Cogent Diol™ column is great for biological samples!

Many of you are analyzing blood samples for applications like clinical trials of pharmaceuticals, forensics testing, and metabolomics research. We have had a number of successful separations of these types of samples with the Diamond Hydride™ in ANP mode. You may be interested in seeing how the latest addition to our TYPE-C Silica™ line of HPLC columns, the Cogent Diol 2.o™, would work in these cases. We selected three test solutes that would be pertinent to blood sample testing: warfarin, hydroxybupropion, and codeine. Let’s take a look at each:

1) Warfarin: An anti-coagulant used for treatment of thrombosis. It does not have an amine group like the other two, which makes its retention lower. Warfarin does exhibit keto–enol tautomerism and its keto form is ionizable. In this form, it has a pKa of 5.0 due to an acidic hydrogen located between two electron-withdrawing carbonyl groups. Its retention was observed to be the lowest of the three.

2) Hydroxybupropion: The active metabolite of bupropion, an antidepressant and smoking cessation drug. Its main structural feature that influences retention in the ANP mode is a secondary amine. It eluted next, with baseline separation from codeine.

3) Codeine: An opiate used in many pharmaceutical formulations for its analgesic or antitussive properties. It has a tertiary amine which makes it amenable to ANP retention. Therefore it eluted the latest of the three compounds. The peak shape was highly symmetrical, which can sometimes be difficult to obtain for amines.

               We spiked the analytes in a real blood sample and separated them by LC-MS. Using extracted ion chromatograms, interferents from the complex sample matrix could be eliminated. What you will have left are three sharp, well-separated peaks corresponding to the analytes.

               The data you can obtain shows how ANP chromatography is not limited to the Diamond Hydride™ column. Any TYPE-C™ column can be used in the ANP mode. Another interesting feature we discovered is that acetone can be used instead of acetonitrile in the mobile phase. Acetone has the advantages to your laboratory of lower cost as well as lower toxicity.

              Click here for more information on this application.

 

Using Near UHPLC to separate an API from a Prodrug

I found that the Cogent Bidentate C8 2.o™ column is great for separations of mometasone furoate from its active form. The official USP assay method calls for an L7 stationary phase, and this column is ideal for such analyses. The method I used here has some useful features.
               First off, the Bidentate C8 2.o™ column has a small average particle size of 2.2µm, leading to high efficiency and rapid analysis; I was able to separate the prodrug, the API, and excipients in the cream matrix in under four minutes. This is a near-UHPLC phase but doesn’t require the specialized instrumentation of smaller particles. Hence you can take advantage of the benefits smaller particle size columns have to offer but without the associated drawbacks of UHPLC.
               Another convenient feature is that the method conditions are very easy to set up. No gradients or complicated buffers are required. The mobile phase consisted of only isocratic 50/50 acetonitrile/DI water. You can even find this available premixed from some solvent suppliers (just be sure it’s HPLC grade). The flow rate was 0.3 mL/min so solvent consumption was low.
               For sample prep, I weighed 5.0 g of the 0.1% mometasone furoate topical cream in an Erlenmeyer flask with a stirbar. After pipetting 50 mL of methanol, I capped the flask and let it stir for an hour. After filtering with a nylon membrane syringe filter, I had a stock solution that I would use for 1:5 dilutions. I prepared two diluted solutions but used different diluents for each. The first used methanol, and in this case the mometasone furoate prodrug should be present without degradation. This prodrug contains an ester bond which can be hydrolyzed to mometasone, the API. Here I used an acid diluent of 90/10 methanol/1N HCl to catalyze the hydrolysis. You need the methanol component for solubility reasons. Then I heated it in a dry bath at 80 °C. Under these conditions, the active form should be present in the solution.
               In the extract using the methanol diluent, I saw a small peak early in the run corresponding to one or more excipients. Aside from that, there was only one other peak in the chromatogram. In a methanol diluent, there will be no conversion to the active form so this peak should be mometasone furoate. However, with the extract using the 90/10 methanol/1N HCl diluent, I saw an extra peak eluting just before the prodrug. There was also a slight decrease in peak height of the prodrug, which indicates some of the mometasone furoate had been converted to the active form.
               As for the chromatography, the elution order made sense. An ester is a relatively hydrophobic functional group and therefore we would expect the prodrug to elute later than the active form in reversed phase conditions. Indeed, this is what is observed in the data.
               Hence, this column can distinguish between the prodrug and the active form with good resolution and a low run time. This may be useful for various analyses, such as studies investigating the rate at which the prodrug is converted.   
              Click here to read about the full study and see the chromatograms.