| 摘要: |
| 目的 建立一种简便、可靠和灵敏的超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，用于检测白念珠菌分泌的3种群体感应分子（法尼醇、酪醇、3-吲哚乙醇）的含量。方法 样品经乙酸乙酯萃取后，采用Agilent 6470型三重四极杆串联质谱仪，通过正离子监测模式，以卡马西平为内标，在Agilent Poroshell 120 EC-C18色谱柱（3.0 mm×100 mm，2.7 μm）上进行色谱分离。采用梯度洗脱，流动相为0.1%甲酸水溶液（A）和乙腈（B），洗脱梯度为0～3 min 20%～40% B、3～4 min 40%～80% B、4～8 min 80% B，流速为0.5 mL/min，进样量为5 µL，柱温为25 ℃，分析时间为8 min，平衡时间为2 min。采用电喷雾离子源、正离子多反应监测模式进行质谱分析。结果 法尼醇、酪醇、3-吲哚乙醇的专属性和线性关系良好（r均＞0.999），精密度和准确度均良好（日内、日间精密度均＜5%，准确度的绝对值均＜10%），平均回收率为90%～115%；3种条件（室温放置4 h、4 ℃自动进样器放置24 h及冻融3次）下的稳定性均符合方法学要求。在浮游型样品中，3种群体感应分子的含量均随培养时间的延长而增加；而在被膜型样品中，法尼醇和酪醇在被膜成熟阶段含量降低。结论 该UHPLC-MS/MS方法可用于测定白念珠菌分泌的法尼醇、酪醇、3-吲哚乙醇的含量，为真菌群体感应分子的快速、准确检测提供参考。 |
| 关键词: 超高效液相色谱-串联质谱法 白念珠菌 群体感应分子 法尼醇 酪醇 3-吲哚乙醇 |
| DOI：10.16781/j.CN31-2187/R.20220678 |
| 投稿时间：2022-08-19修订日期：2022-12-22 |
| 基金项目:国家自然科学基金（82104127）. |
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| Determination of quorum sensing molecules in Candida albicans by ultra-high performance liquid chromatography-tandem mass spectrometry |
| LI Ling1,Lü Lei2* |
(1. Pharmaceutical Analysis Center, School of Pharmacy, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Pharmacy, The Third Affiliated Hospital of Naval Medical University(Second Military Medical University), Shanghai 200438, China
*Corresponding author) |
| Abstract: |
| Objective To establish a convenient, reliable and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting the content of 3 kinds of quorum sensing molecules (farnesol, tyrosol, and 3-indoleethanol) secreted by Candida albicans. Methods With carbamazepine as the internal standard, the samples were extracted with ethyl acetate and separated by gradient elution on Agilent Poroshell 120 EC-C18 column (3.0 mm×100 mm, 2.7 μm) using Agilent 6470 triple quadrupole tandem mass spectrometry in positive ion monitoring mode. The mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile. The elution gradient was 0-3 min 20%-40% B, 3-4 min 40%-80% B, and 4-8 min 80% B; flow rate was 0.5 mL/min; injection volume was 5 µL; column temperature was 25 ℃; the analysis period of each sample was 8 min; and the equilibrium time was 2 min. The mass spectrometry was performed using electrospray ionization source and positive ion multiple reaction monitoring mode. Results Farnesol, tyrosol and 3-indoleethanol had good specificity and linear relationship (all r＞0.999), and had good precisions and accuracy (intra- and inter-day precisions were ＜5% and absolute values of accuracy were ＜10%). The average recovery rate was 90%-115%. The stability under 3 conditions (4 h at room temperature, 24 h at 4 ℃ with autosampler, and 3 freeze-thaw cycles) met the requirements of the methodology. In the planktonic samples, the contents of the 3 kinds of quorum sensing molecules were increased with the growth of the strains. However, in the biofilm samples, the contents of farnesol and tyrosol were decreased at the mature stage of the biofilm. Conclusion This UHPLC-MS/MS method can be used to determine the contents of farnesol, tyrosol and 3-indoleethanol secreted by Candida albicans, and it provides a reference for rapid and accurate detection of quorum sensing molecules in fungi. |
| Key words: ultra-high performance liquid chromatography-tandem mass spectrometry Candida albicans quorum sensing molecules farnesol tyrosol 3-indoleethanol |
