this receptor site. However, the discovery of endogenous opioid peptides makes the morphine assay less feasible with brain tissue because of the potential for these substances to be present in the tissue extracts. 

For morphine assay, the B.P, extraction is essentially that due to Nicholls, described above. 

The U.S.P, method of morphine assay is somewhat cumbersome but the alkaloid is finally titrated if sufficient sample (100 ml) is available the B,P. method may be followed until evaporation of the chloroform-ethanol mixture and then the alkaloids determined by solution in excess of 0 02N acid and back-titration after boiling the solution, using methyl red as indicator (1 ml 0-02N — 0 005707 g anhydrous morphine). 

Squill contains substances which interfere with the morphine assay but they can be removed by extraction with alcohol-free chloroform. 

Specific IgE Assay. Two radioimmunoassays are available in France using a quaternary ammonium compoimd coupled to Sepharose [30, 31]. The sensitivity of these tests was equivalent at 88%, the specificity reaches 90%. A morphine-based immunoassay has been proposed in Australia [14]. More recently, Ebo et al. [32] investigated a rocuronium ImmimoCAP and set the sensitivity at 85%, the specificity being absolute, provided an assay-specific decision threshold is applied. An ImmimoCAP (Phadia A) is available. 

Colorimetric procedures used In steroid assays are often subject to drug Interference. In the determination of 17-Ketosterolds by the Zimmerman reaction, drugs with the 17-Keto basic structure such as ascorbic acid, morphine and reserplne will cause Increased values. In the determination of 17,21 -dlhydroxysterolds by the Porter-Sllber reaction the dlhydroxy-acetone chain Is the reactive unit. Drugs like meprobamate, chloral hydrate, chloropromazlne and potassium Iodide will Interfere with this reaction and cause elevated values. In the colorimetric determination of vanlllylmandellc acid (VMA) by a dlazo reaction, drugs like methocarbamol and methyl dopa cause... 

Hydromorphone (I) and hydrocodone (II) belong to the morphine group of drugs and are used invariably in combination with other ingredients in a number of proprietory antitussive and analgesic antipyretic mixtures. However, interest in the pharmacokinetics of hydromorphone and hydrocodone in human subjects required an adequate assay for drug levels in plasma. 

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

The analysis of codeine, morphine, 6-monoacetylmorphine (6-MAM, a metabohte of heroin), and cocaine is important for many toxicology labs to determine illicit drug use. When analyzing opiates in urine samples, frequently the matrix chosen for drug screening, the conjugated metabolites must be hydrolyzed however, this process can break down 6-MAM (Christophersen et al., 1987). These compounds can be derivatized to increase sensitivity, and both BCD and NPD are used for these assays. Derivatizations used include reaction with N-methyl-N-trimethylsilyltrifluoroacetamide followed by GC-FID (Lin et al., 1994) or with N,0-bis(trimethylsilyl)trifluoroacetamide (Christophersen et al., 1987 Lee and Lee, 1991), PFPA (Christophersen et al., 1987), or heptafluorobutyric anhydride (HFBA) followed by GC-ECD. All these methods show good sensitivity and selectivity. 

Rotshteyn Y, Weingarten B. 1996. A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatography with electrochemical and fluorescence detection. Ther Drug Monit 18(2) 179-188. 

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