
MATERIALS AND METHODS
Standards and reagents
Method | Compound | RT (min) | Transition | LOQ (pg on-column) | r | Recovery (%) | Intra-Day Precision (%) | Inter-Day Precision (%) | Matrix Effect (%) | Stabilitya | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Low (n = 6) | Medium (n = 6) | High (n = 6) | Low (n = 6) | Medium (n = 6) | High (n = 6) | Low (n = 6) | Medium (n = 6) | High (n = 6) | t = 0 Months (n = 6) | t = 15 Months (n = 6) | |||||||
I | Tetranor PGEM | 3.43 | 327 > 143 | 13 | 0.996 | 106 | 99 | 103 | 10 | 7 | 5 | 5 | 5 | 5 | 101 | 31.1 ± 5.3 | 40.8 ± 1.4 |
Tetranor PGDM | 3.91 | 327 > 143 | 1 | 0.999 | 96 | 95 | 100 | 9 | 11 | 13 | 12 | 11 | 16 | 109 | 8.1 ± 0.3 | 7.9 ± 0.3 | |
2,3-Dinor-8-iso PGF2α | 6.81 | 325 > 237 | 1 | 0.998 | 105 | 99 | 104 | 4 | 6 | 3 | 7 | 5 | 6 | 84 | 4 ± 0.5 | 3.8 ± 0.2 | |
2,3-Dinor-11α-PGF2α | 7.15 | 325 > 145 | 1 | 0.996 | 100 | 97 | 102 | 10 | 2 | 1 | 16 | 18 | 12 | 82 | 4.7 ± 0.5 | 3.3 ± 0.2 | |
6-Keto-PGF1α | 7.24 | 369 > 163 | 1 | 0.998 | 104 | 98 | 98 | 0 | 3 | 4 | 9 | 4 | 4 | 119 | 2.3 ± 0.1 | 2.8 ± 0.2 | |
13,14-Dihydro-15- keto-tetranor-PGE2 | 7.51 | 297 > 109 | 5 | 0.996 | 92 | 107 | 103 | 11 | 7 | 9 | ND | 18 | 12 | 92 | 5.5 ± 0.2 | 4 ± 0.2 | |
13,14-Dihydro-15- keto-tetranor-PGD2 | 8.12 | 297 > 109 | 3 | 0.999 | 102 | 98 | 103 | 8 | 5 | 4 | 6 | 2 | 7 | 92 | 8.1 ± 0.4 | 8.8 ± 0.3 | |
8-Iso-PGF2α | 9.08 | 353 > 193 | 2 | 0.999 | 100 | 97 | 95 | 11 | 4 | 5 | 14 | 6 | 7 | 96 | 2.4 ± 0.3 | 1.8 ± 0.1 | |
11α-PGF2α | 9.49 | 353 > 291 | 2 | 0.999 | 96 | 91 | 94 | 8 | 3 | 4 | 1 | 4 | 1 | 117 | 11.8 ± 0.1 | 15.3 ± 0.3 | |
5-iPF2α-VI | 9.85 | 353 > 115 | 4 | 0.999 | 94 | 93 | 99 | 9 | 3 | 3 | 7 | 2 | 2 | 109 | 8.9 ± 0.3 | 8.5 ± 0.8 | |
PGF2α | 10.91 | 353 > 291 | 2 | 0.999 | 103 | 86 | 88 | 5 | 5 | 2 | 5 | 2 | 2 | 123 | 17.5 ± 1.2 | 15.8 ± 0.8 | |
11-Dehydro-TXB2 | 11.33 | 367 > 161 | 3 | 0.999 | 97 | 92 | 95 | 8 | 2 | 4 | 6 | 3 | 1 | 121 | 7.7 ± 0.3 | 8.7 ± 0.7 | |
PGE2 | 11.40 | 351 > 271 | 1 | 0.998 | 100 | 91 | 94 | 13 | 6 | 4 | 3 | 2 | 1 | 114 | 3.5 ± 0.2 | 3.6 ± 0.3 | |
PGD2 | 12.19 | 351 > 271 | 2 | 0.996 | ND | 79 | 98 | ND | 11 | 10 | ND | 13 | 5 | 122 | 2.1 ± 0.2 | 5.3 ± 0.2 | |
PGE1 | 12.25 | 353 > 273 | 10 | 0.999 | ND | 85 | 91 | ND | 5 | 4 | ND | 6 | 2 | 113 | 12.2 ± 0.8 | 12.4 ± 0.3 | |
13,14-Dihydro-15-keto-PGE2 | 13.98 | 351 > 175 | 2 | 0.999 | 90 | 90 | 95 | 8 | 4 | 2 | 10 | 2 | 1 | 113 | 9.1 ± 0.3 | 10 ± 0.5 | |
8,12-Iso-iPF2α-VI | 14.12 | 353 > 115 | 1 | 0.999 | 100 | 91 | 91 | 6 | 4 | 4 | 2 | 1 | 1 | 126 | 11.6 ± 0.4 | 11.3 ± 0.7 | |
13,14-Dihydro-15-keto-PGF2α | 14.19 | 353 > 291 | 3 | 0.999 | 101 | 90 | 94 | 8 | 4 | 5 | 2 | 2 | 2 | 125 | 20.2 ± 0.5 | 21.2 ± 0.7 | |
13,14-Dihydro-15-keto-PGE1 | 14.86 | 353 > 209 | 2 | 0.997 | 93 | 92 | 88 | 7 | 5 | 4 | 27 | 10 | 4 | 124 | 10.1 ± 0.2 | 10.5 ± 0.6 | |
13,14-Dihydro-15-keto-PGD2 | 15.04 | 351 > 175 | 3 | 0.998 | 97 | 90 | 90 | 11 | 6 | 1 | 6 | 5 | 3 | 102 | 12.1 ± 0.5 | 16.4 ± 0.9 | |
II | 2,3-Dinor-6-keto-PGF1α | 3.12 | 370 > 232 | 10 | 0.999 | 106 | 104 | 104 | 6 | 4 | 6 | 12 | 14 | 11 | 117 | 82.1 ± 5.8 | 68.2 ± 7.8 |
2,3-Dinor-TXB2 | 3.45 | 370 > 155 | 3 | 0.999 | 108 | 97 | 100 | 3 | 3 | 3 | 5 | 2 | 2 | 115 | 21.7 ± 2.3 | 21.3 ± 0.9 | |
TXB2 | 3.81 | 398 > 169 | 1 | 0.999 | 97 | 92 | 95 | 7 | 3 | 3 | 3 | 3 | 3 | 118 | 3.5 ± 0.3 | 3.4 ± 0.1 | |
III | 20-Carboxy-LTB4 | 0.85 | 365 > 195 | 3 | 0.996 | ND | 89 | 86 | ND | 6 | 5 | ND | 25 | 27 | 5 | 0.2 ± 0.1 | 4.7 ± 0.4b |
20-Hydroxy-LTB4 | 0.90 | 351 > 195 | 3 | 0.997 | 85 | 82 | 82 | 0 | 6 | 6 | 9 | 18 | 18 | 5 | 0.2 ± 0.1 | 0.1 ± 0.1 | |
EXC4 | 1.55 | 624 > 306 | 4 | 0.999 | 90 | 69 | 80 | 6 | 15 | 13 | 19 | 12 | 4 | 76 | 2.2 ± 0.3 | 0.9 ± 0.1 | |
LTD4 | 1.68 | 495 > 187 | 3 | 0.997 | 74 | 65 | 68 | 8 | 9 | 8 | 16 | 8 | 9 | 136 | 1.3 ± 0.1c | 3 ± 0.2 | |
EXE4 | 1.87 | 438 > 333 | 3 | 0.999 | 74 | 75 | 76 | 13 | 6 | 6 | 2 | 5 | 3 | 173 | 2.7 ± 0.2 | 3 ± 0.2 | |
LTC4 | 2.29 | 624 > 306 | 4 | 0.999 | 93 | 75 | 83 | 10 | 8 | 14 | 9 | 7 | 7 | 80 | 1.6 ± 0.2 | 0.8 ± 0.1 | |
LTE4 | 2.35 | 438 > 333 | 3 | 0.999 | 82 | 81 | 83 | 2 | 3 | 4 | 3 | 3 | 1 | 131 | 5.1 ± 0.3 | 4.6 ± 0.4 | |
6-trans-LTB4 | 3.23 | 335 > 195 | 2 | 0.997 | 84 | 82 | 83 | 2 | 1 | 0 | 4 | 5 | 5 | 140 | 2.7 ± 0.1 | 2.8 ± 0.2 | |
LTB4 | 3.46 | 335 > 195 | 2 | 0.999 | 77 | 76 | 77 | 2 | 1 | 1 | 5 | 5 | 6 | 129 | 4.3 ± 0.2 | 3.4 ± 0.1 | |
RT, retention time; r, linearity; Low, low concentration; Medium, medium concentration; High, high concentration; ND, not detected. | |||||||||||||||||
a Data are shown as the concentration (nanograns per milligram) of the indicated compound in the fortified laboratory reference material at the time of preparation (t = 0 months) or after 15 months storage at −80°C (t = 15 months). | |||||||||||||||||
b The observed reduced stability of this compound is most likely a combination of large matrix effects, low precision, and low recovery for quantification. | |||||||||||||||||
c The initial timepoint is t = 4 months for this compound. |
Solid phase extraction
PGMsIPs.
CysLTs.
LC-MS/MS analysis
Volume optimization
Normalization
- Fraselle S.
- De Cremer K.
- Coucke W.
- Glorieux G.
- Vanmassenhove J.
- Schepers E.
- Neirynck N.
- Van Overmeire I.
- Van Loco J.
- Van Biesen W.
- et al.
Glucuronidation
- Medina S.
- Miguel-Elízaga I.D.
- Oger C.
- Galano J.M.
- Durand T.
- Martínez-Villanueva M.
- Castillo M.L.
- Villegas-Martínez I.
- Ferreres F.
- Martínez-Hernández P.
- et al.
Method validation
Clinical samples
Data analysis and statistics
RESULTS AND DISCUSSION
Volume optimization
Parent Compound | Eicosanoid Metabolite | %RSD | %RSD (15 months) | %RSD [Balgoma et al. ( 21. )] |
---|---|---|---|---|
PGE2 | PGE2 | 6.7 | 12.4 | 6.6 |
Tetranor PGEM | 15.0 | 28 | 22.9 | |
PGD2 | 2,3-Dinor-11-α-PGF2α | 6.3 | 18.2 | 14.4 |
Tetranor PGDM | 5.2 | 9.1 | 8.5 | |
PGF2α | PGF2α | 8.5 | 8.4 | 9.7 |
13,14-Dihydro-15-keto-PGF2α | 3.0 | 7.8 | Not included | |
PGI2 | 6-Keto-PGF1α | 6.0 | 10.7 | Not included |
2,3-Dinor-6-keto-PGF1α | 5.7 | 13.8 | 15.7 | |
TXA2 | TXB2 | 7.4 | 11.8 | 7.8 |
11-DehydroTXB2 | 5.6 | 6.7 | 12.1 | |
2,3-Dinor-TXB2 | 11.2 | 7.1 | 15.1 | |
Isoprostanes | 8-IsoPGF2α | 8.3 | 12.6 | 8.8 |
2,3-Dinor-8-isoPGF2α | 7.9 | 14.3 | 8.7 | |
5-iPF2α-VI | 3.9 | 9.0 | Not included | |
8,12-Iso-iPF2α-VI | 2.7 | 7.3 | 4.0 | |
LTE4 | LTE4 | 4.1 | 10.1 | 8.4 |
Normalization
Glucuronidation
- Medina S.
- Miguel-Elízaga I.D.
- Oger C.
- Galano J.M.
- Durand T.
- Martínez-Villanueva M.
- Castillo M.L.
- Villegas-Martínez I.
- Ferreres F.
- Martínez-Hernández P.
- et al.
- Fu J.
- Schoeman J.C.
- Harms A.C.
- van Wietmarschen H.A.
- Vreeken R.J.
- Berger R.
- Cuppen B.V.J.
- Lafeber F.P.J.G
- van der Greef J.
- Hankemeier T.
Validation results

Urinary eicosanoids increase following allergen challenge

Methodological improvements
- Armstrong M.
- Liu A.H.
- Harbeck R.
- Reisdorph R.
- Rabinovitch N.
- Reisdorph N.
- Fu J.
- Schoeman J.C.
- Harms A.C.
- van Wietmarschen H.A.
- Vreeken R.J.
- Berger R.
- Cuppen B.V.J.
- Lafeber F.P.J.G
- van der Greef J.
- Hankemeier T.
- Armstrong M.
- Liu A.H.
- Harbeck R.
- Reisdorph R.
- Rabinovitch N.
- Reisdorph N.
- Armstrong M.
- Liu A.H.
- Harbeck R.
- Reisdorph R.
- Rabinovitch N.
- Reisdorph N.
- Fu J.
- Schoeman J.C.
- Harms A.C.
- van Wietmarschen H.A.
- Vreeken R.J.
- Berger R.
- Cuppen B.V.J.
- Lafeber F.P.J.G
- van der Greef J.
- Hankemeier T.
- Fu J.
- Schoeman J.C.
- Harms A.C.
- van Wietmarschen H.A.
- Vreeken R.J.
- Berger R.
- Cuppen B.V.J.
- Lafeber F.P.J.G
- van der Greef J.
- Hankemeier T.
- Armstrong M.
- Liu A.H.
- Harbeck R.
- Reisdorph R.
- Rabinovitch N.
- Reisdorph N.
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Article info
Publication history
Footnotes
This work was supported by Swedish Heart-Lung Foundation Grants 20170603 and 20170734, Swedish Research Council Grant 2016-02798, AllerGen National Center of Excellence Grant GxE4, Stockholm County Council Research Funds (ALF), and the Centre for Allergy Research Highlights Asthma Markers of Phenotype (ChAMP) consortium, which is funded by the Swedish Foundation for Strategic Research, the Karolinska Institutet, AstraZeneca Canada and Science for Life Laboratory Joint Research Collaboration, and the Vårdal Foundation. C.E.W. was supported by Swedish Heart-Lung Foundation Grant 20180290. C.C. was supported by the Canada Research Chairs program and the AstraZeneca Canada Chair in Occupational and Environmental Lung Disease.
The online version of this article (available at http://www.jlr.org) contains a supplement.
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