Abstract
Alkaline phosphatase (ALP) is an important native enzyme of milk which is an established indicator for testing the adequacy of milk pasteurization. In the present study, a HPLC-UV method was standardized for the determination of ALP activity in pasteurized milk in terms of p-nitrophenol concentration liberated from the enzyme substrate di-sodium p-nitrophenyl phosphate. p-Nitrophenol was extracted by clarification, centrifugation, evaporation, and solvent exchange. The analyte was separated by a reversed-phase C18 column using an isocratic mobile phase composed of distilled water and acetonitrile (1:1) in a low-pressure gradient elution mode with a total run time of 12 min. The UV detection was achieved at a wavelength of 319 nm. Values obtained for different validation parameters confirmed that the method was fit to serve the purpose because of good specificity, linearity demonstrated under various experimental conditions (r 2 > 0.9), very low limit of detection (LOD, 0.024 mg/L) and limit of quantitation (LOQ, 0.072 mg/L), accuracy (>86 %), and precision (percent relative standard deviation (% RSD) < 6.152). Validation studies also clearly demonstrated the ability of the HPLC method to detect as low as 0.1 mg/L of p-nitrophenol and 0.0006 % of raw milk. Laboratory evaluation of the HPLC method in comparison with an international reference method (ISO method) showed that the HPLC method has advantages over the ISO method due to lower LOD and LOQ, ability to detect very low levels of raw milk contamination, and comparatively high accuracy and precision.
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References
Almasi A, Fischer E, Perjesi P (2011) HPLC quantification of p-nitrophenol and its conjugated metabolites from bile. Sci Pharm 79:837–847
Aschaffenburg R, Mullen JEC (1949) A rapid and simple phosphatase test for milk. J Dairy Res 16:58–67
ATSDR (1992) Toxicological profile for nitrophenols: 2-nitrophenol and 4-nitrophenol. ATSDR. U.S. Public Health Service 50
Dimou AD, Sakellarides TM, Vosniakos FK, Giannoulis N, Leneti E, Albanis T (2006) Determination of phenolic compounds in the marine environment of Thermaikos Gulf, Northern Greece. Intern J Environ Anal Chem 86:119–130
EU (2002) Commission decision of 12th August, 2002 implementing council directives 96/23/EC concerning the performance of analytical method and interpretation of results. Off J European Commu L221/8
GonzalezToledo E, Prat MD, Alpendurada MF (2001) Solid-phase microextraction coupled to liquid chromatography for the analysis of phenolic compounds in water. J Chromatogr A 923:45–52
Haynes WM (2014) CRC handbook of chemistry and physics, 94th edn. CRC Press LLC, Boca Raton, p 172
ISO (2004) Milk and dried milk, buttermilk andbuttermilk powder, whey and wheypowder-Detection of phosphatase activity ISO/TS 6090:2004(E) IDF/RM 82:2004(E), 1st edition International Organisation for Standardisation, Switzerland
ISO (2007) Milk and milk products—determination of alkaline phosphatase activity. Part I: fluorimetric method for milk and milk-based drinks. ISO 11816–1 IDF 155–1, 2nd Edn. International Organization for Standardization, Switzerland
NIST (2015) Phenol, 4-Nitro-, NIST webbook. Available at http://webbook.nist.gov/cgi/cbook.cgi?ID=C100027&Units=SI&Mask=400#UV-vis-Spec. Accessed on 24.3.2015
O’Keefe M (2000) Residue analysis in food: principles and applications. Harwood Academic Publishers, The Netherlands, p 5
Payne C, Wilbey R (2009) Alkaline phosphatase activity in pasteurized milk: a quantitative comparison of Fluorophos and colourimetric procedures. Int J Dairy Technol 62:308–314
Prescott LM (1999) Microbial nutrition, growth and control and microbial diseases and their control. In: Prescott LM, Harley JP, Klein DA (eds) Microbiology. WCB/McGraw Hill, Boston, pp 107–110
Rocco RM (1990) Fluorometric determination of alkaline phosphatase in fluid dairy products: collaborative study. J AOAC 73:842–849
Rocco RM (2004) Alkaline phosphatase methods. In: Wehr HM, Frank JF (eds) Standard methods for the examination of dairy products, 17th edn. APHA, Washington, pp 341–362
Scharer H (1938) A rapid phosphomonoesterase test for control of dairy pasteurization. J Dairy Sci 21:21–34
Van Bever AK (1943) The influence of temperature and pasteurizing time upon the enzymes and bacteria in milk. Enzymologia 11:7–18
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No funding. (The research paper is based on a postgraduate research work of students of the Department of Veterinary Public Health and Epidemiology, LUVAS, Hisar, India—125004. Grants were not provided, specifically, for this research work.)
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The authors declare that they have no competing interests.
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The present research work was conducted on milk samples, and animals or human subjects were not used at any stage of experimentation.
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This study is part of a M.V.Sc thesis submitted to Lala Lajpat Rai University of Veterinary and Animal Sciences, Haryana, India.
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Lakra, S., Jadhav, V.J. & Garg, S.R. Development of a Chromatographic Method for the Determination of Alkaline Phosphatase Activity in Pasteurized Milk. Food Anal. Methods 9, 2002–2009 (2016). https://doi.org/10.1007/s12161-015-0380-7
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DOI: https://doi.org/10.1007/s12161-015-0380-7