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

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

Development of a barley reference material for gluten analysis.

Celiac disease (CD) can be triggered in susceptible individuals by the consumption of gluten, a complex storage protein mixture present in wheat, rye and barley. There is no specific reference material (RM) available for barley and this leads to inaccurate quantitation of barley gluten in supposedly gluten-free foods. Therefore, the aim was to select representative barley cultivars to establish a new barley RM. The relative protein composition of the 35 barley cultivars averaged 25% albumins and globulins, 11% d-hordeins, 19% C-hordeins, and 45% B/γ-hordeins. The mean gluten and protein content was 7.2 g/100 g and 11.2 g/100 g, respectively. The prolamin/glutelin ratio (1:1) commonly used in ELISAs to calculate the gluten content was found to be inappropriate for barley (1.6 ± 0.6). Eight cultivars suitable as potential RMs were selected to ensure a typical barley protein composition and improve food safety for CD patients.

Prediction of wheat gluten composition via near-infrared spectroscopy.

Gluten composition is an important quality parameter for wheat flour, because it is strongly correlated to baking quality. Wheat proteins are commonly extracted stepwise and analysed using RP-HPLC-UV to determine the gluten composition. This procedure is very time-consuming and labour-intensive. Therefore, a new, fast and easy method to quantitate gluten proteins was established using NIR spectroscopy (NIRS). PLS-regression models were calculated containing 207 samples for calibration and 169 for test set validation. Albumin/globulin (ALGL), gluten, gliadin and glutenin content was predicted with a root mean square error of prediction (RMSEP) of 2.01 mg/g, 6.09 mg/g, 4.25 mg/g and 3.50 mg/g, respectively. High-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) were predicted with a RMSEP of 1.12 mg/g and 2.38 mg/g. The relative error was too high for ALGL, LMW-GS and HMW-GS, but that of gluten, gliadins and glutenins was in a range comparable to the reference method. Therefore, the new NIRS method can be used to estimate the gluten composition of wheat flour, including the gliadin/glutenin and the LMW-GS/HMW-GS ratio.

Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine / 中国生物制品学杂志

@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

Development and verification of pre-column derivatization reverse phase-high performance liquid chromatography for determination of Glycine and Histidine content in recombinant proteins / 中国生物制品学杂志

@#Objective To develop and verify a pre-column derivatization reverse phase-high performance liquid chromatography(RP-HPLC) method for determination of Glycine and Histidine content in recombinant proteins.Methods AccQ Tag-C 18(3.9 mm × 150 mm,4 μm) column was used as chromatographic column,6-aminoquinolyl-N-hydroxysccinimidyl carbamate(AQC) was used as pre-column derivatization reagent,while α-aminobutyric acid as internal standard.AccQTag Eluent A solution,acetonitrile solution and high-purity water were used as mobile phases.The UV detection wavelength was 248 nm,injection volume was 10 μL,flow rate was 1.0 mL/min,and column temperature was 37 ℃.The contents of Glycine and Histidine in samples were determined by the internal standard method,and the specificity,linearity,detection limit,quantitative limit,precision,accuracy and stability of the method were verified.Results The developed method effectively separated Glycine,Histidine and internal standard α-aminobutyric acid with high specificity.The standard curves of Glycine in the range of 2.25～11.25 μg/mL and Histidine in the range of 72.85～364.24 μg/mL showed good linearity,each correlation coefficient(R~2) 0.99.The detection limits were 2.25 μg/mL for Glycine and 18.21 μg/mL for Histidine.The quantitative limits were 4.69 μg/mL for Glycine and 32.86 μg/mL for Histidine.The relative standard deviation(RSD) of 6 replicates with the same concentration of Glycine and Histidine were 4.6% and 5.0%,and the RSD of recovery rate in intermediate precision test was 6.9% and 2.0%,respectively.The content of Glycine was close to the quantitative limit,and the average recoveries of high,medium and low concentrations of samples were within 75.9%～111.7%;The recoveries of Histidine ranged from 88.9% to 97.3%.The RSD of Glycine content and Histidine content was 7.7% and 3.3% respectively at 0,12,18,24,30 and 48 h in the same sample.Conclusion The pre-column derivatization RP-HPLC method has accurate and reliable results with high precision,which might be used for quality control of Glycine and Histidine content in recombinant proteins.

Development and validation of reverse phase-high performance liquid chromatography for determination of residual N-hydroxy succinimide content in semaglutide / 中国生物制品学杂志

@#Abstract： Objective To develop and validate a reverse phase⁃high performance liquid chromatography（RP⁃HPLC） method for determination of residual N⁃hydroxy succinimide（NHS）content in semaglutide. Methods A RP⁃HPLC method was developed based on the screening of chromatographic column and optimization of mobile phase（phosphate concentration and the ratio of acetonitrile），validated for specificity，suitability，accuracy，reproducibility and stability， and determined for linear range，limit of quantitation（LOQ）and limit of detection（LOD）. The NHS contents in three batches of semaglutide were determined by the developed method. Results The optimal condition for RP⁃HPLC was as follows：CAPCELL PAK ADME column（4. 6 mm × 150 mm，3 μm）was adopted，serving 0. 05 mol／L potassium dihy⁃ drogen phosphate solution⁃acetonitrile（98∶2）as mobile phase A，and 70% acetonitrile as mobile phase B with gradient elution（0 min，0% B；10 min，0% B；19 min，90% B；19. 1 min，0% B；25 min，0% B）at a flow rate of 0. 8 mL／min. The detection wave length was set at 260 nm，while the column temperature was 30 ℃. The developed method showed good specificity and systemic suitability，of which the linear range was 0. 2 ~ 3. 0 μg／mL（R2 = 1. 000 0），while the LOD and LOQ were 4. 8 and 9. 6 ng respectively. The RSD of recovery rates of NHS samples at three concentrations was 0. 58%， indicating a high accuracy. The RSD of NHS contents in six test samples was 0. 16%，indicating a high reproducibility. The RSD of peak areas of NHS after storage at room temperature for 0，4，8，12，16，20 and 24 h was 0. 34%，indicating a high stability. No NHS was detected in three batches of semaglutide by the developed method. Conclusion The developed RP⁃HPLC method is simple and sensitive，which may be used for the determination of NHS content in semaglutide.

Identification of RNAi hypoallergic bread wheat lines for wheat-dependent exercise-induced anaphylaxis patients.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is one of the most severe forms of wheat allergy. It occurs in patients when they exercise after ingesting wheat-containing foods. Nowadays, the only possible alternative for WDEIA patients is to avoid such foods. This study investigated the potential of six RNA of interference (RNAi) wheat lines with low-prolamin content as alternatives for WDEIA patients. For that purpose, a high performance-liquid chromatography (HPLC) analysis was performed to evaluate differences in gluten protein fractions among these lines. Next, western blots were conducted to measure the immunoglobulin E (IgE) reactivity to wheat proteins in sera from five WDEIA patients. Additionally, monoclonal antibodies (moAb) recognition sites and the IgE binding sites were searched in all peptides identified by LC-MS/MS after protein digestion. The results showed a 61.4%-81.2% reduction in the gliadin content of the RNAi lines, accompanied by an increase in their high-molecular weight (HMW) glutenin content compared to the wild type bread wheat line (WT). In all cases, the reduction in gliadin content correlated with a decrease in IgE reactivity observed in the sera of WDEIA patients, highlighting the E82 and H320 lines. These two RNAi lines exhibited a ≤90% reduction in IgE reactivity. This reduction could be attributed to an absence of IgE binding sites associated with α- and ω5-gliadins, which were present in the WT. Overall, these lines offer a potential alternative for foodstuff for individuals with WDEIA.

Chromatographic separation of glycated peptide isomers derived from glucose and fructose.

Amino groups in proteins can react with aldehyde groups in aldoses or keto groups in ketoses, e.g., D-glucose and D-fructose, yielding Schiff bases that rearrange to more stable Amadori and Heyns products, respectively. Analytical strategies to identify and quantify each glycation product in the presence of the corresponding isomer are challenged by similar physicochemical properties, impeding chromatographic separations, and by identical masses including very similar fragmentation patterns in tandem mass spectrometry. Thus, we studied the separation of seven peptide families, each consisting of unmodified, glucated, and fructated 15mer to 22mer peptides using reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). In RP-HPLC using acidic acetonitrile gradients, unglycated peptides eluted ~ 0.1 to 0.8 min after the corresponding glycated peptides with four of seven peptides being baseline separated. Isomeric glucated and fructated peptides typically coeluted, although two late-eluting peptides were partially separated. Neutral eluents (pH 7.2) improved the chromatographic resolution (Rs), especially in the presence of phosphate, providing good and often even baseline separations for six of the seven isomeric glycated peptide pairs with fructated peptides eluting earlier (Rs = 0.7 to 1.5). Some glucated and unmodified peptides coeluted, but they can be distinguished by mass spectrometry. HILIC separated glycated and unmodified peptides well, whereas glucated and fructated peptides typically coeluted. In conclusion, HILIC efficiently separated unmodified and the corresponding glycated peptides, while isomeric Amadori and Heyns peptides were best separated by RP-HPLC using phosphate buffered eluents.

Comparative Study on Gluten Protein Composition of Ancient (Einkorn, Emmer and Spelt) and Modern Wheat Species (Durum and Common Wheat).

The spectrophotometric Bradford assay was adapted for the analysis of gluten protein contents (gliadins and glutenins) of spelt, durum wheat, emmer and einkorn. The assay was applied to a set of 300 samples, including 15 cultivars each of common wheat, spelt, durum wheat, emmer and einkorn cultivated at four locations in Germany in the same year. The total protein content was equally influenced by location and wheat species, however, gliadin, glutenin and gluten contents were influenced more strongly by wheat species than location. Einkorn, emmer and spelt had higher protein and gluten contents than common wheat at all four locations. However, common wheat had higher glutenin contents than einkorn, emmer and spelt resulting in increasing ratios of gliadins to glutenins from common wheat (< 3.8) to spelt, emmer and einkorn (up to 12.1). With the knowledge that glutenin contents are suitable predictors for high baking volume, cultivars of einkorn, emmer and spelt with good predicted baking performance were identified. Finally, spelt, emmer and einkorn were found to have a higher nitrogen partial factor productivity than common and durum wheat making them promising crops for a more sustainable agriculture.

Characterization of polyethermethylsiloxanes using ultra-high performance liquid chromatography-electrospray ionization and time-of-flight mass spectrometry.

Polyethermethylsiloxanes (PEMSs) are silicone based polymers formed by attaching one or more ethylene oxide (EO)/propylene oxide (PO) chains to a siloxane chain. As the siloxane chain is lengthened, the polarity of the PEMSs are reduced. Little research has been conducted on the use of ultra-high performance liquid chromatography (UHPLC)-mass spectrometry (MS) to analyze PEMS oligomers with more than 4 siloxane groups because of their low polarity and poor solubility in water and acetonitrile (ACN). In this study, we developed a high chromatographic resolution method for PEMS and polyether oligomers using the water-ACN-isopropanol (IPA) tertiary solvents gradient. PEMSs oligomers containing one EO pendant chain and 3-13 silicone groups were analyzed. More than 102 PEMS oligomers and 21 polyether oligomers were separated within 45 min and identified by accurate molecular masses. During this gradient elution process, different chromatographic modes were used: both precipitation-redissolution and adsorption mode for PEMSs, adsorption mode for EO chains in polyethers, and exclusion mode for EO chains in PEMSs. This efficient separation method for PEMSs would broaden the characterization of silicone surfactants. Also, it is beneficial to establish the relationship between surfactant structure, synthetic route and performance optimization.

Application of Natural Deep Eutectic Solvents in the Extraction of Quercetin from Vegetables.

Quercetin is a phytochemical with disease prevention and health promotion activities that has attracted significant research attention. In this study, choline chloride and betaine-based natural deep eutectic solvents were prepared using a heating method. Their physical and chemical properties were also tested. Then, they were applied to extract quercetin from onion and broccoli with ultrasonic-assisted solid liquid method coupled with HPLC. Three factors (temperature, amount, and time) were considered for the optimization of the extraction assays. In the optimal conditions, the extraction recoveries were 88.91-98.99%, 88.45-99.01%, and 89.56-98.74% for quercetin, isorhamnetin, and kaempferol. Tailor-made natural deep eutectic solvents could be applied as sustainable and safe extraction media for biochemical applications.

[Impurity profile of macitentan in tablet dosage form using a stability-indicating high performance liquid chromatography method and forced degradation study].

Macitentan (MAC) is a pulmonary arterial hypertension (PAH) drug marketed as a tablet and often has stability issues in the final dosage form. Quantitative determination of MAC and its associated impurities in tablet dosage form has not been previously reported. This study quantified impurities present in Macitentan tablets using a binary solvent-based gradient elution method using reversed phase-high performance liquid chromatography. The developed method was validated per International Conference on Harmonization (ICH) guidelines and the drug product was subjected to forced degradation studies to evaluate stability. The developed method efficiently separated the drug and impurities (48 min) without interference from solvents, excipients, or other impurities. The developed method met all guidelines in all characteristics with recoveries ranging from 85%-115%, linearity with r2 ≥ 0.9966, and substantial robustness. The stability-indicating nature of the method was evaluated using stressed conditions (hydrolysis:1 N HCl at 80℃/15 min; 1 N NaOH at 25℃/45 min; humidity stress (90% relative humidity) at 25℃ for 24 h, oxidation:at 6% (v/v) H2O2, 80℃/15 min, thermolysis:at 105℃/16 h and photolysis:UV light at 200 Wh/m2; Fluorescent light at 1.2 million luxh). Forced degradation experiments showed that the developed method was effective for impurity profiling. All stressed samples were assayed and mass balance was>96%. Forced degradation results indicated that MAC tablets were sensitive to hydrolysis (acid and alkali) and thermal conditions. The developed method is suitable for both assay and impurity determination, which is applicable to the pharmaceutical industry.

Comparative Analysis of Protein Quantification Methods for the Rapid Determination of Protein Loading in Liposomal Formulations.

Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties, from small molecules to large biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly, by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.

Determination of ATP, ADP, and AMP Levels by Reversed-Phase High-Performance Liquid Chromatography in Cultured Cells.

Cytoplasmic and mitochondrial Ca2+ signals couple cellular ATP production to activity-related energy demand. In order to accurately determine the bioenergetic effect of Ca2+ signals, cellular energy charge, i.e., the compound ratio of the phosphorylated adenine nucleotides AMP, ADP, and ATP, should be estimated. Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation and quantitation of these molecules. Here we describe a protocol applied in our laboratories to quantify ATP, ADP, and AMP nucleotides in cellular extracts.

Identification and characterization of flavor precursor peptide from beef enzymatic hydrolysate by Maillard reaction.

In this study, beef enzymatic hydrolysate was fractionated by sequential UF/GFC/RP-HPLC. RP-HPLC fractions were subjected to Maillard reaction with xylose. The FD/TD factors of the MRPs were prominent. Sensory evaluation showed that F-3 exhibited intense meaty taste; the kokumi intensity, umami-enhancing capacity in F-4; the umami-enhancing capacity in F-5; the umami intensity and umami-enhancing capacity in F-6. All these were strong in F-7 and F-8. The RP-HPLC fractions were subsequently analyzed by ESI-Q-TOF MS/MS. Of the 21 peptides identified, Leu(Ile)-X, Val-X, Phe-X, and Cys-containing peptides were the major ones. Six peptides (Leu-Cys, Glu-Cys-Gly, Cys-Gly-Val, Val-Met, Phe-Glu, and Phe-Gln) were synthesized and included in the Maillard reaction with xylose. FD factors of MRPs of all these synthesized peptides were significantly greater than those of fraction F-7. The stronger Maillard reactivity demonstrated by the synthesized peptides proves that this work is correct.

Metabolite characterization of powdered fruits and leaves from Adansonia digitata L. (baobab): A multi-methodological approach.

The metabolite profiling of extracts from Adansonia digitata L. (baobab) fruit pulp and leaf, and the quantification of their major components, was conducted by means of reverse-phase, high-performance liquid chromatography with photodiode array detection, coupled to electrospray ion-trap mass spectrometry (RP-HPLC-PDA-ESI-MS/MS) and high field nuclear magnetic resonance (NMR) spectroscopy. Water-soluble metabolites from chemical classes including sugars, amino acids, organic acids, and phenolic compounds, were identified, in addition to metabolites soluble in organic solvents such as triacylglycerides, sterols, and fatty acids, and most of these were quantified. The profiling of the primary and secondary metabolites of baobab fruit and leaves addresses the limited knowledge of the chemical composition of baobab, and helps support and explain the growing evidence on its nutritional and biological properties, and provide suggestions about the possible uses of baobab fruit and leaves by food, pharmaceutical and cosmetic industries.

Reverse phase high-performance liquid chromatography (RP-HPLC) system for analysis of vitamin D3 soft capsules with soybean oil matrix / 复旦学报（医学版）

Objective To develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system.Methods RP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm,5 ttm) with detection wavelength of 265 nm,column temperature of 25 ℃ and flow rate of 1 mL/min.Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase.Firstly,acetonitrile was mixed with different proportions of methanol,water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column.Then,a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study.Results The recovery was only 80.55％-84.37％ with 100％ acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement.Recovery rate was achieved between 98.07 ％ and 103.23 ％ with V (acetonitrile) ∶V (ethanol) =90∶10 as the mobile phase,while improving pealk shape.The method showed good linearity [(0.52-5.2) x 10-4 t mol/L,R2＞0.999] and fine density (RSD＜2.32％) which can be used for determination.For impurities profile,it could be achieved using V (acetonitrile):V (water) =95:5 as the mobile phase which can obviate interference from soybean oil matrix.Conclusions The method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.

Process for production and purification of Lethal Toxin Neutralizing Factor (LTNF) from E. coli and its economic analysis.

BACKGROUND: Purification of peptides offers unique challenges with respect to obtaining the desired process yield and selectivity. Lethal Toxin Neutralizing Factor (LTNF) is a peptide that is known to neutralize snake venom in mice when the peptide is preincubated with the venom prior to intravenous injection. A process for producing highly purified recombinant LTNF has been developed. The process has been modelled in SuperPro designer using laboratory data for a plant capable of producing 10 Kg of purified rLTNF. Economic analysis has been performed for manufacturing 3 ton of purified rLTNF. RESULTS: The process developed produces peptide in the form of concatemer that has been specifically designed to accumulate as insoluble inclusion bodies (IB) during expression in E. coli. A cation exchange chromatography step has been developed to capture the rLTNF concatemer at 140 g/L dynamic binding capacity. Further, the purified concatemer is cleaved completely into monomeric rLTNF using alpha-chymotrypsin enzyme. Finally, a reversed phase high performance liquid chromatography has been designed to purify rLTNF with a recovery of more than 90% and purity greater than 98%. The overall process recovery is 78±2% resulting in 3.36 g of purified product per batch. Techno-economic evaluation of the process has been performed to demonstrate its economic feasibility against currently marketed antivenom products. CONCLUSIONS: The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.

Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method.

Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

Determination of urazamide in pharmaceutical preparation with room temperature ionic liquid.

A high performance liquid chromatographic method was developed and validated for the determination of urazamide in pharmaceutical preparation with novel green aqueous mobile phase modified with room temperature ionic liquids (RTILs). 1-Ethyl-3-methyl-imidazolium tetrafluoroborate ([EMIM][BF4]) was selected as a mobile phase additive to improve retention and avoid baseline disturbances at t0. Various mobile phase parameters such as cation moiety, chaotropic anion moiety, pH and concentration of RTILs were optimized to determine urazamide at the proper retention time. The assay was validated according to International Conference on Harmonization guidelines. The linearity of the calibration curve was good (r2 > 0.999). Intra-day precision varied between 0.50 and 1.23%. Relative standard deviations of inter-day precision ranged between 1.07 and 1.66%. Recoveries in tablets ranged between 99.7 and 101.2% and it was successfully applied to determine urazamide in pharmaceutical preparations.