Determination of AcetyIsaIicyIic Acid, Acetop henetidin, and Caffeine in Dosage Forms by Nonaqueous Titration SONG-LING LIN and MARTIN 1. BLAKE Department o f Pharmacy, University o f Illinois at the Medical Center, Chicago, 111.
b Dosage forms containing acetylsalicylic acid, acetophenetidin, and caffeine (APC) are analyzed by potentiometric nonaqueous titration. The acetylsalicylic acid is determined by direct titration of an aliquot of the powder mass dissolved in dimethylformamide. The titrant is sodium methoxide in benzene-methanol. The acetophenetidin and caffeine are determined in a second aliquot of the powder mass by a differentiating titration in acetic anhydride-chloroformbenzene ( 1 : 1 :9 ) using perchloric acid in acetic acid-acetic anhydride ( 1 : 1 ) as the titrant. When the acetophenetidin-to-caffeine ratio is greater than 4, an additive method is applied.
I
N SPITE OF THE WIDESPREAD USE and
established therapeutic effectiveness of acetylsalicylic acid (aspirin) , acetophenetidin (phenacetin), and caffeine combinations (4PC), the development of a simple and accurate method of analysis for the individual components in drug dosage forms remains the objective of numerous research investigations. A variety of procedures have been proposed for the analysis of APC preparations. These have involved chromatography, volumetric titration, colorimetry and infrared, ultraviolet and NMR spectrometry. These have been noted by Connors (5) in his review of the literature. The British Pharmaceutical Codex ( 2 ) recognizes a gravimetric procedure for the determination of the individual components in compound acetylsalicylic acid tablets after preliminary separation with immiscible solvents. The A.O.A.C. method ( 1 ) utilizes either a spectrophotometric or gravimetric procedure after preliminary treatment of the sample for analysis. The K.F. XI1 assay (11) for APC capsules and tablets is an ultraviolet spectrophotometric procedure involving preliminary separation of the components by column chromatography using purified siliceous earth which has been treated with sulfuric acid and sodium bicarbonate. Although satisfactory results are attainable by these methods, they possess several drawbacks including time-consuming extraction processes and costly instrumentation. Application of nonaqueous titrimetry
to the analysis of this combination of drugs has been limited because of the weakly basic nature of acetophenetidin. Wollish, et al. (16) analyzed APC tablets for acetophenetidin content by titrating the amine, p-phenetidine, which was liberated by hydrolysis of the acetophenetidin with hydrochloric acid. The amine, extracted with chloroform after making the solution alkaline, was titrated with perchloric acid in p-dioxane. The caffeine which was also extracted with the chloroform did not interfere. They suggest that the caffeine be determined on a separate sample by the iodination procedure of Wirth (15). The aspirin was extracted with a chloroform-ether mixture (1 :3) and titrated in dimethylformamide with lithium methoxide in benzene-methanol. Chatten, et al. (4)found that acetophenetidin interfered with the assay of caffeine in certain APC combinations. Nitromethane was the solvent and perchloric acid the titrant. Lin and Blake (10) determined acetophenetidin in a variety of dosage forms by titration in acetic anhydride-chloroform-benzene (1 :1 :9) solvent mixture using perchloric acid in acetic acid-acetic anhydride (1 :1). In the present study a simple nonaqueous titration procedure is presented for the analysis of acetylsalicylic acid, acetophenetidin and caffeine mixtures in a number of dosage forms. EXPERIMENTAL
Apparatus. Titrations were performed potentiometrically with a Fisher titrimeter, Model 35, equipped with a glass electrode (Beckman No. 11-505-346) and a sleeve-type calomel electrode (Beckman NO. 11-505-80). The calomel electrode was used as such for the determination of acetylsalicylic acid. For the differentiating titrations the electrode was modified by replacing the aqueous bridge in the calomel cell with a 0.1M solution of anhydrous lithium perchlorate in acetic anhydride as the supporting electrolyte as described by Wimer (14). Reagents. Acetylsalicylic acid, U.S.P. (Mallinckrodt), was dried a t 60" C. for 4 hours and was used without further purification. Analysis by U.S.P. assay indicated a purity of better than 99.50j0. Acetophenetidin, U.S.P. (American Pharmaceutical Co.), was dried a t 60' C.
for 2 hours and used without further purification. Analysis by U.S.P. assay (for tablets) indicated a purity of better than 99.0%. Caffeine, U.S.P. (Mallinckrodt), was dried at 80' C. for 5 hours and used without further purification. Analysis by U.S.P. assay indicated a purity of better than 99.0%. Sodium methoxide, O . 1 N in benzenemethanol, was prepared and standardized as described by Fritz and Lisicki (8)* Perchloric acid, 0.LV in glacial acetic acid or acetic acid-acetic anhydride (1 :I), was prepared and standardized as described by Lin and Blake (IO). Determination of Acetylsalicylic Acid in Presence of Acetophenetidin and Caffeine. Approximately 0.6 meq. of acetylsalicylic acid and 0.6 meq. of acetophenetidin or 0.6 meq. of caffeine, or 0.6 meq. of both acetophenetidin and caffeine, accurately weighed, were dissolved in 50 ml. of dimethylformamide with the aid of magnetic stirring for about 15 minutes. The solution was titrated potentiometrically with 0.1N sodium methoxide in benzene-methanol. The end point in the titration was determined from the inflection in the curve obtained by plotting mv. us. ml. of titrant added. During the titration process, the titration beaker was covered with a rubber plate having holes for the passage of the electrodes and the buret tip. The solvent blank was determined and subtracted from the volume consumed in the titration. Dosage forms containing acetylsalicylic acid, acetophenetidin , and caffeine were analyzed for acetylsalicylic acid content by the above procedure. Twenty tablets were weighed and powdered or twenty capsules were emptied as completely as possible and the contents weighed. An amount of the powder mass equivalent to about 0.6 meq. of acetylsalicylic acid was accurately weighed and assayed as described above. Differentiating Titration of Acetophenetidin and Caffeine. Approximately 1.0 meq. of acetophenetidin and 1.0 meq. of caffeine, accurately weighed, were dissolved in 50 ml. of solvent or solvent mixture with the aid of magnetic stirring for a t least 20 minutes. The solution was titrated potentiometrically with 0.1N perchloric acid in glacial acetic acid or in acetic acid-acetic anhydride (1 :1) solvent mixture. The titration solvents used in this study included glacial acetic acid, acetic anhydride, and acetic anVOL. 38,
NO. 4,
APRIL 1966
@
549
357 L""
h h r j i l b O . f N Sodium Methoxido hl.)
Figure 1 . Typical titration curve for the analysis of acetylsalicylic acid in the presence of acetophenetidin and caffeine in synthetic mixtures and dosage forms
hydride-chloroform-benzene (1:1 :9) solvent mixture. Effect of Varying the Benzene Concentration in the Titration Solvent. Approximately 1.0 meq. of both acetophenetidin and caffeine, accurately weighed, was dissolved in 75 ml. of solvent mixture. The solution was stirred magnetically for a t least 25 minutes and then titrated with 0.1N perchloric acid in acetic acid-acetic anhydride. All titration solvents in this study contained 1 part acetic anhydride and 1 part chloroform. The benzene concentration ranged from 0 to 15 parts. Effect of Caffeine Concentration on Differentiating Titration. A series of differentiating titrations was performed in which the milliequivalent ratio of acetophenetidin to caffeine was varied from 1 to greater than 5. Since APC dosage forms usually contain a significantly greater amount of acetophenetidin than caffeine, the effect on the sensitivity of the differentiating titration was studied. Appropriate amounts of acetylsalicylic acid, acetophenetidin and caffeine to give the desired milliequivalent ratio of components were weighed accurately and dissolved in 50 inl. acetic anhydridechloroform-benzene solvent mixture. The solution was titrated with 0.1N perchloric acid in acetic acid-acetic anhydride. Table I. Determination of Acetylsalicylic Acid in the Presence of Acetophenetidin and Caffeine
Mixture Acetylsalicylic acid
Acetylsalicylic acid Acetophenetidin Acetylsalicylic
Amount weighed, meq. Recovery, 70 0.5
100.08 i 0.86"
0.6
100.15 =t 1.04
0.6
acid 0 . 6 100.51 3~ 0.93 Caffeine 0.6 Acetylsalicylic acid 0.6 99.54 =t 0.37 Acetophenetidin 0.6 Caffeine 0.6 a Standard deviation based on a t least 5 determinations.
550
ANALYTICAL CHEMISTRY
Additive Rocedure for Caffeine Determination. When the milliequivalent ratio of acetophenetidin to caffeine in a commercial tablet or capsule was greater than 4 , a known amount of pure caffeine was added to the mixture to make the ratio more favorable for a differentiating titration. Twenty APC tablets were weighed and powdered, or twenty capsules were emptied as completely as possible and the contents weighed. A sample of the powder mass, equivalent to about 1.0 meq. of acetophenetidin was accurately weighed and transferred to a 150-ml. beaker. Sufficient pure caffeine powder, U.S.P., accurately weighed, was added to the powder mass to bring the caffeine content to 0.4-0.6 meq. The sample was dissolved in 75 ml. of acetic anhydride-chloroformbenzene solvent mixture. The solution was titrated with 0.W perchloric acid in acetic acid-acetic anhydride. RESULTS AND DISCUSSION
The acetylsalicylic acid content of APC dosage forms was determined by titrating a sample of the powder mass in dimethylformamide with sodium methoxide in benzene-methanol as the titrant. By the proposed procedure preliminary extraction of the acetylsalicylic acid, as in the method of Wollish, et al. (16) was not necessary. However, stearic acid, if present in a commercial variety of APC tablets, would interfere in the proposed procedure. If present, the stearic acid may be removed by the technique of Wollish, et al. in which the powder mass is treated with petroleum ether. The data reported in Table I indicate that acetophenetidin and caffeine do not interfere with the titration. Preliminary studies indicated that the presence of salicylic acid, a degradation product of acetylsalicylic acid, can be determined by a differentiating titration by the proposed procedure and, therefore, did not represent an interfering substance. A typical titration curve is shown in Figure 1. 4 major objective of this study was to develop a differentiating titration procedure for the weak bases acetophenetidin and caffeine. In preliminary experiments a series of solvents and combinations of solvents were employed as titration solvents. These included : acetonitrile, chloroform, pyridine, methyl isobutyl ketone, methyl ethyl ketone, acetone, t-butyl alcohol and nitrobenzene. These have been used successfully by other workers in differentiating mixtures of acids or bases. They were not applicable in the present investigation. Acetic anhydride as a solvent for nonaqueous titrations and solvent mixtures containing acetic anhydride have proved useful for the determination of compounds too weakly basic to permit titration in glacial acetic acid. Wimer
1000~
\
A
2ooi 100 J
5
IO
7 15
20
25
30
0.1N Perchloric Acld (ml.)
Figure 2. Typical curves for the titration of acefophenetidin and caffeine, individually and in combination, with 0.1N perchloric acid in glacial acetic acid A.
B.
C. D.
E.
Acetophenetidin and caffeine in glacial acetic acid Acetophenetidin and caffeine in acetic anhydride Acetophenetidin and caffeine in acetic anhydride-chloroform-benzene (1 :1:9) Caffeine in acetic anhydride Acetophenetidin in acetic anhydride
(14) titrated a variety of amides and acylated amines in acetic anhydride using perchloric acid in glacial acetic acid or dioxane as the titrant. Streuli (13) determined caffeine and acetanilid, among other organic bases, in acetic anhydride as the titration solvent. The titrant was perchloric acid in acetic acid-acetic anhydride (1:1). Cowell and Selby (6) titrated certain semicarbazones and substituted hydrazones in a similiar manner. They indicate that the active species is probably the acetyl cation, CHsCO+, rather than the less acidic solvated proton. Gremillion (9) titrated a number of weak organic bases in acetic anhydride using perchloric acid in acetic acid or acetic anhydride as the titrant. Fritz and Fulda (7) titrated weak bases such as caffeine and nicotinamide in nitromethane-acetic anhydride (4:1) with perchloric acid in glacial acetic acid. In a previous report (10) a procedure was described for the determination of acetophenetidin in dosage forms. The titration solvent was acetic anhydridechloroform-benzene (I : 1:9) and the titrant was perchloric acid in acetic acid-acetic anhydride (1 : 1). End point detection was performed with a Fisher titrimeter equipped with a modified calomel-glass electrode system. The same technique was applied in this investigation for differentiating the weak bases acetophenetidin and caffeine. Figures 2 and 3 illustrate the effect of titration solvent and titrant solvent on the determination of acetophenetidin and caffeine, individually and in combination. The titration
E
200 I
5
IO
15
20
25
30
0.1N Perchloric Acid (ml.)
Figure 3. Typical curves for the titration of acetophenetidin and caffeine, individually and in combination, with 0.1N perchloric acid in acetic acidacetic anhydride (1: 1 ) A.
B. C. D.
E.
Acetophenetidin and caffeine in glacial acetic acid Acetophenetidin ond caffeine in acetic anhydride Acetophenetidin and caffeine in acetic anhydride-chloroform-benzene ( 1 :1:9) Caffeine in acetic anhydride-chloroform-benzene (1 :1:9) Acetophenetidin in acetic anhydridechloroform-benzene (1 :1:9)
curves in Figure 2 were obtained with perchloric acid in glacial acetic acid as titrant Lvhile those in Figure 3 are for perchloric acid in acetic acid-acetic anhydride as titrant. Acetophenetidin and caffeine, titrated individually in acetic anhydride (Figure 2 , curves D and E), gave satisfactory end points as indicated by the sharp breaks in the titration curves. I n acetic anhydridechloroform-benzene solvent mixture (Figure 3, curves D and E), a greater sensitivity in end point detection was noted for the individual components. When the combination of drugs was titrated (Figures 2 and 3, curve B) in acetic anhydride, however, a single end point was obtained corresponding to the total amount of base present. Apparently, in acetic anhydride, acetophenetidin and caffeine are leveled to the same basic strength and differentiation is therefore not possible. The ionization constants of the bases in this solvent do not differ sufficiently to permit a differentiating titration. With glacial acetic acid as the solvent (Figures 2 and 3, curve A) no detectable end point was apparent when acetophenetidin and caffeine were titrated with perchloric acid. Although glacial acetic acid is extremely useful for titrating stronger bases such as amines, it is not applicable for very weak bases as those studied here. Of the solvents and solvent mixtures tested for differentiating acetophenetidin and caffeine, the most satisfactory system was found to be acetic anhydridechloroform-benzene (1 : 1 :9). Typical differentiating titration curves are
shown in Figures 2 and 3, curve C. Two distinct inflections are apparent; the first corresponding to the caffeine content and the second representing the acetophenetidin end point. The sensitivity of the end points is improved somewhat when the titrant is perchloric acid in acetic acid-acetic anhydride. A series of ten mixtures containing acetophenetidin and caffeine was analyzed by differentiating titration, using acetic anhydride-chloroformbenzene (1: 1 :9) as sample solvent and perchloric acid in acetic acid-acetic anhydride (1:l) as titrant. The data reported in Table I1 indicate satisfactory precision for both components. Figure 4 demonstrates the effect of varying the benzene concentration in the titration solvent on the sensitivity of the end points in a differentiating titration. Only one inflection in the curve was obtained when the titration solvent was acetic anhydridechloroform (1: 1). This end point corresponds to the total base present. Two end points become apparent as the proportion of benzene in the solvent is increased. The optimum solvent composition for the differentiating titration was acetic anhydride : chloroform : benzene, 1:1:9, When the proportion of benzene was greater than 15 parts, fluctuations in voltage scale readings were experienced. Pifer, et al. (12) point out that the inclusion in the titration solvent system of an excess of a solvent having a low dielectric constant can increase markedly the sensitivity in the detection of the end point and may also aid in the solubilization of the sample. The incorporation of benzene in the solvent system contributed these properties to the titrations performed in this investigation. A number of electrode systems were tested in preliminary experiments but the best results were obtained with the glass-calomel electrode pair in which the aqueous bridge in the calomel cell was replaced with 0.1M anhydrous lithium perchlorate in acetic anhydride. The sleeve-type calomel electrode was superior to the fiber-type electrode. The electrodes mere bathed continuously in acetic anhydride when not in use. The calomel electrode was frequently recharged with fresh lithium perchlorate in acetic anhydride throughout the course of study. In commercial APC dosage forms the acetophenetidin content is usually 5 times that of the caffeine content. Therefore, the effect of disproportionate concentrations of components on the sensitivity of the differentiating titration was studied. The data for a series of titrations in which the ratio of acetophenetidin to caffeine was varied are reported in Table I11 and typical curves are shown in Figure 5. Two inflections in the titration curves (Figure 5, curves
““A
300-
‘I
,
,
100
5
,
+
IO 15 PO 25 0.1N Perchloric Acid (ml.)
30
Figure 4. Effect of varying benzene concentration in solvent mixture on the differentiating titration of acetophenetidin and caffeine. The ratios above the curves indicate relative volumes of acetic anhydride : chloroform : benzene
A through D) were obtained when the ratio of acetophenetidin to caffeine ranged from 1 to 4. When the acetophenetidin-to-caffeine ratio was greater than 4, one end point corresponding to the total base present was obtained as in the case of curve E, and no discernible end point was obtained as shown in curve F, G. The concentration of the components represented by titration curve E approximates that found in a typical unit dosage form. When double or triple the concentrations were titrated, as shown in curve F, G ; no inflection in the curve was obtained even when the titration was continued far beyond the theoretical end point for total base present. The attempt here was to determine the caffeine content in the presence of large amounts of acetophenetidin and acetylsalicylic acid. The fact that there was no inflection in the titration curve can not be explained at this time. I t may be Table II. Differentiating Titration of Acetophenetidin and Caffeine
Amount weighed, grams Acetophenetidin Caffeine 0.1832 0.1809 0.1801 0.1801 0.1563 0.1628 0.1570 0.1630 0.1359 0.1008 0.1348 0.0997 0.1144 0.0823 0.1152 0.0811 0.0990 0.0810 0.0977 0.0806
Recovery, yo
Aceto-
phenetidin 99.56 100.62 99.11 100.05 100.24 99.88 99.70 100.68 100.31 99.93 Av. 100.01 Std. dev. f 0 . 4 8
VOL. 38, NO. 4, APRIL 1 9 6 6
Caffeine 100.07 100.43 101.08
100.72 98.95 99.98 100.32 100.16 99.61 100.80 100.21 &0.62 551
Table 111.
Effect of Acetophenetidin-to-Caffeine Ratio on the Sensitivity of Differentiating Titration of APC Mixtures
Amount weighed, meq. AcetylCurve salicylic AcetoRecovery, 70 reference acid phenetidin Caffeine Acetophenetidin Caffeine A 1.00 1.00 1.00 99.78 f 1.42” 99.03 f 1.71 B 1.00 1.oo 0.75 99.91 f 0.74 100.28 f 0.75 C 1 .oo 1.00 0.50 99.91 f 0.70 100.98 f 1.15 D 1.oo 1.00 0.25 99.55 f 1.35 99.98 f 2.94 E 1.26 0.89 0.17 one end point * F 2.52 1.78 0.34 no end point no end point G 3.78 2.67 0.51 a Standard deviation based on a t least 4 determinations. * Corresponds to acetophenetidin plus caffeine.
F,G
LL 1 100J
I
5
10
15
20
25
1 -
70
0.1N Perchloric Acid (ml.)
Table IV.
Determination of Acetylsalicylic Acid, Acetophenetidin, and Caffeine in Dosage Forms
Label claim found, % Dosage form” Acetylsalicylic acid Acetophenetidin Caffeine 99.98 f 0.46 100.06 f 0.79 Tablet A 102.80 f 1.06b Tablet B 102.20 f 0.50 100.65 f 0.51 99.36 f 1.77 Tablet C 99.07 f 0.54 i o i . i 5 f 0.66 99.81 5 i . 9 9 101.62 f 0.80 98.90 f 1.70 Tablet D 99.05 f 0.49 Tablet E 100.45 f 1.50 99.96 f 1.98 100.26 f 1.20 100.63 f 0.48 101.24 f 1.81 100.25 f 1.16 Tablet F 99.43 f 0.83 98.42 f 1.12 100.21 f 0.62 Capsule A a Each unit dosage form was labeled to contain: acetylsalicylic acid, 226.8 mg.; acetophenetidin, 162.0 mg.; caffeine, 32.4 mg. Standard deviation based on a t least 5 determinations.
Table V. Collaborative Study of Differentiating Titration of Acetophenetidin and Caffeine in APC Dosage Form by Additive Method
Label claim found, Yo AcetoCaffeine phenetidin 99.02 f 0.57” 99.90 f 0.22 97.84 f 0.67 99.83 f 0.32 100.09 f 1.46 100.11 f 0.23 99.17 f 0.91 103.20 f 1 . 8 7 98.86 f 1.79 100.05 f 0.52 100.14 f 0.46 100.38 f 0.83 97.74 f 0.58 100.55 f 0.66 100.38 f 0.14 99.80 f 1.00 99.79 f 0.13 100.87 f 0.84 J 98.95 f 0.12 100.93 f 0.81 a Standard deviation based on 4 determinations. Collaborator A B C D E F G H I
due to the inhomogeneity of the solution caused by a large amount of solute present in a limited amount of solvent mixture. This problem is under further study. As reported in Table 111, excellent precision and accuracy were obtained when the acetophenetidin-tocaffeine ratio was 4 or less. Since the usual dosage forms contain acetophenetidin and caffeine in the ratio of about 5 to 1, an additive procedure was employed. This involved the addition of a known amount of pure caffeine to the weighed aliquot of powder mass taken for analysis to alter the acetophenetidinto-caffeine ratio to a more favorable one ranging from 1.5 to 2.5. This technique yielded satisfactory differentiating titration curves.
552
ANALYTICAL CHEMISTRY
Seven commercially available .4PC dosage froms were analyzed for acetylsalicylic acid content by titration with sodium methoxide as described earlier. The acetophenetidin and caffeine were titrated differentially with perchloric acid using the additive procedure. The results of the analyses are reported in Table IV. The applicability of the additive procedure for determining acetophenetidin and caffeine was further tested by a collaborative study in which ten analysts performed 4 determinations on a particular brand of APC tablets representing the same batch. The results of these analyses are reported in Table V. Additives such as excipients, coloring agents, fillers and lubricants used in the preparation of the dosage forms studied here apparently did not interfere with the analyses. While the presence of additives may appear to limit the usefulness of the proposed procedure, it has been shown by Chatten and Illainville (8) that tablet additives do not constitute an important source of interference in the nonaqueous titration of medicinal agents even when the additives themselves are titratable. The proposed nonaqueous procedure for APC dosage forms is a relatively simple one requiring two titrations on separate aliquots of the powder mass; the direct titration of acetylsalicylic acid with sodium methoxide and the differentiating titration of the acetophenetidin and caffeine with perchloric acid using a n additive procedure. Preliminary treatment of the sample is
Figure 5. Effect of acetophenetidinto-caffeine ratio on sensitivity of differentiating titration of APC mixtures. Data for curves are recorded in Table 111. The letters above the curves correspond to those in Table 111
unnecessary and tedious techniques are obviated.
extraction
ACKNOWLEDGMENT
The authors extend their gratitude to Burroughs Wellcome and Co., Eli Lilly and Co., Rexall Drug Co. and the Upjohn Co. for generously supplying samples of APC dosage forms. The authors also acknowledge the analysts who contributed their efforts to the collaborative study. LITERATURE CITED
(1) Assoc. Offic. Agr. Chemists, “Officia Methods of Analysis,” 9th ed., p. 486
Washington, 1960. (2) “British Pharmaceutical Codex,” Pharmaceutical Press, p. 1234, London, 1963. (3) Chatten, L. G., Nainville, C. A., J. Pharm. Sci. 52, 146 (1963). (4) Chatten, L. G., Pernarowski, M., Levi, L., J. Am. Pharm. Assoc., Sci. E d . 44, 332 (1955). (5) Connors, K. A., “Pharmaceutical Analysis,” T. Higuchi and E. BrochmannHanssen, edk, p. 561, Interscience, New York-London, 1961. (6) Cowell, D. B., Selby, B. D., Analyst 88. 974 (1963). ( 7 ) Fritz, ‘J. -S,’, Fulda, bI. O., ANAL. CHEM.25, 1837 (1953). (8) Fritz, J. S., Lisicki, ? AI., I.Ibid., 23, 589 (1951). (9) . . Gremillion. -4. F.. Ibid.,, 27., 133 (1955). ’ (10) Lin, S. L., Blake, M. I., J . Pharm. Sci. 54, 1512 (1965). (11) “National Formulary,” American Pharmaceutical Association, 12th ed., D. 42. 196,5.
RECEIVED for review September 14, 1965. Accepted January 14,1966.
