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Infrared Analysis of Active Ingredients in Ointments
Infrared Analysis of Active Ingredients in Ointments* 11.
Pilocarpine Hydrochloride and Phenacaine Hydrochloride By WILLIAM H. WASHBURN
An infrared method for the analysis of pilocarpine hydrochloride and phenacaine hydrochloride i n ointments is described. Pilocarpine hydrochloride i n the amount of 2 per cent may be determined in a 1-gram sample with an accuracy of f 4 per cent. Phenacaine hydrochloride in the amount of 1% may be determined i n a I-gram sample n i t h an equal accuracy.
(1) infrared methods were described for atropine sulfate and scopolamine hydrobromide i n ointments. T h e infrared method is an improvement over t h e usual titrimetric methods for this type of compound i n t h a t i t is specific and practical for t h e assays of small amounts of active ingredients. T h e method has now been applied to the determination of pilocarpine hydrochloride and phenacaine hydrochloride in ointments.
I
N A PREVIOUS paper
tative determination of pilocarpine. For phenacaine, the band at 9.52 p was chosen. The ointment base in these preparations consists of lanolin, petrolatum, and mineral oil. Working curves were constructed as follows: several portions of pure pilocarpine hydrochloride were dissolved in water and made slightly alkaline by the addition of 15% ammonium hydroxide. The pilocarpine base was then extracted with four 10-cc. portions of chloroform, and the chloroform extracts were evaporated to dryness. The residues were diluted in chloroform to known concentrations ranging from 3-8 mg./cc., and using a cell of 0.5 mm. thickness, absorbancies were noted at 9.01 p. Absorbancies were then plotted against concentration.
EXPERIMENTAL Spectra of both pilocarpine base and phenacaine base were recorded. Pilocarpine (Fig. 1). a colorless oily material, was prepared by pressing a drop of material between two rock salt plates. Phenacaine, the spectrum of which is shown in Fig. 2, was prepared by evaporating '/z cc. of a 1% carbori tetrachloride solution on a rock salt plate. Per ccnt transmittance curves were constructed from the single beam records (2). In ointments, water-soluble salts of both pilocarpine and phenacaine are employed. This necessitates conversion of the salt t o free bases in order that a suitable nonpolar solvent may be employed for quantitative infrared work. I n the case of pilocarpine, chloroform was selected as the solvent, and for phenacaine, carbon tetrachloride was found t o be the solvent of choice. The absorption band at 9.01 p was selected for the quatlti-
3 4 6
6
7 8
9 10 11
12
13
14
W A V E LENGTH I N MICRONS
Fig. 1 .-Infrared
absorption spectra of pilocarpine.
* Received March 2 5 . 1953, from the Control Department, Abbott Laboratories, North Chicago, Illinois.
a
1
1
1
1
I
I
~
I
II I
I
I
7 8 9 1 0 1 1 12 13 14 W A V E LENGTH IN MICRONS
3 4 5
Fig. Z.-Infrared
6
absorption spectrum of phenacaine.
In the construction of a working curve for phenacaine hydrochloride, the same procedure was used with the following exceptions: The aqueous phase was made alkaline with 1 N sodium hydroxide, the rcsidues were taken up in carbon tetrachloride, and absorbancy measurements were made at 9.52 p. Beer's law is followed closely in both cases. Throughout this study the measurements were made with a Perkin-Elmer model 12C infrared recording spectrometer equipped with sodium chloride optics and a three-speed slit drive. Quantitative Infrared Determination of Pilocarpine Hydrochloride 2% in Ointment.-Transfer about 1 Gm. of ointment (accurately weighed) to a suitable flask. Dissolve in 15 cc. of ether and transfer to a separatory funnel. Rinse flask with two 10-cc. portions of ether and add to the separatory funnel. Extract with five 10-cc. portions of water, rinsing each portion first through the original flask and collect the aqueous phase in a second separatory funnel. Render the aqueous phase alkaline with 15% ammonium hydroxide, and extract with four 10-cc. portions of chloroform. With the aid of a stream of nitrogen evaporate the chloroform to dryness in a 50-cc. flask. Dissolve the residue in 5 cc. of 2% hydrochloric acid and heat on a steam bath. Transfer the solution t o a separatory funnel. Rinse the original flask with two 10-cc. portions of 1% hydrochloric
SCIENTIFIC EDITION
November, 1953
acid, and add the rinsings to the separatory funnel. Wash the aqueous solution with four 10-cc. portions of ether. Render the aqueous phase alkaline with 15% ammonium hydroxide, and extract with four 10-cc. portions of chloroform. Filter the combined extracts through a chloroform soaked pledget of cotton into a small flask, evaporate to dryness under a stream of nitrogen, and dry for one hour in a vacuum desiccator. Dissolve the residue in 2 cc. of chloroform and using a sealed cell of 0.5 mm. thickness, determine the absorbancy at 9.01 p. Refer the observed absorbancy t o the working curve and determine the concentration of the solution. Confirm the identity of pilocarpine by scanning the spectrum from 8-10 p Table I lists a series of determinations on ointments of known strength and on commercial preparations.
699
ditional purification of phenacaine is not necessary as it is in the case of pilocarpine, because the absorption of any ointment base carried over is negligible a t 9.52 p . ) Dissolve the residue in 2 cc. of carbon tetrachloride. (If the residue does not dissolve entirely within three minutes, apply heat gently t o the tightly stoppered flask.) When solution has been effected, immediately determine the absorbancy of the sample at 9.52 p i n a sealed cell of about 0.5 mm. thickness. Refer absorbancy to the working curve and determine the concentration of the solution. The identity of phenacaine may be confirmed by scanning the spectrum from 9-10 p. Table I1 lists a series of determinations on ointments of known strength and on commercial preparations.
TABLE II.-RESULTS OF ANALYSES OF PHENACAINE HYDROCHLORIDE OINTMENT 1% TABLEI.-RESULTS OF ANALYSES OF PILOCARPINE HYDROCHLORIDE OINTMENT 2% Laboratory Preparations Sample Theoretical. Found, Recovery. Laboratorv Preparations No. Sample No.
1 2 3 4 5
6 7
Theoretical, Mg.
Found, Mg.
10.2 10.25 10.0 9.99 10.0 9.78 10.0 10.43 9.75 9.8 9.75 10.15 9.8 10.1 Commercial Ointments
Recovery,
%
100.5 99.9 97.8 104.3 IQ?.5 104.1 103.1
Sample No.
Theoretical, Mg.
Found.
Label Claim,
Mg.
%
1
10.07 10.41 10.02 10.18 10.15 9.92 I0 37 10.46 10.36 10.36
10.35 10.60 8.85 8.76 9 5 8 9 in 2 10.45 10,75 10.30
2 3 4 5
102.8 101.9 88.3 88.0 93 6 89.7 98.4 99 9 103.8 99.4
Quantitative Determination of Phenacaine Hydrochloride 1% in Ointment.-Transfer about 0.5 Gm. of ointment (accurately weighed) to a suitable flask. Dissolve in 15 cc. of ether and transfer t o a separatory funnel. Rinse the flask with two 10-cc. portions of ether and add t o the separatory funnel. Extract with five 10-cc. portions of water, rinsing each portion first through the original flask, and collect the aqueous phase in a second separatory funnel. Render the aqueous phase alkaline with 5 cc. of 1 N sodium hydroxide, and extract with four 10-cc. portions of chloroform. Collect the chloroform phase in a second separatory funnel. Wash the chloroform extracts with one 10-cc. portion of 0.05 N sodium hydroxide and drain the chloroform to a small flask. Evaporate t o dryness under a stream of nitrogen and dry under vacuum for one hour. (Ad-
Mg.
1 7 3
4 6 6 c
I
Mg.
10.0 10.08 10.9 10.84 10.9 11.3 10.7 10.9 10.1 9.8 9.6 10.0 10.7 10.9 Commercial Ointments
Sample No.
Theoretical, Me.
Found, Mg.
I
10.22 10.12 In.06 i n . 39 in.22 10.17 10.4 10.i2 10.16 10.30
10.06 9.7 9.89 10.n6 9.23 9.16 11.55 11.59 9.94 10.13
2 3
4 5
%
100.8 99.4 103.7 101.9 97.0 96.0 101.9 Label Claim,
% 98.4 95.8 98.3 96.8 90.3 9n. 1 111.1 108.1 97.8 98.3
SUMMARY AND CONCLUSIONS 1. An infrared method has been developed for the determination of small amounts of pilocarpine hydrochloride a n d phenacaine hydrochloride i n ointments. 2. Accuracy of the method for each determination is i n t h e range of *4Oj,. 3. The method is specific i n t h a t both pilocarpine a n d phenacaine may be identified by their characteristic infrared absorption. REFERENCES I ) Washburn. W. H., THISJOURNAL, 16, 602(1952). 2) Willis, H. A,. and Philpotts. A. R.. Trans. Faraday SOC.,41, 187(1845).
Pilocarpine Hydrochloride and Phenacaine Hydrochloride By WILLIAM H. WASHBURN
An infrared method for the analysis of pilocarpine hydrochloride and phenacaine hydrochloride i n ointments is described. Pilocarpine hydrochloride i n the amount of 2 per cent may be determined in a 1-gram sample with an accuracy of f 4 per cent. Phenacaine hydrochloride in the amount of 1% may be determined i n a I-gram sample n i t h an equal accuracy.
(1) infrared methods were described for atropine sulfate and scopolamine hydrobromide i n ointments. T h e infrared method is an improvement over t h e usual titrimetric methods for this type of compound i n t h a t i t is specific and practical for t h e assays of small amounts of active ingredients. T h e method has now been applied to the determination of pilocarpine hydrochloride and phenacaine hydrochloride in ointments.
I
N A PREVIOUS paper
tative determination of pilocarpine. For phenacaine, the band at 9.52 p was chosen. The ointment base in these preparations consists of lanolin, petrolatum, and mineral oil. Working curves were constructed as follows: several portions of pure pilocarpine hydrochloride were dissolved in water and made slightly alkaline by the addition of 15% ammonium hydroxide. The pilocarpine base was then extracted with four 10-cc. portions of chloroform, and the chloroform extracts were evaporated to dryness. The residues were diluted in chloroform to known concentrations ranging from 3-8 mg./cc., and using a cell of 0.5 mm. thickness, absorbancies were noted at 9.01 p. Absorbancies were then plotted against concentration.
EXPERIMENTAL Spectra of both pilocarpine base and phenacaine base were recorded. Pilocarpine (Fig. 1). a colorless oily material, was prepared by pressing a drop of material between two rock salt plates. Phenacaine, the spectrum of which is shown in Fig. 2, was prepared by evaporating '/z cc. of a 1% carbori tetrachloride solution on a rock salt plate. Per ccnt transmittance curves were constructed from the single beam records (2). In ointments, water-soluble salts of both pilocarpine and phenacaine are employed. This necessitates conversion of the salt t o free bases in order that a suitable nonpolar solvent may be employed for quantitative infrared work. I n the case of pilocarpine, chloroform was selected as the solvent, and for phenacaine, carbon tetrachloride was found t o be the solvent of choice. The absorption band at 9.01 p was selected for the quatlti-
3 4 6
6
7 8
9 10 11
12
13
14
W A V E LENGTH I N MICRONS
Fig. 1 .-Infrared
absorption spectra of pilocarpine.
* Received March 2 5 . 1953, from the Control Department, Abbott Laboratories, North Chicago, Illinois.
a
1
1
1
1
I
I
~
I
II I
I
I
7 8 9 1 0 1 1 12 13 14 W A V E LENGTH IN MICRONS
3 4 5
Fig. Z.-Infrared
6
absorption spectrum of phenacaine.
In the construction of a working curve for phenacaine hydrochloride, the same procedure was used with the following exceptions: The aqueous phase was made alkaline with 1 N sodium hydroxide, the rcsidues were taken up in carbon tetrachloride, and absorbancy measurements were made at 9.52 p. Beer's law is followed closely in both cases. Throughout this study the measurements were made with a Perkin-Elmer model 12C infrared recording spectrometer equipped with sodium chloride optics and a three-speed slit drive. Quantitative Infrared Determination of Pilocarpine Hydrochloride 2% in Ointment.-Transfer about 1 Gm. of ointment (accurately weighed) to a suitable flask. Dissolve in 15 cc. of ether and transfer to a separatory funnel. Rinse flask with two 10-cc. portions of ether and add to the separatory funnel. Extract with five 10-cc. portions of water, rinsing each portion first through the original flask and collect the aqueous phase in a second separatory funnel. Render the aqueous phase alkaline with 15% ammonium hydroxide, and extract with four 10-cc. portions of chloroform. With the aid of a stream of nitrogen evaporate the chloroform to dryness in a 50-cc. flask. Dissolve the residue in 5 cc. of 2% hydrochloric acid and heat on a steam bath. Transfer the solution t o a separatory funnel. Rinse the original flask with two 10-cc. portions of 1% hydrochloric
SCIENTIFIC EDITION
November, 1953
acid, and add the rinsings to the separatory funnel. Wash the aqueous solution with four 10-cc. portions of ether. Render the aqueous phase alkaline with 15% ammonium hydroxide, and extract with four 10-cc. portions of chloroform. Filter the combined extracts through a chloroform soaked pledget of cotton into a small flask, evaporate to dryness under a stream of nitrogen, and dry for one hour in a vacuum desiccator. Dissolve the residue in 2 cc. of chloroform and using a sealed cell of 0.5 mm. thickness, determine the absorbancy at 9.01 p. Refer the observed absorbancy t o the working curve and determine the concentration of the solution. Confirm the identity of pilocarpine by scanning the spectrum from 8-10 p Table I lists a series of determinations on ointments of known strength and on commercial preparations.
699
ditional purification of phenacaine is not necessary as it is in the case of pilocarpine, because the absorption of any ointment base carried over is negligible a t 9.52 p . ) Dissolve the residue in 2 cc. of carbon tetrachloride. (If the residue does not dissolve entirely within three minutes, apply heat gently t o the tightly stoppered flask.) When solution has been effected, immediately determine the absorbancy of the sample at 9.52 p i n a sealed cell of about 0.5 mm. thickness. Refer absorbancy to the working curve and determine the concentration of the solution. The identity of phenacaine may be confirmed by scanning the spectrum from 9-10 p. Table I1 lists a series of determinations on ointments of known strength and on commercial preparations.
TABLE II.-RESULTS OF ANALYSES OF PHENACAINE HYDROCHLORIDE OINTMENT 1% TABLEI.-RESULTS OF ANALYSES OF PILOCARPINE HYDROCHLORIDE OINTMENT 2% Laboratory Preparations Sample Theoretical. Found, Recovery. Laboratorv Preparations No. Sample No.
1 2 3 4 5
6 7
Theoretical, Mg.
Found, Mg.
10.2 10.25 10.0 9.99 10.0 9.78 10.0 10.43 9.75 9.8 9.75 10.15 9.8 10.1 Commercial Ointments
Recovery,
%
100.5 99.9 97.8 104.3 IQ?.5 104.1 103.1
Sample No.
Theoretical, Mg.
Found.
Label Claim,
Mg.
%
1
10.07 10.41 10.02 10.18 10.15 9.92 I0 37 10.46 10.36 10.36
10.35 10.60 8.85 8.76 9 5 8 9 in 2 10.45 10,75 10.30
2 3 4 5
102.8 101.9 88.3 88.0 93 6 89.7 98.4 99 9 103.8 99.4
Quantitative Determination of Phenacaine Hydrochloride 1% in Ointment.-Transfer about 0.5 Gm. of ointment (accurately weighed) to a suitable flask. Dissolve in 15 cc. of ether and transfer t o a separatory funnel. Rinse the flask with two 10-cc. portions of ether and add t o the separatory funnel. Extract with five 10-cc. portions of water, rinsing each portion first through the original flask, and collect the aqueous phase in a second separatory funnel. Render the aqueous phase alkaline with 5 cc. of 1 N sodium hydroxide, and extract with four 10-cc. portions of chloroform. Collect the chloroform phase in a second separatory funnel. Wash the chloroform extracts with one 10-cc. portion of 0.05 N sodium hydroxide and drain the chloroform to a small flask. Evaporate t o dryness under a stream of nitrogen and dry under vacuum for one hour. (Ad-
Mg.
1 7 3
4 6 6 c
I
Mg.
10.0 10.08 10.9 10.84 10.9 11.3 10.7 10.9 10.1 9.8 9.6 10.0 10.7 10.9 Commercial Ointments
Sample No.
Theoretical, Me.
Found, Mg.
I
10.22 10.12 In.06 i n . 39 in.22 10.17 10.4 10.i2 10.16 10.30
10.06 9.7 9.89 10.n6 9.23 9.16 11.55 11.59 9.94 10.13
2 3
4 5
%
100.8 99.4 103.7 101.9 97.0 96.0 101.9 Label Claim,
% 98.4 95.8 98.3 96.8 90.3 9n. 1 111.1 108.1 97.8 98.3
SUMMARY AND CONCLUSIONS 1. An infrared method has been developed for the determination of small amounts of pilocarpine hydrochloride a n d phenacaine hydrochloride i n ointments. 2. Accuracy of the method for each determination is i n t h e range of *4Oj,. 3. The method is specific i n t h a t both pilocarpine a n d phenacaine may be identified by their characteristic infrared absorption. REFERENCES I ) Washburn. W. H., THISJOURNAL, 16, 602(1952). 2) Willis, H. A,. and Philpotts. A. R.. Trans. Faraday SOC.,41, 187(1845).