Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection.

The analysis of morphine in biological fluids is of vital interest in monitoring opiate abuse and in drug abuse research. Although methods for analysis of morphine and its metabolites are well established, studies are still being carried out to improve sample preparation procedures as well as detection levels of morphine in biological samples. In this study, morphine-specific immunosorbents were developed to concentrate morphine prior to HPLC analysis. Urine (0.1 ml) was diluted 10-fold with phosphate-buffered saline, pH 7.4 (PBS), loaded onto a solid-phase immunoextraction column and washed with 15 ml PBS followed by elution with 2 ml of elution buffer (40% ethanol in PBS, pH 4). The eluted fraction was analysed for morphine by HPLC-electrochemical detection using a cyanopropyl (CN) analytical column with 25% acetonitrile in phosphate buffer-sodium lauryl sulphate, pH 2.4 as the mobile phase. Duration of the extraction procedure was approximately 40 min. Calibration graphs were linear from 100 ng ml-1 to 500 ng ml-1 in urine. The inter-assay R.S.D. was < 10% and the recovery of morphine from urine was > 98%. Immunocolumns demonstrated remarkably high specificity towards morphine showing minimal binding with other opiate metabolites such as codeine, normorphine, norcodeine, morphine-3-glucuronide, morphine-6-glucuronide.

Enzyme immunoaffinity chromatography--a rapid semi-quantitative immunoassay technique for screening the presence of isoproturon in water samples.

Immunochromatography devices based on the principles of affinity chromatography and enzyme immunoassay have been developed to illustrate the possibility of providing extra-laboratory 'in the field' tests for pesticide monitoring. Isoproturon was chosen in this study as an example though other pesticides could have been used provided a suitable antiserum existed. The test system was prepared by immobilising isoproturon antibodies to porous silica which were then packed into disposable columns (immunoaffinity columns). The addition of chromagen-substrate 3,3',5,5'-tetramethylbenzidine into the immunoaffinity columns, after the application of a mixture containing an equal volume of isoproturon samples or isoproturon standard solutions with a fixed concentration of isoproturon labelled with horseradish peroxidase, allowed the development of a colorimetric portable assay capable of screening samples qualitatively for the presence of isoproturon in water. Samples with contamination levels of 0.12 microgram l-1 isoproturon and above were visually identified as positive samples in comparison to the zero standard sample. However, samples with concentrations below this level would be considered as negative (no isoproturon present at this limit of detection). The duration of the sample screening procedure on the column was less than 25 min making the technique highly suitable for a rapid estimate of isoproturon concentrations. Furthermore, the system could estimate isoproturon concentrations in water from various sources with no requirement for sample preparation. Therefore, the technique is ideally suited for monitoring the presence of pesticides in water. Further refinement of the assay could result in a test suitable for use by non-skilled personnel in extra-laboratory locations (e.g., a mobile or field laboratory).

Competitive enzyme-linked immunosorbent assay for the determination of the phenylurea herbicide chlortoluron in water and biological fluids.

A competitive ELISA method suitable for the monitoring of the herbicide chlortoluron [N-(3-chloro-4-methylphenyl)-N'-dimethylurea] in different types of water and biological fluids was developed. The production of the immunogen utilized in this work was achieved by covalently coupling bovine thryroglobulin with the synthesized hapten (N'-3-chloro-4-methylphenyl-N-carboxypropyl urea) using the N-hydroxysuccinimide active ester method. The chlortoluron antibody, raised in sheep after immunization with the immunogen, showed no cross-reactivity with a large range of pesticides, although some cross-reactivity was displayed with various phenylurea herbicides (i.e., chlorbromuron, isoproturon and metoxuron). The limit of detection of the chlortoluron ELISA method was 0.015 microgram l-1, well below the legal European limit for individual pesticides in drinking water (the EC maximum admissible concentration, 0.1 microgram l-1). In addition, reproducible and quantitative recovery of chlortoluron from water, obtained from various sources, and biological fluids was possible without any sample preparation. The ELISA technique for chlortoluron developed and described here proved to be rapid, sensitive and specific, fulfilling the needs of present legislation relating to the use and levels of pesticides in the environment.

Development and evaluation of a chemiluminescent immunoassay for chlortoluron using a camera luminometer.

The feasibility of using an enhanced chemiluminescent immunoassay for screening water samples was adapted to a Dynatech Microlite camera luminometer and assessed to provide a semiquantitative assay based on a photographic record of the luminescent end-point. An enhanced chemiluminescent immunoassay of the herbicide chlortoluron was chosen in this instance as an example, although other pesticide compounds could equally well have been used provided that suitable antisera were available. This luminescent assay has shown considerable potential by providing a rapid, simple and portable means of monitoring multiple water samples for the presence of chlortoluron. The assay was able to identify samples containing the herbicide at or above the European limit for individual pesticides in drinking water [the EU Maximum Admissible Concentration (MAC) = 0.1 microgram l-1]. A 100% accuracy was obtained in the analysis of samples containing chlortoluron at concentrations above the range of 0.07-0.12 microgram l-1; however, for concentrations within this range the results were 65% accurate. Consequently, the method could greatly facilitate increased monitoring for the presence of chlortoluron in water supplies and in other areas of environmental analysis.

Development of an enzyme-linked immunosorbent assay for isoproturon in water.

A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon [3-(4-isopropylphenyl)-1,1-dimethyl urea] in water has been developed. A derivative of isoproturon [3-(4-isopropylphenyl)-1-carboxypropyl-1-methyl urea] has been synthesized and linked to thyroglobulin using the N-hydrosuccinimide reaction. The immunogen was used to immunize two sheep, which both responded by producing specific antibodies to isoproturon with little cross-reactivity to various structurally related and unrelated pesticides. The enzyme label was prepared by coupling the hapten to horseradish peroxidase using the above reaction. The coated-antibody competitive ELISA, which has a sensitivity of 0.03 microgram l-1, permits the direct determination of isoproturon in various water matrices and can facilitate the monitoring of water quality in drinking-water supplies.