Retinoic acid recovery from plasma

RECOVERY. A dual label recovery experiment, from plasma and PBS, was performed using 3H-all trans and 14C-cis retinoic acid. The normal extraction procedure was followed up to and including the HPLC purification step. No LC/MS analysis was performed. Aliquots were taken and total radioactivity determined after extraction and derivatization. Fractions (0.5 ml) from the HPLC were collected and counted. Counting was performed using a Beckman Model LC3801 liquid scintillation counter. Radioactivity was corrected for spillover and quench. 

Table I. Recovery of 14C-13-c1s and 3H-all trans retinoic acid from PBS or normal human plasma...

There appears to be a differential recovery of cis over trans retinoic acid throughout all the steps of the extraction procedure. However, it is more pronounced after HPLC purification, especially in the plasma samples. In these samples the recovery of all trans retinoic acid was less than that from the PBS samples. 

ACCURACY. Assay accuracy was examined by performing a standard addition experiment. To plasma was added either 0, 5 or 10 ng/ml of 13-cis or all trans retinoic acid, followed by extraction (n=10). A mixing experiment (n=4), in which plasma was fortified with both cis and trans retinoic acid, was also performed. No mixing effect was observed. The internal standard was 20 ng/ml throughout. Table II shows the results of this experiment. The observed values correlate quite well for 13-cis retinoic acid. However, the observed values for all trans retinoic acid were less than those calculated. This is most likely due to the somewhat poorer recovery of the all trans isomer from plasma. 

We have developed a very sensitive assay which can quantify both 13-cis and all trans retinoic acid in the same plasma sample. Only 1 ml of plasma is necessary for analysis, with a limit of quantification of 0.5 ng/ml. The assay is linear for both cis and trans retinoic acid, and there is virtually no interconversion of the two isomers by assay manipulations. However, the assay does slightly underestimate the amount of all trans retinoic acid present due to the differential recovery of this isomer from plasma as opposed to recovery from PBS. This will be corrected in future work by the addition of a stable isotope labelled all trans retinoic acid internal standard for quantification. 

---