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Highlights • Using HPTLC plates for solvent-free nanomole-scaled on-surface synthesis. • Miniaturized synthesis, workup and analysis performed on one HPTLC plate. • Tool for rapid impurity analysis of an anti-cancer drug. • On-surface synthesis enabled impurity signal highlighting in HPTLC-HRMS. Abstract On-surface reactions were introduced as a new strategy for rapid structure elucidation. This strategy is illustrated by the miniaturized synthesis-guided identification of two new degradation products (impurities) occurring in a pharmaceutical formulation of the anti-cancer drug ifosfamide, especially in the presence of urea. Synthesis on the silica gel surface bypassed the need for solvents, as the large nm-porous surface favoured a fast conversion of the reaction partners. For the on-surface synthesis of the impurities, the respective reagents were accurately and automatedly applied in the nanomole scale on a high-performance thin-layer chromatography (HPTLC) silica gel plate. After a fast reaction, the workup of the reaction mixture was performed by development of the HPTLC plate followed by online high-resolution mass spectrometry. As proof of concept and for benchmarking, a reaction mixture obtained from conventional preparative synthesis in a round-bottom flask was analysed in parallel as well as different formulations. The use of adsorbents as inert layer turned out to be highly efficient for a rapid generation and confirmation of impurities, as the synthesis on the HPTLC layer revealed them within 10 min. Image evaluation was simply performed by videodensitometry. The advantageous combination of all steps on one HPTLC plate and its resulting efficiency made surface synthesis on chromatographic phases an optimal tool for signal highlighting in mass spectrometry, and thus for the assignment of impurities in drugs. The chemistry process scale was miniaturized down to the μg-level per synthesis (in total 30-60 μg chemicals/reaction), setting a new state-of-the-art standard. All material savings clearly contribute to green chemistry, and this strategy substantially reduces the consumption of chemicals and greatly enhances the analytical efficiency, when adapted by scientists for the quality control of any other chemical product. The combination of synthesis, workup and detection in a miniaturized process, contributes to optimized workflows in a lean laboratory. [ABSTRACT FROM AUTHOR]

Abstract: Combining the most powerful separation techniques, i.e. liquid chromatography (LC) or capillary electrophoresis (CE) with a information rich detection system – the mass spectrometer or the nuclear magnetic resonance (NMR) spectrometer – has been pursued for more than three decades. This compilation shall provide an overview of the advantages and limitations of the LC–NMR hyphenation in the light of its most valued application–the unequivocal analyte identification. Especially the post LC trapping of analytes with an in-line solid phase extraction (SPE) device prior to transferring the analyte of interest to the NMR spectrometer (LC–SPE–NMR) proved to be a robust installation allowing a significant cut-down of the amount of analyte needed for the generation of high quality heteronuclear NMR shift correlation data. Different available technical realizations will be discussed and typical application examples from natural product research and from industrial settings will be given. [Copyright &y& Elsevier]

Abstract: Monofunctional maleimide polyethylene glycol (mono-mal-PEG) with average molecular weight up to 40kDa can be used as a raw material for the PEGylation of therapeutic proteins. A possible impurity in this raw material which needs to be controlled is the bisfunctional maleimide-PEG, which has a similar average molecular weight to mono-mal-PEG. Chromatographic separation and detection of low level bis-mal-PEG in mono-mal-PEG presents a major challenge because of the polydispersity of the analytes and the minor difference between the desired mono-mal-PEG and the bis-mal-PEG impurity. In this study, linear mal-PEGs were first derivatized with a specially designed cys-peptide containing a UV chromophore and multiple ionizable sites. Separations were then carried out by reversed-phase HPLC with UV detection at 360nm. Mono-mal-PEG and bis-mal-PEG were well resolved using a Gemini C18 column with an aqueous-acetonitrile mobile phase. Retention times increased as PEG molecular weight increased from 10kDa to 40kDa, while selectivities decreased as PEG molecular weight increased. Results from systematically designed studies for optimization of critical parameters including gradient slope, column temperature, and acidic modifier in the mobile phase led to the selection of the final separation conditions. The developed method conditions were specific, accurate, and sensitive for detecting bis-mal-PEG as an impurity in mono-mal-PEG with limit of quantitation of 0.2% and may be used to assess the quality of mono-mal-PEG as a raw material for protein PEGylation. [Copyright &y& Elsevier]

Abstract: While supercritical fluid chromatography (SFC) has received great popularity in chiral separation and purification, it has rarely been used for trace level pharmaceutical impurity analysis, partially due to the limitation of instrument sensitivity. In this study, a packed column SFC method has been developed for the quantitative analysis of mometasone furoate and its trace level impurities. The UV detection was optimized to improve the sensitivity by 2–4 fold. In combination with an increased sample concentration, this SFC method is capable of trace level (0.05% of the active) analysis of the impurities. The SFC method used a silica column and a mobile phase consisting of CO2 and methanol. The new method provides an orthogonal selectivity complementary to the reversed phase HPLC (RP-HPLC) method. All of the impurities and the active were baseline separated within 12min on SFC, which is less than one third of the RP-HPLC method run time. The method was also partially validated for linearity, accuracy, precision (repeatability), and limit of quantitation. This study demonstrated that the SFC method, with improved sensitivity, can be a valuable tool to provide orthogonal selectivity for trace level impurity separation. With further validation, the method may be suitable for release testing and stability testing for mometasone furoate drug substance. [Copyright &y& Elsevier]

Abstract: Numerous combinations of one-, two-, and three-chiral-component microemulsions have been previously prepared in our group, using N-dodecoxycarbonylvaline (DDCV), 2-hexanol, and ethyl acetate, dibutyl tartrate, or diethyl tartrate. A few results of the various formulations investigated suggested the possible presence of minor impurities in one or more components of the microemulsion. In this study, the purity of the current lots of R- and S-surfactant were measured, as was the subsequent effect of minor impurities on the relevant chromatographic figures of merit (CFOMs) that describe a chiral separation, i.e., efficiency, enantioselectivity, retention, migration window (elution range), and resolution. Two related methods are proposed for correcting enantioselectivities measured in the presence of chiral impurities in the chiral microemulsion. [Copyright &y& Elsevier]

Abstract: A versatile procedure has been developed and validated for the determination of triphenylphosphine oxide (TPPO) at low levels in various active pharmaceutical ingredients (APIs). This procedure incorporates the use of the novel hollow-fiber liquid-phase microextraction (LPME) for the measurement of this potential process-related impurity in aqueous solutions of APIs. A small volume (40μL) of 1-octanol contained within a hollow polypropylene fiber is used for the extraction of TPPO from low pH aqueous API solutions. More than a 100-fold increase in the TPPO concentration is obtained without additional evaporation of the extract. Experimental parameters of the extraction procedure were investigated to optimize extraction efficiency and minimize sample matrix interference. Using HPLC/UV as the end analysis technique, the procedure was validated for TPPO in the concentration range of 3–16μg/L with an API present at 1500mg/L. The versatility of the method was demonstrated by applying the procedure to the analysis of APIs with different molecular structures. This simple LPME procedure is inexpensive and offers appropriate sensitivity for the intended use while providing several advantages over other analysis methods for pharmaceutical samples. [Copyright &y& Elsevier]

• Power function setting of Charged Aerosol Detector is optimized to linearize signal • Empirical vs. mathematical power function value (PFV) optimization approach • Results of both approaches in good agreement with the experimental optimal PFV • Accuracy of an impurity analysis method is substantially improved by adjusting PFV • PFV optimized methods can be employed in a GMP regulated environment Charged aerosol detection (CAD) is an universal technique in liquid chromatography that is increasingly used for the quality control of drugs. Consequently, it has found its way into compendial monographs promoted by its simple and robust application. However, the response of CAD is inherently nonlinear due to its principle of function. Thus, easy and rapid linearization procedures, in particular regarding compendial applications, are highly desirable. One effective approach to linearize the detector's signal makes use of the built-in power function value (PFV) setting of the instrument. The PFV is basically a multiplication factor to the power law exponent of the equation describing the CAD's response, thereby altering the detector's signal output to optimize the quasi-linear range of the response curve. The experimental optimization of the PFV for a series of analytes is a time-consuming process, limiting the practicability of this approach. Here, two independent approaches for the determination of the optimal PFV based on an empirical model and a mathematical transformation in each case, are evaluated. Both approaches can be utilized to predict the optimal PFV for each analyte solely based on the experimental results of a series of calibration standards obtained at a single PFV. The approaches were applied to the HPLC-UV-CAD impurity analysis of the drug gabapentin to improve the observed nonlinear response of the impurities in the range of interest. The predicted optimal PFV of both approaches were in good agreement with the experimentally obtained optimal PFV of the analytes. As a result, the accuracy of the method was significantly improved when using the optimal PFV (90 – 105% versus 81 – 115% recovery rate for quantitation by either single-point calibration or linear regression) for the majority of the analytes. The final method with a PFV adjusted to 1.30 was validated with respect to ICH guideline Q2(R1). [ABSTRACT FROM AUTHOR]