Anal. Chem. 1907, 59, 2773-2776
across the “hole”, i.e., from the leading to trailing edge of the double peak. For HC1 samples below 0.02 M, the peaks were below the starting base line. This is probably due to a leveling of the acid sample plug by the buffer and dilution of the buffer by the sample plug.
CONCLUSIONS Selecting the readout (peak height or width) is a powerful tool for increasing the range and extending the detection limits of any FIA method. Titrations are a special case in that an equivalence point is sought for peak width evaluation. When acid or base concentration is determined as in the present work, selection of readout is a choice between titrimetric assay and assay based on pH measurement. Technically, the present work resulted in the development of a sturdy, simple system which allows monitoring at a high sample frequency of 100-120 samples per hour in real time (with a delay of less than 30 s, which is the time between sample injection and availability of the readout), low sample consumption with 30 FL of sample injected (less than 0.5 mL required totally), and low reagent consumption (less than 0.5 mL) per assay. Additionally, both acids and bases can be determined with this system. The successful application of the optosensing technique to the determination of the pH of “acid rain” is the topic of a paper that is currently in preparation.
2773
The use of FIA as a method to evaluate materials for the construction of chemical systems has also been demonstrated.
LITERATURE CITED (1) Ruzlcka, J.: Hansen, E. H.: Mosbak, H. Anal. Chlm. Acta 1977, 92, 235-249. (2) Astrom, 0. Anal. Chlm. Acta 1979, 105, 67-75. (3) Rarnsing, A. U.; Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1981, 129, 1-17. (4) Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 198$, 173, 3-21. (5) Ishibashi. N.; Imato, T. Frezenlus’ Z . Anal. Chem. 1986, 323, 244-248. (6) Imato, I.; Ishibashi, N. Anal. Scl. 1985, 1 , 481-482. (7) Wolcott, D. K.; Hunt, D. 0. Presented at the 11th FACSS Meetlng, Philadelphia, PA, 1984; paper 353. (8) Rwlcka, J.; Hansen, E. H. f l o w In/ectlon Analysis; Wiiey: New York, 1981; pp 138-141. (9) Ruzicka, J.: Hansen, E. H. Anal. Chlm. Acta 1985, 173, 3-21. (10) Woods, E. A.; Ruzlcka, J.; Christlan, 0. D.; Charison, R. J. Anal. Chem. 1986, 58, 2496-2502. (11) Haj-Hussein, A. T.: Chrlstian, G. D. Mlcrochem. J. 1986, 34, 67-75. (12) Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1984, 161, 1-25. (13) Reijn, J. M.; van der Linden, W. E., Poppe, H. Anal. Chlm. Acta 1981, 123, 229-237. (14) Rosetti, F. J. C.; Rosetti, H. J. Chem. Educ. 1965, 42(7), 375-378. (15) Perrin, D. D.; Dempsey, E. Buffers for pH and Metal Ion Control; Chapman and Hail: London, 1974. (16) Rhee, J. S.; Dasgupta, P. K. Mkrochlm. Acta 1985, I I I , 107-122. (17) Rhee, J. S.; Dasgupta, P. K. Mlkrochlm. Acta 1985 IIZ, 49-64. (18) Ruzicka. J., private communication, Seattle, WA, July 1988.
RECEIVED for review March, 6, 1987. Accepted August 14, 1987.
Determination of Ingredients of Antipyretic Analgesic Preparations by Micellar Electrokinetic Capillary Chromatography Shigeru Fujiwara Pharmaceuticals Research Center, Kanebo, Ltd., Miyakojima-ku, Osaka 534, J a p a n
Susumu Honda* Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka 577, J a p a n
The principal ingredients of antipyretic analgesic preparations were determined simultaneously by micellar capillary eiectrokinetlc chromatography wlth sodium dodecyl sulfate as the anionic surfactant. All of these compounds mlgrated to the cathode and were well-resolved within ca. 20 min between the aqueous and micellar phases, wlth number of theoretical plates values ranglng from 70000 to 130000. On-column detection at 214 nm wlth ethyl p-aminobenzoate as the internal standard allowed accurate and reproducible determlnation of these compounds. Application to a commerclai antlpyretlc analgesic tablet demonstrated the usefulness of this method.
Electrophoresis in an open capillary tube (capillary zone electrophoresis, CZE), has brought forth many advantages regarding the separation of ionic substances. Electroosmotic flow having a flat profile perpendicular to the capillary axis carries ions rapidly with high column efficiency (1-5). Oncolumn detection allows simultaneous microanalysis of the 0003-2700/87/0359-2773$01.50/0
component ions of a sample with the number of theoretical plates (NTP) values reaching several hundred thousands or more. Recently, Terabe et al. (6, 7) introduced the micellar solubilization technique to CZE and extended its applicability to nonionic substances. Introduction of a sample composed of lipophilic components to a fused silica capillary tube filled with a buffer solution containing an ionic surfactant such as sodium dodecyl sulfate (SDS),followed by application of a high voltage between both ends of the tube, causes distribution of the components between the aqueous and micellar phases, which migrate toward the cathode. Since the distribution is kinetic, dependent on the concentration of the surfactant, good separation can be obtained for multiple components by changing the concentration of the surfactant. This new analytical tool of micellar electrokinetic capillary chromatography (MECC) has been successfully applied to the separation of the phenolic compounds (7), phenylthiohydantoin derivatives of amino acids (8))and purines (9). In this paper, we have described the application of this method to the analysis of the ingredients of antipyretic analgesic preparations, with an emphasis on quantitative aspects. 0 1987 American Chemical Society
2774
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
40
t
30 1
2-
0
.,
7
9
/
11
PH
Figwe 1. Retention times of principal ingredients of antipyretic analgesics as a function of the pH of the carrier: 0, methanol; A,AP; V,ASA; A,CA; E B 0,SA. Conditions: capillary, fused s i k (80 cm, 0.1-mm i.d.); carrier, 0.02 M phosphate solution; applied current 100 PA; wavelength for detection, 214 nm; each sample applied as a 1.0 X M methanoiic solution.
EXPERIMENTAL SECTION Reagents. Sodium dodecyl sulfate (SDS), acetylsalicyclic acid
(ASA), anhydrous caffeine (CA), p-acetamidophenol (AP), salicylamide (SA), and ethyl p-aminobenzoate ( E m ) were obtained from Wako Pure Chemicals (Dosho-machi, Higashi-ku, Osaka, Japan). o-Ethoxybenzamide (EB) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Nihonbashi-Honcho, Chuo-ku, Tokyo, Japan). All these compounds except SDS were used as methanol solutions. All other chemicals were of the highest grade commercially available. Phosphate solutions were prepared by mixing M disodium hydrogen phosphate with 2.0 X M 2.0 X potassium dihydrogen phosphate in appropriate proportions (for pH 7 and 9) or by adding 1 M sodium hydroxide to the former solution (for pH 11). SDS-carriers were prepared by dissolving 2.5-10 mmol of SDS in 100 mL of the phosphate solutions obtained above. Apparatus. A high-voltage dc power supplier of a Shimadzu IP-SA isotachophoresis apparatus capable of supplying up to 30 kV and a Shimadzu SPD-2A double-beam, variable-wavelength detector were used for MECC. Peak area was measured by C h r ~ m & C-R1B ~ p ~ (Shimadzu, Ni~hinokyo,Nakakyo-ku,Kyoto, Japan). Separation was performed by using a fused silica c a p w tube (80 cm, 0.1-mm i.d.; 0.15-mm 0.d.) obtained from Shimadzu. Detection was carried out by on-column measurement of UV absorption at a position 50 cm from the anode. The polyimine resin coated on the capillary tube was partly removed by burning at the point of detection, and the uncovered portion of the tube was set on the detector block with a handmade slit (0.2mm X 1 mm). Procedure for MECC. The capillary tube was filled with a carrier solution by suction,and both ends of the tube were dipped into electrode solutions having the same composition as that of the carrier. The surface of both electrode solutionswere adjusted to the same level. To introduce a sample solution into the tube, the anodic end of the tube was rapidly moved into the sample solution, whose level was then lifted up 5 cm higher than the level of the cathodic solution and maintained at this level for 5 s. The end of the tube was returned to the anodic solution, and a high voltage was applied in the constant current mode. Procedure for the Determination o f the Ingredients of a Commercial Antipyretic Analgesic Tablet. Acidic methanol for extraction of the ingredients was prepared by adding 10 mL of 0.1 N hydrochloric acid to 1L of methanol. Five tablets were weighed and ground. One-fifth of the resultant powder was weighed accurately and 75 mL of acidic methanol was added. After sonication for 5 min followed by shaking for 10 min, acidic methanol was added to make the volume of the mixture exactly 100 mL. The mixture was centrifuged for 10 min at 2500g. A 5-mL portion of the supernatant was taken out, to which was added 5 mL of a 1.0 X 10-2M methanolic solution of EAB (internal standard), and then methanol was added to make the volume exactly 50 mL. Authentic samples (amounts equivalent to those in a tablet) of AP,CA, and EB were weighed accurately and dissolved in acidic methanol to make the volume exactly 100 mL. To a 5mL portion of the resultant solution was added 5 mL of the EAB (internal standard) solution, and the volume was adjusted to 50 mL by an addition of methanol. This solution was used as the standard solution.
OLl
0
0.m
I
0.05
0,075
0,lO
concentratlon o f SDS (M) Figure 2. Effect of SDS concentration on retention time. The carrier was 0.02 M phosphate Sdutlohs, pH 11, containing 0.025-0.10 M SDS. Other experimental conditions were the same as those described in Figure 1. The symbols ate also the same as those in Figure 1.
The sample and standard solutions were introduced into the capillary tube and analyzed by MECC according to the procedure described above. The peak area ratio of each compound to the internal standard was measured, and the content of each sample in a tablet was calculated.
RESULTS AND DISCUSSION O p t i m i z a t i o n of Separation. Figure 1 shows the pH dependence of the retention times of ASA, AP, CA,EB, and SA, which are principal ingredients of antipyretic analgesics, in 0.02 M phosphate solutions. Since SDS was absent in the carrier under these conditions, separation was simply due to CZE. Rapid electroosmotic flow was generated by the interaction of the carrier and the inner wall of the tube in the strong electric field. All compounds were driven from the anodic inlet to the cathodic outlet. Anionic species were additionally pulled back by electrophoresis with different velocities dependent on the charge and weight and hence were slower than the electroosmotic flow. The carboxyl group of ASA was dissociated in the pH range examined. Therefore, it was more retarded than methanol due to electrophoresis and its retention time was not changed significantly over the whole pH range. On the other hand, retention times of AP and SA were increased with increasing pH values, because the phenolic hydroxyl groups were dissociated to form the phenolate ions above their pK values and the resultant phenolate ions were more strongly pulled back than the undissociated forms by electrostatic force. They were better separated from each other, as pH increased. As a result, ASA, AP, and SA were well-resolved a t pH 11. However, neutral compounds (CA and EB) were eluted at almost the same retention time as that of methanol a t every pH value examined. The retention times of methanol and these neutral compounds became slightly longer as pH increased. This was due to a gradual decrease in the velocity of electroosmotic flow with increasing pH value, presumably as a result of the decrease in the { potential with the pH change (IO). When SDS was added to the carrier, nonionic substances were separated by MECC and they were distributed between the aqueous solution and the micelles, which were moving at different velocities in the capillary tube. Anionic substances were migrated by the combination of electroosmosis and electrophoresis. As a result, both ionic and nonionic substances were eluted between methanol (insolubilized solute) and micelles, provided that appropriate conditions were selected. Figure 2 shows the effect of the SDS concentration on the retention time of each species,obtained for the SDS-containing phosphate solutions a t pH 11. Retention times of nonionic substances (CA and EB) were increased with increasing SDS
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
2775
2ot
Table I. Reproducibility of the Determination of Authentic Specimen by the Internal Standard Methods"
peak area or peak height ratio to the internal standard (n = 5) AP ASA CA EB SA Peak Area Ratio Mode fb SC
s/f,d %
0.81 0.014 1.7
0.56 0.010 1.8
1.35 0.015 1.1
2.95 0.025 0.8
1.97 0.033 1.7
5t I
Peak Height Ratio Mode f* SC
s/f,d %
1.83 0.047 2.6
1.05 0.038 3.6
1.79 0.028 1.6
2.95 0.041 1.4
2.53 0.050 2.0
" The sample solution contained approximately equimolar amounts (ca. 1 X M) of authentic specimen of these compounds and the internal standard. b Mean. Standard deviation. Relative standard deviation.
I
I
I
7
9
11
PH
Flgure 3. Effect of canier pH on retention time. The carrier was 0.02 M phosphate solution containing 0.05 M SDS. Other experimental conditions were the same as those described in Figure 1. The symbols are also the same as those in Figure 1. 4
Table 11. Result of Recovery Test"
excipient
AP
ASA
recovery CA EB
Avicel cornstarch lactose
101.0
100.8 99.5 98.9
98.8 100.0 99.4
%
a
100.3 98.7
99.0 99.7 100.6
SA 100.2 100.3 100.4
Each value is the mean of three experiments.
concentrations with greater rates than those of the ionic substances, consistent with the result reported by Otsuka et al. (8). E B was more retarded than CA, because the former was more easily incorporated into SDS due to its higher lipophilicity. At a SDS concentration of 0.05 M all compounds examined were well-resolved. In 0.075 M SDS separation of Ap and CA was poor. In 0.10 M SDS the elution order of Ap and CA was reversed, but separation of all these compounds was complete. However, retention times of all these compounds were longer than those obtained in 0.05 M SDS. Figure 3 shows the plot of retention time vs pH, obtained by keeping SDS and the phosphate concentrations constant at 0.05 M and 0.02 M, respectively. The retention time of every compound was increased gradually with increasing pH values, though the elution order of AP and CA, as well as that of ASA and SA, were reversed between pH 9 and 11. The best separation was obtained at pH 11. On the basis of the foregoing results, the 0.02 M phosphate solution, pH 11, containing SDS a t a concentration of 0.05 M was used for quantification of these compounds. Retention time could be shortened by increasing the applied current, but separation of SA and E B became incomplete above 150 PA. Under these conditions, EAB as the internal standard was also well-separated from the compounds examined. Figure 4 shows an electropherogram of these compounds together with the internal standard. The values of numbers of theoretical plates (NTP, 5 . 5 4 t ~ ~ where ~ ~ / ~t R- is~ retention , time of a compound, and wlj2 is peak width at half the maximal response) of CA, AP, ASA, SA, and E B were 70 000,130 000,
I
l
0
5
U
I
I
I
I
20 25 Retention tlme (mln) 10
15
Figure 4. Separation of principal ingredients of antipyretic analgesics. The carrier was 0.02 M phosphate solution, pH 11, containing SDS at a concentration of 0.05 M. The experimental conditions were the same as those described in Figure 3. Peak assignments: 1, CA; 2, AP; 3, ASA; 4, SA; 5, E B 6, EAB (internal standard).
90 000, 80 000, and 70 000, respectively.
Quantification. Reproducibility of the determination of these compounds was investigated by repeated introduction of a sample solution containing equimolar amounts of the authentic specimen of these compounds and the internal standard. As observed from Table I, the peak area ratio mode gave a higher reproducibility than the peak height ratio mode, the relative standard deviation being in the range 0.8-1.8%. The calibration curves (not shown) of all these compounds obtained by the peak area ratio mode gave excellent linearity in the range of 0.5-2.0, and all of them passed through the origin, when the amount of the internal standard was fixed at 1.0 X M. This linearity range might be slightly altered by the use of narrower slits than those used in this study (0.2-mm i.d.1, due to the change in the sensitivity by a reduction in the stray light. Table I1 gives the recoveries of these compounds added to various excipients. Samples were made by adding 1 mmol of each compound to 200 mg of an excipient. Extraction of the samples with methanol acidified with dilute hydrochloric
Table 111. Accuracy and Reproducibility of the Determination of the Ingredients of a Commercial Antipyretic Analgesic Tablet
ingredient
amount added, mg
amount found, mg
f"
Sb
s/f,C %
(foundladded) X lo2
CA
25.0 150.0 132.5
25.61,24.81,25.09,24.88,25.40 149.7,149.7,147.0,148.4,150.3 133.2,131.4,130.9,130.1,133.7
25.16 149.0 131.9
0.34 1.3 1.5
1.4 0.87 1.1
100.6 99.3 99.5
AP
EB 'Mean. Standard deviation. Relative standard deviation.
2776
Anal. Chem. 1987, 59, 2776-2780 5
U L
I
I
I
'
5 10 15 20 Retention time (mln)
0
Flgure 5. Electropherogram of the extract of a commercial tablet of an antipyretic analgesic. The experimental conditions were the same as those described in Figure 3. Peak assignments are the same as those in Flgure 4.
acid and determination of the components by the proposed procedure of MECC indicated that every compound was recovered almost completely. Determination of the Ingredients of a Commercial Antipyretic Analgesic Tablet. On the basis of the aforementioned results, we applied this method to the determination of the ingredients of a commercial antipyretic analgesic tablet containing AP, CA, and EB. A typical chromatogram is shown in Figure 5. The accuracy and reproducibility of the determination of these ingredients are given in Table 111. It is indicated that the error was within 0.7% and the relative standard deviation was less than 1.4% for these three ingredients.
There are a number of papers on high-performance liquid chromatography (HPLC) of antipyretic analgesics, in which several ingredients of tablets were separated and quantified (11-13). The proposed MECC method also allowed rapid simultaneous determination of five ingredients of antipyretic analgesic preparations with high accuracy and reproducibility. It is remarkable that a variety of compounds having such a wide range of polarity and ionic nature could be analyzed simultaneously, simply by selecting appropriate pH and SDS concentration. The column efficiency and resolution were rather higher. Thus, the proposed method is considered to he an alternative to the HPLC methods. Registry No. 2-HO2CCeH40C0CH3,50-78-2; caffeine, 58-08-2; p-acetamidophenol, 103-90-2; salicylamide, 65-45-2; o-ethoxybenzamide, 938-73-8; sodium dodecyl sulfate, 151-21-3.
LITERATURE CITED Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M. J. Chromatoor. 1979. 169. 1-10, Jorgekon, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53,1298-1302. Jorgenson, J. W.; Lukacs, K. D.J. Chromatogr. 1981, 278, 209-216. Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272. Lauer, H. H.; McManlgill, D. Anal. Chem. 1986, 58, 166-170. Terabe, S.;Otsuka. K.; Ichikawa, K.; Ando, T. Anal. Chem. 1984, 56, 111-113. Terabe, S.;Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. Otsuka, K.; Terabe, S.;Ando, T. J. Chromatogr. 1985, 332,219-226. Burton. D. E.: Sepaniak, M. J.; Maskarinec, M. P. Chromatographia 1986, 21, 583-566. Lukacs, K. D.;Jorgenson, J. W. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Commun. 1985, 8 , 407-41 1. Gupta, V. D. J. Pharm. Sci. 1980, 69, 110-113. Thomis, R.; Roets, E.; Hoogmartens, J. J . Pharm. Sci. 1984, 73, 1830-1833. Mamolo. M. G.; Vio, L.; Maurich, V. J. Pharm. Homed. Anal. 1985, 3, 157-164.
RECEIVED for review May 18, 1987. Accepted July 30, 1987.
Application of Chelate-Forming Resin and Modified Glassy Carbon Electrode for Selective Determination of Iron(I II)by Liquid Chromatography with Electrochemical Detection Pawel J. Kulesza,* Krystyna Brajter, and Ewa Dabek-Zlotorzynska Department of Chemistry, University of Warsaw, Pasteura I , 02-093 Warsaw, Poland
A thln fHm of a mixed-metai NGFe(CN), deposit on a glassy carbon electrode is demonstrated to be a stable and durable electrocatalyst for the falrly Irreverslble, and often lrreproducible, reduction of Fe(II1) at the bare substrate. By use of an lndlcator coated with such nickel( I I ) hexacyanoferrate mlcrostructures, the scope of ampermetry can be extended to Include monitoring of Fe( III)without the necessity of oxygen removal. A Uquld chromatography procedure comblned wlth electrochemical detection has been developed for Fe( I I I).A hlghty selective separation of Fe( III)was achieved by the use of a tboroMoaded common anlon-exchange resin. The most satlrtactory results were obtained wlth the 1 M HCI-1 M KCI eluent and wlth an electrolysis potentlal of 0.20 V V.I SCE. The chromatography peak current was proportional to the Fe(II1) concentration In the sample over the range 2 X lo-' to 9 X lo-' M Fe(II1). The detection limit, without preconcentratlon, was about 2-3 ppb Fe( II I). The system was free of interference from metal cations and common anions.
The determination of inorganic cations by liquid chromatography, LC, has received increased attention over recent years. This has been commonly achieved with the use of ion-exchange high-performance liquid chromatography (HPLC) columns and various detection systems that are based upon conductivity (1-3), coulometry ( 4 ) ,spectrophotometry (5-7), either in the ultraviolet (UV) or visible range and sometimes coupled with the postcolumn derivatization, and, more recently, on potentiometry (8)measurements. Although the electrochemical/amperometric detection is coming into widespread use for trace determinations of electroactive organic compounds ( S l l ) to , date, there have not been many such studies of metal ions or metal complexes (11-13). Indeed, the performance of an electrochemical detector varies widely with the analyte and chromatographic conditions (11-15). The majority of metal cations would have to be determined via reduction steps that a t common electrode materials frequently introduce problems associated with oxygen interference, electrode stability, and/or the effects related
0003-2700/87/0359-2776$01.50/0 0 1987 American Chemical Society
