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compared the chromatographic and colorimetric methods for HMF determination and found that about 70% of the HMF measured by the colorimetric method was due to interferences. Hence, the TBA method is not a reliable measurement of HMF content, although it is still used as a quick, cost-effective measurement of the heat load of milk products. Since the development of the Keeney and Bassette method (96), a distinction has been made between free HMF and the measurement of the so-called total HMF content described earlier. To determine the latter, the milk sample is heated in 0.3 N oxalic acid to release HMF. Potential HMF is the sum of the HMF precursors (lactulosyllysine, 1-2 enolized products, etc.), plus free HMF. Free HMF is determined by omitting the heating step. Later, Morales et al. (122) described an approach to estimate the potential HMF formed only from the Amadori rearrangement product (ARP) degradation in milk-resembling systems, which could be applied as an indirect determination of the ARP in milk-based products. Spectrophotometric Methods
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Spectrophotometric determinations of HMF can be carried out either by direct measurement of the absorption, or by preparing a derivative (indirect methods). White and Siciliano modi ed the previous Winkler method by recording the absorbance at 284 and 336 nm with a spectrophotometer (45); a plate reader can also be used to expedite measurement (123). However, experimental errors are expected if other compounds that also absorb at 284 nm and react with hydrogensul te are present at appreciable concentrations. In an indirect method, HMF reacts with thiosemicarbazide to give the corresponding thiosemicarbazones that absorb at 322 nm (73). The use of derivative spectra, rst to fourth, and the use of partial least squares multivariate calibration enhance the selectivity and have been applied for the determination of HMF in citrus fruit juices (100, 123). In summary, both the colorimetric and spectrophotometric methods have several drawbacks. They are often tedious, prone to interference especially in strongly colored foods, and employ toxic or hazardous chemicals. Further, they require a strict control of both reaction time and temperature as the instability of the reaction product may lead to low recoveries and hence a high variability in the results. Chromatographic Methods
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Today, LC techniques are preferably used for accurate and reliable measurement of furanic compounds in several food products. These techniques can determine HMF and furfural speci cally, and the formation of a colored derivative is not required because of the strong UV absorption of furfurals at approximately 280 nm. For many analyses, a detection wavelength of 280 nm is chosen because it is located between the maxima of HMF (284 nm) and furfural (277 nm). HMF shows a band at 284 nm (18,000 molar absorptivity) and a less intense band at 230 nm. Reversed-phase LC (RP/LC) methods have
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been widely used to determine the contents of 5-HMF and furfural in many food items, such as apple juices and concentrates (124), commercial brandies and caramels (86), coffee (111), milk (64), infant formulas (20a), breakfast cereals (34), tomato products (81), and jams (6b). HMF is often eluted isocratically with mobile phases containing 5 10% acetonitrile or methanol in water, acidi ed water (0.1% acetic acid), or sodium-acetate buffers (pH 3.6), although gradient elution was reported for coffee and whiskies (7, 20b). Some authors reported a derivative of the 2,4-dinitrophenylhydrazone (DNPH) of HMF in order to enhance both the selectivity and sensitivity of the method in fruit juices and beers (62). The reaction between DNPH and carbonyl compounds is highly speci c. The UV/Vis spectra of HMF hydrazone reveal three maxima at 287, 331, and 352 nm. DNPH should be in excess, and a ratio 1 : 20 is considered suf cient. The derivatives are formed in 25 min and are stable at room temperature for several days. Colored or uorescent hydrazones are detected photometrically in acidic medium or uorimetrically after an extraction step or after dialysis into methanol (125, 126). Yuan and Chen (14) described an ion-exchange LC/PDA (photodiode array detection) approach for the separation of HMF, 2,5-dimethyl-4-hydroxy3(2H)-furanone (DMHF), furoic acid (FA), furfural, 2-acetylfuran, and furfuryl alcohol in several fruit juices. The results of HMF from honey correlated well with the Winkler method, but poorly with RP/LC (cation-exchange LC) (110). Recently, G kmen and Senyuva (22) proposed the use of positive atmospheric pressure chemical ionization/mass spectrometry (APCI/MS) for the analysis of HMF. Characteristic are the precursor ion [M+1] and the analytespeci c ion [C6H5O2] due to loss of water from the protonated molecule. Both ions at m/z of 127 and 109 were used to monitor HMF in the selected ion monitoring (SIM) mode. Furthermore, the ratio of these ions (responses of ions 127/109 = 1.12) con rmed the purity of the HMF peak. Importantly, when using traditional LC/UV methodologies, it is recommended to determine the purity of the HMF peak (19). Capillary Electrophoresis Methods
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Nowadays, capillary electrophoresis (CE) has been proven a powerful and promising technique for food analysis, mainly because of its resolving power, small sample, and buffer requirements. Moreover, CE demands less rigorous sample cleanup, which is advantageous over traditional separation techniques such as LC (113). Electromigration can be conducted in an uncoated fusedsilica capillary with phosphate buffer (50 mM, pH 7.5) containing sodium dodecyl sulphate as electrolyte; separation is achieved within 5 min and HMF can be recorded at 280 nm and con rmed by its UV spectrum (26). Other Methods
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Reyes-Salas et al. (101) described an electrochemical approach for the analysis of HMF in honey. HMF presented a single, well-de ned reduction signal at
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