Chasing Peaks
Don’t be alarmed by the title– I haven’t taken up hiking or started climbing actual mountains. I’m talking about a different kind of peaks – those on the chromatogram… Now that’s more Smilangi-fitting.
Last week marked the beginning of Phase 2 of my project: Solubility studies, where I narrowed down several potential stabilizers via qualitative characteristics. This week, I was able to quantify their potential through High Performance Liquid Chromatography (HPLC).
We compared several variations of the base solution, each containing different excipients. Using the HPLC, I analyzed the percent of Temozolomide remaining in each solution at the end of the week (this potency is technically referred to as assay) and identified any impurities or degradants that could compromise the solution’s effectiveness. The goal of this comparative study was to isolate the formulation variation that remained the most stable over time. Quantitatively, this would be the solution with the highest Temozolomide assay at the end of the week and fewest detected impurities. Generally, acceptable assay values range from 90 – 110%, with the extra 10% accounting for human and technological error – but a 100% yield is optimal.
The HPLC outputs a graph with peaks that represent different compounds at their respective retention times. However, it does not automatically label them, so it is up to us to determine which peak corresponds to each compound in the solution. Since our project relies on calculating the assay of TMZ retained, correctly identifying the TMZ peak is crucial.
In order to distinguish the API from the excipients of the base solution, we conducted the following control experiment:
- We ran pure Temozolomide through the HPLC and recorded its retention time (3.5 minutes). This established that the peak around 3.5 minutes would correspond to TMZ in future trials.
- We ran a blank base formulation with all the excipients but without Temozolomide. Since no peak appeared at 3.5 minutes, we were able to affirm that the excipients wouldn’t interfere with the TMZ output readings. Had there been a peak at 3.5 minutes without the presence of Temozolomide, a new method would have been required.
- We ran the full base solution with all the excipients and TMZ, adding extra TMZ. This resulted in a graph with a taller peak at 3.5 minutes, confirming that TMZ’s retention time is indeed 3.5 minutes.
To match the remaining peaks with their corresponding compounds in the base solution, we used a Diode Array Detector (DAD). The DAD is a UV spectrum reader attached to the HPLC, that provides a unique UV fingerprint for each peak. I compared these fingerprint values to published absorbance spectra values for Miglyol 812 N, Propylene Glycol, Labrasol, and Transcutol HP to identify each excipeint’s peak.
However, Propylene Glycol and Miglyol 812 N have such low absorbance that they are quantified as UV silent. They could not be detected at the standard 254 nm (as expected) and only became visible when scanning across a border range of 190 – 400nm.
After labelling each peak with the known ingredients, we tested each variation of the base solution individually. All solutions contained the same API and excipients, with the only difference being the stabilizer added. Since we already identified which peaks corresponded to the API and excipients, we used the process of elimination to identify new peaks as impurities.
The assay of TMZ was calculated as follows:
Assay = (Area under TMZ peak in each trial/ Area under the TMZ peak for pure Temozolomide) * 100
In this way we collected impurity and assay value for several solutions before finalizing Alpha-tocopherol as the primary stabilizer!