Bing Mao

Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

Examination of rofecoxib solution decomposition under alkaline and photolytic stress conditions

Rofecoxib is a highly active and selective cyclo-oxygenase II inhibitor. A stability-indicating method for the assay of rofecoxib has been developed using reverse-phase high-performance liquid chromatography (HPLC). Stress testing of rofecoxib was conducted during the method development and validation. HPLC analysis of rofecoxib solutions stressed under alkaline and photolytic conditions revealed the presence of several degradates. Two main degradates were determined to be the cyclization product formed by photo-cyclization and the dicarboxylate formed by ring opening in the presence of base and oxygen. The identities of these degradates were confirmed by comparison of UV spectra and HPLC retention time with the independently synthesized products. The mechanistic pathways for the formation of these degradates are discussed. Further improvement of the HPLC methods ruggedness has been made based on these studies.

Development and Validation of an HPLC Method for the Impurity and Quantitative Analysis of Etoricoxib

Abstract Etoricoxib (5‐chloro‐6′‐methyl‐3[4‐(methanesulfonyl)phenyl]‐2,3′‐bipyridine) is a highly active and selective cyclo‐oxygenase II inhibitor. A single, stability‐indicating HPLC method has been developed and validated for both the impurity and quantitative analysis of etoricoxib. Method development incorporated the optimization of stationary phase, pH, temperature, and mobile phase composition for the resolution of thirteen process impurities and three major degradation products. Further optimization of pH and mobile phase composition was aided by the use of DryLab®, a computer‐based simulation program. The stability‐indicating capability of the method was proven through the identification of photolytic and oxidative decomposition products. Method validation produced excellent results for linearity, precision, limit of quantitation and limit of detection, specificity, accuracy, recovery, and robustness. The identities of etoricoxib decomposition products were confirmed by UV, LC/MS, and NMR spectra.

Improving the In Vivo Therapeutic Index of siRNA Polymer Conjugates through Increasing pH Responsiveness

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.

Application of Fast Reversed Phase Liquid Chromatography for Analysis of Pharmaceutical Related Boronic Acid and Boronic Pinacol Ester Functionalized Compounds

Abstract Boronic acid and boronic pinacol ester functionalized compounds are the basis for Suzuki coupling and Petasis reactions that are widely used in pharmaceutical synthetic schemes. The purity analysis of these compounds utilizing traditional reversed phase liquid chromatography methodology is complicated by the potential of on‐column hydrolysis. Therefore, a fast liquid chromatography method was developed to minimize on‐column hydrolysis effects. The method was applied as an in‐process control method for the separation of three process related impurities from a boronic pinacol ester functionalized intermediate with no appreciable on‐column hydrolysis observed. The method was then optimized to successfully resolve ten different boronic acid and boronic pinacol ester functionalized compounds within five minutes. Thus, the wide range applicability of fast liquid chromatographic technology to this specific class of compounds was demonstrated.

Evaluation of core-shell particle columns for ion-pair reversed-phase liquid chromatography analysis of oligonucleotides.

An investigation into the use of core-shell particle columns for separation of short (∼21 base pairs) RNA oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC) showed improved resolution for a number of test analytes relative to conventional (fully-porous) reversed-phase columns. The best resolutions were obtained using columns packed with smaller sub-2μm core-shell particles.

LCMS using a hybrid quadrupole time of flight mass spectrometer for impurity identification during process chemical development of a novel integrase inhibitor

LCMS incorporating a quadrupole time of flight mass spectrometer was used to identify impurities found in a chemical process development sample of a novel integrase inhibitor, raltegravir. The combination of accurate mass measurement in full scan mode followed by construction of targeted masses for further MSMS interrogation allowed for the determination of atomic composition and connectivity. The fragmentation pattern of raltegravir was used as a model compound, and the product ion spectra of an impurity was compared to both the model fragmentation pattern and the atomic composition generated in the full scan experiment to deduce a structure.

On‐Column Hydrolysis Kinetics Determination of Boronic Pinacol Ester Intermediates for Use in Optimization of Fast HPLC Methods

Abstract Boronic acid and boronic ester intermediates are the basis for Suzuki coupling and Petasis reactions that are widely used in pharmaceutical synthetic schemes. The analysis of these compounds utilizing traditional reversed phase liquid chromatography (RPLC) is complicated by the potential of on‐column hydrolysis. In order to effectively develop and optimize RPLC methods for accurate analysis of these compounds, a better understanding of the potential on‐column hydrolysis needs to be achieved. Kinetic studies of this type of on‐column hydrolysis were performed utilizing a stop flow kinetic approach. The rate of on‐column hydrolysis was determined as a function of initial organic composition, mobile phase pH, and column temperature. In addition, the Arrhenius activation energy was calculated. A fast reversed phase liquid chromatography method was then developed and optimized to minimize on‐column hydrolysis effects based on the garnered information. The method was applied to successfully resolve ten different boronic acid and boronic pinacol ester functionalized compounds within five minutes. Thus, the wide range applicability of fast RPLC technology for accurate analysis of this specific class of compounds was demonstrated.

Mechanistic Study of the Gas-Phase In-Source Hofmann Elimination of Doubly Quaternized Cinchona-Alkaloid Based Phase-Transfer Catalysts by (+)-Electrospray Ionization/Tandem Mass Spectrometry

AbstractAn unusual in-source fragmentation pattern observed for 14 doubly quaternized cinchona alkaloid-based phase-transfer catalysts (PTC) was studied using (+)-ESI high resolution mass spectrometry. Loss of the substituted benzyl cation (R1 or R2) was found to be the major product ion [M2+ – R1+ or R2+]+ in MS spectra of all PTC compounds. A Hofmann elimination product ion [M – H]+ was also observed. Only a small amount of the doubly charged M2+ ions were observed in the MS spectra, likely due to strong Columbic repulsion between the two quaternary ammonium cations in the gas phase. The positive voltage in the MS inlet but not the ESI probe was found to induce this extensive fragmentation for all PTC diboromo-salts. Compound 1 was used as an example to illustrate the proposed in-source fragmentation mechanism. The mechanism of formation of the Hofmann elimination product ion [M – H]+ was further investigated using HRMS/MS, H/D exchange, and DFT calculations. The proposed formation of 2b as the major Hofmann elimination product ion was supported both by HRMS/MS and DFT calculations. Formation of product ion 2b through a concerted unimolecular Ei elimination pathway is proposed rather than a bimolecular E2 elimination pathway for common solution Hofmann eliminations. Graphical Abstractᅟ

Quantitation of Ammonium Cations in Pharmaceutical Samples by Nonaqueous Capillary Electrophoresis with Indirect UV Detection

Abstract A nonaqueous capillary electrophoresis (CE) method with indirect UV detection was developed specifically for quantitation of ammonium cations in pharmaceutical samples. Employing a methanol‐based background electrolyte system with options of using up to 60% of acetonitrile or 30% of tetrahydrofuran (THF) as buffer additives, allows for injection of a variety of sample matrices with organic solvents. Consequently, ammonium in most samples that have poor solubility in water can be determined by the CE method. Apparent pH* of the background electrolyte was optimized to achieve a close mobility match between the ammonium cation and the UV probe, imidazole. This provided symmetrical ammonium peak shape, and consequently, improved detection sensitivity, injection reproducibility, and resolution from possible interferences. A buffer additive, 15‐crown‐5 ether, significantly improved the resolution of ammonium from potassium interference in the methanol‐based background electrolyte system, which allowed for quantitation of ammonium in a sample that contained 10,000 times higher levels of potassium. The CE method was validated according to current pharmaceutical industry standards. The injection reproducibility and linearity in the target range were determined to be acceptable. The limit of detection (LOD) and limit of quantitation (LOQ) of ammonium cations in solutions were determined to be 50 ppb and 0.5 ppm, respectively. The method has been successfully employed for quantitation of the ammonium impurity in pharmaceutical samples.