retinoic acid assay (350 nm)

[Mathieu]

I am a Msc student and I work on the all-trans-retinoic acid (RA) metabolism in a RA sensitive cell line culture. We extract RA from cell culture medium and from cell homogenates with mixtures of hexane and ethyl acetate ... then we analyse the extracts with high performance liquid chromatography coupled to a UV/VIS detector setted to 350 nm. The chromatography is done with a isocratic solvent containing acetonitrile:methanol:THF:TFA:water (16% water). The column is a reverse-phase Inertil C18 250 x 4.6mm ; 5μm particles. We are able to detect RA and its metabolites however, with each extraction, an unknown giant peak is obtained to a retention time just before RA : RA has a retention time of about 22 min and the unknown peak has a retention time of about 21 min. We recover this peak in medium and cells, and we recover it also in medium and cells that do not contain RA. In fact, when we inject a cell medium extract that do not contain RA, we only recover retinol (it comes from the serum added to the culture medium) with a retention time of about 19,5 min, and the unknown peak to 21 min. Does anybody know a molecule (lipid, protein, vitamin...) that absorbs strongly to 350 nm and that is present in cell culture medium (not the retinol)? We bought a new HPLC column and now, we have coelution problems between this unkown peak and RA!!!

[Jumpshooter]

Mat, you have some molecule in your prep that has a very similar adsorption/elution chromatography profile as the "all-trans RA" analyte. I hypothesize that the 'unknown peak' is some isomer of RA--possibly the usual suspects:
1) cis-retinoic acid
2) dehydro retinol
3) some side product of beta carotene
4) some partially degraded molecular form of all-trans RA.

Try adding an 'end-capping' reagent to your mobile phase in order to reduce the k value of the presumptive contaminant. In that way the "contaminant" should elute 3 to 5 minutes before your RA. Let me know if this works!

[Jumpshooter]

Oh, I was curious why you are using ABS = 350 nm ? All-trans RA has an ABS maxima ~ 325 nm. This may also be part of your experience problem in that the contaminant is having an ABS maxima of 350 nm while the all-trans RA has ABS maxima = 325 nm. Try programming your UV Detector to 325 nm +/-2 nm. Let us know if this works! :

[Mathieu]

Hi!

About the used wavelength … we selected 350 nm because all-trans-RA has a λmax = 350 nm (Barua and Furr). All-trans retinol has a maximum absorbance to 325 nm, but it is not an interesting molecule for our study (we are able to detect it to 350 nm). I’ve also hypothesize that the unknown peak was an isomer of all-trans-RA or another related molecule. However we don’t have all the standards in our laboratory. We identified 13-cis RA, dehydroretinol, retinaldehyde and anhydroretinol but their retention times dot not correspond with this unknown peak. There are a lot of isomers of RA and derived molecules (9cis-RA, 9,13-di-cisRA, 18-OH-RA, etc…) but the retention times screening of these molecules would be very laborious. The contaminant (unkown peak about 21 min) seems to be present in the MEM medium (not in the serum) : alpha MEM or DMEM. However, maybe this contaminant is formed during the extraction procedure with hexane? What is an end-capping reagent? Is it the same thing of a derivatization procedure?

Mathieu

[Mark Tracy]

Try collecting the fraction with the offending peak and getting a mass spec of it.

[Jumpshooter]

1) use of an 'end capping reagent": this is an additive that you would add to your mobile phase sol'n to prevent binding of the isomer onto the active site on the column. The isomer is prevented from binding to the column and would therefore be eluted off early in the run. In my own Vitamin A set up I add some ethyl palmitate to the mobile phase--this occupies some of the column binding site and keeps the retinyl palmitate (Vit A) from sticking to the column and accumulating with each subsequent injection of sample (std or unk).

2) as Mark said---you can elute it and mass spec it to make more determinations of it.

3)*But the quizzical aspect of your problem here is the fact that: you said that this peak shows up in the BLANK Serum (a.k.a. "negative control'').
Please answer these 2 questions:

A) Does the offending peak show up in your clean reference std (RA)? Given this occurence, it seems that the offending peak could be some additive that the manufacturer put in the solution--perhaps some stabilizer compound to protect RA from photodegradation.

B) Does the peak show up when you inject only blank hexane?

c) does the peak show up when you inject your reagent blank?