Retinoid determination - Wang et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPLC-MS 5.1.1 HPLC (Series 1100) 5.1.2 MS G1946A LCMSD (Hewlett-Packard) 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Recovery 6.6 Linearity 7 Internal Standard 8 Reference

Abstract

In order to investigate the effect of neonatal estrogen exposure on RA and retinol levels in rat prostate, Wang et al. developed a positive APCI LC-MS method for the simultaneous measurement of RA and retinol. The method provides excellent sensitivity and linearity of response for RA quantification. Advantageously, only small amounts of tissue samples (8 mg) are required and the sample preparation consisted simply of ethanol extraction.

Analysed Matrices

tissues

Analytes

• all-trans-retinoic acid (atRA)
• 9-cis-retinoic acid (9cRA)
• 13-cis-retinoic acid (13cRA)
• all-trans retinol (atROH)

Sample Preparation

• weighting (8 mg) of tissue samples and homogenization with 1 ml H2O (deionized) on ice
• addition of 3 volumes of EtOH (ice-cold), vortexing, placed on ice (15 min)
• centrifugation (5 min, 2205 g, 4 °C)
• washing the precipitate with one volume EtOH, centrifugate once more
• evaporation of the combined EtOH-supernatants under vacuum
• redissolvation of residue in 0,5 ml EtOH (ice-cold), sonification for 5 min, placed on ice for 10 min
• centrifugation (5 min, 2205 g, 4 °C)
• collection of EtOH layer, evaporation to dryness
• redissolvation of residue in 10 μl IS-solution (1,23 pmol/μl IS in solvent A) and 40 μl solvent A

All extractions were carried out in duplicate under subdued light!

Analytical Method

HPLC-MS

HPLC (Series 1100)

columns: Micra non-porous silica C18 ( 4,6 mm * 33 mm); YMC porous silica C30 (2,1 mm* 100 mm); HP Hypersil ODS porous silica (2,1 mm * 100 mm)

binary pump; automatic solvent degasser; thermostatically controlled column compartment and autosampler

mobile phase A: H2O/MeOH/CH3COOH (50/50/0,5); mobile phase B: MeOH/MTBE/CH3COOH (50/50/0,5)

linear gradient:

8-16 % B (0-16 min)

16-28 % B (16-28 min)

95 % B ( 28-31 min)

8 % B (31-39 min)

injection volume: 50 μl; flow rate: 0,25 ml/min; T (column): 25 °C

MS G1946A LCMSD (Hewlett-Packard)

quadrupole

sources: ESI, APCI (positive ion APCI)

dwell times: 889 ms

scan modus: SIM

nebulizer gas: 30 psi; curtain gas: 6; collision gas: 6; nebulizer current: 3; T (vaporizer): 350 °C; T (N2-drying gas): 300 °C (6 l/min); capillary voltage: 3500 V; corona current: 5,5 μA; fragmentor voltage: 70 V

m/z 269 ([atROH-H2O]+ and [IS-AcOH]+)

Method Validation

Accuracy

n.d.

Precision

6,4 % CV (atRA); 6,7 % CV (atROH)

LOD

n.d.

LOQ

702 fmol for atRA; 2,14 pmol for retinol

Recovery

> 86 % (RA and retinol)

Linearity

1,2- 965 pmol (atRA); 0,936- 750 pmol (atROH)

Internal Standard

all-trans-retinyl acatate

Reference

• Y. Wang, W. Y. Chang, G. S. Prins, R. B. van Breemen Journal of Mass Spectrometry, 2001, 36, 882-888.

Department of Medical Chemistry and Pharmacognosy; University of Illinois at Chicago; College of Pharmacy; 833 S. Wood Street; Chicago; IL 60612; USA.

e-mail: breemen@uic.edu