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High-resolution tandem quadrupole time-of-flight mass analysers enable new automated workflows for untargeted data evaluation of complex samples like drug products. An example of such procedure is the so-called general unknown comparative screening (GUCS), which is used for software-assisted, automated identification of components that are only present in a sample and not in a reference. The GUCS approach has been employed for the first time to detect both degradation products and reaction products in drug products. Two different carbocisteine containing syrup prototypes - one with sucrose and the other with artificial sweeteners - were selected as examples after nine months of storage at 40 °C and 75% relative humidity. The samples were analysed chromatographically using a Coresep SB mixed-mode column and high-resolution MS and MS/MS data were recorded in information dependant acquisition mode on a Sciex X500R quadrupole time-of-flight mass spectrometer. Data analysis was considerably facilitated using the corresponding placebo formulation as reference samples. With the GUCS approach two hitherto unknown degradation products of carbocisteine, i.e. the carbocisteine lactam of the sulfoxides and the disulfide between l-cysteine and thioglycolic acid, were detected at low concentrations in both of the syrup formulations. The presumed structures were confirmed by in silico analysis of the fragment spectra and high-resolution LC-MS experiments with reference substances. Two additional impurities were found in the sucrose-containing sample and identified as the N-glycosides of carbocisteine and its lactam, respectively, using binary mixtures with a 13 C-labelled monosaccharide., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rasmus Walther reports equipment, drugs, or supplies was provided by A Nattermann und Cie GmbH., (Copyright © 2023 Elsevier B.V. All rights reserved.) more...

The charged aerosol detector (CAD) is frequently employed in liquid chromatography for the analysis of small polar and ionizable compounds such as amino acids and amino sugars, which provide a weak chromophore only. Separation of these compounds is achieved by means of ion pair chromatography (IPC), and, more recently, hydrophilic interaction chromatography (HILIC) techniques. However, as the CAD's response is highly dependent on the mobile phase composition, the substantial differences in the mobile phase composition of IPC and HILIC have a distinct impact on the detector's performance. This study was aimed at systematically comparing the performance of IPC and HILIC when coupled to the CAD. Therefore, the separation techniques characterized by their specific mobile phase compositions were evaluated for their influence on the CAD response and the signal-to-noise ratio (S/N) of the amino acids L-alanine, L-leucine, and L-phenylalanine applying the response surface methodology (RSM). The RSM results derived from flow injection analysis (FIA) indicated that the CAD response and thus the obtainable S/N are significantly higher in HILIC compared to IPC where the S/N decreased with the chain length of the applied ion-pairing reagent. In addition, an IPC and a HILIC method, respectively, were developed for the impurity profiling of the branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. The beneficial effects of the HILIC conditions on the S/N observed under FIA conditions were partly offset by moderate column bleed effects when using an amide functionalized column, which facilitates the separation in the HILIC method. Satisfactory LOQs (3-10 ng on column) were obtained with both methods; however, the HILIC method was found to be slightly superior in terms of sensitivity and separation efficiency., Competing Interests: Declaration of Competing Interest None of the authors of this paper does have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2021. Published by Elsevier B.V.) more...

The quantitative structure-retention relationship (QSRR) models are not only employed in retention behaviour prediction, but also in an in-depth understanding of complex chromatographic systems. The goal of the present research is to enable the comprehensive understanding of retention underlying the separation in β-cyclodextrin (CD) modified reversed-phase high performance liquid chromatography (RP-HPLC) systems, through the development of mixed QSRR models. Moreover, the amount of β-CD adsorbed on the stationary phase surface (β-CD A ) is added as the model's input in order to evaluate its contribution to both model performances and retention. Nuclear magnetic resonance (NMR) experiments were conducted to confirm the predicted inclusion complex structures and support the application of in silico tools. The most significant descriptors revealed that retention is governed by the steric factors 7.5 Å distant from the geometrical centre of a molecule, 3D arrangement of atoms determining the molecular size and shape, lipophilicity indicated by topological distances, as well as the unbound system's energy, related to the inclusion complex formation. In addition, a notable effect of the pH of the aqueous phase on the retention of ionizable analytes was shown. In the case of pH of the aqueous phase and β-CD A the change in retention behaviour of the studied analytes was observed only at the highest β-CD A value (5.17 μM/m 2 ), but it was not related to the ionization state of analytes. When the analytes did not change the ionization form across the investigated studied pH range, and the acetonitrile content in the mobile phase was 25% (v/v), the retention factor had low values regardless of the β-CD A ; under these circumstances the retention is probably acetonitrile driven., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.) more...

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)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...

High performance liquid chromatography (HPLC) methods with UV/vis detection are the most widespread analytical procedures in modern pharmaceutical applications, but reach their limitations when it comes to non-chromophore molecules. Hence, instead of using tiresome derivatization procedures, many liquid chromatography methods make use of the so-called aerosol-based universal detectors, namely the evaporative light scattering detector (ELSD), the condensation nucleation light scattering detector (CNLSD) and the charged aerosol detector (CAD). Amongst these, the CAD, being the youngest (introduced in 2005) of these three options, is often described as the most easy-to-use detector and is stated to exhibit sufficient sensitivity and good linearity of signal in a dedicated range of concentration. Therefore, this review sets its focus on the recent applications of the CAD for active pharmaceutical ingredients, excipient analysis as well as botanical applications. Alongside the post-column solvent addition techniques, the new CAD's ability to adjust the evaporation temperature and the possibility to use an integrated power function for signal linearization are reviewed as previously unavailable, new parameters for optimization., Competing Interests: Declaration of Competing Interest The authors are in a cooperative relationship with Thermo Fisher Scientific® and also were generously provided with a Sedere® ELSD by ERC® GmbH for trial experiments., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...

The analysis of polysorbate 80 is a challenge because all components lack a chromophore. Here, an ultra-high-performance liquid chromatography system equipped with a charged aerosol detector (UHPLC-CAD) was used to study the effect of systematic variation of the CAD settings, namely evaporation temperature, filter constant and power function value (PFV), on the detector response of fatty acid standards and manufacturing batches of polysorbate. Evaporation temperature and filter constant strongly affect the detection limits described by signal-to-noise (S/N) ratios. Although evaporation temperature can be increased to improve signal to noise ratios, analyte volatility at higher temperatures is an important limiting factor. The PFV was found to be a strong tool for optimizing response linearity, but the optimal PFV differed depending on analyte volatility. Because PFV optimization required some additional measurement time and because double-logarithmic transformation at the default PFV of 1.0 yielded satisfying universal results with less measurement time over a range of two orders of magnitude for every homologue fatty acid from C14 to C18, use of the log-log transformation is the favored linearization strategy. Possible optimization procedures for semi volatile substances are presented. Overall, this new UHPLC method method offers improved detection limits, as well as time savings of over 75% and eluent savings of more than 40% compared to the previously published HPLC-CAD method for polysorbate analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

High performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) is a versatile, easy to use and inexpensive alternative when it comes to the analysis of substances lacking a chromophor for UV detection. However, in pharmaceutical analysis injection of highly concentrated test solutions are normally required to control impurities at low levels. Under these conditions spike peaks were observed in the chromatograms of the test solutions making a proper evaluation of the impurity profile impossible. The influence of different eluent and ELSD parameters such as eluent composition, eluent flow-rate, ELSD scavenger gas flow-rate and evaporation temperature on the appearance of spike peaks was investigated. It could be shown that spike peaks can be avoided when selecting elevated eluent flow-rates and ESLD scavenger gas flow-rates. Moreover, eluents containing high amounts of organic modifier seem to foster the appearance of spike peaks., (Copyright (c) 2010 Elsevier B.V. All rights reserved.) more...

In this study a reversed phase ion-pair high-performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed and fully validated for the pharmaceutical quality control of L-aspartic acid (Asp). With a slight modification, the method also allows the evaluation of related substances in L-alanine (Ala). The method enables simultaneous control of related amino acids and of possibly occurring organic acids contaminants. A minimum limit of quantification of 0.03% could be achieved for all occurring related substances. Moreover, the detector sensitivity of the CAD was compared with an evaporative light scattering detector (ELSD). Depending on the analyte the CAD was found to be 3.6-42 times more sensitive than the ELSD. The HPLC method was applied to the purity testing of 8 samples of pharmaceutical grade and reagent grade Asp and of 12 samples of Ala supplied by various manufacturers. Both substances were found to be of high purity (greater than 99.8% for Asp and greater than 99.9% for Ala). Malic acid and Ala were the major impurities in Asp. Asp and glutamic acid (Glu) were the only detectable impurities in Ala., (2009 Elsevier B.V. All rights reserved.) more...

A chiral microemulsion electrokinetic chromatography method has been developed for the enantiomeric separation of 3,4-dihydroxyphenylalanine (dopa), its precursors phenylalanine and tyrosine, and the structurally related substance methyldopa. The separations were achieved using an oil-in-water microemulsion, which consisted of the oil-compound ethyl acetate, the surfactant sodium dodecylsulfate (SDS), the co-surfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 or 2.0 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The resolution of each racemate was optimized by varying the concentration of the buffer and all components of the microemulsion. Enantioseparation could be achieved for dl-dopa, dl-phenylalanine and dl-tyrosine within 13 min with a resolution of 4.3, 3.1 and 3.3, respectively, and for methyldopa in 17 min (Rs: 1.4). The established methods allowed the detection of dopa, phenylalanine, tyrosine and methyldopa with a limit at 0.5, 1.0, 0.2 and 2.0 microg/ml. more...

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the measurement of erythromycin A (EA) and related substances in commercial samples was developed and validated. The samples were chromatographed on a reversed-phase column with a polar endcapping and analyzed by ion trap tandem mass spectrometry in the multiple reaction monitoring (MRM) mode using positive electrospray ionization. The method showed high recovery (>or=98.82%), high sensitivity (lower limit of quantitation of 0.25 ng/mL for EA and less than 7.3 ng/mL for the related substances) and high precision (or=0.991) with a run time of only 13 min. The method was successfully applied to the determination of EA and related substances in commercial samples. Moreover, using the advanced data-dependent acquisition capability of the ion trap software two new unexpected EA related substances could be detected and possible structures for these substances were postulated. more...

Chromatographic separation and quantification methods of tropa alkaloids were often described. In order to separate atropine from its degradation products ion-pair chromatography (IPC) has been frequently applied. Beside long equilibration times IPC often suffers from poor robustness. The aim of this study was to develop robust and simple HPLC methods for both stability testing of atropine solutions and limitation of related substances in atropine from plant material. Using a hydrophilic embedded RP18 column and a gradient elution gave baseline separation of all components. more...

The enantiomers of methylephedrine, pseudoephedrine and ephedrine showed a different migration behavior in capillary electrophoresis in the presence of beta-cyclodextrin (beta-CD), heptakis(2,3-O-diacetyl)-beta-cyclodextrin and heptakis(2,3-O-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS). Utilizing UV, MS and NMR spectroscopy, in particular rotating frame Overhauser experiments, an attempt was made to elucidate the chiral recognition mechanism. In the case of the neutral CDs 1:1 complexes were formed with ephedrine and methylephedrine characterized by the inclusion of the phenyl ring in the cavity and the side chain pointing out of the wider rim. In contrast, manifold complexes were formed with HDAS, which on average are characterized by an upside down inclusion of the phenyl ring in the cavity and the side chain pointing out of the narrow rim. This complex geometry is likely be stabilized by an ion-ion interaction between the positively charged nitrogens of the ephedrine derivatives and the negative charges of HDAS. In addition, an attachment of the ligand to the outside of HDAS and other complex stoichiometries are also possible. more...

A new single isomer beta-cyclodextrin, the heptakis(2-N,N-dimethylcarbamoyl)-beta-cyclodextrin (HDMC-beta-CD), has been synthesized and spectroscopically characterized. The outstanding feature of the new neutral beta-CD derivatives is their high solubility in water (>100 mM) and methanol. The resolution of chiral drugs can take advantage of this property. The resolution power of the new HDMC-beta-CD is demonstrated by a selection of acidic and basic compounds using standard conditions. more...

The racemates of newly synthesized 4-aryl-1,4-dihydropyridine derivatives attracting interest in the treatment of coronary insufficience were resolved via the formation of diastereomeric salts. In order to check the quality of the preparative resolution, capillary electrophoresis using neutral cyclodextrins (CDs) was developed. In particular, the alpha-CD was found to be a powerful discriminator of the enantiomers. Additionally, taking amlodipine and nicardipine into consideration, a mechanism of the chiral recognition with alpha-CD could be proposed. more...