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Eicosanoids method

Sample preparation

  1. 取400μL 样品到2ml EP管
  2. 加入10μL 内标 (0.5 ng/mL in ethanol), 涡旋
  3. 用1N HCl调节PH至3.0 (500ul组织研磨液加10ul)
  4. 1.2mL of Hexane : MTBE (50:50, v/v) into the tube, then vortex for 1min
  5. Centrifuge 5 minutes at 5000g
  6. 转移上清至新的2ml tube
  7. 重复步骤4, 5
  8. 合并上清液，吹干
  9. Reconstitute with 50 µl of mobile phase A , vortex, 超声5min,14000g离心取35ul上清至进样瓶

组织样品离心后上清液有脂质出现，转移时要避开
2ml EP管离心后不好吸, 可转移至1.5mlEP管
转移的上清不能有沉淀, 可用20ul枪吸两次

Mobile phase A: Water:Acetonitrile:Formic acid = 63:37:0.02
Mobile phase B: Acetonitrile:Isopropyl alcohol = 50:50

Column: Kinetex 2.6 μm C18, 100 x 2.1 mm

Inject volumn: 5 μl (tissue)

Method Time (minutes) Mobile phase A% Mobile phase B% Flow Rate (μL/min)
0.00 100 0 350
6.00 92 8 350
6.50 45 55 350
10.00 45 55 350
13.00 0 100 350
14.00 0 100 350
14.50 100 0 350
17.00 100 0 350

Figure 1. Major enzymatic pathways for the synthesis of arachidonic acid-derived eicosanoids
Figure 1. Major enzymatic pathways for the synthesis of arachidonic acid-derived eicosanoids

Figure 2. Representative chromatogram of arachidonic acid-derived eicosanoids in the targeted metabolomic analysis by liquid chromatography – mass spectrometry
Figure 2. Representative chromatogram of arachidonic acid-derived eicosanoids in the targeted metabolomic analysis by liquid chromatography – mass spectrometry

ID # chemical 事件号 RT m/z
1 15d-PGJ2 1:MRM(-) 7.8 315.1000>271.1000
2 15d-PGJ2-D4 2:MRM(-) 0.1 319.1000>275.1000
3 20HETE 3:MRM(-) 8.45 319.1500>289.1000
4 20HETE-D6 4:MRM(-) 8.45 325.1000>295.1000
5 5,6-EET 5:MRM(-) 8.25 319.2000>219.1000
6 8,9-EET 6:MRM(-) 8.5 319.2000>123.2000
7 14,15-EET 7:MRM(-) 8.25 319.2000>219.2000
8 14,15-EET-D11 8:MRM(-) 0.1 330.2000>219.2000
9 15HETE 9:MRM(-) 8.45 319.1500>219.1000
10 15HETE-D8 10:MRM(-) 0.1 327.1500>226.2000
11 12HETE 11:MRM(-) 8.45 319.2000>179.2000
12 12HETE-D8 12:MRM(-) 8.45 327.2000>184.2000
13 11,12-EET 13:MRM(-) 8.4
14 5HETE 14:MRM(-) 8.7 319.2000>115.2000
15 14,15-DHET 15:MRM(-) 7.6 337.2000>207.1000
16 11,12-DHET 16:MRM(-) 7.7 337.2000>167.1000
17 8,9-DHET 17:MRM(-) 7.7 337.2000>127.1000
18 5,6-DHET 18:MRM(-) 8 337.2000>145.1000
19 5HETE-D8 19:MRM(-) 0.1 327.2000>116.2000
20 5-IPF2A-VI 20:MRM(-) 1.69 353.1000>115.0000
21 AA 21:MRM(-) 11.6 303.6500>259.2500
22 13HODE 22:MRM(-) 8.1 295.1000>195.0000
23 13HODE-D4 23:MRM(-) 8.1 299.3000>198.2000
24 9HODE 24:MRM(-) 8.2 295.1010>171.0000
25 PGJ2 25:MRM(-) 5.51 333.2000>233.1000
26 PGE2-D4-2.15 26:MRM(-) 2 355.1000>193.0000
27 PGD2-D4-2.6 27:MRM(-) 0.1 355.1000>275.1000
28 PGE2-2.15 28:MRM(-) 2 351.1000>271.1000
29 8-ISO-PGF2A 29:MRM(-) 1.45 353.1000>193.0000
30 8-ISO-PGF2A-D4 30:MRM(-) 1.45 357.2000>197.2000
31 8-ISO-PGF1A 31:MRM(-) 1.45 355.2000>311.2000
32 PGF2A 32:MRM(-) 1.6 353.1000>309.1000
33 PGF2A-D4 33:MRM(-) 0.001 357.1000>313.3000
34 PGFM 34:MRM(-) 2.6 353.2000>183.2000
35 PGI2 35:MRM(-) 0.91 369.1000>163.0000
36 PGI2-D4 36:MRM(-) 0.1 373.2000>249.1000
37 LTE4 37:MRM(-) 5.57 438.1000>333.0000
38 LTC4 38:MRM(-) 5.01 624.2000>272.0000
39 LTC4-D5 39:MRM(-) 0.1 629.2000>272.3000
40 LTE4-D5 40:MRM(-) 0.1 443.2000>338.2000
41 PGFM-D4 41:MRM(-) 2.6 357.2000>187.2000
42 PGD2-2.61 42:MRM(-) 2.6 351.1000>271.1000
43 15D-PGD2 43:MRM(-) 7.3 333.1000>271.1200
44 LTB4 44:MRM(-) 7.46 335.1000>195.0000
45 LTB4-D4 45:MRM(-) 0.1 339.2000>197.2000
46 11-dehydro-TXB2 46:MRM(-) 2.41 367.1000>305.0000
47 11-dehydro-TXB2-D4 47:MRM(-) 0.1 371.2000>309.2000
48 TXB2 48:MRM(-) 1.3 369.2000>169.0000
49 TXB2-D4 49:MRM(-) 0.1 373.1000>173.2000
50 AA-D8 50:MRM(-) 0.1 311.6500>267.2500

未使用内标

ID # chemical 事件号 RT m/z
2 15d-PGJ2-D4 2:MRM(-) 0.1 319.1000>275.1000
8 14,15-EET-D11 8:MRM(-) 0.1 330.2000>219.2000
10 15HETE-D8 10:MRM(-) 0.1 327.1500>226.2000
19 5HETE-D8 19:MRM(-) 0.1 327.2000>116.2000
27 PGD2-D4-2.6 27:MRM(-) 0.1 355.1000>275.1000
33 PGF2A-D4 33:MRM(-) 0.001 357.1000>313.3000
39 LTC4-D5 39:MRM(-) 0.1 629.2000>272.3000
40 LTE4-D5 40:MRM(-) 0.1 443.2000>338.2000
45 LTB4-D4 45:MRM(-) 0.1 339.2000>197.2000
47 11-dehydro-TXB2-D4 47:MRM(-) 0.1 371.2000>309.2000
49 TXB2-D4 49:MRM(-) 0.1 373.1000>173.2000
50 AA-D8 50:MRM(-) 0.1 311.6500>267.2500
Internal standard Concentration (ng/μL)
8-ISO-PGF2A-D4 0.5
12HETE-D8 0.5
PGE2-D4-2.15 0.5
13HODE-D4 0.5
PGFM-D4 0.1

各内标出峰时间截图 alt text
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modified version

STDMIX的配制

商品内标均用methanol稀释到500ng/mL
然后等体积混合
最终得到STDMIX, 每种内标的浓度均为100ng/mL

贴壁细胞

  1. 用2mLEP管收集刮下来的细胞
  2. 离心去上请, 100μL H2O重悬细胞, 取3μL用于测BCA
  3. 继续添加300μL H2O
  4. 加入10μL STDMIX, 涡旋
  5. 用1N HCl调节PH至3.0 (理论上1N HCl稀释1000倍, pH等于3.0)
  6. 1.2mL of Hexane : MTBE (50:50, v/v) into the tube, then vortex for 1min
  7. Centrifuge 5 minutes at 5000g
  8. 转移上清至新的2ml tube
  9. re-extraction
  10. 合并上清液，吹干
  11. Reconstitute with 50 µl of mobile phase A , vortex, 超声5min,14000g离心取35ul上清至进样瓶