Department of Biomedical and Pharmaceutical Science, Graduated School, Kyung Hee University, Seoul 02447, Republic of Korea.
Kyung Hee Drug Analysis Center, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea.
Molecules. 2024 Sep 23;29(18):4505. doi: 10.3390/molecules29184505.
A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at 310.2 → 96.0 for KTF and 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC-MS/MS system. Analytes were separated on a Luna Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (/) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC-MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup.
建立并验证了一种用于从比格犬血浆中定量测定酮替芬（KTF）的高准确度、高精密度和简单的液相色谱-串联质谱（LC-MS/MS）方法，以酮替芬-d3（KTF-d3）为内标（IS）。KTF 和 IS 在 API 4000 质谱仪上，以电喷雾电离（ESI）正离子模式的多重反应监测（MRM）模式进行检测。监测到的跃迁分别为 KTF 的 310.2→96.0 和 IS 的 313.2→99.1。KTF 和 IS 采用甲基叔丁基醚进行液-液萃取，从血浆中提取，然后用提取样品（7 μL）在 3 分钟内分析进入 LC-MS/MS 系统。分析物在 Luna Hilic 柱（50×2.0mm i.d.，3μm）上分离，使用 Nexera X2 HPLC。流动相 A 由 10mmol/L 甲酸铵（pH3.0）组成，而流动相 B 由乙腈中的 0.05%甲酸组成。流动相的比例为 5：95（/），流速为 0.2mL/min。该方法已根据韩国食品药品安全部和美国食品药品管理局制定的生物分析方法验证指南进行了全面验证，涉及选择性、定量下限、线性、拖尾、精密度、准确度、回收率、基质效应和稳定性。该开发的 LC-MS/MS 方法在比格犬口服酮替芬糖浆后用于评估酮替芬的生物等效性方面得到了有效应用。
