Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC.

Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.

Determination of Polar Lipids in Wheat and Oat by a Complementary Approach of Hydrophilic Interaction Liquid Chromatography and Reversed-Phase High-Performance Liquid Chromatography Hyphenated with High-Resolution Mass Spectrometry.

Cereals contain lipids that fulfill important physiological roles and are associated with stress in the plant. However, many of the specific biological roles of lipids are yet unknown. Comprehensive analysis of these polar lipid categories in whole grain wheat and oat, cereals highly relevant also in nutrition, was performed. Hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with high-resolution mass spectrometry using electrospray ionization in both positive and negative ionization mode was used. Exploiting the different separation mechanisms, HILIC was used as a screening method for straightforward lipid class assignment and enabled differentiation of isomeric lipid classes, like phosphatidylethanolamine and lyso-N-acylphosphatidylethanolamine, while RP-HPLC facilitated separation of constitutional isomers. In combination with data-dependent MS/MS experiments, 67 lipid species belonging to nine polar lipid classes could be identified. Furthermore, with both ionization modes, fatty acyl chains directly connected to the lipid headgroups could be assigned. This work focused on the four lipid classes N-acylphosphatidylethanolamines, acyl-monogalactosyldiacylglycerols, digalactosyldiacylglycerols, and monogalactosyldiacylglycerols as they were less studied in detail in the past. Applying the complementary approach, the relative lipid species compositions in these lipid classes was investigated in detail.

New N-acyl Thiourea Derivatives: Synthesis, Standardized Quantification Method and In Vitro Evaluation of Potential Biological Activities.

New N-acyl thiourea derivatives with heterocyclic rings have been synthesized by first obtaining isothiocyanate, which further reacted with a heterocyclic amine, characterized by (FT-IR, NMR spectroscopy and FT-ICR) and tested for their in vitro antimicrobial, anti-biofilm and antioxidant activities to obtain a drug candidate in a lead-optimization process. From the tested compounds, those bearing benzothiazole (1b) and 6-methylpyridine (1d) moieties revealed anti-biofilm activity against E. coli ATCC 25922 at MBIC values of 625 µg/mL. Compound 1d exhibited the highest antioxidant capacity (~43%) in the in vitro assay using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Considering the in vitro results, the highest anti-biofilm and antioxidant activities were obtained for compound 1d. Therefore, a reversed-phase high-performance liquid chromatography (RP-HPLC) method has been optimized and validated for the quantitative determination of compound 1d. The detection and quantitation limits were 0.0174 µg/mL and 0.0521 µg/mL, respectively. The R2 correlation coefficient of the LOQ and linearity curves were greater than 0.99, over the concentration range of 0.05 µg/mL-40 µg/mL. The precision and accuracy of the analytical method were within 98-102%, confirming that the method is suitable for the quantitative determination of compound 1d in routine quality control analyses. Evaluating the results, the promising potential of the new N-acyl thiourea derivatives bearing 6-methylpyridine moiety will be further investigated for developing agents with anti-biofilm and antioxidant activities.

Comprehensive analysis of alkenones by reversed-phase HPLC-MS with unprecedented selectivity, linearity and sensitivity.

Alkenones are among the most widely used paleotemperature biomarkers. Traditionally, alkenones are analyzed using gas chromatography-flame ionization detector (GC-FID), or GC-chemical ionization-mass spectrometry (GC-CI-MS). However, these methods encounter considerable challenges for samples that exhibit matrix interference or low concentrations, with GC-FID requiring tedious sample preparations and GC-CI-MS suffering from nonlinear response and a narrow linear dynamic range. Here we demonstrate that reversed-phase high pressure liquid chromatography-mass spectrometry (HPLC-MS) methods provide excellent resolution, selectivity, linearity and sensitivity for alkenones in complex matrices. We systematically compared the advantages and limitations of three mass detectors (quadrupole, Orbitrap, and quadrupole-time of flight) and two ionization modes (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) for alkenone analyses. We demonstrate that ESI performs better than APCI as response factors of various unsaturated alkenones are similar. Among the three mass analyzers tested, orbitrap MS provided the lowest limit of detection (0.4, 3.8 and 8.6 pg injected masses for Orbitrap, qTOF and single quadrupole MS, respectively) and the widest linear dynamic range (600, 20 and 30 folds for Orbitrap, qTOF and single quadrupole MS, respectively). Single quadrupole MS operated in ESI mode provides accurate quantification of proxy measurements over a wide range of injection masses, and with its modest instrument cost, represents an ideal method for routine applications. Analysis of global core-top sediment samples confirmed the efficacy of HPLC-MS methods for the detection and quantification of paleotemperature proxies based on alkenones and their superiority over GC-based methods. The analytical method demonstrated in this study should also allow highly sensitive analyses of diverse aliphatic ketones in complex matrices.

Exploring the variable space of shallow machine learning models for reversed-phase retention time prediction.

Peptide retention time (RT) prediction algorithms are tools to study and identify the physicochemical properties that drive the peptide-sorbent interaction. Traditional RT algorithms use multiple linear regression with manually curated parameters to determine the degree of direct contribution for each parameter and improvements to RT prediction accuracies relied on superior feature engineering. Deep learning led to a significant increase in RT prediction accuracy and automated feature engineering via chaining multiple learning modules. However, the significance and the identity of these extracted variables are not well understood due to the inherent complexity when interpreting "relationships-of-relationships" found in deep learning variables. To achieve both accuracy and interpretability simultaneously, we isolated individual modules used in deep learning and the isolated modules are the shallow learners employed for RT prediction in this work. Using a shallow convolutional neural network (CNN) and gated recurrent unit (GRU), we find that the spatial features obtained via the CNN correlate with real-world physicochemical properties namely cross-collisional sections (CCS) and variations of assessable surface area (ASA). Furthermore, we determined that the discovered parameters are "micro-coefficients" that contribute to the "macro-coefficient" - hydrophobicity. Manually embedding CCS and the variations of ASA to the GRU model yielded an R2 = 0.981 using only 525 variables and can represent 88% of the ∼110,000 tryptic peptides used in our dataset. This work highlights the feature discovery process of our shallow learners can achieve beyond traditional RT models in performance and have better interpretability when compared with the deep learning RT algorithms found in the literature.

A simple and reliable isocratic high-performance chromatographic assay for the simultaneous determination of hydrophilic benzophenone-4 and lipophilic octocrylene in sunscreens.

OBJECTIVE: This work aimed to develop a simple HPLC method for the simultaneous quantitative determination of the ultraviolet (UV) filters, hydrophilic benzophenone-4 and lipophilic octocrylene, in the presence of three other commonly used UV filters, avobenzone, octisalate and homosalate. METHODS: Reverse-phased HPLC was performed on a C18 column. A scouting gradient was initially used to determine the approximate mobile phase composition required for efficient analyte elution and separation before further optimization. The assay was validated with regard to specificity, linearity, intra- and inter-day accuracy and precision, limits of detection and limits of quantification. An ultrasound dispersion extraction method for the UV filters from a commercial sunscreen was developed, and the extraction efficiencies from spiked samples were calculated. RESULTS: An acetonitrile-methanol-water mixture (20:67:13, v/v/v), where the water component contained 0.2% trifluoroacetic acid (v/v), was found to be the optimal mobile phase at a flow rate of 1.0 mL/min. The assay was linear between 1.0-100 µg/mL for both benzophenone-4 and octocrylene (both correlation coefficients were above 0.999). There was no interference from the excipients of the sunscreen nor from the three other UV filters. The intra- and inter-day accuracy was between 90.0-104.6% for both analytes. Extraction recoveries from a spiked commercial sunscreen were between 95.4 ± 2.1% to 98.5 ± 2.1% for benzophenone-4, and between 87.3 ± 2.3% and 98.9 ± 3.1% for octocrylene. All validation parameters were within the acceptance criteria set out in the International Council for Harmonization (ICH) guidelines. The HPLC assay showed the extracted quantities of benzophenone-4 and octocrylene from the commercial sunscreen closely matched claimed quantities. CONCLUSION: The developed isocratic HPLC method was suitable for simultaneously determining the hydrophilic benzophenone-4 and lipophilic octocrylene in the presence of other commonly used UV filters. Additionally, the extraction method was simple and effective for accurately quantifying the UV filters in a commercial sunscreen.

OBJECTIF: Ces travaux visaient à développer une méthode de chromatographie en phase liquide à haute performance simple pour la détermination quantitative simultanée de la benzophénone-4 hydrophile et de l'octocrylène lipophile, des filtres ultraviolets (UV), en présence de trois autres filtres UV couramment utilisés, l'avobenzone, l'octisalate et l'homosalate. MÉTHODES: Une chromatographie en phase liquide à haute performance en phase inverse a été réalisée sur une colonne C18. Un gradient de référence a été initialement utilisé pour déterminer la composition approximative de la phase mobile requise pour une élution et une séparation efficace de l'analyte avant une optimisation plus poussée. Le dosage a été validé en termes de spécificité, de linéarité, d'exactitude et de précision intra- et inter-journalières, de limites de détection et de limites de quantification. Une méthode d'extraction par dispersion ultrasonique des filtres UV d'une crème solaire commerciale a été mise au point, et les efficacités d'extraction des échantillons artificiellement traités ont été calculées. RÉSULTATS: Un mélange acétonitrile-méthanol-eau (20:67:13, v/v/v), où la composante eau contenait 0,2 % d'acide trifluoroacétique (v/v), s'est avéré être la phase mobile optimale à un débit de 1,0 ml/min. Le dosage était linéaire entre 1,0 et 100 µg/ml pour la benzophénone-4 et l'octocrylène (les deux coefficients de corrélation étaient supérieurs à 0,999). Aucune interférence n'a été observée entre les excipients de l'écran solaire et les trois autres filtres UV. La précision intra et inter-journalière était comprise entre 90,0 et 104,6 % pour les deux analytes. Les récupérations par extraction à partir d'une crème solaire commerciale artificiellement traitée étaient comprises entre 95,4 % ± 2,1 % et 98,5 % ± 2,1 % pour la benzophénone-4, et entre 87,3 % ± 2,3 % et 98,9 % ± 3,1 % pour l'octocrylène. Tous les paramètres de validation étaient conformes aux critères d'acceptation définis dans les lignes directrices du Conseil international d'harmonisation (ICH). Le dosage par chromatographie en phase liquide à haute performance a montré que les quantités extraites de benzophénone-4 et d'octocrylène de la crème solaire commerciale correspondaient étroitement aux quantités revendiquées. CONCLUSION: La méthode de chromatographie en phase liquide à haute performance isocratique mise au point a permis de déterminer simultanément la benzophénone-4 hydrophile et l'octocrylène lipophile en présence d'autres filtres UV couramment utilisés. En outre, la méthode d'extraction était simple et efficace pour quantifier avec précision les filtres UV dans une crème solaire commerciale.

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.

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.

A method of isolating and analysing drugs from cancer cells for preclinical research.

The presented paper describes a new isolation method of recovery and analysis of selected drugs developed for preclinical research. The method uses the RP-HPLC technique (in a single chromatographic separation) and serves the recovery and analysis of selected drugs from neoplastic cells. It enables the determination of cytostatics statins, fibrates, and pioglitazone. Chromatographic separations of the tested compounds were carried out on a Gemini-NX 5 µ C18 (4.6 × 150 mm i.d.) column, in a gradient system with a mobile phase consisting of ACN (0.1% TFA) and water (0.1% TFA) at ambient temperature. The separations were carried out at a flow of 1 ml/min and UV detection of 220 nm. The inter-day and intra-day precision and accuracy of the method were determined. Extending the extraction time at reduced temperature resulted in a significant increase in the recovery of the pharmaceuticals in comparison with traditional extraction methods. The presence of the tested pharmaceuticals at defined retention times was confirmed by mass spectrometry. A recovery procedure for the tested compounds from biological material (medium, cell pellets) was developed at a level ranging between 93 and 99%. The utility of the new HPLC method has been confirmed in drug absorption studies as screening tests for the analysis of the new therapeutic compositions on melanoma cell lines.

A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations.

Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and control the quality of LNPs. The present study is focused on the development and validation of a high-performance liquid chromatography - diode array detector -evaporative light scattering detector (HPLC-DAD/ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs: ionizable lipids, the neutral co-lipid cholesterol, phospholipids, hydrophilic polymer-lipids for colloidal stability (e.g., a PEGylated lipid). In particular, this study focuses on two innovative synthetic lipids: a switchable cationic lipid (CSL3) which has demonstrated in vitro and in vivo siRNA transfection abilities, and the palmitic acid-grafted-poly(ethyloxazoline)5000 (PolyEtOx), used as an alternative polymer to address allergic reactions attributed to PEGylated lipids. The HPLC separation was achieved on a Poroshell C18 column at 50 °C using a step gradient of a mobile phase composed of water/methanol mixtures with 0.1% (v/v) trifluoroacetic acid (TFA). This method was validated following ICH Q2(R1) & (R2) guidelines in terms of linearity (R² ≥ 0.997), precision (relative standard deviation on peak areas < 5% for intermediate repeatability), accuracy (recoveries between 92.9% and 108.5%), and sensitivity. Indeed, low detection and quantitation limits were determined (between 0.02 and 0.04 µg and between 0.04 and 0.10 µg, respectively). Due to its high selectivity, this method allowed the analysis of lipid degradation products produced through degradation studies in basic, acidic, and oxidative conditions. Moreover, the method was successfully applied to the analysis of several liposome formulations at two key steps of the development process. Consequently, the reported HPLC method offers fast, versatile, selective and quantitative analysis of lipids, essential for development optimization, chemical characterization, and stability testing of LNP formulations.

Rapid determination of key impurities in high purity bisphenol A with reversed phase separation and triple quadrupole mass spectrometry.

Liquid chromatography with diode array detection (DAD) or ultraviolet spectroscopic (UV) detection as the most important analytical technique for the accurate quantification of impurities in bisphenol compounds normally requires long analysis time for baseline separation of all components as well as highly concentrated sample solutions for the detection of trace levels. To expand the application possibilities to all stages of polymerisation processes, an easy and robust reversed phase separation for 7 known impurities of bisphenol-A (BPA) including 4-isopropenylphenol and its dimeric isomers, o, p-bisphenol-A and trisphenol was established in this work. The method has been validated for the detection with triple quadrupole mass spectrometry (qqqMS) and DAD. In the investigated concentration range 0.5 - 100 mg/kg, the linearity is verified for both detection techniques. The limit of quantification (LOQ) for each impurity is with 0.5 - 1.5 mg/kg for qqqMS and 15 mg/kg for DAD sufficient for the evaluation of BPA as a raw material for polymerisation processes. The separation time for all impurities is 10 min whereas earlier reported methods need a minimum of 25 to 40 min. In addition the necessary sample concentration of BPA could be reduced to 5 mg/mL compared to existing methods where the sample concentration typically is > 50 mg/mL. For all those reasons the validated method can be efficiently applied for frequent process monitoring. Furthermore, 4 additional impurities were detected and identified. Mainly these are reaction products from the isopropenylphenol structure in combination with confirmed impurities as trisphenol or chroman. The quantification of these structures was established with trisphenol as reference and two structures were detected in all BPA qualities of this study in a concentration range from 20 - 400 mg/kg.

Separation and detection of D-/L-serine by conventional HPLC.

D-serine has a role as an endogenous allosteric agonist of N-methyl-D-aspartate (NMDA) receptor in the mammalian brain. In this study, we present a detailed description of our method that measures D-/L-serine by using conventional high performance liquid chromatography (HPLC). • We reacted D-serine and L-serine with ortho-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) to form diastereomeric isoindole derivatives, then we separated and detected them by conventional reversed phase HPLC with electrochemical detector (ECD). • We present typical measurement data of rat brain homogenate as an example of a convenient, appropriate method for measuring brain concentrations of D-serine. • Since many peaks appear in biological samples, we confirmed that the peaks were derived from serine by treating the sample with D-amino oxidase and catalase to decompose D-serine. As a results, one peak disappeared, suggesting that it is derived from D-serine.

Determination of Chiral Impurity of Naproxen in Different Pharmaceutical Formulations Using Polysaccharide-Based Stationary Phases in Reversed-Phased Mode.

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (Rs) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a Rs = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.

Stability-indicating HPLC method development and validation for simultaneous estimation of metformin, dapagliflozin, and saxagliptin in bulk drug and pharmaceutical dosage form.

A simple, precise, and rapid stability-indicating reversed-phase-HPLC method was developed and validated for the estimation of metformin (MET), dapagliflozin (DAP), and saxagliptin (SAX) combination in bulk and tablet dosage forms. The proposed method uses a Kromasil C18 column (150 × 4.6 mm, 5 µm) with column oven temperature of 30°C and mobile phase containing a mixture of 60% phosphate buffer (pH = 3) and 40% acetonitrile. The flow rate was set at 1.0 mL/min, and the injection volume was 10 µL. The detection was carried out at 230 nm using a photodiode array detector, and the total run time was 4 min. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines for specificity, linearity, precision, accuracy, robustness, and solution stability. The method is linear over the range of 125-750 µg/mL for MET, 1.25-7.5 µg/mL for DAP, and 0.625-3.75 µg/mL for SAX. The observed correlation coefficients (R2 ) for MET, DAP, and SAX are >0.999. The proposed method is precise, and the percentage relative standard deviation was found to be between 0.4 and 0.8. The observed percentage recoveries were between 98.51 and 100.80 for all three compounds. The product was subjected to stress conditions of acid, base, oxidative, thermal, and photolytic degradation. The product was found to degrade significantly in oxidative, acid, and base hydrolysis degradation conditions, and the degradation products were well determined from the active peaks, thus proving the stability-indicating power of the method. The developed and validated stability-indicating reversed-phase-HPLC method was appropriate for quantitative determination of these drugs in pharmaceutical preparations and also for quality control in bulk manufacturing.

Development of a retention prediction model in ion-pair reversed-phase HPLC for nucleoside triphosphates used as mRNA vaccine raw materials.

The International Conference on Harmonization guidelines for quality on pharmaceutical development recommends a systematic development approach including robustness studies which assure performance of manufacturing and analytical method development of drug product. It was demonstrated that the retention prediction model for nucleoside triphosphates (NTPs) on ion-pair reversed-phase HPLC was developed by a highly accurate Kawabe's model which supports the development of robust HPLC methods. As NTPs and its derivatives are typically used for Messenger ribonucleic acid (mRNA) vaccine production, adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), 5-methylcytidine-5'-triphosphate (m5-CTP), uridine-5'-triphosphate (UTP), 5-methyluridine-5'-triphosphate (m5-UTP), pseudouridine-5'-triphosphate (Ψ-UTP) and N1-methylpseudouridine-5'-triphosphate (m1Ψ-UTP) were applied for prediction model development. By a comparison of the predicted retention factor in eight studied samples with the retention factor measured under six isocratic conditions, the absolute prediction error was 0.075 and also the prediction error (%) was 2.70%. In practical examples, analytical method for residual ATP, GTP, CTP, and m1Ψ-UTP in the commercial mRNA-based drugs and purity method for UTP derivatives were optimized by QbD approach. The design space for the minimum resolution between adjacent peaks was simulated with the models developed to evaluate the robustness of peak separation, and the optimal mobile phase condition was also simulated. As a conclusion, the desired peak was successfully separated under the optimized condition, and we thought that these retention models could optimize the mobile phase condition of the NTP analysis method for applying to various quality tests, such as quantity, purity and identity test for NTPs and its derivates in the mRNA-based drugs.

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone.

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.

Stability-indicating reversed-phase-HPLC method development and validation for sacubitril/valsartan complex in the presence of impurities and degradation products: Robustness by quality-by-design approach.

According to current regulatory guidelines, a stability-indicating method has been developed to determine the impurities in sacubitril (SCB) and valsartan (VLS) tablet dosage forms and perform robustness studies using the design of experiments approach. The present study was initiated to understand quality target product profile, analytical target profile, and risk assessment for method variables that affect the method response. A reversed-phase-HPLC system was equipped with a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 µm) and a photo diode array detector. A gradient mobile phase was used in this research work. The detection was performed at 254 nm; the flow rate was 1.5 mL/min, and the column temperature was maintained at 30°C. The proposed method was validated per the International Council for Harmonisation Q2 (R1) guidelines. The coefficient of correlation was >0.999 for all impurities. The limits of detection and quantification were evaluated for SCB, VLS, and all impurities. The precision and accuracy were obtained for SCB, VLS, and their related impurities. Intra- and inter-day relative standard deviation values were less than 10.0%, and the recoveries of impurities varied between 90.0 and 115.0%. Based on the validation results, the proposed DoE method can estimate SCB and VLS impurities in the finished dosage form.

Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.