Simultaneous estimation of paclitaxel and bortezomib via LC-MS/MS: pharmaceutical and pharmacokinetic applications

<b>Aim &amp; Objective:</b> This study evaluates the potential of combining paclitaxel (PTX) and bortezomib (BTZ) for breast cancer therapy. <b>Materials &amp; Methods:</b> The nanoformulation was optimized via Box-Behnken Design (BBD), with method validation adhering to US-FDA guidelines. <b>Results:</b> Multiple reaction monitoring transitions for PTX, BTZ and internal standard were m/z 855.80→286.60, 366.80→226.00 and 179.80→110.00, respectively. Elution done on C18 Luna column with 0.1% FA in MeOH:10 mM ammonium acetate. The size of nanoformulation was 133.9 ± 1.97 nm, PDI 0.19 ± 0.01 and zeta potential -19.20 ± 1.36 mV. Pharmacokinetics showed higher C_max for PTX-BTZ-NE (313.75 ± 10.71 ng/ml PTX, 11.92 ± 0.53 ng/ml BTZ) versus free PTX-BTZ (104 ± 13.06 ng/ml PTX, 1.9 ± 0.08 ng/ml BTZ). <b>Conclusion:</b> Future findings will contribute to the treatment of breast cancer using PTX and BTZ. The importance of paclitaxel (PTX) and bortezomib (BTZ) in combination with chemotherapy is demonstrated by recently published studies. An obstacle to measuring the results of translational research is the lack of an analytical or bioanalytical approach for simultaneously estimating PTX and BTZ. A gap in the scientific literature has been filled by the development and validation of an LC-MS/MS method for the co-estimation or simultaneous measurement of PTX and BTZ. Both moieties were resolved in a Phenomenex Luna (C18), (7.5 × 4.6 mm, 3μ) column with a 0.1% FA in MeOH:10 mM ammonium acetate, 80:20 (% v/v) mobile phase at 0.6 ml/min flow rate. According to the US-FDA, the developed method was validated in female Sprague–Dawley rats plasma for different characteristics, such as linearity, precision and accuracy, LLOQ and stability studies. The oral P.K profile of PTX and BTZ co-loaded in a nanoformulation has been investigated in young female Sprague–Dawley rats to support the applicability of the established LC-MS/MS methodology. With an LLOQ of 1.0 ng/ml for each drug and a run time of 6.0 min, the calibration curve was found linear for the 1–600 ng/ml concentration range. The proposed LC-MS/MS method was confirmed to be repeatable, precise and accurate. The developed LC-MS/MS method was successfully illustrated for the co-estimation or simultaneous estimation of both drugs in rat plasma to generate an oral P.K profile. US-FDA Guidance of Industry and Bioanalytical Method Validation criteria was followed for the co-estimation of PTX and BTZ and was applied to nanoformulation development and bioanalytical investigations.

**研究目的**：本研究旨在评估紫杉醇（Paclitaxel，PTX）与硼替佐米（Bortezomib，BTZ）联合用于乳腺癌治疗的潜力。
**材料与方法**：本研究通过Box-Behnken设计（Box-Behnken Design，BBD）优化纳米制剂，并遵循美国食品药品监督管理局（US-FDA）指南进行方法学验证。
**结果**：紫杉醇、硼替佐米及内标的多反应监测离子对分别为m/z 855.80→286.60、366.80→226.00及179.80→110.00。色谱分离采用C18 Luna色谱柱，流动相为含0.1%甲酸（Formic Acid，FA）的甲醇（Methanol，MeOH）与10 mM乙酸铵混合溶液。纳米制剂的粒径为133.9±1.97 nm，多分散指数（Polydispersity Index，PDI）为0.19±0.01，Zeta电位（Zeta potential）为-19.20±1.36 mV。药代动力学结果显示，紫杉醇-硼替佐米纳米乳（PTX-BTZ-NE）的血药峰浓度（Cmax）显著高于游离紫杉醇-硼替佐米组：紫杉醇浓度为313.75±10.71 ng/ml，硼替佐米浓度为11.92±0.53 ng/ml；而游离组紫杉醇仅为104±13.06 ng/ml，硼替佐米为1.9±0.08 ng/ml。
**结论**：本研究后续结果将为紫杉醇与硼替佐米联合治疗乳腺癌提供支持。近期发表的研究已证实紫杉醇与硼替佐米联合化疗的临床价值。然而，转化研究中难以同时定量检测紫杉醇与硼替佐米，这一问题长期阻碍了相关转化研究的进展。本研究建立并验证了液相色谱-串联质谱（LC-MS/MS）法，可同时定量检测紫杉醇与硼替佐米，填补了该领域的研究空白。两种药物成分在Phenomenex Luna C18色谱柱（7.5×4.6 mm，3μm）上实现分离，流动相为含0.1%甲酸的甲醇-10 mM乙酸铵混合溶液（体积比80:20），流速为0.6 ml/min。本研究按照美国食品药品监督管理局（US-FDA）指南，对建立的方法在雌性Sprague-Dawley大鼠血浆中进行了方法学验证，包括线性、精密度、准确度、定量下限（Lower Limit of Quantitation，LLOQ）及稳定性等指标。为验证所建立的LC-MS/MS方法的适用性，本研究在年轻雌性Sprague-Dawley大鼠体内考察了共载紫杉醇与硼替佐米的纳米制剂的口服药代动力学特征。两种药物的定量下限均为1.0 ng/ml，分析时长仅6.0 min，校准曲线在1~600 ng/ml浓度范围内线性良好。所建立的LC-MS/MS方法具有良好的重复性、精密度与准确度。本研究成功将该LC-MS/MS方法应用于大鼠血浆中紫杉醇与硼替佐米的同时定量检测，并获得了口服给药后的药代动力学曲线。本研究在紫杉醇与硼替佐米的同时定量检测以及纳米制剂开发与生物分析研究中，严格遵循了美国食品药品监督管理局（US-FDA）的行业指南与生物分析方法验证标准。