Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices

Enrofloxacin has been increasingly used in veterinary medicine to treat microbial infections. A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography (HPLC) is sensitive but labor-intensive. This paper reports an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) and the development of a rapid test kit based on immunochromatography. The detection limits using the ELISA were 10 ppb for chicken liver and muscle, and 1 ppb for cattle milk, respectively. The mean recovery values were 77.3-96.0% for chicken liver, 72.4-92.0% for chicken muscle and 84.0-99.0% for cattle milk. The detection limits using the kit were ca. 100 ppb for chicken muscle and ca. 10 ppb for cattle milk, respectively. All ELISA results for assay of chicken liver, chicken muscle and cattle milk were confirmed using HPLC which is used as the routine assay. The HPLC (x) and ELISA (y) results showed close correlation for chicken liver (y = 8.7 + 0.85x, r2 = 0.99, n = 25), chicken muscle (y = -3.9 + 0.94x, r2 = 0.98, n = 25) and cattle milk (y = 18.4 + 0.92x, r2 = 0.99, n = 25).     

Analysis of acrolein and acrylonitrile in aqueous solution by membrane introduction mass spectrometry

Acrolein and acrylonitrile can be quantified directly at low levels in aqueous solution using membrane introduction mass spectrometry. Electron impact was used to generate positively charged ions and electron capture of the O-(2,3,4,5,6-pentafluorobenzyl)hydroxyl amine (PFBOA) derivative was used to generate negatively charged ions of acrolein in aqueous solutions. The origins of all ions in the mass spectra and product MS/MS spectra recorded using both ionization methods were assigned and a reaction scheme is given which accounts for the fragmentation of the PFBOA derivative. Detection limits were measured using multiple reaction monitoring in both the methods. With electron capture detection, acrolein could be detected without preconcentration at 10 ppb levels. Electron impact ionization and multiple reaction monitoring both allowed the measurement of acrylonitrile at levels as low as 10 ppb.     

High-performance liquid chromatographic analysis of glutathione and its thiol and disulfide degradation products

A rapid and sensitive high-performance liquid chromatographic method for quantitation of picomole levels of glutathione, glutathione disulfide, cysteine, cystine, cysteinylglycine, cysteinylglycine disulfide and cysteine glutathione-mixed disulfide in biological samples is described. The compounds were separated isocratically on a reversed-phase column by ion-pair chromatography. The mobile phase consisted of an aqueous buffer containing 0.1 M monochloroacetic acid and 3.3 mM 1-heptanesulfonic acid (pH 2.60)-methanol-N,N-dimethylformamide (96.5:3.0:0.5). After chromatographic separation, the disulfides were reduced by a potential (-1.0 V) from a battery, with subsequent detection of all thiols by electrochemical oxidation (+0.15 V) with a dual gold-mercury electrode. Thiol and disulfide concentrations were determined in tissue extracts (liver and kidney) and fluids (bile and plasma) from control rats and rats treated with acivicin, an inhibitor of gamma-glutamyltranspeptidase. A marked increase in biliary glutathione concentration was observed in treated animals with a corresponding decrease in cysteine and cysteinylglycine concentrations. The results demonstrate that this method is useful for measuring glutathione and its degradation products in tissues and fluids.     

A HPLC Screening Procedure for Sulfamethazine Residues in Pork Tissues

Abstract

A sensitive and specific screening procedure is described for the quantitative detection of sulfamethazine residues in pork kidney, liver and muscle. Initial screening is by both the Bratton-Marshall reaction, and by high pressure liquid chromatography (HPLC); quantitation is by HPLC; identification is then confirmed by means of thin-layer chromatography (TLC) of the derivatized standards and the unknown from the Bratton-Marshall reaction. Only one extraction of a 50g sample is needed, one portion (10g tissue equivalent) is used for the colorimetric reaction and TLC confirmation, and another portion (25g tissue equivalent) is used for quantitative HPLC determination. Standard curves for sulfamethazine are constructed for each tissue at 50, 100, 200 and 500 ppb levels. The average mean recovery for all tissues at all levels is 78.2%. The method is verified by a 150 sample survey using 50 samples of each tissue from local supermarkets. Approximately 4% of the samples show contamination ranging from a level of 100 ppb to 3 ppm.     

Multiresidue determination of sulfonamides in a variety of biological matrices by supported liquid membrane with high pressure liquid chromatography-electrospray mass spectrometry detection

A high performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) was used for a simultaneous determination of 16 sulfonamide compounds spiked in water, urine, milk, and bovine liver and kidney tissues. Supported liquid membrane (SLM) made up of 5% tri-n-octylphosphine oxide (TOPO) dissolved in hexyl amine was used as a sample clean-up and/or enrichment technique. The sulfonamides mixture was made up of 5-sulfaminouracil, sulfaguanidine, sulfamethoxazole, sulfamerazine, sulfamethizole, sulfamethazine (sulfadimidine), sulfacetamide, sulfapyridine, sulfabenzamide, sulfamethoxypyridazine, sulfamonomethoxine, sulfadimethoxine sulfasalazine, sulfaquinoxaline, sulfadiazine, and sulfathiazole. Some of these compounds, such as, sulfaquinoxaline, sulfadiazine, sulfabenzamide, sulfathiazole and sulfapyridine failed to be trapped efficiently by the same liquid membrane (5% TOPO in hexylamine). The detection limits (DL) obtained were 1.8 ppb for sulfaguanidine and sulfamerazine and between 3.3 and 10 ppb in bovine liver and kidney tissues for the other sulfonamides that were successfully enriched with SLM; 2.1 ppb for sulfaguanidine and sulfamerazine and between 7.5 and 15 ppb in cow’s urine, whereas the DL values in milk were 12.4 ppb for sulfaguanidine and sulfamerazine and between 16.8 and 24.3 for the other compounds that were successfully enriched by the membrane. Several factors affecting the extraction efficiency during SLM enrichment, such as donor pH, acceptor pH, enrichment time and the membrane solvent were studied.     

Hypoglycemic and hypolipidemic effects of polyphenols from burs of Castanea mollissima Blume

Substantial evidence suggests that phenolic extracts of Castanea mollissima spiny burs (CMPE) increase pancreatic cell viability after STZ (streptozotocin) treatment as a result of their antioxidant properties. In the present study, the hypoglycemic and hypolipidemic activities of CMPE were studied in normal and STZ-induced diabetic rats CMPE were orally administrated at doses of 150 and 300 mg/kg twice a day for 12 consecutive days. Serum glucose, triglyceride, total cholesterol, HDL- and LDL-cholesterol levels, malondialdehyde (MDA) level and SOD activity in liver, kidney, spleen and heart tissues were measured spectrophotometrically. In normal rats, no significant changes were observed in serum glucose, lipid profiles and tissue MDA and GSH levels after orally administration of CMPE. In diabetic rats, oral administration of CMPE at a dose of 300 mg/kg caused significant decreases in serum glucose, triglyceride, total cholesterol, LDL-cholesterol levels, as well as MDA and GSH levels in spleen and liver tissues. However, the 300 mg/kg dosage caused a significant body weight loss in both normal and diabetic rats. The observed effects indicated that CMPE could be further developed as a drug to prevent abnormal changes in blood glucose and lipid profile and to attenuate lipid peroxidation in liver and spleen tissues.     

LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine

The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.     

Protective effects of Ocimum sanctum on lipid peroxidation and antioxidant status in streptozocin-induced diabetic rats

The antioxidant effects of Ocimum sanctum in experimental streptozocin-induced diabetic rats was evaluated in this study. Streptozocin, 55?mg?kg(-1) body weight, was injected intraperitoneally once daily for 30 days to induce diabetes mellitus in rats. Streptozocin-induced diabetic rats were orally treated with an aqueous extract of O. sanctum once daily for 30 days. After the experimental period, thiobarbituric acid reacting substances (TBARS) and antioxidant enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase were measured. Administration of O. sanctum to streptozocin-induced diabetic rats for 30 days significantly reduced the plasma level of TBARS and improved the status of the antioxidant enzymes catalase, SOD and glutathione peroxidase in vital organs such as the liver and kidney. These results confirmed that the Indian medicinal plant O. sanctum has a protective effect and it may be useful in controlling complications resulting from diabetes.     

Preconcentration of trace nickel with the ion pair of disodium 1-nitroso-2-naphthol-3,6-disulfonate and tetradecyldimethylbenzylammonium chloride on microcrystalline naphthalene or by the column method and determination by third derivative spectrophotometry

Nickel is quantitatively retained by disodium 1-nitroso-2-naphthol-3,6-disulfonate (nitroso-R salt) and tetradecyldimethylbenzylammonium chloride (TDBA(+)Cl(-)) on microcrystalline naphthalene in the pH range 5.4-12.1 from large volumes of aqueous solutions of various alloys and biological samples. After filtration, the solid mass consisting of the nickel complex and naphthalene was dissolved with 5 ml of dimethylformamide (DMF) and the metal was determined by third derivative spectrophotometry. Nickel complex can alternatively be quantitatively adsorbed on tetradecyldimethylbenzylammonium-naphthalene adsorbent packed in a column and determined similarly. The detection limit is 10 ppb (signal to noise ratio 2) and the calibration curve is linear from 30 to 5.4x10(3) ppb in dimethylformamide solution with a correlation coefficient of 0.9997 by measuring the distance d(3)A/dlambda(3) between lambda(1) (537 nm) and lambda(2) (507 nm). Eight replicated determinations of 2.5 mug of nickel in 5 ml of dimethylformamide solution gave a mean intensity (peak-to-peak signal between lambda(1) and lambda(2)) of 0.339 with a relative standard deviation of +/-0.87%. The sensitivity of the method is 0.677 ml/mug found from the slope (d(3)A/dnm(3)) of the calibration curve. Various parameters such as the effect of pH, volume of aqueous phase and interference of a number of metal ions on the determination of nickel has been studied in detail to optimize the conditions for nickel determination in various alloys and biological samples.     

Confirmatory analysis of sulfonamide antibacterials in bovine liver and kidney: extraction with hot water and liquid chromatography coupled to a single- or triple-quadrupole mass spectrometer

A simple, specific, and rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in bovine liver and kidney is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography/mass spectrometry (LC/MS) with an electrospray ion source. The method was tailored for use with both single-quadrupole MS (I) and triple-quadrupole MS (II) instruments. After acidification and filtration of the aqueous extract, a 250-microL aliquot was injected into instrument I while only 25 microL was analyzed by instrument II. With instrument I MS data acquisition was performed in the selected ion monitoring (SIM) mode, selecting at least three ions for each target compound. With instrument II the selected reaction monitoring (SRM) mode with three fragmentation reactions for each compound was chosen. With the exception of sulfaquinoxaline (SQX), recovery of the analytes at the 50 ppb level in both liver and kidney was 72-96% with relative standard deviations (RSDs) ranging between 3 and 11%. The very poor recovery of SQX was due to its rapid enzymatic oxidation when in contact with the two tissues. With instrument I, limits of quantification (LOQs, S/N = 10) were 5-14 ppb of SAs. Even lower LOQs (1-8 ppb) were estimated by using instrument II, even though the extract volume analyzed was ten times lower than that with instrument I. With both matrices and using instrument I, severe ion signal suppression was experienced for the early-eluted SAs when trying to fractionate analytes by using a short chromatographic run time. This effect was traced to polar endogenous co-extractives eluted in the first part of the chromatographic run that interfered with gas-phase ion formation for SAs. Adopting more selective chromatographic conditions minimized this effect.     

Determination and confirmation of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment using high-performance liquid chromatography/tandem mass spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.     

Evaluation of Chromatographic Methods for the Determination of Isocyanates in Air

Abstract

Chromatographic methods for the determination of toluene diisocyanates (TDI) in air, at concentrations of 20–40μg/m³, were evaluated. Test atmospheres were generated by a gas-phase permeation principle. The GC method was based on sampling in impingers containing an acidic aqueous solution (0.4 M HC1), where the isocyanates are hydrolysed into the corresponding amines. The pentafiuoropropionic acid derivatives of the amines were analysed using glass capillary GC with thermionic detection. The HPLC methods were based on reaction with the amine reagents 9-(N-methylaminomethyl)-anthracene (1 × 10^?4M in toluene) and 1-(2-methoxyphenyl)-piperazine (2 × 10^?4M in toluene). The urea derivatives were determined using UV detection. The sampling efficiency for gaseous TDI with toluene as absorbing solution was 97%. Sampling efficiencies in various acidic aqueous solutions were 83–88%. The relative standard deviation for the various methods was ca. 6%. No sampling losses for TDI, due to influences of interfering substances (diethylamine, dimethylethylamine, N-methylmorpholine, DABCO, aniline, ethanol, phenol) using acidic aqueous 0.4 M HC1 were obtained. Diethylamine, N-methylmorpholine and DABCO affected the analytical results when toluene solutions of the amine reagents MAMA and MPP were used.     

A PLS-based extractive spectrophotometric method for simultaneous determination of carbamazepine and carbamazepine-10,11-epoxide in plasma and comparison with HPLC

Carbamazepine (CBZ) undergoes enzyme biotransformation through epoxidation with the formation of its metabolite, carbamazepine-10,11-epoxide (CBZE). A simple chemometrics-assisted spectrophotometric method has been proposed for simultaneous determination of CBZ and CBZE in plasma. A liquid extraction procedure was operated to separate the analytes from plasma, and the UV absorbance spectra of the resultant solutions were subjected to partial least squares (PLS) regression. The optimum number of PLS latent variables was selected according to the PRESS values of leave-one-out cross-validation. A HPLC method was also employed for comparison. The respective mean recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 (+/-0.25)% and 103.00 (+/-0.09)% for PLS and 99.40 (+/-0.15)% and 102.20 (+/-0.02)%. The concentrations of CBZ and CBZE were also determined in five patients using the PLS and HPLC methods. The results showed that the data obtained by PLS were comparable with those obtained by HPLC method.     

Anti-hyperuricemia activity and toxicity prediction of a novel xanthine oxidoreductase inhibitor

A potent xanthine oxidoreductase inhibitor (LS087) was recently proved to exhibit a similar hypouricemic potency to febuxostat. A hyperuricemia model induced by potassium oxonate and hypoxanthine was proposed in specific pathogen-free male Kunming mice, and the serum urea nitrogen, creatinine and uric acid levels were measured after oral administration of LS087. Furthermore, renal histopathology was conducted by staining with hematoxylin and eosin, periodic acid–Schiff and Masson's trichrome stains, respectively. The results showed that the levels of serum urea nitrogen and uric acid significantly decreased compared with the model group, but the level of creatinine showed no significant changes. The pathological abnormalities in kidney tubules were improved after LS087 administration. Ten metabolites (M1–M10) of LS087 were identified after a single oral dosing of 10 mg/kg in rats. M6 was the primary LS087 metabolite in vivo with a pathway of methylation. The toxicity and potential risks of LS087 and its metabolites were predicted using the ProTox-II software. LS087 and the major metabolites (M2, M3, M5, M6, M7 and M8) were predicted to have no potential hepatotoxicity, but some metabolites with a total rate of <1% (M1, M4, M9, and M10) showed potential hepatotoxicity. M1 and M8 showed potential carcinogenicity. The LS087 biotransformation pathway in rat was well characterized.     

Effects of Lycium barbarum aqueous and ethanol extracts on high-fat-diet induced oxidative stress in rat liver tissue

This study evaluated the protective effects of aqueous extract of Lycium barbarum (LBAE) and ethanol extract of Lycium barbarum (LBEE) on blood lipid levels, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities and liver tissue antioxidant enzyme activities in rats fed a high fat diet (HF). The rats were randomly divided into seven groups of ten rats each and fed a different diet for eight weeks as follows: One group (NC group) was fed a standard diet, one group was fed a high-fat diet (HF group), one group was fed a high-fat diet and orally fed with 20 mg/kg b.w. simvastatin (HF + simvastatin group), and the other group was fed the high fat diet and orally fed with 50 mg/kg b.w. or 100 mg/kg b.w. LBAE (HF + LBAE), or 50 mg/kg b.w. or 100 mg/kg b.w. LBEE (HF + LBEE), respectively. After eight weeks, the HF diet caused deleterious metabolic effects. Rats fed the HF diet alone showed increased hepatocellular enzyme activities in plasma, a significant decline in antioxidant enzyme activities, and elevated liver lipid peroxidation indices. LBAE and LBEE administration significantly reduced liver damage and oxidative changes, and brought back the antioxidants and lipids towards normal levels. These data suggest that these antioxidants protect against toxicity parameters in HF rats.     

Rapid Analysis for Codeine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid codeine in pharmaceutical preparations and in body fluids. The minimum detectable concentrations for body fluids were 5ppb and 7ppb respectively for urine and whole blood with an analysis time of under 5 min. A RP-8 Spheri-5 guard column and a RP-8 Lichrosorb-10 column were used and codeine was detected at its absorption maximum wavelength of 212 nm using an eluting system of methanol: 0.5% w/v aqueous ammonium acetate (70:30) at a pH of about 7.0.     

A facile and sensitive chemiluminescence detection of amino acids in biological samples after capillary electrophoretic separation

It was found that native amino acids enhanced the chemiluminescence (CL) reaction between luminol and BrO(-) in an alkaline aqueous solution. This has led to the development of a facile and highly sensitive CL detection scheme for the determination of amino acids in biological samples after capillary electrophoretic (CE) separation. The CE-CL conditions were optimized. An electrophoretic buffer of 2.5 x 10(-2) M sodium borate (pH 9.4) containing 1 x 10(-4) M luminol was used. The oxidizer solution of 8 x 10(-4) M NaBrO in 0.1 M sodium carbonate buffer solution (pH 12.5) was introduced post-column. Under the optimal conditions, the detection limits were 1.0 x 10(-7) M for glutamic acid (Glu) and 1.3 x 10(-7) M (S/N = 3) for aspartic acid (Asp). The relative standard deviations (RSDs) of peak area and migration time were in the ranges of 3.8-4.3% and 1.4-1.6%, respectively. The present method was applied to the determination of excitatory amino acids (i.e., Asp and Glu) in rat brain tissue and monkey plasma. The levels of these major excitatory amino acids in monkey plasma were quantified for the first time and found to be 1.17 +/- 0.17 x 10(-5) M (mean +/- SD, n = 6) for Glu and 1.64 +/- 0.19 x 10(-6) M for Asp, which were comparable with the levels in human plasma.     

Use of capillary electrophoresis in drug quality assessment of synthetic porcine secretin

The purity profile for porcine secretin attributable to contamination by equilibrium products such as aspartoyl(3) secretin has been shown to be dependent on the pH of the analytical system. Capillary zone electrophoresis (CZE) methods have been developed for the efficient separation of synthetic porcine secretin, its equilibrium products and other impurities in aqueous solutions at both acidic and alkaline pH. These conditions are more representative of those used for the reconstitution and administration of porcine secretin, and good results cannot be achieved using HPLC due to poor peak shape above pH 5.8. The influence of various CZE operational parameters was systematically examined. The methods were validated for accuracy, precision, linearity, LOD and LOQ. A comparative evaluation of the stability of test solutions was determined using CZE and HPLC over a range of pH values. HPLC and CZE methods produced similar results at low pH.     

Comparison of Programmable versus Single Wavelength Fluorescence for the Detection of Three Fluoroquinolone Antibacterials Isolated from Fortified Chicken Liver Using Coupled On Line Microdialysis and HPLC

A recently introduced programmable fluorescence detector was compared with a single wavelength fluorescence detector for quantification of fluoroquinolone (FQ) antibacterial agents, which have widely varying spectral characteristics. The two detectors were connected in parallel to an HPLC system to test their performance characteristics. With single wavelength detection, two FQs, flumequine and oxolinic acid could be detected at an emission wavelength of 368 nm in a single chromatogram while a third FQ, sarafloxacin, was not observed at that wavelength. Similarly, when the detector was optimized for sarafloxacin emission at 440 nm, the other two compounds were undetected. In contrast, all three FQs were quantified at their individual maxima in a single run using the programmable fluorescence detection. The applicability of an HPLC – programmable fluorescence detector, in combination with on-line microdialysis, also was evaluated using chicken liver fortified at low ppb levels with the three FQs. After on-line microdialysis sample clean up, the resultant HPLC chromatograms were free of background interference enabling the programmable detector to optimize the quantitation of the three analytes in a single run. The limit of quantification (LOQ) determined for each FQ was 1.0 ppb and the limit of detection (LOD) was 0.2 ppb, an order lower in magnitude than was obtainable with single wavelength detection.     

Green biosynthesis of Pt-nanoparticles from Anbara fruits: Toxic and protective effects on CCl4 induced hepatotoxicity in Wister rats

Platinum Nanoparticles (PtNPs) are synthesized from the Anbara fruit (Phoenix dactylifera L.) and are characterized using various spectroscopic analytical methods. These PtNPs were used to study the Hepatotoxic and Hepatoprotective effects on acute liver damage caused by CCl₄ in Wister rats. Seventy-two rats of both sexes are divided into twelve groups and are treated with PtNPs and aqueous Anbara extract (AAE). Histopathological examinations were performed to identify the toxic effects on the vital organs of the rats. Hepatoprotective activity was monitored by observing the serobiochemical and hematological parameters and the intensity of hepatic marker enzymes alanine transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) in the organs such as liver, intestine and kidney. Considerable experimental results were obtained when compared with the standard drug Silymarine. The PtNPs and AAE were proven to have protective activity of enzymes in the liver of Wister rats.