Derek Stevenson

Molecular imprinted polymers for solid-phase extraction

Solid-phase extraction (SPE) has become the method of choice in many laboratories for the analysis of complex samples. Recently, highly selective extraction based on antibody columns or molecular imprinted polymers (MIPs) has been developed. To date biological antibodies have shown better specificity but MIPs are easier to produce. Several procedures using MIPs for SPE are reviewed along with the preparation and evaluation of a MIP for SPE of atenolol.

Protein crystallization and biosensor applications of hydrogel-based molecularly imprinted polymers.

We have characterized the imprinting capability of a family of acrylamide polymer-based molecularly imprinted polymers (MIPs) for bovine hemoglobin (BHb) and trypsin (Tryp) using spectrophotometric and quartz crystal microbalance (QCM) sensor techniques. Bulk gel characterization on acrylamide (AA), N-hydroxymethylacrylamide (NHMA), and N-isopropylacrylamide (NiPAM) gave varied selectivities when compared with nonimprinted polymers. We have also harnessed the ability of the MIPs to facilitate protein crystallization as a means of evaluating their selectivity for cognate and noncognate proteins. Crystallization trials indicated improved crystal formation in the order NiPAM<AA<NHMA. QCM studies of thin film MIPs confirm this trend with N-hydroxymethyl acrylamide MIPs exhibiting best discrimination between MIP and NIP and also cognate/noncognate protein loading. Equivalent results for acrylamide MIPs suggested that the cavities were equally selective for both proteins, while N-isopropylacrylamide MIPs were not selective for either cognate BHb or noncognate BSA. All BHb MIP-QCM sensors based on AA, NHMA, or NiPAM were essentially nonresponsive to smaller, noncognate proteins. Protein crystallization studies validated the hydrophilic efficacy of MIPS indicated in the QCM studies.

Development and optimisation of an immunoaffinity-based solid-phase extraction for chlortoluron

Abstract The determination of trace organics such as pesticides in biological and environmental samples require rapid, easy-to-use, reliable sample preparation procedures. Solid-phase extraction using silica or bonded silicas has proven useful for broad-range screening. We have used antisera to chlortoluron immobilised onto silica as a “tailor-made” solid-phase extraction system. The chlortoluron can be selectively retained and eluted using a simple phosphate buffered saline/ethanol mixture. Preliminary studies have demonstrated that the immuno column has a high volume breakthrough (at least 11 of water) and can retain up to 500 ng of chlortoluron. Quantitative recovery from tap water, river water, drinking water, plasma and urine is achieved along with an HPLC trace free from co-eluting compounds at the chlortoluron retention time.

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.

Rapid method for the determination of organochlorine pesticides in milk

This method involved one step solvent extraction of milk with ethyl acetate-acetone-methanol by ultrasonication. The supernatants were further cleaned-up and enriched by solid-phase extraction using octadecyl (C18)-bonded silica cartridges, then assayed by capillary gas-liquid chromatography with electron capture detection. The recoveries of eleven organochlorine pesticides (OCPs) from raw milks were quantitative, ranging from 90-110% at 10 times the limit of detection (LOD). The LOD ranged from 0.5 micrograms/l whole milk for alpha-hexachlorocyclohexane to 2.5 micrograms/l whole milk for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. The day-to-day variation of the method was evaluated over 7 days using 3 different pools of spiked cow milks (at the LOD, 5 and 10 times the LOD). The coefficient of variations (C.V.s) were 16 +/- 6, 10 +/- 2 and 9 +/- 3% (mean S.D.), respectively. The method showed no emulsion problems common with conventional non-polar solvent extraction, and the use of solid-phase extraction considerably reduced the sample clean-up process compared with the existing methods. The method also showed that OCPs in milk could be extracted quantitatively without extraction of total fat, and that OCPs spiked into cows milk could be used to construct calibration curves for human milk determinations.

Highly selective antibody-mediated extraction of isoproturon from complex matrices

SummaryFor the analysis of pesticides such as isoproturon in complex matrices the rate determining step is sample preparation. A novel solid-phase extraction system for isoproturon based on antibody mediated extraction has been developed. The isoproturon can be retained while the immuno-extraction column is washed with phosphate buffered saline and eluted using phosphate buffered saline/ethanol at low pH. The column can preconcentrate isoproturon from up to at least 1000 mL water and elute in 1 mL. Quantitative recovery of isoproturon from water, plasma and urine can be obtained and the proposed water assay can detect as low as 50 ng L−1 with a 50 mL sample, or 5 ng L−1 with a 1 L sample.

Effect of template size on the selectivity of molecularly imprinted polymers for phenylurea herbicides

SummaryFour different methacrylic acid-based, molecularly imprinted, polymers, using fenuron or isoproturon as template and acetonitrile or toluene as porogen, were prepared in order to evaluate the template size effect on the selectivity and the porogen effect on the affinity of recognition in molecular imprinting solid-phase extraction. The results of different tests applied to both polymers have shown that fenuron-polymers were highly selective whereas isoproturon-polymers were able to recognise several structurally related compounds (metoxuron, fenuron, metobromuron, chlortoluron, linuron and chlorbromuron). On the other hand, the polymers prepared using toluene as porogen showed a higher affinity for the tested compounds than those prepared in acetonitrile. Accordingly, the isoproturon-imprinted polymer prepared in toluene has been evaluated as a new sorbent in solid-phase extraction procedures for the trace-enrichment and clean-up of phenyureas in ground water and soil samples.

Solid phase extraction of clenbuterol from plasma using immunoaffinity followed by HPLC.

An immuno-extraction column for clenbuterol has been prepared. Optimum conditions for the selective retention and elution of clenbuterol have been developed, based on a modification of our earlier work on morphine, chlortoluron and isoproturon. Clenbuterol could be retained on the immuno-column then eluted in one x one ml fraction using 50% methanol in phosphate buffered saline pH 2. On columns containing antisera (but not to clenbuterol) the clenbuterol was removed in the washing step. HPLC-UV determination gave clean traces. Day-to-day reproducibility was improved by precipitating the plasma proteins with acetonitrile.

Determination of tamoxifen and five metabolites in plasma

Tamoxifen is an anti-estrogenic agent widely used in the treatment of breast cancer. It is usually administered at a dose of 20-40 mg daily. Methods for the determination of tamoxifen (TX) and its major metabolite N-desmethyltamoxifen (DMTX) in plasma have usually been based on the conversion of TX and DMTX to their fluorescent phenanthrene derivatives followed by HPLC [l] or TLC [2]. The reaction has been carried out both pre[l, 31 and post[4] column. Gas liquid chromatography-mass spectrometry has also been used [5]. The majority of previous work has been carried out on TX and DMTX only. Several other metabolites of TX have been identified [6] and attention has recently focused on these. Several thousand plasma samples have been assayed for TX and DMTX in the Authors’ laboratories [7, 81 using a modification of the method of Sternson. The aim of the present work was to adapt this method to allow the determination of four further metabolites of tamoxifen: 4-hydroxytamoxifen (B); N-desdimethyltamoxifen (Z); 4hydroxyethoxytriphenylethylethylene (Y); and 4-hydroxytriphenylethylethylene (E): the designations B, Z, Y and E are from the literature [6]. The structures are shown in Fig. 1. The method was used to determine plasma levels of these metabolites from patients at steady state taking 30 mg TX daily and also in a study of patients taking a loading dose (160 mg) of TX before taking 30 mg daily.

Serum elimination half-life of tamoxifen and its metabolites in patients with advanced breast cancer

SummaryIn breast cancer patients discontinuing chronic tamoxifen therapy, the serum elimination of metabolites X, Y and E paralleled that of tamoxifen, whereas that of metabolite Z did not. The serum elimination of tamoxifen and metabolites X and B was increased by amino-glutethimide treatment, whereas that of metabolites Z, Y, and E was not.