Eicosanoid determination - Masoodi et al.

From LipidomicsWiki

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 Reference 7 Contact

Analytes

• 27 Eicosanoids:

PGD₁; PGE₁; PGF₁α; 6-keto-PGF₁α; PGB₂; PGD₂; PGE₂; PGF₂α; PGJ₂; ∆¹²-PGJ₂; 15-desoxy-∆^12,14-PGJ₂; PGD₃; PGE₃; PGF₃α; TXB₂; TXB₃; 8-iso-PGE₂; 8-iso-15-keto-PGE₂; 8-iso-PGF₂α; 8-iso-15-keto-PGF₂α; 13,14-dihydro-PGE₁; 13,14-dihydro-15-keto-PGE₁; 13,14-dihydro-PGF₁α; 13,14-dihydro-15-keto-PGF₁α; 13,14-dihydro-15-keto-PGE₂; 13,14-dihydro-PGF₂α; 13,14-dihydro-15-keto-PGF₂α;

• 1 Internal Standard (IS):

PGB₂-d₄

Sample Preparation

• homogenisation of tissue samples (in H₂O)
• adjust to 15 % MeOH (final volume: 3 ml)
• addition of 40 ng PGB₂-d₄ (IS)
• incubation (on ice, 30 min)
• centrifugation (3000 rpm, 5 min)
• acidification (0.1 M HCl; pH = 3.0)

SPE

column	equilibration	wash	elution
C18-E (500 mg, 6 ml, Phenomenex)	20 ml MeOH; 20 ml H2O	200 μl MeOH (10 %); 20 ml H2O; 10 ml Hexan	15 ml HCOOMe

• evaporation of solvents
• addition of 100 μl EtOH (stored at -20 °C)

Material	Material used	Internal Standard(s)	Internal Standard(s) added
rat brain tissue
rat liver tissue
rat plasma
rat urine

Instrumentation and Method

Autosampler

• Type: 2690 (Waters)
• Injection volume: 5 μl
• Wash solvent: n.d.
• T (autosampler chamber): 8 °C

HPLC

• Type: Alliance 2695 (Waters)

• Column: Gemini C-18 (2.0 * 150 mm, 5 μl, Phenomenex)

• T column: n.d.

• Solvents:

A: CH₃CN / H₂O / CH₃COOH (45 / 55 / 0.02)

B: CH₃CN / H₂O / CH₃COOH (90 / 10 / 0.02)

• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0-8	0,2	100	0
8,1-12	0,2	50	50
12,1-20	0,2	30	70
20,1-30	0,2	100	0

• Run time: 30 min (10 min wash cycle included)

Mass Spectrometer

• Type: triple quadrupole Quattro Ultima MS (Waters)
• Ionization mode: ESI negative
• Capillary voltage: 3500 V
• T (source): 120 °C
• T (desolvation): 360 °C
• Cone voltage: 35 V

• MS/MS-conditions:
  • Scan modi: Q1, MS2, MRM
  • Collision energy: optimized (12 - 38 eV)
  • Dwell time: 0.2 s
  • MRM table:

Analyte	MRM [m/z]	Collision energy [eV]
PGD1	353 -> 317	15
PGE1	353 -> 317	15
PGF1α	355 -> 311	25
6-keto-PGF1α	369 -> 163	23
PGB2	333 -> 175	20
PGB2-d4	337 -> 179	20
PGD2	351 -> 271	17
PGE2	351 -> 271	17
PGF2α	353 -> 193	25
PGJ2	333 -> 271	15
Δ 12-PGJ2	333 -> 271	15
15-deoxy-Δ 12,14-PGJ2	315 -> 271	15
PGD3	349 -> 269	15
PGE3	349 -> 269	15
PGF3α	351 -> 193	25
TXB2	369 -> 169	17
TXB3	367 -> 169	15
8-iso-PGE2	351 -> 315	15
8-iso-15-keto-PGE2	349 -> 113	23
8-iso-PGF2α	353 -> 193	25
8-iso-15-keto-PGF2α	351 -> 315	15
13,14-dihydro-PGE1	355 -> 337	15
13,14-dihydro-15-keto-PGE1	353 -> 335	12
13,14-dihydro-PGF1α	357 -> 113	38
13,14-dihydro-15-keto-PGF1α	355 -> 193	32
13,14-dihydro-15-keto-PGE2	351 -> 333	12
13,14-dihydro-PGF2α	355 -> 311	30
13,14-dihydro-15-keto-PGF2α	353 -> 113	28

Data analysis and quantification

Data handling

Isotope correction

Calibration and quantification

Method Validation

Accuracy

Precision

LOD

n.d.

LOQ

n.d.

Recovery

Linearity

n.d.

Reference

• M. Masoodi, A. Nicolaou Rapid Commun. Mass Spectrom. 2006, 20, 3023-3029.

Contact

• School of Pharmacy; University of Bradford; Richmond Road; Bradford BD7 1DP; UK.

• e-mail: a.nicolaou@bradford.ac.uk