Mikrochimica Acta [Wien] 1979 I, 81--86 9 by Springer-Verlag 1979

Department of Vegetable Crops, Punjab Agricultural University, Ludhiana, India

Colorimetric Determination of Capsaicin in Capsicum Fruits With the Folin-Ciocalteu Reagent

By

K. L. Bajaj and Gurdeep Kaur

(Received February 14, 1978)

Scvcral colorimctric methods 1-12 for determination of capsaicin, the pungent principle in capsicum fruits, are based on various extractions and use of chromogenic reagents which involve the phenolic group of capsaicin. Most of the colorimetric methods suffer from instability of the colour developed and from turbidity formation 13. The pigments and other interfering substances present in capsicum fruits should be removed in all colorimetric methods 11, but in some this is not done 1,4,s,lz and in others elaborate procedures are used which make them inconvenient for routine analysis~, 3. Methods involving diazonium salts as reagents require the preparation of these in situ 5, v on account of their instability. Some of the methods which involve the measurement of spot size or visual comparison of colour are only semi-quantitative 7,1~ Recently a method using VOC13 has been reported 12 in which the colour formed was stable for only 1--1.5 min, which is rather a short for a colorimetric method. The present communication describes a colorimetric method in which intcrfering substances are removed on a basic alumina column and capsaicin is determined by means of the Folin-Ciocalteu reagent which forms a highly stable blue colour.

Experimental
Reagents Folin-Ciocalteu reagent. To 700 ml of water add 100 g of sodium tungstate (Na2WO4 "2H20), 25 g of sodium molybdate (Na2MoO4 92H20), 50 ml of 85% phosphoric acid and 100 ml of conc. hydrochloric acid.
6 M i k r o c h i m . Acta 1979 I/1~2

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K.L. Bajaj and G. Kaur: Table I. Total interfering substances + capsaicin, %

Extraction procedure

(Reference)

Aqueous methanol, 50% . . . . . . . . . . . . . . . . Acetone (refluxing for 1 hr) . . . . . . . . . . . . . Acetone (refluxing for 1 hr)* . . . . . . . . . . . . Acetone (shaking for 10 min)** . . . . . . . . . . . Aqueous ethanol, 70% . . . . . . . . . . . . . . . . . . Ethyl acetate** . . . . . . . . . . . . . . . . . . . . . . . . .

(10) (13) (9) (1, 4, 8) (5, 7) (12)

1.76 2.00 1.02 0.68 1.23 0.70

* Further purification by Norit treatment (Ref. 9) ** Recommended procedures.

Reflux for 10 hr. Add 150 g of lithium sulphate (Li2SOa'H20), 50 ml of water and 3 drops of bromine. Boil off the excess of bromine. Cool and dilute to 1 litre with water. Filter and store in a brown bottle.

Saturated sodium carbonate solution. To 100 ml of water add 35 g of anhydrous sodium carbonate, dissolve at 70--80 o C and allow to cool overnight. Seed the supersaturated solution with crystals of Na2COa" 10H20 and after the crystallisation is complete, filter the mother liquor through glass wool. Standard capsaicin solution. Dissolve 10 mg in 100 ml of methanol. Solvents. Dried and distilled before use. Calibration curve. Take 0--1.5 ml (in appropriate steps) of standard capsaicin solution in small beakers. Evaporate to less than 0.5 ml, if necessary, at a temperature below 650 C (methanol interferes if more than 0.5 ml is present). Add 0.5 ml of Folin-Ciocalteu reagent and 6.5 ml of distilled water, let stand for 3 min, add 1.0 ml of saturated sodium carbonate solution and mix well. Transfer quantitatively to 10-ml standard flasks, and dilute to the mark with water. Measure the absorbance at 760 nm after 1 hr at room temperature. Method
E x t r a c t 0.5 g of dried, p o w d e r e d ( 3 0 - 4 0 mesh) c a p s i c u m fruits with 25 ml of a c e t o n e or 25 ml of ethyl acetate. If a c e t o n e is used, shake the m i x t u r e for 10 rain, let stand for 2 hr and then filter t h r o u g h a g l a s s - w o o l plug in a s h o r t - s t e m m e d funnel. If ethyl acetate

Colorimetric Determination of Capsaicin in Capsicum Fruits Determination of Capsaicin Capsaicin, Min. % Average By other methods (Reference)

83

By present method Max.

Interfering substances, %

Not applicable 0.67 0.46 0.52 0.51 0.53 Not applicable 0.52 0.54
0.63

0.65 0.50 0.52 0.53

0.55 0.50 0.51 0.51 0.55 0.51

(6) (7) (7) (8) (5) (12)

1.21 1.50 0.52 0.16 0.78 0.17

is used, keep the extract at r o o m temperature for 24 hr, then filter as for acetone extracts. Make up to 25 ml. Use 2 g of material for low capsaicin content (less than 0.1%). Pass 2 ml of the extract through a basic alumina column (Brockmann grade I, 80--200 mesh), 1.5 g in a 10.0 0.9 cm column, washed with 5 ml of acetone. Wash the column with three 5-ml portions of acetone after loading. Elute pure capsaicin with methanol-acetone-water mixture (75 : 25 : 1) 13, collecting 50 ml. Evaporate 10 ml (or other suitable volume) to dryness (temperature ~<65 ~ C) and develop the colour as for the calibration curve.
Calculation

If the absorbance of 10 ml of eluate from the column corresponds to x mg of capsaicin, then 0.5 g of capsicum fruit contains 62.5 x mg of capsaicin, i. e. 1.25 x %.

Results and Discussion

Capsaicin was extracted by various methods reported in the literature, and its content was determined by the present and earlier methods. The results are given in Table I (averages of six replicates). The sum of capsaicin + interfering substances was determined as capsaicin prior to column chromatography, and the pure capsaicin content was determined after the chromatography. The data show that m a x i m u m extraction of interfering substances was
6*

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K.L. Bajaj and G. Kaur:

achieved by refluxing for 1 hr with acetone, and the minimum by using shaking with acetone for 10 rain or extracting with ethyl acetate. The results also show that some of the methods which do not involve prior removal of interfering substances do not give the true value of capsaicin content. The main disadvantage with the VOCI3 method 12 seemed to be instability of the colour and susceptibility towards moisture, making it difficult to get reproducible results. The results show that chromatography of the acetone or ethyl acetate extracts on a basic alumina column gives correct and consistent results. Extracts containing large quantities of interfering substances are not suitable for this method, however, since they give high results, probably because of elution of some other pigments along with capsaicin. Correct capsaicin values were obtained when these pigments were partially removed by treatment with Norit 9. However, this method is not recommended since the amount of Norit to be added for clarification of the extract has been found to be very critical. The recovery of capsaicin from the alumina column was found to be 100 _+0.6%, in close agreement with that reported earlier for purification of capsaicin prior to its GLC deterruination la. If 50% aqueous methanol or 70% aqueous ethanol is used for extraction, the capsaicin cannot be purified in this way, owing to inactivation of the alumina. It is concluded that only the acetone or ethyl acetate extract can be purified by using a basic alumina column 9,13. With phosphomolybdic acid or Folin-Denis reagent, formation of a turbidity was the main problem, causing irreproducible results. Reproducible results were obtained with the Folin-Ciocalteu reagent, which gave a blue colour stable for 48 hr, without turbidity formation. The method is quite sensitive, i. e. 0.5 #g/ml can be detected. The coefficient of variation found was 1.7%. Analysis of variance of the capsaicin content found by the present method and other methods indicated no significant difference between the results. It is recommended that capsaicin should be extracted from capsicum fruits at room temperature by shaking with acetone for 10 min or standing with ethyl acetate for 24 hr, and purified on a basic alumina column; the Folin-Ciocalteu reagent should be used for colour development. Acknowledgement This work was done as part of the All India Coordinated Project for Improvement of Vegetable Crops, financed by the Indian Council of Agricultural Research, New Delhi, India.

Colorimetric Determination of Capsaicin in Capsicum Fruits

85

Summary
Colorimetric Determination of Capsaicin in Capsicum Fruits With the Folin-Ciocalteu Reagent
A colorimetric method for determination of capsaicin in capsicum fruits is described. T h e acetone or ethyl acetate extract (prepared at r o o m temperature) is purified on a basic alumina (Brockmann Grade I) column and pure capsaicin is eluted with methanol-acetonewater (75 : 25 : 1); the capsaicin content is determined after colour development with Folin-Ciocalteu reagent, the absorbance being measured at 760 nm. T h e m e t h o d is reproducible and results are in good agreement with those obtained by existing methods.

Zusammenfassung
Eine kolorimetrische Methode zur Bestimmung yon Capsaicin in Paprikafriichten wurde beschrieben. Der bei Zimmertemperatur gewonnene Aceton- oder Essigesterextrakt wird in einer Sfiule aus basischem Aluminiumoxid (Brockmann I) gereinigt und das reine Capsaicin durch Farbreaktion mit Folin-Ciocalteu-Reagens und Messung bei 760 nm durchgefiihrt. Die reproduzierbaren Ergebnisse stimmen mit denen anderer Methoden gut iiberein.

References
1 K. Csedo, M. P. Horvfith, and S. Nagy, Orvose Szemle 6, 235 (1960); Chem Abstr. 54, 23185 a (1960). 2 H. North, Analyt. Chemistry 21, 934 (1949). 3 A. S. L. Tirimanna, Analyst 97, 372 (1972). 4 L. Quagliotti, Hort. Res. 11 (2), 93 (1971). 5 R. Adamski and A. Socha, Farm. Pol. 23, 603 (1967); Chem. Abstr. 68, 98677 c (1968). 6 W. Debska and B. Okulicz-Kozarynowa, Chem. Analit. (Warsaw) 18, 291 (1973); Chem. Abstr. 79, 83497 x (1973). 7 M.S. Karawya, S.I. Balbaa, A.N. Girgis, and N . Z . Youssef, Analyst 92, 581 (1967). 8 j. Jonczyk, Herba Pol. 13 (3), 120 (1967); Chem. Abstr. 69, 12908 g (1967). 9 j.I. Suzuki, F. Tausig, and R. E. Morse, Food Technol. 11, 100 (1957). l0 A. Laszlo and M. Laszlo, Acta Aliment. 4 (2), 113 (1975); Analyt. Abstr. 30, SF 38 (1976).

86 K.L. Bajaj et al.: ColorimetricDetermination of Capsaicin in CapsicumFruits 11 p. Spany~ir and M. Blazovich, Analyst 94, 1084 (1969). 12 j. j. R. Palacio, J. Assoc. Offic. Analyt. Chem. 69, 970 (1977). 13 j. j. DiCecco, J. Assoc. Offic. Analyt. Chem. 59, 1 (1976).

Correspondence and reprints: Dr. K. L. Bajaj and Mrs. Gurdeep Kaur, Department of Vegetable Crops, Punjab Agricultural University, Ludhiana-141004, India.