Mobile Phase Buffer Research Articles

Optimization and Validation of an HPLC-UV Method for Determination of Benzoic Acid and Sorbic Acid in Yogurt and Dried-Yogurt Products Using a Design of Experiment

A method for the determination and analysis of benzoic acid and sorbic acid in yogurt and dried-yogurt products has been developed. This method was based on the use of a simple solid-liquid extraction method, followed by the high-performance liquid chromatography with a UV detector (HPLC–UV), enhanced with the aid of response surface methodology and design of experiment (DOE). The method excludes the use of complicated procedures, time-consuming and labor-intensive pre-treatment processes. Separation of the benzoic acid and sorbic acid with higher selectivity and sensitivity, and within reasonable retention time was performed by using an isocratic mobile phase of acetate buffer (pH 5.6)-methanol 60:40 at a column temperature of 25 °C. Optimization of sample preparation and analytical conditions gave recoveries in the range of 81 to 111% at spike levels of 2–20 mg/L and the relative standard deviations (RSDs) was lower than 9% in all cases. The intra-day precision and inter-day precision results were in the range of 8.4–8.5% and 10.4–11.0%. Additionally, the limits of detection (LOD) were 0.66 and 0.51 mg/L and the limits of quantification (LOQ) were 1.3 and 1.0 mg/L for benzoic acid and sorbic acid, respectively.

Trace Phosphate Improves ZIC-pHILIC Peak Shape, Sensitivity, and Coverage for Untargeted Metabolomics.

Existing hydrophilic interaction liquid chromatography (HILIC) methods, considered individually, each exhibit poor chromatographic performance for a substantial fraction of polar metabolites. In addition to limiting metabolome coverage, such deficiencies also complicate automated data processing. Here we show that some of these analytical challenges can be addressed for the ZIC-pHILIC, a zwitterionic stationary phase commonly used in metabolomics, with the addition of trace levels of phosphate. Specifically, micromolar phosphate extended metabolome coverage by hundreds of credentialed features, improved peak shapes, and reduced peak-detection errors during informatic processing. Although the addition of high levels of phosphate (millimolar) as a HILIC mobile phase buffer has been explored previously, such concentrations interfere with mass spectrometric (MS) detection. We show that using phosphate as a trace additive at micromolar concentrations improves analysis by electrospray MS, increasing signal for a diverse set of polar standards. Given the small amount of phosphate needed, comparable chromatographic improvements were also achieved by direct addition of phosphate to the sample during reconstitution. Our results suggest that defects in ZIC-pHILIC performance are predominantly driven by electrostatic interactions, which can be modulated by phosphate. These findings constitute both a methodological improvement for untargeted metabolomics and an advance in our understanding of the mechanisms limiting HILIC coverage.

The influence of addition of ion-pairing acid and organic modifier of the mobile phase on retention and migration of peptides in pressurized planar electrochromatography system with octadecyl silica-based adsorbent

In our previous papers we have investigated the influence of the mobile phase composition on mechanism of retention, selectivity and efficiency of peptide separation in various high-performance thin-layer chromatography (HPTLC) systems with commercially available silica-based adsorbents. We have also investigated the influence of pH of the mobile phase buffer on migration and separation of peptides in pressurized planar electrochromatography (PPEC). Here we investigate the influence of concentration of ion-pairing additive, and concentration and type of organic modifier of the mobile phase on migration of peptides in PPEC system with octadecyl silica-based adsorbent, and with the same set of the solutes as before. We compare our current results with the results obtained before for similar HPTLC and PPEC systems, and discuss the influence of particular variables on retention, electrophoretic mobility of solutes and electroosmotic flow of the mobile phase. We show, that the final selectivity of peptide separation results from co-influence of all the three factors mentioned. Concentration of organic modifier of the mobile phase, as well as concentration of ion-pairing additive, affect the retention, the electrophoretic mobility, and the electroosmotic flow simultaneously. This makes independent optimization of these factors rather difficult. Anyway PPEC offers much faster separation of peptides with quite different selectivity, in comparison to HPTLC, with similar adsorbents and similar mobile phase composition. However, we also present and discuss the issue of extensive tailing of peptide zones in the PPEC in comparison to similar HPTLC systems.

UPLC/MS-MS assay development for estimation of mozavaptan in plasma and its pharmacokinetic study in rats.

Mozavaptan is a nonpeptide vasopressin receptor antagonist approved for the treatment of ectopic antidiuretic hormone secretion syndrome. A simple, rapid and fully validated UPLC/MS-MS method was developed for the quantitation of mozavaptan in rat plasma. The chromatographic separation was conducted on an Acquity UPLC BEH™ C18 column with an optimum mobile phase of 10mM ammonium acetate buffer and 0.1% formic acid in acetonitrile (30:70 v/v) at a flow rate of 0.3ml/min. The multiple reaction monitoring transitions were performed at m/z 428.16→119.03 for mozavaptan and m/z 237.06→179.10 for carbamazepine (internal standard). The method was effectively applied for the determination of mozavaptan pharmacokinetic parameters after the oral administration of 3mg/kg mozavaptan in rats.

Mixed-Mode Liquid Chromatography on Core Shell Stationary Phases based on Layer-By-Layer Nanodiamond/Polyamine Architecture

Background: The use of diamond as a chromatographic stationary phase has been studied for more than 45 years now. Detonation nanodiamonds (DND) have been regularly used to increase the low specific surface area of these non-porous stationary phases. Recently, core-shell particles with multiple layers of polyallylamine (PAAm) and DND have been reported for liquid chromatographic separations and solid phase extractions. However, retention mechanisms and selectivity of such core-shell particles are not well -understood due to their complex architecture. Objective: The objective of this work was to perform a detailed evaluation of the mixed mode behavior of three structural analogues of new nanodiamond (ND)/polyamine based core-shell stationary phases. Method: Particles were composed of a glassy carbon core coated with poly(allylamine) (PAAm) and ND, deposited using a layer-by-layer (LbL) approach, with the outer PAAm layer treated with a mixture of a hydrophobic cross-linker and modifier to introduce C8/C18 functional groups. Three types of the core-shell stationary phases having different shell thicknesses and purity of NDs were investigated. Type II was prepared with fewer PAAm/ND bilayers as compared to Type I, whereas Type III was prepared with a same number of bilayers but with an additionally purified form of ND as compared to Type II. Ion-exchange interactions of the phases were tested with a set of model anionic, cationic and ampholytic analytes at different eluent pHs. The relative degree of anionexchange and hydrophobic interactions was also evaluated using a set of dansylated amino acids (Dns-AAs) at varied mobile phase buffer concentrations and organic solvent contents. Results: Anion-exchange properties were noted for all phases at low pH, with the Type I phase exhibiting stronger retention of anionic analytes due to higher PAAm content in its shell. Typical reversed-phase behaviour was observed for the hydrophobic Dns-AAs (Phe, Ile, Leu, Trp, Val and Pro), while the retention selectivity for hydrophilic Dns-AAs (Gln, Asn, Thr, Ser) indicated mixed mode mechanism. Values of methylene selectivity obtained for homologues of carboxylic acids indicated relatively low hydrophobicity for all studied phases. Conclusion: All three columns demonstrated low to medium hydrophobicity, and the solute selectivity was dominated by their ion-exchange property. These nanodiamond columns result in lower performance as compared to conventional analytical columns, however, the inherently stable nanodiamond platform ascertains that nanodiamond columns offer better stability at extreme conditions.

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE IGURATIMOD

Analytical method with accuracy, precision and stability indicting was developed for Iguratimod, achieved the required chromatographic separation by High pressure liquid chromatography (HPLC) techniqu e. HPLC method with accuracy, precision, robustness and stability indicating was developed and optimized for quantitative evaluation of Iguratimod as active pharmaceutical ingredient. Chromatographic separation for Iguratimod and its related compounds was achieved by using analytical column with stationary phase Inertsil ODS-3 (Dimension: length 150mm, internal diameter 4.6mm and particle size 5µ) and mobile phase (Buffer and acetonitrile in gradient proportion) at flow rate 1.0ml/minute with detection at 257nm. Squared correlation coefficient (r2) was observed more than 0.999 with concentration window of 12.5 to 75µg/ml. Iguratimod was retained at about 19.0±2.0 minutes. Forced degradation study for Iguratimod was carried out under the stress condition to get required degradation by using acid, base, thermal, photolytic and oxidation by hydrogen peroxide. Degradation products which are generated during stress condition did not interfere to active ingredient, Iguratimod. This analytical method optimized, validated for all the parameters specified in ICH guideline, to get results selective, accurate and precise in presence of degradation impurities.

THE USE OF A CHROMATOGRAPHIC COLUMN PACKED WITH AMIDE SORBENT FOR THE ANALYSIS OF PARABENS

The article investigates the applicability of the amino column XBridge Amide 150×4.6 mm for separation of 4-hydroxybenzoic acid esters (parabens) — based on experience with hydrophilic interaction liquid chromatography used for analysis of various compounds. The study analysed the retention times, elution order and resolution between the peaks of parabens and impurity A (4-hydroxybenzoic acid) while using acetonitrile : phosphate buffer mobile phases containing 7–10 % of acetonitrile and acetonitrile : water mobile phases containing 96–99 % of acetonitrile, and the above-mentioned column. It was demonstrated that reversed-phased chromatography was preferable for separation of paraben homologues, while hydrophilic interaction chromatography was preferable for analysis of 4-hydroxybenzoic acid impurity. When analysing potential mechanisms of retention of compounds by the XBridge Amide column it was suggested that, unlike amino groups, amide groups of the sorbent do not have affinity for phenolic hydroxyl groups of parabens in hydrophilic interaction liquid chromatography.

Comparative Analysis of the Distribution of Copper in Green and Brown Brazilian Propolis Extracts

We proposed here a novel analytical procedure for copper speciation in green and brown propolis extracts using SEC—HPLC—GFAAS with 0.5% m v−1 SDS in 2.5 m mol L−1 Tris–HCl (pH 7.4) as the mobile phase buffer solution. Both basic (0.05 mol L−1 NaOH) and acid (0.05 mol L−1 HCl) conditions were evaluated for sample extraction. Depending on the extraction procedure, differences in copper distribution were identified. Copper was mainly associated with high-molecular-weight (HMW) fractions in green propolis extract when extracted with basic solution, whereas with acid extraction solution, only low-molecular-weight (LMW) fractions were obtained in both samples. Furthermore, combined analysis of results obtained using SEC-UV and GF AAS confirmed the association of copper with LMW and HMW species.

Characterization of Retention Mechanisms in Mixed-Mode HPLC with a Bimodal Reversed-Phase/Cation-Exchange Stationary Phase

Mixed-mode HPLC (MM-HPLC), combining different interactions or retention modes in a single column, can be an interesting alternative to reversed-phase HPLC, notably to achieve the combined retention of polar and non-polar species. In the present fundamental study, we have selected one bimodal stationary phase allowing for both reversed-phase and weak cation-exchange retention modes (Acclaim mixed-mode WCX-1 LC). First, the mobile phase buffer composition (buffer pH ranging from 5 to 7 and concentration ranging from 20 to 100 mM) was explored with a small set of probe compounds (15 acids, bases and neutrals) to ensure adequate retention and peak shapes for the target compounds, and to evaluate the relative contributions of reversed-phase and ion-exchange mechanisms. Second, retention values measured for 63 probe compounds with various proportions of acetonitrile (ranging from 30 to 80%) served to establish linear solvation energy relationships based on (a) the usual and (b) a modified version of the solvation parameter model comprising additional descriptors to take account of interactions with ionizable species to bring some insights into the retention mechanisms. Finally, temperature effects at the low (30%) and high (60%) proportions of acetonitrile were observed between 20 and 40 °C (with 5 °C increments) and Van’t Hoff plots were drawn to measure the changes in interactions energies when the mobile phase composition changed.

A New Simple RP-HPLC Method for Simultaneous Estimation of Meropenem and Vaborbactam in Tablet Dosage Form

A simple, accurate, economical, rapid, selective, reverse phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous estimation of Meropenem and Vaborbactam in its tablet dosage form. The separation was carried out using a mobile phase of buffer and acetonitrile in the ratio of 50: 50 pumped at a flow rate of 1 ml/min along with 250nm as a UV detection wavelength. The stationary phase used was column Kromasil 250 x 4.6 mm, 5μ. Meropenem and Vaborbactam were eluted at a retention time of Meropenem 2.47min and Vaborbactam 3.31 min. The method was developed and validated as per ICH guidelines by considering the parameters such as precision, accuracy, linearity, specificity, robustness and degradation studies. The developed RP-HPLC method can be used for routine analysis of Meropenem and Vaborbactam in combinational dosage form.

Validated Reversed-Phase Liquid Chromatographic Method for Simultaneous Determination of Dextromethorphan and Chlorpheniramine in Non-biological and Biological Matrices Using PDA Detector

A simple, precise and sensitive reversed-phase HPLC method using Photo-diode array detector for simultaneous determination of dextromethorphan (DXM) and chlorpheniramine (CLP) in various matrices had been developed and validated. The separation was achieved within 8 minutes on a C-18 column (15 cm x 4.6 mm x 5 μm) using methanol/ pH 3.0 potassium dihydrogen phosphate buffer (60:40, v/v) mobile phase in an isocratic elution mode with flow rate 0.8 mL/min and UV-detection at 230 nm. External standard was employed for quantification. The current method demonstrated good linearity ranged 1-200 µg/mL for CLP (retention time 3.83 ± 0.10 min) with r2 of 0.9994 and 2-1000 µg/mL for DXM (retention time 5.02 ± 0.10 min) with R2 of 0.9993. The limits of detection and quantification were 0.25 µg/mL and 1.0 µg/mL for CLP and 0.50 µg/mL and 1.5 µg/mL for DXM. No interference from matrices was observed and samples remained stable in the HPLC injector for 12 h. The developed method was accurate that employed a simple liquid/liquid extraction procedure with recovery ranged 90.0%-101.9% for both analytes. Intra and inter-day precision were less than 2.5%. The method was proved robust and reproducible that is applicable to pharmaceutical (active raw materials, syrups) and biological (blood) matrices.

Retention Mechanism of Proteins in Hydroxyapatite Chromatography - Multimodal Interaction Based Protein Separations: A Model Study.

Background: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE).Materials and Methods: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces.Conclusion: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient

The influence of pH on retention and migration of peptides in systems with octadecyl silica-based adsorbent by high-performance thin-layer chromatography and pressurized planar electrochromatography techniques

In our paper we investigate the influence of pH of the mobile phase buffer, on retention of peptides in both: normal phase (NP) and reversed phase (RP) high-performance thin-layer chromatography systems (HPTLC), using C18 silica-based adsorbent. We also compare the influence of pH on retention and/or migration distance of peptides in separation systems of HPTLC and PPEC (pressurized planar electrochromatography) techniques. Our results show, that the change of pH of the mobile phase buffer can be used for two-dimensional HPTLC separation of peptides, but much stronger change of separation selectivity can be obtained by overall inversion of separation system type (NP/RP), as we have described before (Gwarda et al., 2016). In PPEC influence of the mobile phase pH on selectivity of peptide separation is very clear and strictly different than that in HPTLC (including overall inversion of direction of solutes migration), what results from the significant share of electrophoretic effect in overall mechanism of separation. Migration of peptides in two opposite directions results in better selectivity of separation by PPEC. In separation systems of both HPTLC and PPEC techniques, the change of pH clearly affects the efficiency of the separation system, so it can be used for optimization of separation conditions. Our results also show, that some modifications of PPEC equipment and materials could be useful for application of this technique in the field of peptide analysis.

QbD APPROACH FOR STABILITY INDICATING HPLC METHOD FOR DETERMINATION OF ARTEMETHER AND LUMEFANTRINE IN COMBINED DOSAGE FORM

The present paper reports a highly selective, sensitive and robust stability indicating RP-HPLC method using QbDapproach, developed and validated for determination of Artemether and Lumefantrine in combined dosage form. Theexperimental design describes the scouting of the key HPLC method components including mobile phase and pH.Their interrelationships are studied and optimized conditions are obtained for each combination of mobile phase andpH with the help of design expert 10.0 version. Optimal chromatographic conditions were obtained usingPhenomenex, Gemini 5u C-18 column combined with a mixture of Acetonitrile: Buffer (35:65 v/v) mobile phase, pHadjusted to 2.5 with buffer H3PO4. The flow rate was 1.5ml/min. The injection volume was 10μl. Retention time forArtemether and Lumefantrine were found 4.44 and 7.84 min respectively. The method was linear with 2-6μg/ml and12-36μg/ml for Artemether and Lumefantrine respectively. The LOD and LOQ were 0.000278 μg/ml and 0.000841μg/ml respectively. This method found to have a good percentage recovery in forced degradation study using acid,base, oxidation, photolytic, thermal and neutral conditions indicates well separation of both the drugs with otherdegradation products. All validation parameters were within the acceptance limits.&nbsp

Characterization of the Retention of Artificial Sweeteners by Hydrophilic Interaction Liquid Chromatography

ABSTRACTThe aim of this work was to study the chromatographic behavior of polar artificial sweeteners on three hydrophilic interaction liquid chromatography (HILIC) columns: Kinetex HILIC (unbounded silica, core shell), ZIC HILIC (sulfoalkylbetain) and TSKgel Amide-80 (amide). The studied analytes were dipeptides (aspartame, neotame, advantame, alitame), sulfamates (acesulfame, saccharin, cyclamate), and sugars (sucralose, neohesperidin dihydrochalcone). Critical parameters such as the mobile phase pH, buffer concentration, column temperature, and organic modifier were studied for the investigation of the retention mechanism. Partition and adsorption models were also used. The retention mechanism of dipeptides seems to be partition for the aqueous layer and weak electrostatic interactions or hydrogen bonds. For the sulfamates, on Kinetex and ZIC HILIC columns, it is likely that partitioning to the aqueous layer is combined with the ERLIC phenomena at pH 5.5. For the TSKgel Amide, the partition and the hydrogen bonds formed between the analytes and carbamoyl groups of the stationary phase play an important role in the retention mechanism. For sucralose, partition seems to be the predominant mechanism in every column, while, for neohesperidin dihydrochalcone, both partition to aqueous layer and hydrogen bonding seems to contribute to the retention. This is also the first report of a method for the chromatographic determination of advantame, a new sweetener.

Consequences of transition from liquid chromatography to supercritical fluid chromatography on the overall performance of a chiral zwitterionic ion-exchanger

Major differences in the chromatographic performance of a zwitterion ion-exchange type (ZWIX) chiral stationary phase (CSP) in supercritical fluid chromatography (SFC) and high-performance liquid chromatography (HPLC) have been observed. To explain these differences, transition from HPLC to SFC conditions has been performed. The amount of a protic organic modifier in supercritical carbon dioxide (scCO2) was stepwise increased and the effect of this change studied using acidic, basic and ampholytic analytes. At the same time, the effect of various basic additives to the mobile phase and transient acidic buffer species, formed by the reaction of scCO2 with the organic modifier and additives, was assessed. Evidence is provided that a transient acid together with the intrinsic counter-ions present in the ZWIX selector structure drive the elution of analytes even when no buffer is employed. We show that the tested analytes can be enantioseparated under both SFC and HPLC conditions; the best conditions for the resolution of ampholytes are in the so-called enhanced-fluidity mobile phase region. As a consequence, subcritical fluid and enhanced-fluidity mobile phase regions seem to be chromatographic modes with a high potential for operating ZWIX CSPs.

Qualitative and quantitative measurement of cannabinoids in cannabis using modified HPLC/DAD method

This study presents an accurate and high throughput method for the quantitative determination of various cannabinoids in cannabis plant material using high pressure liquid chromatography (HPLC) with a diode array detector (DAD). Sample extraction and chromatographic analysis conditions for the measurement of cannabinoids in the complex cannabis plant material matrix were optimized. The Agilent Poroshell 120 SB-C18 column provided high resolution for all target analytes with a short run time (10minutes) given the core shell technology. The aqueous buffer mobile phase was optimized with ammonium acetate at pH 4.75. The change in the mobile phase and the new column ensured a separation between cannabidiol (CBD and cannabigerol (CBG) along with cannabigerol and tetrahydrocannabinolic acid (THCA), which were not well separated by previous publications, improved buffering capacity, and provided analytical performance stability. Moreover, baseline drifting was significantly minimized by the use of a low concentration buffer solution (25mM ammonium acetate). In addition, evaporation and reconstitution of the sample residue with a methanol-organic pure (OP) water solution (65:35) significantly reduced the matrix interference. The modified extraction produced good recoveries (>91%) for each of the eight cannabinoids.The optimized method was validated for specificity, linearity, sensitivity, precision, accuracy, and stability. The combined relative standard deviation (%RSD) for intra-day and inter-day precision for all eight analytes varied from 2.5% to 5.2% and 0.28% to 5.5%, respectively. The %RSD for the repeatability study varied from 1.1% to 5.5%. The recoveries from spiked cannabis matrix samples were greater than 90% for all analytes, except delta-8-tetrahydrocannabinol (Δ8-THC), which was 80%. The recoveries varied from 81% to 107% with a precision of 0.7-8.1%RSD. Delta-9-tetrahydrocannabinol (Δ9-THC) in all of the cannabis samples (n=635) was less than 10%, which is in compliance with the NJ Medicinal Marijuana regulation. Analysis of samples from two cultivars, which included ten individual samples, four composite samples, seven calibration standards, and four quality control standards, can be performed within 24hours by this high throughput method.

User-Friendly HPLC Method Development and Validation for Determination of Enalapril Maleate and Its Impurities in Enalapril Tablets.

The official method for the determination of Enalapril Maleate and its related substances in European Pharmacopoeia (EP) is a gradient liquid chromatographic method. The method used styrene-divinylbenzene copolymer column, mobile phase buffer pH 6.8 and column oven temperature 70°C. In this method, the separation between main component Enalapril and Ph. Eur. Imp-A was not completed hence the achieving system suitability requirement is a tough task and it requires quite often adjustment in chromatographic parameters. Moreover, column oven temperature 70°C is not user friendly to HPLC instruments and users. In this study, several changes were introduced to the method in order to improve the separation, peak shapes and to overcome the column oven temperature. A new user-friendly stability-indicating RP-HPLC method was developed for Enalapril related substances analysis. The developed method uses a ZORBAX Eclipse XDB-C18 column with column oven temperature at 55°C and mobile phase containing acetonitrile and a phosphate buffer at pH 3.0. The method is capable of separating all the known impurities with resolution more than 3.5, which is much better than that obtained with the existing monograph methods. The optimized method was validated and demonstrated to have acceptable specificity, sensitivity, linearity, accuracy, precision, robustness, solution stability and equivalency to the EP method. The developed method proved to be applicable to a wide number of C18 reversed-phase columns. In addition, the Enalapril assay method also presented with 20 min run time.

Separation, optimization, and quantification of cytokinins by a recently developed amide-embedded stationary phase

ABSTRACTIn this study, some plant growth regulators known as cytokinins [kinetin (K), zeatin (Z), thidiazuron (TDZ), benzylaminopurine (BAP), and dimethylallylaminopurine (AAP)] were separated by HPLC using an amide-embedded mixed-mode stationary phase which was synthesized by Aral et al. in recent years. The effect of mobile phase content, mobile phase pH, buffer concentration, and temperature on separation process was studied. In addition, a quantitative determination of cytokinins from Salvia limbata extract was studied, and some validation parameters such as limit of detection (LOD), limit of quantification (LOQ), and relative standard deviation (RSD) were calculated as a range of 0.03–0.1, 0.1–0.26 mg/L, and 0.03–0.08, respectively.

Quantitative analysis of acid-catalyzed levulinic acid product mixture from cellulose by mixed-mode liquid chromatography

A mixed-mode weak anion-exchange/reversed-phase liquid chromatography (LC) column was successfully applied for the analysis of levulinic acid (4-oxopentanoic acid, LA) product mixture derived from cellulose. Due to the existence of ionic and neutral byproducts, the analysis of the product mixture usually requires ion chromatography, LC and gas chromatography simultaneously. The new method enables accomplishment of the analysis in one LC run within 6min. LC mobile phase of 10mM phosphate buffer containing 5% acetonitrile with pH=5.5 was used. The linear regression coefficients for the UV signal of standard compounds with the corresponding mass concentrations were greater than 0.999. The method recoveries were between 98.57–103.48%. The limits of quantification were 5, 10,1000, 1500 and 3000ng/mL for 5-hydroxymethylfurfural, furfural, acetic acid, formic acid and LA respectively. The mixed-mode column exhibits comprehensive separation mechanism of both reversed-phase and ion-exchange interactions. The mobile phase with different pH, organic modifier solvent and buffer concentration provided flexible LC method for the sample with different complexity.