Analytical Study of Chemiluminescence from the Vitamin BI2Luminol System Terry L. Sheehan and David M. Hercules* Department of Chemistry, University of Georgia, Athens, Ga.30602
Chemilurnlnescencefrom the Vltamln BI2-luminol system has been studied to develop a sensltlve analytlcal method for Vitamin B12.The method described has been applled successfully to pills contalnlng BI2. CL is achieved from the reduced form of BI2 produced by an in-line Jones Reductor. Oxygen constitutes an interference and must be removed from all solutions. Diffusion of oxygen into flow llnes was ellmlnated by use of a dry box fllled with nitrogen. Parameters of the CL-tlme curves were studied, such as background noise and peak shape. Experimental condltlons were optimized; uslng optlmum M BI2, the linear condltlons, the llmit of detection was 2 X range was 10-g-10-6 M. Assay of multicomponent vitamin capsules was unsuccessful, due to interference by large amounts of riboflavin. It was not possible to remove the rlboflavln without at the same time affecting the B12signal.
Several important metal ions “catalyze” the hydrogen peroxide oxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) in basic aqueous solution to produce chemiluminescence (CL). The metal ion is not a true catalyst since its oxidation state is changed in the reaction. Reagent concentrations can be adjusted to use the metal-luminol systems for trace analysis. In the presence of excess luminol and hydrogen peroxide, CL intensity is proportional to the limiting concentration of metal ion. With relatively simple and inexpensive instrumentation, part-per-billion (and lower) detection limits have been demonstrated for Cu(II),Ni(II), Cr(III), Mn(II), and Fe(I1) with linear ranges of roughly three orders of magnitude. The most efficient metal catalyst is Co(I1) which has a detection limit below M and a linear range up to M Co(I1) (1-3).
Complexation of a metal will reduce or completely eliminate the metal’s ability to catalyze the oxidation of luminol, for example, EDTA has been used to quench CL in the Co(II), Fe(II), and Cu(I1) systems. A notable exception to organic complexation quenching is hemin. Although 5 of 6 iron coordination sites are strongly complexed, hemin (also hemoglobin) is an excellent catalyst of the luminol CL reaction ( 4 ) . The efficiencies of Co(1I) and hemin prompted a study of CL from the luminol-vitamin B12 system. The structure of vitamin Blz is similar to that of hemin, the major exception being a cobalt central atom instead of iron. However, cobalt exists in the 3+ oxidation state in vitamin Blz but Co(II1) is not a catalyst of the luminol CL reaction. Thus, reduction of the Bl2 was necessary. Although several methods for reduction were studied, a Jones reductor column was ultimately selected. This permitted development of a sensitive method for Blz based on catalysis of the luminol reaction by the reduced form of the vitamin B12.
EXPERIMENTAL Instrumentation. Chemiluminescence for the vitamin Bizrluminol system was followed in the continuous flow system shown in Present address, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pa. 15260. 446
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Figure 1.The system uses four 50-ml plastic syringes, containing luminol dissolved in 0.1 M H~BOS-KOH buffer, EDTA, hydrogen peroxide, and water. The syringes are driven simultaneously by a Harvard Apparatus Co. Syringe drive. Each syringe is connected to a Chromatronix CAV-3 three-way valve which allows the syringes to be filled from respective reservoirs (pump withdrawal mode) or emptied into the CL flow system (pump infusion mode). Samples are introduced into the water (background) flow line by a Chromatronix SV-8031 sample injection valve. Either the aqueous background or the sample slug flows into the Jones reductor column. The column is a -8 cm Pyrex tube grooved to accommodate Chromatronix column fittings. Before entering the reaction cell, the column effluent is combined with the flow from the EDTA syringe by a Chromatronix tee connector. The flows from the luminol and hydrogen peroxide syringes are initially combined by another Chromatronix tee connector; a small platinum coil is placed in the line immediately after the tee to enhance mixing of the luminol and hydrogen peroxide solutions. Cell volume is approximately 1.3 ml. The cell is made of Pyrex glass with an i.d. of 9 mm and a total length of 40 mm. An exit sidearm is positioned 20 mm from the bottom of the cell. Although the sidearm has an i.d. of 3 mm, the junction point of the sidearm and cell body is enlarged slightly to facilitate steady flow out this exit. The top of the cell is fitted with a Teflon stopper which also serves to align the mixing paddle. The paddle is a 7 mm Teflon disk with 5 small holes drilled through its face. The paddle is connected to a Corning IM-2 vertical vibrating mixer by a stainless steel rod. The portion of the rod which contacts the CL reaction mixture is triple spray-coated with Teflon. The base of the cell is constructed with two symmetrically positioned glass nipples. Teflon tubing (I&-inch 0.d.) is fitted through these nipples and approximately 2 mm into the cell. The arrangement ensures mixing toward the center of the cell and the small tube size reduces the possibility of solution back up into the tubing. Only Teflon tubing is used in the flow system. Reagents. Luminol (Eastman Kodak) was converted to the sodium salt by slow addition of reagent grade NaOH (Fisher Scientific Co.). The sodium salt of luminol was purified by filtration through decolorizing carbon. Luminol was precipitated by the addition of dilute H2S04. A 8 X M luminol stock solution was prepared by dissolving 1.42 g of luminol, 78 g KOH (J. T. Baker), and 61.8 g of boric acid (Fisher) in 11. of water. In later studies, 3.72 g of disodium EDTA were added to the stock solution to reduce background emission. The luminol stock solution was allowed to stand for three days to stabilize before use. Luminol stock solutions were always stored in the dark. Hydrogen peroxide solutions were prepared by diluting a 3% H202 stock solution (Baker) with water. All EDTA solutions were prepared with the disodium salt of EDTA (Fisher). Vitamin BIZ (Sigma M) was prepared by dissolving Chemical Co.) stock solution approximately 1.35 g of crystalline BIZin 100 ml of water. The exact concentration of the stock solution was measured spectrophotometrically (361 nm). The Jones reductor column was packed with 20 mesh amalgamated zinc (Fisher). Before amalgamation, the zinc was washed in 1 N HC1 to remove dust and clean the surface. The washed zinc was treated with a 2% solution of Hg (N03)2 (Baker) that contained a small amount of HN03. The zinc and mercuric ions were allowed to react for 15 minutes after which time the amalgamated zinc was washed with five 100-ml volumes of water. All other reagents were of analytical grade. All solutions were prepared using water from a Contenental Water conditioning Company deionization system. Procedure. Samples were introduced into the glove box through the side chamber. After the chamber was opened to air, it was purged with nitrogen a t least 30 minutes before the initiation of B12 runs. During this purging period, the Jones Reductor column was flushed with 100-150 ml of background solution. When purging was completed, all valves were set to direct the flow of reagents into the cell.
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Figure 1. Diagram of the flow system for the chemiluminescentdetermination of vitamin BI2
With the pump in continuous operation, samples were loaded into the 1-ml sample loop and injected onto the column. Calibration curves were prepared by standard additions to a 500-ml bottle of deionized water. Standard additions were delivered t o the sample bottle by a Grunbaum micropipet. The light emission of the CL reaction is detected by a RCA 1P21 photomultiplier, positioned directly beside the reaction cell. The signal is amplified by a Princeton Applied Research (P.A.R.) model 270 D.C. photometer-preamplifier in conjunction with a P.A.R. model 280 power supply. The response is recorded on a Hewlett-Packard model 7101 B potentiometric recorder. The area of the recorder response vs. time (chart speed) was determined with a Keuffel and Esser compensating polar planimeter. The dashed line in Figure 1Pepresents a glove box enclosure. All portions of the flow system within the dashed line are maintained in a nitrogen atmosphere. This includes the photomultiplier tube housing and the Harvard pump which are not shown. The N2 lines which enter the glove box are regulated with a system of tee joints and valves. The Nz gas system allows individual or simultaneous performance of the following operation: 1)flushing the box atmosphere, 2) purging EDTA water reservoirs, and 3) purging 1 to 6 vitamin BIZ sample solutions simultaneously. The external nitrogen flow system and the dimensions of the glove box are shown in Figure 2. Copper turnings are heated at 450-500 “C to remove traces of oxygen from the nitrogen flow. The gas flow system is designed to permit regeneration of the Cu turnings with hydrogen gas. A water jacket immediately follows the oven to cool the gas before it enters the glove box. The portion of line surrounded by the water jacket is filled with glass beads to improve the cooling efficiency. Valves are positioned in the flow system to allow flusining or evacuation of the static side chamber. All “out” gas lines have traps to prevent backup of oxygen into the glove box. The ‘“2 out” line a t the bottom left corner of the box not only allows nitrogen to escape from the glove box during flushing and purging but also uses the gas flow to force out liquid wastes from the CL cell. The left side of the box has a 9 inch X 12 inch plate with the following connections: I) high voltage and anode current for the photomultiplier inside the box, 2) EDTA and two water lines to fill the internal reservoirs, and 3) lines from external luminol and hydrogen peroxide reservoirs to the respective 3-way valves. Teflon lines carrying the respective solutions are held tightly by brass Swagelok fittings. Small amounts of grease are spread on the fittings to improve the gas seal of the connections.
RESULTS AND DISCUSSION Reduction Methods. Although the literature describes several procedures for the reduction of BIZ, few were applicable to the CL analysis. Reduction by catalytic dehydrogenation or heating in ethanol was too slow. Chromous acetate ( 5 ) gave a good reduction rate, but the Cr3+ and Cr2+ ions interfered with the luminol-hydrogen peroxide reaction. Mercaptoacetic acid also interfered with chemiluminescence by reaction with hydrogen peroxide. The Jones reductor was immediately applicable to BIZreduction. Perhaps the most appealing characteristic of the Jones reductor was the ease with which a reductor column could be placed in the basic CL flow system. Reduction of Bl2
Figure 2.
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I N2 Out
External gas system and glove box dimensions
in the flow system eliminated many difficulties which were anticipated in handling solutions of reduced BIZ. In preliminary studies, the flow of the background/sample line was not mixed with EDTA before entering the reaction cell. The steady bleed of zinc from the column during flow and the surge of zinc released upon Bl2 reduction resulted in a suppressed CL background. The suppression of background was the result of flocculent Zn(OH)2 which had formed a t the CL reaction pH of 10.5. Mixing the column effluent with EDTA readily eliminated this zinc interference. EDTA served the additional function of complexing traces of interfering metal ions which might be found in the Blz samples. EDTA could not be mixed with the sample before reduction on the column. If EDTA is passed through the column, background stability is destroyed. Jones reductors have a tendency to slowly form hydrogen gas, especially if the zinc was not heavily amalgamated. As the gas coated the zinc particles, the effective surface area was decreased. This problem was eliminated by heavier amalgamation and thorough column washing at the start of each day of assays. New reductor columns required conditioning before application to quantitative I 3 1 2 assay. Columns packed with freshly amalgamated zinc increased background noise until washed with 300-500 ml of water. Although literature on the Jones reductor recommended an acid wash between samples, the acid wash could not be used since it freed too much zinc from the column. After a new column was properly washed, it still required conditioning to Biz. Although peaks could be recorded immediately after washing, the reproducibility and linearity of the results were improved by passing several concentrated ( 5 X M) slugs of Bl2 through the column. Oxygen Interference. During tests of the flow system a t low BIZ concentrations, it became evident that dissolved oxygen constituted an interference. The Bl2 samples, the background water, and the solution of EDTA had to be thoroughly purged with nitrogen before Blz could be measured M. a t concentrations below 5 X Even though purging improved the limit of detection to approximately 5 X M, reproducibility was poor; also as the sample concentration approached the limit of detection, light emission deviated from linearity. These problems were the result of oxygen and the entire flow system was placed in the glove box. The nitrogen atmosphere resulted in increased sensitivity, improved reproducibility (approximately &I%), and linearity through the zero intercept. Background Noise. I t became necessary to decrease flow system noise if the limit of detection was t o exceed 1X M. The efficiency of reagent mixing was demonstrated to be a major factor in the noise. A nitrogen bubbler was originally used to mix the reagents in the reaction cell. A vertical motion ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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Figure 3. Chemiluminescent response of the luminol-H202 reaction to vitamin B12r Numbers above the peaks represent B T 2concentration X M. Numbers below the peaks represent relative peak area. Conditions: 1 X M EDTA, 1X M H202,4 X M lurninol, reaction pH 10.5.and a flow rate of 1.91 ml/minute/syringe
-5
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Flgure 5. Chemiluminescent intensity vs. luminol concentration Conditions: 1 X M EDTA, 2 X M H202,1 X 10.8,and flow rate of 1.91 ml/mlnute/syringe
M BIZ, reaction pH
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2000
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Figure 4. Chemiluminescentintensity vs. H202concentration Conditions: 1 X M EDTA, 4 X M iuminol, 1 X pH 10.6, and flow rate of 1.91 ml/minute/syringe
M B12, reaction
(vibrating) mixer yielded more efficient and more consistent mixing patterns and dower noise. Efficient mixing of the luminol-hydrogen peroxide flow and the background-EDTA flow was equally important. Large i.d. (2 mm) glass Y connectors were not acceptable unless a small platinum coil was inserted into the Y to enhance mixing. Chromatronix tee connectors with $&-inch orifices were substituted for the glass Y connectors since the tee connectors demonstrated a slight reduction in system noise. The smoothness of the pump-syringe delivery directly influences system noise. The Harvard pump was extremely steady in its rate of delivery if the syringes were in good condition. Disposable plastic syringes, however, had a tendency to bind if used repeatedly without lubrication. Syringe binding resulted in drops of the background light level followed by small peaks as the pump pressure suddenly overpowered the binding. Lubrication with silicone grease eliminated the binding problems. The design of the reaction cell was also found to influence the pattern of the background and sample emissions. The original cell design did not allow steady flow out ?f the sidearm junction point with the cell wall. Surface tension would allow a “head” to build up in the cell above the sidearm exit. The increase in effective cell volume yielded a proportional in448
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Figure 8. Chemiluminescent intensity vs. reaction pH Conditions: 1 X IO-’ M EDTA, 2 X lo-* M H202,8 X M luminol, 1 X M B12,and flow rate of 3.82ml/rninute)syringe
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80
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FLOW RATE
Figure 7. Chemiluminescentintensity vs. flow rate Conditions: 1 X M EDTA, 2 X M 812, and reaction pH 10.5
M HZ02,8 X
M luminol, 5 X
Table I. Relative Chemiluminescent Response of Different Vitamins Chemiluminescence responseu Without column Without column With column Vitamin without luminol with luminol without luminol
With column with luminol
Vitamin B1zb 0 0 5 980 Thiamine HC1‘ 26 120 43-4 2800-280 75 8500 23 1600 Riboflavin 1 46 3 17 Pyridoxine‘ 0 12 0 3 Folic acid/ 3 321 43 374 Nicotinic acid8 M EDTA, reaction pH 10.5, and flow rate of 3.82 M H202,8 X M luminol (when used), 1 X a Other conditions: 5 X ml/minute/syringe. 3 p g / 5 0 ml. 3 mg/50 ml. 6 mg/50 ml. e 19 pg/50 ml. 2 pg/50 ml. 1 mg/80 ml.
crease in the light collected by the photomultiplier. When the surface tension was broken, the effective cell volume and total light emitted dropped sharply. This situation was manifested as a “choppy” sine wave on the background emission. Peak Shape. Typical data are shown in Figure 3. Note significant tailing of the peaks. This tailing was partially due to sample mixing with water background as the sample flowed through the lines and column. Another factor which contributed to the tailing was the adsorption of B1:! on the amalgamated zinc. Although previous trace metal studies (2) have used peak height of the recorded light emission as a relative measure of concentration, peak height measurements deviated from linearity at concentrations above 5 X lods M BIZ.Peak areas, however, were found to be linear to at least X lo-’ M B12. Optimization of t h e Luminol-HzOz Reaction. The sensitivity of the reaction was shown to be markedly affected by the concentration of H202 and luminol and the reaction pH. Holding pH, flow rate, and luminol concentration constant, a profile of CL intensity vs. H202 concentration was developed for the range 2 X 10-3 M to 2.5 X 10-1 M as shown in Figure 4. Maximum light emission occurred at 6 X M H202. The width of the background noise increased slightly with increasing H202. The effect of luminol concentration on CL intensity is shown in Figure 5. Background noise increased dramatically with increasing luminol concentration. Although a maximum areahoise ratio occurred at 2 X M luminol, the absolute level of light emission did not begin to decline until 8 X M luminol. The effect of reaction pH on CL intensity is shown in Figure 6. Although maximum light emission occurred at pH 10.8, maximum signalhoke was at pH 10.5. The signal/noise ratio had another interesting feature in that it wiis relatively flat over a pH range of 10.2-11.0. The light output of the free cobalt catalyzed reaction (maximum pH 11.2) is much more sensitive to the cell pH (1). The effect of flow rate on relative light emission is shown in Figure 7. Total light emission increased very rapidly at flow rates lower than 1.91 ml/minute. The reason for the increased emission is not apparent. Although the increase could be attributed to more complete reduction of the B12 sample, this explanation is not consistent with the leveling of response above a flow rate of 4 ml/minute. The most important aspect of flow rate study was the areahoke ratio. Although a 7.64 ml/minute rate showed the best areahoise ratio, the 3.82 ml/minute rate was used in all studies since it allowed more time between the introduction of sample. At 3.82 ml/minute, the operator could easily run 5-7 samples/50 ml of background solution. With conditions optimized for the luminol-H202 reaction, the linear range of vitamin Bl2 was 3 X to 5 X M. The peak height to background noise ratio equaled 2 a t 3 x
M Bl2. Reproducibility in peak area was determ’ined by measuring ten replicates of 2 X M BIZ. The relative standard deviation in peak area was f1.2%. ANALYSIS OF VITAMIN PILLS The limit of detection of the CL method is an order of magnitude higher than the normal serum concentration of vitamin BIZ;however, the sensitivity of the method makes it potentially applicable to pharmaceutical preparations. Studies were performed to determine the applicability of the method to pill samples using only one pill per analysis. Interferences. CL analysis using the luminol-H202 reaction is susceptible to interferences, since the reaction does not differentiate between different catalyst. For example, Co(I1) cannot be quantitated in the presence of Cu(I1) since Cu(I1) also catalyzes the CL emission. In the analysis of pharmaceutical preparations, it should be easy to separate B12 from interfering metal ions, but possible organic interferences exist. Interference was expected from riboflavin, since riboflavin shows chemiluminescence in the presence of H202 and Cu(I1) ( 6 )and is present in vitamin pills at concentrations -lo4 those of Biz. Five vitamins were investigated as interference: thiamine HC1 (vitamin Bl), riboflavin (vitamin Bz), nicotinic acid, folic acid, and pyridoxin (vitamin BG).Since the objective was to use only one vitamin pill per assay, each vitamin was studied a t the concentration expected if one pill were dissolved in 50 ml of water. Four separate experiments were run to investigate potential interferences: 1)without the reductor column or luminol, 2) without the reductor column but with luminol, 3) with the reductor column but without luminol, and 4) with the reductor column and luminol. The results are summarized in Table I. The magnitude of the pyridoxine and folic acid peaks was less than 5% of the BIZresponse. Thiamine emission was not reproducible with the reductor column in the flow line. Consecutive thiamine peaks displayed decreasing intensities, which is characteristic of the buildup of some CL inhibiting species in the reaction cell. Nicotinic acid caused a fairly strong response with luminol and H202, approximately one third of the BIZresponse. Removal of Interferences. Because riboflavin demonstrated the greatest potential as an interference, most studies were directed toward the removal of riboflavin from solutions containing only B12 and riboflavin. Adsorbents such as florisil, fuller’s earth, decolorizing carbon, Cab-o-Sil, and Celite 545 were unsuccessful in completely separating Bl2 and riboflavin. Solvent extractions using cresol and 2,4-dichlorophenol were also unsuccessful since Bl2 and riboflavin have very similar solubilities. The dicyanide-benzyl alcohol extraction (7) was studied since the procedure was reported to remove ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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90% of the species which interfere in the spectrophotometric assay. Although very little riboflavin could be seen in the final extract, B12 results lacked reproducibility and correlation with pure standards. Attempts were made to remove riboflavin by conversion to lumiflavin and subsequent CHC13 extraction (8).The efficiency and reproducibility of the conversion was poor, f80-90%. The separation of B12 from other compounds by gel filtration has been reported, but the studies involved fairly concentrated solutions of Blz (9).When attempted at our levels of concentration, no B12 was detected in the eluant. The absence of detectable Bl2 could be due to dilution of the column to a concentration below the detection limit or loss by adsorption of Blz on the Sephadex. A study was also performed to test the possible separation of Bl2 by affinity chromatography on a column of Sepharose-Intrinsic Factor. Although the Sepharose-I.F. is normally discarded after its use in the radiosorbent method, B12 can be separated from the I.F. at high pH, without destruction of either the Blz or the I.F. (10, 11). In order to test the basic principle of the affinity column, a batch process was developed, but the solutions treated showed no correlation with Blz standards. While studying the removal of riboflavin by Zn(OH)z,it was found that ionic strength and pH of the sample greatly influenced the reproducibility of the results. The effect of ionic strength stabilized only after 8 consecutive samples had been assayed. The degree of stability achieved by running consecutive samples was lost if the column was not used for over 30 minutes. The effect of pH is probably related to an attack of the hydrogen ions on the reductor material, the background CL decreasing with increasing pH. If the ionic strength of the samples and the background solution was constant, peaks from the different pH's were fairly reproducible. The minimum background region was pH 4.5-6.0. Reproducible peaks (f3%) were generated for samples of B1z having KCl concentrations of 10-3-10-1 M and pH's of 4.5-6.0; however, this required a constant ionic strength and pH for each sample and an equivalent ionic strength and pH for the background solution. Even by carefully maintaining constant pH and ionic strength of all samples and background, a poor correlation was achieved with Bl2 standards. This control of sample composition was extremely tedious and still resulted in an occasional point with an error of 30-40%.
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The only pills which were successfully assayed by the CL method were Redisol (Merck Sharp and Dohme) tablets. The tablets were composed of only vitamin BIZ(25 kg/tablet) and binders. Tablets dissolved in 10 ml of deionized water were concentrated enough for spectrophotometric assay; a portion of the concentrated solution was diluted by a factor of 20 for the CL assay. The results of the spectrophotometric assay was 25.10 f 0.34 wg/tablet where the CL method gave 25.03 f 0.18 pg/tablet. The only direct application of the Blz CL method would be to tablets such as the Redisol which contain no other contaminations or possibly to injectable preparations of Blz (which are also pure). Using a separation procedure to separate Bl2 from the multitude of possible interferences, the CL method would then be applicable to multitomponent vitamin tablets. If preconcentration techniques would be devised to concentrate BIZby a factor of ten, the CL method would be applicable to serum analyses.
ACKNOWLEDGMENT The authors thank W. R. Seitz, and P. W. Carr for helpful discussions. LITERATURE CITED (1) W. R. Seitz and D.M. Hercules in "Chemiiuminescence and Bioluminescence". M. J. Cormier. D.M. Hercules. and J. Lee.. Ed... Plenum Press. New York, 1973,pp 427-449. (2) W. R. Seitz, W. W. Suydam, and D. M. Hercules, Anal. Chem., 44, 957 (1972). (3) W. R. Seitz and D.M. Hercules, Anal. Chem., 44, 2143 (1972). (4) W. Specht, Angew. Chem., 50, 155 (1937). (5) R. N. Boos, J. E. Carr, and J. 6. Conn, Science, 117, 603(1953). (6) R. H. Steel, Biochemistry, 2, 529 (1963). (7) G. 0. Rudkin and R. J. Taylor, Anal. Chem., 24, 1155 (1952). (8) "Vitamin Methods", Vol. 1, I.P. Gyorgy, Ed., Academic Press, New York, 1950, p 124. (9) T. Buchman, F. Kennedy, and J. Wood, Biochemistry, 8, 4437 (1969). (10) R. Grasbeck, U. Stenman, L. Puutula, and K. Visuri, Biochim. Biophys. Acta, 158, 295 (1968). (11) H. Shum. B. J. O'Neill, and A. M. Streeter, J. Clin. Pathol., 24, 239 (1971).
RECEIVEDfor review August 30, 1976. Accepted December 3, 1976. This work was supported by the National Institute of General Medical Sciences, NIH, under Grant No. GM17913-05.
