Colorimetric Determination of Thiamine in Pharmaceutical Products F. J. BANDELIN AND J. \-.TUSCIIHOFF Research Laboratories, F l i n t , Eatora a n d Co., Decatur, 111.
The commonly used thiochrome method has certain disadvantages for the determination of thiamine in pharmaceutical products. The high concentrations of relatively pure thiamine usually encountered in these preparations make unnecessary the great dilutions and low-level determinations inherent in the thiochrome method. A more direct colorimetric method involves the precipitation of thiamine as the reineckate, solution of the thiamine reineckate in acetone, and colorimetric determination at 525 mp. Precipitation is carried out in 33970
methanol, which renders the method specific for thiamine in mixtures with other B complex vitamin. The method may be specific for the intact thiamine molecule, as the usual degradation products of thiamine do not yield insoluble reineckates under the conditions of the procedure. The method has the advantage of being rapid, simple, and applicable in a convenient range of concentration for thiamine in pharmaceutical products and without numerous dilutions of the sample. The accuracy and reproducibility of the method are within 25Zo.
T
HE generally used and official ( 5 )thiochrome method for the
determination of thiamine in pharmaceutical products has certain disadvantages. illthough it is the chemical method of choice for the determination of exiguous quantities of thiamine in biological tissues, it is someivhat cumbersome and time-consuming when employed for relatively high concentrations of pure, synthetic thiamine encountered in pharmaceutical products. It is not uncommon for tablets or capsules of various vitamin formulas to contain as muchaslomg. of thiamine in each, either alone or in combination with other vitamins or therapeutic substances, while certain injectable solutions may contain as much as 100 mg. of thiamine hydrochloride per ml. When thiamine is to be determined by the thiochrome method in such preparations, numerous serial dilutions are necessary to obtain a final working solution containing approximately 0.2 microgram per ml. required for the determination. Aside from the inconvenience of such dilutions, the possibility of error through manipulation cannot be overlooked. Furthermore, the oxidation of thiamine to thiochrome is not a quantitative reaction and although a certain mean reproducibility is attained through careful standardization of the method, alkalinity, amount of ferricyanide, time of reaction, extraction of the thiochrome with isobutyl alcohol, and exposure to ultraviolet light necessary to produce fluorescence are all variables which may affect the end result. A simpler and more direct analytical method for relatively high conceiitrations of thiamine might indeed be attractive to the pharniac*euticalcontrol chemist. Earlier work indicated that certain heterocyclic amines and quateriiary ammonium salts could be quantitatively precipitated from aqueous solutions as the insoluble reineckate by ammonium reineckate ( 2 - 4 ) . ilfter isolation and solution in acetone, the reincckate could be utilized for the colorimetric determination of the organic base. I t qeenied not unlikely that thiamine, being both B heterocyclic amine and a quaternary salt, might be determined in a similar manner in pharmaceutical products. Preliminary investigations, using pure, synthetic thiamine hyLirochloride, indicated that thiamine could, in fact, be quantitatively precipitated as the reineckate from aqueous solution. In the course of this work it was noted that certain other synthetic vitamins of the B complex-namely, niacin, niarinarnide, and pyridoiine-interfered under certain conditions by also protlucing insoluble reineckates. Fui ther investigation indicated that thiamine reineckate could be selectively precipitated in a buffered solution having a pH of i 5 Under conditions of the method developed, thiamine could lle Qeparated from mixtures of other
vitamins of the B complex and the determination made specific for thiamine. COYSTRUCTIOK OF STANDARD CURVE FOR THIAMIKE REINECKATE
Into each of five 20-mI. beakers containing 5 mI. of acetate buffer is pipetted 1, 2, 3, 4, or 5 ml. of standard thiamine hydrochloride solution, and the volume of each is adjusted to 10 ml. by the addition of distilled water. Thiamine reineckate is precipitated by the addition of ammonium reineckate solution as described above. A standard curve is constructed by plotting transmittance against concentration on semilog graph paper.
B number of determinations were made by this method, using thiamine hydrochloride at the 3-mg. level (Table I). Standard deviations
(S.D.
=
4;)
of the determinations in
Table I was found to be f 0 . 0 1 mg. REAGENTS
Ammonium reineckate reagent, 27, ammonium reineckate in anhydrous methanol. Standard solution of thiamine hydrochloride containing 1 mg. per ml. was prepared by dissolving 500 mg. of thiamine hydrochloride, U.S.P. reference standard (previously dried over anhydrous calcium sulfate in a vacuum desiccator), in sufficient 1% hydrochloric acid to make 500 m!. Acetate buffer, pH 4.5, was made by adding 114 ml. of analytical reagent grade 0.2 A-. Acetic acid to 86 ml. of 0.2 sodium acetate. K a s h solution, 2 ml. of ammonium reineckate reagent in sufficient distilled water to make 1 liter. .Icetone, analytical reagent. PROCEDURE
To a solution of from 2 to 5 mg. of thiamine hydrochloride in 10 ml. of acetate buffer, pH 4.5, in a 20-ml. beaker is added 5 ml.
1198
Table I. Recovery. 3.03 3.00 3 01 2.99 3.00 2.99 3.00 2 99
2.99 3 01)
Deviations in Determinations of Thiamine H: rlrochloride in 3-\Ig. Aliquots Ng. Recovery, i7c Recovery, M g . Recovery, % 0 0 3 7 100 0
