- Email: [email protected]
Colorimetric determination of vitamin A with trichloroacetic acid
ANALYTICAL
BIOCHEMISTRY
39,
Calorimetric
282-287
(1971)
Determination with
of
Trichloroacetic
Vitamin
A
Acid
R. F. BAYFIELD Veterinary Research Station, Glenfield, New South Received
Department Wales 2167 April
of Agriculture, Australia
10, 1970
The estimation of vitamin A by methods which have a calorimetric finish generally provides the analyst with some problems, such as detection of end-point and sensitivity of the reagent. Undoubtedly the method most widely used is that employing the Carr-Price reagent (1)) but others have been developed (2,3). Reactions depending on the strong oxidizing properties of the Lewis acid type of compound with vitamin A have been investigated (1,4) and analytical procedures using trifluoroacetic acid (TFA) have been described (45). There appears to be only brief mention in t.he literature concerning the use of trichloroacetic acid (TCA) as a reagent for vitamin A determination (1,6) although its strong oxidizing capabilities are well recognized. The use of TCA as a reagent for vitamin A determination was investigated because it is readily available, is comparatively easy to handle in preparation (readily soluble in chloroform) and in cleaning of apparatus (readily soluble in water), and has possible advantages over TFA (4,7) and SbCl, from a toxicity viewpoint. Moisture problems encountered with SbCI, (4,5) are also eliminated with TCA. PROCEDURE
A saturated solution of TCA crystals in chloroform was prepared immediately before use by dissolving approximately 50 gm TCA (AR) in 25 ml chloroform (alcohol-free and recently distilled). This yielded approximately 50 ml reagent. Standard solutions of vitamin A acetate were prepared in chloroform from the USP reference capsules, so that the solution finally measured contained from 1 to 7 pg in a total of 3 ml chloroform. In practice, a strong solution of the reference oil was made by dis252
VITAMIN
A DETERMINATION
WITH
TCA
283
solving 100 mg in 10 ml chloroform. This was then diluted 1 in 50 to give a concentration of about 7 pg./ml. Aliquots up to 1 ml were taken and sufficient chloroform was added to make 1 ml, followed by 2 ml of TCA reagent. The calorimetric measurements were made in tubes 1 cm in diameter and approximately 11 cm long in a Unicam SP 350 spectrophotometer. The reagent was added from a fast-delivery 2 ml pipet, and the optical density reading taken at maximum deflection of the galvanometer spot at wavelength 620 nm. Samples of serum and liver from animals were analyzed by dissolving the appropriate residue in chloroform and treating an aliquot of 1 ml with the reagent, the concentration being read off the calibration graph prepared from the standards. Sheep serum containing minimal amounts of vitamin A (deficiency) was extracted by shaking 15 ml with 15 ml ethyl alcohol and 30 ml light petroleum, as described elsewhere (8). An aliquot of 25 ml was evaporated under vacuum and the residue dissolved in 1.25 m chloroform. Treatment of 1 ml of the chloroform solution with the TCA reagent permitted the estimation of vitamin A in 10 ml serum. For higher vitamin A concentrations, appropriately smaller amounts of serum were extracted. To ascertain the value of TCA for the determination of vitamin A in composite serum extracts, the reagent was applied to each serum component, after separation by paper chromatography, using a modification of a previously published method (8). RESULTS
The wavelength of maximum absorption for the color reaction was found to lie in the region of 616-620 nm (Fig. 1) and the linearity of the relationship between concentration and absorbance at this wavelength is shown in Fig. 2. Intensity of optical density values obtained with the TCA reagent for standard solutions of vitamin A acetate USP reference oil was identical with that obtained when SbCl, was used as reagent. Identical results for both reagents were also obtained when standard curves were prepared from vitamin A alcohol and vitamin A palmitate. When pure vitamin A alcohol was assayed by the method described, results corresponding to 98% of the known concentration were obtained. After storage at -20” for 1 week, this value dropped to 95% and after 2 weeks to 92%. Dilute solutions of vitamin A in chloroform, freshly prepared from a stock solution of vitamin A acet,ate, stored under deep frozen con-
284
B. F. BAYFIELD
540
MO
580
600
620
640
660
680
Wavelength (nm) FIG.
1. Absorption
spectrum of blue species produced with TCA and Vitamin
I
2
3.
4
5
b
A
1
Vitamin A - ug13ml Final Solution FIG.
2.
Relationship
between
absorbance
and concentration
of Vitamin
A.
VITAMIN
A DETERMINATION
Vitamin
Animal NO.
not
detected
in any
285
TCA
TABLE 1 A in Sheep Seruma
Deficient state bg/lOO ml)
After grazing for 1 month
5.0 11.0 9.0 1.0 5.5 4.5 6.0
29.0 30.5 29.0 24.0 20.0 24.0 25.5
54 1 8 70 37 38 59 0 Carotene
WITH
of those
samples.
ditions (-20”) in the absence of light, gave remarkably consistent results over a period of 4 weeks. A commercial sample of vitamin A concentrate assayed by the SbCl, and TCA reagents gave similar results: 8930 I.U.,/gm (SbCl,) and 8725 I.U./gm (TCA). When the TCA reagent was allowed to stand with daylight excluded, no differences were observed in the absorbance of standard solutions at zero time and 5 hr. However, optical density values showed a tendency to decrease over extended periods of time up to 24 hr. The decline in absorbance was more striking when the reagent was exposed to bright diffuse light over several hours, ultimately reaching zero, and showing no color for the reaction. To date, the method has been applied to the determination of vitamin A in animal serum and tissues, both normal and deficient. A selection of results of such materials from a series of more than 100 analyses, is reported in Tables 1 and 2 to show the capability of the method. Vitamin Animal No. 26 81
(i)
20 81 (ii) 9 48
a Vitamin
A separated
TABLE 2 A in Pig Livera
Remarks
a/gm 13.2 17.8 19.9 103.9 59.7 2.9
by
chromatography
Piglet Piglet Piglet Mature pig Mature pig Mature pig (suspected deficiency) before
assay
with
TCA.
286
R. F. BATFIELD
The method allows good duplication, which results shown in Table 3 for an experimental When the components of sheep serum were matography, th:> only one which gave the responding to true vitamin A, the location chromatography of the pure vitamin. TABLE
Serum Vitamin Class Weaners
3
A in Experimental
1
18.0,M.O 16.0, 13.5 22.0,22.0 22.0,21.0
1 2
Ewes
Sheepa Vitamin Ab bd100 ml)
Group
2
Hoggetts
is illustrated by the set of group of sheep. separated by paper chroblue color was that corof which was checked by
1
16.0, 15.5
2
16.0,
16.0
a Carotene was not detected in the serum from these animals. b Duplicate values determined on pooled samples from each group. DISCUSSION
The wavelength of maximum absorption of the blue colored species produced by the reaction between TCA and vitamin A was similar to that reported for SbCl,, TFA, and other Lewis acids (4). For strong solutions of vitamin A, the color of the reaction mixture was a deep blue, which faded rapidly through mauve to pink, and later to a pale yellow solution. Maximum blue color was reached in approximately 5 sec. These color changes follow closely those obtained with SbCl,. Although the TCA remained stable for several hours under controlled conditions of light, changes were noted in its reactivity towards vitamin A over extended periods of time. This corresponds with observations concerning TFA (4) and suggested that, in agreement with earlier workers (6), the reagent should always be prepared immediately before use. Under these conditions, consistent and successful results were obtained with the TCA reagent over a period of 1 year, as opposed to the mixed results obtained with this reagent by others (1). As noted earlier (6), the reagent may be prepared in light petroleum. However, compared with the color obtained when chloroform is used as solvent, there is a loss in intensity of about 607’&, which of course precludes its use for quantitative purposes. Interference
from
carotenoids
and
other
substances
with
the
Carr-
VITAMIN
A DETERMIR’ATIOn’
WITH
TCA
287
Price reagent has been reported (2,9). In this laboratory, highly colored solutions containing carotenoids resulted in difficulty in determining the point of maximum deflection, there being a slow drift from low to higher optical density values. This occurred when either the SbCl, or the TCA reagent was used, and would suggest that care is necessary when assessing vitamin A by a gross measurement to which a correction factor is applied. At this stage, therefore, for extracts from natural sources, the method is best confined to mat’erials which are relatively free of carotenoid-like substances. Alternatively, separation of the vitamin A component by chromatography is recommended. The acceptabilit’y of the method rests on the following points: (i) obedience to Beer’s law over the concentration range indicated; (ii) the excellent comparison obtained in absorbance in standard curves prepared with vitamin A est’ers or alcohol using either TCA or the conventional SbCl, as reagent; (iii) analysis of known pure and commercial samples of vitamin A to acceptable standards; (iv) repeatability of results from standards maintained under acceptable conditions of storage; (v) serum vitamin A levels similar to the normal range for grazing animals (10) (Table 1) and the good degree of duplication (Table 3) ; and (vi) chromatographic verification of vitamin A as the principal colorproducing component in composite serum extracts. SUMMARY
Vitamin A was determined in pure samples and animal tissues using trichloroacetic acid as the chromogenic agent’. Results were similar to those obtained with SbCl,, but the TCA reagent is generally more convenient to use and is less toxic. REFERENCES 1. CARR,
F. H., AND PRICE, E. A., Biochem. J. 20, 497 (1926). A. E.. AND SNOW. S. D.. J. Bill. Chem. 171, 617 (1947). P., Proc. Sot. Exptl. Biol. Med. 84, 148 (1953). DUGAN, R. E., FRIGERIO, N. A., AND &BERT, J. M., Anal. Chem. 36, 114 (1964). NEELD, J. B., JR., AND PEARSON, W. N., J. Nut?. 79, 454 (1963). ROSENHEIM. O., AND DRUMMOND, J. C.. B&hem. J. 19, 753 (19%). OLSEN, J. E., Vitumins Hormones (N. Y.) 26, 9 (1968). BAYFIELD. R. F., FALK, R. H., AND BARRETT, J. D.. J. Chromatog. 36, 54 (1968). AVAMPATO, J. E., AND EATON, H. D.. J. Dairy Sci. 26, 783 (1953). PEIRCE, A. W., Australian J. Exptl. Biol. Med. Sci. 24, 231 (1946).
2. SOBEL, 3. FLESCH, 4. 5. 6.
7. 8. 9.
10.
BIOCHEMISTRY
39,
Calorimetric
282-287
(1971)
Determination with
of
Trichloroacetic
Vitamin
A
Acid
R. F. BAYFIELD Veterinary Research Station, Glenfield, New South Received
Department Wales 2167 April
of Agriculture, Australia
10, 1970
The estimation of vitamin A by methods which have a calorimetric finish generally provides the analyst with some problems, such as detection of end-point and sensitivity of the reagent. Undoubtedly the method most widely used is that employing the Carr-Price reagent (1)) but others have been developed (2,3). Reactions depending on the strong oxidizing properties of the Lewis acid type of compound with vitamin A have been investigated (1,4) and analytical procedures using trifluoroacetic acid (TFA) have been described (45). There appears to be only brief mention in t.he literature concerning the use of trichloroacetic acid (TCA) as a reagent for vitamin A determination (1,6) although its strong oxidizing capabilities are well recognized. The use of TCA as a reagent for vitamin A determination was investigated because it is readily available, is comparatively easy to handle in preparation (readily soluble in chloroform) and in cleaning of apparatus (readily soluble in water), and has possible advantages over TFA (4,7) and SbCl, from a toxicity viewpoint. Moisture problems encountered with SbCI, (4,5) are also eliminated with TCA. PROCEDURE
A saturated solution of TCA crystals in chloroform was prepared immediately before use by dissolving approximately 50 gm TCA (AR) in 25 ml chloroform (alcohol-free and recently distilled). This yielded approximately 50 ml reagent. Standard solutions of vitamin A acetate were prepared in chloroform from the USP reference capsules, so that the solution finally measured contained from 1 to 7 pg in a total of 3 ml chloroform. In practice, a strong solution of the reference oil was made by dis252
VITAMIN
A DETERMINATION
WITH
TCA
283
solving 100 mg in 10 ml chloroform. This was then diluted 1 in 50 to give a concentration of about 7 pg./ml. Aliquots up to 1 ml were taken and sufficient chloroform was added to make 1 ml, followed by 2 ml of TCA reagent. The calorimetric measurements were made in tubes 1 cm in diameter and approximately 11 cm long in a Unicam SP 350 spectrophotometer. The reagent was added from a fast-delivery 2 ml pipet, and the optical density reading taken at maximum deflection of the galvanometer spot at wavelength 620 nm. Samples of serum and liver from animals were analyzed by dissolving the appropriate residue in chloroform and treating an aliquot of 1 ml with the reagent, the concentration being read off the calibration graph prepared from the standards. Sheep serum containing minimal amounts of vitamin A (deficiency) was extracted by shaking 15 ml with 15 ml ethyl alcohol and 30 ml light petroleum, as described elsewhere (8). An aliquot of 25 ml was evaporated under vacuum and the residue dissolved in 1.25 m chloroform. Treatment of 1 ml of the chloroform solution with the TCA reagent permitted the estimation of vitamin A in 10 ml serum. For higher vitamin A concentrations, appropriately smaller amounts of serum were extracted. To ascertain the value of TCA for the determination of vitamin A in composite serum extracts, the reagent was applied to each serum component, after separation by paper chromatography, using a modification of a previously published method (8). RESULTS
The wavelength of maximum absorption for the color reaction was found to lie in the region of 616-620 nm (Fig. 1) and the linearity of the relationship between concentration and absorbance at this wavelength is shown in Fig. 2. Intensity of optical density values obtained with the TCA reagent for standard solutions of vitamin A acetate USP reference oil was identical with that obtained when SbCl, was used as reagent. Identical results for both reagents were also obtained when standard curves were prepared from vitamin A alcohol and vitamin A palmitate. When pure vitamin A alcohol was assayed by the method described, results corresponding to 98% of the known concentration were obtained. After storage at -20” for 1 week, this value dropped to 95% and after 2 weeks to 92%. Dilute solutions of vitamin A in chloroform, freshly prepared from a stock solution of vitamin A acet,ate, stored under deep frozen con-
284
B. F. BAYFIELD
540
MO
580
600
620
640
660
680
Wavelength (nm) FIG.
1. Absorption
spectrum of blue species produced with TCA and Vitamin
I
2
3.
4
5
b
A
1
Vitamin A - ug13ml Final Solution FIG.
2.
Relationship
between
absorbance
and concentration
of Vitamin
A.
VITAMIN
A DETERMINATION
Vitamin
Animal NO.
not
detected
in any
285
TCA
TABLE 1 A in Sheep Seruma
Deficient state bg/lOO ml)
After grazing for 1 month
5.0 11.0 9.0 1.0 5.5 4.5 6.0
29.0 30.5 29.0 24.0 20.0 24.0 25.5
54 1 8 70 37 38 59 0 Carotene
WITH
of those
samples.
ditions (-20”) in the absence of light, gave remarkably consistent results over a period of 4 weeks. A commercial sample of vitamin A concentrate assayed by the SbCl, and TCA reagents gave similar results: 8930 I.U.,/gm (SbCl,) and 8725 I.U./gm (TCA). When the TCA reagent was allowed to stand with daylight excluded, no differences were observed in the absorbance of standard solutions at zero time and 5 hr. However, optical density values showed a tendency to decrease over extended periods of time up to 24 hr. The decline in absorbance was more striking when the reagent was exposed to bright diffuse light over several hours, ultimately reaching zero, and showing no color for the reaction. To date, the method has been applied to the determination of vitamin A in animal serum and tissues, both normal and deficient. A selection of results of such materials from a series of more than 100 analyses, is reported in Tables 1 and 2 to show the capability of the method. Vitamin Animal No. 26 81
(i)
20 81 (ii) 9 48
a Vitamin
A separated
TABLE 2 A in Pig Livera
Remarks
a/gm 13.2 17.8 19.9 103.9 59.7 2.9
by
chromatography
Piglet Piglet Piglet Mature pig Mature pig Mature pig (suspected deficiency) before
assay
with
TCA.
286
R. F. BATFIELD
The method allows good duplication, which results shown in Table 3 for an experimental When the components of sheep serum were matography, th:> only one which gave the responding to true vitamin A, the location chromatography of the pure vitamin. TABLE
Serum Vitamin Class Weaners
3
A in Experimental
1
18.0,M.O 16.0, 13.5 22.0,22.0 22.0,21.0
1 2
Ewes
Sheepa Vitamin Ab bd100 ml)
Group
2
Hoggetts
is illustrated by the set of group of sheep. separated by paper chroblue color was that corof which was checked by
1
16.0, 15.5
2
16.0,
16.0
a Carotene was not detected in the serum from these animals. b Duplicate values determined on pooled samples from each group. DISCUSSION
The wavelength of maximum absorption of the blue colored species produced by the reaction between TCA and vitamin A was similar to that reported for SbCl,, TFA, and other Lewis acids (4). For strong solutions of vitamin A, the color of the reaction mixture was a deep blue, which faded rapidly through mauve to pink, and later to a pale yellow solution. Maximum blue color was reached in approximately 5 sec. These color changes follow closely those obtained with SbCl,. Although the TCA remained stable for several hours under controlled conditions of light, changes were noted in its reactivity towards vitamin A over extended periods of time. This corresponds with observations concerning TFA (4) and suggested that, in agreement with earlier workers (6), the reagent should always be prepared immediately before use. Under these conditions, consistent and successful results were obtained with the TCA reagent over a period of 1 year, as opposed to the mixed results obtained with this reagent by others (1). As noted earlier (6), the reagent may be prepared in light petroleum. However, compared with the color obtained when chloroform is used as solvent, there is a loss in intensity of about 607’&, which of course precludes its use for quantitative purposes. Interference
from
carotenoids
and
other
substances
with
the
Carr-
VITAMIN
A DETERMIR’ATIOn’
WITH
TCA
287
Price reagent has been reported (2,9). In this laboratory, highly colored solutions containing carotenoids resulted in difficulty in determining the point of maximum deflection, there being a slow drift from low to higher optical density values. This occurred when either the SbCl, or the TCA reagent was used, and would suggest that care is necessary when assessing vitamin A by a gross measurement to which a correction factor is applied. At this stage, therefore, for extracts from natural sources, the method is best confined to mat’erials which are relatively free of carotenoid-like substances. Alternatively, separation of the vitamin A component by chromatography is recommended. The acceptabilit’y of the method rests on the following points: (i) obedience to Beer’s law over the concentration range indicated; (ii) the excellent comparison obtained in absorbance in standard curves prepared with vitamin A est’ers or alcohol using either TCA or the conventional SbCl, as reagent; (iii) analysis of known pure and commercial samples of vitamin A to acceptable standards; (iv) repeatability of results from standards maintained under acceptable conditions of storage; (v) serum vitamin A levels similar to the normal range for grazing animals (10) (Table 1) and the good degree of duplication (Table 3) ; and (vi) chromatographic verification of vitamin A as the principal colorproducing component in composite serum extracts. SUMMARY
Vitamin A was determined in pure samples and animal tissues using trichloroacetic acid as the chromogenic agent’. Results were similar to those obtained with SbCl,, but the TCA reagent is generally more convenient to use and is less toxic. REFERENCES 1. CARR,
F. H., AND PRICE, E. A., Biochem. J. 20, 497 (1926). A. E.. AND SNOW. S. D.. J. Bill. Chem. 171, 617 (1947). P., Proc. Sot. Exptl. Biol. Med. 84, 148 (1953). DUGAN, R. E., FRIGERIO, N. A., AND &BERT, J. M., Anal. Chem. 36, 114 (1964). NEELD, J. B., JR., AND PEARSON, W. N., J. Nut?. 79, 454 (1963). ROSENHEIM. O., AND DRUMMOND, J. C.. B&hem. J. 19, 753 (19%). OLSEN, J. E., Vitumins Hormones (N. Y.) 26, 9 (1968). BAYFIELD. R. F., FALK, R. H., AND BARRETT, J. D.. J. Chromatog. 36, 54 (1968). AVAMPATO, J. E., AND EATON, H. D.. J. Dairy Sci. 26, 783 (1953). PEIRCE, A. W., Australian J. Exptl. Biol. Med. Sci. 24, 231 (1946).
2. SOBEL, 3. FLESCH, 4. 5. 6.
7. 8. 9.
10.