Chromatography Forum

I have been working on the determination of cholecalciferol (Vitamin D3) in an ointment (Lipidic ointment based on lanoline). For the extraction of the vitamin, the following saponification process has been tested:

1. Weigh 4 grams of the ointment in a erlenmeyer flask with screwed cap.
2. Add 20 mL of ethanol and 16 mL of KOH 5N
3. Proceed to cover the flask with the cap, and put into a thermoregulated bath at 50°C and 70 rpm for 60 minutes.
4. When the saponification process have been concluded, add 20 mL of water and 20 mL of n-Hexane (HPLC Quality).
5. Wait until decantation and take a portion of the top layer of the 2 phases that just formed.
6. Filter the solution through a 0,22 filter and inject in the hplc

Column: Kromasil Si- 250 mm x 4,6 mm x 5um
Column temperature: 40°C
Injection Volume: 50 uL
Flow: 1,0 mL/min
Mobil phase: Hexane/Isopropanol [99:1]
The problem that we have here, is that we cannot obtain a clean chromatography, and the peak of vitamin D3 is impure, the reason of that is the matrix effect of lanoline (which presents many peaks). So, I want to know if any of you have been working with something similar.

Can you post a view of your chromatogram, with the major VD3 peak marked ?

Also, why do you perform an alkaline hydrolysis? Surely the VD3 is an additive, not a constituent, so does not need to be released chemically.

Regards,
JMB

What is your detector? What is your expected concentration? Why not just dilute and shoot (why saponify)?

In my opinion/experience, a saponification for this analysis is meaningful in a lipid matrix.

In step 2, did you check (visually) if the sample disperses in ethanolic KOH? We sometimes have issues with samples high in fat clumping together in this step. If this happens, we use an ethanolic KOH solution with lower water content.

In step 3-4, do you shake the samples after adding hexane?

If the concentration is high enough, I would repeat the procedure with less sample.

I can also recommend to perform a back-extraction after extracting your analyte to hexane: transfer (part of) the hexane phase to a new recipient, add fresh water/ethanol 50/50, shake for +-1min and settle. Continue with filtration and injection of hexane phase.

It is possible for vitamin D3 to perform RPLC if you have issues related to NPLC. Dry the hexane extract under nitrogen and dissolve in methanol. Elute on a conventional RPLC column with a gradient/isocratic program starting with high % organic.

Thank you for your information, regarding to your first comment, yes, we verified the dispersion of the sample, and that's fine, but we can try another type of alcohol to improve this.

Regarding to your second comment, I forgot to report that we proceeded to shake the sample with hexane for 10 minutes at 200 rpm in a digital orbital shaker. The concentration of vitamin D3 in my product is 5000 IU / 100 g, and I weight 4 g (which represents a lot of volume), therefore, in my sample I only have 200 IU of vitamin D3.

The point of retro-extraction is interesting, i will try that. On the other hand, i already tried a RPLC method, the problem was the recovery of the sample and the precipitation of the sample into the vial, we try a lot of diluents (Methanol, Isopropanol, Acetonitrile, THF, etc.), and with every one of them we had the same problem. The risk of clogging the column or the HPLC was too high

I have to fulfill a recovery from the vitamin d3 between 75-125%. An the only way i obtain something near to that was with NPLC. I wish to try a solvent that retain the lanoline but not the vitamin D3, but I still can't find it.

In my opinion/experience, a saponification for this analysis is meaningful in a lipid matrix.

In step 2, did you check (visually) if the sample disperses in ethanolic KOH? We sometimes have issues with samples high in fat clumping together in this step. If this happens, we use an ethanolic KOH solution with lower water content.

In step 3-4, do you shake the samples after adding hexane?

If the concentration is high enough, I would repeat the procedure with less sample.

I can also recommend to perform a back-extraction after extracting your analyte to hexane: transfer (part of) the hexane phase to a new recipient, add fresh water/ethanol 50/50, shake for +-1min and settle. Continue with filtration and injection of hexane phase.

It is possible for vitamin D3 to perform RPLC if you have issues related to NPLC. Dry the hexane extract under nitrogen and dissolve in methanol. Elute on a conventional RPLC column with a gradient/isocratic program starting with high % organic.

The concentration of vitamin D3 in my product is 5000 IU / 100 g, and I weight 4 g (which represents a lot of volume), therefore, in my sample I only have 200 IU of vitamin D3.

If i calculate correctly, there's supposed to be 1.25ppm D3 in your sample? Is your detection UV/DAD? It's low, but not impossible by HPLC-UV/DAD.

On the other hand, i already tried a RPLC method, the problem was the recovery of the sample and the precipitation of the sample into the vial, we try a lot of diluents (Methanol, Isopropanol, Acetonitrile, THF, etc.), and with every one of them we had the same problem. The risk of clogging the column or the HPLC was too high

If you dry the hexane and redissolve with for instance MeOH, it's likely that not everything redissolves nicely. As long as your analyte dissolves (vortex/shake well) it's OK, you still have to filter after this solvent exchange step. You're right, injecting undissolved stuff is not done in liquid chromatography.

For some specific fat matrices, using a syringe filter as the final step in this type of analysis, in my experience the speed matters at which the liquid is pushed through the filter. Simply said, the slower the better.