Microchip isotachophoresis for green and sustainable pharmaceutical quality control: Method validation and application to complex pharmaceutical formulations.

Universal microchip isotachophoresis (µITP) methods were developed for the determination of cationic and anionic macrocomponents (active pharmaceutical ingredients and counterions) in cardiovascular drugs marketed in salt form, amlodipine besylate and perindopril erbumine. The developed methods are characterized by low reagent and sample consumption, waste production and energy consumption, require only minimal sample preparation and provide fast analysis. The greenness of the proposed methods was assessed using AGREE. An internal standard addition was used to improve the quantitative parameters of µITP. The proposed methods were validated according to the ICH guideline. Linearity, precision, accuracy and specificity were evaluated for each of the studied analytes and all set validation criteria were met. Good linearity was observed in the presence of matrix and in the absence of matrix, with a correlation coefficient of at least 0.9993. The developed methods allowed precise and accurate determination of the studied analytes, the RSD of the quantitative and qualitative parameters were less than 1.5% and the recoveries ranged from 98 to 102%. The developed µITP methods were successfully applied to the determination of cationic and anionic macrocomponents in six commercially available pharmaceutical formulations.

Impact of structural similarity on the accuracy of retention time prediction.

Quantitative Structure-Retention Relationships offer a valuable tool for de-risking chromatographic methods in relation to newly formed or hypothetical compounds, arising from synthetic processes or formulation activities. They can also be used to identify optimal separation conditions, or in support of structural elucidation. In this contribution, we provide a systematic study of the relationship between the accuracy of the retention model, the size of the training set and its structural similarity to the predicted compound. We compare structural similarity expressed either on a fingerprint basis (e.g., Tanimoto index), or by Euclidean distance calculated from of subset of molecular descriptors. The results presented indicate that accurate and predictive models can be built from a small dataset containing as few as 25 compounds, provided that the training set is structurally similar to the test compound. When the training set contains compounds selected by minimizing the Euclidean distance calculated from 3 descriptors most correlated with the retention time, root mean square error of 0.48 min and correlation coefficient of 0.9464 were observed for the test sets of 104 compounds. Moreover, these models meet the Tropsha predictivity criteria. These findings potentially bring the prediction of retention times within the practical reach of pharmaceutical analysts involved in chromatographic method development. We also present an optimisation approach to select algorithm settings in order to minimize the prediction error and ensure model predictivity.

Development of Microchip Isotachophoresis Coupled with Ion Mobility Spectrometry and Evaluation of Its Potential for the Analysis of Food, Biological and Pharmaceutical Samples.

An online coupling of microchip isotachophoresis (µITP) with ion mobility spectrometry (IMS) using thermal evaporation interface is reported for the first time. This combination integrates preconcentration power of the µITP followed by unambiguous identification of trace compounds in complex samples by IMS. Short-chain carboxylic acids, chosen as model analytes, were first separated by the µITP in a discontinuous electrolyte system at pH 5-6, and subsequently evaporated at 130 °C during their transfer to the IMS analyzer. Various parameters, affecting the transfer of the separated sample components through the evaporation system, were optimized to minimize dispersion and loss of the analytes as well as to improve sensitivity. The following analytical attributes were obtained for carboxylic acids in the standard solutions: 0.1-0.3 mg L-1 detection limits, 0.4-0.9 mg L-1 quantitation limits, linear calibration range from the quantitation limit to 75 mg L-1, 0.2-0.3% RSD of the IMS response and 98-102% accuracy. The analytical potential of the developed µITP-IMS combination was demonstrated on the analysis of various food, pharmaceutical and biological samples, in which the studied acids are naturally present. These include: apple vinegar, wine, fish sauce, saliva and ear drops. In the real samples, 0.3-0.6% RSD of the IMS response and 93-109% accuracy were obtained.

Potential of microchip electrophoresis in pharmaceutical analysis: Development of a universal method for frequently prescribed nonsteroidal anti-inflammatory drugs.

A novel microchip electrophoresis method with conductivity detection for the determination of nonsteroidal anti-inflammatory drugs (NSAIDs) in several pharmaceutical formulations was developed. The three frequently used NSAIDs - acetylsalicylic acid, diclofenac and ibuprofen were baseline separated on a poly(methyl methacrylate) microchip with coupled separation channels. Elimination of matrix components such as excipients, was realized through online combination of isotachophoresis (ITP) with zone electrophoresis (ZE). ITP-ZE hyphenation can also facilitate preconcentration of target analytes. ITP was carried out in the first separation channel at pH 6.5, while the second channel of the microchip was used for ZE separation and detection of the analytes at pH 7.0. The developed ITP-ZE method was demonstrated to be applicable for direct and reliable determination of NSAIDs in eleven pharmaceutical formulations. The noticeable advantage of this approach is that only simple sample pretreatment (filtration and dilution) is necessary. The method performance parameters, such as linearity (20-250% of nominal concentration of studied NSAIDs in the test samples), accuracy (98-102%) and precision (less than 2% RSD) were obtained. This universal approach is suitable for the determination of frequently used NSAIDs in a single analysis in less than 15 min. In addition to simple sample pretreatment, low running costs and minimal environmental impact could make this method attractive for the analysis of pharmaceutical preparations.

Structure Driven Prediction of Chromatographic Retention Times: Applications to Pharmaceutical Analysis.

Pharmaceutical drug development relies heavily on the use of Reversed-Phase Liquid Chromatography methods. These methods are used to characterize active pharmaceutical ingredients and drug products by separating the main component from related substances such as process related impurities or main component degradation products. The results presented here indicate that retention models based on Quantitative Structure Retention Relationships can be used for de-risking methods used in pharmaceutical analysis and for the identification of optimal conditions for separation of known sample constituents from postulated/hypothetical components. The prediction of retention times for hypothetical components in established methods is highly valuable as these compounds are not usually readily available for analysis. Here we discuss the development and optimization of retention models, selection of the most relevant structural molecular descriptors, regression model building and validation. We also present a practical example applied to chromatographic method development and discuss the accuracy of these models on selection of optimal separation parameters.

Advantages and Pitfalls of Capillary Electrophoresis of Pharmaceutical Compounds and Their Enantiomers in Complex Samples: Comparison of Hydrodynamically Opened and Closed Systems.

Several research disciplines require fast, reliable and highly automated determination of pharmaceutically active compounds and their enantiomers in complex biological matrices. To address some of the challenges of Capillary Electrophoresis (CE), such as low concentration sensitivity and performance degradation linked to the adsorption and interference of matrix components, CE in a hydrodynamically closed system was evaluated using the model compounds Pindolol and Propranolol. Some established validation parameters such as repeatability of injection efficiency, resolution and sensitivity were used to assess its performance, and it was found to be broadly identical to that of hydrodynamically opened systems. While some reduction in separation efficiency was observed, this was mainly due to dispersion caused by injection and it had no impact on the ability to resolve enantiomers of model compounds even when spiked into complex biological matrix such as blood serum. An approximately 18- to 23-fold increase in concentration sensitivity due to the employment of wide bore capillaries was observed. This brings the sensitivity of CE to a level similar to that of liquid chromatography techniques. In addition to this benefit and unlike in hydrodynamically opened systems, suppression of electroosmotic flow, which is essential for hydrodynamically closed systems practically eliminates the matrix effects that are linked to protein adsorption.

Microchip isotachophoresis coupled to surface-enhanced Raman spectroscopy for pharmaceutical analysis.

A novel online coupling of microchip isotachophoresis (µITP) with surface-enhanced Raman spectroscopy (SERS) for the analysis of complex samples is presented. Polymeric microchip with coupled channels was used for µITP-SERS analysis of four structurally similar Raman active synthetic dyes (brilliant black BN, carmoisine, ponceau 4R, and sunset yellow FCF) in pharmaceuticals. The µITP separation and simultaneous pre-concentration of the analytes were performed in the first channel of the microchip at pH 6.0 with the aid of non-Raman active discrete spacers (acetate, butyrate, glutarate, pantothenate, and valerate). Silver nanoparticles used for Raman enhancement were present in the second channel, and individual SERS spectra of the dyes were acquired by a mini Raman spectrometer operating at 532 nm. The analytical enhancement factors for silver nanoparticles were 1-5 × 104. The microchip with coupled channels enabled independent µITP separation and SERS detection, and eliminated any adverse impact of nanoparticles on the separation. The developed approach allowed reliable online SERS identification and detection of dyes with limits of detection ranging from 12 to 62 nM. Synthetic dyes were successfully separated, identified, and quantified in pharmaceutical preparations within 7 min without the need for complex or time-consuming sample pretreatment. The results were in good agreement with those obtained by an independent analytical method reported for studied dyes. Graphical abstract.

Microchip Isotachophoresis: Analysis of Pharmaceuticals.

Microchip isotachophoresis (µITP) is a miniaturized version of conventional isotachophoresis (ITP) characterized by low sample and buffer consumption and reduced waste production. µITP with universal conductivity detection is suitable for quantitative analysis of relatively simplified samples that contain analyte(s) at relatively high concentration, e.g., pharmaceutical preparations. Here we describe in detail a principle of µITP in terms of reaching highly precise results. A practical use of µITP is shown on the analyses of various pharmaceutical preparations for content of major constituents including active pharmaceutical ingredients as well as pharmaceutical counterions. The pharmaceuticals are treated only minimally prior to the ITP run on a microchip with coupled channels and sample injection channel with 0.9 µL volume. Developed method is suitable for rapid (analysis time up to 10 min), precise (less than 1% RSD of analyte zone length), and accurate (recovery of 98-101%) determination of major pharmaceutical ingredients using a method of internal standard for data evaluation.

Quantitative aspects of microchip isotachophoresis for high precision determination of main components in pharmaceuticals.

Although microchip electrophoresis (MCE) is intended to provide reliable quantitative data, so far there is only limited attention paid to these important aspects. This study gives a general overview of key aspects to be followed to reach high-precise determination using isotachophoresis (ITP) on the microchip with conductivity detection. From the application point of view, the procedure for the determination of acetate, a main component in the pharmaceutical preparation buserelin acetate, was developed. Our results document that run-to-run fluctuations in the sample injection volume limit the reproducibility of quantitation based on the external calibration. The use of a suitable internal standard (succinate in this study) improved the repeatability of the precision of acetate determination from six to eight times. The robustness of the procedure was studied in terms of impact of fluctuations in various experimental parameters (driving current, concentration of the leading ions, pH of the leading electrolyte and buffer impurities) on the precision of the ITP determination. The use of computer simulation programs provided means to assess the ITP experiments using well-defined theoretical models. A long-term validity of the calibration curves on two microchips and two MCE equipments was verified. This favors ITP over other microchip electrophoresis techniques, when chip-to-chip or equipment-to-equipment transfer of the analytical method is required. The recovery values in the range of 98-101 % indicate very accurate determination of acetate in buserelin acetate, which is used in the treatment of hormone-dependent tumors. This study showed that microchip ITP is suitable for reliable determination of main components in pharmaceutical preparations.

Direct Liquid Sampling for Corona Discharge Ion Mobility Spectrometry.

We present a new technique suitable for direct liquid sampling and analysis by ion mobility spectrometry (IMS). The technique is based on introduction of a droplet stream to the IMS reaction region. The technique was successfully used to detect explosives dissolved in methanol and oil as well as to analyze amino acids and dipeptides. One of the main advantages of this technique is its ability to analyze liquid samples without the requirement of any special solution.