Mobile Phase Buffer Research Articles

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Determination of glyphosate in surface water with high organic matter content

In this paper, we investigate the sample preparation and analysis process in order to achieve adequate results for surface water collected from rivers that flow through swamps and are consequently rich in organic matter. We show that matrix effects in glyphosate determination can be reduced by optimizing sample volume, liquid chromatography (LC) mobile phase buffer concentration and pH as well as gradient speed. Also, aspects of derivatization procedure (borate buffer concentration, fluorenylmethyloxycarbonyl chloride concentration) and their influence on accuracy are considered in detail. We encountered a cross-talk effect in the mass spectra, interfering with quantization during analysis, which was removed by optimizing MS parameters. As a result it was demonstrated that isotope-labelled internal standard with just one 13C atom is sufficient for the analysis.All these aspects were found to strongly impact the accuracy of the glyphosate determination but have received little or no attention in earlier works. We propose a reliable solid phase extraction and LC/ESI/MS/MS method for determination of glyphosate in organic-rich waters and demonstrate that LoD can be decreased by about two times using an ESI nebulizer with a modified design.

Stability Indicating Simultaneous Estimation of Metformin and Empagliflozin in Pharmaceutical Tablet Dosage form by RP-HPLC

A simple, rapid, accurate, precise, specific, sensitive and stability indicating RP-HPLC method has been developed and validated for the simultaneous estimation of Metformin and Empagliflozin in bulk drug and pharmaceutical dosage form. The chromatographic separation was performed on the Kromasil C18 column (250mm×4.6mm, 5μm particle size), using a mobile phase of Buffer: Acetonitrile taken in the ratio 45: 55 v/v, at a flow rate of 1.1 ml/min at an ambient temperature of 30°C with the detection wave length at 226nm. The retention times of Metformin and Empagliflozin were 2.182 min and 2.908 min respectively. The linearity was performed in the concentration range of 125–750 ppm, 3.12–18.75 ppm each of Metformin and Empagliflozin with a correlation coefficient of 0.999 and 0.999 for Metformin and Empagliflozin respectively. The percentage purity of Metformin and Empagliflozin was found to be 100.59% and 100.07% respectively. The proposed method has been validated for specificity, linearity, range, accuracy, precision and robustness were within the acceptance limit according to ICH Q2 (R1) guidelines and the developed method can be employed for routine quality control analysis in the bulk and combined pharmaceutical dosage form of Metformin and Empagliflozin

A Validated Stability-Indicating HPLC Method for Simultaneous Determination of Amoxicillin and Enrofloxacin Combination in an Injectable Suspension.

The combination of amoxicillin and enrofloxacin is a well-known mixture of veterinary drugs; it is used for the treatment of Gram-positive and Gram-negative bacteria. In the scientific literature, there is no high-performance liquid chromatography (HPLC)-UV method for the simultaneous determination of this combination. The objective of this work is to develop and validate an HPLC method for the determination of this combination. In this regard, a new, simple and efficient reversed-phase HPLC method for simultaneous qualitative and quantitative determination of amoxicillin and enrofloxacin, in an injectable preparation with a mixture of inactive excipients, has been developed and validated. The HPLC separation method was performed using a reversed-phase (RP)-C18e (250 mm × 4.0 mm, 5 μm) column at room temperature, with a gradient mobile phase of acetonitrile and phosphate buffer containing methanol at pH 5.0, a flow rate of 0.8 mL/min and ultraviolet detection at 267 nm. This method was validated in accordance with the Food and Drug Administration (FDA) and the International Conference on Harmonisation (ICH) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, ruggedness, and system suitability results within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method can also be used for purity and degradation evaluation of these formulations.

An Innovative Stability Indicating RP-HPLC Assay Method for the Determination of Caroverine in Pharmaceutical Bulk and Tablets

An innovative, quick and easy reversed phase high performance liquid chromatographic method is elaborated and authenticated for the quantitative determination of caroverine in pharmaceutical bulk material and tablets. Shimpack CLC-ODC (C18) column was used. The mobile phase acetonitrile and buffer solution (30:70) pH 4.9 is transmitted at a flow rate of 1 mL/min. The eluent was observed using UV detector at 225 nm. The recent developed RP-HPLC method is specific, accurate, precise and linear (R2>0.998) within the range of 2-150 μg/mL concentration. The limit of detection and quantification is 0.068 μg/mL and 0.201 μg/mL respectively. The newly proposed method is applied to determine caroverine in pharmaceutical tablet formulations. The developed method is optimized using samples generated by forced degradation studies. Results of analysis are validated statistically. The method was selective, precise, accurate and can be used for routine analysis of caroverine in quality control laboratories of pharmaceutical industries.

QUALITY BY DESIGN-BASED OPTIMIZATION AND VALIDATION OF NEW REVERSE PHASE-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF LEVOFLOXACIN HEMIHYDRATE AND AMBROXOL HYDROCHLORIDE IN BULK AND ITS PHARMACEUTICAL DOSAGE FORM

ABSTRACTObjective: Innovative application of quality by design (QbD) technique for simultaneous estimation of levofloxacin and ambroxol hydrochloride (HCL)in bulk and its pharmaceutical dosage form using reverse phase-high-performance liquid chromatography (RP-HPLC) method.Method: A method has been developed for the separation of levofloxacin and ambroxol HCL using RP-HPLC on C18 column (250 4.6 mm, 5 ml) withultraviolet detection at 306 nm. Experimental designs were applied for multivariate optimization of the experimental conditions of RP-HPLC method.Three independent factors: Acetonitrile content in the mobile phase composition, buffer pH, and flow rate were used to design mathematical models.Here, central composite design (CCD) experimental design was used to study the response surface technique and to study in depth the effects ofthese independent factors. Derringer’s desirability function was applied to simultaneously optimize the retention time of last eluting peak (ambroxolhydrochloride) and resolution between levofloxacin and ambroxol hydrochloride.Result and Discussion: The predicted optimum assay condition consisted of acetonitrile, potassium dihydrogen phosphate buffer (pH 5.00;potassium dihydrogen phosphate), and methanol in a proportion of 20:70:10% v/v, respectively, as the mobile phase at a flow rate of 1.2 ml/minute.Using this optimum condition, baseline separation of both drugs with good resolution and a run time of &lt;5 minutes were achieved. The optimizedassay condition was validated according to the ICH guidelines to confirm specificity, linearity, accuracy, and precision.Keywords: Levofloxacin, Ambroxol hydrochloride, Experimental design, Response surface methodology, Derringer’s desirability, Quality by designapproach.

Development and validation of a quantitative assay for the determination of cinacalcet and its main metabolites in human plasma using RP-HPLC method

A novel and sensitive methodology for the quantitative determination of cinacalcet (CIN), and its main metabolites (M2a-Glu, M2b-Glu, M5, and M6) in plasma sample is suggested. The analytical assay is based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. The method has been fully validated at range covering the plasma concentrations in patients receiving therapeutic dosages of CIN. In this investigation, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NI-ETFMS) was used as a pre-column derivation agent according to interaction of amine and carboxylate groups of CIN and its metabolites with NI-ETFMS. The sample pretreatment consists of protein precipitation with acetonitrile using only 100μL of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100Å column with mobile phase of acetonitrile, methanol, and 50mM phosphate buffer (40:20:40v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5ngmL−1), linearity (r2>0.997), sensitivity (LOD=0.05 to 0.75ngmL−1 and LOQ=0.36 to 2.52ngmL−1), intra and inter-days accuracy (−4.6 to 8.1%), precision (<7.4%), and robustness (<3.5%). As results, the proposed method can be useful in the routine analysis for the determination of CIN and its main metabolites in human plasma samples.

Stabilitty indicating method development and validation for the quantification of chlorphenamine maleate related subtances using hplc technique

Chlorpheniramine Maleate (CPM) is an antihistamine used for the treatment of allergic disorders and common cold since, the method for determination of CPM related substances in pharmaceutical products is not described in current pharmacopoeias; the aim of this work was to develop and validate a precise, accurate and robust method. Separation was achieve on a RP C-18 column (250 x 4.6 mm, 5 µm particle size), column temperature kept at 40°C. The mobile phase content A: 80 volumes buffer solution (pH 3.00) and Mobile phase B 20 volumes of Acetonitrile, and UV detection wavelength at 225nm. The method was validated by ICH guidelines and validation studies proved that the method is stability indicating and used for determination of related substances of CPM tablets.

Thermodynamic study of HPLC enantioseparations of some sulfur-containing amino acids on teicoplanin columns in ion-pairing reversed-phase mode

ABSTRACTHomocysteine is an essential amino acid produced in the metabolism of methionine. It can be regulated through two metabolic routes either to cysteine or back to methionine. The imbalance in the methionine or homocysteine metabolism is connected with many disorders. In this work, HPLC enantioseparations of cysteine, homocysteine, and methionine were performed. Enantiomers were separated on natural teicoplanin and teicoplanin aglycone chiral stationary phases when buffered mobile phase with the presence of an ion-interaction reagent was used. The column temperature effect on the enantiomeric retention, selectivity, and resolution was studied in the range of 5–45°C at 5°C increment. Thermal stability of homocysteine standard was investigated by X-ray powder-diffraction measurements at elevated temperature in the range of 25–100°C at 5°C increment in air. Moreover, the thermodynamic analysis was used to quantify contributions of entropic and enthalpic terms on separations of investigated amino acid enantiomers. The elution order of enantiomers was determined in all cases.

UPLC-MS/MS determination of ptaquiloside and pterosin B in preserved natural water.

The naturally occurring carcinogen ptaquiloside and its degradation product pterosin B are found in water leaching from bracken stands. The objective of this work is to present a new sample preservation method and a fast UPLC-MS/MS method for quantification of ptaquiloside and pterosin B in environmental water samples, employing a novel internal standard. A faster, reliable, and efficient method was developed for isolation of high purity ptaquiloside and pterosin B from plant material for use as analytical standards, with purity verified by 1H-NMR. The chemical analysis was performed by cleanup and preconcentration of samples with solid phase extraction, before analyte quantification with UPLC-MS/MS. By including gradient elution and optimizing the liquid chromatography mobile phase buffer system, a total run cycle of 5min was achieved, with method detection limits, including preconcentration, of 8 and 4ng/L for ptaquiloside and pterosin B, respectively. The use of loganin as internal standard improved repeatability of the determination of both analytes, though it could not be employed for sample preparation. Buffering raw water samples in situ with ammonium acetate to pH ∼5.5 decisively increased sample integrity at realistic transportation and storing conditions prior to extraction. Groundwater samples collected in November 2015 at the shallow water table below a Danish bracken stand were preserved and analyzed using the above methods, and PTA concentrations of 3.8 ± 0.24μg/L (±sd, n = 3) were found, much higher than previously reported. Graphical abstract Workflow overview of ptaquiloside determination.

Influence of Some Variables on Separation of Diastereomeric FVDA-Amino Alcohol Derivatives in High Performance Thin Layer Chromatography and Pressurized Planar Electrochromatography Systems

Separation of five -blocker 1-fluoro-2,4-dinitrophenyl-5-L-valine amide (FVDA) derivatives i.e. acebutolol, atenolol, metoprolol, oxprenolol and pindolol by means of high-performance thin layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques and with the use of HPTLC RP-18W plates as well as the acetonitrile - buffer mobile phase is presented. The influence of concentration of acetonitrile and pH of the mobile phase buffer, and temperature on migration distance of the solute zones was investigated. It has been observed that an increase of acetonitrile content of the mobile phase affects the solute zone migrations and retentions in HPTLC and PPEC. Whereas rise of eluent buffer pH enhances predominantly the isomer migration distances in PPEC. The temperature increase results the enhancement of solute migration distances in PPEC and diminishes isomer retention in HPTLC. Moreover, it improves the diastereomer pair zones resolution in both investigated systems. What is more, the separation selectivity in PPEC and HPTLC system differs. The former system presents higher efficiency in comparison with the latter. In addition separation of FVDA-oxprenolol and FVDA-pindolol isomers is presented for the first time. Keywords: Diastereomers, amino alcohols, separation, variables, HPTLC, PPEC.

The hydrophilicity vs. ion interaction selectivity plot revisited: The effect of mobile phase pH and buffer concentration on hydrophilic interaction liquid chromatography selectivity behavior

This work systematically investigates the selectivity changes on many HILIC phases from wwpH 3.7–6.8, at 5 and 25mM buffer concentrations. Hydrophilicity (kcytosine/kuracil) vs. ion interaction (kBTMA/kuracil) selectivity plots developed by Ibrahim et al. (J. Chromatogr. A 1260 (2012) 126–131) are used to investigate the effect of mobile phase changes on the selectivity of 18 HILIC columns from various classes. “Selectivity change plots” focus on the change in hydrophilicity and ion interaction that the columns exhibit upon changing mobile phase conditions. In general, the selectivity behavior of most HILIC columns is dominated by silanol activity. Minimal changes in selectivity are observed upon changing pH between wwpH 5 and 6.8. However, a reduction in ionic interaction is observed when the buffer concentration is increased at wwpH≥5.0 due to ionic shielding. Reduction of the wwpH to<5.0 results in decreasing cation exchange activity due to silanol protonation. Under all eluent conditions, the majority of phases show little change in their hydrophilicity.

Investigation of the Retention Mechanism of Cephalosporins by Zwitterionic Hydrophilic Interaction Liquid Chromatography

The importance of cephalosporin antibiotics in medicine requires the development of high performance separation methods for their analysis. In this paper cefazolin, cefuroxime, cefoxitin, cefaclor and ceftriaxone were used as model compounds to investigate the retention mechanism under zwitterionic HILIC conditions. The analysis was performed using a ZIC®-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 μm, 200 A) with isocratic elution. The mobile phase was a mixture of acetonitrile and ammonium acetate or formate buffer. To get a better insight into the retention mechanism on ZIC®-HILIC columns the main factors influencing the retention and selectivity of the analytes have been thoroughly investigated. It is shown that the type of the salt and its concentration along with the volume fraction of water in the mobile phase and pH of the aqueous buffer greatly affect the retention of the tested cephalosporins due to their structures and physicochemical properties. The method was applied successfully to the analysis of injection dosage forms containing cefazolin, cefuroxime and cefoxitin.

QbD-Driven Development and Validation of a HPLC Method for Estimation of Tamoxifen Citrate with Improved Performance.

The current studies entail Quality by Design (QbD)-enabled development of a simple, rapid, sensitive and cost-effective high-performance liquid chromatographic method for estimation of tamoxifen citrate (TMx). The factor screening studies were performed using a 7-factor 8-run Taguchi design. Systematic optimization was performed employing Box-Behnken design by selecting the mobile phase ratio, buffer pH and oven temperature as the critical method parameters (CMPs) identified from screening studies, thus evaluating the critical analytical attributes (CAAs), namely, peak area, retention time, theoretical plates and peak tailing as the parameters of method robustness. The optimal chromatographic separation was achieved using acetonitrile and phosphate buffer (pH 3.5) 52:48 v/v as the mobile phase with a flow rate 0.7 mL/min, an oven temperature 40°C and UV detection at 256 nm. The method was validated as per the ICH recommended conditions, which revealed high degree of linearity, accuracy, precision, sensitivity and robustness over the existing liquid chromatographic methods of the drug. Also the method was applied for the estimation of TMx in nanostructured formulations, which indicated no significant change in the retention time. In a nutshell, the studies demonstrated successful development of the HPLC method of TMx with improved understanding of the relationship among the influential variables for enhancing the method performance.

Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

Selective separation and characterisation of stress degradation products and process impurities of prucalopride succinate by LC-QTOF-MS/MS

The present study reports the degradation behaviour of a new prokinetic agent, Prucalopride succinate, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A (R2)). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions followed by characterization of the degradation products (DPs) and process related impurities (IMPs). A rapid, precise, accurate and robust reverse phase high performance liquid chromatography (RP-HPLC) method has been developed involving mobile phase of 20mM ammonium bicarbonate buffer and acetonitrile: methanol (80:20v/v) on a Waters Xbridge-C8 (150mm×4.6mm i.d., 3.5μm) column using gradient elution. The drug was found to be degraded in hydrolytic (acidic) and oxidative conditions, whereas it was stable under basic and neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to LC-ESI-QTOF-MS/MS for the structural characterization of DPs and process related IMPs. Structural characterization was carried out based on the generated molecular formula of DPs and its fragment ions. It has been observed that two major DPs were formed under each acid hydrolysis and oxidative stress conditions. The most probable mechanisms involved in the formation of DPs were also proposed. Finally, the method was validated in the term of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines, Q2 (R1).

AN LC- MS/MS METHOD FOR THE DETERMINATION OF OMEPRAZOLE ON PROTON PUMP INHIBITOR IN HUMAN PLASMA

A sensitive and selective liquid chromatographic method coupled with tandem mass spectroscopy (LC-MS/MS) was developed for the quantification of omeprazole in human plasma. Lansoprazole was used as internal standard with plasma samples, extracted using 10mM ammonium acetate. A centrifuged upper layer was then evaporated and reconstituted with Acetonitrile: mobile phase buffer 70:30%v/v. The reconstructed samples were injected into a C 18 column purospher star 5µ. The mobile phase was composed of ACN: mobile phase buffer (5mm ammonium bicarbonate buffer) in the ratio of 70:30%v/v with flow rate 1.0mL/min. The mass spectrometer was operated using positive ion mode and turbo electro spray ionisation. Nitrogen was used as the nebulizer, curtain, collision and auxiliary gases. Using MS/MS with multiple reactions monitoring (MRM) mode, omeprazole was detected without severe interferences from plasma matrix. Detection of omeprazole in human plasma was accurate and precision. This method has been successfully applied to the study of omeprazole in human specimens Keywords: Proton pump inhibitor, omeprazole, lansoprazole, LC-MS/MS, liquid liquid extraction

High-performance thin-layer chromatography and pressurized planar electrochromatography of some diastereomeric amino acid derivatives in reversed-phase system with carboxylic acid mobile phase buffers

The separation of 1-fluoro-2,4-dinitrophenyl-5-l-valine amide (FVDA) diastereomeric derivatives of aspartic acid, cysteine, and histidine by means of high-performance thin-layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques in systems with HPTLC RP-18W plates and the various acetonitrile—buffer mobile phases is presented. The influence of the mobile phase components, i.e., acetonitrile concentration and buffer kind on migration distance of the solute zones, was investigated. The effect of mono (formic) and various dicarboxylic acids (oxalic, malonic, maleic, malic, succinic, tartaric, and pimelic) as the mobile phase buffer components on the solute retention was studied. It is observed that an increase of acetonitrile content of the mobile phase affects the solute zone migration and retention in PPEC and HPTLC. What is more, the separation selectivity in the latter and former techniques differs. The PPEC mode presents a higher efficiency in comparison with HP...

Planar Electrochromatography and thin-layer chromatography of tropane alkaloids fromDatura innoxiaMill. extract in pseudo-reversed-phase systems

In the presented paper, the influence of the static electric field on retention, and in consequence separation, of tropane alkaloids present in Datura innoxia Mill. extract was demonstrated. Thin-layer electrochromatography (ETLC) on initially dry layers was used as the investigation method. The results obtained by ETLC were compared with the data obtained in thin-layer chromatography (TLC) analogous systems. Pseudo-reversed-phase systems (stationary phase — silica gel, mobile phase buffer at different pH and methanol mixture with different content of the methanol) were used. The presence of the electric field influences the retention parameters of the investigated compounds. The influence depends on the pH of the buffered mobile phase, composition of the mobile phase, and turn of the electric field lines in relation to the mobile phase migration direction. In the mobile phase, in which some of the alkaloids are in the ionic form, the significant differences between retention in cathode side and anode sid...

Thin-layer chromatography and pressurized planar electrochromatography of amino acids in systems with silica gel and water mobile phase

Thin-layer chromatography (TLC) and pressurized planar electrochromatography (PPEC) of amino acids in normal-phase system is presented. The results have been obtained for various normal-phase high-performance thin-layer chromatography (HPTLC) and PPEC systems with the mobile phase that comprised acetonitrile in the concentration ranges 40–90% and 20–90%, respectively, and HPTLC silica gel 60 F254s plates from Merck. The data obtained show differences in separation selectivity between HPTLC and PPEC systems. The respective separation selectivities have been obtained for HPTLC and PPEC systems with the mobile phase buffer pH in the range 2.6–9.0. The retention of amino acids in HPTLC systems has demonstrated minor dependence on buffer pH, while, in analogous PPEC systems, migration distances of the solutes have shown considerable changes. The differences of separation selectivity in HPTLC and PPEC systems are interpreted in terms of solute partition in the former and solute partition and electrophoresis in ...