Anal. Chem. 1997, 69, 4278-4282
Postcolumn Immunodetection Following Conditioning of the HPLC Mobile Phase by On-Line Ion-Exchange Extraction Kamlakshi Shahdeo, Clark March, and H. Thomas Karnes*
Department of Pharmacy and Pharmaceutics, Virginia Commonwealth University/Medical College of Virginia, P.O. Box 980533, Richmond, Virginia 23298-0533
Postcolumn immunodetection can provide enhancement of selectivity and sensitivity when used in combination with HPLC. Typical reversed-phase HPLC employs a variety of organic modifiers and buffers, and immunoassays generally perform best in a low ionic strength aqueous matrix with a pH between 7 and 8. There is therefore a need for removal of an analyte from a typical mobile phase into an environment compatible with an immunoreactor. An on-line system utilizing an ionexchange column along with a switching valve to sequester an analyte from a HPLC mobile phase and apply a counterion eluent solution was designed to provide the proper matrix for immunoassay. An on-line HPLC-ionexchange system was developed for the model analyte procainamide hydrochloride. The eluents from the online HPLC and ion-exchange system were collected and analyzed off-line using fluorescence polarization immunoassay. The fluorescence polarization immunoassay was calibrated using standards in a buffer matrix, and the system was validated using controls at 2.0, 7.0, and 15.0 µg/mL in mobile phase. Procainamide and its metabolite were extracted from spiked plasma using liquid-liquid extraction, and validation of the system with plasma samples demonstrated 15.60, 15.63 and 15.70% (n ) 6) RSD for the three controls and accuracies of 6.42, 10.30, and 17.70% (n ) 6) difference from spiked, respectively. On-line coupling of an immunoreactor and high-performance liquid chromatography can result in enhanced selectivity and sensitivity. Reversed-phase HPLC is employed for analysis of most commonly used drugs. Reversed-phase HPLC makes use of several different organic modifiers and buffers in mobile phases. Immunoassays on the other hand, function best in an aqueous matrix. Thus, coupling of immunoassays and HPLC results in problems with compatibility. In order to solve this problem, the drug molecule in the HPLC eluent can be extracted on-line into an aqueous matrix and then applied to the immunoreactor. This can be achieved by coupling an ion-exchange column between the HPLC column and the immunoreactor. Immunoassays involve the interaction of antibodies and antigens. Antibodies recognize an epitome of the antigen and not the entire antigen.1 Thus, antigens having similar epitomes may interact with the antibody resulting in “false positives”. Therefore,
immunoassays are very selective but their selectivity is not complete. Coupling HPLC prior to an immunoassay may result in separation of components with similar epitomes by a theoretically different selectivity limitation and, thus, lead to enhancement of selectivity overall. Immunological determinations by this approach have been performed off-line. Eluents from HPLC are collected and then assayed immunologically.2-5 This approach provides added selectivity, but the immunoassays may require multiple steps such as incubation and washing, which makes them time consuming. On-line coupling of postcolumn immunoreactors with HPLC can help in increasing the speed of these assays.6-12 The presence of organic modifiers such as acetonitrile and methanol in such assays is a major concern because protein reagents can be altered by these solvents. In developing a liquid chromatographic immunological detection system for leukotrienes, Irth et al. changed the HPLC column from a C18 reversedphase to a C4 bonded silica column to reduce the effect of the organic modifier.9 Frequent regeneration of restricted access columns due to the presence of organics needed to separate bound and free antigen complexes has also been a problem. Krull et al. developed a postcolumn immunodetection system for bovine growth hormone releasing factor.10 In order to decrease the effect of the organic modifier, the HPLC eluent was diluted with an aqueous buffer before application to the immunodetection system thus reducing sensitivity of the system. A solution to these problems is the introduction of an ion-exchange column between the HPLC and immunodetection system. The eluent from the HPLC containing the analyte is applied to the ion-exchange column followed by extraction of the analyte, by a change of pH or use of counterion, into an aqueous
* To whom correspondence should be addressed. E-mail: Tom.Karnes@ VCU.EDU. (1) De Frutos, M.; Regnier, F. E. Anal. Chem. 1993, 65, 17A-20A.
(2) Stone, J. A.; Soldin, S. J. Clin. Chem. 1988, 34, 2547-2551. (3) Gelpi, E.; Ramis, I.; Hotter, G.; Bioque, G.; Bulbena, O.; Rosello, J. J. Chromatogr. 1989, 492, 223-250. (4) Meyer, H. H. D.; Hartmann, F. X.; Rapp, M. J. Chromatogr. 1989, 489, 73-180. (5) Rapp, M.; Meyer, H. H. D. J. Chromatogr. 1989, 489, 181-189. (6) Irth, H.; Oosterkamp, A. J.; van der Welle, W.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 633, 65-72. (7) Oosterkamp, A. J.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1994, 66, 4295-4301. (8) Oosterkamp, J.; M. Villaverde Herraiz, T.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 1201-1206. (9) Oosterkamp, A. J.; Irth, H.; Heintz, L.; Marco-Varga, G.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 4101-4106. (10) Cho, B. Y.; Zou, H.; Strong, R.; Fisher, D. H.; Nappier, J.; Krull, I. S. J. Chromatogr. 1996, 743, 181-194. (11) Miller, K. J.; Herman, A. C. Anal. Chem. 1996, 68, 3077-3082. (12) Irth, H.; Oosterkamp, A. J.; Tjaden, U. R.; van der Greef, J. Trends Anal. Chem. 1995, 14, 355-361.
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matrix that could then be applied to the immunochemical detection system. In this case, the analyte may be eluted in a smaller volume due to a concentration step which may enhance sensitivity. The objective of this work was to develop an on-line ion-exchange approach to remove organic solvents which would be followed by postcolumn fluorescence polarization immunoassay to show the feasibility of the system. EXPERIMENTAL SECTION Materials. Procainamide and N-acetylprocainamide (NAPA), Tris(hydroxymethyl)aminomethane hydrochloride (Tris), ammonium chloride, calcium chloride, sodium phosphate, sodium hydroxide and potassium chloride were purchased from Sigma (St. Louis, MO). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Fisher Scientific (Fair lawn, NJ). Carboxymethyl (CBA) weak cationic exchange cartridge was purchased from Varian Separations (Harbor City, CA). Blank plasma was purchased from Biological Specialties (Colmar, PA). Methanol, acetonitrile, methylene chloride, and tetrahydrofuran were of analytical grade and purchased from Baxter (B&J brand, Muskegon, MI). Triethylamine was obtained from Aldrich (Milwaukee, WI). Procainamide and N-acetylprocainamide reagent kits, controls, and standards were purchased from Abbott Laboratories (North Chicago, IL). All solutions were prepared in deionized water distilled in the laboratory with a Corning Mega Pure automatic purification system (Corning, USA). Apparatus. All mobile phases were filtered through a 0.45 µm nylon filter purchased from Alltech (Deerfield, IL) and helium sparged prior to use. The HPLC system consisted of a Gilson pump Model 302 with a manometric module Model 802B from Gilson Medical Electronics (Middleton WI). HPLC columns used were a Beckman C18 Ultrasphere column (250 mm × 4.6 mm i.d., 5-µm particle size) and a Supelcosil ABZ+ Plus C18 column (150 mm × 2.1 mm i.d., 5-µm particle size) from Beckman (Beckman Instruments, Brea, CA) and Supelco (Bellefonte, PA), respectively. UV detection was performed using a Shimadzu SPD 6A UV spectrophotometric detector at a wavelength of 280 nm (Shimadzu Instruments, Columbia, MD). The detector used for off-line ionexchange extractions was a Perkin Elmer Lambda 2S UV/Visible spectrometer (Perkin Elmer, Norwalk, CT). A Rheodyne Model 7161 manual injector equipped with a 20-µL loop for HPLC and a 1.0-mL loop for flow injection ion-exchange were used (Cotati, CA). An Autochrom switching module equipped with a six-port Rheodyne Model 7000 valve (Autochrom, Milford, MA) was used. The ion-exchange system consisted of a Gilson pump and manometric module. The column used was a 30 mm × 2.1 mm i.d., PEEK column from Perseptive Biosystems (Framingham, MA). A packing device for packing the ion-exchange column was purchased from Perseptive Biosystems. Injections were performed manually with Hamilton syringes (Reno, NV), 100-µL and 1.0-mL volumes. Off-Line Ion-Exchange Extractions. Off-line ion-exchange extractions were carried out to test the feasibility of the system. Procainamide was used at a concentration of 2.5 × 10-5 M. A carboxymethyl weak cationic exchanger with a pKa of 4.8 was used for the ion-exchange extraction and remained ionized at the working pH of 7.0. The buffer used for the extraction was 0.005 M Tris (pH 7.0). Sodium, potassium, ammonium, and calcium were evaluated as counterions. Extractions were performed to determine the optimum concentration and minimum volume of counterion solution required to reproducibly and completely elute
the analyte. The system was evaluated using 20% methanol/80% Tris, 20% acetonitrile/80% Tris, and 10% tetrahydrofuran/90% Tris as sample matrix solutions. The effect of 0.5% triethylamine as amine modifier with the methanol/Tris sample matrix solution was also evaluated. Reusability of the cartridges and reproducibility of the extraction were also evaluated.13 Extraction Procedure. The cartridge was conditioned with 1.0 mL of methanol. It was then washed with 2.0 mL of 0.005 M Tris buffer (pH 7.0). 1.0 mL of sample solution was added, the displaced liquid was collected, and the absorbance was measured at the 279-nm maximum. The cartridge was washed with 1.0 mL of Tris buffer, and the absorbance of the wash was also measured. This was followed by sequential 1.0-mL additions of counterion solution in Tris buffer. The eluent was collected, the volume was measured, and the absorbance determined at 279 nm. This was repeated until no significant absorbance was observed. Calculations were carried out to determine the percent recovered. HPLC and Flow Injection Ion Exchange. Methanol as the organic modifier was evaluated with Tris buffer to achieve baseline resolution for procainamide and NAPA. In order to separate and transfer the procainamide peak to the ion-exchange column, HPLC was followed by a “heart-cut” column switch. The flow rate was kept at 1.0 mL/min. A second column, Supelcosil ABZ+ Plus C18 (150 mm × 2.1 mm i.d., 5-µm particle size), was evaluated at a flow rate of 0.2 mL/min. The ion-exchange column was packed with silica-based carboxymethyl cationic exchange extraction sorbent with a pKa of 4.8 and 40-µm particle size that was obtained from the off-line cartridges. Procainamide was extracted from the flow injection ion-exchange column in a procedure similar to that in the off-line ion-exchange extraction. Procainamide solution (2.5 × 10-6 M) in a matrix of 20% methanol/80% 0.005 M Tris buffer (pH 7.0) was injected, and the analyte was extracted with 1.0 mL of 0.5 M calcium chloride in Tris buffer. Similar extractions were carried out at 1.0 × 10-6 M and 7.5 × 10-7 M. Fluorescence Polarization Immunoassay. The fluorescence polarization immunoassay was calibrated with 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µg/mL procainamide standards prepared in 0.5 M calcium chloride in Tris buffer (pH 7.0), to simulate the ion-exchange elution matrix, according to the instructions provided by Abbott Laboratories.14 The controls used were 2.0, 7.0, and 15.0 µg/mL. Each standard and control was diluted 20 times, and the calibration was performed using a sample volume of 20 µL, rather than the usual 1.0 µL required for the procainamide assay, to provide concentrations of 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µg/mL for the standards and 2.0, 7.0, and 15.0 µg/mL for the controls. This was done because the system is designed to measure human plasma therapeutic concentrations of procainamide, which are between 4 and 10 µg/mL, and at these concentrations, the HPLC and ion-exchange columns were overloaded. Validation of On-Line HPLC-Ion-Exchange and Off-Line Immunoassay System. Three procainamide controls were prepared in the mobile phase at concentrations that would provide 2.0, 7.0, and 15.0 µg/mL in the final solution. The immunoassay sample volume was 20 µL, the volume of the ion-exchange eluent was 1.5 mL, and the HPLC injection loop was 20 µL, which resulted in an overall dilution factor of 1:3.75. In order to provide the final concentration required for the immunoassay, the con(13) Shahdeo, K.; Karnes, H. T. Pharm. Res. 1996, 13, S-27. (14) TDx FLx TDx Assays Manual, Abbott Laboratories, 1994.
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centration of procainamide injected into the HPLC system was calculated on the basis of this dilution factor. Methanol was injected to condition the ion-exchange column. At 3.0 min the valve was switched to the ion-exchange column then switched back at 6.5 min. Tris was allowed to flow until 8.0 min. A 1.0-mL aliquot of 0.5 M calcium chloride in 0.005 M Tris (pH 7.0) was injected onto the ion-exchange column, and 1.5 mL of eluent was collected from 8.5 to 16.0 min. A second injection of calcium chloride was made to clear the column. The eluents were then analyzed using fluorescence polarization immunoassay.15 Extraction from Plasma and Validation. Plasma was spiked with procainamide and N-acetylprocainamide to obtain standards of 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µg/mL and controls of 2.0, 7.0, and 15 µg/mL. To 375 µL of spiked plasma, 75 µL of 1.0 N NaoH was added followed by addition of 3.0 mL of methylene chloride. The mixture was vortexed for 5 s and centrifuged for 5 min at 3000 rpm. The organic layer was separated and washed with 750 µL of deionized distilled water. The extract was evaporated to dryness and the residue reconstituted in 100 µL of mobile phase and injected onto the system.16 Similar extractions were carried out for blank human plasma from three different sources. Six replicates of each of the controls were prepared, run through the entire system, and analyzed using the fluorescence polarization immunoassay. The procainamide eluents from the HPLC-ionexchange system were analyzed using the fluorescence polarization immunoassay and the N-acetylprocainamide reagent kit as a test of selectivity. Two blanks and two each of the three procainamide controls collected from the HPLC-ion-exchange system and two NAPA controls were assayed using the fluorescence polarization immunoassay. RESULTS AND DISCUSSION The immunoreactor tolerates pH between 7 and 8 for antigenantibody binding and a low salt concentration (
