Kanjarla Narasimha, Katta Balaraju
Chaitanya (Deemed to be University), Department of Pharmacy, Hyderabad, Telangana, India.
Turk J Pharm Sci. 2025 Aug 1;22(3):191-206. doi: 10.4274/tjps.galenos.2025.70718.
A novel, high-throughput liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique has been developed that uses Etravirine (ETR) as the internal standard (IS) to simultaneously quantify Doravirine (DOR), Lamivudine (LAM), and Tenofovir Disoproxil Fumarate (TDF) in human plasma. The procedure employs a precipitation extraction technique to analyze analytes from human plasma. This study aims to develop and validate a novel and reliable stability-indicating UPLC-MS/MS method for the simultaneous determination of DOR, LAM, and TDF in human plasma, using ETR as the IS.
ETR, based on its stable-isotopic nature and structural and physicochemical similarity to the analytes of interest, was used as an IS. Precipitation extraction was the technique used to prepare samples. An agilent zorbax XDB C18 analytical column (2.1 × 50 mm, 3.5 μm) was used for chromatographic separation, and its isocratic mobile phase consists of acetonitrile and buffer (5 mM of ammonium formate with 0.1 % formic acid) in the ratio 80:20, , at a flow rate of 0.120 mL/min.
The parent-to-product ion transitions for the drugs were as follows : LAM: m/z 231.08 amu → 112.00 amu, TDF: m/z 288.33 amu → 176.17 amu, DOR: m/z 426.38 amu → 112.02 amu, and IS ETR: m/z 437.36 amu → 164.97 amu. These transitions were observed using a triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) positive ion mode. The compound's basic group content determined which positive mode to choose. For DOR, LAM and TDF, the method was validated throughout concentration ranges of 2.5-1000 ng/mL with correlation coefficients (r) values obtained were found to be 0.99. From spiked plasma samples, the mean recovery outcomes were observed and found to be DOR, LAM, and TDF was 83.39%, 87.33%, and 85.56%. With a 3.0-minute total run time, the approach was shown to be reliable and quick.
A triple quadrupole mass spectrometer running in the MRM positive ion mode was used to track these transitions. The compounds' functional group content served as the basis for choosing the positive mode. The mean recovery values were obtained for three APIs from spiked plasma samples. The run times were found to be both reliable and quick. The method was proven to produce precise and specific results for the determination of selected drugs through the current study. The method is stable when exposed to various stress conditions, demonstrating minimal degradation. The current method was validated as per the ICH M10 guidelines and was found to meet the desired acceptance criteria. The developed bioanalytical method, validated in accordance with ICH M10 guidelines, demonstrated high accuracy, precision, and reproducibility for the simultaneous quantification of DOR, LAM, and TDF. Its streamlined design and reliable performance make it a valuable tool for routine analysis.
已开发出一种新型的高通量液相色谱串联质谱法（超高效液相色谱-质谱/质谱法，UPLC-MS/MS），该方法使用依曲韦林（ETR）作为内标（IS），同时定量测定人血浆中的多拉韦林（DOR）、拉米夫定（LAM）和替诺福韦酯富马酸盐（TDF）。该程序采用沉淀萃取技术分析人血浆中的分析物。本研究旨在开发并验证一种新型且可靠的稳定性指示UPLC-MS/MS方法，以ETR作为内标，同时测定人血浆中的DOR、LAM和TDF。
基于ETR的稳定同位素性质及其与目标分析物的结构和物理化学相似性，将其用作内标。采用沉淀萃取技术制备样品。使用安捷伦zorbax XDB C18分析柱（2.1×50 mm，3.5μm）进行色谱分离，其等度流动相由乙腈和缓冲液（5 mM甲酸铵与0.1%甲酸）按80:20的比例组成，流速为0.120 mL/min。
药物的母离子到产物离子的转变如下：LAM：m/z 231.08 amu→112.00 amu，TDF：m/z 288.33 amu→176.17 amu，DOR：m/z 426.38 amu→112.02 amu，内标ETR：m/z 437.36 amu→164.97 amu。这些转变是在三重四极杆质谱仪的多反应监测（MRM）正离子模式下观察到的。化合物的碱性基团含量决定了选择哪种正离子模式进行检测。对于DOR、LAM和TDF，该方法在2.5 - 1000 ng/mL的整个浓度范围内进行了验证，获得的相关系数（r）值为0.99。从加标血浆样品中观察到平均回收率结果，发现DOR、LAM和TDF的平均回收率分别为83.39%、87.33%和85.56%。总运行时间为3.0分钟，该方法被证明是可靠且快速的。
使用在MRM正离子模式下运行的三重四极杆质谱仪来跟踪这些转变。化合物的官能团含量作为选择正离子模式的依据。从加标血浆样品中获得了三种活性成分的平均回收率值。发现运行时间既可靠又快速。通过本研究证明，该方法在测定所选药物时能产生精确且特异的结果。该方法在暴露于各种应激条件下时是稳定的，降解最小。当前方法按照ICH M10指南进行了验证，发现符合预期的验收标准。所开发的生物分析方法按照ICH M10指南进行了验证，在同时定量测定DOR、LAM和TDF方面表现出高准确性、精密度和重现性。其简化的设计和可靠的性能使其成为常规分析的有价值工具。
