Sweeteners HPLC methods

CF, colors preservatives, sweeteners HPLC method. Column Altex Ultrasphere-TM-ODS. Mobile phase ammonium acetate -MeOH. 

HPLC method for simultaneous determination in intense sweeteners in foodstuffs. 

To provide a HPLC method for the simultaneous determination of permitted sweeteners in a range of foodstuffs. 

The purpose of this section is to provide a review of HPLC methods available for the determination of sweeteners in foods. First, general information of the various modes of HPLC and on sample preparation procedures available for the determination of intense sweeteners is described. Then information is given on each individual sweetener. 

Since cyclamate has poor UV absorbing characteristics, HPLC methods for the analysis of this sweetener require specific detection systems, such as indirect photometry or conductivity. Herrmann et al. (24) used indirect photometry for the detection of cyclamate at 267 nm against a UV-absorbing mobile-phase component, p-toluenesulphonate. Biemer (17) and Wu et al. (47) used a conductivity detector for the determination of cyclamate. According to Biemer (17) the use of this detector offers distinct advantages, since compounds coeluting with cyclamate may not exhibit an electrochemical response and, hence, not appear in the chromatogram. 

Table 3 HPLC Methods for the Simultaneous Determination of Intense Sweeteners... 

MacArthur, R. et al. (2002) Development and validation of an HPLC method for simultaneous determination of intense sweeteners in food stuffs (A01012), DEFRA Central Science Laboratory, York. 

Benzoic and sorbic acids are now normally assayed using HPLC. As discussed in the section on the analysis of sweeteners, some of the HPLC methods developed for soft drinks actually allow the separation of both sweeteners and preservatives in one ran, for example, Williams (1986), Hagenauer-Hener et al., (1990) and the EU method for sweeteners (Anon, 1999a), although the preservatives were not included in the collaborative trial of the method. The separation of benzoic and sorbic acids can sometimes be difficult and care should be taken that the system will actually resolve these two preservatives if they are present otherwise spurious results can be obtained. The pH of the solvent is a critical feature that allows the separ ation of these two preservatives. 

The analytical methods proposed for acesulfame-K, cyciamate, and saccharin determination in foods, drinks, dietary products, and pharmaceuticals can be grouped into methods for the determination of an individual artificial sweetener [21-27] and multianalyte approaches [28-38], sometimes also including other sweeteners and/or other food additives, such as colorants or preservatives [39-43]. High-performance liquid chromatography (HPLC) is the most frequently used technique for the determination of these sweeteners, and this is selected by international standard methods because of its multianalyte capability, compatibility with the physicochemical properties of sweeteners, high sensitivity, and robustness [44-47]. However, cyciamate requires chemical derivatization to make it detectable by the most commonly employed UV-absorption detector due to a lack of a chromophore, by conversion to dichlorohexylamine for UV detection or to a fluorescence derivative for fluorimetric detection. Another alternative for cyciamate detection is the postcolumn ion-pair extraction where the eluted sweetener is mixed with an appropriate dye (methyl violet or crystal violet), being detected by visible absorption. Furthermore, cyciamate can be detected directly by refractive index [4]. For this, few HPLC methods for the concurrent determination of these sweeteners exist and... 

Scotter, M. J., L. Castle, D. P. T. Roberts, R. MacArthur, P. A. Brereton, S. K. Hasnip, and N. Katz. 2009. Development and single-laboratory validation of an HPLC method for the determination of cyclamate sweetener in foodstuffs. Food Addit. Contam. A 26 614-622. 

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

Since the publication of the first edition of this book, a few more validated methods for the analysis of soft drinks ingredients have been documented. When the first edition was published in 1998, only a handful of methods for the analysis of soft drinks ingredients had been collaboratively tested in the Association of Official Analytical Chemists (AOAC) official methods manual, and only two of these were modern HPLC approaches. At that time, no methods could be found in the British Standards catalogue. Inspection of the British Standards website (http //www.bsi-global.com) now shows that there are two standardised approaches for the analysis of high-intensity sweeteners in soft drinks, both of which use HPLC. This overall lack of standardisation of methods is probably because a soft drink s matrix is relatively straightforward, without many of the problems associated with other areas of food analysis, and so the industry has not felt the need to standardise the test methods. 

There are a large number of other published procedures for the separation of a number of sweeteners and preservatives at one time these are all based on reverse-phase HPLC. Perhaps one of the most startling is the method published by Williams (1986). This uses a small particle size (3 xm) C8 column and allows the separation of a range of colours, sweeteners and preservatives in less than 5 min. The materials separated were amaranth, quinoline yellow, quinine sulphate, sunset yellow, caffeine, aspartame, saccharin, vanillin, sorbic acid, benzoic acid and green S. 

The very commonly used sorbic acid has been detected with low recoveries in raw beef (52-84%) by photometry, after distillation and extraction. Better recoveries ( 100%) and less manipulation have been achieved by the fluorescence technique in determination of 4-hydroxybenzoic methyl ester in nonalcoholic beverages. Additives can be present in combinations, and chromatographic methods are often used for selective individual or joint determination. Sorbic and benzoic acids have also been determined by thin-layer chromatography in beverages. This method involves minimal sample manipulation. HPLC is often the preferred method for determining additives present in mixtures, which are usually only just volatile. Simultaneous determination of additives, such as sweeteners, preservatives, and colorings in soft drinks is usually done by HPLC with UV... 

HPLC with reversed-phase (RP) or ion-pair RPLC and UV detection is the most popular choice for the determination of aspartame, saccharin, and acesulfame-K. However, the lack of a chromophore in the cyclamate molecule makes its determination difficult using the common HPLC-UV detection mode. A high-performance anion-exchange chromatographic method with UV and conductivity detectors connected in series offers an attractive alternative for simultaneous determination of the four sweeteners and also citric acid. 

Sweeteners used in the food industry typically are limited to the bulk sweeteners, sucrose, fructose, glucose, and com syrups, or the high potency sweeteners, saccharin, aspartame, sucralose, and acesulfame k. While various enzymatic and colorimetric methods may be used, high performance liquid chromatography is the most commonly used technique in this analysis [101]. HPLC offers speed, sensitivity, accuracy, and precision to the analyst. Several types of HPLC columns (anion and cation... 

Saccharin is also determined with FIA methodologies, which allow multianalyte determination. These methodologies include methods cited previously for acesulfame-K and/or cyclamate a method based on electrochemical detection of these three artificial sweeteners using stabilized systems of filter-supported BLMs [72] online dialysis for sample pretreatment prior to the simultaneous determination of saccharin and caffeine, benzoic acid, and sorbic acid by HPLC-FID [76] and molecular spectroscopic methods in the UV region based on the transient retention of analytes on solid phases, silica Cjg [74], and quaternary amine ion exchanger [75]. 

An efficient and accurate analytical method was developed for the simultaneous determination of 20 synthetic food additives using HPLC with a photodiode array detector (20). These additives include sweeteners, food colorants, S mthetic preservatives and caffeine. 

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