Retinoid determination - Kane et al.

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A direct measurement of endogeneous RA is important for the investigation of the relationship between RA and memory formation, cognitive dysfunction, aging and various neurological diseases, such as Alzheimer' disease, Parkinson's disease, Huntington's disease, schizophrenia and depression. In this paper Kane et al. reported a direct, specific and sensitive LC/MS/MS assay to quantify RA in small biological samples, and which distinguishes atRA and 13cRA from other retionoids.

Analysed Matrices

tissue, serum


retinoic acid (RA) all-trans-retinoic acid (atRA) 13-cis-retinoic acid (13cRA) 9-cis-retinoic acid (9cRA)

Sample Preparation

Measurements were made in tissues of 2-4 month-old male wild-type or CRBP-null mice (liver, kidney, testis, whole brain and serum) and 12-month-old female wild-type mice (whole brain and brain sections).

  • tissues: after dissection: placing tissues in ice-cold saline, pat dry, weight, homogenize (25 % homogenate)
  • serum: centrifuging blood (10 min, 7800 g)

liquid-liquid extraction:

  • addition of 15μl IS (50 nM in CH3CN) to 500 μl tissue (200 μl serum)
  • addition of 1ml KOH (0,025 M in EtOH) and vortex-mixing
  • addition of 10 ml hexane, vortex-mixing, centrifugation (1-3 min, 60 rev./min)
  • removal of hexane layer
  • addition of 60 μl HCl (4 M) to the aqueous phase and mixing
  • again addition of 10 ml hexane, vortex-mixing, centrifugation (1-3 min, 60 rev./min)
  • phase seperation and removal of solvent from the hexane phase
  • dissolving the residue in 60-100 μl CH3CN

Due to the adhesion of retinoids to plastic only glass ware should be used!

Analytical Method


HPLC (Agilent 1100)

column: Supelco ABZ+C-16alkylamide (100 mm * 2,1 mm, 3 μm)

Agilent 1100 series: binary pump; vacuum degasser; thermostatically controlled column compartment; diode-array detector

mobile phase A: CH3CN/MeOH/H2O/HCOOH (40/30/30/0,1); mobile phase B: CH3CN/MeOH/H2O/HCOOH (55/30/15/0,1)

linear gradient:

100 % A to 100 % B (0-5 min)

100 % B (5-19 min)

100 % B to 100 % A (19-20 min)

100 % A (20-30 min)

injection volume: 20 μl; flow rate: 0,2 ml/min; T (column): 25 °C; T (autosampler): 10 °C

MS API 3000 (Applied Biosystems)

triple quadrupole

sources: ESI, APCI (APCI positive mode)

scan modus: SRM

nebulizer gas: 4; curtain gas: 6; collision gas: 6; nebulizer current: 3; T: 400 °C

optimized parameters: DP (30 V); EP (4 V); CE (20 V); CXP (4 V)

dwell times: 150 ms

AB software: Analyst (1.3.2)

m/z 329.4→151 (IS); m/z 301.1→205 (RA)

Method Validation


> 97 %


0,5 -2,9 % CV


10 fmol (0,15 ng/ ml)


20 fmol (0,3 ng/ ml)


80 ± 2 % (tissues); quantitative: 101 ± 2 % (serum)


20 fmol-10 pmol (0,3 ng/ml -150 ng/ml)

Internal Standard

4,4-dimethyl RA


  • M. A. Kane, N. Chen, S. Sparks, J. L. Napoli Biochem. J. 2005, 388, 363-369.

Department of Nutritional Sciences and Toxicology; University of California Berkeley; CA 94720-3104; USA.


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