Total Cholesterol and Desmosterol Contents in Raw, UHT, Infant Formula Powder and Human Milks Determined by a New Fast M
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Total Cholesterol and Desmosterol Contents in Raw, UHT, Infant Formula Powder and Human Milks Determined by a New Fast Micro-HPLC Method Heryka Myrna M. Ramalho & Susana Casal & M. Beatriz Prior Pinto Oliveira
Received: 28 August 2010 / Accepted: 8 November 2010 / Published online: 9 December 2010 # Springer Science+Business Media, LLC 2010
Abstract A simple, fast, sensitive, and environmental friendly micro-method is described for the determination of desmosterol and cholesterol in milk samples using isocratic normal phase high performance liquid chromatography and diode array detection. After sample micro-saponification, the chromatographic separation is achieved in 10 min, further reduced to 6 min when only cholesterol is expected to be present. The method is accurate, presenting cholesterol and desmosterol recoveries >96% and inter-day RSD lower than 3%. Quantification limits were clearly below the requirements for any milk sample, with 40 and 20 μg/100 mL for cholesterol and desmosterol, respectively. Several raw and UHT commercial milk samples, infant powder formulas, and human breast milks were analyzed, with cholesterol and desmosterol (present only in the latter) contents within reported amounts. The proposed method proved to be simple and feasible for determination of a large number of samples, requiring less solvent consumption (9–13 mL of hexane for all saponification/extraction/chromatographic steps) and simpler equipment than most ones reported, and a further possibility of partial solvent recycling due to the isocratic chromatographic mode. Keywords Cholesterol . Desmosterol . Raw and UHT milk . Human milk . Validation . HPLC-DAD micro-method
Introduction Cholesterol (5-cholesten-3β-ol; Fig. 1), in particular, is a key compound in most biological systems. It is an essential H. M. M. Ramalho : S. Casal : M. B. P. P. Oliveira (*) REQUIMTE-Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal e-mail: [email protected]
compound in cellular membrane functions of animals and precursor of important endogenous substances such as corticosteroids, sex hormones, and bile acids (Bragagnolo 2008). In humans, cholesterol is obtained from two sources: endogenous synthesis and exogenous ingestion from food (human milk during the breastfeeding period, followed by milk and dairy products, meat, or eggs; Xu et al. 2002). Although of vital biological importance, some health concerns have highlighted the controversy, being consumers currently aware of cholesterol and lipid contents present in foodstuffs (Bragagnolo 2008). Bovine milk is indeed among the controversial foods, with cholesterol generally regarded as an undesirable milk component and being a significant external source for regular consumers. Cholesterol is the major sterol in milk. Its level changes during the lactation period and is related with fat content. Despite the inexistence of scientifically supported evidence that moderate intake of milk and dairy products increases disease risk (Haug et al. 2007; Pearce 1996; McNa
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