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New micro test for vitamin A
CLINICA
NEW
393
CHIMICA ACTA
MICRO TEST
S. TH.4BET
IND OLYMPIA
Chemistry Defxrtment, Beirut (Lebanon) {Received
FOR
VITAMIN
A
TABIBIAN
American
University ofBeirut,
May zgth, 1965)
SUMMARY
o-Aminophenol reacts with vitamin A aldehyde (retinal) in acid medium to produce an intense red colour. A method is proposed for the micro detection of vitamin A (retinol) through its conversion to the corresponding aldehyde. B-Carotene does not interfere.
B-Carotene interferes with the detection and determination of vitamin A. A method is proposed whereby a test responds to vitamin A and not to b-carotene. The test is based on an observation that vitamin A aldehyde (retinal) reacts with o-aminophenol in an acidic medium to form an intense red colour. Sondheimer et al.’ reported that manganese dioxide is a selective oxidizing agent for allylic alcohols converting them to the corresponding carbonyl compounds. In the proposed test, vitamin A (retinol) is converted with manganese dioxide to the corresponding a-/l unsaturated aldehyde (retinal) which in turn reacts with o-aminophenol in an acidic medium to produce an intense red colour. The colour formed could be due to a combined effect of an extended conjugation involving nine double bonds in the Schiff base and to resonance. p-carotene does not interfere. EXPERIMENTb4L
Reagents I. Saturated solution of freshly sublimed o-aminophenol in chloroform. 2. Solid manganese dioxide freshly prepared and stored in a desiccator. The manganese dioxide may be prepared* by placing 55 g of manganous sulfate monohydrate and 150 ml of distilled water in a 2-1, three-neck flask equipped with a stirrer. The mixture is heated with stirring to 86” and a solution of 60 g of potassium permanganate in 400 ml of water (75-85O) is added in portions of 30-35 ml over about an hour and a half keeping the temperature at 75-85”. After completion of the addition the mixture is heated with stirring for 20 min more and filtered while hot. * E. P. Papadopoulos
and C. H. Issidorides,
personal
communication. Clin. Chim. Acta, 13 (1966) 393-394
S. THABET,
394
0. TABIBIBN
The solid is washed with distilled water until the reaction for sulfate in the filtrate becomes quite faint. The solid is then dehydrated in the oven for 48 h at IIO-IZO’. An easily powdered brown solid is obtained. 3. Chloroform. 4. Concentrated hydrochloric acid. Procedure
To one drop of the vitamin A solution in a centrifuge tube add very little solid manganese dioxide. The test tube is placed in a water bath at 60-80” for at least 6 min with intermittent additions of a few drops of chloroform. For limit determination this period is increased to IO min. One to 2 ml of chloroform are then added and the solution well centrifuged. The clear solution is pipetted off and boiled down to I-Z drops; 2 drops of saturated o-aminophenol are then added; the solution heated for about IO set in a water bath followed by the addition of 5-6 drops of concentrated hydrochloric acid. A red colour depending on the concentration of the vitamin is obtained which is extractable in chloroform. A better separation of the colour is obtained if a few drops of water are added. Limit of identification:
4 ,ug vitamin A.
1: 12 500. The limit of identification was made on a standard solution of vitamin A the purity of which was determined by measuring the optical density of the solution at a wavelength of 324-326 m,u. The vitamin was found to be 77.7% pure. The limit of identification for vitamin A aldehyde (retinal) using the above procedure is 0.5 ,ug. This suggests that the conversion of the retinol to the corresponding aldehyde is not complete. A better sensitivity would therefore be obtained if complete oxidation of the retinol were achieved. With the above procedure the following results are obtained: Limit of dilution:
Vitamin 400 /G
A
COlOW deep violet red pink
40 iug 4 P”g
red
p-carotene
Colour
500 Leg 50 Pg
pale yellow pale yellow pale yellow
5 0%
A mixture consisting of a drop of 4 ,ug vitamin A in the presence of a drop of 500 pg B-carotene responds to the test indicating that 4 ,ug vitamin can be detected in the presence of 125 times its concentration of p-carotene. In addition to retinal the following E-B unsaturated aldehydes were treated with o-aminophenol as described above : acrolein, crotonaldehyde, tiglaldehyde, citral, z-hexen-r-al and cinnameldehyde. Only cinnameldehyde gave a reaction similar to retinal. ACKNOWLEDGEMENT
One of us (O.T.) wishes to thank the Arts and Sciences Research Fund of the American University of Beirut for financial support. REFERENCES I F. SONDHEIMER,O.MANCERA,M.URQUI~AANDG.ROSENKRANZ,J. 4145. Clin.
Chim.
Acta,
I.? (1966) 393-394
Am.Chem..Soc.,
77(1955)
NEW
393
CHIMICA ACTA
MICRO TEST
S. TH.4BET
IND OLYMPIA
Chemistry Defxrtment, Beirut (Lebanon) {Received
FOR
VITAMIN
A
TABIBIAN
American
University ofBeirut,
May zgth, 1965)
SUMMARY
o-Aminophenol reacts with vitamin A aldehyde (retinal) in acid medium to produce an intense red colour. A method is proposed for the micro detection of vitamin A (retinol) through its conversion to the corresponding aldehyde. B-Carotene does not interfere.
B-Carotene interferes with the detection and determination of vitamin A. A method is proposed whereby a test responds to vitamin A and not to b-carotene. The test is based on an observation that vitamin A aldehyde (retinal) reacts with o-aminophenol in an acidic medium to form an intense red colour. Sondheimer et al.’ reported that manganese dioxide is a selective oxidizing agent for allylic alcohols converting them to the corresponding carbonyl compounds. In the proposed test, vitamin A (retinol) is converted with manganese dioxide to the corresponding a-/l unsaturated aldehyde (retinal) which in turn reacts with o-aminophenol in an acidic medium to produce an intense red colour. The colour formed could be due to a combined effect of an extended conjugation involving nine double bonds in the Schiff base and to resonance. p-carotene does not interfere. EXPERIMENTb4L
Reagents I. Saturated solution of freshly sublimed o-aminophenol in chloroform. 2. Solid manganese dioxide freshly prepared and stored in a desiccator. The manganese dioxide may be prepared* by placing 55 g of manganous sulfate monohydrate and 150 ml of distilled water in a 2-1, three-neck flask equipped with a stirrer. The mixture is heated with stirring to 86” and a solution of 60 g of potassium permanganate in 400 ml of water (75-85O) is added in portions of 30-35 ml over about an hour and a half keeping the temperature at 75-85”. After completion of the addition the mixture is heated with stirring for 20 min more and filtered while hot. * E. P. Papadopoulos
and C. H. Issidorides,
personal
communication. Clin. Chim. Acta, 13 (1966) 393-394
S. THABET,
394
0. TABIBIBN
The solid is washed with distilled water until the reaction for sulfate in the filtrate becomes quite faint. The solid is then dehydrated in the oven for 48 h at IIO-IZO’. An easily powdered brown solid is obtained. 3. Chloroform. 4. Concentrated hydrochloric acid. Procedure
To one drop of the vitamin A solution in a centrifuge tube add very little solid manganese dioxide. The test tube is placed in a water bath at 60-80” for at least 6 min with intermittent additions of a few drops of chloroform. For limit determination this period is increased to IO min. One to 2 ml of chloroform are then added and the solution well centrifuged. The clear solution is pipetted off and boiled down to I-Z drops; 2 drops of saturated o-aminophenol are then added; the solution heated for about IO set in a water bath followed by the addition of 5-6 drops of concentrated hydrochloric acid. A red colour depending on the concentration of the vitamin is obtained which is extractable in chloroform. A better separation of the colour is obtained if a few drops of water are added. Limit of identification:
4 ,ug vitamin A.
1: 12 500. The limit of identification was made on a standard solution of vitamin A the purity of which was determined by measuring the optical density of the solution at a wavelength of 324-326 m,u. The vitamin was found to be 77.7% pure. The limit of identification for vitamin A aldehyde (retinal) using the above procedure is 0.5 ,ug. This suggests that the conversion of the retinol to the corresponding aldehyde is not complete. A better sensitivity would therefore be obtained if complete oxidation of the retinol were achieved. With the above procedure the following results are obtained: Limit of dilution:
Vitamin 400 /G
A
COlOW deep violet red pink
40 iug 4 P”g
red
p-carotene
Colour
500 Leg 50 Pg
pale yellow pale yellow pale yellow
5 0%
A mixture consisting of a drop of 4 ,ug vitamin A in the presence of a drop of 500 pg B-carotene responds to the test indicating that 4 ,ug vitamin can be detected in the presence of 125 times its concentration of p-carotene. In addition to retinal the following E-B unsaturated aldehydes were treated with o-aminophenol as described above : acrolein, crotonaldehyde, tiglaldehyde, citral, z-hexen-r-al and cinnameldehyde. Only cinnameldehyde gave a reaction similar to retinal. ACKNOWLEDGEMENT
One of us (O.T.) wishes to thank the Arts and Sciences Research Fund of the American University of Beirut for financial support. REFERENCES I F. SONDHEIMER,O.MANCERA,M.URQUI~AANDG.ROSENKRANZ,J. 4145. Clin.
Chim.
Acta,
I.? (1966) 393-394
Am.Chem..Soc.,
77(1955)