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Optimization and Validation of a Rapid Method to Determine Citrate and Inorganic Phosphate in Milk by Capillary Electrophoresis

Dairy Products Technology Center California Polytechnic State University San Luis Obispo, 93407

Corresponding author:
R. Jimenez-Flores; e-mail: rjimenez{at}calpoly.edu.

Quantification of phosphate and citrate compounds is very important because their distribution between soluble and colloidal phases of milk and their interactions with milk proteins influence the stability and some functional properties of dairy products. The aim of this work was to optimize and validate a capillary electrophoresis method for the rapid determination of these compounds in milk. Various parameters affecting analysis have been optimized, including type, composition, and pH of the electrolyte, and sample extraction. Ethanol, acetonitrile, sulfuric acid, water at 50°C or at room temperature were tested as sample buffers (SB). Water at room temperature yielded the best overall results and was chosen for further validation. The extraction time was checked and could be shortened to less than 1 min. Also, sample preparation was simplified to pipet 12 µl of milk into 1 ml of water containing 20 ppm of tartaric acid as an internal standard. The linearity of the method was excellent (R² > 0.999) with CV values of response factors <3%. The detection limits for phosphate and citrate were 5.1 and 2.4 nM, respectively. The accuracy of the method was calculated for each compound (103.2 and 100.3%). In addition, citrate and phosphate content of several commercial milk samples were analyzed by this method, and the results deviated less than 5% from values obtained when analyzing the samples by official methods. To study the versatility of the technique, other dairy products such as cream cheese, yogurt, or Cheddar cheese were analyzed and accuracy was similar to milk in all products tested. The procedure is rapid and offers a very fast and simple sample preparation. Once the sample has arrived at the laboratory, less than 5 min (including handling, preparation, running, integration, and quantification) are necessary to determine the concentration of citric acid and inorganic phosphate. Because of the speed and accuracy of this method, it is promising as an analytical quantitative testing technique.

Key Words: citric acid • phosphate • milk • capillary electrophoresis

Abbreviation key: CCP = calcium colloidal phosphate, CE = capillary electrophoresis, CTAB = hexadecyltrimethylammonium bromide, f = response factor, IS = internal standard, KHP = potassium hydrogen phthalate, MWCO = molecular weight cut off, PDC = 2,6-pyridinedicarboxylic acid, p.s.i. = pound square inch, SB = sample buffer

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