Comparison of supercritical fluid chromatography and reverse phase liquid chromatography for the impurity profiling of the antiretroviral drugs lamivudine/BMS-986001/efavirenz in a combination tablet.

Dual and triple combinations of antiretroviral drugs are a cornerstone of human immunodeficiency virus type 1 (HIV-1) treatment. Supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) methods have been developed for the impurity profiling of a prototype combination tablet containing three such drugs: lamivudine, BMS-986001 and efavirenz. Separation by SFC was achieved using a Princeton 2-ethyl pyridine stationary phase and a mobile phase B consisting of methanol with 10 mM ammonium acetate and 0.1% isopropyl amine. This combination of mobile phase additives was required for both the separation of minor components and to minimize peak tailing of the active pharmaceutical ingredients (APIs). Separation by RPLC was achieved using a Discovery HSF5 stationary phase and a mobile phase consisting of 10 mM ammonium acetate, pH 5.5 and methanol. Mobile phase gradient elution was employed in each case to elute components with a wide range of polarities. Both these methods were found to have advantages and disadvantages. Out of the three APIs and 13 possible impurity/degradation products selected, all were resolved by RPLC. By SFC, 15 peaks were resolved with one co-eluting pair and a high degree of orthogonality was achieved relative to RPLC. A more even distribution of peaks across the separation space, a non-sloping baseline and fewer system peaks were significant advantages associated with the SFC method. Particular attention had to be paid to optimizing the reverse phase diluent strength/initial mobile phase composition to avoid distortion of the peak shapes for early eluting components. This was not an issue with SFC, as the diluent of choice (methanol) was also the solvent of choice (in combination with ≤20% water) for the dissolution of the triple combination tablet. As with RPLC, SFC was found to exhibit the required sensitivity for successful quantitation of potential impurities/degradation products at the 0.05-0.1 area% level.

Evaluation of mobile phase gradient supercritical fluid chromatography for impurity profiling of pharmaceutical compounds.

The use of gradient supercritical fluid chromatography (SFC) for the impurity profiling of pharmaceutical products is not widely practiced. Historically, the limited advancement in SFC instrumentation and the lag in column development have resulted in marginal sensitivity, selectivity and reproducibility when compared with high performance liquid chromatography (HPLC). Using a recently developed commercial module, which allows an ordinary HPLC to be converted to a SFC system, a significant improvement in sensitivity (up to ~12-fold) has been obtained over previous studies. This has allowed for the first time a "real-world" head-to-head comparison of SFC to HPLC for impurity profiling of pharmaceutical products in a regulated environment. Retention time reproducibility and low level impurity detection were found to be comparable to reversed phase liquid chromatography (RPLC), that is, single digit %relative standard deviations (RSDs) were obtained for impurities present at less than 0.1 area%. Furthermore, these results were obtained with drug loading levels (≤2 mg/mL) that are not only comparable to those employed with HPLC, but are dictated by the limited solubility of many drug candidates. The elution of impurities was generally found to be orthogonal to that obtained with RPLC, but it was still challenging to find SFC conditions that would separate all of the components in the mixtures studied. In terms of enhancing selectivity, small amounts of mobile phase additives (0.1-1%) and temperature optimization were found to have a greater impact in SFC method development versus RPLC. However, unlike gradient RPLC, the relative changes in baseline noise and slope were found to be a complex function of the experimental conditions, with the largest differences in noise levels being generally observed for the widest and steepest gradients. It is likely that this gradient related noise is more apparent now because other sources of noise in SFC have been reduced significantly.

Modifying conventional high-performance liquid chromatography systems to achieve fast separations with Fused-Core columns: a case study.

The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 µm HALO C(18) Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3mm ID × 100 mm Fused-Core column approximately 70% of the maximum plate count (∼84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ∼4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ∼3-3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).

A stepwise strategy employing automated screening and DryLab modeling for the development of robust methods for challenging high performance liquid chromatography separations: a case study.

A stepwise method development strategy has been employed to develop a robust HPLC method to resolve several closely eluting structurally related impurities in an active pharmaceutical ingredient (API). This strategy consisted of automated column screening, optimization of the most critical chromatographic parameters, DryLab(®) modeling, and experimental verification of optimized separation conditions. DryLab(®) was used to predict an optimized gradient profile and separation temperature and these predictions were verified experimentally. A discussion of the accuracy of these predictions is presented. The robustness of the method was verified and the ability of DryLab(®) to predict, with reasonable accuracy, the outcome of such robustness studies was also examined. Once the robustness was established by the DryLab(®) predictions the remainder of the subsequent verification by experiment becomes a simple reiterative exercise. This study also demonstrates that factors such as column chemistry and critical chromatographic parameters can have a profound and oftentimes interrelated effect on the chromatographic separation of isomers, bromo analogs and other structurally very similar impurities. Therefore, it is critical to adopt a rational strategy, as demonstrated here, to evaluate the interplay of these factors, thereby greatly enhancing method development efficiency.

Direct chiral resolution of 15-deoxyspergualin using a cellobiohydrolase liquid chromatographic column.

An LC method for the direct determination of the enantiomers of 15-deoxyspergualin was developed, optimized and validated. A commercially available LC column containing a cellulase enzyme (cellobiohydrolase I) stationary phase was used. The effect of various parameters on the separation and validation data are discussed.

Determination of sun-screen agents in cosmetic products by micellar liquid chromatography.

A micellar liquid chromatographic method was developed for the quantitative determination of sun-screen agents in cosmetic products. The qualitative analysis of parabens is also feasible. Excellent linearity was obtained (r = 0.999) and recoveries were generally greater than 98%. A variety of commercial formulations were analyzed.