An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products.

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.

Molecularly imprinted polymer with salicylaldehyde-Cu(OAc)2 as template.

In the report molecularly imprinted polymer (MIP) with salicylaldehyde-Cu(OAc)(2) as the template was synthesized and characterized by SEM, porosity and elemental analysis. Copper acetate was introduced since salicylaldehyde alone cannot display imprinting effect for its intramolecular hydrogen bond. The strong coordination interaction between salicylaldehyde and copper acetate made the complex have high retention on the HPLC column based on the SAD-Cu(OAc)(2) imprinted polymer. Several structural analogues such as salicylaldoxime, sulfosalicylic acid, p-hydroxybenzaldehyde and their complexes with copper acetate were chosen to study the selectivity of the MIPs. The influence of acetic acid and H(2)O in methanol mobile phase was studied. The experimental results showed that small amount of either acetic acid or H(2)O in mobile phase would weaken the interaction between the complex and the polymer, therefore, the retention of the complex was lowered to a large extent, but that of salicylaldehyde remained almost unchanged. The polymer imprinted with the complex showed high selectivity to both the acetate and copper (II). In addition, the MIP showed an enhanced selectivity to its template compared with the polymer prepared without copper acetate.

Sulfamethoxazole-imprinted polymer for selective determination of sulfamethoxazole in tablets.

A sulfamethoxazole (SMO)-imprinted polymer (MIP) was prepared in acetonitrile using the mixture of acrylamide and 4-vinylpyridine as functional monomers. The molecular recognition properties of the polymer was evaluated in both acetonitrile and aqueous acetonitrile mobile phases. SMO contents in two kinds of tablets were determined satisfactorily using the MIP packed HPLC column with aqueous mobile phase.

The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering.

For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 microg ml(-1) for herring sperm DNA (hsDNA), 0.02-10.0 microg ml(-1) for calf thymus DNA (ctDNA), 0.02-20.0 microg ml(-1) for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml(-1), respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2 x 10(5) M(-1). IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.

Pharmacokinetics and tissue distribution of iv injection of polyphase liposome-encapsulated cisplatin (KM-1) in rats.

AIM: The pharmacokinetics and biodistribution of cisplatin encapsulated in polyphase liposome (KM-1) were compared with those of free drug in rats. METHODS: The platinum levels in serum and normal organs, after a single dose of iv injection of free or encapsulated cisplatin to rats, were determined by induced coupled plasma atomic emission spectrometry. RESULTS: Serum platinum concentration-time curve after a single iv dose of KM-1 4.5 mg/kg in rats was fitted with an open three-compartment model. The pharmacokinetic parameters were as follows: Vc=0.10 L/kg, T1/2pai=0.3 h, T1/2alpha=3.5 h, T1/2beta=2.7 h, AUC=265 mg.h.L(-1), and CL(s) =0.02 g.L.h(-1). KM-1 was cleared from the circulation much more slowly than free cisplatin. Liver and spleen had the highest concentration of platinum after KM-1 treatment. CONCLUSION: KM-1 remained in the bloodstream longer than its free drug, and was taken mainly by the reticuloendothelial system.

Flow injection determination of gaseous sulfur dioxide with gas permeation denuder-based online sampling and preconcentration.

A simple and novel flow injection method for the determination of gaseous SO(2) is described based on gas permeation denuder (GPD) online sampling and preconcentration. The GPD is easily prepared with poly(vinylidene) difluoride microporous membrane as gas permeable material and two Perspex blocks with smooth and flat interface and rectangular engraved channels of mirror image. The sample gas is on one side of the membrane and phosphate buffer of pH 7.0 as the absorbing solution is on the other side. Gaseous SO(2) permeates partially through the gas permeable membrane and dissolves in the absorbing solution. After preconcentration for 5.0 or 8.0 min, the solution is injected into the flow of 5.0 x 10(-4) mol L(-1) 5,5'-dithiobis(2,2'-dinitrobenzoic acid) (DTNB) in 0.025 mol L(-1) phosphate buffer. The resulting product formed between DTNB and absorbed SO(2) is spectrophotometrically monitored at 410 nm with a charge coupled device (CCD) fiber optic spectrometer. The calibration graphs for preconcentration of 5.0 and 8.0 min are linear up to 4.0 and 3.2 mg m(-3) with detection limits of 50 and 35 micro g m(-3), respectively. The corresponding analysis speeds are 8.5 and 6 samples h(-1). The method is selective and just suffer from interference of hydrogen sulfide at higher than 1% of SO(2) level (in m/V) with an error >+10%. The assay just uses cheap and common membrane and reagents and shows potential application in the monitoring of atmospheric SO(2).

Cyclic voltammetry characterization of metal complex imprinted polymer.

Polymer capable of specific binding to Cu(2+)-2, 2'-dipyridyl complex was prepared by molecular imprinting technology. The binding specificity of the polymer to the template (Cu(2+)-2, 2'-dipyridyl complex) was investigated by cyclic voltammetric scanning using the carbon paste electrode modified by polymer particles in phosphate buffer solution. Factors that influence rebinding of the imprinted polymer were explored. The results demonstrated that cyclic voltammetry was an efficient approach to explore interactions between template and imprinted polymers.

Production and characterization of anti-estrone monoclonal antibody.

OBJECTIVE: Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay. METHODS: 3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). RESULTS: Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164,000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2 x 10(8) L/mol. The linear range for free E1 determined by CI-ELISA was 10 pg/mL-10 ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank). CONCLUSION: The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.

A new competitive enzyme-linked immunosorbent assay (ELISA) for determination of estrogenic bisphenols.

Bisphenol A and other hydroxylated diphenylalkanes (generally known as bisphenols) have been identified as potential estrogenic substances. In this paper, a specific polyclonal antibody was produced against these compounds by immunization of rabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serum albumin (BHPVA-BSA). The polyclonal antibody showed specific recognition of the bisphenol structure, while the cross reactions of other common phenolic compounds such as phenol, hydroquinol and p-hydroxybenzoic acid were all lower than 1%. The linear range of bisphenol A calibration curve was 1-10 000 ng ml(-1). Real water samples and mouse serum samples were spiked with known amount of bisphenol A and measured by the competitive ELISA. Recoveries were between 92 and 105%. The detection limits were found to be 0.1 ng ml(-1) for real water samples and 2 ng ml(-1) for serum samples. The method is very useful for monitoring bisphenol compounds in environmental and biological samples.