目的: 建立超高效液相色谱串联质谱(UPLC-QQQ MS/MS)同时测定不同品种和产地的松针中14个成分的含量测定方法。方法: 运用UPLC-QQQ MS/MS技术,采用Waters ACQUITY UPLC BEH HILIC(100 mm×2.1 mm,1.7 μm)色谱柱,柱温35 ℃,以0.1%的甲酸-乙腈溶液(A)和0.1%的甲酸-水溶液(B)为流动相,梯度洗脱,流速0.2 mL·min-1;电喷雾离子源,多反应监测模式。各成分的监测离子对分别为m/z 125.0/79.1(没食子酸)、m/z 105.1/77.2(对羟基苯甲醇)、m/z 109.1/91.1(原儿茶酸)、m/z 93.1/65.2(对羟基苯甲酸)、m/z 190.9/85.2(绿原酸)、m/z 151.8/108.2(香草酸)、m/z 135.1/89.2(咖啡酸)、m/z 167.0/122.9(丁香酸)、m/z 119.2/93.2(对香豆酸)、m/z 208.1/193.0(芥子酸)、m/z 82.0/77.2(苯甲酸)、m/z 91.3/65.3(苯乙酸)、m/z 93.1/65.2(水杨酸)、m/z 103.2/77.2(肉桂酸)。结果: 所测定的12份松针样品中各成分含量范围分别为没食子酸0.34~3.42 mg·g-1、对羟基苯甲醇1.32~9.76 mg·g-1、原儿茶酸0~6.32 mg·g-1、对羟基苯甲酸0~19.06 mg·g-1、绿原酸0~18.78 mg·g-1、香草酸0.16~3.81 mg·g-1、咖啡酸0~6.68 mg·g-1、丁香酸0.09~4.64 mg·g-1、对香豆酸0.10~9.90 mg·g-1、芥子酸0.98~19.01 mg·g-1、苯甲酸1.28~18.21 mg·g-1、苯乙酸0.95~20.72 mg·g-1、水杨酸0~3.25 mg·g-1、肉桂酸0~0.27 mg·g-1,各成分在测试范围内线性关系良好,平均加样回收率均在98.0%~101.7%,14个成分定量限介于0.01~0.4 ng。结论: 本方法适用于常见松针中酚酸类成分的含量测定,其中对羟基苯甲醇、绿原酸、香草酸、丁香酸、对香豆酸、芥子酸、苯甲酸、苯乙酸、水杨酸9个成分为首次在松针中发现和测定,可为松针中多种酚酸类成分含量测定提供参考。
Objective: To establish a content determination method for 14 components in pine needles of different varieties and origins simultaneously by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-QQQ MS/MS). Methods: The UPLC-QQQ MS/MS was adopted. A Waters ACQUITY UPLC BEH HILIC (100 mm×2.1 mm,1.7 μm) chromatographic column was used, with a column temperature of 35 ℃. The mobile phase was a mixture of 0.1% formic acid-acetonitrile solution (A) and 0.1% formic acid-aqueous solution (B) for gradient elution, with a flow rate of 0.2 mL·min-1. Electrospray ionization was used, and multiple reaction monitoring mode was employed. The monitored ion pairs of each component were m/z 125.0/79.1(gallic acid), m/z 105.1/77.2(p-hydroxybenzyl alcohol), m/z 109.1/91.1(protocatechuic acid), m/z 93.1/65.2(p-hydroxybenzoic acid), m/z 190.9/85.2(chlorogenic acid), m/z 151.8/108.2(vanillic acid), m/z 135.1/89.2(caffeic acid), m/z 167.0/122.9(syringic acid), m/z 119.2/93.2(p-coumaric acid), m/z 208.1/193.0(sinapic acid), m/z 82.0/77.2(benzoic acid), m/z 91.3/65.3(phenylacetic acid), m/z 93.1/65.2(salicylic acid), and m/z 103.2/77.2(cinnamic acid). Results: The content ranges of each component in the 12 determined pine needle samples were as follows: gallic acid 0.34-3.42 mg·g-1, p-hydroxybenzyl alcohol 1.32-9.76 mg·g-1, protocatechuic acid 0-6.32 mg·g-1, p-hydroxybenzoic acid 0-19.06 mg·g-1, chlorogenic acid 0-18.78 mg·g-1, vanillic acid 0.16-3.81 mg·g-1, caffeic acid 0-6.68 mg·g-1, syringic acid 0.09-4.64 mg·g-1, p-coumaric acid 0.10-9.90 mg·g-1, sinapic acid 0.98-19.01 mg·g-1, benzoic acid 1.28-18.21 mg·g-1, phenylacetic acid 0.95-20.72 mg·g-1, salicylic acid 0-3.25 mg·g-1, and cinnamic acid 0-0.27 mg·g-1. Each component had a good linear relationship within the test range, and the average sample recovery rate was between 98.0% and 101.7%. The quantitative limits of the 14 components were between 0.01 and 0.4 ng. Conclusion: This method is applicable for the determination of phenolic acids in common pine needles, in which 9 compounds, including p-hydroxybenzyl alcohol, chlorogenic acid, vanillic acid, syringic acid, p-coumaric acid, sinapic acid, benzoic acid, salicylic acid, are determined for the first time in pine needles, which can provide reference for the determination of various phenolic acids in pine needles.
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