Help with clean up SPE

[gvportari]

Dear All,
I'm trying to validate a technique to measure the metabolite of vitamin D3, 25(OH)D3, using published methodology by Kand'ar and Zakova (2009) with some differences between brands of column and SPE resin C18. The authors used Discovery DSC-18 SPE tube with a bed weight of 500 mg and a column volume of 3 mL. I'm using an in house made SPE tube with Sepra C18-E (50 um, 65A) - bed weight of 500 mg and vol. of 3 mL.
The procedure is as follows:
The column washed with methanol (2 mL)
The column equilibrated with ethanol–water (1:3 v/v) (2x2 mL)
Diluted supernatant applied to the column (Discard effluent)
The column washed with water (2x2 mL) (Discard effluents)
The column washed with methanol –water (2:3 v/v) (2x2 mL) (Discard effluents)
25(OH)D3 eluted with 100% methanol (2x200 uL)
Actually, I'm working with standards.
I can't see any growing peak in the chromatograms. Around 3 minutes, detection falls bellow the baseline.



well, I don't know what may be happening! I appreciate any help.
Guilherme

[oscarBAL]

Hi Gv.
I can't give you a straight answer maybe someone else have more experience but I can tell you what I would do.
1) First thing is inject your STD direct form the flask to the column without SPE that will help you to identify your Rt with this new column.
2) Collect and inject every effluent after your load your SPE is important to know if you are not losing it in Wash or any intermedium step.
3) Once you r sure your analyte is being retained I could think...

The column washed with methanol (2 mL) (this step allows the cartridge to wet)
The column equilibrated with ethanol–water (1:3 v/v) (2x2 mL) (Ethanol -water displace Metanol from internal pores of the resin; now the "mobile phase” into the resin is more polar and the analyte will prefer C18)
Diluted supernatant applied to the column (Discard effluent) (load with sample; you have to be sure that you are not losing any here)
The column washed with water (2x2 mL) (Discard effluents) (very polar, your analyte should stick to the C18, )
The column washed with methanol –water (2:3 v/v) (2x2 mL) (Discard effluents) (medium polarity compounds should elute here)
25(OH)D3 eluted with 100% methanol (2x200 uL) (if doesn’t elute here your polarity is high and your sample is still retained then you should try with a less polar like a mix of...)

AND I ALMOST FORGET IT: What solvent are you using to dilute samples and Std before SPE??

[che313]

Hi Gv.

I found this protocol from Chromabond for vitamine D3 metabolites using c18 /3ml/500mg may help u:
cond. methanol (2x3ml), water (2x3ml)
after laoding sample wash column with (2x0.5ml) methanol:0.1N HCl (7:3, V/V)
elute ur analytes with dichloromethane (2x0.5 ml)

The column equilibrated with ethanol–water (1:3 v/v) (2x2 mL) (Ethanol -water displace Metanol from internal pores of the resin; now the "mobile phase” into the resin is more polar and the analyte will prefer C18)

Can I have more explanation about this point: the mobile phase is ethanol:water and it will leave column once u applied the sample, how the analyte will prefere c18?
and why the analyte will prefere c18 because it is polar or nonpolar?
I used C18 for extraction morphine & THC-COOH from urine. As I knew morphine is nonpolar and THC-COOH is polar and both of them can be retained by C18. How do you explaine this?