Characterization of positional isomers of drug intermediates by off-line RPLC x SFC hyphenated to high resolution MS.

Many steps are needed in the synthesis of a new active pharmaceutical ingredient (API). In a practical case proposed by a French pharmaceutical company, an intermediate synthesis step, needed to protect 8 hydroxyl groups before oxidation, could produce a mixture of neutral compounds containing up to 652 structures being positional isomers of 18 molecular formulas. Some mixtures allowed obtaining the desired API, others did not. An efficient analytical method was needed to characterize these neutral positional isomers and identify the mixtures to reject. Two samples were provided by the pharmaceutical company: Sample A was conform, Sample B was not. 8 RPLC columns were used with different gradients to screen Sample A. Next, the best RPLC separation was used as the second dimension fast analysis in a comprehensive 2D-RPLC systems. Two columns were used as first dimension: a fluorinated one and a zirconium based one. An order of magnitude was gained in peak capacity, but a better sample characterization was still needed. An off-line RPLC x SFC x Q-TOF/MS analysis was performed collecting 96 RPLC fractions and analyzing them by SFC with Q-TOF/MS detection. A home-made software associated the 96 SFC MS chromatograms to produce either base peak (BPC) or extract ion (EIC) contour plots that allowed for a satisfying characterization of the samples. Subtracting the EIC of expected m/z compounds from the Sample B BPC contour plot produced a unique new contour plot clearly pointing out unexpected compounds explaining the failure of the synthesis and possibly allowing improving the synthesis process.

Quantitative aspects in on-line comprehensive two-dimensional liquid chromatography for pharmaceutical applications.

On-line comprehensive two-dimensional liquid chromatography (on-line LCxLC) is expected to offer impressive effective peak capacity. That makes it an attractive technique for the analysis of complex samples such as pharmaceuticals, for which impurities are often unknown both in number and in structure, and can be either totally different or similar to the active substance. A study on comprehensive on-line RPLCxRPLC (reversed phase liquid chromatography in both dimensions) with respect to quantitative aspect at low concentration levels was carried out with the objective of finding conditions able to meet regulatory requirements for the control of pharmaceutical impurities. Preliminary studies showed that the best approach for quantitative purpose was to relate the impurity concentration to the sum of the second dimension peak areas. RPLCxRPLC conditions were optimized according to a Pareto-optimality approach with a focus on sensitivity, thereby favouring low dilution factor. The resulting RPLCxRPLC separation led to a detection limit below 0.05% for impurities (compared with API). Furthermore, the properties of this analytical method was found to be satisfying in terms of linearity (R2 > 0.999), repeatability (RSD < 1% for second dimension retention times) and precision (RSD < 1.5% for the sum of second dimension peak areas). Finally, this on-line RPLCxRPLC approach was applied to the analysis of a batch of drug substance in the early stages of drug development. The method was able to detect impurities at concentrations as low as 0.04% (signal-to-noise ratio of 3). All these results clearly demonstrate the power of on-line RPLCxRPLC for the quantitative analysis of pharmaceutical samples.

On-line coupling of achiral Reversed Phase Liquid Chromatography and chiral Supercritical Fluid Chromatography for the analysis of pharmaceutical compounds.

On-line selective comprehensive two-dimensional chromatography combining Reversed Phase Liquid Chromatography and Supercritical Fluid Chromatography (sRPLCxSFC) was investigated for the analysis of chiral pharmaceutical compounds. Preliminary studies were carried out with the aim of overcoming instrumental constraints which are related to such 2D-coupling. The impact of both injection solvent and injection volume on the chiral SFC second separation was assessed with a view to limiting injection effects due to mobile phase compatibility issues between both dimensions. The resulting on-line sRPLCxSFC system was applied to the achiral x chiral analysis of a pharmaceutical sample. Using an Acquity BEH C18 column in the first dimension and a Chiralpak IC column in the second one, both chemical (achiral) and enantiomeric (chiral) purities could be evaluated in less than 50 min within a single run. Under such conditions, a detection limit of about 0.5% for R-enantiomer could be obtained with UV detection. The results obtained in sRPLCxSFC were compared to those obtained in conventional chiral 1D-SFC. Baseline resolution was obtained in both cases and the linearity in the detector response was on the same order of magnitude (R²â€¯> 0.99). Finally, despite current instrumental limitations (no commercially available system for sLCxSFC, large dwell volume and large extra-column volume in SFC), the on-line coupling of RPLC and SFC appears to be attractive and promising for rapid achiral/chiral analysis of complex pharmaceutical samples.

Comprehensive two dimensional liquid chromatography as analytical strategy for pharmaceutical analysis.

Comprehensive on-line two-dimensional liquid chromatography (LCxLC) is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceuticals. A comparative study of different sets of chromatographic conditions including stationary phase, pH additive and organic modifier was carried out with two real pharmaceutical samples in order to find out the best analytical conditions for implementation of one or several generic on-line LCxLC separations. Our choice was based on the evaluation of both degree of orthogonality and practical sample peak capacity under linear gradient conditions. The potential of 190 combinations of chromatographic systems was compared. A set of 3 RPLCxRPLC configurations was found to be very attractive for both samples and in good agreement with the findings of a previous study carried out with 17 model compounds, thereby supporting the idea of using generic LCxLC conditions in the pharmaceutical area. The three selected 2D-systems were implemented for the on-line RPLCxRPLC-UV/MS analysis of two pharmaceutical samples. It was shown, for each sample, that these 2D-systems were able to generate an effective peak capacity close to 1000 in less than 50min. For each sample, baseline separation was obtained for every known compound and furthermore a large number of unknown impurities could also be separated and identified. Finally, in the proposed conditions, the total number of compounds detected was significantly improved from one RPLC separation to one RPLCxRPLC separation. Only a small additional gain was observed by performing a second RPLCxRPLC separation or even a third one.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.