| heal.abstract |
In the current paper, a method is introduced to determine lactoferrin in sweet whey using reverse-phase HPLC without any pretreatment of the samples or use of a separation technique. As a starting point, the most common HPLC protocols for acid whey, which included pretreatment of the whey along with a sodium dodecyl sulfate-PAGE step, were tested. By skipping the pretreatment and the separation steps while altering the gradient profile, different chromatographs were obtained that proved to be equally efficient to determine lactoferrin. For this novel 1-step reverse-phase HPLC method, repeatability was very high over a wide range of concentrations (1.88% intraday to 5.89% interday). The limit of detection was 35.46 μg/mL [signal:noise ratio (S/N) = 3], whereas the limit of quantification was 50.86 μg/mL (S/N = 10). Omitting the pretreatment step caused a degradation of the column's lifetime (to approximately 2,000 samples). As a result, the lactoferrin elution time changed, but neither the accuracy nor the separation ability of the method was significantly influenced. We observed that this degradation could be easily avoided or detained by centrifuging the samples to remove fat or by extensive cleaning of the column after every 5 samples. © 2014 American Dairy Science Association. |
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