[14] Simultaneous determination of thiamine and pyrithiamine

[14]

THIAMINE AND PYRITHIAMINE DETERMINATION

81

The mean relative error of the method is 3.25% (see table). The ions of some heavy metals as well as amines interfere with the determination, but do not occur in usual mixtures with thiamine. DETERMINATION OF ERROR

Thiamine

Error

Taken (mg)

Found (mg)

Absolute (mg)

Relative (%)

0.200 0.400 0.600 0.800 1.000

0.196 0.427 0.623 0,807 0,972

-0.004 +0.027 +0.023 +0,007 -0,028

2.00 6.75 3.84 0.87 2.80

Conclusion The method for determination of thiamine by means of lithium picrolonate has a satisfactory accuracy and is very simple to carry out. As little as 0.1 mg of thiamine can be determined.

[14] Simultaneous

Determination

of Thiamine

and Pyrithiamine 1

By R. L. AIRTH and G. ELIZABETH FOERSTER Thiamine + K3Fe(CN)~ ---*"thiochrome" (hexclt = 385 m/z; ~emlt = 440 m/z) Pyrithiamine + K~Fe(CN)6 ---*"pyrichrome" (~,¢xclt= 410 m/z; ~emit = 4 8 0 m/z)

Assay Method Principle. A spcctrophotofluorometric assay of a mixture of thiamine and pyrithiamine based upon the difference in excitation and emission characteristics of their respective oxidation products, thiochrome and pyrichrome, is presented. Reagents and Apparatus Trichloroacctic acid, 1.53 M Potassium acetate, 4 M 1R. L. Airth and G. E. Foer~ler, Anal. Biochem. 3, 383 (1962).

82

THIAMINE: PHOSPHATES AND ANALOGS

[14]

NaOH, 7.5 M K3Fe(CN)6, 0.059 M; 0.65 ml of the 0.059 M solution is diluted to 10.0 ml with 7.5 M NaOH. These solutions are made within 1 hour of use. Hydrogen peroxide, 30% Isoamyl alcohol, redistilled over activated carbon (128-129 ° fraction) and stored saturated with distilled water Thiamine hydrochloride. A stock solution of 1 mg/ml in 0.1 N HC1 is prepared, and appropriate dilutions are made of this standard. Pyrithiamine hydrobromide. A stock solution of 1 mg/ml in distilled water is prepared, and appropriate dilutions are made of this standard. Quinine sulfate. A stock solution of 10.0 rag/100 ml in 0.1 N H~S04 is prepared; from this a working standard of 250 mt~g/ml of 0.1 N H2SO4 is used to calibrate the spectrophotofluorometer. Glass-redistilled water is used throughout. All compounds are analytical reagent grade unless specified otherwise. All glassware must be cleaned in nitric acid, well rinsed with glass redistilled water, and completely dry prior to each use. A major, but easily avoidable, source of error in this procedure is improperly prepared glassware. Any spectrophotofluorometer capable of exciting at 385 mu and 410 mu and measuring fluorescence at 435 and 480 mt~ is suitable. The instrument utilized for the initial development of this procedure was the AmincoBowman spectrophotofluorometer with slits 1, 3, and 4 having }~ inch diameter and the remainder ~'{6 inch. The values reported have not been corrected for photocell sensitivity. Procedure. The oxidation of vitamin B, or its analog was ostensibly accomplished by combining the methods recommended by Burch et al3 and Mickelsen and Yamamoto. 3 To a reaction volume of 3.0 ml, 0.24 ml of 1.53 M trichloroacetic acid is added. Any protein precipitating at this stage is removed by centrifugation. To a 2.0-ml aliquot of this mixture is added 3.40 ml of 4 M potassium acetate and then 0.20 ml of alkaline ferricyanide. The contents of the tube are mixed and left standing for 5 minutes at room temperature; then 0.10 ml of 30% H202 is added. The reaction is then extracted into 10.0 ml of isoamyl alcohol by mixing vigorously. The isoamyl alcohol phase is siphoned off and dried with approximately 6 g of anhydrous granular sodium sulfate. An appropriate series of thiamine and pyrithiamine standards are prepared and processed simultaneously with each batch of unknown 2 H. B. Burch, I). A. Bessey, R. A. Love, and O. H. Lowry, J. Biol. Chem. 198, 477 (1952). O. Mickelsen and R. S. Yamamoto, Methods Biochem. Anal. 6, 191 (1958).

[14]

THIAMINE

AND P Y R I T H I A M I N E

DETERMINATION

83

reactions. After the oxidation and extraction steps, care must be taken to avoid undue exposure of the extracts to light and contamination with dust. A 2.0-ml aliquot of the isoamyl alcohol extract is used for the fluorometric measurements. Appropriate reagent blanks are simultaneously prepared. The emission of the isoamyl alochol extract is measured at 435 and 480 mg with an excitation wavelength of 385 mg. The emission is also measured at the same wavelengths with an excitation light of 410 mg. Calculation of the Results The emission spectra of thiochrome (h,xoit = 385 rag) and pyrichrome (Xo~¢it = 410 m~) overlap. Thus equations reflecting this fact are utilized. Table I gives the relative molar fluorescence of thiochrome and pyrichrome as determined on the Aminco-Bowman spectrophotofluorometer. The values given are the average for three separate determinations. TABLE I RELATIVE MOLAR FLUORESCENCE OF THIOCHROME AND PYRICHROME AT DIFFERENT EXCITATION AND EMISSION WAVELENGTHS Xoxcit= 385 mz

Conditions Thiochrome, from thiamine reactions Pyrichrome, from pyrithiamine reaction Thiochrome Thiochrome extracted with reagent blank

),excit = 4 1 0 m z

~cmit

~emit

~emit

~kemit

= 435 m ~ X 10 4

= 480 m~ X 10 4

= 435 m ~ X 10 4

= 480 m ~ X 10 4

26339

9685

6419

2816

210

1022

252

1709

47200 41300

17620 16050

7720

2679 3040

7720

It is evident that thiochrome is about 15 times more fluorescent than pyrichrome under optimal conditions. Sealock and White, 4 using the ferricyanide oxidation method and measuring the resulting fluorescence of the aqueous phase, found thiamine to be 5.7 times as fluorescent as pyrithiamine. The inadequacy of measuring the fluorescence of the aqueous phase for quantitative determinations has been reviewed by Mickelsen and Yamamoto 3 and reconfirmed in this laboratory. Values are also presented for the relative molar fluorescence of thiochrome as determined by using a known concentration of this compound dissolved in isoamyl alcohol. An estimate of the partition coefficient of thiochrome between isoamyl 4 R . R . S e a l o c k a n d H . W h i l e , J. Biol. Chem. 1 8 1 , 3 9 3 (1949).

84

[14]

THIAMINE: PttOSPItATES AND ANALOGS

alcohol and the reagent blank m a y be obtained from the final values presented. In this case a 10.0-ml isoamyl alcohol-thiochrome solution is extracted with the aqueous phase of a reagent blank, and the fluorescence of the alcohol phase is then determined. I t is readily apparent t h a t there is a reduction in fluorescence, although not sufficient to account for the fluorescence of the oxidation products. Hence, the assumption of complete extraction of thiochrome---under the experimental conditions--is unjustified. This lille of reasoning assumes t h a t no quenching agent is extracted from the aqueous phase of the reagent blank. However, with standard amounts of thiamine or pyrithiamine, repeated determinations give sufficiently reproducible results to justify the method. Whether pyrichrome has a corresponding partition coefficient between isoamyl alcohol and the aqueous phase of the reagent blank is not known. The values of Table I were used to generate Eqs. (1) and (2) to calculate thiamine and pyrithiamine concentration. T = 4.12 X 10-2 E435/3s5 -

0.85 X 10-2 E~s0/3s5 = millimicromoles t h i a m i n e / a s s a y

P = 1.06 E4s0m5 - 0.39 E43~/ss5 = millimicromoles p y r i t h i a m i n e / a s s a y

(1) (2)

Where E = emission, T = thiamine concentration, and P = pyrithiamine concentration. The subscripts describe the conditions under which the emission is measured, the n u m e r a t o r representing the emission wavelength and the denominator the excitation wavelength. I t should be noted t h a t these equations have been formulated on the basis of the volumes outlined in the procedure. One of the chief sources of error is the presence of isoamyl alcohol-soluble fluorescent contaminants in the sample. If the contamination is slight, blank corrections will suffice. If this approach proves inadequate, the adsorption of thiamine and pyrithiamine on such adsorbents TABLE II PYRITHIAMINEAND A AMOUNT OF THIAMINE (1.48 MILL1MICROMOLES)

I~ECOVER]ES OF VARYING AMOUNTS OF

Pyrithiamiae present (m/zmoles)

Molar ratio, pyrithiamine: thiamine

5.95 11.9 23.8 34,5 47,6 59.5 83.9

4.0 8.0 16 23 32 40 57

Pyrithiamine recovered

CONSTANT

Thiamine recovered

m~moles

%

mumoles

%

4.91 11.4 22.3 32.7 45.0 53.8 94.1

83 96 94 95 95 90 105

1.46 1.46 1.49 1.46 1.41 1.36 1.17

99 99 101 99 95 92 80

[14]

THIAMINE AND PYRITHIAMINE DETERMINATION

85

TABL],; III PtECOVFRIFS OF VARYING AMOUNTS OF TIIIAMINE AND A CONSTANT AMOUNT OF PYRITIIIAMINE (35.7 ]~IlI,LIMICROMOLES)

Thiamiue present (mumoles)

Molar ralio, pyrithiamine : I hiamine

Thiamine recovered mumoles

%

mumoles

%

0.296 0.741 1.48 2.22 2.96 7.41 11.1

124 55 25 17 12 5.0 3.2

0.128 0.540 1.38 1.99 2.gg 6.83 11.4

43 73 93 90 97 92 102

36.1 39.2 39.4 40.1 40.5 44.8 50.4

101 110 110 112 113 126 149

Pyrit hiamine recovered

as kieselguhr, l)ecalso, or Amberlite I R C - 5 0 m a y be employed. T h e efficacy of this latter a p p r o a c h has been reviewed? T h e limits of detection and reliability of the m e t h o d are indicated in Tables I I - I V . TABLE IV RECOVERIES OF VARYING AMOUNTS OF TIIIAM1NI'] AND A CONSTANT AMOUNT OF PYRITIIIAMINE (11.9 5IILLIMICROMOLES)

Thiamine recovered

Pyrithiamine recovered

Thiamine present (mtmmles)

Molar rat io, pyrit hiamine : thiamine

mt~moles

%

m#moles

%

0.296 0.741 1.48

40 16 8 5.4 4.0 1.6 1.1

0.270 0.702 1.35 2.01 2.80 6.96 8.24

91 95 92 91 95 94 75

13.0 13.0 13.9 13.4 13.5 13.6 15.0

109 109 117 113 113 114 126

2.22

2.96 7.41

11.1

I t m a y be seen t h a t the sensitivity and reliability of the m e t h o d is determined both b y the absolute a m o u n t of thiamine and pyrithiamine and by their m o l a r ratio. I n practice this difficulty m a y be circumvented, in some instances, b y carrying o u t the assay on a dilution series of the sample to be measured. One s h o r t c o m i n g of these equations is the fact t h a t the constants employed are d e p e n d e n t u p o n such factors as the spectrophotofluorometer used, p u r i t y of the s t a n d a r d s (thiamine, pyrithiamine, and quinine sulfate), and, p r o b a b l y most important, the presence of fluorescent materials of biological origin which are isoamyl alcohol soluble. This difficulty m a y be c i r c u m v e n t e d in part, by using a generalized solution for Eqs. (1) and (2), i.e.:

80

T = [d/(ad-

THIAMINE: PHOSPIIATES AND ANALOGS

bc)]E435/3s5- [ b / ( a d -

[15]

bc)]E4so;3s5

= m~moles thiamine/assay I ~ = [a/(ad -

bc)]E4~o/3s5 -

[c/(ad -

bc)]E435:3s5

= mumoles pyrithiamine/assay where a and b equal the relative molar fluorescence of thiochrome and pyrichrome when excited at 385 m~ and emissions are measured at 435 mu. Constants c and d equal the respective relative molar fluorescence of thiochrome and pyrichrome when excited at the same wavelength, but emissions are measured at 480 mu. Values for a, b, c, and d are measured by separately determining the fluorescence of known concentrations of pyrith]aminc and thiamine with each group of assays carried out, thus permitting the ready calculation of the constants to be applied for each set of assays.

[15] T h i n - L a y e r C h r o m a t o g r a p h y for t h e S e p a r a t i o n of Thiamine, N'-Methylnicotinamide, and Related Compounds By

Z. Z. ZIPOmN and P. P. WANING

By the use of labeled and nonlabeled thiamine, it has been possible to demonstrate that products of thiamine metabolism in rat and man appear in the urine. Approximately 20-22 different labeled spots have been separated from the urine of rats and man given pyrimidine- or thiazole-labeled thiamine.' .2 Quantitatively, many of these fractions represent insignificant amounts of the ingested thiamine, leaving relatively few substances which must be considered in the investigation of urinary excretion of thiamine metabolites. These are: pyramina (a term used by Mickelsen to represent a "pyrimidine-like component of the thiamine molecule which is excreted in the urine," and which enhances CO2 production in the yeast fermentation assay for thiamine); thiamine disulfide, thiochrome, and thiazole4; and 2-methyl-4-amino-pyrimidinecarboxylic acid. 5 However, when studies are conducted requiring isolation and identification of metabolites, in addition to the metabolites listed above, it is necessary to consider other compounds: (a) pyrimidinesulfonic acid; (b) a-hydroxyethylthiamine (HET); (c) N'-methylnicotinamide (NMN). The pyrimidinesulfonie acid is the 1 R. A. Neal and W. N. Pe'~rson, J. N~tr. 83, 343 (1(.t64). M. Balaghi and W. N. l)earson, J. N~dr. 91, (.) (1967). O. Mickelsen, W. O. Casler, and A. Keys, J. Biol. Chem. 168, 415 (1947). 4 j. M. Iacono and B. C. Johnson, J. Am. Chem. Soe. 79, 6321 (1957). 5 R. A. Neal and W. N. Pearson, J. Nulr. 83, 351 (1964).