Method to determine 4‐oxo‐retinoic acids, retinoic acids and retinol in serum and cell extracts by liquid chromatography/diode‐array detection atmospheric pressure chemical ionisation tandem mass spectrometry

Abstract

Retinoic acid isomers, 4-oxo-retinoic acid isomers and retinol are present in the serum of mammals. In this study a high-performance liquid chromatography (HPLC) separation, sample preparation and tandem mass spectrometry (MS/MS) method was established for quick and easy sample preparation and sensitive determination of retinoids such as all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid and retinol in serum and cell extracts. Serum samples were simply treated with three times the volume of isopropanol, dried under vacuum, taken up in the HPLC solvent and immediately put into the autosampler for an automated single-run HPLC analysis. With this MS/MS method we were able to detect 7 pg and quantify 20 pg of all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid and retinol directly on-column and were able to determine a concentration as low as 0.2 ng/mL in ethanolic standards and in biological samples. This method allows ultra-sensitive detection, excellent selectivity and a very simple sample preparation to determine retinoic acids, 4-oxo-retinoic acids and retinol in serum and cell extracts for the study of endogenous retinoids.

Synergistic induction of lipid catabolism and anti-inflammatory lipids in white fat of dietary obese mice in response to calorie restriction and n-3 fatty acids

“…Lipidomic analysis In total, 24 lipid molecular species were quantified in epididymal fat and liver extracts using HPLC MS-MS analysis [31] (see ESM).…”

A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS

“…A lack of analytical separation power may have confounded individual isomer detection, whereas a lack of good MS/MS sensitivity has prevented selective quantifi cation of individual retinoids. Previous methods have been developed using LC-MS/MS ( 9,(16)(17)(18)(19)(20)(21), GC/MS ( 22 ), and LC/diode array detector-atmospheric pressure chemical ionization/MS/MS ( 23 ). However, none of these methods has incorporated measurement of hydroxylated RA metabolites at endogenous levels in serum.…”

Quantitative Profiling of Endogenous Retinoic Acid in Vivo and in Vitro by Tandem Mass Spectrometry

“…[26][27][28]30,34 Recent reports of LC/MS methods limited to serum/ plasma that separate only 13cRA from atRA would produce inflated values for one or both, because 9,13dcRA (and if present, 9cRA) comigrate with 13cRA and/or atRA. 33,35 Gradient 1 (12 min run time) provides slightly better sensitivity in half the analysis time, relative to gradient 2. Gradient 1 works well with cultured cell extracts, subcellular fractions, or samples with a less complex matrix, such as serum ( Figure 3A).…”

“…This method provides the best sensitivity to date for detection and quantification of RA, with a 20-fold lower LOD than our previous work 30 and a 13-300-fold lower LOD than other published methods. [32][33][34][35][36][37][38] A representative calibration curve for atRA using gradient 1 shows the extensive linear dynamic range ( Figure 5). Note the agreement to the best fit line below 50 fmol.…”

“…Therefore, the suggestion that DAD can be used as a routine codetector with MS/MS to confirm peak identity overlooks the very large sensitivity differences between the two techniques and, thus, eliminates the rationale for progressing from optical to MS detection. 33 If DAD or single-wavelength UV had the sensitivity to quantify endogenous RA in readily accessible sample sizes, there would be limited need for the more expensive and complex MS/MS. …”