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Anal. Chem. 1085, 57,2264-2267
Elimination of Matrix Related Interferences in Indirect Photometric Chromatography David Brown, Robert Payton, and Dennis Jenke* Travenol Laboratories, 6301 North Lincoln Avenue, Morton Grove, Illinois 60053
The removal of UV-absorbing matrlx lnterferents In the tltle technlque can be accompllshed either by exploltlng differences In the UV spectral proflles of the lnterferent and the moblle phase or by using postcolumn flber suppressor eluant modlflcatlon. I n the former case, the presence of the lnterferent 18 masked spectroscoplcally. I n the latter case, the lnterferent Is chemlcally removed from the moblle phase prlor to detectlon. Both techniques are demonstrated herein by the removal of amlno acld type lnterferents from pharmaceutlcal type matrices.
Indirect photometric or "vacancy" chromatography is an analytical methodology whereby analyte separation by ion exchange is coupled with indirect photometric detection. Since its introduction in 1982 by Small and Miller (1, 2), it has gained popularity as evidenced by various published applications (3-10). Chromatographic separations can be accomplished with a variety of resin/mobile phase couples including conventional ion chromatography and ion-pairing/reversephase column systems. Indirect analyte detection involves measuring the absorbance decrease as an analyte displaces a UV-active component in the mobile phase. Advantages of the method include its instrumental simplicity [a singlecolumn approach requiring conventional HPLC hardware], sensitivity [in many applications a t least twice as sensitive as the more conventional conductometric detection (11)], ease of calibration [via a "standardless" type approach (12, 13)], and versatility. Like other chromatographic methods, indirect photometric chromatography (IPC) can accurately quantitate analyte concentrations if individual species are resolvable from each other and from other components of the sample matrix. In cases where resolution cannot be achieved by the chromatographic process, other detection strategies can be used to isolate the analyte from potential interferents. These alternative strategies are of some importance in analyzing pharmaceutical samples, where inorganic characterization by IPC is frequently rendered difficult because of interference between the inorganic analytes and organic components of the sample. Because many of these organic species (amino acids and drugs) are present at high concentrations and possess large molar absorptivities in the UV spectral region, their interference is manifested as reverse peaks in the chromatogram. A minimal interference thus results when the organic interferent prevents the accurate identification of the base line for the analyte peak (adversely affecting quantitation). In an extreme case, the interferent response can completely mask the analyte peak. It is the purpose of this paper to describe two methods that can be used to alleviate or prevent this type of interference. In one method, differences in the absorption spectra of the interferent and eluant species are exploited to effectively mask the interferent. In the other, the utilization of postcolumn eluant suppression chemistry actually removes the interfering species from the mobile phase prior to detection. EXPERIMENTAL SECTION The chromatographicsystem consisted of a Perkin-Elmer Series 3B pump, Dionex (Sunnyvale,CA) AG-1guard and AS-1 separator 0003-2700185/0357-2264$01.50/0
columns,a Rheodyne Model 7125 loop injector, a Waters Lambda Max Model 481 LC spectrophotometer and a Linear (Reno, NV) strip chart recorder. Injection loop size was 0.020 mL. All chromatography was performed at ambient (22 & 1 O C ) temM perature. The eluant used was a mixture containing 1 x M sodium potassium hydrogen phthalate (KHP) and 0.75 X borate. The pH of the mixture was adjusted to 9.1. The flow rate for all experiments was 3 mL/min. The eluant was prepared by dissolving appropriate amounts of KHP and sodium borate decihydrate in deionized water; the pH of the resultant mixture was adjusted to 9.1 by the addition of 0.1 M KOH. For experiments that employed a suppressor system, the AFS-1 fiber suppressor manufactured by Dionex was used. The regenerant employed was 0.025 N H2S04,supplied by gravity feed at a flow rate of between 3.0 and 3.5 mL/min. Prior to use, both the mobile phase and the regenerant were filtered and degassed through 0.22-pm polycarbonate media. Unless otherwise stated, the detector was operated with air as the reference, at a wavelength of 265 nm and with a sensitivity of 0.05 AUFS. The samples used in this study represent two high-volume pharmaceuticalproduct types presently on the market and include dopamine hydrochloride in 5% dextrose injection (Abbott Laboratories, North Chicago, IL, lot no. 62022DR), an artificially prepared solution containing dopamine hydrochloride [4-(2aminoethyl)-1,2-benzenediol)],dextrose, sodium bisulfite,and HC1 (as a pH adjuster), and 8.5% Travasol with electrolytes (Travenol Laboratories, Deerfield, IL, lot no. 170012x7). The artificial sample was prepared to contain (initially) 1.6 mg/mL dopamine hydrochloride, 5% by weight dextrose, and 500 mg/L NaHS03. After formulation, sufficient HCl was added to produce a final solution pH of 3.5 (required to aid in stabilization). While the dopamine-containingsolutionswere analyzed untreated, the 8.5% Travasol solution was diluted 1to 50 with deionized water prior to injection. All salts and reagents used were reagent grade. Deionized water was produced with a Millipore Milli-Q cartridge system. UV spectral scans were produced with a Perkin-Elmer 552 spectrophotometer, which had a slit width of 1 nm and was equipped with l-cm quartz cells (Fisher Scientific, no. 14-3859096). Samples containing 15 mg/L dopamine hydrochloride dissolved in a water matrix and a 10-fold dilution of the mobile phase were used to generate the scans contained herein.
RESULTS AND DISCUSSION In addition to the pharmaceutical importance of catecholamine-containing injection solutions, the dopamine-type matrix serves as a useful example of the utility of the two methods suggested for matrix interference reduction for three reasons: (1) under the chromatographic conditions used herein, the dopamine has some affinity for the column; (2) the dopamine is present in sufficiently high concentrations to interfere with the quantitation of the inorganic analytes (Cl-, SOa2-, SO,2-); and (3) its spectroscopic and chemical properties are such that both methods of interference reduction suggested herein can be employed. Focusing on its spectroscopic properties, Figure 1 documents the spectral behavior of both dopamine in water and the IPC mobile phase. While this diagram does not demonstrate the relative differences in molar absorptivities in a quantitative sense, it is useful in qualitatively demonstrating those wavelengths a t which the dopamine interference can be minimized. One notes at 280 nm (a wavelength commonly chosen for IPC measurements with a phthalate-based mobile phase) that the 0 1985 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12,OCTOBER 1985
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Flgure 1. UV spectral profile of (A) mobile phase and (B) dopamine hydrochloride. Concentrations used to generate thls diagram are M potassium hydrogen phthalate, 0.75 X IO-‘ mobile phase, 1 X M sodlum borate at pH 9.1; and dopamlne hydrochloride, 15 mg/L. Arrow denotes wavelength at which dopamlne interference would be minimal (258 nm).
dopamine species exhibits an absorptivity maximum; thus, a considerable interference is possible at this wavelength. As detector wavelength is decreased to roughly 250 nm, phthalate absorptivity remains constant (and thus sensitivity remains roughly equivalent) while the dopamine absorptivity decreases. Thus, while the same quantity of dopamine interferent is present in samples analyzed at wavelengths of 280 and 250 nm, the magnitude of the interference a t 250 nm will be lessened, while overall instrumental performance will remain relatively unchanged. These qualitative observations are mirrored in Figure 2, which documents the chromatographic response to dopamine matrix samples a t wavelengths of 280, 250, and 240 nm. At 280 nm, the dopamine response is of such duration that not only the C1- species is completely masked but also a suitable base line cannot be established on the leading edge of the sulfite response. The C1- species is also buried in the dopamine peak a t 250 nm and a t 240 nm, but in both of these cases a stable base line is established before the leading edge of the suJfite peak. The poorer base line noise level a t the 240-nm wavelength reflects the fact that because this wavelength occurs on an edge in the absorption spectrum (as opposed to the plateau between 250 and 280 nm), the detector will be more greatly influenced by external sources of short-term noise. The use of the alternative wavelength approach to spectrally diminish the dopamine interference is effective, but two problems exist in this approach. On one hand, while the improvement obtainable is sufficient for the matrix studied,
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Figure 2. Detector response to a sample containing 1.6 mg/mL dopamine hydrochloride and 0.025 weight percent NaHSO, (pH 3.5). Species identifications include: (1)SO:-, (2)SO,*-(oxidation product from SO:-), (3)dopamine, and (4)CI-. Wavelengths monitored include (A) 280 nm, (B) 250 nm, and (C)240 nm. The magnitude of the elution) decreases as the dopamine response (dip prior to monitoring wavelength decreases, resulting in more accurate peak quantitation at lower Wavelengths.
the improvement obtained in sulfite/dopamine resolution is small enough that if the concentration of either species increases, the matrix overlap will occur again. The second problem is that the high phthalate absorptivity at 250 nm makes it difficult to utilize a single-beam UV detector (the conventional single-beam detectors have insufficient electronic capability to “zero” or correct for the high negative base line produced in the indirect method). This is the primary reason that 280 nm (where phthalate absorptivity is lower) is used in most IPC applications. Thus, while the spectral approach to matrix interference reduction is viable, a more effective methodology would be useful. The acid/base chemistry of dopamine, which has both amine and hydroxyl functionalities, is such that it may be chemically removed from the mobile phase as part of the process of eluant suppression. While the details of this process are described elsewhere (14), for anion analysis the process essentially involves reduction of the mobile phase pH as hydrogen ions in a “suppressor” solution replace cationic counterions in the mobile phase (e.g., Na+, K+) via diffusion in a membrane fiber reactor situated between the analytical column and the detector. This methodology is particularly useful for reducing the background conductance of high pH, weak
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Figure 3. Detector response to a sample containing 1.6 mg/mL dopamine hydrochiorlde and 0.025 weight percent NaHSO, (at pH 3.5) at 265 nm with (6) and without (A) the postcolumn fiber suppressor.
acid eluants. As the pH decreases in the suppressor module, the weak acids become protonated and the mobile phase conductance decreases. Although this approach is useful for facilitating conductometric detection, this type of change in mobile-phase chemistry is unimportant in the IPC method. Protonation of the phthalate in the eluant will certainly affect its molar absorptivity to some degree, but this change will have only a limited (and unnecessary) effect on analytical sensitivity. Of more importance is the effect of this pH change on the dopamine interferent. As the dopamine interferent passes through the analytical column, its anionic nature at high pH permits its retention by the resin, resulting in the production of the potential interference. However, when this species reaches the zone of decreasing pH in the fiber reactor, it becomes protonated and eventually becomes cationic at the amine site. That this occurs in the fiber suppressor is confirmed by the low pH (3.2) of the unit’s effluent. Once the dopamine exists as a cation, it diffuses through the fiber as part of the H+exchange process; thus the interferent is removed from the mobile phase prior to detection. Qualitatively, this mechanism is confirmed by the chromatograms in Figure 3, which document the detector response to dopamine-containingsolutions in the presence and absence of the fiber reactor. This figure illustrates that the dopamine interference is eliminated when the suppressor system is used; mechanistically it is extremely unlikely that simple protonation of the amine site would affect dopamine’s absorptivity at 265 nm sufficiently to cause this degree of reduction in the
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Figure 4. Detector response to the dopamine hydrochloride injection sample at 265 nm with (B) and without (A) the postcolumn fiber suppressor. The difference in peak response for both chromatograms corresponds to sample degradations which occurred during system switching.
interference. Under these conditions, the sulfite concentration can easily be quantitated; the remaining difficulty in quantitating the C1- ion concentrationis not related to the presence of dopamine but is caused by the ubiquitous void volume or solvent response that occurs at the leading edge of its peak. As is shown in Figure 4,a similar effect is achieved when the actual dopamine injection product is analyzed. In order to confirm quantitatively the loss of dopamine, the following flow injection type system was used. A 50-pL sample of 500 ppm dopamine solution was injected into a mixed bicarbonate/carbonate mobile phase, which was then directed to the UV detector. The detector was set at a monitoring wavelength of 280 nm and a scale reading of 0.100AUFS. Between the injector and the detector, the mobile phase either went through or was allowed to bypass the suppressor system. In this manner, the concentration of dopamine in the mobile phase with and without suppression can be established. As shown in Figure 5, using the fiber suppressor dramatically reduces the absorbance due to dopamine; qualitatively, more than 95% of the dopamine in the original solution is removed via the suppression process. Because of the potential magnitude of the interference, the determination of C1- in the 8.5% Travasol solution presents a more rigorous test of the suppressor system’s ability to remove matrix interferences. Because the retention characteristics of the C1- ion and the amino acid interferents are so
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Figure 5. Detector response at 280 nm to a sample containing 500 ppm dopamine with (A) and without (B) passage through the fiber suppressor. Eluant flow rate is 2.0 mL/mln, chart speed is 20 cmlmin, and recorder output is set at 100 mV full scale.
similar and the disparity between their relative concentrations in this matrix is so great, the effectiveness of the removal process is of particular importance. The Travasol matrix contains, as a class, approximately 85 g/L of amino acids and only 1 g/L of C1-. The amino acids are essentially evenly distributed among the following: L-leucine, L-phenylalanine, L-methionine, L-lysine, L-alanine, aminoaceticacid, L-arginine, L-lysine, L-isoleucine, L-valine, L-histidine, and L-threonine. As the bottom chromatogram in Figure 6 shows, the Cl- peak is surrounded in the nonsuppressed system by a variety of components that produce both negative and positive detector responses. Quantitation of C1- is hampered primarily by the presence of the species that produce the negative response on the trailing edge of the C1- peak; however, there are interferents on the leading edge as well. Using the suppressor unit effectively allows an accurate base line to be established both before and after the C1- response, but it does not completely remove all potential interferents. While the peak that appears before C1- in the chromatogram has been identified as acetate (which is not removed by the fiber reactor), the identity of the latter peak is presently unknown. In any event, use of the fiber suppressor facilitates the determination by IPC of C1- in matrices containing large quantities of amino acids. The discussion to this point has been limited to anion analysis, but there is no reason why a similar strategy cannot be employed in the determination of cations by IPC. Again with dopamine as a model compound, at the low mobile-phase pH commonly used in monovalent cation separations, the dopamine itself would be cationic and could be retained, producing an analytical interference. As eluant pH is increased in a cationic fiber suppressor (as the counterion of the eluant is exchanged across the membrane for OH- in the “suppressor” solution), the dopamine becomes anionic in nature and once again migrates out of the mobile phase as part of the suppression process. Although this effect has not
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Flgure 6. Detector response to an 8.5% Travasol with electrolyte sample (diluted by 50 in deionized water) with (B) and without (A) the postcolumn fiber suppressor.
yet been demonstrated using an indirect photometric method, fiber suppressor removal of amino acid type interferents has been observed in this laboratory when analyte detection is accomplished with conductivity detection. It should also be noted that although the discussion to this point has considered only amine-type interferents, suppressor removal techniques are applicable to any interferent that is amphotropic in nature. Of course, methods that exploit differences in the spectral characteristics of interferent and eluant ions can be used in any system where such a difference exists and where the magnitude of the difference is sufficiently large to be analytically significant. Registry No. C1-, 16887-00-6; S032-,14265-45-3; Sod2-, 14808-79-8;dopamine, 51-61-6.
LITERATURE CITED Small, H.;Miller, T., Jr. Anal. Chem. 1982,5 4 , 462-469. Small, H.; Miller, T., Jr. United States Patent No. 4414842, 1983. Hagakawa, H.; Migatakl, M. Bunzekl Kagaku 1983,3 2 , 504-505. Cochrane, R.; Hlllman, D. J . Chromatogr. 1982,247, 392-394. (5) Jenke, D.; Raghavan, N. J . Chromatogr. Sci. 1885,2 3 , 75-80. (6) Denkert, M.; Hackzell, L.; Schlll, 0.;Sjogren, E. J . Chromatogr. 1981, 278, 31-43. (7) Dreux, M.; LaFosse, M.; Pequlgnot, M. Chromatographla 1982, 75, 653-655. (8) Larson, J.; Pfelffer, L. J . Chromatogr. 1983,259. 519-521. (9) Perrone, P.; Gant, J. Res. Dev. 1984,26(9), 96-160. (10) Larson, J.; Pfelffer, L. Anal. Chem. 1085,55, 393-396. (11) Jenke, D.; Mitchell, P.; Pagenkopf, G. Anal. Chlm. Acta lg83, 55, 249-285. (12) Wllson, S.; Yeung, E. Anal. Chlm. Acta 1984, 757, 53-63. (13) Jenke, D. Anal. Chem. 1984,56, 2468-2470. (14) Pohl, C.; Johnson, E. J . Chromatogr. Scl. 1980, 78, 442-452. (1) (2) (3) (4)
RECEIVED for review April 15,1985. Accepted June 19,1985.
