Elsevier

Analytica Chimica Acta

Volumes 573–574, 28 July 2006, Pages 466-473
Analytica Chimica Acta

New simple spectrophotometric assay of total carotenes in margarines

https://doi.org/10.1016/j.aca.2006.04.017Get rights and content

Abstract

Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of time needed to complete the analysis. The resulting analytical procedure is characterized in details in terms of the figures of merit. The method is sensitive, precise and accurate; for both, standard additions and calibration in soybean oil, recovery ranges between 98 and 102%. For the most accurate analyses the approach of standard additions is preferred but for quick routine analyses this latter can be replaced by the calibration in soybean oil. Limit of detection value (LOD = 3S.D.B/a, where S.D.B is the standard deviation of the blank, “a” is the slope of calibration line) as low as 12 μg TC/100 g was achieved in soybean oil enabling the sensitive detection. Concentration of TC in margarines declared as being coloured with β-carotene (carotene) ranges between 0.3 and 0.9 mg/100 g while in carrot extract-coloured margarine TC is 0.2 mg/100 g.

Introduction

The carotenoids are widespread naturally occuring antioxidants the importance of which is related to their functions. They act as provitamin A and food colorants [1] and are being extensively studied for their potential role in reducing the risk for cancer and other chronic diseases [2], [3]. The quenching of singlet oxygen is the major antioxidative activity of β-carotene [4].

Due to their system of conjugated double bonds carotenoids are extremely reactive and consequently unstable. Precaution steps taken during the isolation and analysis include the protection from light, avoiding the exposure to oxygen, use of antioxidants (e.g. BHT, pyrogallol, vitamin E), operation at reduced temperatures and the need for completing the analysis in the shortest possible time. For foods with a high fat content the saponification is being employed prior to (or after) extraction in order to hydrolyze the carotenoid esters and remove fatty material. Although this optional step facilitates subsequent separation, identification and quantification of carotenes, it prolongs the time of analysis and might also lead to a degradation of carotenoids. Hence the saponification in the analytical procedure should be omitted whenever possible.

Carotenoids are used as colorants [e.g. β-carotene (E160a), annatto dye-stuffs norbixin and bixin (E160b), lycopene, lutein, capsanthin, etc.], in foodstuffs such as beverages, cookies, cereals, margarines, butter, cheese, etc. Natural carotenoid pigments (carrot extract, palm oil, saffron, annato or paprika) or synthetic carotenoids are used to impart yellow colour to margarines. Antioxidant fortified margarine increases the antioxidant status in humans [5].

Quantification of carotenoid colorants in foodstuffs is therefore very important from nutritional, epidemiological and food quality points of view. High performance liquid chromatography (HPLC) is a demonstrated powerful tool in the field of carotenoid research especially in the analysis of complex mixture of carotenoids [6], [7]. For example, HPLC was applied following the saponification and solvent extraction for the analysis of butter and margarine [8] or cheese [9], for the study of margarine following gel permeation isolation step [10] or cheese after SPE [11]. Accelerated solvent extraction was developed to precede HPLC analysis of carotenoids in various foodstuffs [12]. Likewise, photometric determination of β-carotene in butter after saponification and solvent extraction was reported [13].

In this paper we propose a new, simple and direct spectrophotometric method for determination of total carotenes (TC) in margarines. It should be emphasized that in this study the saponification step was for the first time omitted in the analysis of carotenes, leading to cost saving, shortening of analysis time and preservation of carotenes present in the sample. The method is in extenso characterized in terms of analytical performances.

Section snippets

Chemicals, samples and instruments

Betatene® 20% Soy (Cognis Australia Pty Ltd., Cheltenham, Vic., Australia) and β-carotene 30% FS (Roche, Basel, Switzerland) were used as carotene standards. Betatene® 20% Soy is a suspension of a mixture of β- and α-carotene in soybean oil obtained from extracts of the alga Dunaliella salina (λmax 446–448 nm); β-carotene 30% FS is a suspension in vegetable oil (λmax 450–452 nm). Both standards were gifts: the former from Dr. Christina Gaertner from Cognis GmbH, Düsseldorf, Germany, and the

Results and discussion

Our preliminary results revealed that acceptable, time-saving analyses are achievable even after 15 min extraction (with hexane); however, for highly accurate results the extraction process should be prolonged to 30 min (Table 1, Table 2, Table 3, Fig. 1, Fig. 2). Extraction with hexane has already been utilized for determination of β-carotene, vitamin A and vitamin E in butter, margarine or milk prior to photometric [13] or HPLC measurement [8], [14]; however, in all these cases the

Conclusions

Simple, rapid and reliable spectrophotometric method for determination of total carotenes (TC) in margarines is proposed. Unlike other methods, the novel approach eliminates the need for the time-consuming saponification step. The new method is applicable in the analysis of margarines declared as being coloured with carotenes. Both approaches, the standard additions and calibration in soybean oil are sensitive, accurate and precise enough to allow their application for the quality control of

Acknowledgements

The authors thank Mr. T. Smolić and to Mr. Z. Ladešić, Zvijezda, Development, Zagreb, Croatia, for helpful discussions and for providing reference samples and standard.

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Presented at IMA05, October 2–6, 2005, Iraclion, Crete, Greece.

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