MicrobioIog ica I Assay f o r Pa nt heno I in Pharmaceutica I Vita min Products e
0.D.
BIRD and LAUREL McCREADY
Research Division, Parke, Davis &
Co., Detroit, Mich.
b A microbiological assay for panthenol in multivitamin preparations is based on the inhibition of leuconostoc mesenteroides P-60 under standard conditions, and is applicable directly to most types of pharmaceutical vitamin products without prior treatment of samples. Hydrolytic products of panthenol do not interfere and interfering pantothenic acid, which may b e introduced by extracts of natural materials or other sources, can b e removed by a simple resin treatment. The assay has the precision expected of microbiological methods.
A
used chemical method which has been proposed for assaying panthenol, a,y-dihydroxy-5(3 - hydroxypropyl) - @,p - dimethylbutyramide. in pharniareutical vitamin products (4) has not proved satisfactory for all types of samples, especially those containing sugar in the vehicle. The procedure suggested for eliminating the effects of sugar is cunibersonie and often inadequate because the assay lacks sensitivity. The microbiological assay most widely used ( 3 ) is more sensitive, but the growth of Acetobacter suboxydans is hard to control, and all samples have to be hydrolyzed, and some put through purification steps before away. The microbiological assay described here requires no prior treatment of samples and utilizes an easily controlled organisni frequently used in microbiological assays. This assay, which employs Leuconostoc wiesenteroides P-60, is based on the observation by Snell and Shive ( 2 ) that panthenol inhibits the growth of several lactic acid-producing bacteria. The method is applicable to all the types of pharmaceutical vitamin products on which it has been tried. Small amounts of interfering pantothenic acid introduced by such constituents as liver extract or other sources can be removed easily. WIDELY
daily in the same medium to provide inoculum for assays. Inoculum is prepared by transferring the growth from the daily stab culture to 10 ml. of medium which is made b y diluting the assay medium (Table I) 1 t o 1 with water and adjusting the pantothenic acid content t o 0.01 y per ml. This inoculum is incubated 16 hours a t 37" C. The cells are then centrifuged, washed twice with sterile 0.9% saline, and finally diluted with saline to give a transmittance reading of SO in a n Evelyn colorimeter. One drop of this suspension is added to each assay tube. ASSAY MEDIUM AND PROCEDURE
The assay medium (Table I) is a modification of that used by Craig and Snell (1) for the growth of a variety of lactobacilli, but is given in detail because of several critical features. It contains just enough pantothenic acid to produce maximum growth of L. mesenteroides under the conditions described. If maximum growth does not occur, the calcium D-pantothenate content of the medium should be adjusted u p or down accordingly. Standard and samples are diluted to contain 1 y of D-panthenol per ml. (estimated in the case of the samples). Standard tubes are set up in triplicate to contain 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, and 3.4 ml. per tube. Sample tubes are also set up in triplicate to contain 1.8, 2.1, 2.4, 2.7, and 3.0 ml. per tube. Five milliliters of the assay medium is added to each standard and sample tube and to five additional tubes which serve as blanks. (Four of these extra tubes are inoculated and serve as growth blanks. One is not
Figure 1. Response of leuconostoc mesenferoides P-60to D-wantheno1
inoculated and serves as a medium blank.) Water is added t o all tubes t o give a final volume of 10 ml. and they are plugged loosely with cotton. The Table 1.
Double-Strength Assay Medium
Mg.
20 m-Tryptophan 20 L-Cystine 20 L-Cysteine 20 L-Asparagine 2 Adenine 2 Guanine 12 Uracil 5 ml. Difco Casamino acids solutionn 250 Potassium chloride 250 Sodium phosphate, dibasic Sodium acetate, anhydrous 2 g. Glucose 2 g* Pyridoxal HC1 80 Y Thiamine HC1 80 Y 160 y Riboflavin 160 y Ncotinamide p-Aminobenzoic acid 80 Y Pteroylglutamic acid 6 r Biotin 17, Pantothenic acid (as calcium 1 pantothenate) 2 ml. Tween solutionb 2 ml. Salts Bc Make up to 80 nil., adjust to pH 6.0 (glass electrode), and dilute to 100 ml. Dissolve 1 gram Difco Casamino acids in 8 ml. of water, adjust to pH 3.5 with concentrated HC1, add water to 10 ml., add 100 mg. of Darco G-60, stir 15 minutes, and filter. Repeat Darco treatment. b Dissolve 2 grams of Tween 40 and 20 mg. of oleic acid in water and make to volume of 20 ml. c Dissolve 10 grams of hlgSOa 7Hs0; 0.5 gram of SaC1; 0.5 gram of FeSOl 7&0; 0.5 gram of bInSOl 4&0 in water, add 2 drops concentrated HC1, and make to volume of 250 ml. 0
50 -
ASSAY ORGANISM
Two stab cultures of Leuconostoc mesenteroides, ATCC No. 8042, are made in Difco micro-assay medium each month. Both cultures are incubated 24 hours at 37" C.; one is refrigerated undisturbed, while the other is transferred VOL. 30, NO. 12, DECEMBER 1958
0
2045
tubes are placed in a n autoclave with the jacket preheated, and the inside temperature is rapidlyraised to 116-18.5' C., and held within that range for 7 . 5 minutes. The steam is exhausted rapidly, and the tubes are removed and immediately cooled to room temperature by immersion in cold water. They are then inoculated (except for the medium blank) and incubated 16 hours a t 37" C. in a constant temperature incubator. The amount of cell growth in each tube is determined by making turbidity readings in the colorimeter with a 620-mp filter. A curve (such as shown in Figure 1) is constructed n-hich relates the degree of growth inhibition in each tube of standard to the amount of panthenol contained therein. Readings for the sample tubes are calculated from this standard curve for each sample, averaged, and those varying by more than i10% from the mean are discarded, and a new mean determined by averaging the remainder, provided two thirds still remain. PRECISION OF THE ASSAY
This procedure exhibited the typical precision found with most microbiological assays. Table I1 shows the result obtained in six consecutive assays of a liquid multivitamin preparation. DISCUSSION
Many other modifications were investigated while developing this assay medium. A medium containing no Tween or oleic acid required about four times as much pantothenic acid to give maximum growth. A correspondingly larger amount of panthenol mas required to produce inhibition of growth; consequently the response to panthenol
Table II.
Date
of Assay
Precision of Microbiological Panthenol Assay
Panthenol/Ml. Found
Mg.
Dev. from Mean
18.8 19.0
+0.3 +0.5 -0.7
9/5_ 9/1 i
9/18 9/25 9/30 10/8 Mean Std. dev. ( 8 )
17.8 19.0 18 5 18.0 18.5
z ( x--
$0.5 0
-0.5 0.52
X is individual value X is mean value n is number of values
n a s much less and the assay wa5 less sensitive. Substituting amino acids for acid-hydrolyzed casein gave no advantage. K h e n the panthenol samples were added aseptically to the tubes after the medium had been sterilized, there was greater variation in results. The assay procedure was developed primarily for pharmaceutical preparations containing mixtures of pure vitamins in various non-vitamin-containing menstruums. The presence of extraneous substances, including those containing pantothenic acid, was also investigated. Products resulting from the acid hydrolysis of panthenol gave no response a t a level of 100 y per tube, which is a t least 50 times the concentration of panthenol required to give easily detectable inhibition. Samples containing reasonable amounts of natural supplementary products, such as liver extracts, and which contribute pantothenic acid can be assayed for panthenol
by this niethod, if special precautions are taken. A typical procedure for such products is the following which was used in assaying a dry mixture containing powdered liver extract: The sample was diluted in the customary manner so as to be ready to add to the assay tubes. TKO and a half grams of dmberlite resin JIB-1 (Rohni &- Haas Co.) was added to this diluted sample which contained an estimated 100 y of panthenol and 0.5 y of pantothenic acid in each 100 ml. This mixture was shaken on a shaking machine for half an hour. The resin was allowed to settle and the supernate pipetted into the assay tubes as usual. Further tests were carried out in nhich known mixtures of panthenol and pantothenate were given this resin treatment before being assayed. The resin treatment was successful in overcoming the interfering effect of pantothenate even when the amount of pantothenate present n-as equal to the amount of panthenol. ACKNOWLEDGMENT
The authors are indebted to Barbara Hall and Barbara Peterson for assistance and suggestions in the early part of the work reported here. LITERATURE CITED
(1) Craig, J. A . , Snell, E. E., J . Bacteriol. 61, 283 (1951). (2) Snell, E. E., Shive, W., J . Biol. Chem. 158, 551 (1945).
(3) Weiss, >I. S., Sonnenfeld, I., De Ritter, E., Rubin, S. H., ASAL. CHEW 23,1687 (1951). (4)Wollieh. E. G., Schmall. 11.. Zbid., 22, 1033 (1950). ' RECEIVEDfor review March 13, 1958. iicceptecl J U ~30, Y 1958. ~
Semimicrodetermination of Tantalum with Selenous Acid F. S. GRlMALDl and MARIAN M. SCHNEPFE U. S. Geological Survey, Washington 25, D. C.
b Tantalum is separated and determined gravimetrically by precipitation with selenous acid from a highly acidic solution containing oxalic and tartaric acids. The method is selective for the determination of up to 30 mg. of tantalum pentoxide, and tolerates relatively large amounts of scandium, yttrium, cerium, titanium, zirconium, thorium, vanadium, niobium, molybdenum, tungsten, uranium, iron, aluminum, gallium, tin, lead, antimony, and bismuth. The separation of tantalum from niobium and titanium is not 2046
ANALYTICAL CHEMISTRY
strictly quantitative, and correction is made colorimetrically for the small amounts of niobium and titanium coprecipitating with the tantalum. The method was applied to the determination of tantalum in tantaloniobate ores.
T
interest and activity in the analytical chemistry of the earth acids are shown by the number of papers published recently. Xotable advances in the isolation and separation of the earth acids have been made by ion exchange, partition chromatography, HE INCREASED
extraction. volatilization, and precipitation methods. The literature cited by Belekar and ilthavale (4) includes many of the pertinent references. Recent clevelopnients in precipitation methods include the separation of tantalum from niobium by precipitation of tantalum from a n oxalate medium with sodium hypophosphite ( I ) ; the separation of niobium and tantalum from titanium by Precipitation of the earth acids with selenous acid from 3.V hydrochloric acid solution in the presence of small amounts of tartaric acid
