Automated high-performance liquid chromatographic determination of chloramphenicol in milk and swine muscle tissue using on-line immunoaffinity sample clean-up.

An on-line high-performance liquid immunoaffinity chromatographic (HPLIAC) system for the direct determination of chloramphenicol in milk and swine muscle tissue is described. The system consisted of a dual-column system in which an HPLIAC column was directly coupled to an RP-8 high-performance liquid chromatographic column. Skimmed and deproteinated milk or aqueous muscle tissue extract was directly injected into the HPLIAC column. After a washing step with phosphate-buffered saline, chloramphenicol was desorbed by a glycine-NaCl buffer (pH 2.8) and directly concentrated on the RP-8 column. Next, chromatography was carried out using acetonitrile-sodium acetate buffer as the mobile phase. Chloramphenicol was detected at 280 nm. Mean recoveries from spiked raw milk were 70 +/- 2% (1-50 micrograms/kg) and from spiked swine muscle tissue 64 +/- 2% (10-50 micrograms/kg). The calibration curves were linear in the range 1-200 micrograms/kg spiking levels. Limits of determination were 1 microgram/kg for milk and 10 micrograms/kg for muscle tissue.     

Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination

A commercially available system is described for the fully automated clean-up and high-performance liquid chromatographic (HPLC) analysis of aflatoxins in foods and animal feeds. The system marketed primarily for handling solid-phase extraction columns has modified software to facilitate use with immunoaffinity columns. Sample extract clean-up followed by injection onto an HPLC column with post-column iodination and fluorescence detection is carried out completely unattended. A coefficient of variation of 5.1% for aflatoxin B1 analysis was obtained, and the accuracy of the system was demonstrated by the analysis of peanut butter certified reference material.     

Monoclonal antibody-mediated clean-up procedure for the high-performance liquid chromatographic analysis of chloramphenicol in milk and eggs

A simple, rapid and specific sample preparation method based on antibody-mediated clean-up for the determination of chloramphenicol (CAP) in milk and eggs was developed. Skimmed milk and centrifuged egg homogenates were filtered and directly applied to immunoaffinity columns which were prepared by coupling monoclonal antibodies against CAP to a carbonyldiimidazole-activated support. Using a 0.2 M glycine, 0.5 M NaCl (pH 2.8) solution as an eluent, the immunoaffinity columns can be used more than 30 times without a decrease in column capacity. In subsequent high-performance liquid chromatographic analysis, no matrix interferences were observed. Good recoveries were obtained at spiking levels of 1-100 micrograms kg-1. Due to the high specificity of the clean-up procedure, the limit of detection can be lowered by increasing the test portion. Concerning milk, the limit of detection was successfully lowered to 20 ng kg-1 by increasing the test portion to 11 (recovery 99%). The method was applied to eggs produced by hens treated with CAP. The results are compared with those obtained by solid-phase extraction using silica gel.     

Rapid electrodialytic clean-up of biological samples for high-performance liquid chromatography.

A sample clean-up system employing electrodialysis with size-selective and charge-selective membranes is described. When applied to the treatment of 0.5-ml milk samples containing sulfamethazine, the system produced an undiluted, clear solution in 3 min and eliminated the components in untreated milk that caused column fouling and double peaks. In contrast to conventional liquid- and solid-phase extraction procedures, electrodialytic clean-up is readily automated and uses no organic solvents.     

Automated high-performance liquid chromatographic assay of enzymatic activities

High-performance liquid chromatography (HPLC) is a powerful technique which enables a reliable and quantitative determination of enzyme activities. The purpose of the work reported here was to develop an automatic assay of enzymatic activity. Using an automatic sample processor and injector, a program was developed which allows the complete automation of each step of analysis (calibration, enzymatic reaction, HPLC determination). This program can be adapted to different experimental requirements as each step can be performed independently and each input (time, volume, number of standards) is made by answering questions asked by instrument. Using this approach both kinetic and single-point determinations can be carried out, and in the latter case different samples can be analysed sequentially. This paper reports the automated analysis of trypsin.     

Comparison of different immunoaffinity clean-up procedures for high-performance liquid chromatographic analysis of ochratoxin A in wines

Three immunoaffinity clean-up procedures to analyse ochratoxin A (OTA) in wines were compared. The direct wine clean-up with Ochraprep and OchraTest columns gave equivalent results in terms of recovery and precision if compared with the reference procedure involving a preliminary extraction of OTA with chloroform. OTA quantification limit in wine ranged from 0.020 to 0.045 microg/l. The 'on-flow' OTA emission spectrum (excitation 333 nm) showed a maximum at 460 nm and could be used to confirm the quantitative results. The analysis of 11 red and white wines gave no significant quantitative differences between the three clean-up techniques.     

Automated zone-electrophoretic sample treatment for the analysis of biological samples by high-performance liquid chromatography

The automation of zone-electrophoretic sample treatment for liquid chromatography is described. The procedure is completely controlled from a liquid chromatograph. The carry-over of proteins from human serum under different experimental conditions was studied. The influence of the presence of proteins in the sample is illustrated with the anionic compound salicylic acid and the increase in selectivity for cationic compounds is demonstrated with the determination of ephedrine, norephedrine and amphetamine in urine.     

Automated pre-column high-performance liquid chromatographic method for the investigation of adibendan metabolism

An automated pre-column high-performance liquid chromatographic method has been developed for the isolation of adibendan and metabolites from biological fluids and for their simultaneous quantitative assay. High sensitivities were obtained by the use of a multiple-injection device allowing solid-phase extraction from several successive sample injections with enrichment of metabolite traces on the pre-column. Two metabolites in dog urine were identified as N-oxypyridine (M1) and 2-hydroxypyridine (M2) derivatives of adibendan, while the structure of M3 is still unknown. M1 and M2 are also metabolites in rats, rabbits and humans, and contribute to cardiovascular efficacy. The metabolic profiles were determined in plasma, urine and bile, as a function of dose, route of administration and sex, using radioactivity and ultraviolet detection of the eluates.     

Ultra-rapid high-performance liquid chromatographic screening for phenothiazines in human samples

A high-performance liquid chromatographic method is presented for the simultaneous identification and quantification of six commonly prescribed phenothiazines. Single-step extraction was achieved from alkaline samples with heptane - isoamyl alcohol (98.5 + 1.5), using prochlorperazine as an internal standard. A Spherisorb CN column was used, with a mobile phase of acetonitrile - acetate buffer (95 + 5). Detection was carried out at 254 nm.     

Automated on-line gas chromatographic injection of samples completely liquified by pressure

Summary A modified on-line liquid injection technique with rotary valves for gas chromatography has been developed. Applications for the on-line analysis of 2-methylpropene, cyclohexene and 1-butene are described. All samples were loaded under pressure. The results obtained show excellent reproducibility with less than 0.1% relative standard deviation (r.s.d.; n=6) for the peak areas measured. The method is particularly advantageous for samples containing both gas and liquid ccomponents at one bar.     

Determination of polar aniline derivatives in aqueous environmental samples using on-line liquid chromatographic preconcentration techniques

Summary On-line precolumn sample handling is used to enrich polar aniline derivatives in order to preconcentrate them prior to their separation. Liquid-solid extraction is possible with a cation-exchanger precolumn after acidification of water samples at pH 3 and a clean-up in order to remove the high amounts of inorganic cations present in natural samples. Since inorganic removal cannot be total, overloading of the ion exchanger occurs rapidly. The volume which can be directly percolated through the cation-exchanger precolumn cannot exceed 30 ml and the amount preconcentrated is not sufficient for a determination at the 100 ppt level. A two-step preconcentration procedure is carried out in order to increase the sample volume: the direct percolation of samples through the cation-exchanger precolumn is avoided and the clean-up step is no longer necessary. Aniline derivatives are preconcentrated in their neutral form at pH 7 by a 9-cm long column packed with the copolymer-based PRP-1 sorbent; then, a small volume of water-methanol at pH 2 allows the cationic compounds alone to be desorbed from the PRP-1 column in their protonated form and to be transferred to a 1-cm long cation-exchanger precolumn. This precolumn is then coupled to an analytical C18 column and its content on-line analysed by an acetonitrile gradient. The PRP-1 column acts as a powerful filter to many neutral interferents and aniline derivatives can be thus determined from 150-ml drinking water samples with 10–50 ppt UV detection limits.Dedicated to Roland W. Frei     

Group-selective prefractionation and analysis of nucleosides and catecholamines by high-performance liquid chromatographic techniques

Conclusions The use of borate as ligand in conventional affinity chromatography has found numerous applications in biochemical research [6], as for example for the clean-up of ribonucleosides and catecholamines in physiological fluids, the separation of DNA and RNA, the isolation of glycosidated hemoglobins, separation of aminoacylated RNA from free RNA, ligand mediated (piggyback) chromatographic enrichment of enzymes or the separation of base Q containing tRNA from base Q free tRNA. With the development of borate functionalized silica borate affinity chromatography has also been turned out to work under HPLAC conditions.By use of a column switching technique we could introduce a combined HPLAC/HPLC method particularly suitable for the on-line clean-up and analysis of ribonucleosides in complex matrices. We now have expanded the application of our on-line system for the clean-up and analysis of the adrenergic amines from spiked physiological matrix which means a powerful improvement compared to the system introduced by [7] for the on-line analysis just of one of the dopamine catabolites. The method described should be the method of choice for the majority of applications mentioned above as it greatly decreases the analysis time, is suitable for automation and in conjunction with a data-processing system, is applicable to routine clinical analysis.

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Gruppenselektive Vortrennung und Analyse von Nucleosiden und Catecholaminen mittels hochleistungs-chromatographischer Techniken

     

Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.

An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.     

Automated liquid chromatographic analysis of drugs in urine by on-line sample cleanup and isocratic multi-column separation

A multi-column system has been developed for automated analysis of basic drugs in urine. Two polymeric pre-columns, containing PRP-1 and Aminex A-28, were used to isolate the drugs. A short reversed-phase column, coupled to a 150 x 4.6 mm I.D. silica column, produced the analytical separation. Sample preparation consisted of dilution and centrifugation. The entire procedure required less than 30 min. Careful optimization of mobile phase conditions led to retention of benzoylecgonine and barbiturates. For most drugs, levels of 0.3 mg/l were sufficient to produce peaks that could be matched against stored spectra with a computerized library search program.     

Simultaneous quantification of sulfamethoxazole and trimethoprim in whole egg samples by column-switching high-performance liquid chromatography using restricted access media column for on-line sample clean-up

This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5 mL of acetonitrile into 1.0 mL of blended egg followed by centrifugation. The supernatant was injected (250 microL) directly into the multidimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80 ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX-TMP combination.     

Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.