N-alkylamide profiling of Achillea ptarmica and Achillea millefolium extracts by liquid and gas chromatography–mass spectrometry

Achillea millefolium and Achillea ptarmica are both plants belonging to the Asteracea family and are traditionally used for their medicinal properties. It has already been shown that some N-alkylamides(NAAs)are responsible for these pharmacological actions. Therefore, in the present study, the NAA content of the two plants was analytically characterised. Different extracts were prepared from the roots, the leaves, the stems and the flowers. The structures of NAAs have been assigned in ethanolic extracts of Achillea millefolium and Achillea ptarmica using high performance liquid chromatography – electrospray ionisation – mass spectrometry(HPLC–ESI–MS) and gas chromatography – electron impact – mass spectrometry(GC–EI–MS). Using both analytical techniques, the structures of 14 and 15 NAAs have been assigned in Achillea ptarmica and Achillea millefolium, respectively. Structures of two new NAAs, previously never observed in Achillea ptarmica,were assigned: deca-2E,6Z,8E-trienoic acid 2-methylbutylamide(homospilanthol) or a related isomeric compound and deca-2E,4E-dienoic acid N-methyl isobutylamide. The structure of homospilanthol or a related isomeric compound was also assigned in Achillea millefolium for the first time.

Derringer desirability and kinetic plot LC-column comparison approach for MS-compatible lipopeptide analysis

Lipopeptides are currently re-emerging as an interesting subgroup in the peptide research field, having historical applications as antibacterial and antifungal agents and new potential applications as antiviral, antitumor, immune-modulating and cell-penetrating compounds. However, due to their specific structure, chromatographic analysis often requires special buffer systems or the use of trifluoroacetic acid, limiting mass spectrometry detection. Therefore, we used a traditional aqueous/acetonitrile based gradient system, containing 0.1% （m/v） formic acid, to separate four pharmaceutically relevant lipopeptides （polymyxin B1, caspofungin, daptomycin and gramicidin A1）, which were selected based upon hierarchical cluster analysis （HCA） and principal component analysis （PCA）.In total, the performance of four different C18 columns, including one UPLC column, were evaluated using two parallel approaches. First, a Derringer desirability function was used, whereby six single and multiple chromatographic response values were rescaled into one overall D-value per column. Using this approach, the YMC Pack Pro C18 column was ranked as the best column for general MS-compatible lipopeptide separation. Secondly, the kinetic plot approach was used to compare the different columns at different flow rate ranges. As the optimal kinetic column performance is obtained at its maximal pressure, the length elongation factor λ（Pmax/Pexp） was used to transform the obtained experimental data （retention times and peak capacities） and construct kinetic performance limit （KPL） curves, allowing a direct visual and unbiased comparison of the selected columns, whereby the YMC Triart C18 UPLC and ACE C18 columns performed as best. Finally, differences in column performance and the （dis）advantages of both approaches are discussed.

Analytical quality-by-design approach for sample treatment of BSA-containing solutions

The sample preparation of samples conlaining bovine serum albumin（BSA）,e.g..as used in transdermal Franz diffusion cell（FDC） solutions,was evaluated using an analytical qualily-by-design（QbD）approach.Traditional precipitation of BSA by adding an equal volume of organic solvent,often successfully used with conventional HPLC-PDA,was found insufficiently robust when novel fused-core HPLC and/or UPLC-MS methods were used.In this study,three factors（acetonitrile（%）.formic acid（%） and boiling time（min）） were included in the experimental design to determine an optimal and more suitable sample treatment of BSAcontaining FDC solutions.Using a QbD and Derringer desirability（D） approach,combining BSA loss,dilution factor and variability,we constructed an optimal working space with the edge of failure defined as D〈0.9.The design space is modelled and is confirmed to have an ACN range of 83 ± 3% and FA content of 1 ±0.25%.

LC–UV/MS quality analytics of paediatricartemether formulations

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for β-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity： a Prevail organic acid （OA） column （250 mm＆#215;4.6 mm, 5μm）, flow rate set at 1.5 mL/min combined with a linear gradient （where A ？ 25 mM phosphate buffer （pH 2.5）, and B ？ acetonitrile） from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile-water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of β-artemether paediatric formulations. Furthermore, LC-MS on references as well as on stability samples was performed allowing identity confirmation of the β-artemether related impurities. MS-fragmentation scheme of β-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.

A critical quality parameter in quantitative fused-core chromatography: The injection volume

As part of the method development, the injection volume as a critical quality attribute in fast fused-core chromatography was evaluated. Spilanthol, a pharmaceutically interesting N- alkylamide currently under investigation in our laboratory, was chosen as the model compound. Spilanthol was dissolved in both PBS and MeOH/H20 （70/30, v/v） and subsequently analyzed using a fused-core system hereby selecting five chromatographic characteristics （retention time, area, height, theoretical plates and symmetry factor） as responses. We demonstrated that the injection volume significantly influenced both the qualitative and quantitative performance of fused-core chromatography, a phenomenon which is confounded with the nature of the used sample solvent. From 2 ~tL up to 100 laL injection volume with PBS as solvent, the symmetry factor decreased favorably by 20%. Moreover, the theoretical plates and the quantitative parameters （area and height） increased up to 30%. On the contrary, in this injection volume range, the theoretical plates for the methanol-based samples decreased by more than 60%, while the symmetry factor increased and the height decreased, both by 30%. The injection volume is thus a critical and often overlooked parameter in fused-core method description and validation.

Quality evaluation of synthetic quorum sensing peptides used in R&D

Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R＆D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control （QC） revealed a large discrepancy between the purity levels as stated on the supplier＇s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid（s） in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

Reversed-phase fused-core HPLC modeling of peptides

Different fused-core stationary phase chemistries（C18,Amide,Phenyl-hexyl and Peptide ES-C18） were used for the analysis of 21 structurally representative model peptides.In addition,the effects of the mobile phase composition（ACN or MeOH as organic modifier;formic acid or acetic acid,as acidifying component） on the column selectivity,peak shape and overall chromatographic performance were evaluated.The RP-amide column,combined with a formic acid-acetonitrile based gradient system,performed as best.A peptide reversed-phase retention model is proposed,consisting of 5 variables：log SumAA,log Sv,clog P,log nHDon and log nHAcc.Quantitative structure-retention relationship（QSRR） models were constructed for 16 different chromatographic systems.The accuracy of this peptide retention model was demonstrated by the comparison between predicted and experimentally obtained retention times,explaining on average 86% of the variability.Moreover,using an external set of 5 validation peptides,the predictive power of the model was also demonstrated.This peptide retention model includes the novel in-silico calculated amino acid descriptor,AA,which was calculated from log P,3D-MoRSE,RDF and WHIM descriptors.

Risk evaluation of impurities in topical excipients:The acetol case

Pharmaceutical excipients for topical use may contain impurities, which are often neglected from a toxicity qualification viewpoint. The possible impurities in the most frequently used topical excipients were evaluated in-silico for their toxicity hazard. Acetol, an impurity likely present in different topical pharmaceutical excipients such as propylene glycol and glycerol, was withheld for the evaluation of its health risk after dermal exposure. 〈br〉 An ex-vivo in-vitro permeation study using human skin in a Franz Diffusion Cell set-up and GC as quantification methodology showed a significant skin penetration with an overall Kp value of 1.82 ？ 10 ？ 3 cm/h. Using these data, limit specifications after application of a dermal pharmaceutical product were estimated. Based on the TTC approach of Cramer class I substances, i.e. 1800 mg/（day？person）, the toxicity-qualified specification limits of acetol in topical excipients were calculated to be 90 mg/mL and 180 mg/mL for propylene glycol and glycerol, respectively.

Analysis of iodinated quorum sensing peptides by LC–UV/ESI ion trap mass spectrometry

Five different quorum sensing peptides(QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1%(m/v) formic acid as mobile phase. Electrospray ionization(ESI)ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current(TIC) spectra. Non-iodinated peptides and mono-and diiodinated peptides(NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low(2%) to high(57%).