Eicosanoid determination - Harkewitz et al.

From LipidomicsWiki

Contents 1 Analytes 2 Sample Preparation 3 Instrumentation and Method 3.1 Autosampler 3.2 HPLC 3.3 HPLC (chiral) 3.4 Mass Spectrometer 4 Data analysis and quantification 4.1 Data handling 4.2 Isotope correction 4.3 Calibration and quantification 5 Method Validation 5.1 Accuracy 5.2 Precision 5.3 LOD 5.4 LOQ 5.5 Recovery 5.6 Linearity 6 Reference 7 Contact

Analytes

• 75 eicosanoids:

12(S)HHTrE; AA; 15d-∆12,14PGJ2; 5-OxoETE; 12-OxoETE; 15-OxoETE; 20-HETE; 5(R)HETE; 5(S)HETE; 8(R)HETE; 8(S)HETE; 11(R)HETE; 11(S)HETE; 12(R)HETE; 12(S)HETE; 15(R)HETE; 15(S)HETE; ±5,6-EpETrE; ±8,9-EpETrE; ±11,12-EpETrE; ±14,15-EpETrE; tetranor PGEM;5(S)HETE-d8; tetranor PGFM; PGA2; PGB2; PGJ2; ∆12-PGJ2; dhk-PGA2; bicyclo-PGE2; 5(S)6(R)DiHETE; 5(S)6(S)DiHETE;5(S)15(S)DiHETE; 8(S)15(S)DiHETE; LTB4; 5(S)HpETE; 12(S)HpETE; 15(S)HpETE; ±5,6-DiHETrE; ±8,9-DiHETrE;±11,12-DiHETrE; ±14,15-DiHETrE; 2,3-dinor TXB4;15-keto PGE2;PGK2; PGD2; PGE2; PGH2; dhk-PGD2; dhk-PGE2;15-keto PGF2a; 5(S)6(R)15(S)LXA4; 5(S)6(S)15(S)LXA4;5(S)14(R)15(S)LXB4; PGF2a; 11b-PGF2a; PGF2b; dhk-PGF2a; PGD2-d4; PGE2-d4; PGF2a-d4; PGG2; 19(R)hydroxy PGE2; 20-hydroxy PGE2; 6-keto PGE1; 11-dehydro TXB2; TXB2; 6-keto PGF1a; diketo-dihydro PGF1a; PGD2-EA; PGE2-EA; PGF2a-EA; LTE4; LTC4

• 1 Internal Standard (IS):

AA-d8

Sample Preparation

• collection of cultured medium
• centrifugation (3 min, 1000 rpm)
• supplementation with 10 % MeOH

SPE

column	equilibration	wash	elution
Strata-X (60 mg, 3 ml, Phenomenex)	2 ml MeOH; 2 ml H2O	2 ml 10 % MeOH	1 ml MeOH

• evaporation of solvent
• addition of 100 μl LC-buffer A

Material	Material used	Internal Standard(s)	Internal Standard(s) added
mouse macrophage like cells

Instrumentation and Method

Autosampler

• Type:
• Injection volume: 10 μl
• Wash solvent:
• T (autosampler chamber): 4 °C

HPLC

• Type: Shimadzu LC-10AD
• Pump:
• Column: Grace Vydac, RP-C18 (2.1 * 250 mm, 5 μl)
• T column = 35 °C

• Solvents:

A: 63 % H₂O, 37 % CH₃CN, 0,02 % HCOOH

B: 50 % CH₃CN, 50 % iProp

• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0,3	100	0
6	0,3	80	20
6,5	0,3	45	55
11	0,3	28	75
16	0,3	28	75

• Run time: 45 min

HPLC (chiral)

• Column: Chiralpak® AD-H, normal phase (4,6 * 250 mm, Chiral Technologies)
• Guard column: Chiralpak® AD-H
• Solvents:

buffer A: Hex / EtOH / H₂O / HCOOH (96 / 4 / 0.08 / 0.02)

buffer B: EtOH

• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	X	100	0
13	X	90	10
15-25	X	75	25
27-42	X	100	0

Mass Spectrometer

• Type: hybrid triple quadrupole, linear ion trap (4000 QTRAP; Applied Bioscience)
• Ionization mode: negative electrospray mode (CI for chiral chromatography); Turbo V ion source
• QTRAP: CUR: 10 psi
• GS1: 40 psi
• GS2: 40 psi (60 psi for chiral chromatography)
• IS: -4200 V
• collisional activated dissociation: HIGH (-20 to -40 V)
• T: 525 °C (400 °C for chiral chromatography)
• ihe: ON
• DP: -30 V (-60 V for chiral chromatography)
• EP: -15 V
• CXP: -10 V

• MS/MS-conditions:
  • Scan modi: Q1, MS2, MRM
  • Detection table: (see link for the article)

Data analysis and quantification

Data handling

Isotope correction

Calibration and quantification

Method Validation

Accuracy

Precision

LOD

LOQ

Recovery

Linearity

Reference

• R. Harkewitz, E. Fahy, A. Andreyev, E. A. Dennis J. Biolog. Chem. 2007, 282, 2899-2910.

Contact

• Departments of Pharmacology, Chemistry, and Biochemistry; University of California; San Diego; 9500 Gilman Drive; La Jolla; CA 92093-0601. Tel.: 858-534-3055; Fax: 858-534-7390

• e-mail: edennis@ucsd.edu.