Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma.

INTRODUCTION: Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose. OBJECTIVES: The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices. METHODS: Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others. RESULTS: All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column. CONCLUSION: The results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.

Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20-100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.

LC-ESI-HRMS - lipidomics of phospholipids : Characterization of extraction, chromatography and detection parameters.

Lipids are a diverse class of molecules involved in many biological functions including cell signaling or cell membrane assembly. Owing to this relevance, LC-MS/MS-based lipidomics emerged as a major field in modern analytical chemistry. Here, we thoroughly characterized the influence of MS and LC settings - of a Q Exactive HF operated in Full MS/data-dependent MS2 TOP N acquisition mode - in order to optimize the semi-quantification of polar lipids. Optimization of MS-source settings improved the signal intensity by factor 3 compared to default settings. Polar lipids were separated on an ACQUITY Premier CSH C18 reversed-phase column (100 × 2.1 mm, 1.7 µm, 130 Å) during an elution window of 28 min, leading to a sufficient number of both data points across the chromatographic peaks, as well as MS2 spectra. Analysis was carried out in positive and negative ionization mode enabling the detection of a broader spectrum of lipids and to support the structural characterization of lipids. Optimal sample preparation of biological samples was achieved by liquid-liquid extraction using MeOH/MTBE resulting in an excellent extraction recovery > 85% with an intra-day and inter-day variability < 15%. The optimized method was applied on the investigation of changes in the phospholipid pattern in plasma from human subjects supplemented with n3-PUFA (20:5 and 22:6). The strongest increase was observed for lipids bearing 20:5, while 22:4 bearing lipids were lowered. Specifically, LPC 20:5_0:0 and PC 16:0_20:5 were found to be strongest elevated, while PE 18:0_22:4 and PC 18:2_18:2 were decreased by n3-PUFA supplementation. These results were confirmed by targeted LC-MS/MS using commercially available phospholipids as standards.

Cross-column density functional theory-based quantitative structure-retention relationship model development powered by machine learning.

Quantitative structure-retention relationship (QSRR) modeling has emerged as an efficient alternative to predict analyte retention times using molecular descriptors. However, most reported QSRR models are column-specific, requiring separate models for each high-performance liquid chromatography (HPLC) system. This study evaluates the potential of machine learning (ML) algorithms and quantum mechanical (QM) descriptors to develop QSRR models that can predict retention times across three different reversed-phase HPLC columns under varying conditions. Four machine learning methods-partial least squares (PLS) regression, ridge regression (RR), random forest (RF), and gradient boosting (GB)-were compared on a dataset of 360 retention times for 15 aromatic analytes. Molecular descriptors were calculated using density functional theory (DFT). Column characteristics like particle size and pore size and experimental conditions like temperature and gradient time were additionally used as descriptors. Results showed that the GB-QSRR model demonstrated the best predictive performance, with Q2 of 0.989 and root mean square error of prediction (RMSEP) of 0.749 min on the test set. Feature analysis revealed that solvation energy (SE), HOMO-LUMO energy gap (∆E HOMO-LUMO), total dipole moment (Mtot), and global hardness (η) are among the most influential predictors for retention time prediction, indicating the significance of electrostatic interactions and hydrophobicity. Our findings underscore the efficiency of ensemble methods, GB and RF models employing non-linear learners, in capturing local variations in retention times across diverse experimental setups. This study emphasizes the potential of cross-column QSRR modeling and highlights the utility of ML models in optimizing chromatographic analysis.

Rapid analysis of wheat gluten composition using a triple ELISA.

BACKGROUND: Gluten composition is an important quality parameter of wheat flour. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a state-of-the-art method for its analysis. As this is a very labour-intensive and time-consuming procedure, alternative faster methods are desirable. Enzyme-linked immunosorbent assay (ELISA) is a high-throughput method often used for the analysis of gluten traces in gluten-free products. In this proof-of-principle study, we introduce an experimental triple ELISA for the relative quantitation of gliadins, high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) of one wheat flour extract. RESULTS: The results of 80 common wheat flour samples obtained from the triple ELISA and RP-HPLC were correlated. The results for gliadins (r = 0.69) and HMW-GS (r = 0.81) showed a medium and high correlation, respectively. Only a very weak correlation of ELISA and RP-HPLC results was observed for LMW-GS (r = 0.49). Results for glutenins (r = 0.69) and gluten (r = 0.72) had a medium correlation. The gliadin/glutenin ratio (r = 0.47) and LMW-GS/HMW-GS ratio (r = 0.40) showed a weak or no correlation. The gliadin, LMW-GS and gluten contents were lower and the HMW-GS content was higher in the ELISA measurement compared to RP-HPLC. CONCLUSION: The quantitation of gliadins and HMW-GS by the experimental triple ELISA showed comparable results to RP-HPLC, whereas no strong correlation between the results from the two methods was found for LMW-GS. Overall, the experimental triple ELISA is suitable for relative gluten quantitation, especially for the analysis of large sample sets. Further work will focus on improving the experimental procedure of the ELISA. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Acetic Acid Ion Pairing Additive for Reversed-Phase HPLC Improves Detection Sensitivity in Bottom-up Proteomics Compared to Formic Acid.

Despite the general acceptance of formic acid as the additive of choice for peptide reversed-phase LC-MS/MS applications, some still argue that the selection of acetic acid represents a better option. To settle this debate, we investigated both the difference in MS sensitivity and chromatographic behavior of peptides between these two systems. This interlaboratory study was performed using different MS setups and C18 separation media employing both 0.1% formic and 0.5% acetic acid as ion pairing modifiers. Relative to formic acid, we find an overall ∼2.2-2.5× increase in MS signal and a slight decrease in RP LC retention (-0.7% acetonitrile on average) for acetic acid conditions. While these two features have opposing effects on peptide detectability, we find that acetic acid produces up to 60% higher peptide ID output depending on the type of sample. The drop in RPLC retention increases with peptide net charge at acidic pH. MS signal is dependent on the difference between the charge of the precursor ion and the charge of the peptide in solution, favoring species with a low pI. Lower peptide retention under acetic acid conditions demonstrates its higher hydrophilicity and, as expected, leads to composition and sequence-dependent character of the observed retention shift.

Analytical quality-by-design (AQbD) approach for comprehensive analysis of bioactive compounds from Citrus peel wastes by UPLC.

The growing interest in ingredients from natural sources has expanded the need for quality assessments of plant extracts. Analytical quality-by-design (AQbD) has been increasingly applied in regulated environments such as pharmaceutical industries and, more recently, for the bioactive compounds found in botanical materials. This work aimed to obtain qualitative (overall resolution and maximum peak capacity) and quantitative performances for target analytes using AQbD principles. The analytical target profile was elaborated; critical method parameters (independent variables) that affect the critical method attributes (dependent variables) were selected from a risk assessment for a reversed-phase liquid chromatography with diode array detection (RPLC-DAD) method. YMC-Triart C18 (3.0 × 100 mm, 1.9 µm) and a gradient elution using 0.2% acetic acid and methanol:acetonitrile 1:3 (v/v) were chosen as the stationary and mobile phases, respectively. The optimal and robust conditions (temperature at 33.3 °C, flow rate of 0.68 mL.min-1, and a gradient slope of 4.18%.min-1) were established by the method operable design region (MODR). The validation was performed by accuracy profiles using 90% expectation tolerance intervals for the selected compounds found in Citrus spp. using C. japonica as blank matrix. The lower limits of quantification for hesperidin, bergapten, herniarin, and citropten were 5.32, 0.40, 0.49, and 0.52 mg.L-1, respectively (acceptance limit was set at ± 20%). Nobiletin did not show an adequate quantitative performance.

Analytical enantioseparation of N-alkyl drugs by reversed-phase liquid chromatography with carboxymethyl-ß-cyclodextrin as mobile phase additive.

Carboxymethyl-ß-cyclodextrins (CM-ß-CDs) with five kinds of degrees of substitution were synthesized and characterized. Analytical enantioseparation of six basic drugs containing N-alkyl groups, including pheniramine, chlorpheniramine, labetalol, propranolol, venlafaxine, and trans-paroxol, was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) using the synthesized CM-ß-CD as chiral mobile phase additives. Key influence factors were optimized, including organic modifier, pH value, CM-ß-CD with different degrees of substitution, and concentration of CM-ß-CD. The mobile phase was composed of methanol and 10 mmol L-1 of phosphate buffer pH 4.0 containing 10 mmol L-1 of CM-ß-CD. Peak resolution for six racemic drugs was gradually increased with an increasing degree of substitution of the synthesized CM-ß-CD. The stoichiometric ratio and binding constants for the inclusion complex formed by CM-ß-CD and enantiomer were determined, which showed that the stoichiometric ratio for each inclusion complex was 1:1.

Effect of mobile-phase modifiers on the enantioselective retention behavior of methyl mandelate with an amylose 3,5-dimethylphenylcarbamate chiral stationary phase under reversed-phase conditions.

In this study, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone, and tert-butanol were used as organic modifiers in reversed-phase mode chiral liquid-chromatography to systematically investigate the effects of mobile phase components on the enantioselective retention behavior of methyl mandelate with immobilized amylose 3,5-dimethylphenylcarbamate-based sorbent called Chiralpak IA. A two-site enantioselective model was used to obtain information on the recognition mechanisms by observing the dependence of the enantioselectivity and retention factor difference on the modifier content. Similar enantioselective retention behaviors were observed for all modifiers, and characteristic modifier concentration points (PL , PM , and PH ) were identified. At modifier concentrations up to PM , the weakened hydrophobic environment resulted in polymer structural relaxation, which changed the recognition mechanisms. By contrast, at concentrations beyond PH , considerably different enantioselectivity behaviors were observed, indicating that the existence of dipole-dipole interaction, which was stronger at higher modifier concentrations, contributed to the retention mechanisms. The concentrations at which these characteristic points occurred were dependent on the carbon number of the modifier molecule. Modifiers with more carbon numbers facilitated the transition in the enantioselective behaviors. These results demonstrated that the proposed method can provide a physically consistent quantitative description of enantioselective retention behavior in reversed-phase mode.

Selectivity comparison of acetonitrile-methanol-water ternary mobile phases on an octadecylsiloxane-bonded silica stationary phase.

The solvation parameter model was used in this study to investigate various intermolecular interactions that influence retention on the standard C18 stationary phase for the solvent system acetonitrile:methanol (ACN:MeOH, 1:1). In comparison to the organic mobile phase modifiers acetonitrile, acetone, methanol, 2-propanol, and tetrahydrofuran, the solvent strength for the ACN:MeOH (1:1) solvent system was evaluated. To facilitate the interpretation of various intermolecular interactions that contribute to retention on a standard C18 stationary phase for the solvent system ACN:MeOH (1:1), system maps were constructed and compared with those of acetone, tetrahydrofuran, acetonitrile, 2-propanol, and methanol. The solvation parameter models were constructed for the ternary solvent system ACN:MeOH (1:1)-water, and in the models constructed, the coefficient of determination values were from 0.998 to 0.999, the Fisher statistic values for the models were from 1687 to 4015, and the standard error of the estimate values ranged from 0.022 to 0.029. The solvent system ACN:MeOH (1:1) has retention properties more similar to methanol than acetonitrile, indicating methanol's influence is more dominant.

Estimating the hydrophobicity extent of molecular fragments using reversed-phase liquid chromatography.

A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa  < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.

Comparison of supercritical fluid chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco.

This study used reversed-phase liquid chromatography-tandem mass spectrometry and supercritical fluid chromatography-tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10-500 ng/mL showed excellent linearity with R2 ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography-tandem mass spectrometry (86.1-95.7%) as well as supercritical fluid chromatography-tandem mass spectrometry (86.5-94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography-tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography-tandem mass spectrometry.

Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional liquid chromatography-mass spectrometry based on a novel phthalic anhydride bonded stationary phase.

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional liquid chromatography-mass spectrometry was developed. In this two-dimensional liquid chromatography system, a novel phthalic anhydride-bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional liquid chromatography-mass spectrometry, this online two-dimensional liquid chromatography-mass spectrometry system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines.

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.

Development and validation of a stability-indicating, single HPLC method for sacubitril-valsartan and their stereoisomers and identification of forced degradation products using LC-MS/MS.

The aim of this research work was to develop and validate a stability-indicating, single reversed-phase HPLC method for the separation of five impurities, including enantiomers, diastereomers, and degradation products in sacubitril-valsartan tablets. The method was developed using a Chiralcel OJ-RH column (150 × 4.6 mm, 5 µm) at 45°C with a gradient program of (T/%B) 0.01/25, 10.0/25, 25/38, 37.0/45, 39.0/25, and 45.0/25 at a flow rate of 0.8 ml/min. Mobile phase A consisted of 1 ml of trifluoroacetic acid in 1000 ml of Milli-Q water. Mobile phase B consisted of 1 ml of trifluoroacetic acid in a mixture of acetonitrile and methanol in the ratio of 950:50 (v/v). Sacubitril, valsartan, and their five impurities were monitored at 254 nm. Degradation was not observed when sacubitril-valsartan was subjected to heat, light, hydrolytic, and oxidation conditions. In acid degradation study (1 N HCl/60°C/2 h) impurity 1 (m/z 383.44) was formed, and in base degradation study (0.1 N NaOH/40°C/1 h) impurities 1 and 5 (m/z 265.35) were formed; both impurities were confirmed using LC-MS. The degradation products, enantiomers, and diastereomers were well separated from sacubitril and valsartan, proving the stability-indicating power of the method. The developed method was validated per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The inter- and intra-day percentage relative standard deviation for sacubitril, valsartan, and their five impurities was less than 5.2%, recovery of the five impurities was between 93 and 105%, and linearity was ≥0.999. The limit of detection was 0.030-0.048 µg/ml, and the limit of quantification was 0.100-0.160 µg/ml.

Imidazole-octyl mixed-mode stationary phase based on macroporous silica for the purification of ovomucoid and ovotransferrin.

A process-simplified hard template approach was established to synthesize the monodisperse macroporous silica microspheres with homogeneous structures by twice alkali-thermal treatment and calcination routes. Porous vinyl-functionalized polysesquioxane microspheres (V-PMSQ) were synthesized through a hydrolyzation-polycondensation method and used as templates. The template particles with large aperture and high pore volume were obtained by adjusting the pH value and reaction time of the twice alkali-thermal reaction. After calcination, monodisperse silica microspheres with an average pore size of 30 nm, homogeneous pore structures, and narrow particle size distribution were fabricated, which can be directly used as chromatographic matrices without classification. After that, a new reversed-phase/strong anion-exchange (RP/SAX) mixed-mode stationary phase Sil-S-VOIM was prepared by bonding the 1-vinyl-3-octyl-imidazole ligands to the above silica microspheres through a "thiol-ene" click reaction. The performance of the Sil-S-VOIM column was evaluated by one acidic protein (transferrin) and two basic proteins (lysozyme, α-chymotrypsin) and compared to a single imidazole-modified Sil-S-VIM column and an octyl-modified Sil-C8 column, respectively. Due to the synergistic effect of electrostatic repulsion and hydrophobic interactions, baseline separations of the above proteins were observed only on the Sil-S-VOIM column, with resolutions of 2.55 and 2.01 between lysozyme and transferrin, and between transferrin and α-chymotrypsin, respectively, indicating good selectivity and separation ability compared with single-mode stationary phases. It was applied to the isolation of egg white samples with peaks identified by SDS-PAGE and MALDI-TOF-MS. The results showed that the selective retention and isolation of ovomucoid and ovotransferrin were successfully achieved, with yields of 78.8% and 67.2%, respectively. The protocol described in this work is simpler, faster, and has higher protein recovery. Overall, this new mixed-mode stationary phase provided a promising potential for the separation and determination of intact proteins.

A Novel Validated UHPLC Method for the Estimation of Rosuvastatin and Its Complete Impurity Profile in Tablet Formulations.

A key step in the development of medicinal products is the research and validation of selective and sensitive analytical methods for the control of impurities from synthesis and degradation. As most impurities are similar in structure to the drug substance, the achievement of chemo-selective conditions is usually challenging. Herein, a direct and highly selective ultra-high-performance liquid chromatographic method for determining the assay and related substances content in medicinal products containing rosuvastatin calcium salt (RSV) is presented. RSV is used to treat high cholesterol levels and prevent heart attacks and strokes. The most engaging feature of this method was the baseline separation of all organic related substances listed in the European Pharmacopoeia (EP) monograph for the RSV tablets, achieved for the first time in less than 15 min using the Acquity BEH C18 (100 mm × 2.1 mm, 1.7 µm) column under reversed-phase isocratic conditions. The mobile phase adopted for the chemo-selective analysis does not contain buffers but instead contains trifluoroacetic as an acid additive. The chromatographic method was validated according to the guidelines of the International Conference on Harmonization (ICH) and proved to be linear, precise and accurate for determining the content of RSV and related chiral substances in tablet formulations.

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

Prediction of the n-Octanol/Water Partition Coefficients of Basic Compounds Using Multi-Parameter QSRR Models Based on IS-RPLC Retention Behavior in a Wide pH Range.

The n-octanol-water partition coefficient (logP) is an important physicochemical parameter which describes the behavior of organic compounds. In this work, the apparent n-octanol/water partition coefficients (logD) of basic compounds were determined using ion-suppression reversed-phase liquid chromatography (IS-RPLC) on a silica-based C18 column. The quantitative structure-retention relationship (QSRR) models between logD and logkw (logarithm of retention factor corresponding to 100% aqueous fraction of mobile phase) were established at pH 7.0-10.0. It was found that logD had a poor linear correlation with logkw at pH 7.0 and pH 8.0 when strongly ionized compounds were included in the model compounds. However, the linearity of the QSRR model was significantly improved, especially at pH 7.0, when molecular structure parameters such as electrostatic charge ne and hydrogen bonding parameters A and B were introduced. External validation experiments further confirmed that the multi-parameter models could accurately predict the logD value of basic compounds not only under strong alkaline conditions, but also under weak alkaline and even neutral conditions. The logD values of basic sample compounds were predicted based on the multi-parameter QSRR models. Compared with previous work, the findings of this study extended the pH range for the determination of the logD values of basic compounds, providing an optional mild pH for IS-RPLC experiments.