Ruprecht Keller

Photochemical immobilization of heparin, dermatan sulphate, dextran sulphate and endothelial cell surface heparan sulphate onto cellulose membranes for the preparation of athrombogenic and antithrombogenic polymers.

Heparin (HE), dextran sulphate (DX) of molecular weight 40000 and 500000, dermatan sulphate (DS) and endothelial cell surface heparan sulphate (ES-HS) were immobilized covalently onto cellulose membranes (Visking dialysis tubes) using the photochemical heterobifunctional reagent 4-azido-1-fluoro-2-nitrobenzene (AFNB); 120 pmol HE/cm2 and 40 pmol DS/cm2, 3.4 pmol DX 500,000/cm2, 50 pmol DX 40,000/cm2 and 3.6 pmol ES-HS/cm2 were immobilized. The platelet adhesion of the modified membranes was measured in a modified Baumgartner perfusion chamber with citrated human blood at a defined shear rate. Membranes modified with DX 40,000 and DX 500,000 showed 80% and 30% platelet adhesion, respectively, heparinized and DS coated membranes showed 50% and 60% platelet adhesion, respectively, compared with a subendothelial matrix (100% platelet adhesion). ES-HS modified membranes showed no platelet adhesion.

Novel regio- and stereoselective O-6-desulfation of the glucosamine moiety of heparin with N-methylpyrrolidinone–water or N,N-dimethylformamide–water mixtures

The degree of completeness and selectivity of the solvolytic O-6-desulfation reactions of the glucosamine moiety adjacent to the 2-O-sulfoiduronic acid group of heparin was systematically studied. Using solutions of various ammonium salts of heparin (salts of tributylamine, quinoline and pyridine) in mixtures of 9:1 aprotic solvents and water (solvents of medium polarity, in order of decreasing polarity: Me2SO > Me2NCHO > Me2NAc > N-methylpyrrolidinone), the influence of different reaction conditions were studied. The ammonium salt of heparin with a strong base (e.g., tributylamine) in Me2SO showed almost no desulfation, while in Me2NCHO a relatively low degree of completeness of O-6-desulfation (30%) with moderate selectivity (15% [I-2(OS)]-desulfation) was observed. Weak bases like quinoline or pyridine in Me2SO-water resulted in nearly complete [A-6(OS)]-desulfation (95 and 94%, respectively) with low selectivity [I-2(OS)]-desulfation (49 and 35%, respectively). The heparin pyridinium salt in Me2NCHO-water showed both a relatively high degree of completeness and high selectivity (72% [A-6(OS)]- and 8% [I-2(OS)]-desulfation). The highest regioselectivity (i.e., a high degree of completeness accompanied by high selectivity) was achieved using an N-methylpyrrolidinone-water mixture (88% [A-6(OS)]-desulfation and 10% [I-2(OS)]-desulfation). A nearly complete O-6-desulfation (95%), accompanied by a lower selectivity (18% [I-2(OS)]-desulfation), was achieved when the reaction was carried out twice. Lower temperature improved selectivity (5% [I-2(OS)]-desulfation) but reduced the completeness of [A-6(OS)]-desulfation (72%). In comparison with the variety of O-6-desulfations reported to date, the novel reactions presented in this article led to remarkable increase in completeness and regioselectivity of the reactions that were investigated.

Urine labeling with orally applied marker substances in drug substitution therapy.

Abstract The compliance of 581 drug addicts attending six methadone substitution outpatient clinics was determined over a period of 18 months. Urine from these patients was labeled following oral administration of low molecular weight polyethylene glycols as marker substances. These substances were measured in approx. 5800 urine samples. A protocol for applying marker substances and ways to prevent substitution of urine samples were evaluated. Normal values for marker substances in urine were determined. The results suggest that this labeling procedure is a new diagnostictool to prevent manipulation of urine samples by drug addicts receiving substitution therapy.

False-negative results in the immunoassay analysis of drugs of abuse: can adulterants be detected by sample check test?

Background The dilution or adulteration of urine is a serious problem in drugs of abuse testing. Tests to identify adulteration are currently available. This study investigated the ability of the CEDIA® sample check to detect adulteration. Methods Eight different drugs of abuse were added to a urine sample obtained from a healthy, drug-free subject: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 3,4-methylenedioxyamphetamine, benzoylecgonine, D-amphetamine sulphate, ethyl-D-glucuronide, morphine sulphate, oxazepam, (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol. Urine samples were diluted to yield three samples of drugs of abuse concentrations close to general cut-offs as used in methadone treatment centres, by health authorities for psychological tests and in traffic medicine. Aspirin, citric acid, CrO3, H2O2, soap, sodium metaborate, vitamin C were added in three, HCl and NaOH in one, and NaN3 in two concentrations. All samples were measured with commercially available immunological assays shortly after sample preparation and 24 h later. All samples were further analysed with the CEDIA® sample check reaction which may identify adulteration. Results Oxidizing reagents (H2O2 or CrO3) are most effective in interfering in the measurement of benzoylecgonine, EDDP, ethyl-D-glucuronide and morphine sulphate. The measurement of (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol is affected by many adulterants. Adulteration with HCl and NaOH was identified with the sample check reaction. NaN3 generated false negative results for a number of drugs of abuse. Conclusions Urine samples with drugs of abuse concentrations above cut-offs can be successfully tampered with adulterants in a way which cannot be detected with the CEDIA® sample check assay.