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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).
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).
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.
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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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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