Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection

• Published in: Journal of Chromatography A Vol. 828; no. 1; pp. 451 - 460
• Main Authors: Sakhi, A.K, Gundersen, T.E, Ulven, S.M, Blomhoff, R, Lundanes, E
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 18.12.1998; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Electrochemistry; Embryo, Mammalian - chemistry; Fundamental and applied biological sciences. Psychology; Mice; Other biological molecules; Reference Standards; Reproducibility of Results; Retinoic acids; Retinoids; Retinoids - analysis; Sensitivity and Specificity; Terpenes, steroids. Hormones; Retinoic acids; Column switching; Retinoids; Solid phase extraction; Embryo; Chemical analysis; On line; Rodentia; Retinoid; HPLC chromatography; Endogenous substance; Isocratic condition; Electrochemical detector; Chemical enrichment; Vertebrata; Mammalia; Analysis method; Sample preparation; Mouse; Animal; Retinoic acid; Quantitative analysis; Coulometry
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(98)00676-1

An isocratic high-performance liquid chromatographic method for the determination of 9- cis-retinoic acid, 13- cis-retinoic acid, all- trans-retinoic acid and all- trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13- cis-acitretin was used as internal standard. About 370 μl of albumin solution was injected on a 10×2.1-mm I.D. pre-column packed with Bondapak C 18, 37–53-μm particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile–methanol–2% ammonium acetate–glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250×4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86–103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all- trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% ( n=6) for the four retinoids. Inter-assay precision was in the range 3–4% ( n=10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-μl solution containing 100 μl of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all- trans-retinol and all- trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13- cis-retinoic acid and 9- cis-retinoic acid was below the detection limit.