Chromatography Forum

Greetings,
I am trying to develop a method to measure antioxidant leachables. But my drug product (a kind of polar salt) cannot elute out with draft antioxidants HPLC assay.
Could you please help me to have my salt elute out? The following is the comparison of two methods.
Thanks a lot!

Do phenyl columns retain polar chemicals more than ODS columns?

*Drug product assay
Column: Inersil Phenyl 250 mm*4.6 mm*5 um
Mobile phase: 95/5(v/v) pH 3.2 KH2PO4 buffer/ACN
Temperature: Ambient
Injection: 25 uL
Flow: 1.0 mL/min
Detection: 225 nm
Gradient: Isocratic
Run time: 12 min
Elution time (drug product working standard): 7.6 min

*Antioxidants assay
Column: ODS Hypersil 150 mm*4.6 mm*5 mm
Mobile phase: H2O/ACN,
Temperature: 60 °C
Injection: 20 uL
Flow: 1.0 mL/min
Detection: 200 nm
Gradient:
Time %H2O %ACN
0 50 50
11 0 100
25 0 100
30 50 50
35 50 50
Run time: 35 min
Elution time (drug product working standard): Not seen (can not elute out)

If I read your post correctly, the drug product assay is isocratic with 5% ACN. The antioxidants assay is a gradient starting at 50% ACN. Phenyl and C18 columns are not that different; actually, I suspect that your polar drug is unretained and eluting with junk at t0.

Try starting the antioxidants gradient at 5% ACN (lengthen the time appropriately to keep the same slope) and see what happens.

Another issue may be the lack of buffering in the antioxidants method. Is your drug product ionizable? If so, you should probably have some way of controlling the mobile phase pH.

Thank you Tom.

"Try starting the antioxidants gradient at 5% ACN (lengthen the time appropriately to keep the same slope) and see what happens. "

I am going to try this.

"Another issue may be the lack of buffering in the antioxidants method. Is your drug product ionizable? If so, you should probably have some way of controlling the mobile phase pH."
Yes, my drug product is ionizable. If I do not control pH, would the drug product retain or would it elute at t0?
If I control pH, whould my antioxidants, which are not ionizable, be affected?

another question.
If my drug product elutes at t0 with junk, should I see some peaks around t0 with area similar or bigger than my typical working standard peak?

Yes, my drug product is ionizable. If I do not control pH, would the drug product retain or would it elute at t0?
If I control pH, whould my antioxidants, which are not ionizable, be affected?

Could go either way. Most likely decreased retention, but with an ionized base, you might see an increase because of cation exchange with ionized residual silanols. In either case, controlling pH should minimize peak shape and retention reproducibility issues.

If my drug product elutes at t0 with junk, should I see some peaks around t0 with area similar or bigger than my typical working standard peak?

If you compare with / without the drug product, you should see a bigger t0 peak with. Another issue is the different wavelength of the two methods. For most compounds, the "end absorbance" down near 200 nm will be greater than an absorbance "maximum" at 225 nm, but "most" is not "all". If you have a photodiode array detector ("PDA" or "DAD"), you might look at both wavelengths just in case.

I did more study and found out my salt drug molucules are retained. I added salt drug assay program after my antioxidants assay gradient and my drug product elute out.