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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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Simultaneous detection of nitrosamines and monographed impurities in sartans • Novel SFC approach for large group of nitrosamines • High sensitivity and selectivity within extreme short analysis time • Improved reliability and deep understanding by QbD development approach • Both NDMA and NDEA detected in Losartan API Since July 2018, the pharmacological class of "sartans" has been the subject of considerable media and analytical interest, as it became known that they are contaminated with nitrosamines such as N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosodiisopropylamine (NDiPA). Previous compendial methods are not able to detect these new contaminants. Using the latest and innovative Quality-by-Design (QbD) approach, it has now been possible to develop an analytical method that enables to investigate sartans, such as valsartan and losartan. Also a large class of different nitrosamines in the ppb range and sartan-related impurities can thus be determined simultaneously in a single analysis using supercritical fluid chromatography (SFC). By using SFC, a broad spectrum of nonpolar and very polar impurities can be separated and analyzed in under 20 min. The analytical method developed is validated for limit testing according to ICH Q2(R1) and fulfills default thresholds of EMA and FDA for testing of drug substances and genotoxic impurities. Additionally, it can also be adapted to other pharmaceuticals that may be contaminated with nitrosamines, since tetrazole synthesis as the underlying cause of nitrosamine contamination is important for a set of other non-sartan drug substances. [ABSTRACT FROM AUTHOR]

Highlights • Proper correlation between logD and relative retention in LC. • logD calculation is a good basis for starting method screening. • Tool to comply analytical QbD requirements in regards to lifecycle principles. • In silico generation of extended knowledge space possible. • Extreme high robustness of resulting HPLC method with improved reliability and understanding. Abstract The use of trial-and-error principles is a frequently used technique in method development. This may lead to the fact that analytical methods are used routinely without developers and users having gained extensive and well-founded knowledge about the robustness of their analytical methods and the influence of critical key parameters. This very often leads to unnecessary problems for analysts. A simple way in reverse phase chromatography to simulate the effects of pH value changes on the separation and retention of substances is the pH-dependent calculation of the logD value. With this tool, model substances were used to show how the time requirement for method screening can be considerably reduced in silico and, in addition, extended knowledge about the separation mechanics can be generated. Based on this knowledge, a new method for the purity analysis of carbamazepine was developed within a very short period of time, which improves the performance of the official Ph.Eur. monograph by far. Furthermore, the extremely high robustness of the new method was demonstrated. Using the logD based approach, Quality-by-Design is applied in method development and kept pace with the increasing requirements of regulatory authorities in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

• Universal method for nitrosamine analysis. • Novel SFC approach for simultaneous detection of 16 nitrosamines. • High sensitivity and selectivity within extreme short analysis time (separation of nitrosamines in 4 min; total run time 11.5 min). • Case study for lifecycle management with proof of concept. • Development under Quality-by-Design and validation according to ICH Q2. Since the detection of nitrosamines (NA) in valsartan pharmaceuticals, over two years have passed. At present, the occurrence of NAs can be limited to a few drug substances and drug products, but it is already becoming apparent that the issue appears to be much bigger than initially thought. The impact on the global pharmaceutical market has been tremendous and the problem can be attributed mainly to uncritically adopted approval changes and the lack of suitable, modern analytical methods to detect those impurities in time. We hereby demonstrate how lifecycle management (LCM) can be used to develop and improve suitable and universal analytical methods within short time. The resulting SFC-MS/MS method is intended for a universal nitrosamine investigation in drug substances and drug products. Successful NA analysis was demonstrated for seven sartans, metformin, pioglitazone and ranitidine. Additionally, combination drug products, containing also amlodipine, hydrochlorothiazide, vildagliptin and sitagliptin, were analyzed successfully. The method achieved separation of 16 NAs in 4 min with a total run time of 11.5 min, utilizing a Supel Carbon porous graphitic carbon (PGC) column. Carbon dioxide together with 0.1% TFA in methanol as modifier were used as eluents and 0.35% formic acid in methanol as make-up solvent for mass spectrometric NA detection. By implementing LCM in this case study, development time was reduced and knowledge was implemented fast. At the same time, a high adaptability of this "vital" method was achieved, which makes it possible to implement the constantly changing regulatory requirements within the shortest possible time. Supplemental development data, according to the ICH guidelines Q8, Q12 and the proposed Q14 are disclosed, demonstrating the scientific Quality-by-Design (QbD) development approach, the "fitness for use" and the robustness of the analytical procedure. This method contributes to the still ongoing risk assessment process of the pharmaceutical industry and the regulatory agencies, in order to understand root causes of NA formation, maintain the drug supply and prevent drug shortage. [ABSTRACT FROM AUTHOR]