Simultaneous quantitation of water and residual solvents in pharmaceuticals by rapid headspace gas chromatography with thermal conductivity detection (GC-TCD).

In the pharmaceutical industry, an array of analytical testing is performed to demonstrate the safety and efficacy of drug substance and drug products. Among the most critical attributes of release testing are quantitation of residual solvents from the manufacturing process, which pose toxicity concerns, and determination of water content, which can impact potency and shelf life. Residual solvent determination in pharmaceuticals is most commonly performed using headspace capillary gas chromatography (GC) with flame ionization detection (FID), a robust technique that incorporates a mode of detection noteworthy for its sensitivity and wide dynamic range. However, FID responds exclusively to combustible organic species, and does not produce any signal for common gases such as carbon dioxide, ammonia, and notably water. While thermal conductivity detection (TCD) is an alternate, universal mode of detection that has a known response to all GC-appropriate compounds, including water, its use among pharmaceutical companies is uncommon due to the ubiquity of the more sensitive FID and the availability of other techniques for water quantitation such as Karl Fischer titrations (KF). In this work, the use of headspace GC-TCD was successfully demonstrated for the development of a 7.5-minute method for simultaneous quantitation of water, over 25 common residual solvents, and other volatile impurities in small molecule pharmaceutical samples. By carefully controlling sample preparation to minimize the impact of residual water from the diluent, the results for residual solvents and water obtained by this technique were found to be comparable to those of GC-FID and KF, respectively. Headspace GC-TCD improves the throughput of drug testing by greatly reducing the need for KF testing and associated expensive reagents, and helps to conserve samples that are often limited in early stages of development. The technique has desired sensitivity, precision, accuracy and linear dynamic range suitable for pharmaceutical analysis.

Optimization of a two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFS-MS) system to assess "in-vivo" inter-conversion of chiral drug molecules.

Enantioselective analysis is an essential requirement during the pharmaceutical development of chiral drug molecules. In pre-clinical and clinical studies, the Food and Drug Administration (FDA) mandates the assessment of "in vivo" inter-conversion of chiral drugs to determine their physiological effects. In-vivo analysis of the active pharmaceutical ingredient (API) and its potential metabolites could be quite challenging due to their low abundance (ng/mL levels) and matrix interferences. Therefore, highly selective and sensitive analytical techniques are required to separate the API and its metabolites from the matrix components and one another. Additionally, for chiral APIs, further analytical separation is required to resolve the API and its potential metabolites from their corresponding enantiomers. In this work, we demonstrate the optimization of our previously designed two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFC -MS) system to achieve 10 ng/mL detection limit [1]. The first LC dimension, used as a desalting step, could efficiently separate the API from its potential metabolites and matrix components. The API and its metabolites were then trapped/focused on small trapping columns and transferred onto the second SFC dimension for chiral separation. Detection can be achieved by ultra-violet (UV) or MS detection. Different system parameters such as column dimensions, transfer volumes, trapping column stationary phase, system tubing internal diameter (i.d.), and detection techniques, were optimized to enhance the sensitivity of the 2D-LC-SFC-MS system. The limit of detection was determined to be 10 ng/mL. An application is described where a mouse hepatocyte treated sample was analyzed using the optimized 2D-LC-SFC-MS system with successful assessment of the ratio of API to its metabolite (1D-LC), as well as the corresponding enantiomeric excess values (% e.e.) of each (2D-SFC).

High-resolution two-dimensional liquid chromatography analysis of key linker drug intermediate used in antibody drug conjugates.

In this manuscript, the application of high-resolution sampling (HRS) two-dimensional liquid chromatography (2D-LC) in the detailed analysis of key linker drug intermediate is presented. Using HRS, selected regions of the primary column eluent were transferred to a secondary column with fidelity enabling qualitative and quantitative analysis of linker drugs. The primary column purity of linker drug intermediate ranged from 88.9% to 94.5% and the secondary column purity ranged from 99.6% to 99.9%, showing lot-to-lot variability, significant differences between the three lots, and substantiating the synthetic and analytical challenges of ADCs. Over 15 impurities co-eluting with the linker drug intermediate in the primary dimension were resolved in the secondary dimension. The concentrations of most of these impurities were over three orders of magnitude lower than the linker drug. Effective peak focusing and high-speed secondary column analysis resulted in sharp peaks in the secondary dimension, improving the signal-to-noise ratios. The sensitivity of 2D-LC separation was over five fold better than conventional HPLC separation. The limit of quantitation (LOQ) was less than 0.01%. Many peaks originating from primary dimension were resolved into multiple components in the complementary secondary dimension, demonstrating the complexity of these samples. The 2D-LC was highly reproducible, showing good precision between runs with%RSD of peak areas less than 0.1 for the main component. The absolute difference in the peak areas of impurities less than 0.1% were within ±0.01% and for impurities in the range of 0.1%-0.3%, the absolute difference were ±0.02%, which are comparable to 1D-LC. The overall purity of the linker drug intermediate was determined from the product of primary and secondary column purity (HPLC Purity=%peak area of main component in the primary dimension×%peak area of main component in the secondary dimension). Additionally, the 2D-LC separation enables the determination of potential impurities that could impact the downstream process, like ADCs stability, efficacy and patient safety. Peak capacity of this magnitude, sensitivity and reproducibility of 2D-LC for resolving structurally similar impurities co-eluting with the main component has not been demonstrated to date. This application clearly demonstrates the power of 2D-LC in detailed analysis of structurally similar, co-eluting impurities from key linker drug intermediate used in ADCs that is impossible to achieve by conventional 1D-LC.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented.

Assessing stability-indicating methods for coelution by two-dimensional liquid chromatography with mass spectrometric detection.

Chromatographic analysis of trace organic impurities/degradants coeluting in the midst of active pharmaceutical ingredient can be challenging given similarities in their structures and differences in their relative levels/intensities. Conventional detection techniques such as diode array detection and mass spectrometry are often inadequate to detect/identify these residual coeluting impurities and could result in a false negative. Application of two-dimensional chromatography to address/evaluate coelution in conventional chromatography is presented. Areas of interest, usually corresponding to the main component, are transferred to secondary column/s for further separation termed as pseudocomprehensive two-dimensional liquid chromatography. Coelution, if any, in the rest of the chromatogram is monitored using conventional detectors. In this work, the use of similar and complementary phases in both dimensions is presented. The use of the same phase in both dimensions to resolve coeluting impurities (especially in the front and tail of the main component differing by orders of magnitude) is an easier alternative to finding complementary column/s, as hydrophobicity dominates reversed-phase separation. The same phase separation is practical as relative levels of impurities and main component in some transferred fractions are comparable enabling their separation. The results were confirmed using mass spectrometry. This work has significant bearing as a method assessment tool in pharmaceutical and other industries.

Separation orthogonality in temperature-programmed comprehensive two-dimensional gas chromatography.

In a comprehensive two-dimensional gas chromatograph, a thermal modulator serially couples two columns containing dissimilar stationary phases. The secondary column generates a series of high-speed secondary chromatograms from the sample stream formed by the chromatogram eluting from the primary column. This series of secondary chromatograms forms a two-dimensional gas chromatogram with peaks dispersed over a retention plane rather than along a line. The method is comprehensive because the entire primary column chromatogram is transmitted through the secondary column with fidelity. One might expect that a two-dimensional separation in which both dimensions are basically the same technique, gas chromatography, would be inefficient because the two dimensions would behave similarly, generating peaks whose retentions correlate across dimensions. Applying a temperature program to the two columns, however, can tune the separation to eliminate this inefficiency. The temperature program reduces the retentive power of the secondary column as a function of progress of the primary chromatogram such that the retention mechanism of the primary column is eliminated from the second dimension. Retention of a substance in the second dimension is then determined by the difference in its interaction with the two stationary phases. Retention times in the second dimension then fall within a fixed range, and the whole retention plane is accessible. In a properly tuned comprehensive two-dimensional chromatogram, retention times in the two dimensions are independent of each other, and the two-dimensional chromatogram is orthogonal. Orthogonality is important for two reasons. First, an orthogonal separation efficiently uses the separation space and so has either greater speed or peak capacity than nonorthogonal separations. Second, retention in the two dimensions of an orthogonal chromatogram is determined by two different and independent mechanisms and so provides two independent measures of molecular properties.

Evaluation of capillary supercritical fluid chromatography with mass spectrometric detection for the analysis of a drug (mebeverine) in a dog plasma matrix.

Supercritical fluid chromatography with mass spectrometric detection was evaluated as a technique for the analysis of drugs in biological fluids. Dog plasma was spiked with a model drug, mebeverine, and with a deuterium-labeled analog of mebeverine. The spiked plasma was prepared for analysis by solid-phase extraction on octadecylsilane cartridges. Mebeverine levels in the spiked dog plasma samples were determined by interpolation from a standard curve. Accuracy and precision of the analysis were determined within and between days. In general, accuracy was found to be 100 +/- 15% for plasma samples spiked with 6 to 60 ng mebeverine/ml. The relative standard deviation for replicate sample analysis over this concentration range was between 5 and 12.5%.