Eicosanoid determination - Yue et al.

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Contents 1 Analytes 2 Sample Preparation 3 Instrumentation and Method 3.1 Autosampler 3.2 HPLC 3.3 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 Sample data 6.1 Mass spectra 6.2 Chromatogram 7 Reference 8 Contact

Analytes

• 18 Eicosanoids:

14,15-DiHETrE; 11,12-DiHETrE; 8,9-DiHETrE; 5,6-DiHETrE;20-HETE; 15-HETE; 12-HETE; 9-HETE; 8-HETE; 5-HETE; 14,15-EET; 11,12-EET; 8,9-EET; 5,6-EET; PGF₂α; PGE₂; PGD₂; PGJ₂

• 7 Internal Standards (IS):

14,15-EET-d₈; 11,12-EET-d₈; 8,9-EET-d₈; 15(S)-HETE-d₈; PGD₂-d₄; PGF₂α-d₄; AA-d₈

Sample Preparation

• dissection of 20-40 mg cortex brain tissue
• addition of 200 μl MeOH and 2 μl HCOOH
• homogenization with micro unltrasonic cell disrupter
• centrifugation at 0°C (14000 rpm, 10 min)
• dilution of supernatant with 1,8 ml H₂O
• acidification (0.1 M HCl; pH = 3.0)

SPE

column	equilibration	wash	elution
Oasis HLB (30 mg, 30 μm, Waters)	1 ml MeOH 1 ml EE; 1 ml MeOH; 2 ml H2O	200 μl MeOH (10 %); 3 ml H2O; 1 ml MeOH (10 %)	0,5 ml CH3CN; 1,5 ml EE

• addition of 100 pg per IS (except for AA-d₈ = 40 ng)
• evaporation of solvents (gentle stream of Ar, 5.0 grade)
• addition of 20 μl MeOH to the residue

Material	Material used	Internal Standard(s)	Internal Standard(s) added
rat brain tissue

Instrumentation and Method

Autosampler

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

HPLC

• Type: Agilent 1100

• Pump:
• Column: Symmetry C18 (4.6 * 250 mm, 5 μm, Waters)
• T column = 40 °C

• Solvents:

A: 0,1 % HCOOH in H₂O

B: 0,1 % HCOOH in CH₃CN

• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
30	1	40-20	60-80
35	1	20-15	80-85
36	1	15-0	85-100
36-45	1	0	100

• Run time: 45 min

Mass Spectrometer

• Type: quadrupole (SL model)
• Ionization mode: atmospheric ESI negative
• Capillary voltage: 3000 V
• Gas temperature: 120 °C
• Drying gas flow rate: 11 l/min
• Nebulizer pressure: 40 psig

• MS/MS-conditions:
  • Scan modi: SIM
  • Fragmentor voltage: 120 V (0-7 min), 110 V (7- 23 min), 130 V (23- 45 min)
  • Detection table

Analyte	Time [min]	SIM [m/z]
PGF2α	0-7	353
PGD2	0-7	351
PGE2	0-7	351
PGJ2	0-7	333
PGD2-d4	0-7	355
PGF2α-d4	0-7	357
DiHETrEs	7-23	337
HETEs	7-23	319
HETE-d8	7-23	327
EET-d8	7-23	327
AA	23-45	303
AA-d8	23-45	311

Data analysis and quantification

Data handling

Isotope correction

Calibration and quantification

Method Validation

Accuracy

Precision

LOD

5·10^-4 pg (S/N > 4)

LOQ

5·10^-3 pg (S/N > 10)

Recovery

Linearity

• 2-1000 pg (PGs, DiHETrEs, HETEs, EETs)

• 10-2400 pg (PGE₂,PGD₂)

• 20-2000 pg (AA)

Sample data

Mass spectra

Chromatogram

Reference

• H. Yue, S. A. Jansen, K. I. Strauss, M. R. Borenstein, M. F. Barbe, L. J. Rossi, E. Murphy Journal of Pharmaceutical and Biomedical Analysis 2007, 43, 1122-1134.

Contact

• Temple University; Chemistry Department; Analytical Chemistry; 1901 North 13th Street; Philadelphia; PA 19122; USA.

• email: suebee@temple.edu (S.A. Jansen)