5.3 中间精密度试验：由不同的分析人员进行样品处理和含量测定，平行6

5.3 中间精密度试验：由不同的分析人员进行样品处理和含量测定，平行6份，与重复性结果共12次，计算RSD值。
6 加样回收率试验：供试品取半量，同时按测定结果的80%、100%、120%加入对照品溶液（每一浓度3份），进行加样回收率试验，回收率应为95.0%~105.0%，9份的RSD应不得过2.0%。
7 溶液稳定性试验：分别进行对照品溶液与供试品溶液的室温稳定性考察与冰箱冷藏的稳定性考察，为对照品溶液的保存时间及供试品测定时间提供依据。
8 色谱条件的耐用性试验：考察不同初始柱温（±5℃）、不同检测器温度（±5℃）、不同载气流速（±10%）对测定结果的影响及系统适用性情况（拖尾因子、分离度）的影响，确定色谱条件的可接受范围。
9 限度确定；药典规定的含量限度要符合药典要求，其它含量结合不同生产日期样品的测定结果（10批）及其中3批样品的稳定性试验结果确定含量限度。

二 高效液相色谱分析法测定β-谷甾醇
（一） 液相色谱分析条件的摸索
① 色谱柱考察
对Hedera ODS-2 4.6*250mm,5um和AltimaC18 4.6*250mm,5um色谱柱分别进行了考察，后者分离效果明显优于前者，选择后一种规格的色谱柱。
② 溶剂考察
对甲醇和二氯甲烷进行了考察，结果甲醇的峰形较好，所以选择甲醇作为溶剂。
③ 流动相的考察
分别对甲醇-水、甲醇-0.1%甲酸水和乙腈-水进行了考察，甲醇做流动相基线漂移严重，且分离效果不理想，乙腈-水的分离效果最好，拖尾因子小于1.05，分离度大于1.5，且基线较平稳，所以最终选择乙腈-水作为流动相。对乙腈-水不同比例进行了考察，通过改变色谱参数并进行优化流动相比例最终使β-谷甾醇与其他峰的分离度和拖尾因子均达到要求，色谱条件和分析结果见下表，色谱图如下：
色谱条件
仪器 色谱柱 柱温（℃） 流速（mL/min） 进样量（μL） 检测波长（nm）
HPLC-CBM-20A AltimaC184.6*250mm,5um 30 1 20 210

結果 (英文) 1:

5.3 intermediate precision test: by different analysts for sample handling and assay, the parallel 6, and repeatability of results a total of 12 times, and calculate RSD values.6 sampling recovery test: take half dose to be tested, and according to the results of 80%, 100%, 120%, by adding a control solution (concentration 3 each), spotting the recovery test recovery should be 95.0%~105.0%,9 the RSD may not be too 2%.7 solution stability test: separate reference solution for sample stability at room temperature and refrigerate the stability, as the reference solution saves time and provide evidence for determination of Elution times.8 chromatographic conditions for durability test: to determine the initial temperature (± 5 ℃), detector temperature (± 5 ℃), different carrier gas flow rate (± 10%) influence on the measurement results and the system suitability (tailing factor, resolution) the influence of chromatographic conditions are determined the acceptable range.9 limits determined microbacteria content limit to comply with the Pharmacopoeia, other content combined with measurement of samples with different production dates (10) and its stability test results determine the concentration limits of 3 batches of samples.Two high-performance liquid chromatographic method for the determination of β-sitosterol(A) liquid chromatography analysis of conditions of exploration① column studyHedera ODS-2 4.6*250mm,5um and AltimaC18 4.6*250mm,5um column, respectively were studied, which effect is superior to the former, after selecting a chromatographic column specifications.Solvent II reviewFor methanol and methylene chloride were studied and results of methanol forms better, so I chose alcohol as a solvent.③ Mobile phase investigationRespectively of methanol-water, methanol-0.1% investigates the acid water and acetonitrile-water, methanol mobile phase of baseline drift is serious, and the separation is not ideal, the separation of acetonitrile-water works best, trailing factor is less than 1.05, degrees of separation is greater than 1.5, and the baseline is stable, so in the end chose acetonitrile-water as the mobile phase. Different proportions of acetonitrile-water have been investigated by changing the final chromatographic parameters and optimize the mobile phase β-sitosterol and other peaks of the degree of separation and the trailing factor meet the requirements, chromatographic conditions and analysis results shown in the table below, chromatograms are as follows:Chromatographic conditionsInstrument the column temperature (℃) flow rate (mL/min) injection volume (μl) detected wavelengths (nm)HPLC-CBM-20A AltimaC184.6*250mm,5um 30 1 20 210

結果 (英文) 3:

The 5.3 intermediate precision test: sample processing and content were determined by different analysts, parallel 6 copies, and reproducible results a total of 12 times, the calculation of RSD value.
6 recovery test: the sample of half amount, at the same time, according to the determination results of 80%, 100%, 120% add the reference solution (3 copies of each concentration), for recovery of the added sample test, recovery rate should be 95.0%~105.0%, 9 copies of the RSD should be no more than 2%.
7 tests of solution stability were investigated: a reference solution and the stability of the test solution at room temperature stability and the refrigerator, as a reference solution for sample storage time and provide the basis for determination of time.
The durability test of 8 chromatographic conditions: the effects of different initial column temperature (- 5 DEG C), different detector temperature (- 5 DEG C), different carrier gas flow rate (+ 10%) applicable to determination results and systemic conditions (tailing factor, the degree of separation) influence, determine the acceptable range of chromatographic conditions.
9 determination of content of limit; limit of the Pharmacopoeia to meet the requirements of Pharmacopoeia, combined with other content determination results of samples of different production date (the 10 batch) and the stability of the test results of 3 batches of sample to determine the content limit. Determination of beta sitosterol


two by high performance liquid chromatography (a) HPLC analysis conditions of HPLC column study

The Hedera ODS-2 4.6*250mm, 5um and AltimaC18 4.6*250mm, 5um chromatographic column were investigated, the latter separation is better than the former, column a specification selection.

solvent study investigated for methanol and dichloromethane, methanol peak good results, so the choice of methanol as solvent.

the investigation of mobile phase of methanol water, methanol respectively -0.1% formic acid in water and acetonitrile and water were investigated, methanol as mobile phase baseline drift is serious, and the separation effect is not ideal, the best separation effect of acetonitrile water, tailing factor is less than 1.05, the degree of separation is greater than 1.5, and the baseline is more stable,So the final choice of acetonitrile water as mobile phase. In acetonitrile and water in different proportions were studied, finally make the separation degree and the tailing factor beta sitosterol and other peaks met the requirements by changing the chromatographic parameters and to optimize the proportion of mobile phase, chromatographic conditions and the analysis results shown in the table below, the chromatogram chromatographic conditions are as follows:

instrument and column temperature (DEG C) flow velocity (mL/min) of sample amount (L) detection wavelength (nm)
HPLC-CBM-20A AltimaC184.6*250mm 30120, 5um 210