四川大学学报(医学版)
四川大學學報(醫學版)
사천대학학보(의학판)
JOURNAL OF WEST CHINA UNIVERSITY OF MEDICAL SCIENCES
2010年
1期
158-161
,共4页
王希希%胡燕%孙轶卓%别明江%孙成均
王希希%鬍燕%孫軼卓%彆明江%孫成均
왕희희%호연%손질탁%별명강%손성균
高效液相色谱法%水溶性维生素%固相萃取%血清
高效液相色譜法%水溶性維生素%固相萃取%血清
고효액상색보법%수용성유생소%고상췌취%혈청
High performance liquid chromatography%Water-soluble vitamin%Solid phase extraction(SPE)%Human serum
目的 建立血清中水溶性维生素B_1(VB_1)、B_6(VB_6)、烟酸、烟酰胺和叶酸的高效液相色谱同时测定方法.方法 取全血40 μL,用200 μL甲醇沉淀蛋白,冰箱冷冻(-18℃)30 min后离心(8000 r/min,8 min),上清液通过固相萃取柱进一步净化,收集洗脱液,50℃水浴中氮气吹至0.20 mL,取50 μL进样色谱柱分析.对样品的固相萃取条件和色谱条件进行优化后,用于测定人血清样品中5种水溶性维生素的含量.结果 优化后的固相萃取条件;固相萃取柱AGT cleanert~(TM) ODSC_(18)柱;洗脱液:甲醇(2.4 mL)-水(0.6 mL)混合液;洗脱流速:0.5 mL/min.本法的色谱分离条件为:分离柱Phenomenex C18 Luna色谱柱(250×4.6 mm,5 μm);柱温:室温(20℃);流动相:0.05 mol/L KH_2PO_4溶液(pH6.0)-甲醇梯度洗脱;流速:0.9 mL/min;紫外检测波长:266 nm.5种水溶性维生素标准曲线的线性范围为0～100 ng,相关系数均大于0.999,相对标准偏差(RSD)小于5%,方法 检出限(信噪比S/N=2)为0.076～0.170 μg/mL,加标回收率为80.2%～115.0%.人血清样品中除烟酰胺未检出外,其余4种均检出.结论 所建立的方法 快速、简便、灵敏、准确,可用于人血清中水溶性维生素VB_1、VB_6、烟酸和叶酸的同时测定,但烟酰胺的检测灵敏度有待提高.
目的 建立血清中水溶性維生素B_1(VB_1)、B_6(VB_6)、煙痠、煙酰胺和葉痠的高效液相色譜同時測定方法.方法 取全血40 μL,用200 μL甲醇沉澱蛋白,冰箱冷凍(-18℃)30 min後離心(8000 r/min,8 min),上清液通過固相萃取柱進一步淨化,收集洗脫液,50℃水浴中氮氣吹至0.20 mL,取50 μL進樣色譜柱分析.對樣品的固相萃取條件和色譜條件進行優化後,用于測定人血清樣品中5種水溶性維生素的含量.結果 優化後的固相萃取條件;固相萃取柱AGT cleanert~(TM) ODSC_(18)柱;洗脫液:甲醇(2.4 mL)-水(0.6 mL)混閤液;洗脫流速:0.5 mL/min.本法的色譜分離條件為:分離柱Phenomenex C18 Luna色譜柱(250×4.6 mm,5 μm);柱溫:室溫(20℃);流動相:0.05 mol/L KH_2PO_4溶液(pH6.0)-甲醇梯度洗脫;流速:0.9 mL/min;紫外檢測波長:266 nm.5種水溶性維生素標準麯線的線性範圍為0～100 ng,相關繫數均大于0.999,相對標準偏差(RSD)小于5%,方法 檢齣限(信譟比S/N=2)為0.076～0.170 μg/mL,加標迴收率為80.2%～115.0%.人血清樣品中除煙酰胺未檢齣外,其餘4種均檢齣.結論 所建立的方法 快速、簡便、靈敏、準確,可用于人血清中水溶性維生素VB_1、VB_6、煙痠和葉痠的同時測定,但煙酰胺的檢測靈敏度有待提高.
목적 건립혈청중수용성유생소B_1(VB_1)、B_6(VB_6)、연산、연선알화협산적고효액상색보동시측정방법.방법 취전혈40 μL,용200 μL갑순침정단백,빙상냉동(-18℃)30 min후리심(8000 r/min,8 min),상청액통과고상췌취주진일보정화,수집세탈액,50℃수욕중담기취지0.20 mL,취50 μL진양색보주분석.대양품적고상췌취조건화색보조건진행우화후,용우측정인혈청양품중5충수용성유생소적함량.결과 우화후적고상췌취조건;고상췌취주AGT cleanert~(TM) ODSC_(18)주;세탈액:갑순(2.4 mL)-수(0.6 mL)혼합액;세탈류속:0.5 mL/min.본법적색보분리조건위:분리주Phenomenex C18 Luna색보주(250×4.6 mm,5 μm);주온:실온(20℃);류동상:0.05 mol/L KH_2PO_4용액(pH6.0)-갑순제도세탈;류속:0.9 mL/min;자외검측파장:266 nm.5충수용성유생소표준곡선적선성범위위0～100 ng,상관계수균대우0.999,상대표준편차(RSD)소우5%,방법 검출한(신조비S/N=2)위0.076～0.170 μg/mL,가표회수솔위80.2%～115.0%.인혈청양품중제연선알미검출외,기여4충균검출.결론 소건립적방법 쾌속、간편、령민、준학,가용우인혈청중수용성유생소VB_1、VB_6、연산화협산적동시측정,단연선알적검측령민도유대제고.
Objective To establish a method for the simultaneous determination of 5 water-soluble vitamins including VB_1,VB_6,Nicotinic acid,Nicotinamide and Folic acid by high performance liquid chromatography.Methods Methanol(200 μL)was added to 50 μL human blood sample to precipitate proteins.The sample was frozen(-18℃)for 30 min,and then centrifuged at 8000 r/min for 8 min.The supernatant was subsequently applied to the solid-phase cartridge for further purification.The elute was collected and evaporated to 0.20 mL under nitrogen in a water bath at the temperature of 50 ℃ and finally 50 μL of the sample was injected onto HPLC column for analysis.The five water-soluble vitamins were determined under optimized condition of solid phase extraction and chromatograph.Results The optimized condition for solid phase extraction(SPE)was identified as:AGT cleanert~(TM) ODS C_(18) column as SPE column and CH_3OH(2.4 mL)-H_2O(0.6 mL)mixed liquor as eluent,with a flow rate of elution of 0.5 mL/min.The optimized chromatographic condition was identified as:Phenomenex C_(18) Luna column(250×4.6 mm,5 μm)as analytical column under a temperature of 20 ℃ and 0.05 mol/L potassium dehydrogenates phosphate buffer(pH6.0)and methanol as gradient elution with mobile phase with a flow rate of 0.9 mL/min.The detection wavelength was 266 nm.The correlation coefficients for the standard curves were greater than 0.999 in the range from 0 to 100 ng(RSD＜5%).The detection limits(S/N=2)ranged from 0.076 to 0.170 μg/mL.The recovery rate ranged from 80.2%to 115.0%.Except for Nicotinamide,the other four vitamins were detected successfully in the blood sample.Conclusion This quick and convenient method can be applied to the simultaneous determination of 4 water-soluble vitamins in human serum samples.But the sensitivity for detecting Nicotinamide needs to be improved.
