Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids
A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C/sub 18/ reversed-phased analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that HPLC uv only conditions usually would not detect in the circulation and liver of rats under physiological conditions.
- Research Organization:
- Michigan State Univ., East Lansing
- OSTI ID:
- 5698244
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 153:1; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
400102* -- Chemical & Spectral Procedures
550201 -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOLOGICAL MATERIALS
BODY
CARBOXYLIC ACID ESTERS
CHEMICAL ANALYSIS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
ESTERS
EXTRACTION
GLANDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
MATERIALS
ORGANIC COMPOUNDS
ORGANS
QUANTITATIVE CHEMICAL ANALYSIS
RATS
RETINOIC ACID
RODENTS
SEPARATION PROCESSES
SOLVENT EXTRACTION
SPECTRA
STANDARDIZATION
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRAVIOLET SPECTRA
VERTEBRATES
VITAMIN A
VITAMINS