Chromatography Forum

Recently, I am using SPE C18 to pretreat human serum before quantify 2,5 OH vitamin D by RP-HPLC. But the recovery(< 20%) seems not so good. According to literature search, the linear range is around 10ng/ml to 80ng/ml. I spiked 10ng standard to 1ml serum, then precipiated by organic solvent, passing SPE and then dried by vacuum centrifuge and reconstitued back in 100ul MeOH. No peak was observed or the peak is not easily be integrated due to low signal to noise ratio (< 0.01mAU).

Can anyone enlighten me how to improve the recovery from SPE or enhance the signal to noise ratio of the lowest standard point?


Precondition of SPE : 3ml MeOH followed by 3ml water
Sample Load: 1ml-2ml serum or stripped serum with 10ng/ml to 80ng/ml std
Wash: 3ml water
MeOH: 3ml MeOH

All flow rate is due to gravity because I dont have manifold vacuum device and the centrifuge difficultly control the flow rate as too fast (< 1ml/ 30sec -1 min)

There could be two problems. Can't tell which because your description is still a bit too vague.
1. If you do not remove the organic solvent from the precipitated serum, the elution strength of your sample solvent is too strong and the analyte won't stick to the C18. We have done elution protocols with serum on Oasis HLB by first diluting the serum at least 1:1 with acidified water, instead of trying to precipitate the serum. The proteins will wash through the Oasis HLB. The vitamin will stick due to hydrophobic interaction, and should elute with methanol.
2. The elution protocol is not working, and you may need more methanol to elute the analyte. I would run a protocol without serum, sample just dissolved in water or acidified water (see above). You need to check all fractions for the analyte. You should be easily able to quantify, and you can add up the values of the recovered analyte. If something is still missing, methanol is too weak a solvent and you either increase the elution strength by adding some acetonitrile or try to elute with more methanol (You also did not tell us what the size of your SPE is).

I don't see anything wrong in conditioning your spe the way you did.

With methanol, then water, Load the sample, (You might want to dilute the sample with Aq. if it comes from a Protein preciptiation.
Load the sample, wash the sample then elute.

We used to use the centrifuge for this.
We used to condition and load at about 400 to 600 rpm.
Wash steps at 400 to 600 RPM.
Final spin at 3000 rpm for 10 minutes (remove all Aq.)

Elute with High organic (espescially for Vit D.) You might want to try ACN, I have heard it may give less matrix effect (phospholipids)

Dry down your sample to concentrat it and reconstitute in a small volume of something it is soluble it. You might want a little water in it so you don't get distorted peak shape if you are running a organic/Ag mobile phase.

Goog luck.

Alp

The size of SPE is 500mg/3ml, Sepak C18. Is the size too big so that Vit D analyte is difficult to be eluted with 3ml 100% MeOH?

Can I use a smaller size of SPE let say 50mg or 100mg to attain my purpose to concentrate the vit D analyte from serum?

It is difficult to judge without actual experience if the elution volume is insufficient. You need to do some experiments.
Did you read the suggestions by Alp and me? Did you do any of this?