6 11 Vol. 6 No. 11 2015 11 Journal of Food Safety and Quality Nov., 2015-16 游景水 *, 王德伟 (, 519040) 摘要 : 目的 16 方法 6 mol/l, (OPA) 9- (FMOC) ; Agilent Hypersil AA-ODS ; (0.16% ) ; (FLD) ( : 340 nm, : 450 nm; 15 min, : 266 nm, : 305 nm) 结果 16 5 250 pmol/μl (r: 0.99959 1.00000, n=5), 91.6% 104.1%(n=9), (RSD) 0.9% 3.7%, 0.4 1.2 pmol 结论,, 关键词 : ; ; ; ; ; 9- Determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by online pre-column derivazation-reversed-phase high performance liquid chromatography YOU Jing-Shui *, WANG De-Wei (By-Health Co., Ltd., Zhuhai 519040, China) ABSTRACT: Objective To establish a method for determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by reversed-phase high performance liquid chromatography (RP-HPLC). Methods Proteins in the samples were hydrolyzed into amino acids in 6 mol/l hydrochloric acid solution, o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were used as online pre-column derivative reagents for the primary amino acids and secondary amino acids respectively, and the chromatographic experiments were performed on an Agilent Hypersil AA-ODS column. The 0.16% sodium acetate solution was used as mobile phase. Signals were detected by fluorescence detection (FLD) (excitation wavelength: 340 nm, emission wavelength: 450 nm, excitation wavelength changed to 266 nm and emission wavelength changed to 305nm at the 15 min). Results The concentration of sixteen kinds of amino acids demonstrated a good linearity in the range of 5 250 pmol/μl(r: 0.99959 1.00000, n=5). The recoveries were 91.6% 104.1% (n=9). The RSD were 0.9% 3.7%. The limits of detection were 0.4 1.2 pmol. Conclusion This method has advantages of high sensitivity, precision and accuracy. It's suitable for the determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar. KEY WORDS: capsule of sheep placenta and medlar; online pre-column derivazation; amino acid; re- * 通讯作者 :,, E-mail: 752414274@qq.com *Corresponding author: YOU Jing-Shui, Licensed Pharmacist, By-Health Co., Ltd., Zhuhai 519040, China. E-mail: 752414274@qq.com
4672 6 versed-phase high performance liquid chromatography; o-phthaldialdehyde; 9-fluorenylmethyl chloroformate 1 引言, [1], ( ), [2-8],, [9] (GB/T 5009.124-2003) [2], ( (10 pmol) OPA FMOC,, (FLD) [10-15], 2 材料与方法 2.1 主要仪器 Agilent 1200 (, Agilent ), : Chem32; KQ-500E ( ); XW-80A ( ); DHG-9240A ( ); BPZ-6090Lc ( ) 2.2 试剂 OPA (, : BCBF1444); FMOC (, : BCBD9246); (, : BCBF0657); 250 pmol/μl (, : BCBD9914); (, ); (, ); (, ); (, ); (, ); (, ); (, ); (, ) 2.3 色谱条件 : Hypersil AA-ODS (200 mm 2.1 mm, 5 μm, ); : 0.45 ml/min; : 40 ; : 1 µl; : 30 min A(0.16% ): 1.6 g, 1000 ml,, 180 μl,, 2% ph 7.20±0.1, 3 ml,, 0.45 μm, ; B(0.16% ): 1.6 g, 200 ml,, 2% ph 7.20±0.1, 400 ml 400 ml,, 0.45 μm, 1 表 1 梯度洗脱比例 Table 1 Gradient elution ratio (min) (ml/min) A(%) B(%) 0.0 0.45 100 0 17.0 0.45 40 60 18.0 0.45 0 100 18.5 0.80 0 100 23.9 0.80 0 100 24.0 0.45 0 100 25.0 0.45 100 0 30.0 0.45 100 0 : 1 5.0 μl ; 2 1.0 μl OPA ; 3 ( ) 0.0 μl ( ); X 1.0 μl ; 3 ( ) 0.0 μl ( ); 8 μl ;, 6 ; 4 1.0 μl FMOC; 3 ( ) 0.0 μl ( ); 9 μl ;, 3 ; : (FLD) : 0 min, : 340 nm, : 450 nm, PTM : 11; 15 min, : 266 nm, : 305 nm, PTM : 11 2.4 对照品溶液的制备 250 pmol/μl, 0.1 mol/l : 5L 10 50 100 250 pmol/μl
11, : - 16 4673 2.5 样品前处理 0.12 g, 25 ml, 6 mol/l 15 ml, 0.2 g,,,, 110 ±1, 22,,,, 50mL,,, 1 ml 10 ml,, 40 50 ( ), 0.1 mol/l,, 0.45 μm, 3 结果与分析 3.1 线性试验和检出限试验 2.3, 5, (X: pmol/μl), (Y) (S/N)=3 (pmol) 16 2 2 : 16 5 250 pmol/μl (r: 0.99959 1.00000)(n=5), : 0.4 1.2 pmol 表 2 16 种氨基酸的线性方程 相关系数和检出限 Table 2 Linear equation, correlation coefficient and detection limit of the sixteen kinds of amino acids (pmol) Y =18.887 X +26.39 0.99995 0.5 Y =21.853 X 16.05 0.99994 0.5 Y =25.262 X 26.46 0.99996 0.6 Y =14.036 X+38.92 0.99978 0.8 Y =22.876 X+39.18 0.99965 0.6 Y =19.940 X+7.02 1.00000 0.6 Y =23.335 X 14.89 0.99998 0.6 Y =24.975 X+25.50 0.99999 0.5 Y =23.880 X 11.90 0.99996 0.6 Y =24.815 X+39.30 0.99987 0.5 Y =29.005 X 5.40 0.99991 0.5 Y =23.976 X+36.96 0.99963 0.5 Y =25.942 X+53.87 0.99959 0.5 Y =27.078 X+12.87 0.99998 0.5 Y =8.972 X 1.24 0.99997 1.2 Y =48.467 X+41.03 0.99999 0.4 3.2 精密度试验 6,, 2.3,, 16 RSD 0.3% 2.0%, 3, 3, 3.3 加标回收试验 9, 3, 3, 250 nmol/μl 40 50 60 μl,, 2.3, 16 91.6% 104.1%, RSD 0.9% 3.4% 4, 4, 3.4 方法专属性试验 16, 2.3, 16 1 2 3,, 16, 16, 4 讨论 (OPA) 9- (FMOC), : (1), 16 (2), 16, [10,11],,, 16 0.4 1.2 pmol, GB/T 5009.124-2003( ) 10 pmol, 5 结论 16,,,
4674 6 Table 3 表 3 精密度的试验 (n=6) The experiment of the precision (n=6) (mg/g) RSD(%) (mg/g) RSD(%) (mg/g) RSD(%) 22.65 1.5 21.25 1.0 11.92 1.7 44.14 1.3 12.90 0.8 22.94 0.6 7.74 0.3 8.60 0.5 13.87 1.8 4.14 1.8 15.48 1.5 16.13 0.5 22.60 1.5 6.92 1.3 9.89 0.4 10.27 2.0 (mg/g) 251.44 1.4 Table 4 表 4 加标回收率试验 (n=9) The experiment of standard addition recovery (n=9) (%) RSD(%) (%) RSD(%) (%) RSD(%) 91.6 1.1% 95.0 0.9% 98.1 3.4% 94.3 1.8% 96.3 3.1% 94.7 3.3% 98.3 2.7% 99.7 2.4% 99.2 2.7% 93.1 2.6% 96.8 3.7% 99.2 2.1% 104.1 3.0% 100.8 2.3% 100.3 3.1% 98.7 2.4% Fig. 1 1 The chromatogram of blank samples
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