On-line Immunoaffinity Research Articles

Purification and analysis of drug residues in urine samples by on-line immunoaffinity chromatography/high-performance liquid chromatography/continuous-flow fast atom bombardment mass spectrometry.

An automatic system for the on-line extraction and analysis of diethylstilbestrol in the urine of rats and calves is described. Extraction was done by injecting samples directly into an immunoaffinity column containing antidiethylstilbestrol antibodies bound to a Sepharose matrix, and analysis was done by on-line high-performance liquid chromatography with ultraviolet and continuous-flow fast atom bombardment mass spectrometry detectors. The system, consisting of one injector, two switching valves, and three pumps, was operated under computer control and allowed to perform a complete analysis of a sample in 28 min. An accurate quantitation by isotope dilution was also possible, by the use of deuterated diethylstilbestrol as internal standard. The sensitivity of the method, using selected-ion monitoring of the molecular ion of diethylstilbestrol, was 2 ng/mL, injecting 1 mL of urine sample. Results obtained from analyzing the urine of rats and calves treated with diethylstilbestrol are presented.

Determination of drugs from urine by on-line immunoaffinity chromatography—high-performance liquid chromatography—mass spectrometry

A method for rapid extraction and identification of drugs in urine is described. The system utilizes a high-performance protein G immunoaffinity column coupled to a reversed-phase analytical column by use of a trapping column and switching valve. A small amount of antibody (5 μg drug-specific) is used for each analysis to extract either propranolol or lysergic acid diethylamide (LSD) from human urine. Urine diluted with phosphate-buffered saline is pumped directly through the protein G column thus eliminating time- and solvent-consuming sample preparation procedures. On-line ultraviolet or mass spectral analysis provides the means of drug detection and identification. With ultraviolet detection propranolol may be detected in spiked urine at the 250 pg/ml level. A Hewlett-Packard mass spectrometer modified for atmospheric pressure ionization and equipped with an ion spray source allows detection of propranolol in urine at 2.5 ng/ml and LSD at 500 pg/ml using single ion monitoring. The potential applicability of the technique for drug confirmations is discussed.

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 μg/kg) and from spiked swine muscle tissue 64 ± 2% (10–50 μg/kg). The calibration curves were linear in the range 1–200 μg/kg spiking levels. Limits of determination were 1 μg/kg for milk and 10 μg/kg for muscle tissue.

Direct introduction of large-volume urine samples into an on-line immunoaffinity sample pretreatment-capillary gas chromatography system

An immunoaffinity precolumn containing immobilized antibodies raised against the synthetic steroid hormone beta-19-nortestosterone, has been used for the automated sample pretreatment of urine samples containing beta-19-nortestosterone or the related steroids norethindrone and norgestrel. The sample pretreatment system was coupled on-line to a capillary GC. The on-line connection between the immunoaffinity precolumn and the capillary GC was realized with an interface that consisted of a 10 mm X 2 mm i.d. reversed-phase pre-column and a diphenyltetramethyldisilazane-deactivated GC retention gap. After preconcentration on the immunoaffinity precolumn the analytes were eluted and reconcentrated on the reversed-phase precolumn. Subsequently, this precolumn was desorbed with 75 microL of ethyl acetate, which was directly introduced into the retention gap by using partially concurrent solvent evaporation. The system allows the automated pretreatment and GC analysis of 5-25-mL urine samples for the ppt-level determination of 19-norsteroids. The general applicability and potential of on-line immunoaffinity-capillary GC systems are discussed.

On-line immunoaffinity sample pre-treatment for column liquid chromatography: evaluation of desorption techniques and operating conditions using an anti-estrogen immuno-precolumn as a model system

An immunoaffinity precolumn (immuno-precolumn) containing an immobilized antibody directed against estrogen steroids, was used as a model system for the evaluation of different desorbing techniques, suitable for on-line coupling to column liquid chromatography (LC). Desorption of estrogen analytes from the immuno-precolumn proved to be impossible with the buffers and chaotropic solutions frequently used in affinity desorption. Micellar solutions are effective in obstructing the antibody-antigen reaction, but their use as desorbing solutions was not found to be practical because of the large interferences introduced into the chromatograms. Desorption with aqueous solutions at elevated temperature, created by microwave action or conventional heating, although effective is not practical in this instance, because the agarose used in this study as the stationary phase for the immuno-precolumn is prone to heat decomposition. The most effective and practical approach is desorption with a methanol-water mixture (95 + 5 v/v). On-line dilution of the eluate allows the concentration of the desorbed analytes using a reversed-phase LC system with subsequent separation and ultraviolet detection. The performance of the system with spiked urine and plasma samples, which were introduced directly into the system, was evaluated and the results were compared with immunoselective desorption.

Determination of β-19-nortestosterone and its metabolite α-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17β-19-nortestosterone (β-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), β-19-NT and its metabolite 17α-19-nortes-tosterone (α-19-NT) can be determined in biological samples with a detection limit of 0.05 μg/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17β-laurate, the maximum concentrations of β-19-NT (1.3 μg/l) and α-19-NT (3.1 μg/l) were found seven days after intramuscular administration. In a bile sample from this calf only α-19-NT (55 μg/l) was detected. In meat samples from three treated calves, the concentration of β-19-NT varied from 0.1 to 1.6 μg/kg and no α-19-NT could be detected. In liver samples from these calves, the concentrations of β-19-NT and α-19-NT were less than 0.05-0.1 and 0.5-0.9 μg/kg, respectively. In the corresponding kidney samples, the concentrations of β-19-NT and α-19-NT were 0.4-0.5 and 0.5-1.6 μg/kg, respectively. The application of the same immuno precolumn to the determination of 17β- and 17α-trenbolone, two structurally related steroids, is also demonstrated.