Improved Microbiological Assay for Panthenol mcII.mL
s. WEISS, I R M A SONZENFELD,ELMER DE RITTEH,~
s i i L, [I.
Y D
HVBIN
Hoffrnann-La Roche Znc., Sritley, S. J .
The increasing use of panthenol has emphasized the need for a more efficient assay method. Hence, the microbiological assay has been modified to eliminate the ether extraction for removal of pantoyl lactone or the separate assay required to correct for preformed pantoic acid. In the present procedure, these interfering products of hydrolytic cleavage of panthenol are separated from the latter by passing a buffered extract through an ion exchange resin
T
HE microbiological method of De Ritter and Rubin (1)
for assay of panthenol, n,ydihydroxy-Ar-(3-hydroxypropyl)13, ,?-dimethylbutyramide, ha3 been modified to eliminate the ether extraction for separation of pantoyl lactone and the assay before hydrolysis to correct for preformed pantoic acid. Both of these interfering substaiices are removed by pamage of the test extract through a column of ion exchange resin in a manner siniilar to that described by Kollish and S c h n d l (6) for the chemical assay of panthenol. Because the microbiological method is 200 times as sensitive as t,he colorimetric assay, more dilute extract,s may be used in the present procedure : i d there is IIO necessity for preventing breakthrough into the eluate at high flow rates of substances such as ascorbic acid that int,erfere with the color reaction but have no effect 011 the groxth of Acetobacter suboxudans. For hydrolysis of low potency products where the salt, formed 011neutralization may be sufficient to inhibit growth of the test sodium hydroxide is used. organism, 0.1 K rather t h a n 1 a1 medium has been modified to improve both the rate and reproducibility of growth i n the pantoic acid assay. Sonie practical details are given regarding the method of carrying cultures so a8 to maintain t,he semitivity of respoilye of the Acebbacter subozydans. Details of' procedure are givm only when they differ from thore 1)revioudy descrihed ( 1 . 6 ) .
column. To permit assajs at ver) low- wncentrations of panthenol, 0.1 S rather than 1 LV sodium hydroxide is used for hydrolysis to pantoic acid prior to determination with .Icetobacter suboxydans. The basal medium has been improved by adding ethyl alcohol, tripling the dextrose, and reducing the glycerol content. These changes lead to more consistent and rapid growth of the organism and ensure suitable assays in shaken tubes within 16 to 18 hours. the flow rate adjusted to about 18 ml. per minute. As the water level reaches the to of the column, a 50-ml. volumetric flask is placed under the corumn and the test aliquot is pipeted onto the column with the flow rate maintained a t 18 ml. per minute. As soon as no liquid remains above the column, 40 ml. of water are put through a t the same rate of flow. The combined drippings containing the panthenol are collect.ed in the 50-ml. flask and diluted to volume with water. Alkaline Hydrolysis. Both standard and test solutions are hydrolyzed with 0.1 N sodium hydroxide. Exact neutralization of unbuffered solutions such as standards and resin column eluates IS unnecessary. It is sufficient to add 4.5 nil. of 0.2 V . hydrochloric acid to the 10-ml. aliquot, thus allowing a slight excess of alkali. MICROBIOLOGlCAL A S S A Y PROCEDURE
Acetobacter suboxydans, American-Type Culture 62 1 H. The slant and inoculum media and the assay conditions have been described ( I ) . Inocula for shaken assays remain viable when stored for a t least 6 months in a deep freeze ( - 18 to -20' C.) prior to incubation. Suitable growth in the assay may be obtained directly from such a frozen inoculum after shaking 24 hours a t 30" C. and also from subsequent serial transfers. This procedure provides a method of keeping suitable inocula on hand without the necessity of frequent preparation of fresh culture media. Basal Medium. The assay medium ( 2 ) has been modified by raising the glucose level to 15 grams, reducing the glycerol level to 33 grams, and adding 0.2 ml.of ethyl alcohol per liter of doubltsstrength medium.
R E 4GE \ T S
4.8 N Citrate BulTer.
Dissolve 168 grams of citric acid ~H$26H~07 H20) in 200 ml. of water, stir, and cool while titrating with 50% sodium hydroxide to pH 5 and dilute to 500 nil. Dilute 1 to 40 to achieve the desiied concentration of 0.12 S in test solutlolls. TREATMEYT BEFORE MICROBIOLOGICAL A S S A Y
Extraction. Extract samples containing no pantoic acid, pantoyl lactone, or pantothenate in distilled water. Dilute liquid prcyarations to a potency of about 55 micrograms of d-panthenol per nil. Extract tablets or capsules for 5 minutes in a Waring Blcndor and dilute to the same potency. Centrifuge a portion of the extract for 5 minutes a t 2500 revolutions per minute (r.p.ni2 and decant the supernatant. These extracts may be hydrolyzed without preliminary treatment. If the sample contains significant, amounts of pantoic acid, pantoyl lactone, and/or pantothenic acid, it is prepared for resin treatment by extracting andlor diluting in 0.12 A' sodium citrate a t pH 5.0 to a panthenol concentration of 100 micrograms per nil. While this level has proved most convenient in practice, potencies as low as 12 micrograms per ml. may be used if necessary to obtain a suitable extraction ratio. Purification with Ion Exchange Resin. The column of hmberlite IR.4-400, analytical grade, is prepared and charged as previously described ( 6 ) ,but to emure maximum efficiency it is desirable to use fresh resin for earh sample rather than to recharge. If the resin is received as the modified amine base, the preliminary charge with sodium hydroside may he omitted. .L\ 5-mL aliquot of the previously prepared test solution is drawn up into a pipet, the stopcock on the resin tube opened, and
Table 1. Effect of Flow Rate and Buffer Concentration on Panthenol Recoveries in Resin Column Eluate Flow R a t e , Ml.iXIin. ~~~
Sormality of Acetate or Citrate Buffer 0 3 0.12 0 03
16-18
Range 97-106 97-106 89-100
8-10
% Recovery of Panthenol-
Av. 99 100 92
Range 92-97 89-93 83-88
9 -4
-
AY.
Range
Av.
94 92 86
82-Y1
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