Simultaneous determination of thiamine and pyrithiamine

Simultaneous R. L. AIRTH From

the

Determination and Pyrithiaminel AND

Department University

of

Received

of Thiamine

G. ELIZABETH

FOERSTER

Botany and Plant Research of Texas, Austin, Texas September

In.ditutr,

22, 1961

INTRODUCTION

A commonly used method for the detection of thiamine (I) [3-(4amino-2-methylpyridmidyl-5-methyl) -4-methyl-5,/3-hydroxyethylthiazole] is to oxidize it mildly with potassium ferricyanide to thiochrome (II). Thiochrome is a blue fluorescing compound and this property was used by Jansen (1) to determine thiamine concentration quantitatively. Mickelsen and Yamamoto (2) and Metzler (3) have recently reviewed the literature concerning this oxidation reaction. The thiamine analog.

bh0H (III)

pyrithiamine (III) [l- [ (4-amino-2-methyl) -5-pyrimidylmethyl] -2-methyl-3- (/3-hydroxyethyl) pyrimidine] is also reactive in this oxidation. Fujita et al. (4) have isolated and characterized the ferricyanide oxidation product of 2-methyl-4-amino-5-pyrimidylmethylpyridinium bromide ‘This National

investigation Institutes

of

was supported Health.

in

part 383

by

research

grant

RG-5989

from

the

384

AIRTH

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hydrobromide and named it 2-methylpyrichromine. The latter has a greenish yellow fluorescence in butanol. The less specific name, also utilized by Fujita et al., “pyrichrome,” will be used in this publication to refer to the isoamyl alcohol soluble oxidation product of pyrithiamine. The spectral properties of the latter differ from thiochrome and these differences have been used as a basis for estimating amounts of thiamine and pyrithiamine when mixed. This laboratory has been concerned with the enzymic activity of such organisms as Neurospora crassa and Bacillus aneurinolyticus on the substrates thiamine and pyrithiamine. The method described below has been developed within this frame of reference; further applicability would undoubtedly require modification but the principles involved should be applicable. REAGENTS

AND

APPARATUS

(a) Trichloroacetic acid, 1.53 M. (b) Potassium acetate, 4 M. (c) NaOH, 7.5 M. (d) K,Fe (CN) 6, 0.059 M ; 0.65 ml of the 0.059 M solution was diluted to 10.0 ml with 7.5M NaOH. These solutions were made within 1 hr of use. (e) Hydrogen peroxide, 30%, Mallinckrodt. (f) Isoamyl alcohol, redistilled from activated carbon and saturated with distilled water. (g) Thiamine hydrochloride, Mann Research Laboratories. A stock solution of 1 mg/ml of 0.1 N HCl was prepared and appropriate dilutions were made of this standard. (h) Pyrithiamine hydrobromide, California Corporation for Biochemical Research. A stock solution of 1 mg/ml of distilled water was prepared and appropriate dilutions made of this standard. (i) Quinine sulfate, Nutritional Biochemicals Corporation. A stock solution of 10.0 mg/lOO ml of 0.1 N H,SO, was prepared from which a working standard of 250 mpg/ml of 0.1 N H,SO, was used to calibrate the spectrophotofluorometer. (i) Thiochrome, Mann Research Laboratories. Excitation and emission wavelength determinations and intensity measurements were made with the Aminco-Bowman spectrophotofluorometer.Z The values reported have not been corrected for photocell sensitivity. Slits 1, 3, and 4 were l/s-in. diameter and the remainder I/& in. the

‘The authors University

wish to thank Dr. Lester Reed of the of Texas for the use of this instrument.

Department

of Chemistry

of

THIAMINE

AND

PYRITHIAMINE

385

PROCEDURE

The oxidation of vitamin B, or its analog was ostensibly accomplished by combining the methods recommended by Burch et al. (5) and Mickelsen and Yamamoto (2). To a reaction volume of 3.0 ml, 0.24 ml of 1.53 M trichloroacetic acid was added. Any protein precipitating at this stage was removed by eentrifugation. To a 2.0-ml aliquot of this mixture was added 3.40 ml of 4 M potassium acetate and then 0.20 ml of alkaline ferricyanide. The contents of the tube were mixed and left standing for 5 min at room temperature; then 0.10 ml of 30% H,O, was added. The reaction was then extracted into 10.0 ml of isoamyl alcohol by mixing vigorously. The isoamyl alcohol phase was siphoned off and dried with approximately 6 gm of anhydrous sodium sulfate. A 2.0-ml aliquot of the isoamyl alcohol was used for the fluorometric measurements. Appropriate reagent blanks were also utilized. The emission of the isoamyl alcohol extract was measured at 435 and 480 rnp with an exciting wavelength of 385 rnp.. The emission was also measured at the same wavelengths with an exciting light of 410 rnp. EXPERIMENTAL

AND DISCUSSION

The emission properties of the isoamyl alcohol soluble oxidation products of thiamine and pyrithiamine are presented in Pig. 1. The excitation and emission maxima for thiochrome are 385 and approximately 440 mp, respectively. Pyrithiamine, when oxidized and extracted into isoamyl alcohol, yields a fluorescent compound which has an excitation maximum at 410 rnp and maximal emission at 480 mp. The intensity of fluorescence is reported in relative units and the curves have been calculated from known concentrations to give equal emission, thus representing 1.48 X 1O-7M thiochrome and 5.88 X 1O-6 M pyrichrome. Table 1 presents the relative molar fluorescence for triplicate thiamine and pyrithiamine determinations at exciting wavelengths of 385 and 410 rnp and emission at 435 and 480 mp. The values represent the fluorescence, in relative units, of a molar solution of thiochromc and pyrichrome when derived from t’hiamine and pyrithiamine, respectively, by the procedure outlined ahove. It is evident that thiochrome is about, 15 times more fluorescent than pyrichrome under optimal conditions. Yealock and White (6), 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 Mirkelsen and Yamamoto (2) and rccon-

386

AIRTH

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LEGEND -

4xJ

3w

31”

WAVELENGTH

FIQ. 1. Uncorrected excited at 410 mp.

emission of thiochrome

6W

Thiochromr

630

IW

( mp)

excited at 38.5 rnp and pyrichrome

firmed 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 alcohol and the reagent blank may be obtained from the final values presented. In this case a lO.O-ml isoamyl alcohol thiochrome solution was extracted with the aqueous phase of a reagent blank and the fluorescence of the alcohol phase then determined. It is readily apparent that there is a reduction in fluorescence, although not sufficient to account for the fluorescence of the oxidation products. Hence, the assumption of com-

THIAMINE

AND

387

PYRITHIAMINE

TABLE 1 RELATIVE

MOLAR FLUORESCENCE DIFFERENT EXCITATION

OF THIOCHROM~: AND YYRICHROME AND EMISSION WAVELENGTHS hcxe3te = 385 rn,"

Condit.ions

Thiochrome, from thiamine reactions

Pyrichrome, from pyrithiamine reaction

X.mit = 435 nw x 101

X.*oite = 410 In@

Xomi~ = 480111,‘ Aemit = 435~1~ x 1w x 10'

26014 26689 26315

9122 10135

26339 210 210 210 -210

AT

Xcmit = 480mp x 10'

9707

6081 6757 6410

2365 3041 3041

0685

6419

2816

987 1050

252 273 231

1681 1743 1702

1022

253

-1709

1029

--

Thiochrome

47200

17620

7720

2679

Thiochrome, extd. with reagent blanka

41300

16050

7720

3040

a See text.

extraction of thiochrome-under the experimental conditions-is unjustified. This line of reasoning assumes that 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 linear relationship of fluorescence to thiamine concentration may be seen in Fig. 2. In this case, varying amounts of thiamine were used and the emissions at 435 and at 480 mp were studied. These emissions were measured with exciting wavelengths of both 385 and 410 rnp. Proportional fluorescence is obtained with all wavelength combinations. Figure 3 presents the results of a similar set of determinations for pyrithiamine. In this case the fluorescence is also proportional to pyrithiamine concentration no matter what wavelength combination is used. Since the spectral properties and relative molar fluorescence for both thiochrome and pyrichrome are known, the concentration of each in a mixture may be readily obtained. The determination of the concentration of two unknowns in a mixture is possible when the molar fluorescences, at the wavelengths under consideration, are known. The intensity of

plete

388

AIRTH

2

4

AND

FOERSTER

6 THIAMINE/assay

(rnh

moles)

FIG. 2. Proportionality between thiamine concentration and fluorescence. The ratio values represent the exciting wavelengths (denominator) and emission wavelengths (numerator).

emission at any exciting wavelength will be determined by the sum of the fluorescence due to thiochrome plus that due to pyrichrome, As a general rule, the wavelengths selected are those at which the fluorescence is optimal for the particular compound being measured. Thus, the thiamine determination could be carried out at 435-rnp emitting wavelength and excitation at 385 mp; these being the optimal conditions for thiochrome emission. To measure pyrithiamine concentration, on the other hand, one would anticipate that measurement of emission at 480 rnp with an exciting light of 410 rnp would offer the best wavelength combination. In a mixture of thiochrome and pyrichrome, the concentration of each may be determined by solving simultaneous equations using the

THIAMINE

AND

389

PYRITHIAMINE

160

0

FIG.

notation

20

40 PYRITHIAMINE

3. Proportionality between sa.me as Fig. 2.

60

60 /assay

pyrichrome

loo

120

0

(mp, moles)

concentration

and fluorescence. Ratio

relative fluorescence of each at 435 and 480 rnp when excited with 385 or with 410 rnp light. The values of Table 1 were used to set up two sets of simultaneous equations: Ea5,m = 26339 X lo4 T + 210 X lo4 P

(4

Em,,a~ = 9685 X lo4 T + 1022 X lo4 P Eao,m = 2816 X lo4 T + 1709 X lo4 P

@I (4 (4

E435,410

=

6419 X lo4 T + 252 X lo4 P

where E = emission, T = thiamine concentration, and P = pyrithiamine concentration. The subscripts describe the conditions under which the emission is measured, with the numerator representing the emission wavelength and the denominator the excitation wavelength. Solving Eqs. (a) and (b) leads to expressions which determine the thiamine and pyrithiamine concentration when 385 rnp is the exciting light and emission is

390

AIRTH

AND

FOERSTER

measured at 435 and 480 rn,u. Solving Eqs. (c) and hand, leads to expressions which measure thiamine concentration when the exciting light is at 410 rnb and ured at 435 and 480 mp. When equations (a), (b), solved this leads to:

(d) , on the other and pyrithiamine emission is measand (c), (d) are

T = 4.12 X 1O-2 E436,%5- 0.85 X 1O-2 E a0,386= mpmoles thiamine/assay (1) P = 1.06 Em/m - 0.39 E43513gb = mpmoles pyrithiamine/assay (11) T = 16.65 X 1O-2 E436,410- 2.46 X 1O-2 E480,410= mymoles thiamine/assay (III) P = 0.63 E~~o,~M- 0.28 E436j410= m/lmoles pyrithiamine/assay (IV Thus Eqs. (I) and (III) may be used to solve for thiamine concentration and (II) and (IV) for pyrithiamine concentration. It should be stressed that the values used here have been derived from experiments using pyrithiamine and thiamine in the absence of biological materials. When the assay is carried out in their presence, appropriate corrections may be necessary and the corrections will undoubtedly depend upon the type of biological material being used. The chief source of error is the presence of isoamyl alcohol soluble fluorescent material in the sample. If the amount of fluorescent material is comparatively small, blank corrections will suffice. If, on the other hand, this approach proves inadequate then adsorption of thiamine and pyrithiamine on such adsorbents as kieselguhr, Decalso, or Amberlite IRC-50 would have to be employed. The efficacy of this latter approach has been reviewed (2). Measurements were carried out to determine which set of equations were the most applicable. It should be noted in passing that the above values will apply when 10.0 ml of isoamyl alcohol is used in the determination. Should a different volume be used, proportional adjustment of the values would have to be made. The reliability of Eqs. (I) to (IV) was investigated by the following experiments. One series of experiments was carried out in which 1.48 mymoles of thiamine was present in each assay and the pyrithiamine concentration varied; the results are presented in Table 2. Equations (II) and (IV) have been used to calculate the recovery of pyrithiamine and these values have also been reported as per cent recovery. From the values obtained it is evident that Eq. (II) gives a more accurate estimate of the pyrithiamine concentration. The limit of detection for pyrithiamine using this equation is approximately 12 mpmoles per assay. Thiamine recovery is reproducible when Eq. (I) is applied. It should also be noted

THIAMINE

AND

391

PYRITHIAMINE

TABLE 2 RECOVERIES OF VARYING AMOUNTS OF PYRITHIAMINE AND A CaNsTANT AMOUNT OF THIAMINE (1.48 M~MOLES) AS DETERMINED BY USING EQUATIONS (I)To (IV)” Molar

I’yritbiamine present (rnrrn01es)

5.95 11.9 23.8 34.5 47.6 59.5 83.9

Pyrithismine

ratio,, pyrithlamine : thiamine

mpmOle8

4.0 8.0 16 23 32 40 57

4.91 11.4 22.3 32.7 45.0 53.8 94.1

Eq.

(II)

recovered Eq.

%

83 96 94 95 95 90 105

Thiamine (IV)

Eq.

mrmoles

%

4.56 9.80 20.9 31.6 43.9 54.6 90.2

77 82 88 92 92 92 101

recovered Eq.

(I)

mrmoles

%

1.46 1.46 1.49 1.46 1.41 1.36 1.17

99 99 101 99 ‘35 92 80

(III)

mfimoles

%

1.67 1.50 1.39 1.33 1.24 1.49 1.17

131 101

94 90 84 101 80

a Seetext.

that the molar ratio of pyrithiamine to thiamine affects the result no matter what equations are utilized. Table 2 indicates that this approach is applicable only when the ratio of pyrithiamine to thiamine lies between 8 and 40 when assaying a mixture of the two. The results of a reciprocal experiment using a constant amount of pyrithiamine, 35.7 mpmoles per assay, and varying thiamine concentration is presented in Table 3. Equation (I) again gives the most reproTABLE 3 RECOVERIES OF VARYING AMOUNTS OF THIAMINE AND A CONSTANT AMOUNT OF PYRITHIAMINE (35.7 M~MOLES) AS DETERMINED BY USING EQUATIONS (I) TO (IV)a Thiamine present (mpmoles)

0.296

0.741 1.48 2.22 2.96 7.41 11.1

M&r ratio2 pyrithmmine : thiamine

124 55 25 17 13

5.0 3.2

Thiamine Es. mrmoles

0.128 0.540 1.38 1.99 2.88 6.83 11.4

(I)

recovered ES

f&

43 73 93 90 07 9’2 10’2

mrmoles

0.000 0.106 1.12 1.95 2.62 5.37 9.10

Pyrithiamine (III)

Eq. %

0 22 76 88 89 73 83

mcmoles

36.1 39.2 39.4 40.1 40.5 44.8 50.4

(II)

recovered Es.

%

101 110

110 112 1 18

126 149

mpmoles

39.7 40.4 39.9 37.9 36.8 34 .3 34.8

(IV) 7,

111 113 112 106 103

!I6 Y7

a See text,

ducible results and the reliable limit of quantitative thiamine detection is approximately 1.5 mpmoles per assay when the molar ratio of pyrithiamine to thiamine is less than 25. To test whether both the molar ratio

392

AIRTH

AND

FOERSTER

and the absolute amount of thiamine and pyrithiamine determine the limit of thiamine detection, a similar experiment was performed using 11.9 mpmoles of pyrithiamine per assay. Table 4 indicates that in this

RECOVERIES

Thiamine present (m~moles) 0.296 0.741 1.48 2.22 2.96 7.41 11.1

TABLE 4 OF VARYING AMOUNTS OF THIAMINE ANL) A CONSTANT PYRITHIAMINE (11.9 LIPMOLES) AS DETERMINED BY USING EQUATIONS (I) TO (IV)” Molar ratio! pyrithlsmine,: thiamme

Thiamine m~moles

%

m~moles

%

mpmoles

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

0.244 0.856 1.19 1.41 2.01 5.09 7.86

82 115 80 64 68 68 71

13.0 13.0 13.9 13.4 13.5 13.6 15.0

Eq. (1)

recovered

Pyrithiamine

Eq. (III)

Eq. (11)

AMOUNT

OF

recovered Eq. (IV)

70 109 109 117 113 113 114 126

m~moles 12.1 12.5 11.6 11.4 11.2 10.2 9.4

70 102 105 98 96 95 86 79

0 See text.

case 0.3 m,umole of thiamine may be determined while the molar ratio was 40. This limit may be contrasted to that reported in Table 3 of 1.48 mpmoles for thiamine at a molar ratio of 25. Thus the sensitivity and reliability of the method is determined both by the absolute amounts of thiamine and pyrithiamine and by their molar ratio. In practice this difficulty may be circumvented, in some instances, by carrying out the assay on a dilution series of the sample to be measured. Sealock and White (6) have quantitatively measured mixtures of thiamine and pyrithiamine. Thiamine was determined by the diazotization method of Melnick and Field (7) and an alkaline ferricyanide reaction was also carried out on an aliquot to give the concentration of thiamine plus pyrithiamine. Pyrithiamine concentration was then calculated by subtracting the diazo-determined thiamine from the results of the ferricyanide oxidation. The authors report linearity for the method at concentrations varying from zero to 20 mpmoles of pyrithiamine per 5 ml of aliquot analyzed. Difficulty encountered in analyzing unfavorable molar ratios of pyrithiamine to thiamine was also found in the above-mentioned work; a ratio of approximately 4 gave satisfactory results, contrasted to a limit of 8 in the present assay. The latter value is found in the presence of 12 and 1.5 mpmoles of pyrithiamine and thiamine respectively. From the above it is apparent that, Eqs. (I) and (II) are the most

THIAMINE

AND

393

PYRITHIAMINE

reliable for calculating thiamine and pyrithiamine concentrations. One short-coming of these equations is the fact that the constants employed are dependent upon such factors as the spectrophotoffuorometer used, purity of the standards (thiamine, pyrithiamine, and quinine sulfate) and probably most important the presence of fluorescent materials of biological origin which are isoamyl alcohol soluble. This difficulty may be circumvented in part, by using a generalized solution for Eqs. (I) and (II), i.e.:

= mpmoles thiamine/assay

(V>

= mpmoles pyrithiamine/assay

(VI)

where a and b equal the relative molar fluorescence of thiochrome and pyrichrome when excited at 385 my and emissions are measured at 435 mp. Constants c and d equal the respective relat,ive molar fluorescence of thiochrome and pyrichrome when exc,ited at the same wavelength, but emissions are measured at 480 m,p. Values for a, b, c, and d are measured by separately determining t,he fluorescence of known concentrations of pyrithiamine and thiamine with each group of assays carried out, thus permitting the ready calculat’ion of the constants to be applied for each set of assays. The results in Tables 2, 3, and 4 have been determined by Eqs. (Vl and (VI1 and were comparable to those already presented, which is to be expected in view of the fact that the same instrument and standard solutions were used. When determinations for t,hiamine and pyrithiamine were carried out in the presence of biological TABLE PER

CENT

RECOVERY PYRITHIAMINE BIOLOGICAL

(12.31 MATERIAL

Tissw

Me tissue present in assay

Human serum Corn root Achromobacter fkcheri Rat muscle (striated)

1.64 0.14 0.89 1.94

9 See text.

5 OF THIAIWNE

(0.767

Y~MOLES) IN THE USING EQUATIONS

PRESENCE (V) AND

FRO~M MIXTURES

Constants detd. of biological

in prewrap material

RIPMOLES)

AND

OF

(VI)a

Constants detd. in abaencr of biological material

Thiamine

Pyrithiamine

Thiamine

Pyrithiamine

106 98 99 89

107 109 103 106

110 106 109 94

110 121 1’22 130

394

AIRTH

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FOERSTER

material, the per cent recovery varied from 95 to 130 when Eqs. (1) and (II) were applied. When Eqs. (V) and (VI) were utilized with the constants, a, b, c, and d being determined in the presence of the biological material, then recoveries ranging from 89 to 109% were obtained. These results are presented in Table 5. SUMMARY

(1) Alkaline potassium ferricyanide oxidation of thiamine and pyrithiamine yields two different products as judged by excitation and emission characteristics. The former oxidation produces thiochrome and the latter pyrichrome. (2) The differences in spectral characteristics of thiochrome and pyrichrome have been utilized to determine quantitatively thiamine and pyrithiamine concentration in a mixture of the two. (3) Two generalized equations were derived to determine the concentration of thiamine and pyrithiamine in the presence of biological material :

= mpmoles thiamine/assay

= n&moles pyrithiamine/assay where E = emission, P = pyrithiamine, T = thiamine, the numerator of the subscripts the emission wavelength, and the denominator the excitation wavelength. Constants a and b represent the relative molar fluorescence of thiochrome and pyrichrome when excited at 385 rnp and emitting at 435 rnp. Constants c and d represent the relative molar fluorescence of the same two respective compounds with the emission measured at 480 rnp and excitation at 385 mp. (4) The limit of detection for both thiamine and pyrithiamine is determined bot,h by their absolute amounts and by the molar ratio of the two. When the molar ratio of pyrithiamine to thiamine was between 40 and 8, 12 mpmoles of pyrithiamine could be detected with recoveries ranging from 90 to 115%. With a similar range of molar ratios, 1.5 mpmoles of thiamine per assay could be detected with recoveries ranging from 90 t,o 1000/o. The limitations of this approach have been considered. REFERENCES 1. JANSEN, 2. MICBELSEN,

B. C. P., Rec. trav. chim. 55, 1046 (1936). O., AND YAMAMOTO, R. S., Methods of Biochem.

Anal.

6, 191 (1958).

THIAMINE

AND

PYRITHIAhZINE

395

D. E., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbick, Vol. 2, p. 295. Academic Press, New York 1960. s$. FUJITA, A., NOSE, Y., UEDA, K., AND EIICHI, H., J. &IL. Chew. 196, 297 (1952). 5. BURCH, H. B., BF,RSET, 0. A., Low, R. A., .4x1 LOWRY. 0. H., J. Riol. Chem. 198, 477 (1952). 6. SEXLOCX, R. R., AND WHITE, H.> J. Biol. Chem. 181, 393 (1949). 7. MELNICK, D., AND FIELD, H., JR., 1. Riol. Chem. 127, 515 (1939). 3. METZLEK,

eds.),