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• Systematic HILIC assay development of polar analytes remains a challenge. • A new, automated HILIC-DAD-CAD method development workflow for polar analytes. • Screening of 12 columns with sub-2.7 µm fully and superficially porous particles. • Evaluation of different eluent pH and column equilibration time. • Diverse compounds and real-life common pharmaceutical examples were explored. The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time. Herein, we introduce a new automated multicolumn and multi-eluent screening workflow that streamlines the development of new HILIC assays, circumventing the existing tedious 'hit-or-miss' approach. A total of 12 complementary columns packed with sub-2 µm fully porous and 2.7 µm superficially porous particles operated on readily available ultra-high pressure liquid chromatography (UHPLC) instrumentation across a diverse set of commercially available polar stationary phases were investigated. Different mobile phases with pH ranging from pH 3 to 9 were evaluated using different organic modifiers. The gradient and column re-equilibration were judiciously set to ensure a reliable assay screening framework that indicates promising conditions for subsequent method optimization to achieve resolution of challenging mixtures. This UHPLC screening system is coupled with a diode array and charged aerosol detectors (DAD, CAD and mass spectrometry) to ensure versatile detection for a variety of compounds. This fast-screening platform lays the foundation for a convenient generic workflow, accelerating the pace of HILIC method development and transfer across both academic and industrial sectors. [ABSTRACT FROM AUTHOR]

• Development of a purity method by supercritical fluid chromatography for a of late-stage API. • Automation of multifactorial DoE for the development of robust SFC method. • Machine learning method development using a bayesian optimization algorithm on SFC. • SFC method robustness for the KPSS in object vs RPLC method. • Detailed description of green score SFC vs HPLC analytical methods. During drug development, chromatography is frequently used for purity and stability testing of both drug substance and drug product. Reversed phase liquid chromatography (RPLC) is one of the most widely used methodologies due to its wide scope of application. In the later stages of drug development, the specified impurities and degradation products that define the critical quality attribute of the final API, also known as Key Predictive Sample Set (KPSS), are usually well defined and controlled. At this point, a method review enables selecting the most appropriate technique which should be the one providing optimal robustness (ICH-Q14[1]), with the support of Quality by Design (QbD) approaches. Supercritical Fluid Chromatography (SFC) is a preferred technique for its proven diversity in selectivity. The adoption of a technique which presents the most favourable environmental impact, such as, but not limited to, SFC, is also becoming increasingly important as laboratories strive to reduce carbon footprint. Re -developing a method requires high resource-demands in terms of staff, materials, and time. Any step of the process that can be automated can facilitate this approach, speeding up the delivery of the method whilst preserving robustness. In this article we describe how an SFC method was developed for the purity profiling of a late-stage oncology candidate, taking advantage of the superior selectivity of SFC towards structurally similar analytes, owed to the high orthogonality with R2 as low as 0.014 towards the KPSS. We describe two approaches to automate the method development. Firstly, a multifactorial design of experiments (DoE) and secondly, an optimization via a Bayesian algorithm, which was completed in one night, highlighting the potential and limitations, with an insight into the robustness. Both methods achieved baseline separation with varying levels of automation embedded into the process and a large reduction of the resource demands when compared to traditional optimisation methods. Finally, we describe the beneficial environmental impact that implementing SFC methods can yield, with a calculated green score reduced to a value between 17 and 30 % compared to RPLC, depending on the number of runs per sequence. [ABSTRACT FROM AUTHOR]

• Applications of reversed phase in both dimensions of 2D-LC discussed. • Reversed phase offers many advantages compared to other LC separation mechanisms. • 2D-RPLC used in many fields, including food, natural product, environmental analysis, etc. Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics. [ABSTRACT FROM AUTHOR]

Microplastic is ubiquitous in the environment. Recently it was discovered that microplastic (MP, 1 μm−5 mm) contamination is present in the atmosphere where it can be transported over long distances and introduced to remote pristine environments. Sources, concentration levels, and transportation pathways of MP are still associated with large uncertainties. The abundance of atmospheric MP increases with decreasing particle size, suggesting that nanoplastics (NP, < 1 μ m) could be of considerable atmospheric relevance. Only few analytical methods are available for detection of nanosized plastic particles. Thermoanalytical techniques are independent of particle size and are thus a powerful tool for MP and NP analysis. Here we develop a method for analysis of polystyrene on the nanogram scale using pyrolysis gas chromatography coupled to mass spectrometry. Pyrolysis was performed using a slow temperature ramp, and analytes were cryofocused prior to injection. The mass spectrometer was operated in selected ion monitoring (SIM) mode. A lower limit of detection of 1 ± 1 ng and a lower limit of quantification of 2 ± 2 ng were obtained (for the trimer peak). The method was validated with urban ▪ matrices of low (7 μg per sample) and high (53 μg per sample) aerosol mass loadings. The method performs well for low ▪ loadings, whereas high ▪ loadings seem to cause a matrix effect reducing the signal of polystyrene. This effect can be minimized by introducing a thermal desorption step prior to pyrolysis. The study provides a novel analysis method for qualitative and semi-quantitative analysis of PS on the nanogram scale in an aerosol matrix. Application of the method can be used to obtain concentration levels of polystyrene in atmospheric MP and NP. This is important in order to improve the understanding of the sources and sinks of MP and NP in the environment and thereby identify routes of exposure and uptake of this emerging contaminant. • Novel analysis method for atmospheric polystyrene nanoparticles. • Novel pyrolysis temperature profile using a slow temperature ramp. • Detection limit of polystyrene <1 ng. • Matrix effect found at high but not low levels of urban PM 2.5. • A thermal desorption step is recommended for ambient aerosol samples. [ABSTRACT FROM AUTHOR]

• A mixed-mode chromatography method for quantitation of organic bases was developed. • A pH gradient was employed to adjust the retention of charged analytes. • Sufficient sensitivity was obtained without requiring mass spectrometry detection. • Mixed-mode chromatography may be suitable for academic and industrial environments. The use of organic bases is ubiquitous in chemical synthesis, yet quantifying these compounds with traditional HPLC methodologies is often hampered by poor peak shape, low retention, and limited UV absorption. When employed in the manufacture of an active pharmaceutical ingredient (API), these compounds must be controlled to levels that are safe for human consumption, requiring robust analytical methods with sufficiently low quantification limits. This work describes the development of an HPLC method for the quantification of imidazole and 1,8-Diazabicyclo[5.4.0]undec‑7-ene (DBU) in an API using mixed-mode chromatography. Through control of the pH and organic modifier gradients, the retention of the basic analytes and API can be tuned independently to achieve desirable retention and sensitivity for each compound. The resulting HPLC method exhibits good performance in linearity, accuracy, sensitivity, specificity, and solution stability. Notably, these conditions avoid more complex detection modalities, such as mass spectrometry, while maintaining a system pressure below 400 bar, making the method compatible with a broad range of instruments. This approach to mixed-mode chromatography method development could be extended to different organic bases in the presence of complex molecules to fit the needs of projects in an academic or industrial environment. [ABSTRACT FROM AUTHOR]

• 2D-LC separation of oligonucleotides using IPRP separations in both dimensions. • Elution conditions are systematically discovered through iterative retention modeling. • 2D-LC separations provide both the selectivity and sensitivity needed for impurity profiling. • Application to therapeutic siRNA single strands from solid phase oligonucleotide synthesis. Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations. [ABSTRACT FROM AUTHOR]

• Method development and data processing are essential for a robust GC × GC analysis. • Theoretical modeling improves the quality of chromatographic separation. • Machine learning-based workflows can enhance data processing in GC × GC–MS analyses. • Traditional statistical methods fall short with non-compliant datasets. In recent years, comprehensive two-dimensional gas chromatography (GC × GC) has been gradually gaining prominence as a preferred method for the analysis of complex samples due to its higher peak capacity and resolution power compared to conventional gas chromatography (GC). Nonetheless, to fully benefit from the capabilities of GC × GC, a holistic approach to method development and data processing is essential for a successful and informative analysis. Method development enables the fine-tuning of the chromatographic separation, resulting in high-quality data. While generating such data is pivotal, it does not necessarily guarantee that meaningful information will be extracted from it. To this end, the first part of this manuscript reviews the importance of theoretical modeling in achieving good optimization of the separation conditions, ultimately improving the quality of the chromatographic separation. Multiple theoretical modeling approaches are discussed, with a special focus on thermodynamic-based modeling. The second part of this review highlights the importance of establishing robust data processing workflows, with a special emphasis on the use of advanced data processing tools such as, Machine Learning (ML) algorithms. Three widely used ML algorithms are discussed: Random Forest (RF), Support Vector Machine (SVM), and Partial Least Square–Discriminate Analysis (PLS-DA), highlighting their role in discovery-based analysis. [ABSTRACT FROM AUTHOR]

• SBSE was successfully optimized for the determination of seven phthalates in pisco. • 40 mL of pisco with 20 % NaCl were extracted for 120 min employing PDMS stir bars. • The validation of the method provided good analytical performance characteristics. • The method presented a greener character compared with previous methodologies. • DBP, BEHP and BBP were the most frequent compounds found in pisco samples. In this work, for the first time, a stir bar sorptive extraction (SBSE) coupled with thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) was developed and validated for the determination of seven phthalates in Peruvian pisco. The phthalate compounds considered were dimethyl phthalate (DMP), diethyl phthalate (DEP), bis(2-ethylhexyl) hexahydrophthalate (BEHP), benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), di-isodecyl phthalate (DIDP) and di-isobutyl phthalate (DIBP). The best overall analytical conditions obtained from the optimization were as follow: extraction time of 120 min, size of polydimethylsiloxane (PDMS) twister (20 mm length x 1 mm thickness), NaCl content (20 %) and sample volume (40 mL). The in-house validation of SBSE/TD–GC–MS method was performed taking into account the ISO/IEC 17,025 requirements and EURACHEM/CITAC guideline. Under optimal conditions, very low limits of detection of 1.3–0.21 µg L−1 were obtained. Furthermore, the limits of quantification ranged from 4.2–70 µg L−1, and the correlation coefficients were found to be ≥ 0.991. The method was precise, with relative standard deviations (RSD, %) for inter twister repeatability and the inter day repeatability precisions from 1.1 to 11 and from 6.2 to 15.9, respectively. The pisco samples were analysed with recoveries between 91–124.4%, and DBP, BEHP, and BBP were the most commonly found compounds in the samples. The optimized methodology was also evaluated in terms of green character, and it obtained almost the best AGREE score when it was compared with other previous methods for the analysis of phthalates in alcoholic beverages. Therefore, the SBSE/TD–GC–MS method has proved to be suitable for routine practice because it is simple, less laborious, economical, precise, accurate and green, and it would be applicable for pisco safety regulations. [ABSTRACT FROM AUTHOR]

Highlights • Peak distortion due to the activity of carbonyls in RP-LC separation was studied. • A simple and effective solution to this problem was proposed for the first time. • The corresponding mechanism was explicitly revealed and demonstrated. Abstract Peak distortion is frequently encountered for compounds containing active carbonyl groups during reversed-phase (RP) LC separation. However, as being commonly overlooked or misdiagnosed, this problem is rarely reported in the literature and lacks an effective solution. In the present study, six pharmaceutical-related compounds containing keto or aldehyde groups, that exhibited severe peak distortion in early method development, were selected as the model compounds for further investigation. Systematic pH-screening experiments and a series of LC quadrupole-time of flight (Q-TOF) MS and NMR experiments were conducted on each model compound. The underlying chemical behavior of this type of compound during RP-LC separation was explicitly revealed. The formation of gem-diol/hemiketal/hemiacetal species via nucleophilic addition with H 2 O/MeOH will occur in a low pH eluent, but will be completely suppressed when the pH is higher than an analyte-specific value. As further validated by twelve other pharmaceutical-related compounds belonging to this type, we confirmed that increasing the eluent pH using buffers without nucleophilicity is a simple and effective method to solve this peak distortion problem. [ABSTRACT FROM AUTHOR]

Highlights • Rapid separation-direct analysis method analyzing 48 elements in a single sample. • The average isotopic detection limit is at the low picogram level. • Chromatogram peak areas used to measure isotope ratios. • Lanthanide elemental concentrations rapidly determined within a 1–2% uncertainty. • Silicon-based matrix has little effect on detection limits or retention times. Abstract An automated separation–direct analysis scheme has been developed to determine both the concentration and isotopic composition of a suite of elements down to the low picogram level in a complex silicon-based matrix. With the ultimate goal of performing rapid analysis of materials with non-natural isotopic compositions, RAPID (Rapid Analysis of Post-Irradiation Debris) consists of a high-pressure ion chromatography system directly coupled to an inductively coupled plasma mass spectrometer. The RAPID method achieves matrix exclusion and direct online analysis of the elementally separated components, yielding precise isotopic compositions for up to 40 elements in less than one hour per sample. When combined with isotope dilution, this approach shows the potential to yield elemental concentrations with low uncertainties, providing a rapid analytical method that encompasses group I and II metals, transition metals, refractory metals, platinum group metals, lanthanides, and actinides. The method development, robustness, sensitivity, uncertainties, and potential applications in nuclear and environmental measurements will be discussed in this paper. [ABSTRACT FROM AUTHOR]

Highlights • Generic ion-exchange method for analytical and preparative separation of nucleotides. • Chromatographic resolution of over 20 nucleotides in a single experimental run. • The use of a cost-effective resin available in bulk format for preparative separation is demonstrated. • Aqueous ammonium bicarbonate-based eluent profile allows convenient drying process. • Fully implemented for preparative separation of nucleotides in multicomponent reaction mixtures. Abstract Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5P W resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those investigated in this study. [ABSTRACT FROM AUTHOR]

Highlights • Selectivity and retention changes in HILIC upon addition of 1–20 mM electrolyte to mobile phase. • Cationic increase in retention and anions decrease in retention. • Magnitude of retention changes follow Hofmeister series. • A new tool in method development for a broad range of HILIC applications. Abstract This work investigates the selectivity and retention changes on four classes of HILIC columns upon addition of 1–20 mM electrolyte. The effect of four electrolytes (Na+Cl−, Na+ClO 4 −, Na+PF 6 −, and Na+CF 3 CO 2 −) were tested on neutral, cationic, anionic, and zwitterionic analytes under HILIC conditions (70–90% ACN). These electrolytes alter the retention and selectivity on silica, zwitterionic and diol columns through ion exchange and ionic screening mechanisms. Neutral analytes were unaffected by addition of 1–20 mM electrolyte, indicating minimal change to the retentivity of the water layer. Cationic and anionic analytes increase and decrease in retention, respectively (Na+PF 6 − ≈ Na+ClO 4 − > Na+CF 3 CO 2 − > Na+Cl−). Chaotropic electrolytes accumulate at the mobile phase/water layer interface, enhancing the ion exchange and ionic screening effects of the mobile phase electrolyte. Altering the buffer cation (Li+, Na+, K+) caused small but statistically significant changes in retention. [ABSTRACT FROM AUTHOR]

Highlights • Systematic errors in t r,2 prediction result when modeling thermal GC × GC modulation. • t r,2 modeling error is a function of analyte temperature and mobile phase velocity when eluting from the second dimension column. • Derived modeling correction factor applies to several analyte functionalities. Abstract Thermodynamic retention modeling to a thermally modulated comprehensive two-dimensional gas chromatography (GC × GC) system run under constant flow is performed. Significant errors in modeled second dimension retention time (t r,2) were observed, in line with past work on thermally modulated GC × GC modeling. A comprehensive study of t r,2 modeling error for alkane separations across a wide range of heating ramp rates and carrier gas flow rates was performed. Modeling errors were found to be systematic and a function of analyte elution temperature and mobile phase velocity. A model to account for these systematic errors was generated, and associated coefficients were determined which reduced average t r,2 retention time error in 144 hydrocarbon separations by an order of magnitude resulting in significant improvement in prediction accuracy. The model was used to correct the separation of 139 Grob mix analyte separations, providing an average t r,2 modeling error of 0.030 ± 0.022 s. The model successfully predicted the separation of n-alkanes on a longer second dimension column configuration. [ABSTRACT FROM AUTHOR]

Highlights • A new LC–MS method is described for CIT and OTA in several food and feed matrices. • The method was validated for recovery, linearity, precision, matrix effect and LOD/LOQ. • The method was successfully applied to 9 food groups and feed matrices. • CIT and OTA co-occurred in chicken and pig feedstuffs at low concentrations (

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]

Highlights • Certification of a new ginsenosides calibration solution SRM 3389. • Column selectivity optimization of flow rate, mobile phase, and column temperature. • Development of a LC/UV and LC/MS methods for the determination of 12 ginsenosides. Abstract The research presented here describes the development of two analytical methods for use in the certification of a ginsenoside calibration solution Standard Reference Material (SRM) 3389 consisting of seven ginsenosides: Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd. The new methods utilized the liquid chromatographic (LC) separation of ginsenoside mixtures with absorbance detection (UV) and mass spectrometry (MS). Ginsenosides Rb3, Rg2, Rg3, Rh1, and Rh2 were evaluated for use as internal standards for LC/MS measurements. The 12 ginsenosides were baseline resolved by gradient elution LC/UV, with an initial mobile phase composition of 22% acetonitrile and 78% water, flow rate of 0.7 mL/min, and column temperature of 25 °C. The work presented here includes a detailed investigation into the optimization of the chromatographic conditions to minimize measurement biases that result from unresolved constituents. Temperature and mobile phase composition are known to play a significant role in column selectivity; however, flow rate is expected to influence primarily the separation efficiency and detection sensitivity. In the current study, column selectivity changed with changes in flow rate and the relative retention of ginsenoside Rg2 and Rh1 changed as the flow rate increased from 0.6 mL/min to 1.0 mL/min. [ABSTRACT FROM AUTHOR]

• A new method development strategy for unified chromatography is introduced. • Innovative use of mobile phase additives based on their chaotropic/kosmotropic properties. • New method development approach identifies best conditions in less than two hours. • Unified chromatography shows high usefulness in the context of drug development. Recent trends in supercritical fluid chromatography (SFC) introduced an innovative gradient profile called Unified Chromatography (UC), which pushes the amount of liquid modifier up to 80–100 % of the total mobile phase composition. These new conditions allow the full transition from a supercritical to a liquid state, unifying the benefits of both SFC and liquid chromatography. However, to facilitate the use of UC for industrial drug development, a stronger effort is needed to streamline and simplify its method development and optimization. In this work, a quick and novel method development procedure for UC is introduced, enabled by the first-time use of novel additives in SFC/UC that exploit chaotropic/kosmotropic properties. A comprehensive view on some fundamental properties, such as the amount of liquid modifier blended with supercritical CO 2 (scCO 2) and the percentage of water added in the mobile phase is given, to clarify the benefits of using either a chaotropic salt (NaClO 4), kosmotropic (HCOONa) or salt with mixed properties (NaOMs – sodium methanesulfonate). With this expanded knowledge, challenging separations of nucleosides, nucleotide, indoles, triazoles and related derivates have been accomplished with UC. Finally, we provide an example of UC delivering a faster and better method for an AbbVie pipeline compound under accelerated stability study. The combined use of scCO 2 -based chromatography and the novel additive NaClO 4 ensures the retention and elution of all degradation species generated at different conditions, where RP-HPLC failed to provide satisfactory performance. [ABSTRACT FROM AUTHOR]

• An interface for automated closed-loop method development of LC × LC was designed. • Retention times were predicted in shifting gradients in the second dimension. • Within a few iterations, a separation of a complex mAb sample was improved. • The algorithm is modular by design and can be applied to other samples. Method development in comprehensive two-dimensional liquid chromatography (LC × LC) is a complicated endeavor. The dependency between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, renders method development by "trial-and-error" time-consuming and highly dependent on user experience. In this work, an open-source algorithm for the automated and interpretive method development of complex gradients in LC × LC-mass spectrometry (MS) was developed. A workflow was designed to operate within a closed-loop that allowed direct interaction between the LC × LC-MS system and a data-processing computer which ran in an unsupervised and automated fashion. Obtaining accurate retention models in LC × LC is difficult due to the challenges associated with the exact determination of retention times, curve fitting because of the use of gradient elution, and gradient deformation. Thus, retention models were compared in terms of repeatability of determination. Additionally, the design of shifting gradients in the second dimension and the prediction of peak widths were investigated. The algorithm was tested on separations of a tryptic digest of a monoclonal antibody using an objective function that included the sum of resolutions and analysis time as quality descriptors. The algorithm was able to improve the separation relative to a generic starting method using these complex gradient profiles after only four method-development iterations (i.e., sets of chromatographic conditions). Further iterations improved retention time and peak width predictions and thus the accuracy in the separations predicted by the algorithm. [ABSTRACT FROM AUTHOR]

Highlights • Selectivity of mixed-mode reversed-phase/weak-anion-exchange columns were evaluated. • RP and IEX retention mechanisms were investigated for different compound classes. • A novel model combining mixed-mode retention mechanisms was proposed and validated. Abstract The current study comprises a systematic investigation to assess retention properties and selectivity of a mixed-mode reversed-phase/weak-anion-exchange (RP/WAX) stationary phase to aid method development. Retention was investigated for different compound classes which vary in hydrophobicity, van der Waals surface area, and charge as function of organic content, pH, and ionic strength of the mobile phase. The linear-solvent-strength model was successfully applied for aromatic hydrocarbons to obtain retention-time predictions based on log P values and van der Waals surface area values. For phenols, predictions were based on log P values and data from a single scouting run performed in isocratic mode to estimate the S parameter; the deviations between experimental and predicted retention times were smaller than 6%. To describe the mixed-mode (RP/WAX) retention behavior of singly and doubly negatively-charged aromatic acids, a novel model combining the linear-solvent-strength and the empirical stoichiometric-displacement-net-charge models is proposed and validated. Using combinations of three scouting runs that are not linearly dependent, the maximum prediction error was 11% and changes in selectivity were correctly forecasted when altering the mobile-phase composition, i.e. , either organic modifier content or ionic strength. When using nine scouting runs in combination with a least-squares regression approach to determine the model parameters, the maximum prediction error was 6%. [ABSTRACT FROM AUTHOR]

In various fields of endocrinology, the determination of steroid hormones synthesised by the human body plays an important role. Research on central neurosteroids has been intensified within the last years, as they are discussed as biomarkers for various cognitive disorders. Their concentrations in cerebrospinal fluid (CSF) are considered to be regulated independently from peripheral fluids. For that reason, the challenging matrix CSF becomes a very interesting specimen for analysis. Concentrations are expected to be very low and available amount of CSF is limited. Thus, a comprehensive method for very sensitive quantification of a set of analytes as large as possible in one analytical aliquot is desired. However, high structural similarities of the selected panel of 51 steroids and steroid sulfates, including numerous isomers, challenges achievement of chromatographic selectivity. Since decades the analysis of endogenous steroids in various body fluids is mainly performed by gas chromatography (GC) coupled to (tandem) mass spectrometry (MS(/MS)). Due to the structure of the steroids of interest, derivatisation is performed to meet the analytical requirements for GC–MS(/MS). Most of the laboratories use a two-step derivatisation in multi-analyte assays that was already published in the 1980s. However, for some steroids this elaborate procedure yields multiple isomeric derivatives. Thus, some laboratories utilize (ultra) high performance liquid chromatography ((U)HPLC)–MS/MS as alternative but, even UHPLC is not able to separate some of the isomeric pairs. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to GC and (U)HPLC may help to overcome these issues. Within this project the two most promising methods for endogenous steroid profiling were investigated and compared: the “gold standard” GC–MS and the orthogonal separation technique SFC-MS/MS. Different derivatisation procedures for gas chromatographic detection were explored and the formation of multiple derivatives described and confirmed. Taken together, none of the investigated derivatisation procedures provided acceptable results for further method development to meet the requirements of this project. SFC with its unique selectivity was able to overcome these issues and to distinguish all selected steroids, including (pro‐)gestagens, androgens, corticoids, estrogens, and steroid sulfates with appropriate selectivity. Valued especially in the separation of enantiomeric analytes, SFC has shown its potential as alternative to GC. The successful separation of 51 steroids and steroid sulfates on different columns is presented to demonstrate the potential of SFC in endogenous steroid profiling. [ABSTRACT FROM AUTHOR]

Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer’s disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell particles using H 3 PO 4 in H 2 O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities. [ABSTRACT FROM AUTHOR]

Computer-aided method-development programs require accurate models to describe retention and to make predictions based on a limited number of scouting gradients. The performance of five different retention models for hydrophilic-interaction chromatography (HILIC) is assessed for a wide range of analytes. Gradient-elution equations are presented for each model, using Simpson’s Rule to approximate the integral in case no exact solution exists. For most compound classes the adsorption model, i.e. a linear relation between the logarithm of the retention factor and the logarithm of the composition, is found to provide the most robust performance. Prediction accuracies depended on analyte class, with peptide retention being predicted least accurately, and on the stationary phase, with better results for a diol column than for an amide column. The two-parameter adsorption model is also attractive, because it can be used with good results using only two scanning gradients. This model is recommended as the first-choice model for describing and predicting HILIC retention data, because of its accuracy and linearity. Other models (linear solvent-strength model, mixed-mode model) should only be considered after validating their applicability in specific cases. [ABSTRACT FROM AUTHOR]

• LC-MS and data science employed for pharmaceutical analysis research. • MS response scouted using 240 small molecular weight probes. • Surface area and surface charge main compound features impacting ionization. • Methanol, acetonitrile with formic acid or ammonium acetate best average response. Mobile phase selection is of critical importance in liquid chromatography – mass spectrometry (LC-MS) based studies, since it affects retention, chromatographic selectivity, ionization, limits of detection and quantification, and linear dynamic range. Generalized LC-MS mobile phase selection criteria, suitable for a broad class of chemical compounds, do not exist thus far. Here we have performed a large-scale qualitative assessment of the effect of solvent composition used for reversed-phase LC separations on electrospray ionization (ESI) response for 240 small molecular weight drugs, representing various chemical compound classes. Of these 240 analytes 224 were detectable using ESI. The main chemical structural features affecting ESI response were found to all be surface area or surface charge-related. Mobile phase composition was found to be less differentiating, although for some compounds a pH effect was noted. Unsurprisingly, chemical structure was found to be the dominant factor for ESI response for the majority of the investigated analytes, representing about 85% of the replicating detectable complement of the sample data set. A weak correlation between ESI response and structure complexity was observed. Solvents based on isopropanol, and those containing phosphoric or di- and trifluoracetic acids, performed relatively poorly in terms of chromatographic or ESI response, whilst the best performing 'generic' LC solvents were based on methanol, acetonitrile using formic acid and ammonium acetate as buffer components, consistent with current practice in many laboratories. [ABSTRACT FROM AUTHOR]

Volatile amines are among the most frequently used chemicals in organic and pharmaceutical chemistry. Synthetic route optimization often involves the evaluation of several different amines requiring the development and validation of analytical methods for quantitation of residual amine levels. Herein, a simple and fast generic GC-FID method on an Agilent J&W CP-Volamine capillary column (using either He or H 2 as the carrier gas) capable of separating over 25 volatile amines and other basic polar species commonly used in pharmaceutical chemistry workflows is described. This 16 min method is successfully applied to the analysis and quantitation of volatile amines in a variety of pharmaceutically-related drugs and synthetic intermediates. Method validation experiments showed excellent analytical performance in linearity, recovery, repeatability, and limit of quantitation and detection. In addition, diverse examples for the application of this method to the simultaneous determination of other amine-related chemicals in reaction mixtures are illustrated, thereby indicating that these GC-FID method conditions can be effectively used as starting point during method development for the analysis of other basic polar species beyond the validated list of amines described in this study. [ABSTRACT FROM AUTHOR]

Internal standards are essential in electrospray ionization liquid chromatography-mass spectrometry (ESI-LC–MS) to correct for systematic error associated with ionization suppression and/or enhancement. A wide array of instrument setups and interfaces has created difficulty in comparing the quantitation of absolute analyte response across laboratories. This communication demonstrates the use of primary standards as operational qualification standards for LC–MS instruments and their comparison with commonly accepted internal standards. In monitoring the performance of internal standards for perfluorinated compounds, potassium hydrogen phthalate (KHP) presented lower inter-day variability in instrument response than a commonly accepted deuterated perfluorinated internal standard (d3-PFOS), with percent relative standard deviations less than or equal to 6%. The inter-day precision of KHP was greater than d3-PFOS over a 28-day monitoring of perfluorooctanesulfonic acid (PFOS), across concentrations ranging from 0 to 100 μg/L. The primary standard trometamol (Trizma) performed as well as known internal standards simeton and tris (2-chloroisopropyl) phosphate (TCPP), with intra-day precision of Trizma response as low as 7% RSD on day 28. The inter-day precision of Trizma response was found to be greater than simeton and TCPP, across concentrations of neonicotinoids ranging from 1 to 100 μg/L. This study explores the potential of primary standards to be incorporated into LC–MS/MS methodology to improve the quantitative accuracy in environmental contaminant analysis. [ABSTRACT FROM AUTHOR]

A novel mixing unit is proposed for the serial coupling of orthogonal columns to analyze polar and non-polar compounds in a single run. The principle relies on the isolation of unretained peaks eluting from a first dimension column in a sample loop, before directing them to a second column for separation. Since the mobile phases employed in highly orthogonal separations are not directly compatible, a mixing unit is required to alter the mobile phase composition before executing the second dimension separation. The mixing unit proposed in this work is based on the use of two restriction capillaries with different flow resistances to dilute the mobile phase eluting from the first dimension with a solvent appropriate for the second dimension separation. The restriction capillaries are implemented in an ultra-high performance liquid chromatography set-up using three high-pressure switching valves and two T-pieces. It is demonstrated that the dilution ratio can be adequately predicted using the law of Hagen-Poiseuille and can be adjusted easily by changing the dimensions of the restriction capillaries. The dilution volume required to obtain acceptable recoveries is investigated and the use of different column diameters in the first and second dimension is proposed to increase the sensitivity of the analysis. Under optimum dilution conditions, recoveries ranging between 82% and 99% are always obtained, while repeatability values are excellent. The proof-of-concept of the different set-ups is demonstrated for the separation of 20 pharmaceuticals with log D-values ranging between −5.75 and 4.22. [ABSTRACT FROM AUTHOR]

The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC. Another important advantage of the logarithmic gradient, is its peak focusing effect for the DAR0 species, which is particularly useful to improve the quantitation limit of DAR0. Finally, the logarithmic behavior of DAR species of ADC in HIC was modelled using two different approaches, based on i) the linear solvent strength theory (LSS) and two scouting linear gradients and ii) a new derived equation and two logarithmic scouting gradients. In both cases, the retention predictions were excellent and systematically below 3% compared to the experimental values. [ABSTRACT FROM AUTHOR]

Large pressure gradients are generated in ultra-high-pressure liquid chromatography (UHPLC) using sub–2 μm particles causing significant temperature gradients over the column due to viscous heating. These pressure and temperature gradients affect retention and ultimately result in important selectivity shifts. In this study, we developed an approach for predicting the retention time shifts due to these gradients. The approach is presented as a step-by-step procedure and it is based on empirical linear relationships describing how retention varies as a function of temperature and pressure and how the average column temperature increases with the flow rate. It requires only four experiments on standard equipment, is based on straightforward calculations, and is therefore easy to use in method development. The approach was rigorously validated against experimental data obtained with a quality control method for the active pharmaceutical ingredient omeprazole. The accuracy of retention time predictions was very good with relative errors always less than 1% and in many cases around 0.5% ( n = 32). Selectivity shifts observed between omeprazole and the related impurities when changing the flow rate could also be accurately predicted resulting in good estimates of the resolution between critical peak pairs. The approximations which the presented approach are based on were all justified. The retention factor as a function of pressure and temperature was studied in an experimental design while the temperature distribution in the column was obtained by solving the fundamental heat and mass balance equations for the different experimental conditions. We strongly believe that this approach is sufficiently accurate and experimentally feasible for this separation to be a valuable tool when developing a UHPLC method. After further validation with other separation systems, it could become a useful approach in UHPLC method development, especially in the pharmaceutical industry where demands are high for robustness and regulatory oversight. [ABSTRACT FROM AUTHOR]

• Developed a workflow to prioritize single and tandem-column pairs for RPLC separations. • Separation predictions based upon hydrophobic subtraction model (HSM) parameters. • Methodology applied to separation of Linrodostat and 10 related impurities. • Prediction of potentially successful columns greatly simplifies method development. • For the Linrodostat study, tandem-column LC outperformed single column LC under HSM conditions. The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (R c) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. R c values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental R c values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database. [Display omitted] [ABSTRACT FROM AUTHOR]

• Separations were conducted at 1500 bar using 1.5 µm core-shell particles. • The effects of flow rate and gradient steepness on peak capacity have been assessed. • The statistical overlap theory has been applied to assess resolving power. The present study discusses UHPLC method development allowing to establish ultra-high-resolution separations in gradient mode while operating at the kinetic performance limits, targeting the analysis of complex residual multi-class antibiotic samples in food products. The peak capacity and gradient occupation have been systematically assessed at different flow rates and gradient duration. The small particle size (1.5 µm core-shell particles) used in this study limits the mass-transfer contribution to band broadening when operating at high flow rate. As a result, for high-throughput analysis, high-pressure (1500 bar) operation leads to high resolving power where the gradient steepness dominates the peak capacity generation vs mass-transfer resistance. To reach the highest possible resolving power within a practically acceptable analysis time, one should use coupled-column systems at 1500 bar and adjust the gradient steepness correspondingly. Coupling four columns and applying a shallow gradient at 1500 bar led to a sample peak capacity of 379 in 140 min, allowing to resolve 71% of the analytes in a mixture composed of 61 milk antibiotics. [ABSTRACT FROM AUTHOR]

The challenge of fully optimizing LC × LC separations is horrendous. Yet, it is essential to address this challenge if sophisticated LC × LC instruments are to be utilized to their full potential in an efficient manner. Currently, lengthy method development is a major obstacle to the proliferation of the technique, especially in industry. A program was developed for the rigorous optimization of LC × LC separations, using gradient-elution in both dimensions. The program establishes two linear retention models (one for each dimension) based on just two LC × LC experiments. It predicts LC × LC chromatograms using a simple van-Deemter model to generalize band-broadening. Various objectives (analysis time, resolution, orthogonality) can be implemented in a Pareto-optimization framework to establish the optimal conditions. The program was successfully applied to a separation of a complex mixture of 54 aged, authentic synthetic dyestuffs, separated by ion-exchange chromatography and ion pair chromatography. The main limitation experienced was the retention-time stability in the first (ion-exchange) dimension. Using the PIOTR program LC × LC method development can be greatly accelerated, typically from a few months to a few days. [ABSTRACT FROM AUTHOR]

Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7 ≤ cLogP ≤ 2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO 2 -methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO 2 -methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7 min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance. [ABSTRACT FROM AUTHOR]

Supercritical fluid chromatography (SFC) has recently benefited of new instrumentation, together with the availability of many ultra-high performance columns (sub −2 μm fully porous particles or sub −3 μm superficially porous particles), rendering it more attractive than ever. Most of these columns commonly used in SFC were initially developed for HPLC use, with an increasing number of stationary phases specifically designed for SFC. While the availability of different stationary phase chemistries is an advantage to achieve successful SFC separations, selecting a column for method development remains difficult. For this reason, we have previously developed a classification of stationary phases dedicated to SFC use. It is based on linear solvation energy relationships (LSER) with Abraham descriptors (for neutral species). While current interest in SFC is strong in the pharmaceutical industry, the need to take account of interactions occurring with ionisable species is pressing. We have previously shown how a modified version of the solvation parameter model, adapted to take account of ionic and ionizable species, could be applied to the characterization of SFC systems. In the present paper, based on this modified LSER model, and on the analysis of 109 neutral and ionisable species, we propose an improved classification of 31 ultra-high performance stationary phases to facilitate method development with SFC. [ABSTRACT FROM AUTHOR]

Flow-Field Flow Fractionation (Flow-FFF), coupled with online detection systems is one of the most promising tools available for the separation and quantification of engineered nanoparticles (ENPs) in complex matrices. To correctly relate the retention of nanoparticles in the Flow-FFF-channel to the particle size, ideal separation conditions must be met. This requires optimization of the parameters that influence the separation behavior. The aim of this study was therefore to systematically investigate and evaluate the influence of parameters such as the carrier liquid, the cross flow, and the membrane material, on the separation behavior of two metallic ENPs. For this purpose the retention, recovery, and separation efficiency of sterically stabilized silver nanoparticles (AgNPs) and electrostatically stabilized gold nanoparticles (AuNPs), which represent two materials widely used in investigations on environmental fate and ecotoxicology, were investigated against a parameter matrix of three different cross-flow densities, four representative carrier solutions, and two membrane materials. The use of a complex mixture of buffers, ionic and non-ionic surfactants (FL-70 solution) together with a medium cross-flow density provided an acceptable compromise in peak quality and recovery for both types of ENPs. However, these separation conditions do not represent a perfect match for both particle types at the same time (maximized recovery at maximized retention). It could be shown that the behavior of particles within Flow-FFF channels cannot be predicted or explained purely in terms of electrostatic interactions. Particles were irreversibly lost under conditions where the measured zeta potentials suggested that there should have been sufficient electrostatic repulsion to ensure stabilization of the particles in the Flow-FFF channel resulting in good recoveries. The wide variations that we observed in ENP behavior under different conditions, together with the different behavior that has been reported in published literature for the same NPs under similar conditions, indicate a need for improvement in the membrane materials used for Flow-FFF analysis of NPs. This research has shown that careful adjustment of separation conditions can result in acceptable, but not ideal, separation conditions for two fundamentally different stabilized materials, and that it may not be possible to separate a set of different particles under ideal conditions for each particle type. This therefore needs to be taking into account in method development and when interpreting FFF results from complex samples. [ABSTRACT FROM AUTHOR]

In this study, a new supercritical fluid chromatography–mass spectrometry (SFC–MS) method has been developed for the separation of nine vitamin D metabolites within less than eight minutes. This is the first study of analysis of vitamin D and its metabolites carried out by SFC–MS. Six columns of orthogonal selectivity were examined, and the best separation was obtained by using a 1-aminoanthracene (1-AA) column. The number and the position of hydroxyl groups in the structure of the studied compounds as well as the number of unsaturated bonds determine the physiochemical properties and, thus the separation of vitamin D metabolites that is achieved on this column. All D 2 and the D 3 forms were baseline separated with resolution values > 1.5. The effects of pressure, temperature, flow rate and different gradient modes were studied. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared in positive mode, both by direct infusion and after SFC separation. The results showed that the sensitivity in APCI + was higher than in ESI + using direct infusion. In contrast, the sensitivity in APCI + was 6-fold lower than in ESI + after SFC separation. The SFC–MS method was validated between 10 and 500 ng/mL for all analytes with coefficient of determination (R 2 ) ≥ 0.999 for all calibration curves. The limits of detection (LOD) were found to range between 0.39 and 5.98 ng/mL for 24,25-dihydroxyvitamin D 3 (24,25(OH) 2 D 3 ) and 1-hydroxyvitamin D 2 (1OHD 2 ), respectively. To show its potential, the method was applied to human plasma samples from healthy individuals. Vitamin D 3 (D 3 ), 25-hydroxyvitamin D 3 (25OHD 3 ) and 24,25(OH) 2 D 3 were determined in plasma samples and the concentrations were 6.6 ± 3.0 ng/mL, 23.8 ± 9.2 ng/mL and 5.4 ± 2.7 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

Analyses of complex samples of cosmetics, such as creams or lotions, are generally achieved by HPLC. These analyses are often multistep gradients, due to the presence of compounds with a large range of polarity. For instance, the bioactive compounds may be polar, while the matrix contains lipid components that are rather non-polar, thus cosmetic formulations are usually oil-water emulsions. Supercritical fluid chromatography (SFC) uses mobile phases composed of carbon dioxide and organic co-solvents, allowing for good solubility of both the active compounds and the matrix excipients. Moreover, the classical and well-known properties of these mobile phases yield fast analyses and ensure rapid method development. However, due to the large number of stationary phases available for SFC and to the varied additional parameters acting both on retention and separation factors (co-solvent nature and percentage, temperature, backpressure, flow rate, column dimensions and particle size), a simplified approach can be followed to ensure a fast method development. First, suited stationary phases should be carefully selected for an initial screening, and then the other operating parameters can be limited to the co-solvent nature and percentage, maintaining the oven temperature and back-pressure constant. To describe simple method development guidelines in SFC, three sample applications are discussed in this paper: UV-filters (sunscreens) in sunscreen cream, glyceryl caprylate in eye liner and caffeine in eye serum. Firstly, five stationary phases (ACQUITY UPC 2 ) are screened with isocratic elution conditions (10% methanol in carbon dioxide). Complementary of the stationary phases is assessed based on our spider diagram classification which compares a large number of stationary phases based on five molecular interactions. Secondly, the one or two best stationary phases are retained for further optimization of mobile phase composition, with isocratic elution conditions or, when necessary, two-step gradient elution. The developed methods were then applied to real cosmetic samples to assess the method specificity, with regards to matrix interferences, and calibration curves were plotted to evaluate quantification. Besides, depending on the matrix and on the studied compounds, the importance of the detector type, UV or ELSD (evaporative light-scattering detection), and of the particle size of the stationary phase is discussed. [ABSTRACT FROM AUTHOR]

This paper reports a new, easy, cheap, and fast MEPS–HPLC–PDA method for the simultaneous analysis of ciprofloxacin and levofloxacin, two fluoroquinolones (FLQs) commonly used for the treatment of pulmonary infections in cystic fibrosis (CF) patients. The FLQs were resolved on a Discovery C 8 column (250 mm × 4.6 mm; 5 μm particle size) using an isocratic elution with a run time of 15 min, without further purification. The method was validated over concentrations ranging from 0.05 to 2 μg/mL for both analytes in human sputum, and enrofloxacin was used as internal standard. This method was successfully tested to detect FLQs in sputum collected from CF patients. The MEPS–HPLC–PDA method was validated using biological samples collected from CF patients orally or intravenously injected with FLQs. The resultant data showed that the method is selective, sensitive and robust over range of concentrations for both FLQs. The limit of quantification of the method was 0.05 μg/mL for both analytes (comparable to more complex and expensive instrument configurations), weighted-matrix-matched standard curves showed a good linearity up to 2 μg/mL, and parallelism tests were also successfully assessed. The intra- and inter-day precision (RSD%) values were ≤10.4% and ≤11.1%, respectively, for all range of analysis. The intra- and inter-day trueness (Bias%) values are ranged from −11.8% to 7.25% for both antibiotic drugs. At the best of our knowledge, this is the first MEPS–HPLC–PDA based method that uses MEPS procedure for simultaneous determination of ciprofloxacin and levofloxacin in human sputum. The method was tested successfully on real sputum samples by following a conventional drug administration. Furthermore, the MEPS–HPLC–PDA based method provides more advantages to detect and analyze quickly the antibiotic drugs in biological matrices than other analytical procedures reported in literature. [ABSTRACT FROM AUTHOR]

• This paper features a first-time reported sample preparation chemistry for analysis of MIC-tyrosine adducts. • Base-catalyzed hydrolysis of hemoglobin-MIC-tyrosine adducts forms phenylmethyl carbamate. • LC-MS/MS was used to detect the analyte from MIC-exposed rats. • The method has excellent sensitivity, linearity, and dynamic range. • The method also produced remarkable accuracy and precision compared to existing methods. Methyl isocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, is a toxic industrial chemical that causes irritation and damage to the eyes, respiratory tract, and skin. Due to the high reactivity of MIC, it binds to proteins to form protein adducts. While these adducts can be used as biomarkers to verify exposure to MIC, methods to detect MIC adducts are cumbersome, typically involving enzymatic (pronase) or strong acid (Edman degradation) hydrolysis of hemoglobin. Hence, in this study, a simple method was developed which utilizes base hydrolysis of MIC-tyrosine adducts from isolated hemoglobin to form phenyl methyl carbamate (PMC), followed by rapid liquid-liquid extraction, and liquid chromatography tandem mass spectrometry analysis. The hydrolysis chemistry is the first report of base hydrolysis of a tyrosine-β-C-hydroxo phenol bond in aqueous solution. The method produced excellent sensitivity (detection limit of 0.02 mg/kg), linearity (R2 = 0.998, percent residual accuracies > 96), and dynamic range (0.06‒15 mg/kg). The accuracy and precision (100 ± 9% and < 10% relative standard deviation, respectively) of the method were outstanding compared to existing techniques. The validated method was able to detect significantly elevated levels of PMC from hemoglobin isolated from MIC-exposed rats. [ABSTRACT FROM AUTHOR]

Pentafluorophenyl-bonded silica (PFP) phases employed in high-performance liquid chromatography (HPLC) often provide very different results depending on the column manufacturer. PFP phases also provide significantly different selectivity from non-fluorinated aromatic phases. As all HPLC columns can also be employed with carbon dioxide-based mobile phases, PFP phases can also be useful to supercritical fluid chromatography (SFC). However, whether they provide adequate retention and selectivity in SFC conditions is necessary for them to find applicability with this technique. In our laboratory, a column classification for packed column SFC was designed, based on the solvation parameter model, which currently comprises data for about eighty different columns. In this paper, we present the characterization of eleven PFP phases provided by different manufacturers used with carbon dioxide–methanol mobile phases. The columns are compared to fifteen other non-fluorinated phenyl and diphenyl phases in terms of their retention and separation characteristics assessed by the solvation parameter model with five Abraham descriptors. The latter is insufficient for an accurate description of retention mechanisms on the PFP phases, thus two extra terms accounting for ionic interactions with anions and cations ( D − and D + ), previously developed for HPLC in hydrophilic interaction mode (HILIC), are introduced. While some approximations are necessary regarding the true pH and p K a values in CO 2 -based mobile phases, the retention models are significantly improved by this addition, allowing to integrate ionizable analytes in the test set for evaluation of ionic interactions in the chromatographic systems. [ABSTRACT FROM AUTHOR]

The identification of a suitable stationary phase in supercritical fluid chromatography (SFC) is a major source of difficulty for those with little experience in this technique. Several protocols have been suggested for column classification in high-performance liquid chromatography (HPLC), gas chromatography (GC), and SFC. However, none of the proposed classification schemes received general acceptance. A fair way to compare columns was proposed with the sum of ranking differences (SRD). In this project, we used the retention data obtained for 86 test compounds with varied polarity and structure, analyzed on 71 different stationary phases encompassing the full range in polarity of commercial packed columns currently available to the SFC chromatographer, with a single set of mobile phase and operating conditions (carbon dioxide–methanol mobile phase, 25 °C, 150 bar outlet pressure, 3 ml/min). First, a reference column was selected and the 70 remaining columns were ranked based on this reference column and the retention data obtained on the 86 analytes. As these analytes previously served for the calculation of linear solvation energy relationships (LSER) on the 71 columns, SRD ranks were compared to LSER methodology. Finally, an external comparison based on the analysis of 10 other analytes (UV filters) related the observed selectivity to SRD ranking. Comparison of elution orders of the UV filters to the SRD rankings is highly supportive of the adequacy of SRD methodology to select similar and dissimilar columns. [ABSTRACT FROM AUTHOR]

Countercurrent chromatography (CCC) is a form of liquid–liquid chromatography. It works by running one immiscible solvent (mobile phase) over another solvent (stationary phase) being held in a CCC column using centrifugal force. The concentration of compound in each phase is characterised by the partition coefficient ( K d ), which is the concentration in the stationary phase divided by the concentration in the mobile phase. When K d is between approximately 0.2 and 2, it is most likely that optimal separation will be achieved. Having the K d in this range allows the compound enough time in the column to be separated without resulting in a broad peak and long run time. In this paper we report the development of quantitative structure activity relationship (QSAR) models to predict log K d . The QSAR models use only the molecule's 2D structure to predict the molecular property log K d . [ABSTRACT FROM AUTHOR]

The multi-modal retention mechanism in supercritical fluid chromatography (SFC) results in a non-linear dependency of log( k ) on the fraction of organic solvent φ and log( φ ). In the present study, the possibility of retention modeling for method development purposes in SFC was investigated, considering several non-linear isocratic relationships. Therefore, both isocratic and gradient runs were performed, involving different column chemistries and analytes possessing diverse physico-chemical properties. The isocratic retention data of these compounds could be described accurately using the non-linear retention models typically used in HILIC and reversed-phase LC. The interconversion between isocratic and gradient retention data was found to be less straightforward than in RPLC and HILIC because of pressure effects. The possibility of gradient predictions using gradient scouting runs to estimate the retention parameters was investigated as well, showing that predictions for other gradients with the same starting conditions were acceptable (always below 5%), whereas prediction errors for gradients with a different starting condition were found to be highly dependent on the compound. The second part of the study consisted of the gradient optimization of two pharmaceutical mixtures (one involving atorvastatin and four related impurities, and one involving a 16 components mixture including eight drugs and their main phase I metabolites). This could be done via individual retention modeling based on gradient scouting runs. The best linear gradient was found via a grid search and the best multi-segment gradient via the previously published one-segment-per-component search . The latter improved the resolution between the critical pairs for both mixtures, while still giving accurate prediction errors (using the same starting concentrations as the gradient scouting runs used to build the model). The optimized separations were found in less than 3 h and 8 h of analysis time (including equilibration times), respectively. [ABSTRACT FROM AUTHOR]

The development of a novel set-up for the sequential analysis of compounds with a large variety in polarity on HILIC and reversed-phase columns, coupled in series, is discussed. For this purpose, a commercially available ultra-high performance LC system, equipped with two switching valves is employed. The switching valves allow connecting the HILIC and reversed-phase columns either in series or in parallel to the system. An interface to couple the HILIC and reversed-phase columns is developed and optimized. The sample is first injected onto a HILIC column. Apolar compounds in the sample are not retained and will elute close to or within the void volume of the HILIC column. Accurate switching of the valves allows redirecting these compounds towards a trap loop while more polar compounds are retained and separated on the HILIC column. After separation and detection of the polar compounds, the configuration of the valves is switched again to direct the apolar compounds from the trap loop towards a reversed-phase column for separation. To deal with the incompatibility of the mobile phases of HILIC and reversed-phase column separations, commercially available Jet weaver mixers are included in the set-up to allow for an intermediate solvent exchange. The proof-of-concept is demonstrated for the analysis of pharmaceuticals that can be found in waste water and surface water. It is demonstrated that the set-up provides robust analyses with peak capacities that are intermediate to one-dimensional and two-dimensional separations. [ABSTRACT FROM AUTHOR]

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying t G / t 0 = 5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient ( t G / t 0 = 10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion. [ABSTRACT FROM AUTHOR]

Vitamin C is one of the most important vitamins, so reliable information about its content in foodstuffs is a concern to both consumers and quality control agencies. However, the heterogeneity of food matrixes and the potential degradation of this vitamin during its analysis create enormous challenges. This review addresses the development and validation of high-performance liquid chromatography methods for vitamin C analysis in food commodities, during the period 2000–2014. The main characteristics of vitamin C are mentioned, along with the strategies adopted by most authors during sample preparation (freezing and acidification) to avoid vitamin oxidation. After that, the advantages and handicaps of different analytical methods are discussed. Finally, the main aspects concerning method validation for vitamin C analysis are critically discussed. Parameters such as selectivity, linearity, limit of quantification, and accuracy were studied by most authors. Recovery experiments during accuracy evaluation were in general satisfactory, with usual values between 81 and 109%. However, few methods considered vitamin C stability during the analytical process, and the study of the precision was not always clear or complete. Potential future improvements regarding proper method validation are indicated to conclude this review. [ABSTRACT FROM AUTHOR]

Polymeric materials are widely employed to build up tunable nanomasks for nano-patterning technologies. Ultrathin polymer layers are involved in this process. A Thermo Gravimetric Analysis–Mass Spectrometry (TGA-GC-MS) method was optimised, validated and successfully applied to investigate the thermal behavior of ultrathin poly(styrene- r -methylmethacrylate) random copolymer layers P(S-r-MMA) grafted to a silicon wafer surface. The interface between TGA and MS is highly versatile since many instrumental parameters (i.e. loop volumes, pulsed sampling frequencies, acquisition modalities, carrier gases, flow rates) can be easily tuned. Samples featuring substantial scale difference, i.e. bulk materials, thick films (few μm thickness), thin and ultrathin films (few nm thickness) can be analyzed without any instrumental modification or sample pretreatments. The TGA-GC-MS analysis was used to highlight subtle differences in samples featuring different thicknesses, in the 2–6 nm range, and subjected to various thermal treatments, thus indicating that this hyphenated technique could be successfully applied to the investigation of ultrathin polymer films. [ABSTRACT FROM AUTHOR]

This paper reports a new MEPS-HPLC-PDA method for the simultaneous analysis of seven non-steroidal anti-inflammatory drugs (Furprofen, Indoprofen, Ketoprofen, Fenbufen, Flurbiprofen, Indomethacin, and Ibuprofen) in human plasma and urine. NSAIDs were resolved on a Gemini C 18 column (4.6 mm × 250 mm; 5 μm particle size) using a gradient elution mode with a run time of 25 min, comprising re-equilibration, without further purification. The method was validated over the concentration range from 0.1 to 10 μg/mL for all the analytes both in human plasma and urine, using Benzyl 4-hydroxybenzoate as the internal standards. This method was successfully tested to NSAIDs analyses in real matrices, in order to check the method potentiality and the correct response. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 μg/mL for all analytes, and weighted-matrix-matched standard curves showed a good linearity up to 10 μg/mL. In order to check the correct response for over-range samples, parallelism tests were also assessed. In the entire analytical range the intra and inter-day precision (RSD%) values were ≤7.31% and ≤13.5%, respectively. For all the analytes the intra and inter-day trueness (Bias%) values ranged from −11.3% to 10.2%. To our knowledge, this is the first MEPS-HPLC-PDA based method that uses MEPS procedure for simultaneous determination of these seven NSAIDs in plasma and urine samples. [ABSTRACT FROM AUTHOR]

• Convolutional neural network (CNN) for automatic peak detection. • An unprocessed chromatogram is used as input. • Predicted locations, probabilities and areas of the peaks are returned. • CNN can be trained entirely on simulated chromatograms. • CNN works well for real chromatograms similar as those it has been trained on. Although commercially available software provides options for automatic peak detection, visual inspection and manual corrections are often needed. Peak detection algorithms commonly employed require carefully written rules and thresholds to increase true positive rates and decrease false positive rates. In this study, a deep learning model, specifically, a convolutional neural network (CNN), was implemented to perform automatic peak detection in reversed-phase liquid chromatography (RPLC). The model inputs a whole chromatogram and outputs predicted locations, probabilities, and areas of the peaks. The obtained results on a simulated validation set demonstrated that the model performed well (ROC-AUC of 0.996), and comparably or better than a derivative-based approach using the Savitzky-Golay algorithm for detecting peaks on experimental chromatograms (8.6% increase in true positives). In addition, predicted peak probabilities (typically between 0.5 and 1.0 for true positives) gave an indication of how confident the CNN model was in the peaks detected. The CNN model was trained entirely on simulated chromatograms (a training set of 1,000,000 chromatograms), and thus no effort had to be put into collecting and labeling chromatograms. A potential major drawback of this approach, namely training a CNN model on simulated chromatograms, is the risk of not capturing the actual "chromatogram space" well enough that is needed to perform accurate peak detection in real chromatograms. [ABSTRACT FROM AUTHOR]

Several offline and online 2D HPLC methods were investigated for the reversed phase resolution of a complex mixture of closely related warfarin and hydroxywarfarin isomers. By combining reversed phase achiral/chiral HPLC separation with UV-triggered fraction collection and subsequent chiral/achiral reversed phase HPLC analysis of collected fractions, complete resolution of all 12 components of the mixture was possible. In addition, a faster method was developed from online 2D HPLC analysis where multicomponent fractions from the first dimension are simultaneously chromatographed in the second dimension. [ABSTRACT FROM AUTHOR]