2010•Journal of clinical and experimental medicineRequires access
High-performance liquid chromatographic analysis of serum free fatty acid compositions by direct derivatization method
Zhao Xin‐feng
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Abstract
Objective Fatty acids intake and metabolism may play a role in the pathogenesis of essential hypertension, but the methods of determining serum free fatty acid (FFA) compositions have not been unified. The aim of this study was to investigate the validity of high performance liquid chromatogramin by direct derivatization method.Methods Subjects aged between 35 and 59 years from communities including 103 essential hypertensives and 128 normotensives were selected. Fasting blood (12 hours) was collected, separated and dispensed into two 1.5-ml aliquots. Serum FFA was dissolved in ethanol (75%, v/v). Without commonly used isolation steps, fatty acids in serum were directly derivatized by treatment with 2-nitrophenylhydrazine hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(1-EDC·HCl), which produces their non-volatile hydrazine derivatives. The derivatized fatty acids were separated from other acid hydrazides and interfering components by a simple solvent extraction (which were extracted into n-hexane), and were eluted isocratically on a reversed-phase C18 column within 30 min.Results A total of 9 serum fatty acids from C16:0 to C22:6 were determined. Using margaric acid (C17:0) as internal standard, each fatty acid could be quantitated with linearity over the range of 2.6～200 μmol/L. Analytical recoveries ranged from 87.8% to 120.51% and coefficients of variation were less than 10%. Compared with normotensives, hypertensives had lower levels of serum polyunsaturated fatty acids (PUFAs,P=0.004)), and polyunsaturated/saturated fatty acids ratio (P/S ratio, P=0.000), which were in good agreement with those in other reports.Conclusion The analytical results showed good recovery and reproducibility using each internal standard. Due to its excellent selectivity and sensitivity, the present method can serve as a useful tool for routine determinations of FFA compositions in serum.