目的 建立测定抗真菌药物伏立康唑、泊沙康唑及两性霉素B3种药物浓度的高效液相色谱-串联质谱方法,满足临床对其进行常规治疗用药监测的需要。方法 色谱采用Agilent Poroshell 120SB C₁₈色谱柱(规格:4.6 mm×50 mm,2.7 μm),流动相为甲醇/0.2%乙酸溶液,梯度洗脱;质谱采用电喷雾离子源,多反应监测(MRM),正离子模式进行。以对乙酰氨基酚为内标,伏立康唑:m/z 350.1→281.3,泊沙康唑:m/z 701.2→127.0,两性霉素B:m/z 924.0→743.1,对乙酰氨基酚:m/z 152.0→110.0。样本经甲醇沉淀蛋白后进样分析,进样量5 μl,分析时间为9.0 min。结果 样本中内源性物质对测定无干扰,3种药物在各自线性范围内线性关系良好,r均大于0.997。3种药物高、中、低质控样本各自批内、批间精密度(RSD)及准确度(RE)均小于10%,各自提取回收率均在90%以上,基质效应较小,稳定性好。所测48例伏立康唑样本的谷浓度波动在0.4~11.0 μg/ml,受肝肾功能影响较大;所测11例泊沙康唑样本的谷浓度波动在0.31~4.58 μg/ml,透析可能会影响其药物浓度;所测3例两性霉素B制剂样本的谷浓度分别为47.65 μg/ml、6.58 μg/ml和3.84 μg/ml,个体差异较大。结论 建立的方法具有较高的精密度和准确度,提取回收率高,基质效应小,能够满足对3种药物进行高通量测定。

Objective To establish a high-performance liquid chromatography-tandem mass spectrometry method for antifungal drug concentration determination of voriconazole, posaconazole and amphotericin B so as to meet the needs of routine therapeutic drug monitoring in clinical practice. Methods The chromatography was performed using Agilent Poroshell 120SB C18 chromatography column (4.6 mm×50 mm, 2.7 μm). The mobile phase was methanol/0.2% acetic acid solution, with gradient elution. The mass spectrometry was carried out using an electrospray ion source, multiple reaction monitoring (MRM), and positive ion mode. Acetaminophen was used as the internal standard, voriconazole: m/z 350.1→281.3, posaconazole: m/z 701.2→127.0, amphotericin B: m/z 924.0→743.1, acetaminophen: m/z 152.0→110.0. The sample was first subjected to protein precipitation with methanol before injection analysis, with an injection volume of 5 μl. The analysis time was 9.0 minutes. Results There was no interference to the determination of endogenous substances in the samples, and the three drugs had good linear relationships within their respective linear ranges, with r values greater than 0.997. The precision (RSD) and accuracy (RE) of the high, medium, and low quality control samples of the three drugs were all less than 10%, and the recovery rates were all above 90%. The matrix effect was small and the stability was good. The trough concentrations of voriconazole of 48 samples ranged from 0.4 to 11.0 μg/ml, greatly affected by liver and kidney function. The trough concentrations of posaconazole of 11 samples ranged from 0.31 to 4.58 μg/ml, dialysis may affect its drug concentration. The trough concentrations of amphotericin B of 3 samples were 47.65 μg/ml, 6.58 μg/ml and 3.84 μg/ml respectively, with significant individual differences. Conclusions The established method has high precision and accuracy, high extraction recovery and small matrix effect, and can meet the high-throughput determination of the three drugs.