Separation Of Impurities Research Articles

Separation and identification of degradation impurities of esomeprazole sodium.

Drug impurities are important factors that affect drug safety and efficacy. The aim of this study is to separate and confirm the structure of two degradation impurities of esomeprazole sodium, designated X and Y. The impurities X and Y were successfully isolated using preparative HPLC by developing separation methods with the help of ACD/Labs AutoChrom software. There was a steady increase in X and Y impurities in forced esomeprazole sodium degradation. Impurity X was confirmed as 6-methoxy-1h-benzo[d]imidazole-2-yl-4-amino-3,5-dimethylpyridinecarboxylate and impurity Y as 6-methoxy-1h-benzo[d]imidazole-2-yl-4-hydroxy-3,5-dimethylpyridinecarboxylate using nuclear magnetic resonance spectrometry, infrared spectroscopy, and high-resolution mass spectrometry. These findings provide a comprehensive understanding of the impurity profile of esomeprazole sodium because these impurities are reported for the first time. Based on our results, active pharmaceutical ingredient manufacturers can further control process parameters to reduce impurity generation, and drug production manufacturers can optimize the packaging and storage conditions of esomeprazole sodium.

Clean and cost-efficient preparation of vanadium electrolyte from the vanadium-rich solution of black shale by solvent extraction

This study proposes a clean and cost-efficient preparation process for vanadium electrolytes from the vanadium-rich solution of black shale. The introduction of saponified D2EHPA promoted vanadium recovery and further separation of impurities from vanadium-rich solutions. The preparation of a high-purity vanadium electrolyte was achieved by a four-stage countercurrent extraction at an initial pH as low as 1.0, 8 min, D2EHPA concentration of 30%, saponification degree of 60%, and phase ratio of 1:1, as well as a three-stage countercurrent stripping with 4 mol L−1 H2SO4 at phase ratio (O/A) of 6:1, 30 min. The developed electrolyte showed qualified electrochemical and charge-discharge performance, with coulombic efficiency, voltage efficiency, and energy efficiency reaching 91.07%, 93.44%, and 85.10%, respectively. This method saves 30.28% in preparation costs over the traditional method and produces no ammonia, nitrogen, or harmful gases. The technology is technologically advanced, economically sensible, and ecologically friendly, and is expected to be applied to large-scale production and promote the development of VRFB and new energy.

LC-MS-Compatible Chromatographic Method for Quantification of Potential Organic Impurities of Elagolix Sodium in Tablet Dosage Form with Identification of Major Degradation Products.

Elagolix is a gonadotropin-releasing hormone (GnRH) modulator and used for pain relief from endometriosis. The present research was performed to develop and validate a simple, novel, fast, sensitive, and cost-effective LC-MS-compatible chromatographic method for quantification of all prominent organic impurities of elagolix sodium in tablet formulation with identification of major degradation products. The optimum separation of the organic impurities of elagolix sodium was achieved on an ACE C18-PFP (250 mm × 4.6 mm, 5 µm) column by employing pH 5.6 acetate buffer-acetonitrile (95 + 5, by volume) as mobile phase A, and acetonitrile-methanol (90 + 10, by volume) as mobile phase B. UV detection of the drug and impurities was carried out at 210 nm. A forced degradation study was carried out by employing acid, alkali, oxidative, thermal, and photolytic stress conditions on elagolix sodium drug substance and its drug product. The major degradation products observed during the stress study were identified by using mass spectrometry. Elagolix sodium and its prominent organic impurities were resolved in the developed method through a gradient elution program of 46 min at a flow rate of 1.3 mL/min. Significant degradation was observed during alkali hydrolysis and oxidative stress conditions with a mass balance of more than 97.0%. The method was validated in line with present International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The forced degradation study suggested that the developed method is specific and stability-indicating and can be used for related substance analysis of elagolix drug substance and its dosage forms. This is the first research paper which describes a simple and sensitive (LOD 0.08 µg/mL) HPLC method for quantification of all probable impurities of elagolix in tablet dosage forms. The noticeable feature of the developed method is resolution of impurities of similar structures in a short time using routine solvents which are easily available in the QC laboratory.

Separation and characterization of the polymerized impurities in oxacillin sodium by 2D HPSEC and HPLC IT-TOF MS.

The polymerized impurities in oxacillin sodium can induce allergic reaction, which can seriously threaten the health of patients. Gel filtration chromatography (GFC) is currently widely used for the analysis of polymerized impurities, but it has drawbacks. To effectively control the polymerized impurities in oxacillin sodium, a high-performance size exclusion chromatography (HPSEC) method and a reversed-phase high performance liquid chromatography (RP-HPLC) method were established to replace the classical GFC method. By studying the chromatographic behavior of polymerized impurities and small molecular weight impurities in both methods with different chromatographic separation mechanisms, the polymerized impurities in oxacillin sodium were separated and detected effectively. Column-switching two-dimensional liquid chromatography was applied to eluted polymerized impurities from the HPSEC method for oxacillin sodium. Ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and unknown impurities eluted from the HPSEC/RP-HPLC method for oxacillin sodium. The structures of 25 unknown impurities in oxacillin sodium were elucidated based on the high-resolution massn data. Thirteen polymerized impurities were found and characterized. The corresponding relationship of impurities between the two methods was established and the specificity of the two methods was compared. The RP-HPLC method for analysis of the polymerized impurities not only has higher column efficiency and more specificity than the HPSEC method, but also higher sensitivity. The mechanisms of the formation of degradation impurities in oxacillin sodium were studied. The newly established RP-HPLC methods could effectively separate and detect polymerized impurities and unknown impurities in oxacillin sodium. The study of the impurity profile in oxacillin sodium provided a scientific basis for the improvement of official monographs in pharmacopoeias.

Optimizing the preparative capacity of two-dimensional liquid chromatography based on analytes retention behaviors

In this work, a solute retention optimization method (SRO) was proposed to exploit the purification potential of two-dimensional liquid chromatography (2D-LC). According to our findings, the complementarity of 2D-LC correlates with some specific impurities. In the two methods used in 2D-LC, the retention order of these impurities and target compound is completely opposite. Taking full advantage of the complementarity is crucial to enhance the saturation capacity (wmax) of 2D-LC by SRO. For the purpose of validating the effectiveness of SRO, a reverse-phase liquid chromatography (RPLC) coupled with hydrophilic interaction chromatography (HILIC) was developed to purify p-chlorobenzoic acid from substituted benzenes. By using the overloading effects of analytes as indicators, the wmax of RPLC × HILIC was determined by the bisection method, and finally defined by the extremely high loading volume of 4.9 mL. A touch-peak separation of impurities and the target compound occurred precisely during the secondary separation. The effectiveness of SRO was also verified by the greater purification efficiency of RPLC × HILIC than that of HILIC × RPLC. Subsequently, a RPLC × RPLC method was developed by SRO to prepare the reference materials of caffeine from tea extracts. Only by an analytical C18 column, 15.6 mg of caffeine with the purity of 98.3% was obtained at once with the recovery up to 82.3%. However, without the aid of SRO, the purity rapidly decreased to 62.0%. Compared to other methods, SRO-based 2D-LC offers certain advantages in terms of purity, recovery, and the purification efficiency, suggesting that it is particularly effective in developing preparative 2D-LC facing complex matrices.

Processing of serpentenites of the Arvaten deposit of Uzbekistan with the use of ammonium sulphate

The article presents the results of a study of the processes of obtaining magnesium oxide by fusing serpentinites of the Arvaten deposit in the presence of ammonium sulfate, followed by grinding and treatment with water to separate silica from the suspension, neutralizing the filtrate with ammonia to pH 8.5 for precipitation and separation of impurity metal ions, precipitation and separation of ions magnesium from solution using ammonia and ammonium carbonate, thermal decomposition of magnesium corbanate to obtain magnesium oxide. It has been established that the use of ammonium sulfate for fusion of serpentite at a ratio of serpentenite: (NH4)2SO4 = 100: 201.77 and further processing of the resulting solutions allows you to obtain magnesium oxide with a composition (wt.%): MgO - 97.55; Fe2O3 - 0.86; A12O3 - 0.12; CaO - 0.18; SO3 - 0.48.

Application of Chemical Transformation Driven Impurity Separation in Experiments Teaching: A Novel Method for Purification of α-Fluorinated Mandelic acid

Application of Chemical Transformation Driven Impurity Separation in Experiments Teaching: A Novel Method for Purification of α-Fluorinated Mandelic acid

Algorithm for Determining the Purity of Pure Organic Phthalates by the Indirect Mass Balance Method

Pure organic substances are the first and key link in the chain of metrological traceability in organic analysis. In most cases, purity determination of the organic substance is a non-trivial task, the solution of which requires preliminary theoretical study and understanding of the capabilities of instrumental methods for developing a pertinent analytical procedure.Within the study, an algorithm for determining the purity of a separate group of organic compounds – ortho-phthalic acid esters (phthalates) was developed based on the mass balance model and the general algorithm for characterizing substances suitable for isolation or purification by distillation. An additional classification of probable impurities (into five types A – E) depending on their qualitative and quantitative characteristics is proposed, as well as suitable analytical methods for measuring impurities and confirming the identity of the main component are identified.The proposed algorithm was implemented on the State Primary Standard GET 208 to determine the purity of six priority phthalates: dimethyl phthalate, diethyl phthalate, di-(n-butyl) phthalate, benzyl butyl phthalate, di-(2-ethylhexyl) phthalate, and di-(n-octyl) phthalate. Certified pure phthalates were used to create a certified reference material for the composition of a solution of orthophthalic acid esters (phthalates) in methanol (6Ftlt-VNIIM) GSO 11366–2019.The study revealed that the adapted algorithm considers the specifics of work with the phthalates group and the features of the separation of probable impurities within the MB model, and is universal for all congeners of the group, but does not exclude the natural constraints of the mass balance method.

A Combined Hydro-Mechanical and Pyrometallurgical Recycling Approach to Recover Valuable Metals from Lithium-Ion Batteries Avoiding Lithium Slagging

Meeting the increasing demand for energy storage based on lithium-ion batteries (LIB) is not only a question of resource availability but also an issue of resource conservation and efficient recycling management. In this respect, sustainable recycling concepts play a central role in mindful interactions with valuable materials. Based on this approach, a process interconnection of hydromechanical preparation, flotation, and pyrometallurgical treatment was investigated. The hydromechanical preparation showed promising results in achieving highly pure mixtures of LIB-active material. It was found that a pre-opening step could achieve an even better separation of impurities for downstream processes such as Cu and Al to avoid excessive particle size reduction. According to an optimized mixing stage during flotation, the C amount was reduced from 33 wt.% to 19.23 wt.%. A Li-free metal alloy was obtained through the subsequent pyrometallurgical treatment, and evidence for Li removal via the gas phase was provided. Furthermore, heating microscope trials confirmed the results of the process interconnection and showed that further optimization steps for the pre-treatment are necessary for favorable product quality. Therefore, a high-stratification plot was created, which allows a quick future statement about the suitability of the input material for use in the process.

Purification of hydrophobic complex antibody formats using a moderately hydrophobic mixed mode cation exchange resin

Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography. In the present study, both of these challenges were addressed through the development of an innovative mixed mode resin with 2-amino-4methylpentanoic acid ligands that combines weak cation exchange with moderate hydrophobic interactions. Supported by high throughput partition coefficient screens for identification of preferable pH and salt concentration ranges in bind and elute mode, this mixed mode resin successfully demonstrated efficient impurity separation from an extremely hydrophobic bispecific antibody with a single unit operation. High purity (>97%) was obtained as a result of significant reduction of product-related impurities as well as process-related host cell proteins (>3 log scale), while maintaining satisfactory recovery (70%). This also supports that highly hydrophobic antibody formats can be efficiently purified using a resin with moderate hydrophobic characteristics. Studies involving additional antibodies possessing different formats and a wide range of hydrophobicity confirmed the broad applicability of the new resin. In view of its high selectivity and robust operating ranges, as well as the elimination of the need for an additional column step, the novel resin enables simplified downstream processing and economic manufacturing of complex antibody formats.

Degradation pathways and impurity profiling of the anticancer drug apalutamide by HPLC and LC-MS/MS and separation of impurities using Design of Experiments.

Apalutamide, an androgen receptor inhibitor, is used to treat prostate cancer. A stability-indicating high-performance liquid chromatography method was developed for the estimation of assay and organic impurities of apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30 min using Atlantis dC18 , 100 × 4.6mm, 3.0 μm and the binary gradient program (10 mm KH2 PO4 , pH3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection volume used were 270 nm, 1.0 ml/min, 45°C and 10 μl, respectively. The interaction of independent variables (pH, column temperature and flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection limit, quantitation limit, accuracy, precision and robustness as per the International Conference on Harmonization. A high-quality recovery with good precision (91.7-106.0%) and correlations (r2 > 0.997) within a linear range of 0.12-2.24 μg/ml (0.05-0.3%, w/w) were achieved consistently for assay and organic impurities of apalutamide. The stability-indicating characteristics of the proposed method were assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1-DP5) using LC-MS/MS.

Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry

The aim of this research was to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation and qualitative determination of oligonucleotide impurities, exemplified by synthesis of raw products of the two single strands of patisiran siRNA. The stationary phases with mixed hydrophobic/hydrophilic properties (cholesterol and alkylamide) were firstly used for this purpose with reversed-phased high-performance liquid chromatography. Several different chromatographic parameters were tested for their impact on impurities separation: type, concentration, pH of salt, as well as organic solvent type in the mobile phase. The pH was the most influential factor on the separation and signal intensities in mass spectrometry detection. Finally, the optimized method included the application of cholesterol stationary phase, with mobile phase containing 20 mM ammonium formate (pH 6.5) and methanol. It allowed good separation and the identification of most impurities within 25 min. Since not all closely related impurities could be fully resolved from the main peak in this oligonucleotide impurity profiling, two-dimensional liquid chromatography was used for peak purity determination of the target oligonucleotides. The Ethylene Bridged Hybrid (BEH) Amide column in hydrophilic interaction liquid chromatography was applied in the second dimension, allowing additional separation of three closely related impurities.

Separation of Cu impurity from spent lithium-ion batteries by roasting in air and leaching with (NH4)2CO3 solution of low concentration

Separation of Cu impurities from spent lithium-ion batteries (LIBs) before precipitating other valuable metals is critical to improve the quality of final products. Currently, the main process for separating Cu impurity from spent LIBs by extraction and precipitation has the disadvantage of being high cost and complicated process. In this work, the surface characteristics of spent LIBs materials before and after thermal treatment are explored, and the effects of the concentration of ammonium salts, temperature, and pulp density on the leaching of Cu impurity are investigated. The novel process proposed in this work removes Cu impurities from spent LIBs materials using a solution of (NH4)2CO3 at low concentration. The results suggest that organics on the spent LIBs surface can be effectively removed by heating at a temperature of 505 °C to avoid the influence of organics on the removal of Cu impurities. Furthermore, the results of the leaching experiment demonstrate that (NH4)2CO3 displays an excellent leaching effect on Cu compared to NH4Cl, (NH4)2SO4, and NH4HCO3. The leaching efficiency of Cu at the concentration of 0.25 mol/L (NH4)2CO3, temperature 80 °C and leaching time of 120 min reaches 99.1%. On the contrary, the leaching efficiencies of Ni, Co, and Mn are <4% on average. The thermodynamic analysis proves the potential for removing Cu impurities from spent LIBs materials using a solution of (NH4)2CO3 at low concentration. The process lays the foundation for the subsequent hydrometallurgical process to recover other metals.

Optimization of Screen-Hole-Clearing Devices for Mechanized Residual Film–Impurity Separation

The airflow velocity in some nozzles is low, and the clearing of the nozzle is ineffective because of unreasonable airflow pipe arrangements and the distance from the nozzle to the screen surface of screen-hole-clearing devices for trommel-sieve-type residual film–impurity wind separators. In the present study, the main structure and working parameters affecting the screen hole clogging situation were determined through theoretical analysis and computational fluid dynamics simulations. In addition, a three-factor, three-level quadratic regression orthogonal center of rotation combination test was performed. The distance from the nozzle to the screen surface, fan wind speed, and the number of airflow pipes were selected as test factors, and the ratio of impurities in the residual film and the blockage ratio of the screen holes were selected as the evaluation indexes. The results indicated that the ratio of impurities in the residual film was reduced by 2.42% and the blockage ratio of the screen holes was reduced by 1.92% at a nozzle-to-screen distance of 102 mm, a fan wind speed of 24 m/s, and with four air pipes. The resulting impurity ratio in the film was 5.86%, and the blockage ratio of screen pores was 5.41%. The minimum airflow velocity of 15.8 m/s at each nozzle position of the optimized screen-hole-clearing device satisfied the requirements of screen hole clearing and blockage. Furthermore, the ratio of impurities in the residual film and the blockage ratio of the screen holes remained unchanged during the continuous operation of the device. This indicated that the optimized screen-hole-clearing device had a stable working performance. This study may provide a theoretical framework for the future development of screen-hole--clearing devices.

Purifying Metallurgical-Grade Silicon to 4N with 3D Porous Structure by Integrated Metallurgy-Materials Phase Separation

Silicon (Si) is widely used in photovoltaics, semiconductors, and lithium-ion batteries but high purity is required in most applications. Conversion of metallurgical-grade Si (MG-Si) to Si with 4N purity and desired structure by an economical and environmentally friendly technique is still challenging, albeit desirable. Herein, an integrated metallurgy-materials technique is described to produce high-purity Si (99.99%) with a three-dimensional (3D) porous structure from metallurgical Si. This green and cost-effective strategy involves controllable phase separation of impurities from the Si matrix via Mg alloying, nitriding/dealloying (<800 °C), and acid etching, which can remove metallic impurities (Fe, Al, Ca, et al.) and non-metallic impurities (B, P, et al) with the efficiency of above 90% and 80% respectively. The intermediate product of Mg3N2 serves as both the pore-forming medium and impurity carrier /remover to separate impurities, improve the exposed area, and enhance dissolution of impurities. Different from conventional metallurgical processes, B and P are converted into MgB2 and Mg3P2 resulting in easy removal during phase separation. This green process is effective in purifying Si and forming a porous structure on a large scale as demonstrated by the conversion of metallurgical Si to high-purity Si. Moreover, the integrated metallurgy-materials phase separation technique can be extended to other industrial applications.

Comparison of the Preparation Process of Rare Earth Oxides from the Water Leaching Solution of Waste Nd-Fe-B Magnets’ Sulfate Roasting Products

The new process developed here consisting of sulfurization roasting transformation and water immersion can effectively realize the separation of rare earth elements (REEs) and impurities from spent Nd-Fe-B magnets. For the industrial application of the new process, it is critical to determine how to economically and efficiently prepare rare earth oxide (RExOy) products with higher purity from the obtained water leaching solution. Therefore, according to rare earth sulfate (RE2(SO4)3) solution characteristics, the oxalic acid precipitation–calcination method, sodium carbonate precipitation–calcination method, and double sulfates precipitation–alkali conversion–calcination method were optimized and compared. The results show that the recovery efficiency of REE recovery via the oxalic acid precipitation–calcination method is 99.44%, and the purity of RExOy is 99.83% under optimal technological conditions. However, the cost of oxalic acid precipitation is higher. The process consisting of the double sulfates precipitation–alkali conversion–calcination method is relatively complicated, the recovery efficiency of REEs is 97.95%, and the purity of the RExOy is 98.04%. The recovery efficiency of the REEs and the purity of the RExOy obtained from the sodium carbonate precipitation–calcination method are 99.12% and 98.33%, respectively. Moreover, the recycling cost of sodium carbonate precipitation is the lowest among the three processes for preparing RExOy, so it has industrial application potential. The obtained results for REE recovery from spent Nd-Fe-B magnets in this research can provide theoretical guidance for the innovation of the recycling process for REEs as secondary resources.

A new process to produce battery grade lithium carbonate from salt lake brines by purification, synergistic solvent extraction and carbon dioxide stripping

There are abundant lithium resources in China, and the demand for lithium salts is high. However, lithium resources of the sulfate salt lakes in Tibet, such as Baqiancuo Salt Lake, have not been effectively utilised. This study proposes a synergistic extraction system composed of LIX 54, TRPO, and surfactant ADD-1 for efficient lithium extraction from the brine and separation of impurities. The CO2 gas stripped lithium and produced high-purity lithium bicarbonate solution. Thermal decomposition produced lithium carbonate solid from the loaded strip solution. The comprehensive yield of lithium was higher than 95%, and the quality of the lithium carbonate product reached the battery chemical grade standard. This new process offers a new way for the utilisation of lithium resources in salt lakes.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Characterization of seven new related impurities and forced degradation products of tetracaine hydrochloride and proposal of degradation pathway by UHPLC-Q-TOF-MS

A rapid and highly sensitive method was developed for separation and identification of the related impurities and degradation products in tetracaine hydrochloride by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The chromatographic separation was achieved on an Agilent Infinity Lab Poroshell 120 EC-C18 column (4.6 ×100 mm, 2.7 µm) using gradient elution with mobiles phase of A (10 mM ammonium acetate buffer containing 0.1% formic acid) and B (acetonitrile) at a flow rate of 1.0 mL/min. Forced degradation experiments were also performed under acidic, alkaline, thermal, photolytic, and oxidative stress conditions following ICH guidance. The result revealed that tetracaine hydrochloride is extremely sensitive to oxidation condition and highly sensitive to alkaline/acidic hydrolysis, and susceptible to light condition. In total, five related impurities and seven degradation products were successfully detected in the positive mode of electrospray ionization. The structures of all these impurities were characterized based on the high-resolution MS data and manufacture process, and the fragmentation pathways of tetracaine and these impurities were constructed and discussed. Seven of them have not been reported before, and two of them were specified impurities described in various pharmacopoeias. The fragmentation pathways and plausible mechanisms for the formation of these impurities were proposed.

Separation of phosphorothioate oligonucleotide impurities by WAX HPLC under high organic content elution conditions

The separation of impurities in phosphorothioate diester (PS) oligonucleotides is complicated by (1) the presence of a very large number of diastereoisomers, e.g., 219 for a 20-mer oligonucleotide, (2) peak broadening due to the hydrophobic character of the sulfur atom, and (3) the chemical similarity of the impurities to the parent oligonucleotide and each other. Further difficulties arise due to the chemical nature of oligonucleotides, which display a complex mixture of ionic, hydrophobic, H-bonding, and other functionalities. To minimize hydrophobic interactions and peak broadening due to the PS modification, we have developed a novel method that combines a weak anion exchange (WAX) column with a mobile phase elution system designed to maximize separation by a single ionic/electrostatic interaction. We found that although chaotropes are helpful, the most significant beneficial effect of the hydrophilic WAX column is that high-organic, low-salt mobile phase is required for product elution. Separations are also benefitted by pH gradient effects on stationary phase electrostatic potential and analyte ionization. An extraordinary degree of separation is achieved by the new WAX method in comparison to SAX (strong anion exchange) chromatography. For the first time, the extent of deamination of PS oligonucleotides is directly determined by a chromatography-only method. The approach, representative results, and the mechanisms of separation are discussed.