Method Development for the Quantitative Determination of Lactulose in Heat-Treated Milks by HPAEC with Pulsed Amperometric Detection

Abstract

A robust, rapid, and sensitive high-performance anion-exchange chromatographic method for the separation and quantitative determination of lactulose in heated milks, along with other common milk carbohydrates, has been developed. Complete separation of galactose, glucose, N-acetylgalactosamine, lactose, lactulose, and epilactose was isocratically accomplished in about 22 min by an anion-exchange column eluted with 10 mM NaOH spiked with 2 mM Ba(OAc)2. The within-day repeatability was lower than 2.1% for 10 repetitive injections. Under optimized conditions, there was no need either of post-column addition of strong bases to the eluent for enhancing detection sensitivity or, even more important, for column regeneration between chromatographic runs. Upon 100-fold sample dilution, the amperometric response of lactulose in milk samples was found to be linear up to 100 microM (r = 0.99935) with a limit of detection equal to 1.2 microM (S/N = 3). The lactulose content in ultrahigh-temperature (UHT) and sterilized milks was evaluated by a calibration graph using 2-deoxyglucose as the internal standard, making the proposed method very useful in discriminating among heat-treated milks. Whereas the mean value of lactulose in skimmed, partially skimmed, and whole UHT milks ranged from 10 to 90 mg/100 mL, lactulose content in bottle-sterilized whole milk (two samples) was higher than 140 mg/100 mL. The presence of epilactose, which is another isomer of lactose, was also ascertained in sterilized milk.

Advances in Fractionation and Analysis of Milk Carbohydrates

“…The sensitive and accurate quantification of lactulose in sterilized milk by HPAEC-PAD has been demonstrated, and it makes the differentiation between sterilized and pasteurized milks possible [67]. The separation of monosaccharides (glucose, galactose, and fructose) and disaccharides (lactose, lactulose, epilactose, sucrose, and maltose) in dairy products [68,69] and infant formulas [70,71] by HPAEC-PAD has been described. The quantitative determination of NANA present at the terminal position of many glycoprotein and glycolipid-oligosaccharides by HPAEC-PAD has been reported by Rohrer [72].…”

A review of pulsed electrochemical detection following liquid chromatography and capillary electrophoresis

“…Since the introduction of PED on a microchip Fanguy and Henry [59] in 2002, applications utilizing the technique have included the determination of simple sugars [60,84,85], underivatized amino acids and sulfur-containing antibiotics [60,86] as well as the detection of renal function markers such as creatinine, creatine, and uric acid [61]. Several reviews describing applications of CE coupled with PED have been published recently [55,56,[87][88][89].…”

Amperometric detection of carbohydrates based on the glassy carbon electrode modified with gold nano-flake layer

“…The HPLC analysis usually has to follow the clean-up techniques using precipitating agents e.g., carrez to remove the proteins off milk samples [19][20][21]. By using only carrez agents similar to Cataldi et al [21], the non-oxidative PAD and the nano-Au/GC/ECC were able to maintain their activity throughout the following HPLC detection of lactose in this milk sample. The chromatographic peak areas of lactose showed a good reproducibility with $2% RSD for 6 consecutive injections (Fig.…”