马来酸噻吗洛尔滴眼液中有关物质的测定及杂质定性研究
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篇名: 马来酸噻吗洛尔滴眼液中有关物质的测定及杂质定性研究
TITLE:
摘要: 目的:建立测定马来酸噻吗洛尔滴眼液中有关物质的方法,并对其杂质进行定性。方法:采用高效液相色谱法测定有关物质的含量,色谱柱为ACES C18,流动相为甲醇与4.32 g/L辛烷磺酸钠溶液(冰醋酸调pH至3.0)的混合溶液(50 ∶ 50,V/V)-甲醇(梯度洗脱),流速为1.0 mL/min,检测波长为295 nm,柱温为30 ℃,进样量为20 μL。采用制备液相色谱法制备杂质纯品,色谱柱为YMC-PACK ODS-A,流动相为甲醇-0.01%三氟乙酸(40 ∶ 60,V/V)和甲醇-0.01%三氟乙酸(20 ∶ 80,V/V),流速为8 mL/min,检测波长为295 nm,柱温为20 ℃,进样量为0.8 mL。采用液相色谱串联质谱法进行结构推测,色谱柱为Waters ACQUITY UPLC BEH C18,流动相为甲醇-5 mmol/L甲酸铵溶液(含0.05%甲酸)(梯度洗脱),流速为0.2 mL/min,柱温为20 ℃,进样量为2 μL;采用电喷雾离子源,以质谱全扫描模式检测母离子,以子离子扫描模式检测碎片离子。采用氢谱、碳谱进行结构确证和推测。结果:马来酸噻吗洛尔检测质量浓度线性范围为0.501~10.02 μg/mL(r=0.999 9);定量限为3.012 ng,检出限为1.004 ng;精密度试验的RSD为0.2%,稳定性和重复性试验的RSD均小于5%。确证了杂质Ⅰ、Ⅱ、Ⅲ的结构,并推测了杂质Ⅳ和Ⅴ的结构。结论:该方法快速、准确、专属性好,可用于马来酸噻吗洛尔滴眼液的质量控制。
ABSTRACT: OBJECTIVE: To establish a method for the determination of related substances in Timolol maleate eye drops, and to conduct qualitative analysis of its degradation impurities. METHODS: HPLC method was adopted for determination of related substance. The determination was performed on ACES C18 column with mobile phase consisted of the mixed solution of methanol and 4.32 g/L sodium octane sulfonate (pH adjusted to 3.0 with glacial acetic acid)(50 ∶ 50,V/V)-methanol (gradient elution) at flow rate of 1.0 mL/min. The detection wavelength was set at 295 nm, and column temperature was 30 ℃. The sample size was 20 μL. Preparative LC method was used to prepare pure impurities.  The determination was performed on YMC-PACK ODS-A column with mobile phase consisted of methanol-0.01% trifluoroacetic acid (40 ∶ 60,V/V) and methanol-0.01% trifluoroacetic acid (20 ∶ 80,V/V) at flow rate of 8 mL/min. The detection wavelength was set at 295 nm, and column temperature was 20 ℃. The sample size was 0.8 mL. LC-MS/MS method was adopted. The determination was performed on Waters ACQUITY UPLC BEH C18 column with mobile phase consisted of methanol-5 mmol/L ammonium formate (0.05% formic acid) (gradient elution) at flow rate of 0.2 mL/min. The column temperature was 20 ℃. The sample size was 2 μL. The electrospray ionization source was conducted, detecting patient ion by MS full scan and fragment ion by daughter ion scan. The structure was confirmed and speculated by 1H-NMR and 13C-NMR. RESULTS: The linear range of timolol maleate was 0.501-10.02 μg/mL (r=0.999 9). The limit of quantitation was 3.012 ng, and the limit of detection was 1.004 ng. RSD of precision test was 0.2%. RSDs of stability and reproducibility tests were all lower than 5%. The structures of impurityⅠ, Ⅱ and Ⅲ were confirmed, and those of impurity Ⅳ and Ⅴ were speculated. CONCLUSIONS: The method is rapid, accurate and specific. It can be used for the determination of related substance in Timolol maleate eye drops.
期刊: 2018年第29卷第16期
作者: 刘荷英,程奇珍,周敏,易巧
AUTHORS: LIU Heying,CHENG Qizhen,ZHOU Min,YI Qiao
关键字: 马来酸噻吗洛尔;有关物质;降解杂质;高效液相色谱法;制备液相色谱;液相色谱串联质谱法;氢谱;碳谱;测定;结构推测
KEYWORDS: Timolol maleate; Related substance; Degradation impurity; HPLC; Preparative LC; LC-MS/MS; 1H-NMR; 13C-NMR; Determination; Structure speculation
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