Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed

Retention modeling of therapeutic peptides in sub-/supercritical fluid chromatography

Chromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed-phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies. This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino-derivatized aromatic (Torus 2-PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated

Controlling the elution order of insulin and its analogs in sub-/supercritical fluid chromatography using methanesulfonic acid and 18-crown-6 as mobile phase additives

The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed-phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18-crown-6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH)

Prevalence of nitrosamine contaminants in drug samples: Has the crisis been overcome?

Various drug samples (N = 249; drug substances, tablets, capsules, solutions, crèmes, and more) from the European pharmaceutical market were collected since 2019 and analyzed for 16 nitrosamines (NAs). In 2.0% of the cases, NAs were detected. These findings included four active pharmaceutical ingredients already known for potential NA contamination: losartan (N-nitrosodimethylamine [NDMA] and N-nitrosodiethylamine, simultaneously), valsartan (NDMA), metformin (NDMA) and ranitidine (NDMA). The fifth new finding, which has not been reported yet, discovered contamination of a molsidomine tablet sample with N-nitrosomorpholine (NMor). The tablet contained 144% of the toxicological allowable intake for NMor. NMor was included in our screening from the beginning and is currently the focus of regulatory authorities, but was added to the guidelines only last year. Thus, it may not have been the focus of regulatory investigations for too long. Our results indicate that the majority of drug products in the market are nonhazardous in terms of patient safety and drug purity. Unfortunately, the list of individual affected products keeps growing constantly and new NA cases, such as molsidomine or nitrosated drug substances (nitrosamine drug substance-related impurities [NDSRI]), continue to emerge. We therefore expect nitrosamine screenings to remain a high priority

Risk assessment for nitrosated pharmaceuticals: A future perspective in drug development

Since June 2018, thousands of drug products from around the world had to be recalled due to the unexpected presence of nitrosamines (NAs). Starting with the pharmaceutical group of sartans, antidiabetic drugs, antihistamines, and antibiotics also became the subject of investigation. The occurrence of NAs has shown that pharmaceutical companies and regulatory agencies did not focus on these substances in the past during drug development. In this study, we incorporated a nitrosation assay procedure into high-resolution supercritical fluid chromatography (SFC)–mass spectrometry screening to test the potential of direct nitrosation of active pharmaceutical ingredients (APIs). The forced degradation study was performed with a four-fold molar excess of sodium nitrite, relative to the drug substance, at pH 3–4 for 4 h at 37°C. Chromatographic separation was performed on a porous graphitic carbon column by SFC. The mass analysis then focused on direct N-nitrosation or N-nitroso compounds (NOCs) formed after dealkylation. Substances (n = 67) from various pharmaceutical classes were evaluated and 49.3% of them formed NOCs, of which 21.2% have not yet been reported in the literature. In addition, for two APIs, which are known to form an unidentified NOC, the structure could be identified. A few substances also showed multiple NOCs and even N,N’-dinitroso-species. As NAs are carcinogens, they have to be eliminated or at least limited to prevent cancer in patients, who rely on these drugs. This study contributes a procedure that can be implemented in preapproval drug development and postapproval risk assessment to prevent unexpected findings in the future

Überwindung der Nitrosaminkrise - Superkritische Fluidchromatographie gekoppelt mit Massenspektrometrie zur Bestimmung von unerwarteten Arzneimittelverunreinigungen

The present work addresses the systematic development, enhancement and application of highly sensitive analytical methods for the investigation of nitrosamine impurities in drug substances and drug products. It is demonstrated how these can be effectively brought to the point of application in the interaction of science and regulatory compliance in order to meet the highest standards of scientific and technical knowledge. As a first step, quality-by-design (QbD) principles are used to rapidly and systematically develop a robust and sensitive method. Due to the unique properties of supercritical fluid chromatography (SFC), this method is not only capable of separating ten pertinent nitrosamines and achieving detectability at trace levels, but also enables simultaneous analysis of the drug substances losartan, valsartan and their monographed related substances. Based on this work, it is demonstrated which advantages result from the high adaptability of the QbD development approach for the lifecycle of an analytical method. Thus analytical progression within the existing framework and design space is possible without a complete new development. For this purpose, accepted lifecycle management (LCM) principles are used to evolve the existing compound-specific method into a universally applicable method for nitrosamine determination. The published method is the only validated method to date that can simultaneously separate and detect at least 16 nitrosamines, regardless of the sample matrix. Based on this method, analytical results are subsequently presented from hundreds of marketed drug substance and drug product samples collected over a period of four years as part of the "nitrosamine crisis". In addition, a more sophisticated and standardized testing methodology is presented. It can be used not only to test for the presence of known nitrosamines, but also to investigate the formation of novel, drug-substance-related nitrosamines. These have recently become an increasing concern of the regulatory risk assessment process and hereby the dominant issue for pharmaceutical manufacturers and authorization holders. The present work essentially contributes to the detection and elucidation of known and novel potentially carcinogenic nitrosamines in drugs. It thus protects the health of patients, by enabling contaminated drugs to be withdrawn from the market and prevents novel nitrosamines to occur without knowledge of the responsible manufacturer. It therefore enhances the quality and safety of existing and new drugs. However, the outlined principles of method development, analytical lifecycle management and method application can also be applied to areas outside the trace analysis of nitrosamines or pharmaceutical quality control (e.g. in forensics, bio- and environmental analysis) and are generally applicable

Application of sub-/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed-phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design-of-Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry-compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed-phase liquid chromatography were resolved by applying the final method