A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium.

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Evaluation of the potential of annatto seed powder to reduce the formation of heterocyclic amines in charcoal-grilled and pan-fried beef patties.

Various strategies are being explored to reduce the formation of undesirable compounds during the thermal processing of foods. This study investigates the impact of incorporating annatto seed powder (Bixa orellana L.) into beef patties to reduce the formation of heterocyclic amines (HAs) during charcoal-grilling and pan-frying. A three-level full factorial design was used to assess the effect of both annatto seed powder concentration and cooking times on HAs formation. The results showed that HA formation increased with longer cooking times and decreased with higher concentrations of annatto seed powder. A significant reduction in HA content was observed in both charcoal-grilled and pan-fried beef patties when annatto seed powder was added, with a particularly notable 91 % reduction at the 1 % addition level. These findings demonstrate that the addition of annatto seed powder is a highly effective strategy for reducing HA formation in beef patties. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (PubChem CID: 62275); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (PubChem CID: 104739); 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) (PubChem CID: 104855); 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (PubChem CID: 1530); 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) (PubChem CID: 5284474); 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) (PubChem CID: 5284476); 2-amino-9H-pyrido[2,3-b]indole (AαC) (PubChem CID: 62805); 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) (PubChem CID: 62244); Bixin (PubChem CID: 5281226).

Chiral separation and spectroscopic characterization of mefloquine analogues.

Mefloquine, a widely used antimalarial agent, has spurred ongoing research into the development of derivatives with enhanced efficacy and reduced side effects. In this investigation, we synthesized two compounds containing N-allyl or N-tert-butylacetamid groups. A chiral liquid chromatography with polysaccharide chiral stationary phase was utilized to separate the enantiomers of both derivatives. We employed spectroscopic chiroptical and non-polarizable methods such as electronic and vibrational circular dichroism, infrared absorption and ultraviolet spectroscopies. Combined with density functional theory calculations, the stable conformers were found in solution and their spectra were subsequently simulated. We elucidated the three-dimensional structure of the enantiomerically pure compounds and assigned the absolute configuration of all prepared derivatives using both experimental and simulated spectra.

Examination of primary aromatic amines content in polylactic acid straws and migration into food simulants using SERS with LC-MS.

Polylactic acid (PLA) straws hold eco-friendly potential; however, residual diisocyanates used to enhance the mechanical strength can generate carcinogenic primary aromatic amines (PAAs), posing health risks. Herein, we present a rapid, comprehensive strategy to detecting PAAs in 18 brands of food-grade PLA straws and assessing their migration into diverse food simulants. Surface-enhanced Raman spectroscopy was conducted to rapidly screen straws for PAAs. Subsequently, qualitative determination of migrating PAAs into various food simulants (4 % acetic acid, 10 % ethanol, 50 % ethanol) occurred at 70 °C for 2 h using liquid chromatography-mass spectrometry. Three PAAs including 4,4'-methylenedianiline, 2,4'-methylenedianiline, and 2,4-diaminotoluene were detected in all straws. Specifically, 2,4-diaminotoluene in 50 % ethanol exceeded specific migration limit of 2 µg/kg, raising safety concerns. Notably, PAAs migration to 10 % and 50 % ethanol surpassed that to 4 % acetic acid within a short 2-hour period. Moreover, PLA straws underwent varying degrees of shape changes before and after migration. Straws with poly(butylene succinate) resisted deformation compared to those without, indicating enhanced heat resistance, while poly(butyleneadipate-co-terephthalate) improved hydrolysis resistance. Importantly, swelling study unveiled swelling effect wasn't the primary factor contributing to the increased PAAs migration in ethanol food simulant, as there was no significant disparity in swelling degrees across different food simulants. FT-IR and DSC analysis revealed higher PAAs content in 50 % ethanol were due to highly concentrated polar ethanol disrupting hydrogen bonds and van der Waal forces holding PLA molecules together. Overall, minimizing contact between PLA straws and alcoholic foods is crucial to avoid potential safety risks posed by PAAs.

Stable Isotope Tracing Experiments Using LC-MS.

Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.

Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.

A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.

Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).

Preconcentration of Cinnamic Acid Derivatives in Traditional Chinese Medicines by an Effective Dispersive Liquid-Liquid Microextraction Based on a Hydrophobic Deep Eutectic Solvent.

A dispersive liquid-liquid microextraction based on hydrophobic deep eutectic solvent (hDES) was developed for the extraction and quantification of four cinnamic acid derivatives in traditional Chinese medicines coupled with high-performance liquid chromatography-ultraviolet detection. In this method, a hDES (tetrabutylammonium chloride-hexanoic acid, molar ratio of 1:2) was prepared as the extractant. It only took 15 s to handle multiple samples simultaneously by hand-assisted dispersion. The use of a narrow-bore tube reduced the amount of the hydrophobic extractant with easier recovery. The approach was influenced by several key parameters, including the composition and consumption of the DES, sample phase pH, salt amount, extraction time, and centrifugation time, all of which had been investigated and optimized. Moreover, the formation of the DES was characterized by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Under the optimal conditions, enrichment factors of the target analytes ranged from 135 to 220. Satisfactory linearities (r ≥ 0.9977), detection limits (0.2-0.4 ng/mL), precision (<8.5%), and accuracy (recoveries: 90.0%-104.6%) were obtained. The method has been successfully applied to the simultaneous extraction and preconcentration of four cinnamic acid derivatives in Chinese medicinal samples with rapidness, high efficiency, and convenience.

LC-MS/MS and GC-MS/MS Cross-Checking Analysis Method for 426 Pesticide Residues in Agricultural Products: A Method Validation and Measurement of Uncertainty.

A method was developed to analyze residual pesticides in various vegetables using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). A method can analyze over 200 individual compounds, selectively separate peaks within 30 min, and meet various criteria such as those by the Codex Alimentarius Commission (CODEX). Most compounds showed recovery rates between 70 and 120%, and a relative standard deviation was within 20%. Measurement uncertainty, considering various influencing factors such as instrument precision, method sensitivity, experimental conditions, sample handling, and analytical procedures, was meticulously calculated. The expanded uncertainties of cross-checking 33 available pesticides ranged from 10.1 to 26.2 µg/kg at a confidence level of approximately 95%. Risk assessment of detected pesticides in agricultural products indicated a safety range of 0.00003-2.87240%. The developed method effectively analyzes diverse compounds simultaneously, contributing to agricultural product safety.

Thin film microextraction of apixaban from plasma based on the covalent organic framework coated on a mesh prior to liquid chromatography-tandem mass spectrometry.

In this research, a new covalent organic framework was synthesized and utilized as a coating in thin film microextraction for the extraction of apixaban from plasma samples. This coating was applied to the mesh modified through immersion in a HF solution. The extracted drug was then analyzed using liquid chromatography-tandem mass spectrometry. By combining the high specific surface area and selectivity of the covalent organic framework, along with integrating the innovative thin film microextraction method and a sensitive analysis system, an efficient analytical approach was achieved. The target analyte was preconcentrated and extracted by immersing of the covalent organic framework-coated mesh as an absorbent into the biological sample. Subsequently, a sonication process was conducted for a specific duration. Following this, the extracted analyte was desorbed using acetonitrile as the elution solvent. The effective parameters of the proposed technique were optimized by using "one-parameter-at-a-time" strategy and the optimal conditions were selected. By integrating the developed method notable achievements were made in the terms of low limits of detection and quantification (0.17 and 0.56 µg/L, respectively), a wide linear range (0.05-250 µg/L), intra- and inter day precisions (with relative standard deviations of ≤14 %), as well as satisfactory extraction recoveries (53 % and 54 % in plasma and deionized water, respectively). Hence, it can be concluded that the introduced technique exhibits high efficiency and reliability when applied to biological samples.

Antiproliferative Effects of Methanolic Fruit Extract of Solanum xenthocarpum (L.) on Human Breast Cancer Cells.

Solanum xanthocarpum, a perennial herb native to India, contains steroidal glycoalkaloids with notable anticancer properties. This study investigated the antioxidant and antiproliferative effects of methanolic fruit extract of S. xanthocarpum on human breast cancer cells (MDA-MB-231). Phytochemical screening and LC-HRMS analysis confirmed presence of various primary and secondary metabolites. Antioxidant activity was assessed through DPPH, ABTS radical scavenging, reducing power, and phosphomolybdate assays. The extract demonstrated significant antioxidant potential with EC50 values of 60.10 ± 0.88 µg/mL (DPPH) and 392.29 ± 3.93 µg/mL (ABTS). Cytotoxicity against MDA-MB-231 cells was evaluated via morphological analysis, MTT assays, and IC50 determination (24.19 ± 0.56 µg/L). Apoptosis was confirmed using dual staining techniques (AO/EB, Hoechst 33342/PI, DAPI), revealing condensed nuclei, apoptotic bodies, and reduced mitochondrial membrane potential, as indicated by Rhodamine staining. Additionally, increased reactive oxygen species (ROS) levels were observed using H2-DCF-DA staining. The total phenolic and flavonoid contents of the extract were 127.78 ± 3.547 mg GAE/g and 98.06 ± 4.289 mg QE/g, respectively. These findings suggest that the methanolic fruit extract of S. xanthocarpum possesses strong antioxidant and anticancer activities, indicating its potential role in cancer treatment. Further studies are warranted to explore its bioactive compounds for developing novel anticancer therapies.

Detecting Bisphenol A Leaching from Four Different Commercially Available Clear Aligner Sheets: An Ex Vivo Study.

AIM: The study aimed to detect and quantify bisphenol A (BPA) leaching in salivary samples of patients undergoing clear aligner therapy (CAT) using four different commercially available sheets. MATERIALS AND METHODOLOGY: Four different commercially available clear aligners namely Monoflex®, Erkodur®, Leone®, and Duran® were delivered to 20 volunteers who were grouped into (n = 5) group A, group B, group C, and group D, respectively. Salivary samples were collected immediately before aligner insertion (day 0) and at day 1, day 5, and day 7 after aligner wear. Comparisons were made between baseline (day 0) BPA levels and subsequent time points to assess the leaching kinetics of BPA from the clear aligners by liquid chromatography/mass spectrometry. RESULTS: The overall mean leaching of 0.74 ± 0.33 ppm at T1 (day 1) was observed among four groups of aligners, while no leaching was detected at T5 (day 5) and T7 (day 7). Among the four groups, the highest leaching of 1.24 ppm was detected from Duran at T1 followed by Monoflex (0.76 ppm), Erkodur (0.56 ppm), and Leone (0.43 ppm). CONCLUSION: It can be concluded that leaching only during the first 24 hours of aligner usage was dominant compared to other time intervals. Among the aligners considered, Duran was found to be the least safe followed by Monoflex, Erkodur, and Leone. CLINICAL SIGNIFICANCE: Since aligners are expanding in usage it is important to consider their biocompatibility. Even though the results indicate minimal leaching of BPA, it has a cumulative negative effect when patients undergo prolonged treatment with aligners. How to cite this article: Azhagudurai N, Rajendran R, Aishwarya K, et al. Detecting Bisphenol A Leaching from Four Different Commercially Available Clear Aligner Sheets: An Ex Vivo Study. J Contemp Dent Pract 2024;25(6):535-539.

Expanding cyclodextrin use in normal phase and super/subcritical fluid chromatographic modes for the chiral separation of 1,4-dihydropyridines.

Cyclodextrin-based stationary phases are important chiral selectors in liquid chromatography. These chiral selectors are most commonly used in the reversed-phase mode because native cyclodextrin assumes a torus conformation with a hydrophobic cavity, facilitating inclusion complexation in aqueous environments. However, the value of native and aliphatic-derivatized cyclodextrins in other modes, such as the normal phase liquid chromatography (NPLC) or super/subcritical fluid chromatography (SFC), remains unexplored. In this work, we report chiral separations of pharmaceutically relevant compounds with the 1,4-dihydropyridine (DHP) scaffold on a 2-hydroxypropyl-ß-cyclodextrin (CD-RSP) stationary phase in NPLC and SFC modes. Although CD-RSP is conventionally considered only effective in the reversed-phase mode, we show that these compounds tend to separate better in other modes. This is particularly apparent for analytes with hydrogen-bonding moieties. We propose that the separation mechanism primarily depends on external adsorption rather than inclusion complexation. The negligible impact of a complexation-competitive additive on retention in non-aqueous modes further supports this claim. Additionally, van Deemter analysis demonstrated the efficiency and environmental benefit of using this stationary phase in the SFC mode, further highlighting the promise of aliphatic derivatized cyclodextrin stationary phases for greener separations.

Contents of sensitising rubber accelerators in disposable rubber gloves: A Copenhagen market survey.

BACKGROUND: Rubber gloves contain rubber accelerators that may cause contact allergy. The content of sensitising rubber accelerators in contemporary rubber gloves is not well known. OBJECTIVES: Identify and quantify the content of rubber accelerators in disposable rubber gloves. METHODS: Fifty-one gloves of 49 different brands were collected. Forty-eight of the gloves were disposable and three re-usable. The gloves were analysed for their content of sensitising rubber accelerators, that is, zinc dithiocarbamates, thiurams, thiazoles/benzothiazoles, diphenylguanidine, and thioureas by high-performance liquid chromatography. RESULTS: Rubber accelerators were identified in 43/48 (90%) of the disposable gloves. In total, 39 gloves contained zinc dibutyldithiocarbamate (ZDBC) (0.18-1.96 mg/g), 34 zinc diethyldithiocarbamate (ZDEC) (0.032-2.78 mg/g), three zinc dibenzyldithiocarbamate (0.65-1.4 mg/g), one zinc dimethyldithiocarbamate (0.23 mg/g), and one 1,3-diphenylguanidine (0.21 mg/g). 2-cyanoethyl dimethyldithiocarbamate (CEDMC) was identified in three gloves (<0.052 mg/g). The one glove labelled as accelerator free contained ZDBC (1.07 mg/g). Only few glove packages had the specific content of rubber accelerators labelled. CONCLUSIONS: The most frequent rubber accelerators in rubber gloves are ZDEC and ZDBC. Accelerator-free gloves may contain rubber accelerators. Full labelling of rubber gloves is needed and producers should be sure not to falsely claim that the rubber gloves are free of rubber accelerators.

Combined Quantification and Characterization of Dissolved Organic Matter by Liquid Chromatography-Mass Spectrometry Using Charged Aerosol Detection.

Dissolved organic matter (DOM) is a complex mixture of thousands of molecular formulas comprised of an unknown number of chemical compounds, the concentration and composition of which are critical to ecosystem function and biogeochemical cycling. Despite its importance, our understanding of the DOM composition is lacking. This is principally due to its molecular complexity, which means that no single method is capable of describing DOM in its entirety. Quantification is typically done by proxy (e.g., relative to carbon content) and does not necessarily match well to compositional data, due to incomplete analytical windows and selectivity of different analytical methods. We present an integrated liquid chromatography (LC)-diode array detector (DAD)-charged aerosol detector (CAD)-mass spectrometry (MS) pipeline designed to both characterize and quantify solid-phase extractable DOM (SPE-DOM) in a single analysis. We applied this method to a set of eight Swedish water bodies sampled in the summer and winter. Chromophoric SPE-DOM was proportionally higher in samples with higher SPE-DOM concentrations but remained relatively consistent between sampling occasions. Ionizable SPE-DOM was relatively consistent across sites but was proportionally higher in summer. Overall, the carbon content of DOM was very consistently ∼40% across sites in both summer and winter. These findings suggest that SPE-DOM concentration at these sites is driven by (presumably allochthonous) chromophoric inputs, with an increased relative contribution in summer of material that is more ionizable and less chromophoric and may be either autochthonous or selectively enriched from allochthonous sources. Thus, with minimal additional effort, this method provided further compositional insights not attained by any single analysis in isolation.

Plasma niacin is inversely associated with hyperlipidemia in participants with diabetes among Chinese adults.

Evidence is limited regarding the association of plasma niacin with the risk of hyperlipidemia in participants with diabetes. We aimed to determine the relationship between plasma niacinamide/nicotinic acid and hyperlipidemia in participants with/without diabetes. Plasma niacinamide/nicotinic acid concentrations were measured using high-performance liquid chromatography-tandem mass spectroscopy. Multivariable logistic regression analyses were performed to evaluate the association between plasma niacin and hyperlipidemia in participants with diabetes and nondiabetes in a cross-sectional study. Compared to the first quartile, plasma nicotinamide, nicotinic acid, and niacin (nicotinamide plus nicotinic acid) were associated with a 54%, 50%, and 52% lower risk of hyperlipidemia in diabetic participants, respectively, but no significant association was observed in nondiabetic participants. These inverse associations persisted across subgroups stratified by sex, age, body mass index, smoking status, alcohol consumption, and physical activity. In addition, the fully adjusted odds ratios (95% confidence intervals) for hypercholesterolemia and hypertriglyceridemia among diabetic participants were 0.54 (0.38, 0.77) and 0.61 (0.44, 0.85), respectively, when comparing to the first quartile of plasma niacin concentrations (all Ptrend < .001). This study of 2647 participants observed that plasma niacin was inversely associated with hyperlipidemia in those with diabetes.

Robustness evaluation of weak anion exchange chromatography method for the purity analysis of therapeutic oligonucleotides.

Therapeutic oligonucleotides are becoming an important class of therapeutics. Their manufacturing processes can result in the formation of impurities, particularly truncated species. To ensure the quality and safety of the product, it is crucial to evaluate the presence of these species. Liquid chromatography analysis enables such purity determination. In this context, a recently described weak anion exchange chromatography method was optimized to allow the effective separation of different impurities. The optimization addressed the complexity and instability of the mobile phases, which contained salts and organic compounds. Adjustments were made to the mobile phase composition and gradient to meet the requirements of QC laboratories. Additionally, to ensure the method's reliability, a robustness study was conducted based on a risk assessment. Five factors were considered potential risks and were assessed experimentally on different chromatographic outputs. This led to the definition of a robust space, ensuring the method's reliability for the purity determination of oligonucleotides.