Determination of Panthenol in Pharmaceutical Preparations by Microbiological Assay with Escherichia coli 99-4 C. G. ROGERS and J. A. CAMPBELL Food and Drug laborafories, Depurfmenf of Nafional Health and Welfare, Oftawa, Canada
b A microbiological assay for the determination of panthenol in pharmaceutical preparations is described. The method depends upon the growth response of Escherichia coli 99-4 to pantoic acid, which is produced by alkaline hydrolysis of panthenol. In tests with a variety of pharmaceutical preparations the assay compared favorably in accuracy and precision with the inhibition method using Leuconostoc mesenferoides P-60. Both methods were preferable to the Acefobacter soboxydans assay in ease of handling and stability of response. The E. coli method, which was sensitive to as little as 0.5 pg. of pantoic acid per mi., offers a new approach to the assay of panthenol.
R
are available for the assay of many of the vitamins, but a simple and dependable method for the determination of panthenol 12,4dihydroxy - N - (3 - hydroxypropyl) - 3,3dimethylbutyramide] has not been developed. Several chemical methods have been proposed (8, 9, IS), but none have been widely accepted for the routine assay of products that contain many other vitamins, minerals, and biological extracts. The assay of De Ritter et al. (4) and of Weiss et al. (11)with Acetobacter suboxydam is probably the most widely used method for the determination of panthenol. The alcohol is not measured directly, however, but must be hydrolyzed to pantoic acid (ZJ4-dihydroxy3,3-dimethylbutyric acid), the substance to which the test organism responds. Furthermore, the procedure is laborious and growth of the test organism is difficult to control (1). More recently, a method has been described that is based on the inhibitory effect of panthenol for Leucorwstoc mesentera'des P-60 (1). This method cannot be considered specific for the vitamin because any factor toxic tc the organism would be assayed as panthenol. The present method is similar to the Acetobacter assay which also measures pantoic acid. Escherichia coli 99-4 (2, 6, 7) does not require highly aerobic conditions for growth, is easy to maintain, and yields a linear growth response ELIABLE METHODS
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ANALYTICAL CHEMISTRY
curve to pantoic acid. Pantothenic acid can replace pantoic acid for growth of E. coli 99-4; hence, trace amounts of pantothenic acid, present in some multivitamin preparations, must be removed prior to assay. This paper describes the application of E . coli 99-4 to the assay of panthenol in a variety of multivitamin preparations and presents results of a comparison of thk method with the L. mesenteroides assay. EXPERIMENTAL
Culture Maintenance. Maintain stock cultures of E. coli 99-4 by transfer every 2 weeks on agar slopes of the following composition (6): Ditco yeast extract 0.2'%, enzymatic casein hydrolyzate (Nutritional Biochemicals Corp.) 0.295, agar 1.5%. Incubate cultures 7 hours a t 33' + 0.5' C., and store in .a refrigerator until used. Preparation of Inoculum. Prepare the inoculum broth as directed in Table I with t h e omission of vitamin solutions 1 and 2, adenine, guanine, uracil, xanthine, and vitamin BIZ, but
Table 1. Double-Strength Basal Medium for Pantoic Acid Assay
Potassium phosphate, di1 . 4 grams basic Potassium phosphate, 0.6 gram monobasic 100 mg. Sodium citrate, 3H10 Magnesium sulfate, 7H20 20 mg. Ammonium sulfate 200 mg. Glucose 400 mg. Casein hydrolyzate, enzymatico 10 ml. Vitamin solution I b 4 ml. Vitamin solution 2c 1 ml. Vitamin Blz (10 mpg./ml.) 1 ml. Adenine, guanine, uracil solutionb 2 ml. Xanthine solutionb 2 ml. Dilute to 90 ml. with glass-distilled water; adjust pH to 6.4 and make up t o volume of 100 ml. 5% Solution, Nutritional Biochemicals 0
Corp.
b Prepared according to U.S.P. vitamin Blr assay (10). Dissolve 40 mg. of p-aminobenzoic acid, 40 mg. of pyridoxal.HC1. 40 mg. of pyridoxine, HCI, 16 mg. of - yridoxamhe. 2HC1, and 4 mg. of pteroyf)glutamic acid, in 200 mi. of 25% ethyl alcohol and store in a refrigerator at 6" to 8" C.
with the addition of 20 pg. of 'calcium D-pantothenate per ml. Inoculate 10 ml. of sterile broth by transfer from an agar slope culture which is less than 40 hours old. Incubate the broth culture for 7 to 8 hours a t 33' f 0.5' C. Harvest the cells by centrifugation, wash once in sterile 0.85% saline solution, and resuspend in saline to give a turbidity of 40% transmittance of light in a Coleman Model 11 spectrophotometer with a 540-mp filter (or 70% transmittance in an Evelyn colorimeter). Add one drop of this suspension of each tube. Basal Medium. Prepare the assay medium as shown in Table I. This is a modification of the medium of Davis and Mingioli (3). Standard Solutions. Prepare a stock solution of D-pantheno1 by dissolving 69.2 mg. of pure D-pant,otheriyl alcohol (Hoffmann-La Roche Inc.) in 100 ml. of distilled water (1 ml. contains the equivalent of 500 mg. of pantoic acid). The panthenol is converted to pantoic acid by the method of De Ritter and Rubin (4). Autoclave 1 ml. of a I-to-10 dilution of the panthenol stock solution with 3 ml. of 0.1N sodium hydroxide solution for 30 minutes a t 15 pounds' pressure (121' C.). Cool, add 50 ml. of distilled water, adjust the pH to 6.4, and dilute to 100 ml. (equivalent t o 0.5 pg. 'of pantoic acid per ml.). Preparation of Samples. Prepare the sample by aqueous extraction and dilute the extracts to contain an estimated concentration of 6 to 10 p g . of pantoic acid per ml. If pantothenic acid is present in significant amounts, as in samples containing liver RXtracts, treat a measured amount of the sample extract with Amberlite M B l ion exchange resin (1) before alkaline hydrolysis; then filter the supernatant solution through glass wool to remove fine resin particles from the solutioii. I n a measured aliquot of the filtrate, equivalent to 50 t o 100 pg. of pantoic acid, convert the panthenol to pantoic acid by the method described for the standard solution. Cool the solution, adjust the pH to 6.4, and dilute t o contain an estimated 0.5 pug. of pantoic acid per mi. Assay Procedure. Prepare a series of tubes in quadruplicate t o ccrrtain 0, 1. 3 , 3, 4, and 5 ml. of the standard pantoic acid. Set u p a series of tubes in triplicate t o contain 1, 2, 3, and 4
0
0.5
1.0
M I C R O G R A M S OF
I 5
2.0
PANTOIC A C I D
Figure 1. Growth response of Escherichia coli 99-4 to pantoic acid
ml. of the sample solution. Adjust the volume in all tubes to 5 ml. with water and add 5 ml. of water to each of two tubes to serve as medium blanks which are not inoculated. To each tube add 5 ml. of the pantoic acid assay medium. Cover the tubes and autoclave them for 3 minutes at 15 pounds' pressure (121' C.) in a preheated autoclave. Cool the tubes rapidly, inoculate all tubes except the medium blanks, and incubate for 16 hours a t 33' f 0.5' C. Measure the growth turbidimetrically in a spectrophotometer a t 540 mp. Plot turbidimetric readings for the standard, expressed as absorbance, against concentrations of pantoic acid. Determine the amount of pantoic acid per milliliter of sample by interpolstion from the standard curve. Inhibition Method with L. mesenteroides P-60. Several modifications were made in tests employing the inhibition method of Bird and MCCready (I). These included: the use of Tween 80 (polyoxyethylene sorbitan monooleate, Atlas Powder Co.) in place of Tween 40 and oleic acid (18), and the use of a vitamin-free acid hydrolyzate of casein (Nutritional Biochemicals Corp.) to eliminate charcoal treatment of the casein. Four dosage levels were used for both standard and samples with four replicates at each level of the standard and three replicates a t each level of the samples. Sufficient acid was produced by L. mesenh-oides after 20 hours of incubation to permit the titrimetric assay of samples in which the background color interfered with turbidimetric readings. RESULTS AND DISCUSSION
Standard Curves. A typical growth curve obtained with E . coli 99-4 in response to pantoic acid is shown in Figure 1. The culture exhibited satisfactory sensitivity in the range 0.5 t o 2.5 pg. pantoic acid per tube. A standard curve for the inhibition of growth of L. nesenteroides P-60 is shown in
Figure 2. In the inhibition asjay growth was inversely proportional to panthenol concentration over the range from 0 to 4 pg. of panthenol per tube. With L. nzesenteroides a h e a r relationship waa obtained between absorbance and log dose of panthenol. This waa found to be a preferable way of plotting the standard curve because it permitted the statistical evaluation of potency and precision where necesssry. In repeated tests the growth response of E . coZi to pantoic acid was more reproducible than the inhibition response of L . mesenterto panthenol. The sensitivity of the E. coli method was approximately twice that of the inhibition assay. Recovery Experiments. The E . coli method was compared with the inhibition assay in recovery experiments on a series of 5 multivitamin preparations. Panthenol was added to each
Table II.
70
t
M I C R O G R A M S OF d - P A N T H E N O L
Figure 2. Inhibition of growth of leuconostoc mesenteroides P-60 by panthenol
Recovery of Added Panthenol from Multivitamin Preparations
Sample Composition 1, Coated tablet Live! fraction, minerals, vita-
Daily Doseb 3 tab
3, Capsule
1 tab 6 caps
m s 2, Coated tablet Minerals, vitamins
Liver extract, minerals, vitam s
Mean Recovery", yo f S.E. L. m e n E. Eoli tetoides 99-1
105.4f 1.6
P-60 99.6 f 1.8
98.8 f 1.7 103.8 f 1.2 112.4 f 4.8 95.4 f 2.3
4,Elixir Methionine, choline, vitamins 30 cc. 105.6 f 0.7 107.2 f 2.4 5, Liquid Malt, minerals, vitamins 30 cc. 109.0 f 1.6 89.6 f 1.4 Mean of 5 determinations on separate days. b Standard solution of spantheno1 added to each sample to give the equivalent of 5 mg. per daily dose. 0
of 5 samples at the rate of 5 mg. per recommended daily dose of the product and the mixturea were assayed on 5 separate days. The results, summarized in Table 11, indicate that the two methods compared favorably both in reproducibility and accuracy. It is possible that traces of pantothenic acid produced some of the high values obtained with E . coli and the comparatively lower values obtained with L mesenh-oides as shown for Samples 3 and 5. Potency Estimation. Estimates of potency obtained with the E . coli 99-4 assay and with the inhibition method for a series of 11 multivitamin samples, claimed by label t o contain panthenol, are given in Table 111. Dilutions of a single extraction were used for each method. With most samples, values obtained by the E . coli assay agreed closely with those obtained by the inhibition method. The use of a heavy inoculum increased the reproducibility of results in the E coli assay. In preliminary experiments a sigmoid-shaped recponse curve waa obtained with E. coli in the medium of Davis and Mingioli (3).
Table 111. Panthenol Content of Multivitamin Preparations Determined by Two Methods
Sample Injectable 1 2 3 4 5 6 7 8
Assay Value,a Label Mg. per Tab. or Claim, M1. Mg. per E. L. mesenTab. or coli teroides M1. 99-4 P-60
10.0 10.0 10.0 5.0 5.0 10.0 5.0 5.0
11.2 11.3 13.1 5.1 6.3 2.0 5.0 5.3
12.5 12.3 11.4 5.4 6.3 2.0 6.1 5.6
8.3
7.9
8.1
2.0 2.0
1.5 2.0
1.3 1.8
Liquid drops 9
Coated tabletb 10 11
0 All values shown are single determinations. b Contained liver concentrate; purified with Amberlite MBl resin ( 1 ) .
VOL. 32, NO 12, NOVEMBER 1960
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Supplementation of the medium with casein hydrolyzate, vitamins, purines, and pyrimidines eliminated this effect. Hydrolysis with 3 ml. of 0.1Nsodium hydroxide usually gave complete cleavage of panthenol to pantoic .a&d. However, incomplete hydrolysis may occur a t this concentration of alkali with some samples of low potency. Because of the large sample aliquot that would then be required, sufficient buffering capacity may be developed within the system to neutralize the alkali. With samples of low potency, this difficulty can be avoided by doubling the amount of 0.1N sodium hydroxide used for hydrolysis (6 ml. used in place of 3 ml.). I n tests with pharmaceutical preparations, the E. coli method compared favorably with the inhibition assay as to stability of response, precision, and reproducibility. Both methods were simpler to use and more suitable for
routine analysis than the Acetobacter assay. The E . coli 99-4 method was more sensitive than the inhibition assay and seemed particularly suitable for the routine estimation of panthenol in pharmaceutical preparations. ACKNOWLEDGMENT
The culture of Escherichia coli 99-4 used in this study was obtained through the kindness of Werner K. Maas, Bellevue Medical Center, New York University, New York. The authors acknowledge the technical assistance of Fernand J. Noel. LITERATURE CITED
(1) Bird, 0. D.,McCready, L., ANAL. CHEM.30, 2045 (1958). (2) Davis, B. D.,Proc. Natl. Acad. Sci. U . S. 35, 1 (1949). (3) Davis, B. D.,Mingioli, E. S., J. B a o terzol. 60, 17 (1950).
(4) De Ritter, E., Rubin, S. H., ANAL.
CHEM.21, 823 (1949). (5) Maas, W. K.,Bellevue Medical Cen-
ter, New York, personal communication. 1956. (6) Maas, W. K., Davis, B. D., J . Bacter-
iol. 60, 733 (1950). (7) Maas, W.K.,Vogel, H. J., Zbid., 65, 388 (1953). (8) Schmall; M., Wollisch, E. G., ANAL. CHEM.29, 1509 (1957). (9) Szalkowski, C: R., .Davidson, J. H., Jr., Ibid., 25, 1192 (1953). (10) United States Pharmacopeia, 15th Rev., Mack Publ. Co., Easton, Pa., 1956. (11) Weiss, M. S., Sonnenfeld, I., De Ritter, E., Rubin, S. H., ANAL.CHEM. 23, 1689 (1951). (12) Williams, W. L., Broquist, H. P., Snell, E. E., J . Biol. Chem. 170, 619 (1947). (13) Wollisch, E. G.,Schmall, M., ANAL. CHEM.22, 1033 (1950).
RECEIVED for review March 2, 1960. Accepted August 10, 1960.
Repeated Extractions A Method of Analyzing a Mixture of Labeled Co m pounds GOSTA RUDSTAM CERN, European Organization for Nuclear Research, Geneva, Switzerland
b A method of analyzing a solution containing a mixture of labeled compounds is presented. It can b e used when the compounds at hand are extractable with different partition coefficients into the same solvent (even after addition of complex-forming reagents). The method is of greatest value when the different compounds depend on each other and interchange at a rate which is not negligible in comparison with the time required for the analysis. By following a fixed time schedule during the experiment it i s often possible to correct accurately for interchange between the constituents of the solution during the analysis, and to determine their concentrations a t the beginning of the experiment.
A
COMMON analytical problem in radiochemistry is to analyze a solution containing a mixture of compounds labeled with radioactive tracers. If the different compounds are labeled with different tracer atoms, the radiation characteristics of these may be sufficiently different to allow a direct determination by differential countingfor instance, 7-spectroscopy or absorption methods. However, when the game tracer atom is used to label several
1664
ANALYTICAL CHEMISTRY
compounds, a chemical separation must precede the activity measurement. The method of analysis proposed here is intended primarily for a solution containing a mixture of compounds labeled with the same tracer atom. The solution is extracted repeatedly with fresh portions of a suitable solvent into which the compounds are extractable with different partition coefficients, and the activities of the successive portions are measured. From an analysis of these activity data the composition of the solution can be determined. EXPERIMENTAL
Apparatus. The apparatus required is simple. For extractions with a heavier solvent a simple separation funnel is adequate. As many partition coefficients change rapidly with temperature, the funnel should be thermostated. Furthermore, i t is advisable to use some means for facilitating the addition of measured portions of the solvent. Figure 1, A , shows a suitable arrangement. If the solvent is lighter than the solution t o be extracted, an apparatus similar to the one of Figure 1, B, can be used. The upper phase is easily removed after the extraction. Procedure. Add a measured amount of the solvent to the solution
and stir for a certain time. When the phases have separated, the solvent is removed and a fresh portion is added. The whole procedure is repeated a suitable number of times. It is important to keep a fixed time schedule for the procedure if the constituents of the solution depend on each other. By doing so, it is possible t o correct for interchange between the components during the experiment. The activity of the successive portions of the solvent is measured with any suitable device, such as a scintillation counter. ANALYSIS OF EXTRACTION DATA
Independent Constituents. Consider a case where a liquid phase A containing i independent solutes of concentrations c i is extracted repeatedly with equal volumes (equal to the volume of phase A ) of another phase B. The coefficients for the partition of the solutes between the second phase and the first one are denoted by gi. I n order t o simplify the formulas, the symbols p i are introduced by the relation :
The nth portion of phase B will contain the ith component in concentration (ci)n:
