Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy Kafrelsheikh University, Kafrelsheikh City 33516, Egypt.
School of Pharmacy, Pacific University Oregon, Hillsboro, OR 97123, USA.
Molecules. 2020 Dec 6;25(23):5753. doi: 10.3390/molecules25235753.
In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex C column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, /), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at / 291.9 → 173.0, / 396.9 → 225.1, / 388.9 → 217.0, and / 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/) over a concentration range of 1-1000, 2.5-800, and 5-500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.
在本研究中，开发了一种灵敏且完全验证的用于定量测定三种具有潜在抗肿瘤活性的新型碳酸酐酶抑制剂（CAI）的生物分析高效液相色谱-串联质谱（LC-MS/MS）方法。使用 1.5 mL 乙腈从仅 450 µL 人血浆等分试样中提取分析物和内标（IS），以实现所需的蛋白沉淀。色谱分离在 Phenomenex Kinetex C 柱（100×4.6mm，2.6μm）上使用二元梯度洗脱模式在不到 6 分钟的时间内完成。流动相由溶剂（A）：50%甲醇中的 0.1%甲酸和溶剂 B：乙腈中的 0.1%甲酸（30:70，/）组成，以 0.8 mL/min 的流速泵入。检测采用配备电喷雾电离（ESI）源的三重四极杆串联质谱仪（API 3500）在正离子模式下进行。通过监测 / 291.9→173.0、/ 396.9→225.1、/ 388.9→217.0 和 / 146.9→91.0 的前体-母离子过渡，选择多重反应监测（MRM）模式对 AW-9a、WES-1、WES-2 和香豆素（IS）进行定量，分别。线性度使用加权最小二乘法线性回归法（1/）在 AW-9a、WES-1 和 WES-2 的浓度范围为 1-1000、2.5-800 和 5-500ng/mL 进行计算；分别。根据美国食品和药物管理局（FDA）指南，该生物分析 LC-MS/MS 方法已全面验证，符合线性、准确性、精密度、残留、选择性、稀释完整性和稳定性要求。该方法已成功应用于加标人血浆中所有研究药物的测定，无明显基质效应，这是进一步监测新开发治疗剂治疗药物监测的关键基石。
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