Technical Notes Anal. Chem. 1996, 68, 1067-1070
Liquid Chromatographic Determination of Organic Acids Used as Pharmaceutical Counterions Barbara S. Lord* and Rodger W. Stringham
The DuPont Merck Pharmaceutical Company, Process Research Facility, Chambers Works (S1), Deepwater, New Jersey 08023-0999
The determination of counterions in pharmaceutical salts requires good precision but less than optimal sensitivity. A simple, inexpensive system with direct sample preparation has been developed for this application. A silicabased amine liquid chromatographic column is used with a conductivity detector to separate and quantitate methanesulfonic acid without the use of an ion suppression device. Precision for sample analyses was 1-3% RSD, which was adequate for counterion quantitation. To gain a clear understanding of the parameters controlling retention and detection, four dicarboxylic acids were chromatographed on the system using mobile phases with various pH, ionic strength, and organic levels. The effects of ionic strength and pH on retention and elution order of the probe acids can be predicted when the effective charge of the acids and the elution strength of the buffer are considered. Mobile phase pH also induces changes in the stationary phase. Hydrophobic effects, observed when ionization of either the probe acids or the column was minimized, were moderated by incorporation of organic solvents into the mobile phase. Detector sensitivity was dependent upon pH and buffer concentration. The selection and optimization of chromatographic conditions for other UV-inactive acids can be facilitated by the information gained in this study. A variety of organic acids are used as counterions to isolate and stabilize basic pharmaceutical compounds and intermediates. It is often necessary to quantitate the level of these acids to establish stoichiometry, completeness of salt formation, and mass balance. Many of these acids lack chromophores and are difficult to detect and quantitate. A wide variety of chromatographic approaches have been used to accomplish the separation and detection of UV-inactive acids, including derivatization followed by gas chromatography,1-3 conventional liquid chromatography on a diol column with an ion-pairing, UV-absorbing counterion mobile phase,4 LC using a polymeric reversed phase column and (1) Katoaka, H.; Okazaki, T.; Makita, M. J. Chromatogr. 1989, 473 (1), 276. (2) Panter, R.; Penzhorn, R. D. Atmos. Environ. 1980, 14 (1), 149. (3) Klockow, D.; Bayer, W.; Faigle, W. Fresenius’ Z. Anal. Chem. 1978, 292 (5), 385. (4) Hackzell, L.; Denkert, M.; Schill, G. Acta. Pharm. Suec. 1981, 18 (5), 271. 0003-2700/96/0368-1067$12.00/0
© 1996 American Chemical Society
indirect photometric detection,5,6 and ion chromatography with conventional columns, suppressor device, and conductivity detection.7 Each of these approaches can provide excellent sensitivity in the ppm to ppb range. Ion chromatography (IC) is probably the most widely used means of analyzing UV-inactive ionizable compounds. Recent work has shown that carboxylic acids can be analyzed on gel-type columns with an alcohol modifier to eliminate the need for acid in the mobile phase.8 However, alkanesulfonic acids were not successfully retained and separated by this technique. These approaches are cumbersome and costly and may not provide adequate precision to be useful. Since counterion acids may constitute 20-30 wt % of pharmaceutical salts, the sensitivity afforded by ion chromatography is counterproductive in this application. Small sample weighings and multiple dilutions used to accommodate the low IC column capacity and the detector’s limited linear range contribute significantly to assay imprecision. Use of unsuppressed conductivity detection coupled with a weak ion exchanger should circumvent sample preparation and precision problems. In this work, a silica-based amine column and a conductivity detector were used to separate, detect, and quantitate methanesulfonic acid (MSA) without the use of an ion suppression column. A better understanding of the parameters controlling retention and detection was sought to facilitate the development of analysis conditions for other UV-inactive organic acids. Fumaric, maleic, tartaric, and succinic acids are common counterions, and their more complex ionization behavior should provide more information about controlling parameters in this sytem than MSA. EXPERIMENTAL SECTION Instrumentation. A Hewlett Packard HP1050 liquid chromatograph and a column oven temperature of 50 °C were used except where noted otherwise. The flow rate used was 1.5 mL/ min, and the injection volume was 25 µL. All data were recorded on a Hewlett Packard ChemStation using HP3365 II (DOS version) Rev. 2.0 software and an HP35900 analog-to-digital converter to port the detector signal to the ChemStation. The detector used (5) Pietrzyk, D. J.; Rigas, P. G.; Yuan, D. J. Chromatogr. Sci. 1989, 27 (8), 485. (6) Rigas, P. G.; Pietrzyk, D. J. Anal. Chem. 1987, 59, 1388. (7) Fritz, J. S. J. Chromatogr. 1991, 546, 111. (8) Morris, J.; Fritz, J. S. Anal. Chem. 1994, 66, 2390.
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Figure 1. Chromatogram of methanesulfonic acid. ZORBAX NH2 column; isocratic 5% acetonitrile in 20 mM sodium dihydrogen phosphate + 0.13 mM phosphoric acid, pH 4.2 mobile phase; 1.3 mL/min; 40 °C.
was an Alltech Model 350 conductivity detector. The flow cell was maintained at the factory setting of 35 °C. No detector drift due to thermal effects was observed. The range setting used was 10 µS/cm full scale. Column. Separations were carried out on a DuPont Zorbax NH2 (MAC-MOD Analytical, Inc., Chadds Ford, PA) 5 µm column (250 mm × 4.6 mm). Reagents. Eluants were prepared from HPLC grade methanol and acetonitrile, ACS reagent grade sodium dihydrogen phosphate and phosphoric acid from J. T. Baker and Co., and HPLC grade water from a Milli-Q system. The pH of the eluant was adjusted with 85% phosphoric acid or aqueous 1 N sodium hydroxide solution as needed. Fumaric, tartaric, maleic, and succinic acids (99% or better purity) were all obtained from Aldrich. Isocratic conditions were used in all experiments. The eluants were chosen to explore the effects of organic content, ionic strength, and pH on the chromatography of the probe acids. Parameters were varied systematically to elucidate the factors controlling retention and detection. Solutions of the analytes were prepared in the eluant used for each experiment at about 3-5 mg/mL (where possible). RESULTS AND DISCUSSION Methanesulfonic Acid. Methanesulfonic acid (Figure 1) was successfully determined in samples of three different pharmaceutical intermediates and drug substances. Samples are diluted in the mobile phase to contain between 0.4 and 2 mg/mL MSA. No suppression device is required. Linearity was examined at 5, 2, 1, 0.5, and 0.1 mg/mL methanesulfonic acid. The coefficient of determination (r2) was 0.9997, and the intercept did not vary significantly from zero. Injection precision was 1-2% RSD. The method precision is typically 1-3% RSD. This assay has been used for three different drug substances and has been used in the release of 12 batches of final drug substance. Excellent mass balance of the released batches implies good analytical accuracy for this assay. This assay has also been applied successfully to raw material analysis. Retention Mechanism. A series of experiments was performed to identify the controlling parameters in this system. Jenke9 and Hajos et.al.10 present models for the prediction of retention of multiprotic anions in ion chromatography. Retention (9) Jenke, D. R. Anal. Chem. 1994, 66, 4466. (10) Hajos, P.; Horvath, O.; Denke, V. Anal. Chem. 1995, 67, 434.
1068 Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
Figure 2. Structures of dicarboxylic acid analytes. The pK values for fumaric, maleic, and succinic acids were calculated from K1 and K2 values given in the following: Morrison, R. T.; Boyd, R. N. Organic Chemistry, 2nd ed.; Allyn and Bacon: Boston, 1966; p 907. The pK values for dl-tartaric acid were calculated from K1 and K2 values given in the following: The Merck Index: An Encyclopedia of Chemicals and Drugs, 8th ed.; Stecher, P. G., Ed.; Merck: Rahway, NJ, 1968; p 1014. Table 1. Effective Chargea of Analyte Ions at Several pH Values pH
succinic
fumaric
tartaric
maleic
2.5 3.4 4.3 6.0 6.1
0.020 0.138 0.596 1.707 1.754
0.245 0.802 1.404 1.975 1.980
0.249 0.846 1.523 1.984 1.988
0.760 0.963 1.005 1.334 1.387
a
Effective charge ) proportion HA- + 2[proportion A2].
is a function of the analyte’s effective charge, the eluent’s net charge, and the analyte/eluent affinity of the stationary phase. A complex relationship exists between analyte retention and pH. Analyte effective charge, eluent net charge, and stationary phase charge are all affected by pH, thus altering the relative affinity of the stationary phase for the various analyte and eluent ions. As pH increases, the concentration of divalent phosphate increases, stationary phase charge diminishes, and the analyte effective charge values increase. The pKa values of the analytes and their structures are shown in Figure 2. Calculated effective charges of the probe acids at several pH values are given in Table 1. Effective charge is calculated from the ratios of di-, mono-, and nonprotonated ions predicted by Henderson-Hasselbach equations. Diprotonated ions do not contribute to effective charge, while nonprotonated (doubly charged) ions would be expected to count double. However, the affinity of the stationary phase for divalent phosphate may make it a stronger eluting species than the eluent net charge calculations indicate. The relative proportion of phosphate species at these pH values are given in Table 2. Effect of Ionic Strength. The effect of buffer concentration was examined at pH 2.5 and 6.0 (Table 3). As expected, increasing ionic strength resulted in decreased retention for all analytes at both pH values. Effect of pH. Figure 3 shows the elution times of the probe acids with changing pH. Although the effective charge of the analytes corresponds to elution order at pH 2.5 and 3.4, the relationship did not hold at pH 4.3 and 6.0. The analyte effective charge predicted neither the long retention of fumaric and tartaric acid nor the maleic/succinic elution order. Apparently, the affinity of the stationary phase for analyte and eluent ions is dramatically different at pH 4.3. At pH 6.0, where most of the stationary phasecharge may be lost, separation is minimal, and retention is likely to be due to hydrophobic interactions.
Table 2. Distribution of Phosphate Species and Relative Elution Strength
Table 5. Detector Response: Effect of Varying Mobile Phase Parameters
pK1 ) 2.1a H2PO4-
pK2 ) 7.2a HPO42-
strengthb
0.715 0.952 0.993 0.941 0.926
0.0002 0.001 0.059 0.074
0.715 0.953 0.997 1.177 1.222
detector response (area counts)/(mg/mL) succinic fumaric tartaric maleic
pH
H3PO4
2.5 3.4 4.3 6.0 6.1
0.285 0.048 0.006
a pK values taken from the following: Lehninger, A. L. Biochemistry; Worth Publishers: New York, 1970; p 47. b Relative elution strength ) H2PO4- + 4[HPO42-].
Figure 3. Retention of dicarboxylic acids at different pH values. 9, Fumaric acid; 0, tartaric acid; [, maleic acid; and ], succinic acid. Table 3. Effect of Ionic Strength on Retention Time (min) eluant
tartaric
maleic
(a) pH 2.5 and 20% Methanol 15 mM total phosphate nda 9.1 34.4 mM total phosphate nd 5.6 54.4 mM total phosphate nd 4.6
11.4 6.4 5.6
13.1 8.0 6.5
(b) pH 6.0 and 20% Methanol 30 mM total phosphate 6.8 6.8 34.4 mM total phosphate 6.3 6.2 54.4 mM total phosphate 5.0 4.9
7.6 6.6 5.0
5.5 4.8 4.3
a
succinic
fumaric
Not detected.
Table 4. Effect of Organic Solvent on Retention Time (min) succinic
fumaric
tartaric
maleic
5% methanol 20% methanol
4.1 nda
(a) pH 2.5 12.6 9.1
15.3 11.4
14.3 13.1
5% methanol 20% methanol
4.0 5.0
(b) pH 6.0 3.8 4.9
3.8 5.0
3.9 4.3
a
Not detected.
Effect of Organic Content. The data in Table 4 show the effect of the organic solvent concentration on retention. At pH 2.5 and 15 mM phosphate, longer retention times were observed for all analytes when the organic content of the eluant was reduced to 5% from 20% methanol. This is evidence of the importance of hydrophobic interactions at pH 2.5. At pH 6.0 and 30 mM phosphate, slightly shorter retention times were observed when the organic content of the eluant was reduced to 5% from 20% methanol for all analytes. The loss of retention with decreased organic (increased water) content may reflect a difference in the
pH 2.5 pH 3.4 pH 4.8 pH 6.1
eluant conductivity (µS/cm)
(a) pH: 55 mM NaH2PO4 and 20% Methanol 0.43 9.0 5.6 36 5.3 35 26 60 21 56 44 53 0.99 11 3.0 5.9
(b) Ionic Strength: 20% Methanol and pH 3.4 18 mM 101 451 350 620 NaH2PO4 36 mM 9 58 44 100 NaH2PO4 55 mM 5.3 35 26 60 NaH2PO4
4060 2295 2150 3275 645 1500 2295
degree of ionization of the column packing, the analytes, or the phosphate molecules. Substituting 30% acetonitrile for 20% methanol at the same pH and ionic strength produced no striking changes in chromatographic behavior. Elution order and peak shape were unchanged for these analytes. Sensitivity and Detection. No succinate peak is observed at low pH. The pH behavior of the other probe acids indicates that succinate should elute under these conditions and is unlikely to be detected because it is not well ionized. With a conductivity detector, analyte peaks must impose a sufficient change onto background conductivity to be detected. The influences of pH, buffer concentration, and organic content on detector response for each of the probe acids were studied. The LC column was replaced by a piece of tubing, and the conductivity of various mobile phases was measured. A solution of each probe acid at a known concentration was injected using each mobile phase. The peak area for each injection was divided by the concentration to obtain a response factor (Table 5). The low response for succinate at pH 2.5 explains the failure to detect a peak at this pH. Response factors were maximal at pH 4.8, except for maleic acid. As expected, the relative response of all acids increased as the ionic strength of the buffer decreased. There was little change in the background conductance of the mobile phase at various levels of organic, and the relative response of the probe acids decreased only slightly with increasing organic content. There was no remarkable difference in response when acetonitrile was substituted for methanol. Vacant Peaks. Analyte peak identification was complicated by the presence of vacant peaks that arise when eluant ions are displaced from the column by analyte ions and elute as a peak.11 The retention time of the vacant peak may be shorter or longer than that of the analyte of interest. At pH 4.8 and below, a vacant peak corresponding to H2PO4- was observed. At pH 6.0, two vacant peaks appeared, which correspond to HPO42- and H2PO4-. Diluting the samples in the mobile phase minimized vacant peaks and simplified the interpretation of the chromatograms. Vacant peaks can be identified by injecting a stronger buffer. Injections of mobile phase do not help in identifying vacant peaks because displacement of phosphate will not occur in the absence of analyte. (11) Slais, K.; Krejci, M. J. Chromatogr. 1974, 91, 161.
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CONCLUSION A simple and rugged liquid chromatographic system for determining UV-inactive organic acids at the milligrams per milliliter level is described. A polar bonded phase column, Zorbax NH2, and a conductivity detector were used to detect and quantitate alkane carboxylic and -sulfonic acids without the use of an ion suppression device. The system is inexpensive, and the sample preparation is direct. The precision for determination of methanesulfonic acid as a counterion in pharmaceutical salts is typically 1-3% RSD. The parameters controlling retention were examined by chromatographing fumaric, tartaric, succinic, and maleic acids on the system using mobile phases with various pH, ionic strength, and organic levels. Retention is influenced by the analyte’s effective charge, the eluent’s net charge, and the affinity of the stationary phase for analyte and eluent ions. The column stationary phase charge is pH-dependent and significantly influenced retention. Hydrophobic effects were observed when
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ionization of either the probe acids or the column was minimized. Taking these factors into account, the observed retention behavior of the analytes conformed to the models of Jenke9 and Hajos, et al.10 Analytes were detected only if their conductance exceeded the background conductance of the mobile phase. The effective charge of the probe acids was pH- dependent, and the mobile phase conductance was related to buffer concentration and pHdependent ionization. Therefore, detector sensitivity was dependent upon pH and buffer concentration. The selection and optimization of chromatographic conditions for other UV-inactive acids can be facilitated by the information generated in this study.
Received for review August 14, 1995. Accepted December 18, 1995.X AC9508208 X
Abstract published in Advance ACS Abstracts, February 1, 1996.
