超高效液相色谱-串联质谱法测定医用热塑性聚氨酯中33种芳香伯胺方法的建立及应用^*

Development and Application of a Method for Determining 33 Aromatic Primary Amines in Medical-Grade Thermoplastic Polyurethane by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

摘要

目的 建立超高效液相色谱-串联质谱法（ultra-high performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS），实现对医用热塑性聚氨酯（thermoplastic polyurethane, TPU）中33种芳香伯胺含量的同时测定。方法 分别以80%甲醇和含10%乙醇的PBS溶液为溶剂提取目标化合物，80%甲醇浸提液过滤后稀释至原浓度的1/5；PBS浸提液经HLB小柱（3 mL，60 mg，甲醇+水平衡）处理后，5 mL甲醇洗脱，过滤后甲醇定容至5 mL，采用超高效液相色谱-串联质谱进行检测分析。色谱分离采用Agilent InfinityLab Poroshell 120 EC-C18色谱柱（150 mm×3.0 mm，2.7 μm），以甲醇（A）和含0.1%甲酸的去离子水（B）为流动相。梯度洗脱，柱温为40 ℃；流速为0.2 mL/min；进样体积为1 μL。质谱检测选用电喷雾离子源（electro-spray ionization, ESI），多反应监测（multiple reaction monitoring, MRM）正离子模式，考察该方法在不同浸提条件下的标准曲线与定量限、精密度、回收率及基质效应。结果 33种芳香伯胺在2~100 ng/mL浓度范围内线性关系良好（r>0.998），检出限为0.1~0.5 ng/mL，定量限为0.3~1.7 ng/mL；80%甲醇样本中33种芳香伯胺的回收率为70.7%～116.0%；含10%乙醇的PBS样本中31种芳香伯胺的回收率为74.9%～113.8%；本方法在不同样本中的日内、日间精密度均<10%，样品稳定性良好。采用本方法对样品浸提液中芳香伯胺含量进行分析，结果表明，在初始检测的6份样品中均检出MDA，且在37 ℃模拟浸提及60 ℃加速浸提条件下，共有3份样品持续或延迟释放出MDA；2,4'-MDA在初始极限浸提阶段于2份样品中检出，并在60 ℃加速老化条件下仍可在1份样品中检出，但在37 ℃模拟生理环境中未见释放；2,2'-MDA仅在初始极限浸提的2份样品中检出，在后续模拟及加速浸提过程中均未释放。结论 该UPLC-MS/MS方法灵敏度高、准确度好，不仅可满足医用TPU中33种芳香伯胺的精准检测需求，更为该类材料及制品的芳香伯胺风险评估与质量控制提供了可靠技术支持。

Abstract

Objective To develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 33 aromatic primary amines in medical-grade thermoplastic polyurethane (TPU). Methods The target compounds were extracted using 80% methanol and PBS solution containing 10% ethanol as the solvents. The 80% methanol extract was filtered through and diluted to one-fifth of the original concentration. The PBS extract was processed through an HLB solid-phase extraction column (3 mL, 60 mg, methanol and water equilibrium), eluted with 5 mL methanol, filtered, and diluted to 5 mL with methanol. Then, analysis was performed using UPLC-MS/MS. Chromatographic separation was performed using an Agilent InfinityLab Poroshell 120 EC-C18 column (150 mm × 3.0 mm, 2.7 μm) with methanol (A) and deionized water containing 0.1% formic acid (B) as the mobile phases. Gradient elution was conducted with a column temperature of 40 ℃, a flow rate of 0.2 mL/min, and an injection volume of 1 μL. Mass spectrometry analysis was performed using an electro-spray ionization (ESI) source in multiple reaction monitoring (MRM) positive ion mode. The standard curves, quantification limits, precision, recovery rates, and matrix effects of the method under different extraction conditions were evaluated. Results The 33 aromatic primary amines analyzed in the study showed good linearity (r > 0.998) within the 2-100 ng/mL concentration range. Detection limits ranged from 0.1 to 0.5 ng/mL, and quantification limits ranged from 0.3 to 1.7 ng/mL. Recovery rates for the 33 aromatic primary amines in 80% methanol samples ranged from 70.7% to 116.0%. Recovery rates for 31 aromatic primary amines in PBS samples containing 10% ethanol ranged from 74.9% to 113.8%. The intra-day and inter-day precision for different samples was both less than 10%, indicating good sample stability. Analysis of aromatic primary amine content in sample extracts using this method revealed that methylenedianiline (MDA) was detected in all 6 initially tested samples. Under simulated extraction at 37 ℃ and accelerated extraction at 60 ℃, 3 samples exhibited continuous or delayed MDA release. 2,4'-MDA was detected in 2 samples during the initial limit extraction phase and remained detectable in 1 sample under 60 ℃ accelerated aging conditions, but no release was observed under the 37 ℃ simulated physiological environment. 2,2'-MDA was detected only in two samples during initial limit extraction and was not released in subsequent simulated or accelerated extraction processes. Conclusion The UPLC-MS/MS method demonstrates high sensitivity and accuracy, meeting the requirements for precise detection of 33 aromatic primary amines in medical-grade TPU and providing reliable technical support for risk assessment and quality control of aromatic primary amines in these materials and products.

关键词

超高效液相色谱-串联质谱法 / 医用热塑性聚氨酯 / 芳香伯胺 / 同时测定 / 浸提释放 / 风险评估

Key words

Ultra-high performance liquid chromatography-tandem mass spectrometry / Medical-grade thermoplastic polyurethane / Aromatic primary amines / Simultaneous determination / Extraction and release / Risk assessment

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基金资助

*四川省区域创新合作项目(2025YFHZ0299)