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CN1320354C - Method for analysizing amino acid - Google Patents

Method for analysizing amino acid Download PDF

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CN1320354C
CN1320354C CNB2005100144780A CN200510014478A CN1320354C CN 1320354 C CN1320354 C CN 1320354C CN B2005100144780 A CNB2005100144780 A CN B2005100144780A CN 200510014478 A CN200510014478 A CN 200510014478A CN 1320354 C CN1320354 C CN 1320354C
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amino acid
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CN1749748A (en
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朱彭龄
朱润之
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Tianjin Pharmaceutical Research Institute Co ltd
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Abstract

本发明涉及一种氨基酸的分析方法。在C18柱上，以pH2-3或6-7的磷酸三乙胺(TEAP)/乙腈(ACN)为流动相高效液相色谱分离氨基酸(AA)的2，4-二硝基氟苯(DNFB)衍生物。柱温35℃，360nm紫外检测。本发明使用磷酸三乙胺(TEAP)/乙腈(ACN)流动相，提高了分离结果的重现性。通过选择水相缓冲区间和在区间内微调pH值，使分析方法适用于不同品牌色谱柱和各种试样类型，解决了氨基酸混合物的分析，并给出准确的结果。本发明不需增加设备，即可适合于一般HPLC分析室采用。应用范围包括各种氨基酸的分析以及其它物质转化为氨基酸后的间接分析，还包括除氨基酸以外其它能与DNFB进行定量衍生反应物质的分析。The invention relates to an analysis method of amino acid. On C18 post, with the triethylamine phosphate (TEAP)/acetonitrile (ACN) of pH2-3 or 6-7 as mobile phase high performance liquid chromatography separation amino acid (AA) 2,4-dinitrofluorobenzene ( DNFB) derivatives. The column temperature is 35°C, and 360nm ultraviolet detection. The present invention uses triethylamine phosphate (TEAP)/acetonitrile (ACN) mobile phase to improve the reproducibility of separation results. By selecting the aqueous phase buffer and fine-tuning the pH value within the range, the analysis method is applicable to different brands of chromatographic columns and various sample types, which solves the analysis of amino acid mixtures and gives accurate results. The invention does not need to add equipment, and can be suitable for general HPLC analysis room. The scope of application includes the analysis of various amino acids and the indirect analysis of other substances converted into amino acids, as well as the analysis of substances other than amino acids that can be quantitatively derivatized with DNFB.

Description

氨基酸的分析方法Amino Acid Analysis Methods

技术领域technical field

本发明涉及氨基酸的分析方法，基于以磷酸三乙胺/乙腈为流动相反相分离氨基酸-2，4-二硝基氟苯衍生物，使用通常的高效液相色谱仪和色谱条件，不再需要其它的仪器配置，就可以完成各种氨基酸试样的分析，并给出准确的结果。The present invention relates to an analysis method for amino acids, based on the separation of amino acid-2,4-dinitrofluorobenzene derivatives using triethylamine phosphate/acetonitrile as the mobile reverse phase, using common high-performance liquid chromatography and chromatographic conditions, no longer need Other instrument configurations can complete the analysis of various amino acid samples and give accurate results.

背景技术Background technique

氨基酸(amino acid，AA)试样比较复杂，常见的AA有20多种，色谱分析方法必须有足够的选择性，以分辨试样中所要测定的AA。大多数AA对紫外光无明显的吸收，为了检测，最常用的手段是使AA分子中的氨基与衍生剂反应，生成具有紫外吸收或荧光性质的衍生物。Amino acid (amino acid, AA) samples are relatively complex, and there are more than 20 common AA. The chromatographic analysis method must have sufficient selectivity to distinguish the AA to be determined in the sample. Most AA has no obvious absorption to ultraviolet light. For detection, the most common method is to react the amino group in the AA molecule with a derivatizing agent to generate derivatives with ultraviolet absorption or fluorescence properties.

通常采用两种途径分析AA试样。一种是借助离子交换机理先使AA彼此分离，再用柱后衍生进行检测。这是一般AA分析仪所使用的办法。AA samples are generally analyzed in two ways. One is to separate AA from each other by means of ion exchange mechanism, and then use post-column derivatization for detection. This is the approach used by general AA analyzers.

另一途径是，AA经柱前衍生后，以反相色谱分离AA的衍生物。柱前衍生法分离对仪器设备没有特殊的要求，同时采用反相分离模式，但要求衍生反应能使每种AA几乎完全地生成相应的特定产物，且有足够的检测灵敏度[朱彭龄、云自厚、谢光华，“现代液相色谱”，第22章，兰州大学出版社，1989]。Another approach is to separate AA derivatives by reverse phase chromatography after pre-column derivatization of AA. The pre-column derivatization separation has no special requirements on the equipment, while the reversed-phase separation mode is adopted, but it is required that the derivatization reaction can make each AA almost completely generate the corresponding specific product, and there is sufficient detection sensitivity [Zhu Pengling, Yun Zihou , Xie Guanghua, "Modern Liquid Chromatography", Chapter 22, Lanzhou University Press, 1989].

在早期的文献报道中，AA经2，4-二硝基氟苯(DNFB)柱前衍生，反相分离衍生物AA-DNFB，分离DNFB-AA是以60mM醋酸钠(pH＝4.45)为水相，以甲醇为有机相，在C18柱上进行梯度洗脱[Morton R.C.；Gerber G.E.，Anal.Biochem.，170(1988)220.]。后来，大多使用pH＝6.5-7的醋酸盐缓冲液-乙腈体系作为流动相，使异亮氨酸和亮氨酸之间有足够的分离度[Shang，Zhen-Hua；Yu，Yi-Nian；Guo，Wei；Jiang，Hui；Zhou，Liang-Mo，Chinese Journal of Chemistry，13(1995)163.]。In early literature reports, AA was derivatized by 2,4-dinitrofluorobenzene (DNFB) before the column, and the derivative AA-DNFB was separated by reverse phase. phase, with methanol as the organic phase, gradient elution was performed on a C 18 column [Morton RC; Gerber GE, Anal. Biochem., 170(1988) 220.]. Later, the acetate buffer-acetonitrile system with pH = 6.5-7 was mostly used as the mobile phase to achieve sufficient resolution between isoleucine and leucine [Shang, Zhen-Hua; Yu, Yi-Nian ; Guo, Wei; Jiang, Hui; Zhou, Liang-Mo, Chinese Journal of Chemistry, 13 (1995) 163.].

中国专利CN 1053128A公开了十八种复合氨基酸注射液的高效液相色谱分析方法，它以2，4-二硝基氟苯作衍生试剂，用柱前衍生化法对十八种复合氨基酸注射液进行定量测定。用定量稀释配制检测液，以一定浓度和pH值的流动相乙腈和流动相醋酸钠和醋酸缓冲液作梯度洗脱剂。Chinese patent CN 1053128A discloses a high-performance liquid chromatography analysis method for eighteen kinds of compound amino acid injections. It uses 2,4-dinitrofluorobenzene as a derivatization reagent, and uses a pre-column derivatization method to analyze eighteen kinds of compound amino acid injections. Perform quantitative measurements. The detection solution was prepared by quantitative dilution, and the mobile phase acetonitrile, mobile phase sodium acetate and acetate buffer with a certain concentration and pH value were used as gradient eluents.

实验表明，反相分离AA-DNFB对流动相pH值的变化很敏感，精确控制流动相pH值是至关重要的。当使用接近中性的醋酸盐溶液作为流动相的组分，一个明显的问题是，流动相的酸度远离该缓冲液的缓冲区间，此方法的耐用性(robustness)就得不到保证。不同日期配制的流动相和不同品牌，甚至同一品牌不同批号的色谱柱都可能影响DNFB-AA衍生物的色谱分离结果。Experiments showed that reversed-phase separation of AA-DNFB was sensitive to changes in mobile phase pH, and precise control of mobile phase pH was crucial. When using near-neutral acetate solution as a component of the mobile phase, an obvious problem is that the acidity of the mobile phase is far away from the buffer zone of the buffer, and the robustness of the method cannot be guaranteed. Mobile phases prepared on different dates and different brands, or even columns of the same brand with different batch numbers may affect the chromatographic separation results of DNFB-AA derivatives.

发明内容Contents of the invention

本发明的目的是提供一种氨基酸的分析方法。用磷酸三乙胺/乙腈为流动相反相分离氨基酸-2，4-二硝基氟苯衍生物，可以克服现有技术之不足。本发明以磷酸盐代替原有的醋酸盐，使用磷酸三乙胺(TEAP)/乙腈(ACN)流动相体系，提高了分离结果的重现性。本发明耐用性好，易于为一般实验室所采用。通过选择水相缓冲区间和在区间内微调pH值，使分析方法适用于不同品牌色谱柱和各种试样类型。不需增加设备，即可为一般HPLC分析室采用。The purpose of the present invention is to provide a method for analyzing amino acids. Using triethylamine phosphate/acetonitrile as the mobile reverse phase to separate amino acid-2,4-dinitrofluorobenzene derivatives can overcome the shortcomings of the prior art. The present invention replaces the original acetate with phosphate, and uses a triethylamine phosphate (TEAP)/acetonitrile (ACN) mobile phase system to improve the reproducibility of the separation result. The invention has good durability and is easy to be adopted in general laboratories. By selecting between aqueous phase buffers and fine-tuning the pH value within the range, the analysis method is applicable to different brands of chromatographic columns and various sample types. It can be used in general HPLC analysis room without additional equipment.

本发明氨基酸的分析方法包括：使用高效液相色谱仪，A经2，4-二硝基氟苯(DNFB)柱前衍生，反相分离衍生物AA-DNFB，以磷酸三乙胺/乙腈为流动相梯度洗脱或等度洗脱，在分析柱上反相分离氨基酸-2，4-二硝基氟苯衍生物，再用外标法定量分析计算出氨基酸的含量。The analysis method of amino acid of the present invention comprises: using high performance liquid chromatography, A is derivatized through 2,4-dinitrofluorobenzene (DNFB) before the column, reverse phase separation derivative AA-DNFB, with triethylamine phosphate/acetonitrile as Gradient elution or isocratic elution of the mobile phase, reverse-phase separation of amino acid-2,4-dinitrofluorobenzene derivatives on the analytical column, and quantitative analysis and calculation of the content of amino acids by external standard method.

本发明氨基酸的分析方法包括下述步骤：The analysis method of amino acid of the present invention comprises the following steps:

1)分别用HCl溶液作溶剂制备氨基酸标准溶液和试样溶液；1) using HCl solution as solvent to prepare amino acid standard solution and sample solution;

2)分别取标准溶液和试样溶液于为其体积10倍量的容量瓶中，加入0.5MNaHCO3，标准溶液或试样溶液与NaHCO3的体积比为1∶1；再加入1％2，4-二硝基氟苯的乙腈溶液，与标准溶液或试样溶液的体积比为0.5-1∶1，摇匀；置于60℃下加热60分钟，冷却至室温，用0.1MpH为6.5的磷酸盐缓冲液冲至刻度，摇匀，备用；2) Take the standard solution and the sample solution respectively in a volumetric flask 10 times its volume, add 0.5M NaHCO 3 , the volume ratio of the standard solution or sample solution to NaHCO 3 is 1:1; then add 1% 2, The acetonitrile solution of 4-dinitrofluorobenzene, with a volume ratio of 0.5-1:1 to the standard solution or sample solution, shake well; heat at 60°C for 60 minutes, cool to room temperature, and use 0.1M pH 6.5 Rinse the phosphate buffer to the mark, shake well, and set aside;

3)使用包括泵、混合器、恒温箱、紫外检测器以及工作站所组成的HPLC仪。在C18分析柱上，以的TEAP/ACN为流动相，梯度洗脱或等度洗脱分离AA-DNFB；色谱条件如下：3) Use an HPLC instrument comprising a pump, a mixer, an incubator, an ultraviolet detector and a workstation. On a C 18 analytical column, use TEAP/ACN as the mobile phase, gradient elution or isocratic elution to separate AA-DNFB; the chromatographic conditions are as follows:

分析柱  C18，5μm，250×4.6mmAnalytical column C 18 , 5μm, 250×4.6mm

流动相  A：ACN    B：36mM TEAP，pH2-3或6-7Mobile phase A: ACN B: 36mM TEAP, pH2-3 or 6-7

洗脱方式  梯度洗脱或等度洗脱Elution mode Gradient elution or isocratic elution

检测  UV 300-380nm，Detect UV 300-380nm,

温度  20-45℃Temperature 20-45℃

4)用外标法定量分析计算4) Quantitative analysis and calculation with external standard method

试样中AA的浓度＝(A试样/A标准)×C标准 The concentration of AA in the sample = (A sample /A standard ) × C standard

A试样：试样色谱图中AA的峰面积A sample : the peak area of AA in the sample chromatogram

A标准：标准色谱图中AA的峰面积A standard : the peak area of AA in the standard chromatogram

C标准：标准溶液AA的浓度。C standard : the concentration of standard solution AA.

本发明的具体实施方案包括下述步骤：Specific embodiments of the present invention include the following steps:

1)分别用0.1M HCl作溶剂制备AA标准溶液和试样溶液；1) Prepare AA standard solution and sample solution with 0.1M HCl as solvent respectively;

2)分别取1mL标准溶液和样品溶液于10mL棕色容量瓶中，加入1mL 0.5MNaHCO3，摇匀。再加入0.5-1mL 1％ DNFB的乙腈溶液，摇匀；置于60℃下加热60分钟，冷却至室温，用0.1M pH为6.5的磷酸盐缓冲液冲至刻度，摇匀，备用。2) Take 1mL standard solution and sample solution respectively in a 10mL brown volumetric flask, add 1mL 0.5M NaHCO 3 , and shake well. Then add 0.5-1mL 1% DNFB in acetonitrile solution, shake well; heat at 60°C for 60 minutes, cool to room temperature, wash to the mark with 0.1M phosphate buffer solution with pH 6.5, shake well, and set aside.

3)使用包括泵、混合器、恒温箱、紫外检测器以及工作站所组成的HPLC仪。在C18分析柱上，以的TEAP/ACN为流动相，梯度洗脱或等度洗脱分离AA-DNFB。色谱条件如下：3) Use an HPLC instrument comprising a pump, a mixer, an incubator, an ultraviolet detector and a workstation. On a C 18 analytical column, AA-DNFB was separated by gradient elution or isocratic elution with TEAP/ACN as mobile phase. The chromatographic conditions are as follows:

分析柱  C18，5μm，250×4.6mmAnalytical column C 18 , 5μm, 250×4.6mm

流动相  A：ACN  B：36mM TEAP，pH 2-3或6-7Mobile phase A: ACN B: 36mM TEAP, pH 2-3 or 6-7

洗脱方式  梯度洗脱或等度洗脱Elution mode Gradient elution or isocratic elution

检测  UV 300-380nm，优选360nm，Detect UV 300-380nm, preferably 360nm,

温度  20-45℃，优选35℃；Temperature 20-45°C, preferably 35°C;

4)用外标法定量分析计算4) Quantitative analysis and calculation with external standard method

试样中AA的浓度＝(A试样/A标准)×C标准 The concentration of AA in the sample = (A sample /A standard ) × C standard

A试样：试样色谱图中AA的峰面积A sample : the peak area of AA in the sample chromatogram

A标准：标准色谱图中AA的峰面积A standard : the peak area of AA in the standard chromatogram

C标准：标准溶液AA的浓度。C standard : the concentration of standard solution AA.

本发明采用了磷酸盐代替原有的醋酸盐，提高了方法的耐用性。在该缓冲体系中，三乙胺的阳离子作为磷酸根的对离子，而不是钾或钠离子。磷酸盐的缓冲区间是pH＝2-3和6-8。在所述的AA分析方法中，以36mM磷酸三乙胺，pH＝2-3和6-7水溶液/ACN流动相体系洗脱AA-DNFB(2，4-二硝基氟苯)。其次，流动相的酸度影响分离选择性，以不同pH的TEAP为水相，AA分离选择性显示很大的差异，这为优化色谱条件提供了方便。此外，三乙胺的加入可抑制固定相上残留硅羟基对分离的影响。The invention adopts phosphate to replace the original acetate, which improves the durability of the method. In this buffer system, the cation of triethylamine acts as the counterion of phosphate, rather than potassium or sodium ions. Phosphate buffers are between pH=2-3 and 6-8. In the described AA analysis method, AA-DNFB (2,4-dinitrofluorobenzene) was eluted with 36 mM triethylamine phosphate, pH=2-3 and 6-7 aqueous solution/ACN mobile phase system. Secondly, the acidity of the mobile phase affects the separation selectivity. With TEAP of different pH as the aqueous phase, the separation selectivity of AA shows great differences, which provides convenience for optimizing the chromatographic conditions. In addition, the addition of triethylamine can inhibit the influence of residual silanol on the stationary phase on the separation.

本发明使用磷酸三乙胺(TEAP)/乙腈(ACN)流动相，提高了分离结果的重现性。通过选择水相缓冲区间和在区间内微调pH值，使分析方法适用于不同品牌色谱柱和各种试样类型(例如：注射液类、或原料药中主要成分的测定等)，解决了氨基酸混合物的分析，并给出准确的结果。本发明不需增加设备，即可适合于一般HPLC分析室采用。应用范围扩大，包括各种氨基酸的分析以及其它物质转化为氨基酸后的间接分析，还包括除氨基酸以外其它能与DNFB进行定量衍生反应物质的分析。The present invention uses triethylamine phosphate (TEAP)/acetonitrile (ACN) mobile phase to improve the reproducibility of separation results. By selecting the aqueous phase buffer and fine-tuning the pH value within the range, the analysis method is applicable to different brands of chromatographic columns and various sample types (for example: injections, or the determination of main components in raw materials, etc.), to solve the problem of amino acids Analysis of mixtures and gives accurate results. The invention does not need to add equipment, and can be suitable for general HPLC analysis room. The scope of application is expanded, including the analysis of various amino acids and the indirect analysis of other substances converted into amino acids, as well as the analysis of substances other than amino acids that can be quantitatively derivatized with DNFB.

附图说明Description of drawings

图1是以36mM TEAP，pH 6-7/ACN为流动相肾病注射液色谱图。Figure 1 is a chromatogram of kidney disease injection with 36mM TEAP, pH 6-7/ACN as the mobile phase.

图2是以36mM TEAP，pH 6-7/ACN为流动相肝病注射液色谱图。Figure 2 is a chromatogram of liver disease injection with 36mM TEAP, pH 6-7/ACN as the mobile phase.

图3是以36mM TEAP，pH 2-3/ACN为流动相肾病注射液色谱图。Figure 3 is a chromatogram of kidney disease injection with 36mM TEAP, pH 2-3/ACN as the mobile phase.

图4是以36mM TEAP，pH 2-3/ACN为流动相肝病注射液色谱图。Figure 4 is a chromatogram of liver disease injection with 36mM TEAP, pH 2-3/ACN as the mobile phase.

图5是以36mM TEAP，pH 2-3/ACN为流动相丙谷酰胺酸解液色谱图。Fig. 5 is with 36mM TEAP, pH 2-3/ACN is the mobile phase chromatogram of glutamic acid hydrolyzate.

图6是以36mM TEAP，pH 2-3/ACN为流动相苯丙氨酸色谱图。Fig. 6 is chromatogram of mobile phase phenylalanine with 36mM TEAP, pH 2-3/ACN.

具体实施方式Detailed ways

以下结合实施例进一步说明本发明。Below in conjunction with embodiment further illustrate the present invention.

实施例1  肾病注射液(Amino acid for renal insufficiency， Hoechst Marion Roussel公司生产)和肝病注射液(Amino acid preparation for hepaticinsufficiency， Hoechst Marion Roussel公司生产)中AA的分析Example 1 Analysis of AA in kidney disease injection (Amino acid for renal insufficiency, produced by Hoechst Marion Roussel Company) and liver disease injection (Amino acid preparation for hepatic insufficiency, produced by Hoechst Marion Roussel Company )

1.试剂1. Reagents

1.1.0.1M HCl 4mL盐酸与480mL水相混合。1.1.0.1M HCl 4mL hydrochloric acid mixed with 480mL water.

1.2.1％DNFB 1g DNFB溶于100mL乙腈中。1.2.1% DNFB 1g DNFB was dissolved in 100mL acetonitrile.

1.3.0.5M NaHCO3 2.1g NaHCO3溶于50mL水中。1.3.0.5M NaHCO 3 2.1g NaHCO 3 was dissolved in 50mL of water.

1.4.0.1M磷酸盐缓冲液，pH 6.5称1.74g K2HPO4溶于100mL水中得0.1MK2HPO4。称1.36g KH2PO4溶于100mL水中得0.1M KH2PO4。向0.1M KH2PO4中加0.1M K2HPO4至pH＝6.5。1.4.0.1M phosphate buffer, pH 6.5 Weigh 1.74g K 2 HPO 4 dissolved in 100mL water to obtain 0.1M K 2 HPO 4 . Dissolve 1.36g KH 2 PO 4 in 100mL water to obtain 0.1M KH 2 PO 4 . To 0.1M KH2PO4 was added 0.1M K2HPO4 to pH = 6.5.

1.5.36mM磷酸三乙胺(TEAP)，pH 2.75 在烧杯中取～950mL水，用移液管取5mL三乙胺加至其中。混合后，用磷酸调节pH值至2.75。将溶液转移到1000mL容量瓶中，用水冲至刻度。过滤后使用。1.5.36mM triethylamine phosphate (TEAP), pH 2.75 Take ~950mL water in a beaker, and add 5mL triethylamine to it with a pipette. After mixing, adjust the pH to 2.75 with phosphoric acid. Transfer the solution to a 1000mL volumetric flask and flush to the mark with water. Use after filtering.

1.6.36mM磷酸三乙胺(TEAP)，pH 6.50 在烧杯中取～950mL水，用移液管取5mL三乙胺加至其中。混合后，用磷酸调节pH值至6.50。将溶液转移到1000mL容量瓶中，用水冲至刻度。过滤后使用。1.6.36mM triethylamine phosphate (TEAP), pH 6.50 Take ~950mL water in a beaker, and add 5mL triethylamine to it with a pipette. After mixing, adjust the pH to 6.50 with phosphoric acid. Transfer the solution to a 1000mL volumetric flask and flush to the mark with water. Use after filtering.

2.标准溶液的制备  按肾病注射液和肝病注射液的组成，精确称取AA参照物，以0.1M HCl作溶剂定容。用移液管取AA标准溶液5mL于25mL容量瓶中，以0.1M HCl冲至刻度，摇匀备用。2. Preparation of standard solution According to the composition of Kidney Disease Injection and Liver Disease Injection, accurately weigh AA reference substance, and use 0.1M HCl as solvent to make up the volume. Use a pipette to take 5mL of AA standard solution into a 25mL volumetric flask, flush to the mark with 0.1M HCl, shake well and set aside.

3.试样溶液的制备  用移液管取AA注射液样品1mL于25mL容量瓶中，以0.1M HCl冲至刻度，摇匀备用。3. Preparation of sample solution Use a pipette to take 1mL of AA injection sample into a 25mL volumetric flask, flush to the mark with 0.1M HCl, and shake well for later use.

4.标准溶液和试样溶液的衍生反应  用移液管取稀释后的AA标准溶液和样品溶液各1mL，分别置于10mL棕色容量瓶中，加入1mL 0.5M NaHCO3，摇匀，再加入1mL1％DNFB，摇匀。置于60℃加热块上加热60分钟。冷却后，用0.1M磷酸盐缓冲液(pH 6.5)冲至刻度，摇匀，准备HPLC分析用。4. Derivative reaction of standard solution and sample solution Use a pipette to take 1mL each of the diluted AA standard solution and sample solution, put them in a 10mL brown volumetric flask, add 1mL 0.5M NaHCO 3 , shake well, then add 1mL1 %DNFB, shake well. Place on a 60°C heating block and heat for 60 minutes. After cooling, flush to the mark with 0.1M phosphate buffer (pH 6.5), shake well, and prepare for HPLC analysis.

5.色谱条件5. Chromatographic conditions

5.1.测定天冬、谷、脯、丙、异亮、亮氨酸5.1. Determination of Asparagus, Gu, Preserved, C, Isoleucine and Leucine

分析柱           Kromasil C18，5μm，250×4.6mmAnalytical column Kromasil C 18 , 5μm, 250×4.6mm

流动相           A：ACN B：36mM TEAP，pH 6.50Mobile phase A: ACN B: 36mM TEAP, pH 6.50

梯度             83/71/68/10/10 B％[VB/(VA+VB)]at 0/10/25/30/35minGradient 83/71/68/10/10 B%[V B /(V A +V B )]at 0/10/25/30/35min

平衡时间         10minBalance time 10min

检测             UV 360nmDetection UV 360nm

温度             35℃Temperature 35℃

进样体积         20μLInjection volume 20μL

在此条件下得到如色谱图1(肾病注射液)和2(肝病注射液)的分离结果，出峰顺序为：天冬、谷、精、丝、苏和甘(与试剂峰重叠)、脯、丙、缬、蛋、异亮、亮、色、组、苯丙、赖、酪氨酸Under these conditions, the separation results shown in chromatograms 1 (Kenbing Injection) and 2 (Ganbing Injection) were obtained, and the order of peaks was asparagus, grain, essence, silk, Su and Gan (overlapped with reagent peaks), preserved , acrylic, valerian, egg, isoleucine, bright, color, group, phenylpropanoid, lysine, tyrosine

5.2.测定精、丝、苏、甘、组、蛋、缬、色、苯丙、赖、酪氨酸5.2. Determination of essence, silk, threonine, sweetness, group, egg, valerian, color, phenylpropanoid, lysine, tyrosine

分析柱              Kromasil C18，5μm，250×4.6mmAnalytical column Kromasil C 18 , 5μm, 250×4.6mm

流动相              A：ACN B：36mM TEAP，pH 2.75Mobile phase A: ACN B: 36mM TEAP, pH 2.75

梯度                78/60/35/10/10 B％[VB/(VA+VB)]at 0/20/30/35/40minGradient 78/60/35/10/10 B%[V B /(V A +V B )]at 0/20/30/35/40min

平衡时间            10minBalance time 10min

检测                UV 360nmDetection UV 360nm

温度                35℃Temperature 35℃

进样体积            20μLInjection volume 20μL

在此条件下得到如色谱图3(肾病注射液)和4(肝病注射液)的分离结果，出峰顺序为：精、丝、天冬、谷、苏、甘、丙、脯、组、蛋、缬、色、苯丙、亮+异亮、赖、酪氨酸。Under this condition, the separation results shown in chromatograms 3 (Kenbing Injection) and 4 (Ganbing Injection) were obtained, and the order of peaks was: essence, silk, asparagus, grain, Su, Gan, C, preserved, group, egg , Valerian, color, phenylpropanoid, leuc + isoleic, lysine, tyrosine.

6.计算6. Calculate

试样中AA的浓度＝(A试样/A标准)×C标准 The concentration of AA in the sample = (A sample /A standard ) × C standard

A试样：试样色谱图中AA的峰面积A sample : the peak area of AA in the sample chromatogram

A标准：标准色谱图中AA的峰面积A standard : the peak area of AA in the standard chromatogram

C标准：标准溶液AA的浓度C standard : the concentration of standard solution AA

7.肾病注射液的分析结果、准确度、精密度7. Analytical results, accuracy and precision of kidney disease injection

这里以肾病注射液为例，给出方法的准确度和精密度。肝病注射液也给出相近的结果。Here, the accuracy and precision of the method are given by taking nephrotic injection as an example. Liver Disease Injection also gave similar results.

7.1.准确度：7.1. Accuracy:

表1准确度测定结果(％RSD，相对标准偏差)   天冬氨酸   浓度   50％   75％   100％   125％   150％   ％RSD   1   响应值   107505   159941   212435   264899   317737   线性校正测定值   0.5004   0.7499   0.9997   1.2493   1.5007   回收率100％   99.99   99.97   99.94   0.02   2   响应值   107609   159240   212106   265468   316251   线性校正测定值   0.5009   0.7474   0.9999   1.2547   1.4972   回收率100％   99.66   99.99   100.37   0.36   3   响应值   106905   160295   212177   265850   315048   线性校正测定值   0.4964   0.7521   1.0006   1.2577   1.4933   回收率100％   100.28   100.06   100.61   0.28   谷氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   82767   125208   169925   214174   262019   线性校正测定值   0.5083   0.7453   0.9950   1.2421   1.5093   回收率100％   99.37   99.50   99.37   0.08   2   响应值   81268   123844   167062   211833   257995   线性校正测定值   0.5067   0.7477   0.9924   1.2459   1.5073   回收率100％   99.70   99.24   99.67   0.26   3   响应值   81949   124875   169259   214294   258174   线性校正测定值   0.5035   0.7463   0.9974   1.2522   1.5005   回收率100％   99.51   99.74   100.18   0.34   脯氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   674060   1016544   1349867   1695593   2030976   线性校正测定值   0.4994   0.7518   0.9974   1.2521   1.4992   回收率100％   100.24   99.74   100.17   0.27   2   响应值   671004   1011913   1349929   1699227   2026416   线性校正测定值   0.4993   0.7500   0.9987   1.2557   1.4964   回收率100％   100.01   99.87   100.45   0.30   3   响应值   677057   1013637   1358996   1694995   2037505   线性校正测定值   0.5008   0.7481   1.0019   1.2488   1.5004   回收率100％   99.75   100.19   99.90   0.22   丙氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   1689226   2529193   3358129   4213693   5039937   线性校正测定值   0.5001   0.7505   0.9976   1.2527   1.4990   回收率100％   100.07   99.76   100.22   0.23   2   响应值   1684704   2526283   3357122   4207109   5048707   线性校正测定值   0.5005   0.7507   0.9977   1.2504   1.5006   回收率100％   100.09   99.77   100.03   0.17   3   响应值   1686922   2530517   3370821   4203437   5059251   线性校正测定值   0.5001   0.7506   1.0002   1.2475   1.5016   回收率100％   100.08   100.02   99.80   0.15   异亮氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   2847226   4278219   5702401   7146538   8590133   线性校正测定值   0.5009   0.7501   0.9982   1.2497   1.5011   回收率100％   100.02   99.82   99.98   0.11   2   响应值   2846123   4271169   5711365   7156548   8589588   线性校正测定值   0.5010   0.7489   0.9994   1.2508   1.5000   回收率100％   99.85   99.94   100.06   0.11   3   响应值   2846518   4279660   5713699   7160764   8601874   线性校正测定值   0.5008   0.7497   0.9988   1.2502   1.5005   回收率100％   99.96   99.88   100.01   0.07   亮氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   3877328   5830216   7766820   9760839   11695672   线性校正测定值   0.5006   0.7501   0.9975   1.2523   1.4995   回收率100％   100.01   99.75   100.18   0.22   2   响应值   3871704   5822899   7779548   9744576   11704465   线性校正测定值   0.5006   0.7496   0.9994   1.2502   1.5003   回收率100％   99.95   99.94   100.01   0.04   3   响应值   3885949   5847103   7779066   9760675   11707404   线性校正测定值   0.5002   0.7509   0.9978   1.2511   1.5000   回收率100％   100.11   99.78   100.09   0.19   精氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   835750   1242453   1661765   2095025   2511935   线性校正测定值   0.5044   0.7462   0.9965   1.2530   1.5009   回收率100％   99.49   99.55   100.24   0.42   2   响应值   826133   1244503   1649347   2084369   2492214   线性校正测定值   0.5008   0.7515   0.9940   1.2547   1.4990   回收率100％   100.19   99.40   100.37   0.52   3   响应值   829654   1249113   1660278   2094206   2485311   线性校正测定值   0.4984   0.7507   0.9979   1.2589   1.4941   回收率100％   100.09   99.79   100.71   0.47   丝氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   461403   697391   927173   1171787   1408063   线性校正测定值   0.5019   0.7511   0.9937   1.2519   1.5014   回收率100％   100.14   99.37   100.15   0.45   2   响应值   456751   691675   924844   1167655   1395277   线性校正测定值   0.5002   0.7497   0.9975   1.2554   1.4972   回收率100％   99.97   99.75   100.43   0.35   3   响应值   462885   699799   935278   1170366   1397845   线性校正测定值   0.4976   0.7507   1.0022   1.2533   1.4962   回收率100％   100.09   100.22   100.26   0.09   苏氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   970437   1462010   1942883   2437173   2921780   线性校正测定值   0.4996   0.7515   0.9980   1.2513   1.4997   回收率100％   100.20   99.80   100.10   0.21   2   响应值   969140   1468548   1942414   2443789   2923002   线性校正测定值   0.5002   0.7502   0.9975   1.2536   1.4985   回收率100％   100.03   99.75   100.29   0.27   3   响应值   969102   1458788   1945406   2438242   2925560   线性校正测定值   0.5001   0.7503   0.9990   1.2508   1.4998   回收率100％   100.04   99.90   100.06   0.09   甘氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   894958   1350889   1807642   2289228   2745832   线性校正测定值   0.5029   0.7485   0.9946   1.2540   1.5000   回收率100％   99.80   99.46   100.32   0.44   2   响应值   890634   1325487   1776681   2236567   2671139   线性校正测定值   0.5028   0.7459   0.9981   1.2552   1.4981   回收率100％   99.45   99.81   100.41   0.49   3   响应值   895637   1325477   1787844   2246018   2669270   线性校正测定值   0.5026   0.7430   1.0017   1.2580   1.4948   回收率100％   99.07   100.17   100.64   0.81   组氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   775169   1155863   1521847   1888645   2208544   线性校正测定值   0.4901   0.7543   1.0082   1.2627   1.4847   回收率100％   100.57   100.82   101.02   0.22   2   响应值   769331   1131464   1510620   1871636   2227754   线性校正测定值   0.4991   0.7466   1.0058   1.2526   1.4960   回收率100％   99.55   100.58   100.20   0.52   3   响应值   765605   1139491   1492895   1838605   2158399   线性校正测定值   0.4886   0.7566   1.0100   1.2578   1.4870   回收率100％   100.88   101.00   100.62   0.19   蛋氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   1541638   2315268   3078507   3828190   4612621   线性校正测定值   0.4992   0.7518   1.0011   1.2459   1.5021   回收率100％   100.24   100.11   99.67   0.30   2   响应值   1525963   2280448   3039779   3800354   4568568   线性校正测定值   0.5013   0.7493   0.9989   1.2490   1.5015   回收率100％   99.91   99.89   99.92   0.01   3   响应值   1535531   2285886   3041390   3828625   4575687   线性校正测定值   0.5022   0.7483   0.9961   1.2542   1.4992   回收率100％   99.77   99.61   100.34   0.38   缬氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   2870482   4302568   5634504   7063143   8408206   线性校正测定值   0.4968   0.7555   0.9962   1.2543   1.4973   回收率100％   100.74   99.62   100.34   0.57   2   响应值   2827334   4244625   5665077   7035612   8393130   线性校正测定值   0.4962   0.7507   1.0057   1.2518   1.4955   回收率100％   100.09   100.57   100.14   0.26   3   响应值   2841364   4242346   5644377   7083558   8427622   线性校正测定值   0.4994   0.7493   0.9994   1.2561   1.4959   回收率100％   99.90   99.94   100.49   0.33   色氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   556880   847486   1131498   1409325   1712273   线性校正测定值   0.5000   0.7529   1.0000   1.2418   1.5054   回收率100％   100.38   100.00   99.34   0.53   2   响应值   555125   827557   1111071   1398090   1689129   线性校正测定值   0.5059   0.7458   0.9955   1.2482   1.5045   回收率100％   99.44   99.55   99.86   0.22   3   响应值   553313   838300   1122892   1403467   1683358   线性校正测定值   0.4983   0.7505   1.0023   1.2506   1.4983   回收率100％   100.07   100.23   100.05   0.10   苯丙氨酸   浓度   50％   75％   100％   125％   150％   1   响应值   1393886   2095567   2758770   3465822   4166411   线性校正测定值   0.5004   0.7540   0.9937   1.2493   1.5026   回收率100％   100.53   99.37   99.95   0.58   2   响应值  1374051   2064323   2750768   3438517   4139278   线性校正测定值   0.5006   0.7505   0.9991   1.2481   1.5018   回收率100％   100.07   99.91   99.85   0.12   3   响应值  1381206   2077333   2745871   3440346   4117863   线性校正测定值  0.4985   0.7531   0.9976   1.2515   1.4993   回收率100％   100.41   99.76   100.12   0.33   赖氨酸   浓度  50％   75％   100％   125％   150％   1   响应值  3009342   4493168   5934873   7335943   8679634   线性校正测定值  0.4923   0.7538   1.0078   1.2547   1.4914   回收率100％   100.50   100.78   100.37   0.21   2   响应值  3016029   4473573   5928032   7343220   8683054   线性校正测定值  0.4945   0.7510   1.0069   1.2559   1.4917   回收率100％   100.13   100.69   100.47   0.28   3   响应值  3012738   4500359   5927950   7328485   8678348   线性校正测定值  0.4922   0.7548   1.0068   1.2540   1.4922   回收率100％   100.64   100.68   100.32   0.20   酪氨酸  50％   75％   100％   125％   150％   1   响应值  132634   198614   263986   329785   398749   线性校正测定值  0.5022   0.7508   0.9971   1.2450   1.5049   回收率100％   100.10   99.71   99.60   0.26   2   响应值  131624   195033   262815   329784   400745   线性校正测定值  0.5084   0.7439   0.9956   1.2443   1.5078   回收率100％   99.19   99.56   99.54   0.21   3   响应值  131952   196135   264876   331776   401083   线性校正测定值  0.5059   0.7440   0.9989   1.2471   1.5041   回收率100％   99.19   99.89   99.77   0.37 Table 1 Accuracy measurement results (%RSD, relative standard deviation) aspartic acid concentration 50% 75% 100% 125% 150% %RSD 1 Response 107505 159941 212435 264899 317737 Linearity Corrected Measured Value 0.5004 0.7499 0.9997 1.2493 1.5007 100% recovery rate 99.99 99.97 99.94 0.02 2 Response 107609 159240 212106 265468 316251 Linearity Corrected Measured Value 0.5009 0.7474 0.9999 1.2547 1.4972 100% recovery rate 99.66 99.99 100.37 0.36 3 Response 106905 160295 212177 265850 315048 Linearity Corrected Measured Value 0.4964 0.7521 1.0006 1.2577 1.4933 100% recovery rate 100.28 100.06 100.61 0.28 glutamic acid concentration 50% 75% 100% 125% 150% 1 Response 82767 125208 169925 214174 262019 Linearity Corrected Measured Value 0.5083 0.7453 0.9950 1.2421 1.5093 100% recovery rate 99.37 99.50 99.37 0.08 2 Response 81268 123844 167062 211833 257995 Linearity Corrected Measured Value 0.5067 0.7477 0.9924 1.2459 1.5073 100% recovery rate 99.70 99.24 99.67 0.26 3 Response 81949 124875 169259 214294 258174 Linearity Corrected Measured Value 0.5035 0.7463 0.9974 1.2522 1.5005 100% recovery rate 99.51 99.74 100.18 0.34 proline concentration 50% 75% 100% 125% 150% 1 Response 674060 1016544 1349867 1695593 2030976 Linearity Corrected Measured Value 0.4994 0.7518 0.9974 1.2521 1.4992 100% recovery rate 100.24 99.74 100.17 0.27 2 Response 671004 1011913 1349929 1699227 2026416 Linearity Corrected Measured Value 0.4993 0.7500 0.9987 1.2557 1.4964 100% recovery rate 100.01 99.87 100.45 0.30 3 Response 677057 1013637 1358996 1694995 2037505 Linearity Corrected Measured Value 0.5008 0.7481 1.0019 1.2488 1.5004 100% recovery rate 99.75 100.19 99.90 0.22 Alanine concentration 50% 75% 100% 125% 150% 1 Response 1689226 2529193 3358129 4213693 5039937 Linearity Corrected Measured Value 0.5001 0.7505 0.9976 1.2527 1.4990 100% recovery rate 100.07 99.76 100.22 0.23 2 Response 1684704 2526283 3357122 4207109 5048707 Linearity Corrected Measured Value 0.5005 0.7507 0.9977 1.2504 1.5006 100% recovery rate 100.09 99.77 100.03 0.17 3 Response 1686922 2530517 3370821 4203437 5059251 Linearity Corrected Measured Value 0.5001 0.7506 1.0002 1.2475 1.5016 100% recovery rate 100.08 100.02 99.80 0.15 Isoleucine concentration 50% 75% 100% 125% 150% 1 Response 2847226 4278219 5702401 7146538 8590133 Linearity Corrected Measured Value 0.5009 0.7501 0.9982 1.2497 1.5011 100% recovery rate 100.02 99.82 99.98 0.11 2 Response 2846123 4271169 5711365 7156548 8589588 Linearity Corrected Measured Value 0.5010 0.7489 0.9994 1.2508 1.5000 100% recovery rate 99.85 99.94 100.06 0.11 3 Response 2846518 4279660 5713699 7160764 8601874 Linearity Corrected Measured Value 0.5008 0.7497 0.9988 1.2502 1.5005 100% recovery rate 99.96 99.88 100.01 0.07 Leucine concentration 50% 75% 100% 125% 150% 1 Response 3877328 5830216 7766820 9760839 11695672 Linearity Corrected Measured Value 0.5006 0.7501 0.9975 1.2523 1.4995 100% recovery rate 100.01 99.75 100.18 0.22 2 Response 3871704 5822899 7779548 9744576 11704465 Linearity Corrected Measured Value 0.5006 0.7496 0.9994 1.2502 1.5003 100% recovery rate 99.95 99.94 100.01 0.04 3 Response 3885949 5847103 7779066 9760675 11707404 Linearity Corrected Measured Value 0.5002 0.7509 0.9978 1.2511 1.5000 100% recovery rate 100.11 99.78 100.09 0.19 arginine concentration 50% 75% 100% 125% 150% 1 Response 835750 1242453 1661765 2095025 2511935 Linearity Corrected Measured Value 0.5044 0.7462 0.9965 1.2530 1.5009 100% recovery rate 99.49 99.55 100.24 0.42 2 Response 826133 1244503 1649347 2084369 2492214 Linearity Corrected Measured Value 0.5008 0.7515 0.9940 1.2547 1.4990 100% recovery rate 100.19 99.40 100.37 0.52 3 Response 829654 1249113 1660278 2094206 2485311 Linearity Corrected Measured Value 0.4984 0.7507 0.9979 1.2589 1.4941 100% recovery rate 100.09 99.79 100.71 0.47 serine concentration 50% 75% 100% 125% 150% 1 Response 461403 697391 927173 1171787 1408063 Linearity Corrected Measured Value 0.5019 0.7511 0.9937 1.2519 1.5014 100% recovery rate 100.14 99.37 100.15 0.45 2 Response 456751 691675 924844 1167655 1395277 Linearity Corrected Measured Value 0.5002 0.7497 0.9975 1.2554 1.4972 100% recovery rate 99.97 99.75 100.43 0.35 3 Response 462885 699799 935278 1170366 1397845 Linearity Corrected Measured Value 0.4976 0.7507 1.0022 1.2533 1.4962 100% recovery rate 100.09 100.22 100.26 0.09 threonine concentration 50% 75% 100% 125% 150% 1 Response 970437 1462010 1942883 2437173 2921780 Linearity Corrected Measured Value 0.4996 0.7515 0.9980 1.2513 1.4997 100% recovery rate 100.20 99.80 100.10 0.21 2 Response 969140 1468548 1942414 2443789 2923002 Linearity Corrected Measured Value 0.5002 0.7502 0.9975 1.2536 1.4985 100% recovery rate 100.03 99.75 100.29 0.27 3 Response 969102 1458788 1945406 2438242 2925560 Linearity Corrected Measured Value 0.5001 0.7503 0.9990 1.2508 1.4998 100% recovery rate 100.04 99.90 100.06 0.09 Glycine concentration 50% 75% 100% 125% 150% 1 Response 894958 1350889 1807642 2289228 2745832 Linearity Corrected Measured Value 0.5029 0.7485 0.9946 1.2540 1.5000 100% recovery rate 99.80 99.46 100.32 0.44 2 Response 890634 1325487 1776681 2236567 2671139 Linearity Corrected Measured Value 0.5028 0.7459 0.9981 1.2552 1.4981 100% recovery rate 99.45 99.81 100.41 0.49 3 Response 895637 1325477 1787844 2246018 2669270 Linearity Corrected Measured Value 0.5026 0.7430 1.0017 1.2580 1.4948 100% recovery rate 99.07 100.17 100.64 0.81 Histidine concentration 50% 75% 100% 125% 150% 1 Response 775169 1155863 1521847 1888645 2208544 Linearity Corrected Measured Value 0.4901 0.7543 1.0082 1.2627 1.4847 100% recovery rate 100.57 100.82 101.02 0.22 2 Response 769331 1131464 1510620 1871636 2227754 Linearity Corrected Measured Value 0.4991 0.7466 1.0058 1.2526 1.4960 100% recovery rate 99.55 100.58 100.20 0.52 3 Response 765605 1139491 1492895 1838605 2158399 Linearity Corrected Measured Value 0.4886 0.7566 1.0100 1.2578 1.4870 100% recovery rate 100.88 101.00 100.62 0.19 Methionine concentration 50% 75% 100% 125% 150% 1 Response 1541638 2315268 3078507 3828190 4612621 Linearity Corrected Measured Value 0.4992 0.7518 1.0011 1.2459 1.5021 100% recovery rate 100.24 100.11 99.67 0.30 2 Response 1525963 2280448 3039779 3800354 4568568 Linearity Corrected Measured Value 0.5013 0.7493 0.9989 1.2490 1.5015 100% recovery rate 99.91 99.89 99.92 0.01 3 Response 1535531 2285886 3041390 3828625 4575687 Linearity Corrected Measured Value 0.5022 0.7483 0.9961 1.2542 1.4992 100% recovery rate 99.77 99.61 100.34 0.38 Valine concentration 50% 75% 100% 125% 150% 1 Response 2870482 4302568 5634504 7063143 8408206 Linearity Corrected Measured Value 0.4968 0.7555 0.9962 1.2543 1.4973 100% recovery rate 100.74 99.62 100.34 0.57 2 Response 2827334 4244625 5665077 7035612 8393130 Linearity Corrected Measured Value 0.4962 0.7507 1.0057 1.2518 1.4955 100% recovery rate 100.09 100.57 100.14 0.26 3 Response 2841364 4242346 5644377 7083558 8427622 Linearity Corrected Measured Value 0.4994 0.7493 0.9994 1.2561 1.4959 100% recovery rate 99.90 99.94 100.49 0.33 Tryptophan concentration 50% 75% 100% 125% 150% 1 Response 556880 847486 1131498 1409325 1712273 Linearity Corrected Measured Value 0.5000 0.7529 1.0000 1.2418 1.5054 100% recovery rate 100.38 100.00 99.34 0.53 2 Response 555125 827557 1111071 1398090 1689129 Linearity Corrected Measured Value 0.5059 0.7458 0.9955 1.2482 1.5045 100% recovery rate 99.44 99.55 99.86 0.22 3 Response 553313 838300 1122892 1403467 1683358 Linearity Corrected Measured Value 0.4983 0.7505 1.0023 1.2506 1.4983 100% recovery rate 100.07 100.23 100.05 0.10 Phenylalanine concentration 50% 75% 100% 125% 150% 1 Response 1393886 2095567 2758770 3465822 4166411 Linearity Corrected Measured Value 0.5004 0.7540 0.9937 1.2493 1.5026 100% recovery rate 100.53 99.37 99.95 0.58 2 Response 1374051 2064323 2750768 3438517 4139278 Linearity Corrected Measured Value 0.5006 0.7505 0.9991 1.2481 1.5018 100% recovery rate 100.07 99.91 99.85 0.12 3 Response 1381206 2077333 2745871 3440346 4117863 Linearity Corrected Measured Value 0.4985 0.7531 0.9976 1.2515 1.4993 100% recovery rate 100.41 99.76 100.12 0.33 Lysine concentration 50% 75% 100% 125% 150% 1 Response 3009342 4493168 5934873 7335943 8679634 Linearity Corrected Measured Value 0.4923 0.7538 1.0078 1.2547 1.4914 100% recovery rate 100.50 100.78 100.37 0.21 2 Response 3016029 4473573 5928032 7343220 8683054 Linearity Corrected Measured Value 0.4945 0.7510 1.0069 1.2559 1.4917 100% recovery rate 100.13 100.69 100.47 0.28 3 Response 3012738 4500359 5927950 7328485 8678348 Linearity Corrected Measured Value 0.4922 0.7548 1.0068 1.2540 1.4922 100% recovery rate 100.64 100.68 100.32 0.20 Tyrosine 50% 75% 100% 125% 150% 1 Response 132634 198614 263986 329785 398749 Linearity Corrected Measured Value 0.5022 0.7508 0.9971 1.2450 1.5049 100% recovery rate 100.10 99.71 99.60 0.26 2 Response 131624 195033 262815 329784 400745 Linearity Corrected Measured Value 0.5084 0.7439 0.9956 1.2443 1.5078 100% recovery rate 99.19 99.56 99.54 0.21 3 Response 131952 196135 264876 331776 401083 Linearity Corrected Measured Value 0.5059 0.7440 0.9989 1.2471 1.5041 100% recovery rate 99.19 99.89 99.77 0.37

对所有测试的AA，回收率都在100±1％范围内。The recoveries were within 100 ± 1% for all AAs tested.

7.2.精密度：7.2. Precision:

该方法有较高的精密度，中间精密度和重复性都不大于0.5％，以下是测定结果。The method has high precision, the intermediate precision and repeatability are not more than 0.5%, the following are the measurement results.

7.2.1.中间精密度：7.2.1. Intermediate precision:

表2：中间精密度测定结果   天冬氨酸  浓度   50％   75％   100％   125％   150％   平均值  测定值1   0.5004   0.7499   0.9997   1.2493   1.5007  测定值2   0.5009   0.7474   0.9999   1.2547   1.4972  测定值3   0.4964   0.7521   1.0006   1.2577   1.4933  SD   2.5E-03   2.3E-03   4.8E-04   4.2E-03   3.7E-03  ％RSD   0.50   0.31   0.05   0.34   0.25   0.29   谷氨酸  测定值1   0.5083   0.7453   0.9950   1.2421   1.5093  测定值2   0.5067   0.7477   0.9924   1.2459   1.5073  测定值3   0.5035   0.7463   0.9974   1.2522   1.5005  SD   2.4E-03   1.2E-03   2.5E-03   5.1E-03   4.6E-03  ％RSD   0.49   0.16   0.25   0.41   0.31   0.32   脯氨酸  测定值1   0.4994   0.7518   0.9974   1.2521   1.4992  测定值2   0.4993   0.7500   0.9987   1.2557   1.4964  测定值3   0.5008   0.7481   1.0019   1.2488   1.5004  SD   8.4E-04   1.8E-03   2.3E-03   3.4E-03   2.1E-03  ％RSD   0.17   0.25   0.23   0.28   0.14   0.21   丙氨酸  测定值1   0.5001   0.7505   0.9976   1.2527   1.4990  测定值2   0.5005   0.7507   0.9977   1.2504   1.5006  测定值3   0.5001   0.7506   1.0002   1.2475   1.5016  SD   2.3E-04   9.2E-05   1.4E-03   2.6E-03   1.3E-03  ％RSD   0.05   0.01   0.14   0.21   0.09   0.10   异亮氨酸  测定值1   0.5009   0.7501   0.9982   1.2497   1.5011  测定值2   0.5010   0.7489   0.9994   1.2508   1.5000  测定值3   0.5008   0.7497   0.9988   1.2502   1.5005  SD   1.1E-04   6.5E-04   6.0E-04   5.3E-04   5.4E-04  ％RSD   0.02   0.09   0.06   0.04   0.04   0.05   亮氨酸  测定值1   0.5006   0.7501   0.9975   1.2523   1.4995  测定值2   0.5006   0.7496   0.9994   1.2502   1.5003  测定值3   0.5002   0.7509   0.9978   1.2511   1.5000  SD   2.5E-04   6.3E-04   9.8E-04   1.1E-03   4.1E-04  ％RSD   0.05   0.08   0.10   0.09   0.03   0.07   精氨酸  测定值1   0.5044   0.7462   0.9955   1.2530   1.5009  测定值2   0.5008   0.7515   0.9940   1.2547   1.4990  测定值3   0.4984   0.7507   0.9979   1.2589   1.4941  SD   3.0E-03   2.8E-03   2.0E-03   3.0E-03   3.5E-03  ％RSD   0.60   0.38   0.20   0.24   0.23   0.33   丝氨酸  测定值1   0.5019   0.7511   0.9937   1.2519   1.5014  测定值2   0.5002   0.7497   0.9975   1.2554   1.4972  测定值3   0.4976   0.7507   1.0022   1.2533   1.4962  SD   2.2E-03   6.9E-04   4.3E-03   1.8E-03   2.7E-03  ％RSD   0.43   0.09   0.43   0.14   0.18   0.25   苏氨酸  测定值1   0.4996   0.7515   0.9980   1.2513   1.4997  测定值2   0.5002   0.7502   0.9975   1.2536   1.4985  测定值3   0.5001   0.7503   0.9990   1.2508   1.4998  SD   3.3E-04   7.1E-04   7.7E-04   1.5E-03   7.4E-04  ％RSD   0.07   0.09   0.08   0.12   0.05   0.08   甘氨酸  测定值1   0.5029   0.7485   0.9946   1.2540   1.5000  测定值2   0.5028   0.7459   0.9981   1.2552   1.4981  测定值3   0.5026   0.7430   1.0017   1.2580   1.4948  SD   1.7E-04   2.7E-03   3.5E-03   2.0E-03   2.7E-03  ％RSD   0.03   0.37   0.35   0.16   0.18   0.22   组氨酸  测定值1   0.4901   0.7543   1.0082   1.2627   1.4847  测定值2   0.4991   0.7466   1.0058   1.2526   1.4960  测定值3   0.4886   0.7566   1.0100   1.2578   1.4870   SD   5.7E-03   5.2E-03   2.1E-03   5.1E-03   6.0E-03   ％RSD   1.13   0.70   0.21   0.41   0.40   0.57   蛋氨酸   测定值1   0.4992   0.7518   1.0011   1.2459   1.5021   测定值2   0.5013   0.7493   0.9989   1.2490   1.5015   测定值3   0.5022   0.7483   0.9961   1.2542   1.4992   SD   1.6E-03   1.8E-03   2.5E-03   4.2E-03   1.5E-03   ％RSD   0.32   0.24   0.25   0.34   0.10   0.25   缬氨酸   测定值1   0.4968   0.7555   0.9962   1.2543   1.4973   测定值2   0.4962   0.7507   1.0057   1.2518   1.4955   测定值3   0.4994   0.7493   0.9994   1.2561   1.4959   SD   1.7E-03   3.3E-03   4.9E-03   2.2E-03   9.2E-04   ％RSD   0.34   0.44   0.49   0.17   0.06   0.30   色氨酸   测定值1   0.5000   0.7529   1.0000   1.2418   1.5054   测定值2   0.5059   0.7458   0.9955   1.2482   1.5045   测定值3   0.4983   0.7505   1.0023   1.2506   1.4983   SD   4.0E-03   3.6E-03   3.5E-03   4.6E-03   3.9E-03   ％RSD   0.80   0.48   0.35   0.37   0.26   0.45   苯丙氨酸   测定值1   0.5004   0.7540   0.9937   1.2493   1.5026   测定值2   0.5006   0.7505   0.9991   1.2481   1.5018   测定值3   0.4985   0.7531   0.9976   1.2515   1.4993   SD   1.1E-03   1.8E-03   2.7E-03   1.8E-03   1.7E-03   ％RSD   0.22   0.24   0.27   0.14   0.11   0.20   赖氨酸   测定值1   0.4923   0.7538   1.0078   1.2547   1.4914   测定值2   0.4945   0.7510   1.0069   1.2559   1.4917   测定值3   0.4922   0.7548   1.0068   1.2540   1.4922   SD   1.3E-03   2.0E-03   5.6E-04   9.9E-04   4.1E-04   ％RSD   0.26   0.26   0.06   0.08   0.03   0.14   酪氨酸   测定值1   0.5022   0.7508   0.9971   1.2450   1.5049   测定值2   0.5084   0.7439   0.9956   1.2443   1.5078   测定值3   0.5059   0.7440   0.9989   1.2471   1.5041   SD   3.2E-03   4.0E-03   1.7E-03   1.4E-03   1.9E-03   ％RSD   0.63   0.53   0.17   0.12   0.13   0.31 Table 2: Results of intermediate precision determination aspartic acid concentration 50% 75% 100% 125% 150% average value measured value 1 0.5004 0.7499 0.9997 1.2493 1.5007 measured value 2 0.5009 0.7474 0.9999 1.2547 1.4972 measured value 3 0.4964 0.7521 1.0006 1.2577 1.4933 SD 2.5E-03 2.3E-03 4.8E-04 4.2E-03 3.7E-03 %RSD 0.50 0.31 0.05 0.34 0.25 0.29 glutamic acid measured value 1 0.5083 0.7453 0.9950 1.2421 1.5093 measured value 2 0.5067 0.7477 0.9924 1.2459 1.5073 measured value 3 0.5035 0.7463 0.9974 1.2522 1.5005 SD 2.4E-03 1.2E-03 2.5E-03 5.1E-03 4.6E-03 %RSD 0.49 0.16 0.25 0.41 0.31 0.32 proline measured value 1 0.4994 0.7518 0.9974 1.2521 1.4992 measured value 2 0.4993 0.7500 0.9987 1.2557 1.4964 measured value 3 0.5008 0.7481 1.0019 1.2488 1.5004 SD 8.4E-04 1.8E-03 2.3E-03 3.4E-03 2.1E-03 %RSD 0.17 0.25 0.23 0.28 0.14 0.21 Alanine measured value 1 0.5001 0.7505 0.9976 1.2527 1.4990 measured value 2 0.5005 0.7507 0.9977 1.2504 1.5006 measured value 3 0.5001 0.7506 1.0002 1.2475 1.5016 SD 2.3E-04 9.2E-05 1.4E-03 2.6E-03 1.3E-03 %RSD 0.05 0.01 0.14 0.21 0.09 0.10 Isoleucine measured value 1 0.5009 0.7501 0.9982 1.2497 1.5011 measured value 2 0.5010 0.7489 0.9994 1.2508 1.5000 measured value 3 0.5008 0.7497 0.9988 1.2502 1.5005 SD 1.1E-04 6.5E-04 6.0E-04 5.3E-04 5.4E-04 %RSD 0.02 0.09 0.06 0.04 0.04 0.05 Leucine measured value 1 0.5006 0.7501 0.9975 1.2523 1.4995 measured value 2 0.5006 0.7496 0.9994 1.2502 1.5003 measured value 3 0.5002 0.7509 0.9978 1.2511 1.5000 SD 2.5E-04 6.3E-04 9.8E-04 1.1E-03 4.1E-04 %RSD 0.05 0.08 0.10 0.09 0.03 0.07 arginine measured value 1 0.5044 0.7462 0.9955 1.2530 1.5009 measured value 2 0.5008 0.7515 0.9940 1.2547 1.4990 measured value 3 0.4984 0.7507 0.9979 1.2589 1.4941 SD 3.0E-03 2.8E-03 2.0E-03 3.0E-03 3.5E-03 %RSD 0.60 0.38 0.20 0.24 0.23 0.33 serine measured value 1 0.5019 0.7511 0.9937 1.2519 1.5014 measured value 2 0.5002 0.7497 0.9975 1.2554 1.4972 measured value 3 0.4976 0.7507 1.0022 1.2533 1.4962 SD 2.2E-03 6.9E-04 4.3E-03 1.8E-03 2.7E-03 %RSD 0.43 0.09 0.43 0.14 0.18 0.25 threonine measured value 1 0.4996 0.7515 0.9980 1.2513 1.4997 measured value 2 0.5002 0.7502 0.9975 1.2536 1.4985 measured value 3 0.5001 0.7503 0.9990 1.2508 1.4998 SD 3.3E-04 7.1E-04 7.7E-04 1.5E-03 7.4E-04 %RSD 0.07 0.09 0.08 0.12 0.05 0.08 Glycine measured value 1 0.5029 0.7485 0.9946 1.2540 1.5000 measured value 2 0.5028 0.7459 0.9981 1.2552 1.4981 measured value 3 0.5026 0.7430 1.0017 1.2580 1.4948 SD 1.7E-04 2.7E-03 3.5E-03 2.0E-03 2.7E-03 %RSD 0.03 0.37 0.35 0.16 0.18 0.22 Histidine measured value 1 0.4901 0.7543 1.0082 1.2627 1.4847 measured value 2 0.4991 0.7466 1.0058 1.2526 1.4960 measured value 3 0.4886 0.7566 1.0100 1.2578 1.4870 SD 5.7E-03 5.2E-03 2.1E-03 5.1E-03 6.0E-03 %RSD 1.13 0.70 0.21 0.41 0.40 0.57 Methionine measured value 1 0.4992 0.7518 1.0011 1.2459 1.5021 measured value 2 0.5013 0.7493 0.9989 1.2490 1.5015 measured value 3 0.5022 0.7483 0.9961 1.2542 1.4992 SD 1.6E-03 1.8E-03 2.5E-03 4.2E-03 1.5E-03 %RSD 0.32 0.24 0.25 0.34 0.10 0.25 Valine measured value 1 0.4968 0.7555 0.9962 1.2543 1.4973 measured value 2 0.4962 0.7507 1.0057 1.2518 1.4955 measured value 3 0.4994 0.7493 0.9994 1.2561 1.4959 SD 1.7E-03 3.3E-03 4.9E-03 2.2E-03 9.2E-04 %RSD 0.34 0.44 0.49 0.17 0.06 0.30 Tryptophan measured value 1 0.5000 0.7529 1.0000 1.2418 1.5054 measured value 2 0.5059 0.7458 0.9955 1.2482 1.5045 measured value 3 0.4983 0.7505 1.0023 1.2506 1.4983 SD 4.0E-03 3.6E-03 3.5E-03 4.6E-03 3.9E-03 %RSD 0.80 0.48 0.35 0.37 0.26 0.45 Phenylalanine measured value 1 0.5004 0.7540 0.9937 1.2493 1.5026 measured value 2 0.5006 0.7505 0.9991 1.2481 1.5018 measured value 3 0.4985 0.7531 0.9976 1.2515 1.4993 SD 1.1E-03 1.8E-03 2.7E-03 1.8E-03 1.7E-03 %RSD 0.22 0.24 0.27 0.14 0.11 0.20 Lysine measured value 1 0.4923 0.7538 1.0078 1.2547 1.4914 measured value 2 0.4945 0.7510 1.0069 1.2559 1.4917 measured value 3 0.4922 0.7548 1.0068 1.2540 1.4922 SD 1.3E-03 2.0E-03 5.6E-04 9.9E-04 4.1E-04 %RSD 0.26 0.26 0.06 0.08 0.03 0.14 Tyrosine measured value 1 0.5022 0.7508 0.9971 1.2450 1.5049 measured value 2 0.5084 0.7439 0.9956 1.2443 1.5078 measured value 3 0.5059 0.7440 0.9989 1.2471 1.5041 SD 3.2E-03 4.0E-03 1.7E-03 1.4E-03 1.9E-03 %RSD 0.63 0.53 0.17 0.12 0.13 0.31

7.2.2.重复性：7.2.2. Repeatability:

表3：重复性测定结果(SD标准偏差) AA   六份100％AA溶液试样响应值 平均值 SD   ％RSD  1   2   3   4   5   6   天冬氨酸  282213   282838   281975   282412   283246   283707   282731.8   658.9   0.23   谷氨酸  114212   113094   112932   113514   113567   112901   113370   501.3   0.44   脯氨酸  1511838   1515825   1506534   1506317   1513907   1509467   1510648   3896.6   0.26   丙氨酸  3318444   3322253   3308888   3307867   3319691   3313502   3315108   5947.8   0.18   异亮氨酸  5865799   5864773   5862861   5857414   5858293   5864464   5862267   3557.5   0.06   亮氨酸  8026932   8025250   8022505   8019344   8018902   8025567   8023083   3390.5   0.04   精氨酸  1744130   1725521   1727469   1728244   1730010   1732531   1731318   6711.2   0.39   丝氨酸  951103   949570   951600   945253   952324   950975   950137.5   2558.7   0.27   苏氨酸  1866838   1861626   1864604   1859593   1864654   1863245   1863427   2549.6   0.14   甘氨酸  1780536   1775098   1789397   1775533   1794689   1783383   1783106   7770.5   0.44   组氨酸  1512464   1510513   1523605   1507769   1505902   1520203   1513409   7036.5   0.46   蛋氨酸  3226137   3225164   3228301   3220109   3226790   3229103   3225934   3191.9   0.10   缬氨酸  4997805   5001130   4996398   4992264   4999446   5000763   4997968   3317.3   0.07   色氨酸  1033196   1026753   1036733   1036675   1033353   1035042   1033625   3700.1   0.36   苯丙氨酸  2703400   2706028   2709602   2701132   2697518   2714989   2705445   6234.3   0.23   赖氨酸  5839359   5823697   5821899   5875818   5821530   5810851   5832192   23245.7   0.40   酪氨酸  238750   237775   238836   239670   238830   238934   238799.2   605.0   0.25 Table 3: Repeatability determination results (SD standard deviation) AAA Response values of six 100% AA solution samples average value SD %RSD 1 2 3 4 5 6 aspartic acid 282213 282838 281975 282412 283246 283707 282731.8 658.9 0.23 glutamic acid 114212 113094 112932 113514 113567 112901 113370 501.3 0.44 proline 1511838 1515825 1506534 1506317 1513907 1509467 1510648 3896.6 0.26 Alanine 3318444 3322253 3308888 3307867 3319691 3313502 3315108 5947.8 0.18 Isoleucine 5865799 5864773 5862861 5857414 5858293 5864464 5862267 3557.5 0.06 Leucine 8026932 8025250 8022505 8019344 8018902 8025567 8023083 3390.5 0.04 arginine 1744130 1725521 1727469 1728244 1730010 1732531 1731318 6711.2 0.39 serine 951103 949570 951600 945253 952324 950975 950137.5 2558.7 0.27 threonine 1866838 1861626 1864604 1859593 1864654 1863245 1863427 2549.6 0.14 Glycine 1780536 1775098 1789397 1775533 1794689 1783383 1783106 7770.5 0.44 Histidine 1512464 1510513 1523605 1507769 1505902 1520203 1513409 7036.5 0.46 Methionine 3226137 3225164 3228301 3220109 3226790 3229103 3225934 3191.9 0.10 Valine 4997805 5001130 4996398 4992264 4999446 5000763 4997968 3317.3 0.07 Tryptophan 1033196 1026753 1036733 1036675 1033353 1035042 1033625 3700.1 0.36 Phenylalanine 2703400 2706028 2709602 2701132 2697518 2714989 2705445 6234.3 0.23 Lysine 5839359 5823697 5821899 5875818 5821530 5810851 5832192 23245.7 0.40 Tyrosine 238750 237775 238836 239670 238830 238934 238799.2 605.0 0.25

实施例2  丙谷酰胺(L-丙氨酰-L-谷氨酰胺)原料药(天津天成制药有限公司生产)中主成分丙谷酰胺含量的测定Example 2 Determination of content of main component propylglutamine in the bulk drug (produced by Tianjin Tiancheng Pharmaceutical Co., Ltd.) of propylglutamine (L-alanyl-L-glutamine)

1.试剂1. Reagents

1.1. 参见实施例l。1.1. See embodiment 1.

1.2. 6N HCl  浓盐酸和水按1∶1(V/V)混合。1.2. Mix 6N HCl concentrated hydrochloric acid and water at a ratio of 1:1 (V/V).

2.标准溶液的制备  从干燥器中取出丙谷酰胺基准物，精确称取约15mg于带盖的聚乙烯管(1)中，用移液管加1mL 6N HCl，盖严，摇匀备用。2. Preparation of standard solution Take out the standard substance of Proglutamine from the desiccator, accurately weigh about 15mg into a polyethylene tube with a cover (1), add 1mL of 6N HCl with a pipette, cover tightly, and shake well for later use.

3.试样溶液的制备  精确称取丙谷酰胺试样约15mg于带盖的聚乙烯管(2)中，用移液管加1mL 6N HCl，盖严，摇匀备用。3. Preparation of sample solution Accurately weigh about 15 mg of proglutamine sample into a polyethylene tube with a cover (2), add 1 mL of 6N HCl with a pipette, cover tightly, and shake well for later use.

4.标准溶液和试样溶液的酸解  将聚乙烯管(1)和(2)于105℃加热6小。冷却，摇匀备用。4. Acid hydrolysis of standard solution and sample solution Heat polyethylene tubes (1) and (2) at 105°C for 6 hours. Let cool, shake well and set aside.

5.不经酸解试样溶液的制备  精确称取丙谷酰胺试样约15mg于带盖的聚乙烯管(3)中，用移液管加1mL水，盖严，摇匀备用。5. Preparation of sample solution without acid hydrolysis Accurately weigh about 15 mg of proglutamine sample into a polyethylene tube with a cover (3), add 1 mL of water with a pipette, cover tightly, and shake well for later use.

6.衍生反应  用移液管分别从聚乙烯管(1)、(2)和(3)吸取溶液各20L，置于10mL棕色容量瓶中。分别加1mL 0.5M NaHCO3，摇匀。再加0.5mL 1％ DNFB，摇匀。将溶液置于加热块中，60℃加热60分钟，取出冷却。用0.1M磷酸盐缓冲液(pH 6.5)冲至刻度，摇匀，准备HPLC分析用。6. Derivatization reaction Use a pipette to draw 20L each of the solutions from polyethylene tubes (1), (2) and (3), and place them in a 10mL brown volumetric flask. Add 1mL 0.5M NaHCO 3 respectively and shake well. Add 0.5mL 1% DNFB and shake well. Place the solution in a heating block, heat at 60°C for 60 minutes, remove to cool. Rinse to the mark with 0.1M phosphate buffer (pH 6.5), shake well, and prepare for HPLC analysis.

7.色谱条件7. Chromatographic conditions

分析柱       Kromasil C18，5μm，250×4.6mmAnalytical column Kromasil C 18 , 5μm, 250×4.6mm

流动相       A：ACN    B：36mM TEAP，pH2.75Mobile phase A: ACN B: 36mM TEAP, pH2.75

梯度         78/64.5/60/60B％[VB/(VA+VB)]at 0/15/25/30minGradient 78/64.5/60/60B%[V B /(V A +V B )]at 0/15/25/30min

平衡时间     10minBalance time 10min

检测         UV360nmDetection UV360nm

温度         35℃Temperature 35℃

进样体积     20μLInjection volume 20μL

在此条件下得到如色谱图5的分离结果。Under these conditions, the separation results as in chromatogram 5 were obtained.

8.计算8. Calculate

8.1.当未酸解试样谱图中Ala-Glu、L-Glu衍生物的峰面积小于Ala-Gln衍生物峰面积的0.1％时，就不考虑这些杂质对测定的影响。8.1. When the peak area of Ala-Glu and L-Glu derivatives is less than 0.1% of the peak area of Ala-Gln derivatives in the chromatogram of the unacidified sample, the influence of these impurities on the determination is not considered.

丙谷酰胺的百分含量＝[(A试样：×W标准)/(A标准×W试样)]×100％The percentage content of proglutamine=[(A sample :×W standard )/(A standard ×W sample )]×100%

W试样：酸解试样重(mg)W sample : acid hydrolysis sample weight (mg)

W标准：丙谷酰胺基准物重(mg)W standard : Proglutamine reference weight (mg)

A试样：酸解试样谱图中谷氨酸衍生物峰面积A sample : the peak area of glutamic acid derivatives in the spectrum of the acid hydrolyzed sample

A标准：基准物谱图中谷氨酸衍生物峰面积 Standard A: Peak area of glutamic acid derivatives in the spectrum of reference substances

8.2.当未酸解试样谱图中Ala-Glu、L-Glu的峰面积大于Ala-Gln峰面积的0.1％时，应校正丙谷酰胺的测定结果。丙谷酰胺在酸、碱、氧化剂存在下的降解实验结果表明，Ala-Glu是主要降解物，但没观察到L-Glu。因此，校正时主要考虑试样中原有的Ala-Glu。8.2. When the peak area of Ala-Glu and L-Glu in the chromatogram of the unacid-hydrolyzed sample is greater than 0.1% of the peak area of Ala-Gln, the determination result of proglutamine should be corrected. The degradation experiment results of proglutamine in the presence of acid, alkali and oxidant showed that Ala-Glu was the main degradation product, but no L-Glu was observed. Therefore, the original Ala-Glu in the sample is mainly considered during calibration.

丙谷酰胺的百分含量＝{[A试样×AAla-Gln×W标准]/[(AAla-Gln+AAla-Glu×f)×(A标准×W试样)]}×100％The percentage content of proglutamine={[A sample ×A Ala-Gln ×W standard ]/[(A Ala-Gln +A Ala-Glu ×f)×(A standard ×W sample )]}×100 %

W试样：酸解试样重(mg)W sample : acid hydrolysis sample weight (mg)

W标准：丙谷酰胺基准物重(mg)W standard : Proglutamine reference weight (mg)

A试样：酸解试样谱图中谷氨酸衍生物峰面积A sample : the peak area of glutamic acid derivatives in the spectrum of the acid hydrolyzed sample

A标准：基准物谱图中谷氨酸衍生物峰面积 Standard A: Peak area of glutamic acid derivatives in the spectrum of reference substances

AAla-Gln：：未酸解试样谱图中丙谷酰胺衍生物的峰面积A Ala-Gln :: the peak area of the proglutamine derivative in the spectrum of the unacid-hydrolyzed sample

AAla-Glu：：未酸解试样谱图中丙谷二肽衍生物的峰面积A Ala-Glu :: the peak area of the dipeptide derivative in the spectrum of the unacid-hydrolyzed sample

f：丙谷酰胺和丙谷二肽相对响应因子。f: Relative response factors of proglutamine and proglutamine.

实施例3  丙谷酰胺注射液(四川科伦药业股份有限公司生产)中丙谷酰胺含量的测定Example 3 Determination of Proglutamine Content in Proglutamine Injection (Sichuan Kelun Pharmaceutical Co., Ltd. Production)

1.试剂1. Reagents

1.1. 参见实施例1。1.1. See Example 1.

1.2. 6.7N HCl 167mL浓盐酸用水稀释到300mL。1.2. Dilute 6.7N HCl 167mL concentrated hydrochloric acid to 300mL with water.

2.标准溶液的制备  精确称取约20mg丙谷酰胺或13.5mg谷氨酸基准物于带盖的聚乙烯管(1)中，加100μL水和900μL 6.7N HCl，盖严，摇匀备用。2. Preparation of standard solution Accurately weigh about 20mg of proglutamine or 13.5mg of glutamic acid reference substance into a polyethylene tube with a cover (1), add 100μL of water and 900μL of 6.7N HCl, cover tightly, and shake well for later use.

3.试样溶液的制备  取100μL试样溶液和900μL 6.7N HCl于带盖的聚乙烯管(2)中，盖严，摇匀备用。3. Preparation of sample solution Take 100 μL of sample solution and 900 μL of 6.7N HCl in a polyethylene tube with a cover (2), cover it tightly, and shake well for later use.

4.酸解  将聚乙烯管(1)和(2)置于加热块中，105℃加热6小时后，取出冷却，摇匀备用。4. Acid hydrolysis Put the polyethylene tubes (1) and (2) in a heating block, heat at 105°C for 6 hours, take it out to cool, shake well and set aside.

5.衍生反应  用移液管分别吸取酸解标准溶液和试样溶液各20mL于10mL棕色容量瓶中。分别加1mL 0.5M NaHCO3，摇匀。再加0.5mL 1％ DNFB，摇匀。将容量瓶置于加热块中，60℃加热60分钟，取出冷却。用0.1M磷酸盐缓冲液(pH 6.5)冲至刻度，摇匀，准备HPLC分析用。5. Derivatization reaction Use a pipette to draw 20mL each of the acid hydrolysis standard solution and the sample solution into a 10mL brown volumetric flask. Add 1mL 0.5M NaHCO 3 respectively and shake well. Add 0.5mL 1% DNFB and shake well. Place the volumetric flask in a heating block, heat at 60°C for 60 minutes, take it out and cool. Rinse to the mark with 0.1M phosphate buffer (pH 6.5), shake well, and prepare for HPLC analysis.

6.色谱条件6. Chromatographic conditions

分析柱      Kromasil C18，5μm，250×4.6mmAnalytical column Kromasil C 18 , 5μm, 250×4.6mm

流动相      A：ACN    B：36mM TEAP，pH2.75Mobile phase A: ACN B: 36mM TEAP, pH2.75

梯度        78/64.5/60/60B％[VB/(VA+VB)]at 0/15/25/30minGradient 78/64.5/60/60B%[V B /(V A +V B )]at 0/15/25/30min

平衡时间    10minBalance time 10min

检测        UV360nmDetection UV360nm

温度        35℃Temperature 35°C

进样体积    20μLInjection volume 20μL

在此条件下得到如色谱图5的分离结果。Under these conditions, the separation results as in chromatogram 5 were obtained.

7.计算7. Calculate

7.1.以丙谷酰胺为基准物时，丙谷酰胺的浓度按下试计算：7.1. When proglutamine is used as the reference substance, the concentration of proglutamine is calculated as follows:

丙谷酰胺的浓度(g/100mL)＝A试样×C标准/A标准 Concentration of Proglutamine (g/100mL)=A sample ×C standard /A standard

C标准：丙谷酰胺基准物溶液浓度(以100L溶液中基准物称量计算，g/100mL)C standard : concentration of proglutamine reference substance solution (calculated by weighing the reference substance in 100L solution, g/100mL)

A试样：试样谱图中谷氨酸衍生物峰面积A sample : peak area of glutamic acid derivative in the sample spectrum

A标准：基准物谱图中谷氨酸衍生物峰面积 Standard A: Peak area of glutamic acid derivatives in the spectrum of reference substances

7.2.以谷氨酸为基准物时，丙谷酰胺的浓度按下试计算：7.2. When glutamic acid is used as the reference substance, the concentration of proglutamine is calculated as follows:

丙谷酰胺的浓度(g/100mL)＝(A试样×C标准/A标准)×(MAla-Gln/ML-Glu)Proglutamine concentration (g/100mL) = (A sample × C standard / A standard ) × (M Ala-Gln / M L-Glu )

C标准：丙谷酰胺基准物溶液浓度(以100L溶液中基准物称量计算，g/100mL)C standard : concentration of proglutamine reference substance solution (calculated by weighing the reference substance in 100L solution, g/100mL)

A试样：试样谱图中谷氨酸衍生物峰面积A sample : peak area of glutamic acid derivative in the sample spectrum

A标准：基准物谱图中谷氨酸衍生物峰面积 Standard A: Peak area of glutamic acid derivatives in the spectrum of reference substances

MAla-Gln：丙谷酰胺摩尔量M Ala-Gln : molar amount of proglutamine

ML-Glu：谷氨酸摩尔量。M L-Glu : molar amount of glutamic acid.

实施例4  L-苯丙氨酸原料药中主成分苯丙氨酸含量的测定The determination of main component phenylalanine content in the embodiment 4 L-phenylalanine crude drug

1.试剂  参见实施例1。1. Reagents See Example 1.

2.标准溶液的制备  从干燥器中取出苯丙氨酸基准物，于100mL容量瓶中精确称取约20mg。以0.1M HCl为溶剂溶解并冲至刻度，摇匀。2. Preparation of standard solution Take out the phenylalanine reference substance from the desiccator, and accurately weigh about 20mg in a 100mL volumetric flask. Dissolve in 0.1M HCl as a solvent and rush to the mark, shake well.

3.试样溶液的制备  于100mL容量瓶中精确称取苯丙氨酸试样约20mg。以0.1MHCl为溶剂溶解并冲至刻度，摇匀。3. Preparation of sample solution Accurately weigh about 20 mg of phenylalanine sample in a 100 mL volumetric flask. Dissolve in 0.1M HCl as solvent and rush to the mark, shake well.

4.标准溶液和试样溶液的衍生反应  用移液管分别取标准溶液和样品溶液1mL，置于10mL容量瓶中，加入1mL 0.5M NaHCO3摇匀，再加0.5mL 1％DNFB，摇匀。4. Derivative reaction of standard solution and sample solution Take 1mL of standard solution and sample solution respectively with a pipette, put them in a 10mL volumetric flask, add 1mL 0.5M NaHCO 3 and shake well, then add 0.5mL 1% DNFB, shake well .

将溶液置于60℃水浴上加热60分钟，冷却。用0.1M磷酸盐缓冲液(pH 6.5)冲至刻度，摇匀，准备HPLC分析用。The solution was heated on a 60°C water bath for 60 minutes and cooled. Rinse to the mark with 0.1M phosphate buffer (pH 6.5), shake well, and prepare for HPLC analysis.

5.色谱条件5. Chromatographic conditions

分析柱       Alltima C18，5μm，250×4.6mmAnalytical column Alltima C 18 , 5μm, 250×4.6mm

流动相       ACN/36mM TEAP，pH2.75  体积比：48/52等度洗脱Mobile phase ACN/36mM TEAP, pH2.75 volume ratio: 48/52 isocratic elution

检测         UV 360nmDetection UV 360nm

温度         35℃Temperature 35℃

进样体积     20μLInjection volume 20μL

在此条件下得到如色谱图6的分离结果。Under these conditions, the separation results shown in chromatogram 6 were obtained.

6.计算6. Calculate

试样中的苯丙氨酸的百分含量＝(A试样/A标准)×(W标准/W标准)×100％The percentage content of phenylalanine in the sample=(A sample /A standard )×(W standard /W standard )×100%

A试样：试样色谱图中苯丙氨酸的峰面积A sample : the peak area of phenylalanine in the sample chromatogram

A标准：标准色谱图中苯丙氨酸的峰面积A standard : the peak area of phenylalanine in the standard chromatogram

W标准：苯丙氨酸基准物的称量，mgW standard : weighing of phenylalanine reference substance, mg

W试样：苯丙氨酸基试样的称量，mg。W sample : the weight of the phenylalanine-based sample, mg.

Claims (8)

1, a kind of amino acid whose analytical approach, the step that it comprises is to use high performance liquid chromatograph, and amino acid is through 2,4-dinitrofluorobenzene column front derivation, reverse phase separation derivant amino acid-2,4-dinitrofluorobenzene derivant is characterized in that:
With phosphoric acid triethylamine and acetonitrile is eluent gradient wash-out or isocratic elution, reverse phase separation amino acid-2 on analytical column, and 4-dinitrofluorobenzene derivant calculates amino acid whose content with the external standard method quantitative test again.
2, a kind of amino acid whose analytical approach is characterized in that it comprises the steps:
1) prepares amino acid standard solution and sample solution with hydrochloric acid solution as solvent respectively;
2) get standard solution and sample solution respectively in in the volumetric flask of 10 times of amounts of its volume, add the 0.5M sodium bicarbonate solution, the volume ratio of standard solution or sample solution and sodium bicarbonate solution is 1: 1; Add 1%2 again, the acetonitrile solution of 4-dinitrofluorobenzene, with the volume ratio of standard solution or sample solution be 0.5-1: 1, shake up; Place 60 ℃ of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby;
3) use comprises the high performance liquid chromatograph that pump, mixer, constant temperature oven, UV-detector and workstation are formed; At C 18On the analytical column, be moving phase with phosphoric acid triethylamine/acetonitrile, gradient elution or isocratic elution amino acid separation-2,4-dinitrofluorobenzene; Chromatographic condition is as follows:
Analytical column C 18Chromatographic column, particle 5 μ m, column length 250mm, internal diameter 4.6mm;
Mobile phase A: acetonitrile B:36mM phosphoric acid triethylamine, pH 2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV 300-380nm,
Temperature 20-45 ℃,
4) calculate with the external standard method quantitative test
Amino acid whose concentration=(A in the sample Sample/ A Standard) * C Standard
A Sample: amino acid whose peak area in the sample chromatogram
A Standard: amino acid whose peak area in the standard colors spectrogram
C Standard: the amino acid whose concentration of standard solution.
3, a kind of amino acid whose analytical approach is characterized in that it comprises the steps:
1) prepares amino acid standard solution and sample solution with 0.1M hydrochloric acid as solvent respectively;
2) get 1mL standard solution and sample solution respectively in the brown volumetric flask of 10mL, add 1mL 0.5M sodium bicarbonate solution, shake up.Add 0.5-1mL 1%2 again, the acetonitrile solution of 4-dinitrofluorobenzene shakes up; Place 60 ℃ of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby;
3) use comprises the high performance liquid chromatograph that pump, mixer, constant temperature oven, UV-detector and workstation are formed.At C 18On the analytical column, be moving phase with phosphoric acid triethylamine/acetonitrile, gradient elution or isocratic elution amino acid separation-2,4-dinitrofluorobenzene; Chromatographic condition is as follows:
Analytical column C 18Chromatographic column, particle 5 μ m, column length 250mm, internal diameter 4.6mm;
Mobile phase A: acetonitrile B:36mM phosphoric acid triethylamine, pH 2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV 360nm
35 ℃ of temperature;
4) calculate with the external standard method quantitative test
Amino acid whose concentration=(A in the sample Sample/ A Standard) * C Standard
A Sample: amino acid whose peak area in the sample chromatogram
A Standard: amino acid whose peak area in the standard colors spectrogram
C Standard: the amino acid whose concentration of standard solution.
4, according to claim 2 or 3 said amino acid whose analytical approachs, it is characterized in that the volume ratio of said 36mM phosphoric acid triethylamine and acetonitrile is: 1: 0.01-100.
5, according to claim 2 or 3 said amino acid whose analytical approachs, it is characterized in that said phosphate buffer is potassium hydrogen phosphate and potassium dihydrogen phosphate, or dibastic sodium phosphate and sodium dihydrogen phosphate.
6,, it is characterized in that said sample solution is to contain raw material and the preparation thereof that several amino acids is formed, or major component is xylamidine or phenylalanine bulk drug and preparation thereof according to claim 2 or 3 said amino acid whose analytical approachs.
7,, it is characterized in that the said preparation that contains the raw material of several amino acids composition is ephrosis parenteral solution or hepatopathy parenteral solution according to the said amino acid whose analytical approach of claim 6.
8,, it is characterized in that said major component xylamidine preparation is the xylamidine parenteral solution according to the said amino acid whose analytical approach of claim 6.
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Address after: 300457 Tianjin Binhai New Area Tianjin Development Zone West District Xinye Jiujie north, Xinhuan West Road East

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Address after: 300457 North Xinye 9th Street and East Xinhuan West Road, West District, Binhai New Area Development Zone, Tianjin

Patentee after: Tianjin Pharmaceutical Research Institute Co.,Ltd.

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