超高效液相色谱-串联质谱法快速测定血浆中维奈克拉药物

doi:10.3724/SP.J.1123.2025.06002

摘要/Abstract

摘要：

维奈克拉是一种B细胞淋巴瘤因子-2抑制剂，对急性髓系白血病和慢性淋巴细胞白血病等血液系统恶性肿瘤的临床治疗具有重要作用。维奈克拉呈现显著个体间药代动力学差异，谷浓度和治疗反应显著相关，峰浓度超过警戒浓度又会引发不良反应，因此监测维奈克拉血药浓度变化至关重要。本研究建立了一种快速、灵敏、可靠的超高效液相色谱-串联质谱法（UPLC-MS/MS）用于定量测定血浆中的维奈克拉药物浓度。该方法采用电喷雾电离，正离子模式下多反应监测（MRM）检测维奈克拉及其同位素内标维奈克拉-d₈。前处理采用甲醇进行蛋白沉淀。液相分离使用C18反相色谱柱，用乙腈和0.1%甲酸水溶液作为流动相进行梯度洗脱，维奈克拉保留时间为1.95 min。方法学验证结果显示维奈克拉线性范围为50~10 000 ng/mL，r²>0.999，方法特异性良好，批内精密度和批间精密度分别为1.8%~4.5%和2.7%~6.1%，回收率为100.3%~102.9%，基质效应为88.0%~111.0%，进样最高浓度维奈克拉（10 000 ng/mL）后在空白样本中的残留小于线性范围最低浓度峰面积的20%，满足临床要求。将该方法应用于3例急性髓系白血病患者维奈克拉的治疗药物监测，3例患者血浆维奈克拉的最高峰浓度为5 357.9 ng/mL，最低谷浓度为438.2 ng/mL。上述研究结果表明该方法能够准确、高效地定量维奈克拉血药浓度，有助于解决白血病患者的维奈克拉治疗药物浓度监测问题，指导临床个性化治疗。

关键词: 超高效液相色谱-串联质谱, 维奈克拉, 治疗药物监测, 血浆

Abstract:

Leukemia is a malignant tumor of the hematological system characterized by the uncontrolled proliferation of abnormal hematopoietic cells in the bone marrow. It often presents with anemia， bleeding tendency， infection risk and organ invasion. These clinical symptoms bring severe survival risks to patients. Although traditional chemotherapy regimens are effective in the treatment of some hematological malignancies， their efficacy is limited for elderly patients， those with high-risk genetic characteristics or comorbidities. In recent years， targeted drugs have revolutionized the treatment of leukemia. By selectively inducing tumor cell apoptosis， they have significantly improved the remission rate and survival prognosis of patients with multiple leukemia subtypes. Venetoclax is a B-cell lymphoma 2 （BCL-2） inhibitor and plays an important role in the clinical treatment of hematological malignancies， such as acute myeloid leukemia and chronic lymphocytic leukemia. In addition， it also shows potential efficacy in other hematological malignancies such as multiple myeloma and mantle cell lymphoma. Although the efficacy of venetoclax is remarkable， the individual differences in blood drug concentration are significant due to factors such as drug interactions， polymorphisms of metabolic enzymes， and liver and kidney function. Venetoclax exhibits significant inter-individual pharmacokinetic differences， the trough concentration is significantly correlated with the treatment response， and if the peak concentration exceeds the warning concentration， adverse reactions will be triggered. Clinical trials have reported a variety of adverse events associated with venetoclax， including neutropenia， tumor lysis syndrome， thrombocytopenia， infection， anemia， diarrhea， nausea， upper respiratory tract infection， cough and musculoskeletal pain. Therefore， to minimize the risk of adverse events in the clinical use of venetoclax as much as possible， it is necessary to reasonably guide its clinical dosage. Therapeutic drug monitoring can optimize individual dosing regimens by measuring the steady-state concentration in patients’ blood. This research aims to establish a rapid， sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. This method is used for the quantitative determination of venetoclax in plasma， and its performance was validated. This method employs an electrospray ionization and multiple reaction monitoring （MRM） in the positive ion mode to detect venetoclax and its isotope internal standard venetoclax-d₈. Methanol was used for protein precipitation， C18 reversed-phase chromatographic column was used for liquid phase separation. Gradient elution was performed using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The flow rate was 0.4 mL/min， the run time was 3.5 min， and the retention time of venetoclax was 1.95 min. The analysis time is short， facilitationg the rapid determination of clinical samples. Dosage escalation is commonly adopted in the treatment of venetoclax. In this study， a pretreatment approach involving extraction followed by dilution was used. This methodincreased the upper limit of quantification and expanded the linear range. The linear range of venetoclax was 50–10 000 ng/mL， r²>0.999. The method had good specificity. The intra-run precision and inter-run precision were 1.8%–4.5% and 2.7%–6.1%， respectively， the recoveries were 100.3%–102.9%， the matrix effects ranged from 88.0% to 111.0%， and the carryover was less than 20% of the minimum concentration of linear range. This method was applied to the therapeutic drug monitoring of venetoclax in acute myeloid leukemia （AML） patients， and the peak and trough concentrations of venetoclax were obtained for different patients to monitor their blood drug concentration. The above research results indicate that this method can accurately and robustly quantify venetoclax in human plasma， which helps address the drug monitoring needs of leukemia patients receiving venetoclax treatment and guide clinical personalizd therapy.

Key words: ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）, venetoclax, therapeutic drug monitoring, plasma

中图分类号:

O658

朱颖, 马晓丽, 禹松林, 邱玲. 超高效液相色谱-串联质谱法快速测定血浆中维奈克拉药物[J]. 色谱, 2026, 44(3): 357-362.

ZHU Ying, MA Xiaoli, YU Songlin, QIU Ling. Rapid determination of venetoclax in plasma by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2026, 44(3): 357-362.

图/表 4

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Compound	Precursor ion （m/z）	Product ion （m/z）	Cone voltage/V	Collision energy/eV
Venetoclax	868.3	321.0	30	40
	868.3	636.2^*	30	25
Venetoclax-d₈	876.3	329.1	42	36
	876.3	644.2^*	42	26

Compound	Precursor ion （m/z）	Product ion （m/z）	Cone voltage/V	Collision energy/eV
Venetoclax	868.3	321.0	30	40
	868.3	636.2^*	30	25
Venetoclax-d₈	876.3	329.1	42	36
	876.3	644.2^*	42	26

Sample	Mobile phase	Column	Acquisition time/min	Linear range/ （ng/mL）	Publication year	Ref.
Rat plasma	0.1% formic acid in water -acetonitrile （50∶50， V/V， pH 3.2）	Agilent Eclipse plus C18 column （100 mm×2.1 mm， 3.5 µm）	3.0	5-1000	2023	［13］
Humuan plasma	0.1% formic acid in water and acetonitrile	ACQUITY UPLC BEH C18 column （100 mm×2.1 mm， 1.7 μm）	4.0	25-8000	2022	［14］
Rat plasma	0.1% formic acid in water -acetonitrile （50∶50， V/V， pH 3.2）	Eclipse plus C18 column （100 mm×2.1 mm， 1.8 μm）	2.5	5-1000	2025	［16］
Human cerebrospinal fluid， bone marrow， and plasma	NA	NA	NA	30-8000	2024	［17］
rat plasma	10 mmol/L ammonium formate and 0.1% formic acid acetonitrile and methanol	Phenomenex Kinetex C18 （150 mm×2.1 mm， 2.6 µm）	6.0	5-500	2021	［18］
Human plasma and cerebrospinal fluid	10 mmol/L ammonium acetate containing 0.1% formic acid and acetonitrile	ACQUITY UPLC HSS T3 column （50 mm×2.1 mm， 1.8 μm）	5.0	20-5000	2023	［19］
Human plasma	0.1% formic acid in water and acetonitrile	ACQUITY UPLC BEH C18 column （50 mm×2.1 mm， 1.7 μm）	4.0	125-8000	2023	［20］

Sample	Mobile phase	Column	Acquisition time/min	Linear range/ （ng/mL）	Publication year	Ref.
Rat plasma	0.1% formic acid in water -acetonitrile （50∶50， V/V， pH 3.2）	Agilent Eclipse plus C18 column （100 mm×2.1 mm， 3.5 µm）	3.0	5-1000	2023	［13］
Humuan plasma	0.1% formic acid in water and acetonitrile	ACQUITY UPLC BEH C18 column （100 mm×2.1 mm， 1.7 μm）	4.0	25-8000	2022	［14］
Rat plasma	0.1% formic acid in water -acetonitrile （50∶50， V/V， pH 3.2）	Eclipse plus C18 column （100 mm×2.1 mm， 1.8 μm）	2.5	5-1000	2025	［16］
Human cerebrospinal fluid， bone marrow， and plasma	NA	NA	NA	30-8000	2024	［17］
rat plasma	10 mmol/L ammonium formate and 0.1% formic acid acetonitrile and methanol	Phenomenex Kinetex C18 （150 mm×2.1 mm， 2.6 µm）	6.0	5-500	2021	［18］
Human plasma and cerebrospinal fluid	10 mmol/L ammonium acetate containing 0.1% formic acid and acetonitrile	ACQUITY UPLC HSS T3 column （50 mm×2.1 mm， 1.8 μm）	5.0	20-5000	2023	［19］
Human plasma	0.1% formic acid in water and acetonitrile	ACQUITY UPLC BEH C18 column （50 mm×2.1 mm， 1.7 μm）	4.0	125-8000	2023	［20］

Patient No.	Gender	Age/year	C_max/（ng/mL）	C_min/（ng/mL）
1	male	29	2860.0	1284.3
2	male	59	5357.9	4342.8
3	female	48	3217.4	438.2