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1、Comparison between the two Validated HPLC methods for PMIDA purity determination两种已验证的测定双甘膦纯度的HPLC方法的比较For the purity determination of PMIDA by HPLC technique, two separated methods were developed and validated following procedure were defined in agreement with ICH (International Conference on Harmo
2、nization) rules “Validation of Analytical Method: Definition and Terminology”针对HPLC技术测定双甘膦的纯度，研发并验证了两种不同的方法，按照ICH（国际协调会）条例“分析方法的验证：定义和术语”来确定以下步骤。The two methods, respectively reported in Report 048/11 and 140/11, are based on different chromatographic columns and different eluent solutions.分别报告在报告04
3、8/11 和 140/11中的这两种方法基于不同的色谱柱和不同的洗脱溶液（流动相）。In both the methods, the time of the analysis is comparable and the parameters obtained from the validation are quite comparable.在这两种方法中，分析时间是可对比的并且从验证中获得的参数非常有可比性。A summary of analytical data collected for each of them are following reported: 收集的两种方法的分析数据汇总
4、报告如下：Report 048/11 with Strong Ion Exchange column报告048/11，使用的是强离子交换柱Analytical Procedure 分析步骤Chromatographic conditions 色谱条件Instrument:HPLC Agilent 1100仪器：HPLC 安捷伦 1100Column: ZORBAX SAX 4.6 x 250 mm 5 m柱：ZORBAX SAX 4.6 x 250 mm 5 mMobile phase: 85 % (250mM KH2PO4 pH 2) 15 % (MeOH) mixed流动相：85 % (2
5、50mM 磷酸二氢钾 pH 2) ：15 % (甲醇) 的混合物Flow:1.5 mL/min流量：1.5 毫升/分钟Volume injected:20 mL注入体积：20 mLColumn temperature:25C柱温：25CDetector:UV-visible at 195nm探测器：195纳米的可视紫外线Analysis time:25 min.分析时间：25分钟Blank solution 空白溶液Solvent. 溶剂(85 % (250mM KH2PO4 pH 2) 15 % (MeOH) mixed)85 % (250mM 磷酸二氢钾 pH 2) ：15 % (甲醇)
6、的混合物Reference soltution (S) 参比溶液（S）20 mg of PMIDA standard, accurately weighted, are dissolved into 20 mL of solvent.把精确称取的20mg双甘膦标样溶解在20ml溶剂中。Sample Solution (C) 样品溶液（C）20 mg on the sample, accurately weighted, are dissolved into 20 mL of solvent.把精确称取的20mg样品溶解在20ml溶剂中。Sequence of injections 注入顺序Th
7、e solutions have to be injected in the instrument in the following sequence:溶液必须按照以下顺序注入到仪器中：Blank3 injection空白溶液 3次注入Reference Solution (S)2 injection参比溶液（S） 2次注入Blank1 injection空白溶液 1次注入Sample Preparation (C)2 injection样品溶液（C） 2次注入Retention time 保留时间Chromatogram of PMIDA solution (Conc. PMIDA = 1.
8、048 mg / mL)双甘膦溶液（双甘膦浓度=1.048 mg / mL）的色谱图The peak for the evaluation is eluting at 7.105 min, while the peak at 12.5 min is related to mobile phase interaction with the column support.用于评估的峰在7.105分钟时洗脱出来，在12.5分钟时出现的峰与流动相和柱支撑的相互影响有关。Compound化合物Retention time (min)保留时间（分钟）Relative retention time相对保留时
9、间PMIDA 双甘膦7.105/Analytical Results 分析结果PMIDA 双甘膦Determination coefficient测定系数0.99837Intercept (mAU*s)截距(mAU*s)-151.22Slope (mAU*s*L/mg)斜率(mAU*s*L/mg)636.68Precision (%)精密度(%) 0.71Precision Robustness(%)精密度稳健性(%) 0.42 Instrument Precision (%)仪器精密度(%) 0.18Selectivity/Specificity选择性/特异性The method show
10、a good selectivity and ability to measure accurately the PMIDA in the presence of possible interferences such as known or unknown impurities.该方法表现出很好的选择性而且可以在存在已知或未知干扰物的情况下精确测定双甘膦。Precision:精密度： The degree of repeatability of the method under normal operation conditions, expressed as the percentage
11、relative standard deviation is reported below.在正常操作条件下，该方法可重复性的程度用相对偏差百分比表示为如下：Instrumental precision (%)仪器精密度(%)PMIDA 双甘膦 0.18 The intermediate precision of the data obtained from two populations of area for each analyte obtained from two analysts in separated days, results with a value of t-studen
12、t reported below.从两位分析人员在不同的日子制备的分析物中得到的两个主面积的数据的中间精密度得出如下中间精密度值：t-STUDENT VALUES中间精密度值PMIDA 双甘膦0.545The tabulate t-student value at 95% of interval of confidence with four freedom degrees is 2.776. If t-student value at 95% of interval of confidence with four freedom degrees is below the tabulated
13、value (2.776) it means that the two determinations carried out by two analysts are not significantly different.表格中95%可信度间隔的中间精密度值（具有4种自由度）为2.776；若低于该值（2.776），即说明由两名分析人员进行的两次测试无明显不同。Linearity and Range of applicability线性和应用范围The linearity of the method in the concentration range of the applicability,
14、 results in agreement with the ICH rules. The determination coefficient (r2) of the linearity results higher than 0.99.在应用浓度范围中该方法的线性与ICH（国际协调会）规则一致。线性的测定系数(r2)结果高于0.99。The values of the “t” Students statistics for the two tails tests at 95% of the probability level results or almost symmetrically a
15、rround the intercept value even if this value is not very close to zero. It can be state that the intercept of the regression straight line is different from zero; since the concentration interval of the determination (80 120%) is very far from the zero value. The intercept value result different fr
16、om zero since at high concentration of PMIDA there are constant influence of the mobile phase.两次拖尾试验的中间精密度值的统计结果在95%的概率水平或几乎对称于截距值（即使该值并非很接近零）。由于测定的浓度区间(80 - 120%)离零值很远，所以可以说明回归直线的截距并不为零。因为双甘膦浓度高时，流动相具有恒定的影响，所以产生的截距值不等于零。Stability稳定性As evidence in the instrumental precision test and after stored at
17、room temperature the solutions of PMIDA to be submitted to analyses result stable at least for 24 hours from the preparation.作为在仪器精密度测试中的证据并且在室温下储存后，制备的待分析的双甘膦溶液仍然要静置稳定24小时。Robustness稳健性Little variations in the working condition do not induce difference in the linearity response.工作条件中细小的变化不会导致在线性回复中
18、产生差异。Report 140/11 with C18 Column报告140/11，用碳18柱Analytical Procedure 分析步骤Chromatographic conditions 色谱条件Instrument:HPLC Agilent 1100仪器：HPLC Agilent 1100Column: Kromasil 100 C18 4.6 x 250 mm 5 m柱：Kromasil 100 C18 4.6 x 250 mm 5 mMobile phase: 98 %(120 mM Na2HPO4 pH 7) 2 % (ACN) mixed流动相：98 %(120 mM 磷
19、酸氢二钠 pH 7) 2 % (乙腈)混合物Flow:0.5 mL/min流量：0.5毫升/分钟Volume injected:10 mL注入体积：10 mLColumn temperature:35C柱温：35CDetector:UV-visible at 200 nm探测器：200纳米的可视紫外线Analysis time:10 min.分析时间：10分钟Blank solution 空白溶液Buffer. 缓冲液120 mM Na2HPO4 pH 7 120 mM 磷酸氢二钠 pH 7Reference soltution (S) 参比溶液（S）40 mg of PMIDA standa
20、rd, accurately weighted, are dissolved into 20 mL of buffer.把精确称量的40mg双甘膦标样溶解到20ml缓冲液中。Sample Solution (C) 样品溶液（C）40 mg on the sample, accurately weighted, are dissolved into 20 mL of buffer.把精确称量的40mg样品溶解到20ml缓冲液中。Sequence of injections 注入顺序The solutions have to be injected in the instrument in the
21、 following sequence:溶液必须按照以下顺序注入到仪器中：Blank3 injection空白溶液 3次注入Reference Solution (S)2 injection参比溶液（S） 2次注入Blank1 injection空白溶液 1次注入Sample Preparation (C)2 injection样品溶液（C） 2次注入Retention time 保留时间Chromatogram of PMIDA solution (Conc. PMIDA = 2.002 mg / mL)双甘膦溶液（双甘膦浓度=2.002 mg / mL）的色谱图The typical re
22、tention times detected are reported in the following table.检测出的典型保留时间报告在下表中。The peak for the evaluation is eluting at 4.366 min.用于评估的峰在4.366分钟时洗脱出来。Compound化合物Retention time (min)保留时间（分钟）Relative retention time相对保留时间PMIDA 双甘膦4.366/-Analytical results 分析结果PMIDA 双甘膦Determination coefficient测定系数0.99628
23、Intercept (mAU*s)截距(mAU*s)256.60Slope (mAU*s*L/mg)斜率(mAU*s*L/mg)379.51Precision (%)精密度(%) 0.72Precision Robustness(%)精密度稳健性(%) 0.16Instrument Precision (%)仪器精密度(%) 0.45Selectivity/Specificity选择性/特异性The method show a good selectivity and ability to measure accurately the PMIDA in the presence of poss
24、ible interferences such as known or unknown impurities.该方法表现出很好的选择性而且可以在存在已知或未知干扰物的情况下精确测定双甘膦。Precision:精密度： The degree of repeatability of the method under normal operation conditions, expressed as the percentage relative standard deviation is reported below. 在正常操作条件下，该方法可重复性的程度用相对偏差百分比表示为如下：Instru
25、mental precision (%)仪器精密度(%)PMIDA 双甘膦 0.45 The intermediate precision of the data obtained from two populations of area for each analyte obtained from two analysts in separated days, results with a value of t-student reported below. 从两位分析人员在不同的日子制备的分析物中得到的两个主面积的数据的中间精密度得出如下中间精密度值：t-STUDENT VALUES中间精
26、密度值PMIDA 双甘膦1.964The tabulate t-student value at 95% of interval of confidence with four freedom degrees is 2.776. If t-student value at 95% of interval of confidence with four freedom degrees is below the tabulated value (2.776) it means that the two determinations carried out by two analysts are n
27、ot significantly different.表格中95%可信度间隔的中间精密度值（具有4种自由度）为2.776；若低于该值（2.776），即说明由两名分析人员进行的两次测试无明显不同。Linearity and Range of applicability线性和应用范围The linearity of the method in the concentration range of the applicability, results in agreement with the ICH rules. The determination coefficient (r2) of the
28、linearity results higher than 0.99.在应用浓度范围中该方法的线性与ICH（国际协调会）规则一致。线性的测定系数(r2)结果高于0.99。The values of the “t” Students statistics for the two tails tests at 95% of the probability level results or almost symmetrically arround the intercep value even if this value is not very close to zero. It can be st
29、ate that the intercept of the regression straight line is different from zero; since the concentration interval of the determination (80 120%) is very far from the zero value. The intercept value result different from zero sinca at high concentration of PMIDA there are constant influence of the mobi
30、le phase.两次拖尾试验的中间精密度值的统计结果在95%的概率水平或几乎对称于截距值（即使该值并非很接近零）。由于测定的浓度区间(80 - 120%)离零值很远，所以可以说明回归直线的截距并不为零。因为双甘膦浓度高时，流动相具有恒定的影响，所以产生的截距值不等于零。Stability稳定性As evidence in the instrumental precision test and after stored at room temperature the solutions of PMIDA to be submitted to analyses result stable at
31、least for 24 hours from the preparation.作为在仪器精密度测试中的证据并且在室温下储存后，制备的待分析的双甘膦溶液仍然要静置稳定24小时。Robustness稳健性Little variations in the working condition do not induce difference in the linearity response. 工作条件中细小的变化不会导致在线性回复中产生差异。SUMMARY 总结From the data following reported in comparison, the two methods show
32、the same validation parameters.从以下对比报告的数据来看，这两种方法都表现出相同的验证参数。Little differences in Determination Coefficient or in Instrumental Precision can be related only to the age of the column.测定系数或仪器精密度中的微小差别只和柱的年限有关。The retention time and the selectivity of PMIDA in both the methods are comparable.两种方法的保留时间
33、和双甘膦的选择性是可比的。C18 Column 碳18柱PMIDA 双甘膦Determination coefficient测定系数0.99628Intercept (mAU*s)截距(mAU*s)256.60Slope (mAU*s*L/mg)斜率(mAU*s*L/mg)379.51Precision (%)精密度(%) 0.72Precision Robustness(%)精密度稳健性(%) 0.16Instrument Precision (%)仪器精密度(%) 0.45Strong Ion ExchangeColumn强离子交换柱PMIDA 双甘膦Determination coefficient测定系数0.99837Intercept (mAU*s)截距(mAU*s)-151.22Slope (mAU*s*L/mg)斜率(mAU*s*L/mg)636.68Precision (%)精密度(%) 0.71Precision Robustness(%)精密度稳健性(%) 0.42 Instrument Precision (%)仪器精密度(%) 0.18