Anal. Chem. 1992, 64, 1096-1099
1096
Characterization of Azoniaadamantane-Based Preservatives by Combined Reversed-Phase Cation-Exchange Chromatography with Suppressed Conductivity Detection William Russell Summers Buckman Laboratories International, Inc., 1256 North McLean Boulevard, Memphis, Tennessee 38108-0305
A method has been developed for the characterlzatlon of an Important clam of preservatives based on the atonlaadamantane substructure. These compounds are quaternary amlnes and functlon prlmarily by the release of methylene glycol, which ultlmately donates the formaldehyde to the matrlx In whlch they are Incorporated. The analytkal methodolagy Involves the dlrect determlnatlon of the actlve molecules rather than resorting to chromogenlc derivatlzatlon of the available formaldehyde equivalent. Thls approach provides both for the speclatlon of lndlvldual preservative compounds and for the determlnatlon of thelr lntrlndc stablltty In matrlcw contalnhg not only free formaldehyde but also other preservatlves. Partlal resolution of diastereomers, enhanced vla mathematkal deconvolutlonof thelr overlapplng chromatographic peaks, allows for the elucldatlon of their relatlve stabliltles as well. The method detectlon ilmlt Is around 1 ppm, and the Ilnear dynamk range spans 3 decades. Rdatlve standard deviatlon averages 1.O % .
INTRODUCTION Preservativesbased on the azoadamantane structure I play a significant role in mitigating microbially-induced degradation of a variety of water-based formulations. Typical substances
(IV)
requiring preservation to extend their shelf-life might include paints, cosmetics, and personal-care products. Interestingly, azoadamantane (also known as hexamethylenetetramine, methenamine, and urotropine) exhibits no preservative efficacy in and of itself; rather, it is generally thought to ultimately donate aqueous formaldehyde to the matrix in which it is incorporated, although pertinent empirical studies are sparse.’ Quaternization of I yields several preservatives 11-IV which demonstrate potential for improvement on I with respect to ease of formulation and handling, microbiological activity, stability, patentability, and synergy with the quaternizing reagent. In spite of the commercial success of these preser-
vatives, little analytical methodology has been reported for the direct determination of the intact molecules. Published methods for the characterization of 11-IV have focused instead on the chromogenic derivatization of their equivalent formaldehyde content. This is due to their lack of a chromophore amenable to absorbance detection. Although the role that formaldehyde release plays in the efficacy of these compounds is debatable, they can nevertheless be determined using standard reagents developed for the analysis of formaldehyde. Reagents that have been applied to the determination of 11-IV, as well as numerous other preservatives, include chromotropic acid? 2,4dinitrophenylhydrazine~ and Nash’s reagent: a buffered acetylacetone solution. A major shortcoming of the wet-chemical batch derivatization approach is that it cannot distinguish the source of the formaldehyde response; i.e., the formaldehyde response cannot be unambiguously attributed to free formaldehyde in solution or to one or several formaldehydelabilepreservatives that may be present. The use of hydrazine under conditions chosen to derivatize only free formaldehyde followed by reaction under conditions that derivatize the preservative molecule has been reported for IIks this allows for the determination of the preservative by difference in a fresh solution of known history. The differential formaldehyde response of aged solutions or solutions of unknown history will be convoluted with the formaldehyde release kinetics of the preservative and the formaldehyde uptake kinetics of the matrix. An elegant but little used refinement to batch derivatization is to configure the derivatization step after a separation step, so that the various sources of formaldehyde that may be present in a mixture are resolved prior to detection. Of the reagents previously mentioned, Nash’sreagent is ideally suited to this approach, since the reagent is transparent at the derivative’s wavelength of maximum absorbance (410 nm) and fluorescence (510 nm). Although I11 and IV have been determined by this methodology,6-8the attendant complexity of the instrumentation may offset the utility of this approach somewhat. In light of the cationic nature of 11-IV, an alternative to reversed-phase liquid chromatography with chromogenic derivatization was investigated. Recent advances in ion-exchange chromatography have resulted in the development of mixed-mode columns that incorporate both reversed-phase and cation-exchange characteristics? These columns employ macroreticular polystyrendvinylbenzene polymer beads that have a superficial cation-exchange functionality. Retention in the hydrophobic pores is govemd by typical reversed-phase mechanisms, while the sulfonate surface layer provides cation-exchange sites. Suppressed conductivity detection was utilized for the direct determination of the preservative molecules, thereby avoiding the potentially confounding issue of formaldehyde equivalence. EXPERIMENTAL SECTION The apparatus utilized for this study consisted of a Dionex Model 4000i high-pressure ion chromatograph equipped with suppressed conductivity detection. The Dionex PCX-500 column
0003-2700/92/0364-1096$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
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Flgure 1. Conductivity measured as a function of retention time for 200 ppm Busan 1024.
was used (guard and analytical), and the injection volume was 50 pL. The eluant used was 60% water, 20% acetonitrile, and 20% (50 mM HC1-0.50 mM DAP; see below), with a flow rate of 1.0 mL/min. The Dionex cation micromembrane suppressor was used in-line between the column and Dionex CDM-1 conductivity detector. The regenerant was 100 mM tetrabutylammonium hydroxide, at a flow rate of about 1mL/min (3 psi). Preparation of Eluant. Fisher Optima grade acetonitrilewas used as received from the manufacturer. The water used for this study was obtained from a Barnstead Nanopure water purifier system and was submicron filtered. The conductivity was typically 17.5 MQ. The DAP-HC1 eluant was prepared by diluting 4.15 mL of concentrated hydrochloric acid (Baker Instranalyzed) and dissolving 0.14 g of diaminopropionic acid monohydrochloride (Aldrich 21,963-0) per liter of water. Preparation of Regenerant. The 100 mM TBAOH regenerant was prepared by diluting 69 mL of 40% tetrabutylammonium hydroxide (Aldrich 17,878-0) per liter of water. Preparation of Samples. l-Methyl-3,5,7-triaza-l-azoniaadamantane chloride (11)is available commercially from Buckman Laboratories, Inc., Memphis, TN, under the product name Busan 1024. This product is provided as a water-based formulation nominally 18% in II. A 200 ppm solution was prepared by diluting 200 mg of Busan 1024with 100 mL water and diluting this solution 10-fold. ck-l-(3-Chloroallyl)-3,5,7-triaza-l-azoniaadamantane chloride (111)is a product of the Dow Chemical Co., Midland, MI. It is sold as a powder nominally 95% in I11 under the product names Dowicil-200and Quaternium-15. A 100 ppm solution was prepared by dissolving 100 mg of Dowicil-200in 100 mL water and diluting this solution 10-fold. l-(3-Chloroallyl)-3,5,7-triaza-l-azoniaadamantane chloride (IV) is also a product of the Dow Chemical Co. It is provided as a powder nominally 69% in IV under the product name Dowicil-75. A 200 ppm solution was prepared by dissolving 200 mg of Dowid-75 in 100 mL water and diluting this solution 10-fold. Software. Data were acquired from the 1.0-Vfull-scale output of the conductivity detector using an IBM PC/XT loaded with Interactive Microwave's (State College, PA) Chromatochart-PC, version 2.0. Input sensitivity was 1.0 V full scale. The data acquisition rate was 1.25 Hz. The 30-pS full-scale range was used for the conductivity detection of the concentration range of 50 ppm and higher; the 3-WSscale was used for the concentration range of 25 ppm and lower. Peak integration and deconvolution was performed using Jandel Scientific's (Corte Madera, CA) PeakFit, version 1.0. Chromatographic data were plotted using Jandel Scientific's Sigmalot, version 4.1. Chemical structures were drawn using Molecular Design, Limited's (San Leandro, CA), Isis/Draw, version 1.0. The text was processed on Wordperfect (Orem, UT) version 5.1. Photochemistry. The photochemically induced free radical isomerization experiment employed a General Electric sunlamp Model 7080. The reaction was carried out in a 250-mL quartz Erlenmeyer flask, using methylene chloride as the solvent. Ap-
Flgure 2. Conductivity measured as a function of retention time for 100 ppm Dowlcll-200.
proximately 100 mg of Dowicil-75 was added to 100 mL of methylene chloride,forming a slurry. The reaction was initiated by turning on the UV lamp and adding a catalytic amount (i.e. one drop) of bromine and was allowed to proceed for 2 h. The solution was agitated using a magnetic stir bar, and the thermal output of the sunlamp heated the solution to about 50 "C. Solvent was added periodicallyto replace that lost by evaporation. The Dowicil-75was recovered from the solvent by partition into 100 mL of water in a 250-mL separatory funnel. This solution was diluted 10-fold prior to injection on the chromatograph.
RESULTS AND DISCUSSION Figure 1 is the chromatogram of a 200 ppm solution of Busan 1024 and shows the active ingredient (11) eluting at about 15-min retention time. Compound 11is synthesized via a termolecular condensation of ammonia, formaldehyde, and monomethylamiie (eq 1). The unreacted precursor ammonia and monomethylamine are shown eluting at about 8.5 and 9.5 min, respectively. 0
3
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Figure 2 is the chromatogram of a 100 ppm solution of Dowicil-200; the active ingredient (III)is shown eluting around 21-min retention time. Compound I11 is synthesized from azoadamantane and cis-l,3-dichloropropene (eq 2). The cis 4
isomer of 1,3-dichloropropeneis used as a soil fumigant for the control of nematodes and demonstrates superior efficacy compared to the trans isomer.1° The cis isomer can be fractionally distilled from the 1:l equilibrium cis/trans mixture by virtue of the fact that it boils at about 104 "C, whereas the trans isomer boils at about 112 "C. The resultant cis/trans mixture, depleted in the cis isomer, can be restored to the 1:l equilibrium ratio by a photochemically induced free radical chain reaction;ll this process can be repeated until a point of diminishing returns is reached, thereby resulting in the recovery of an essentially pure cis fraction of l,&dichloropropene. This fraction is used to quaternize the azoadamantane, resulting in a more efficacious preservative that
10@8 ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, I992 10'
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Figure 5. Stablltty of DowkiC75 diastereomers as a function of sample age. Note that the chromatograms have been plotted with nonuniform
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commands a premium when targeted at the upper end of the preservative market, e.g. cosmetics. Figure 3 is a plot of I11 peak area as a function of concentration, spanning the range from the detection limit of about 1ppm to the column capacity at around loo0 ppm. The error bars correspond to the 95% confidence intervals generated from triplicate analysis at each concentration value. The solid line is the linear least-squares fit to the average peak area; the correlation coefficient ( F ) for this line is 0.999. Precision at either extreme is inferior to that observed between 50 and 250 ppm (1% RSD), although the logarithmic compression fails to illustrate this trend. In light of the similarity among the compounds studied, the precision and linearity for I1 and IV should be comparable to that presented here, taking into account the differences in active ingredient concentrations. Figure 4 is a chromatogram of a 200 ppm solution of Dowicil-75, nominally 69% in the active ingredient (IV). Compound IV is synthesized according to eq 3 from azodamantane and a mixture of cis- and truns-l,3-dichloropropene.
is due to sodium arising from the 25% sodium bicarbonate with which the product is formulated. The partial resolution of the diastereomers of IV makes possible the study of their relative stabilities. Figure 5 is an overlay of chromatograms from four batches of Dowicil-75 manufactured over a time frame of approximately 2.8 years; chromatogram a is the same data shown in Figure 4. The batches were stored in their original containers under ambient laboratory conditions. The sample ages correspond to the time elapsed between the date of manufacture and the date of analysis. It should be emphasized that the chromatographic traces are from four distinct batches and therefore this data set does not constitute a stability study in the traditional sense. It should also be pointed out that the sample ages have been spaced nonuniformly. This data set depicts in a qualitative fashion the relative instability of the trans isomer of IV compared to the cis isomer. The growth of the peak at around 15-min retention time implicates I1 (see Figure 1) as a likely degradation product of trans-IV. This degradation mechanism has been observed previously for compound 111using fast atom bombardment mass spectrometry.12The discrepancy between the chromatographic evidence for the degradation of trans-IV to I1 and the mass spectrometric evidence for the degradation of cis-I11 to I1 is intriguing. At this juncture, it was desired to obtain additional corroboration for the cis and trans peak assignments and for the relative stabilities previously discussed. Recall that the cis and trans isomers of 1,3-dichloropropenecan be interconverted in a photochemically induced free radical chain reaction." Subjecting the diastereomeric mixture of IV to this same chemistry should result in observable changes in the relative peak areas previously assigned to each isomer in Figure 4. Equation 4 shows a photochemically generated bromine free
H
By comparison of Figure 4 to Figure 2, it can be deduced that the peak at around 21-min retention time is attributable to the cis isomer; the peak at around 22.5 min is therefore attributable to the trans isomer. The off-scale peak at 6 min
H
radical adding to the double bond of trans-IV. The radical intermediate is free to undergo rotation about the C2-C, bond, whereas rotation about this bond was previously restricted. This intermediate then regenerates the catalytic bromine free
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
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that compound I should be protonated at the pH of the eluant and might therefore elute in the general vicinity of 11. During the course of this investigation, solutions of I were prepared and injected into the system as described and also into a flow injection analysis version. The protonated cation of I was not observed under any circumstances. Therefore, degradation of trans-IV to I, however plausible, remains hypothetical.
CONCLUSION Based on the greater stability of cis-IV demonstrated by the decay study and the photochemical isomerization study, it seems reasonable to propose a conjectural structure that invokes a charge stabilization mechanism between the positively charged quaternary ammonium atom and the electronegative chlorine atom in structure 111. Although relatively inconsequential compared to the charge interaction between the quaternary ammonium cation and the chloride counterion, this charge stabilization interaction would presumably be more significant for the cis isomer than for the trans isomer. The chromatographic conditions employed for this study were optimized for the resolution of the diastereomers of IV in order to examine their relative stability differences and degradation pathways. For applications not requiring this resolving power, retention times can be shortened by approximately 30% (with corresponding improvements in peak shapes) by changing the eluant to 40% water, 20% acetonitrile, and 40% HC1-DAP. The postcolumn chemistry employed for chemical suppression of the eluant is quite mature and is virtually transparent to the analysis. The methodology reported here is therefore readily amenable to automation. Determination of these preservatives in target matrices will entail their extraction into an aqueous miscible solvent compatible with the analytical system. Methanol has been used successfully in the author's laboratory to agglomerate the solids in paints and cosmetics and simultaneously extract the preservative, forming a clear top layer that can be sampled with a syringe and injected directly onto the chromatograph. This approach provides for the study of a preservative's stability in the target matrix without the potential complications associated with the indirect detection of equivalent formaldehyde. Registry No. 11, 76902-90-4;111, 51229-78-8;IV, 4080-31-3; Busan 1024, 129204-80-4;Dowicil-200, 51229-78-8;Dowicil-75, 4080-31-3.
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Flguro 8. Mathematical deconvolutlon of natlve (a) and cls-enriched (b) DowlclC75 dlastereomerlc distribution. The lower traces (dashed lines) are the deconvoived cis and trans peaks. The upper traces are the raw data (dotted line) and the model fit (solid line) generated from the component peaks.
radical and reverts to the original allylic configuration, assuming the stereochemical conformation of lower potential energy. Figure 6a is the 10-30-min time slice of the same data plotted in Figure 4. The partially resolved chromatographic peaks have been fit by a pair of exponentially modified Gaussian peaks, allowing for the quantitative mathematical deconvolution of their relative peak areas. This model indicates 23% cis-IV and 77% trans-IV. Of course, this model is not a unique fit to the empirical data; it does however conform to reality in light of the fundamental chemistry by which IV is synthesized and with respect to the peak shapes and relatively greater peak width for the more retained isomer. Figwe 6b is the chromatographic data from Dowicil-75 that has been subjected to the free radical chemistry previously discussed. The same mathematical model used to fit the data of Figure 6a indicates that the cis isomer now comprises 32% of the IV peak area and the trans isomer 68%. This result is consistent with the catalytic conversion of trans-N to cis-N. It is also consistent with a constant level of cis-IV in conjunction with a degradative loss of tram-IV. The latter possibility is contraindicated by the lack of growth in the primary degradation product peak at 15-min retention time observed in Figure 5. Conversion of cis-111 to a diastereomeric mixture under identical experimental conditions did not occur. The fate of compound I is enigmatic. On the basis of similarities among I through IV, it seems reasonable to expect
REFERENCES Wallhaeusser, K. H. I n Cosmetlc and Lkug Reservatkn; Kabara, J. J., Ed.; Marcel Dekker: New York, 1984;pp 644-645. Sawickl, E.; Hauser, T. R.; McPherson. S. Ana/. Chem. 1982, 34 (ll),
1460-64. Kuwata. K.; Uebori, M.; Yamasaki, H. J . chromarogr. Sc/. 1979, 17,
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Benassl, C. A.; S m " e t o , A.; Zaccarie, F.; Bettero, A. J . chrome togr. 1990, 502,193-200. Marouchoc, S. R. I n Cosmefb end Lkug Reservation; Kabara, J. J., Ed.; Marcel Dekker: New York, 1984;pp 143-164. Stack, A. R.; Davis, ti. M. J . A m . Off. Anal. Chem. 1984. 67(1),
13-15. Engelhardt, H.; Kllnkner, R. Ctuomafc?@raphia1985, 20 (9),559-565. Summers, W. R. Anal. Chem. 1990, 62(14),1397-1402. Pohl. C.A. I n Recent L%vebpmnfs In Ion Exchenge XI; Williams. P. A., Hudson, M. J., Eds.; Elsevier: New York, 1990; pp 243-253. Neudecker, T.; Lutz, D.; Eder. E.; Henschler, D. Blochem. Phamcd. 1980, 29,2611-2617. Ivy, J. B.: Wlllls, Q.Q.;Kelsoe, D. C. US. Patent 3,914,167, Oct. 21,
1975. Fabrls. D.; Traldl, P.; Benassi. C. A.; Pastore, S.; Bettero, A,; Rossato. P. Biol. Mess Specfrom. 1991, 20 (6).361-366.
RECEIVED for review December 3,1991. Accepted February 20, 1992.
