酰胺基亲水作用色谱-高效液相色谱-紫外法测定DHA颗粒中的磷脂酰丝氨酸

目的：建立酰胺基亲水作用色谱-高效液相色谱-紫外法测定DHA颗粒中磷脂酰丝氨酸的含量。方法：以正己烷-异丙醇为提取溶剂对样品进行超声提取；选用Waters XBridge Amide（4.6 mm×250 mm，3.5 μm）色谱柱，以乙腈-异丙醇（7∶3）为流动相，流速1.0 mL·min^-1，柱温45℃，进样量20 μL，紫外检测波长203 nm，单次运行时间10 min。结果：样品中磷脂酰丝氨酸获得很好的分离，在0.1~1.0mg·mL^-1线性关系良好（r=0.999 9）；在高、低浓度的回收率（n=6）分别为101.2%和95.8%，RSD分别为1.3%和2.6%；3批样品中磷脂酰丝氨酸含量分别为10.5、10.3、10.6 mg·g^-1。结论：本方法操作简捷、环保、高效，经方法学验证，本法专属性、重复性好，准确度高，适应性强，为含磷脂酰丝氨酸功能食品的全面质量控制提供一种新的方法。

Objective: To establish an Amide-HPLC-UV method for the determination of phosphatidylserine in DHA granules.Methods: The sample was extracted with n-hexane-isopropanol as the extraction solvent.The chromatographic separation was achieved on a Waters XBridge Amide (4.6 mm×250 mm, 3.5 μm) column using acetonitrile-isopropanol (7:3) as the mobile phase at a flow rate of 1.0 mL·min^-1.The column temperature was 45, the injection volume was 20 μL, the detection wavelength was set at 203 nm, and the single run time was 10 min.Results: Phosphatidylserine in the sample was well separated.The calibration curves of phosphatidylserine showed good linearity in the range of 0.1-1.0 mg·mL^-1 (r=0.999 9).The average recoveries (n=6) at high and low concentrations were 101.2% and 95.8%, respectively;and RSD were 1.3% and 2.6%, respectively.The contents of phosphatidylserine in three batches were 10.5, 10.3, 10.6 mg·g^-1, respectively.Conclusion: This method is simple, environmental and efficient.The established method is proved by methodology validation which is specific, reproducible, accurate and adaptable, that can be applied to overall quality control of phosphatidylserine in functional food.

[1] LIN Q,ZHANG J,PEI WJ,et al.Determination of phosphatidylserine in milk-based nutritional products using online derivatization high-performance liquid chromatography[J].J Chromatogr A,2015(1381):260
[2] GLADE MJ,SMITH K.Phosphatidylserine and the human brain.[J].Nutr,2015,31(6):781
[3] KIDD PM.Phosphatidylserine,membrane nutrient for memory.A clinical and mechanistic assessment[J].Altern Med Rev,1996,1(2):70
[4] MCDANIE MA,MAIER SF,EINSTEIN GO."Brain-specific" nutrients:a memory cure?[J].Nutrit,2003,19(11/12):957
[5] YANG SL,DUAN ZQ.Insight into enzymatic synthesis of phosphatidylserine in deep eutectic solvents[J].Catal Commun,2016,82:16
[6] LIU SH,CHANG CD,CHEN PH,et al.Docosahexaenoic acid and phosphatidylserine supplementations improve antioxidant activities and cognitive functions of the developing brain on pentylenetetrazol-induced seizure model.[J].Brain Res,2012,1451:19
[7] KIM HY.Biochemical and biological functions of docosahexaenoic acid in the nervous system:modulation by ethanol[J].Chem Phys Lipids,2008,153(1):34
[8] KIM HY,BIGELOW J,KEVALA JH.Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species[J].Biochemistry,2004,43(4):1030
[9] 李阅兵,刘承初,谢晶,等.ω-3脂肪酸及磷脂酰丝氨酸的益智作用研究进展[J].中国油脂,2011,36(9):51 LI YB,LIU CC,XIE J,et al.Research advance in the function on intelligence of ω-3 fatty acid and phosphatidylserine[J].China Oils Fats,2011,36(9):51
[10] CHEN,S,CHEN R.Docosahexaenoic acid-containing phospholipids and triglycerides based nutritional supplements[J].Recent Patents Food Nutr Agric,2010,2(3):213
[11] AKITO KK,MASASHI S,RIKA E,et al.Soybean-derived phosphatidylserine improves memory function of the elderly Japanese subjects with memory complaints[J].J Clin Biochem Nutr,2010,47(3):246
[12] MACKENZIE A,VYSSOTSKI M,NEKRASOV E.Quantitative analysis of dairy phospholipids by ³¹P NMR[J].J Am Oil Chem Soc,2009,86(8):757
[13] MACIEL E,FARIA R,SANTINHA D,et al.Evaluation of oxidation and glyco-oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylserine by LC-MS/MS[J].J Chromatogr B,2013, 929:76
[14] BRAUN M,FLUECK B,COTTING C,et al.Quantification of phospholipids in infant formula and growing up milk by high-performance liquid chromatograhy with evaporative light scattering detector[J].J Aoac Int,010,93(3):948
[15] 石丹,王晓彦,魏显纲,等.高效液相色谱检测奶粉中磷脂酰丝氨酸分析方法的建立[J].营养学报,2015,37(1):88 SHI D,WANG XY,WEI XG,et al.Establishment of analytical method for detection of phosphatidylserine in mlik powder by high performance liquid chromatography[J].Acta Nutrim Sina,2015,37(1):88