Gardner, W. E., Hine, M.,J . Am. Chem. SOC.77,594(19.55). (6) Kloetzel, M.C.,“Organic Reactions,” Vol. IV,p. 47, Wiley, New York, 1948. (7) McICusick, €3. C., Heckert, R. E., Cairns, T. L., Coffman, D. D., Mower, H. F., J. Am. Chem. SOC. 80, 2806 (1958). (8) Merrifield, R . E., Phillips, W. TI., Ibid., 80,2778(1958). (9) Middleton, W. J., Hrckert,, R. E., Little, E. I,., Iiwspan, C. G., Ihid., 80, 2783 (1958). (10)Polgar, A., Jungnickel, ,J. L.,
“Or anic Analysis ” Vol. 111, pp. 31015, fnterscience dew York, 1956. (11) Putnam, 5. k., Moss, M. L., Hall, R. T.,IND.ENG.CHEM.,ANAL. ED. 18,628(1846). (12) Rehm, IC., Bodin, J. T., Connors, K. A,, Higuchi, T.,ANAL. CHEnr. 31, 483 (1959). (13) Sairsen, G. N.,Engelhardt, V. A., Middleton, W.J., J. A m . C h m . Soc. 80, 28h5 (1958). (14) Sawicki, E,, Elhcrt, W., Stanley, T.W.,Hauser, T. It., Fox, F. T., ANAL. CIWM.32,811 (1960).
(15) Schenk, G. H.,Oaolins, M., Talnnta 8,109 (1961). (16) F t ’ e v , A. P., Vinogradova, E. V., Hal ern, G. D., Conpl. tend. acad. 8CZ. 8.R.S.8. 4, 267 (1935). (17) Ubaldini, I., Crespi, V., Guerrieri, F.,Ann. chim. appl . 39,77 (1949). (18)Unger, P.; Analyst 80,820( I 955).
RECEIVED for review January 12, 1961. Accepted A ril 13, 1961. Division of Analytical bhernistry, 139th Meeting, ACS, 8t. IJouis, Mo., March 1961.
END OF SYMPOSIUM
Preliminary Solvent Extraction for the Spectrophotometric Determination of Panthenol in Pharmaceutical Products THAVIL PANALAKS and J. A. CAMPBELL Food and Drug laboratories, Deparfment of Nafional Health and Welfare, Ottawa, Canada
b Substances in pharmaceutical preparations which interfere in the chemical determination of d-panthenol are not removed completely by the present method of ion exchange chromatography. However, they were eliminated b y saturation of the aqueous solution with ammonium sulfate, followed by extraction with benzyl alcohol and re-extraction of d-panthenol into water with the aid of toluene. Using this procedure and the 1,2-naphthoquinone-4-sulfonatereaction, a linear relationship was found between absorbance and concentrations of 0.01 to 0.05 mg. of d-panthenol per mi. The method is accurate and precise. Mean recovery of d-panthenol added to a variety of complex multivitamin mixtures was 100%. The proposed method was not influenced b y substances which interfered with two other chemical assays. Results were in good agreement with those obtained by two microbiological methods.
A
d-panthenol (2,4dihydroxy-N-(3-hydroxypropy1)-3, 3dimethylbutyramide], the alcohol form of pantothenic acid, is being used extensively in pharmaceutical vitamin preparations, no single method for ita determination has been generally accepted. Microbiological methods (1, 8, 6, 7) are time consuming and inconvenient for routine assays, and in many hands have not performed too well. Chemical methods have also been used, but are not always applicable to samples containing other vitamins, LTHOUGH
1038
ANALYTICAL CHEMISTRY
minerals, biological extracts, and other ingredients commonly used in pharmaceutical vitamin products. Schmall and Woilish (6) reviewed some of these and proposed two methods based on the reactions of 8-alanol, the product of hydrolysis of d-panthenol, with ninhydrin and with 1,a-naphthoquinone-4-sulfonate. The latter reaction utilized a modification by Frame, Russell, and Wilhelmi ( 4 ) of the original method of Folin (3)for amino nitrogen. Although these two methods were intended for use with fairly complex multivitamin-mineral mixtures, they were not applicable to a large proportion of more complex preparations commonly available. In this laboratory, both of these methods yielded poor recovery of d-panthenol in a number of complex multivitamin preparations, especially those which were highly colored and contained a high concentration of biological extracts. The purpose of the present work IS to describe a solvent extraction procedure for the purification of panthenol sample solutions prior to chromatography, and a modification of the 1,2-naphthoquinone-4-sulfonate reaction for the quantitative determination of &panthenol in various types of pharmaceutical multivitamin products. EXPERIMENTAL
Solvent Extraction. The preliminary purification of d-panthenol by solvent extraction consists of two steps. The panthenol solution is saturated with ammonium sulfate and then extracted into benzyl alcohol by
its greater affinity for the latter, leaving most of the interfering substances in the aqueous phase. For final extraction, d-panthenol is forced into distilled water by the addition of toluene, which reduces the solubility of d-panthenol in benzyl alcohol. To ensure reproducibility of results, a standard is used concurrently with samples. Reagents. Dowex 50-X12 (H), 50 to 100 mesh. Add a volume of 2N sodium hydroxide approximately twice t h a t of Dowex, stir, and let stand I/, hour. Wash three times with liberal amounts of distilled water by decantation. Add a volume of 2N hydrochloric acid approximately equal t o t h a t of sodium hydroxide, stir, and let stand for the =me length of time. Wash by decantation with distilled water until the washing is neutral to litmus. Preserve the resin under distilled water. Amberlite IRA-400(OH), 20 to 50 mesh. Follow the procedure for Dowex but reverse the order of alkali and acid washing. Borate Buffer. Dissolve 75 grams of sodium hydroxide in 500 to 600 ml. of distilled water, and add 150 grams of sodium borate (NazB~0,.10H20). Make up to 1 liter. Naphthoquinone Reagent. Oissolve 250 mg. of 1,2-naphthoquinone-4-sulfonate sodium salt in distilled water, and make up to 50 ml. Prepare fresh before use. Formaldehyde Reagent. Mix, by volume, 3 parts of 3N hydrochloric acid, 2 parts of glacial acetic mid, and 4 parts of 0.3M formaldehyde. Panthenol Standard. Weigh accurately 75 mg. of pure d-panthenol (Hoffmann-LaRoche), dissolve in dis-
tilled water, and make up to 250 ml. Prepare fresh daily. Apparatus. Chromatographic glass tubes, approximately 10 mm. in diameter and 30 cm. in length, fitted with a stopcock. Procedure. Plug the bottom of the chromatographic tube with a small pledget of glass wool, and add the washed Amhcrlite to a height of 7 cm. Cover with a second plcdget of glass wool, and add the washed Dowex t o a height of 7 cm. (Separate columns of the resins may be prepared and used one above the other in the same order as that in the single column. Such columns may be separately regenerated after use by the procedure used in the preparation of the adsorbants.) Pipet 10 ml. of aqueous extract of a sample containing 2 to 4 mg. of dpanthenol into a 40-ml. glass-stoppered centrifuge tube containing 7 grams of ammonium sulfate. Shake for 5 minutes. Add 20 ml. of benzyl alcohol, stopper, and shake on a mechanical shaker for 15 minutes. Centrifuge to break thc emulsion. Pipet a IO-mi. aliquot of the upper benzyl alcohol layer into another centrifuge tube containing 10 ml. of toluene, and add 15 ml. of distilled water. Shakc for 15 minutes and centrifuge. Pipet 10 ml. of the lower aqueous layer onto a previously prepared column. Allow the solution to pass rapidly through the column, and collect the effluent in E 25-ml. volumetric flask. Wash the column three times with 5 ml. of distilled water or until the total volume of the effluent and washings is 25 mi. Transfer half 112.5 ml.) of the solution to a 25-ml. volumetric flask, A , leaving the other half in the same flask, B. T o each add 1 ml. of 2N hydrochloric acid and autoclave both flasks a t 16 pounds for 30 minutes. Cool to room temperature, and to flask A add 1 ml. of borate buffer and 1 ml. of naphthoquinone reagent, and to flask B add 1 ml. of borate buffer. Prepare a third 25-ml. volumetric flask, C, for a reagent blank by adding 12.5 ml. of distilled water, 1 ml. of 2N hydrochloric acid, 1 ml. of borate buffer, and 1 ml. of naphthoquinone reagent. Steam all three ilasks in an autoclave a t 100" C. for 10 minutes. Cool to room temperature, and to each add 0.5 ml. of formaldehyde reagent and 0.5 ml. of 0.2N sodium thiosulfate solution. Let stand for 10 to 30 minutes, and make each solution up to lolume with distilled water. Read the absorbance a t 465 mp in a spectrophotometer, setting the instrument at zero with distilled water. The nct absorbance of the sample is that which has been corrected for sample blank and reagent blank. For each series of determinations treat a standard solution together with its blank and reagent blank in the same manner as the sample solution. RESULTS AND DISCUSSIONS
The reaction of d-panthenol after solvent extraction was conducted ac-
Table
I. Per Cent Recovery of d-Panthenol Added to Complex Multivitamin Preparations
Chemical Method NaphthoSolvent quinone Ninh drin extraction method' mettod'
Sample0 B vitamins, ferrous gluconate, liver fraction B vitamins, C, D, liver extract, ferrous sulfate B vitamins B vitamins, yeast, and rice bean extracts B vitamins, A, C, D B vitamins, A, C, D, bone meal, ferrous sulfate, minerals
B vitamins, ferrous and copper sulfates, liver, and yeast concentrates B vitamins, C, D, ferrous and copper sulfates, liver fraction B vitamins, A, C, D, ferrous sulfate bone meal, minerals
B vitamins, C, gelatin, ferrous sulfate, desiccated liver, stomach substance
104 100 96 97 101 105 95 103 104 97
108
e
129
250
96
110
e
e
105
120
99 99
78
87
97 98
119
162
100 100
68
104
98 97
109
103
102 105 100
82
e
Mean recovery 99 134 a SamFles of coated tableta or capsulea with corn osition given by label declaration; first 5 contain calcium panthothenate. &Panthenor added to aqueous extracta in proportion of 3.00 m per tablet or capsule. b Method of &%mall and Woolish (8). Final solution too highly colored to be aaaayed. Table II.
Sample Injectable
Determination of Panthenol in Multivitamin Preparations
Label Claim, Mg. per Tablet or M1. 10.0
Injectable
5.0
Liquid drops Coated tablet
8.3 2.0
cording to the procedure given by Schmall and Wollish (67, with three To accommodate a modifications. larger aliquot of the test solution, 0.6 ml. each of formaldehyde rmgent and thiosulfate reagent of double strength was used. Neutralization by titration of the test solution after hydrolysis waa eliminated by the addition of borate buffer to bring the p H of the solution within the range of 8.8 and 9.5, which was suitable for the naphthoquinone reaction. Addition of ethyl alcohol to the solution to make up to volume after the color development was unnecessary; distilled water was used instead. With these modifications the
Assayed Value, Mg. per Tablet or M1. Rogers and Bird and Solvent Campbell McCready extraction method (6) method ( 1 ) 11.6 12.2 11.8 4.66 6.16 5.32 4.66 8.18 1.58 1.03
11.2 11.3 13.1 5.1 6.3 5.0 6.3 7.9 1.6 2.0
12.6 12.3 11.4 5.4 6.8 6.1
6.6
8.1 1.3 1.8
effective range of concentration of standard d-panthenol solution wm determined. A linear absorbance-concentration relationship was obtained between 0.01 and 0.05 mg. per ml., approximately similar to the range recommended by Schmall and Wollish. Since the actual amounts of d-panthenol extracted by the solvent separation were about 90%, it was necessary to use a standard with each series of unknowns. The complete method was applied to aliquots of a d-panthenol solution assayed simultaneously and on different days. Reproducibility of the resulte waa good, as indicated by a coefficient of variation of 12 replicates in one assay VOL. 33, NO. 8, JULY 1961
1039
of 0.87%, and for 18 assays conducted on differcnt days, of 2.74%, The accuracy and precision of the modified method were determined by adding known amotints of d-panthrnol to aqueous extracts of samples of two pharmaceutical product!s containing dpanthenol, a liqriid and a capsule prcparation. The mean recovery f standard deviation based on 10 determinations for thc former was 99.9 =t 2.3%, and for the latter was 100.9 f 4.6%. The method was further evaluated by adding d-panthenol to aqueous extracts of each of 10 complex multivitamin samples containing no d-panthenol in the proportion of 3.00 mg. of d-panthcnol per tablet or capsule. Five of the products contained calcium pantothenate. The results of duplicate analyses are given in Table 1 together with values obtained by the two methods of Schmall and Wollish (6). Ap-
proximately 100% recovery for all samples was obtained by the propowd method. Recoveries by the other two methods were markedly poorer in most samples, and, in some instances, the final solutions were so highly colored that they could not be assayed colorimetrically. The ninhydrin reaction appeared to be influenced to a greater extent than the naphthoquinone reagent by highly colored extracts. Another serirs of 10 samples, which had previously been assaycd by Rogers and Campbell in this laboratory using both their microbiological method (6) and that of Bird and McCready (I), gave comparable results by the proposed method, as shown in Table 11. The proposed preliminary solvent extraction togcther with the modified naphthoquinonc procedure is accurate, simplc, and applicable to a wide variety of complex multivitamin preparations.
Most interfering substances present in these preparations are eliminated by ammonium sulfate precipitation or solvent extraction. Although this method requires preliminary extraction, it is suita,ble for routine work. LITERATURE CITED
(1) Bird, 0. D.,McCready, L., ANAL. CHEM.30,2045 (1958). (2) De Ritter, E., Rubin, S. H.,Ibid., 21, 823 (1949). (3) Folk. 0..J . Biol. Chcm. 51. 377 (4) Frame, E.G.,Russell, J. A,, Wilhelmi, A. E.,Ibid. 149, 255 (1943). (5) Campbell, J. A., ANAL. . . nouers, 6. G., CHEM.32, i662’(196oj. (6) Schmall, M.,Wollish, E. G., Ibid., 29, 1,509 _ _ . (1967). . - , (7) Weiss, M. S., Sonnerfeld, J., De Ritter, E., Rubin, S. H.,Ibid., 23, 1687 (1951). \ -
RECEIVED for review January 6, 1961. Accepted April 21, 1961.
Quantitative Analysis by an Automatic Potentiometric Reaction Rate Method Specific Enzymatic Determination of Glucose H. V. MALMSTADT and H. L. PARDUE Departmenf of Chemisfry and Chemical Engineering, University of Illinois, Urbana, 111.
b An automatic potentiometric reaction rate method i s described and shown to b e sensitive, simple, rapid, and accurate for quantitative determinations and directly applicable for the selective determination o f glucose. Changes in electromotive force (e.m.f,) of a concentration cell are followed automatically b y a measurement system, and a value related to concentration of sought-for constituent i s read off a dial. A beaker can serve as the reference compartment o f the cell, and a short test tube immersed in the beaker as the sample compartment. The test tube has a small fiber sealed in the bottom to provide electrical contact between sample and reference solutions, but does not allow mixing. Platinum electrodes sensitive to iodine concentration, one in each compartment, complete the cell for the glucose determination. Glucose is oxidized selectively in the presence of a specific enzyme to produce hydrogen peroxide a t a rate proportional to the glucose concentration. As the hydrogen peroxide forms it reacts immediately with iodide in the presence of a molybdenum catalyst to form iodine, and the change o f iodine concentra1046
ANALYTICAL CHEMISTRY
tion in the sample compartment changes the cell e m f . Commercial equipment and an auxiliary relay system are combined easily to provide automatic results within 1 minute from the start o f the reaction. Glucose concentrations between 5 and 500 p.p.m. in a total volume o f 2 ml. were determined with relative errors of about 1% throughout the whole ran,ge. For samples between 5 and 50 p.p.m., only lo-’ gram of glucose react during the measurement interval, so that the precision in terms of the amount gram. reacted i s about
T
of precision nullpoint potentiometry (PNPP) has demonstrated the uscfulness of precise potentiometric measurements for quantitative determinations, especially in the micro range (6,6). With the PNPP method, it has been possible in many cases to determine constituents a t the part per million level with reproducibility and accuracy of 0.1%. Its high sensitivity and precision led the authors to consider the use of precision potentiometric measurements for following the rate of change of a reactant or product HE DEVELOPMENT
related to the concentration of a soughtfor constituent. Recent work by the authors (6) demonstrated the application of PNPP for iodometric procedures, including its application for the determination of micro amounts of hydrogen peroxide. The work of Malmstadt and Hicks ( 4 ) with B spectrophotomctric reaction-rate procedure showed that within a few seconds after the start of the reaction, the rate of change of hydrogen peroxide from the specific oxidation of glucose was proportional to the glucose concentration, a t least over a fewfold range. I t seemed feasible, therefore, to devise a procedure where the hydrogen peroxide formed in the glucose reaction would combine immediately with iodide to form iodine, which could be determined continuously by a potentiometric technique. It was originally thought that a concentration null point could be maintained continuously during a reaction. For the glucose determination it was plannrd to generate iodine electrolytically in the reference compartment at a rate equal to the rate of formation of iodine by the reaction of glucose in the sample compartment. However, this
