Mobile Phase Buffer Research Articles

Stability indicating HPLC method for the simultaneous analysis of Gatifloxacin and Loteprednol in eyedrop formulation using design of experiment approach

Design of experiment (DOE) assisted simple, rapid, precise and accurate stability indicating HPLC method has been developed for simultaneous estimation of Gatifloxacin (GTF) and Loteprednol (LOT) along with their forced degradation products. The developed method has been optimized and developed by using central composite design (CCD) in response surface methodology (RSM). Trails have been undertaken and ratio of phosphate buffer in mobile phase, pH of buffer and flow rate are selected as factors. Resolution, tailing factor (GTF) and tailing factor (LOTE) are selected for determining the system response in the process of method optimization. The responses have been optimized using the Derringer’s desirability function. The effective separation is achieved on Phenomenex EVO-C18 column (250 mm x 4.6 mm i.d, 5 μm particle size) with mobile composed of 10 mM phosphate buffer, pH 3.5 and organic phase composed of mixture of acetonitrile and methanol 60:40 % v/v, the flow rate was 1.0 mL/min, the signals were detected at 267 nm. The developed method was validated for linearity, accuracy, precision, and robustness. The method was applied successfully for stability samples.

Comparison of Workflows for Milk Lipid Analysis: Phospholipids.

Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed.

High-performance liquid chromatography evaluation of lipophilicity and QSRR modeling of a series of dual DNA gyrase and topoisomerase IV inhibitors

AbstractBacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and represent important antibacterial drug targets. To be successful as drug candidates, newly synthesized compounds must possess optimal lipophilicity, which enables efficient delivery to the site of action. In this study, retention behavior of twenty-three previously synthesized dual DNA gyrase and topoisomerase IV inhibitors was tested in RP-HPLC system, consisting of C8 column and acetonitrile/phosphate buffer (pH 5.5 and pH 7.4) mobile phase. logD was calculated at both pH values and the best correlation with logD was obtained for retention parameter φ0, indicating that this RP-HPLC system could be used as an alternative to the shake-flask determination of lipophilicity. Subsequent QSRR analysis revealed that intrinsic lipophilicity (logP) and molecular weight (bcutm13) have a positive, while solubility (bcutp3) has a negative influence on this retention parameter.

AQbD based green UPLC method to determine mycophenolate mofetil impurities and Identification of degradation products by QToF LCMS

We report an ideal method for quantifying impurities in mycophenolate mofetil drug substances and their oral suspension preparations. We developed a systematic and eco-friendly analytical approach utilizing quality by design (QbD) and green chemistry principles. Initially, the critical method parameters (CMPs) were screened using a D-optimal design. The robust final method conditions were optimized using a systematic central composite design (CCD). Through graphical and numerical optimization, the protocol conditions were augmented. The pH of mobile phase buffer (25 mM KH2PO4) (MP-A), initial gradient composition (% MP-A), flow rate (mL min−1), and column oven temperatures (°C) are 4.05, 87, 0.4, and 30, respectively. The best possible separation between the critical pairs was achieved while using the Waters Acquity UPLC BEH C18 (100 × 2.1) mm, 1.7 µm analytical column. A mixture of water and acetonitrile in the ratio of 30:70 (v/v) was used as mobile phase-B for the gradient elution. The analytical method was validated in agreement with ICH and USP guidelines. The specificity results revealed that no peaks interfered with the impurities and MPM. The mean recovery of the impurities ranged between 96.2 and 102.7%, and the linearity results r > 0.999 across the range of LOQ – 150%. The precision results (%RSD) ranged between 0.8 and 4.5%. The degradation products formed during the base-induced degradation were identified as isomers of mycophenolic acid and sorbitol esters using Q-ToF LC–MS and their molecular and fragment ion peaks. The developed method eco-friendliness and greenness were assessed using analytical greenness (AGREE), green analytical procedure index (GAPI), and analytical eco score, and found it is green.

Pre-column Derivatization¸ Elution Order, Molecular Configuration and Green Chromatographic Separation of Diastereomeric Derivatives of β-Amino Alcohols

A green chromatographic method was developed for the indirect enantio-separation of racemic amino alcohols by introducing a micellar mobile phase for the HPLC. Here, cyanuric chloride-based activated chiral reagents were prepared, characterized, and used to derivatize racemic amino alcohols into diastereomeric derivatives. The chromatographic separation of the prepared diastereomeric derivatives was achieved on the C18 column of the RP-HPLC using a surfactant-based (Brij-35 + SDS) aqueous mobile phase (as an alternative to organic solvents). The impact of the varying concentration of surfactants in the mobile phase and pH of buffer on the chromatographic separation was optimized. Additionally, the DFT calculations were performed, and the absolute configurations and elution orders of the diastereomeric derivatives were investigated. Developed method’s robustness and selectivity was optimized by performing validation studies (LOD = 0.286 ng/mL; LOQ = 0.858 ng/mL). Also, the green assessment score was calculated for the developed method (84 scores; an excellent green method).

Retention behaviour of analytes in reversed-phase high-performance liquid chromatography-A review.

The understanding of principles that drive the separation in reversed-phase chromatography plays an important role in the prediction of the elution of solutes in RP-HPLC. The separation in RP-HPLC is based on the principle of adsorption and partition. In addition, the logP value, the pKa value of the drug and chromatographic parameters like mobile phase pH, buffer concentration, organic modifier and mobile phase additives also influence the retention and selectivity of the analyte. It was found that hydrophobic, electrostatic, hydrogen bonding and other specific interactions between the stationary phase and the solutes, along with the hydrophobicity of an analyte molecule (logP), modify the retention behaviour of the analytes. This article gives special attention to the influence of ionization and ion interaction on the separation of analytes. The drug molecules with different logP values containing protonated and deprotonated acids, bases and zwitterions are selected as examples and this article addresses various issues related to the method development, relationships between analyte retention and mobile phase pH and the pKa value of the analyte. The advances in this regard, with highlights on topics such as mechanisms of retention and various factors that influence the retention behaviour of analytes, are also updated with suitable examples.

RP - HPLC Method Development and Validation for Simultaneous Estimation of Pyrantel Pamoate, Praziquantel, Febantel in Tablet

A simple, rapid, economical, precise and accurate RP-HPLC method for simultaneous estimation of praziquantel, pyrental pamoate and febantel in their combined dosage form has been developed. A validated RP-HPLC method for simultaneous estimation of pyrental pamoate, praziquantel, febantel in tablet dosage form has been developed. The separation was achieved by C18 column (250 x 4.6mm, 5µm particle size) with mobile phase of Buffer: ACN (60:40% v/v) pH 3.0, at a flowrate of 1mL/min and effluent was monitored at 240nm using PDA detector. Retention time of praziqunatel, pyrental pamoate and febantel was found to be 2.587 min, 5.970 min and 8.401 min respectively. The method has been validated for linearity, accuracy, precision, specificity, limit of detection and limit of quanitzation. Linearity was found in the range of 6.25-18.75 µg/ml, 18-54 µg/ml and 18.75-56.25 µg/ml for praziquantel, pyrental pamoate and febantel. The recoveries for praziquantel, pyrental pamoate and febantel were found to be 99.76 ± 0.211%, 99.77 ± 0.490% and 99.61± 0.241 respectively in tablet. The percentage assay of three drugs were found to be 96.82%,97.66% and 96.92% for Praziquantel, Pyrental pamoate and Febantel respectively. Developed method was found to be new, accurate, precise and robust for simultaneous estimation of praziquantel, pyrental pamoate and febantel in tablet dosage form. The proposed method enables rapid quantification and simultaneous analysis of three drugs from commercial formulations without any excipients interference.

Response Surface Method for the Simultaneous Estimation of Atorvastatin and Olmesartan

The simultaneous estimation of Atorvastatin Calcium (ATS) and Olmesartan Medoxomil (OLM) in bulk and pharmaceutical dosage forms, a new, quick, and cost-effective Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed. The experimental design was used to achieve multivariate optimization of the RP-HPLC experimental conditions. Three independent variables were used to create mathematical models: Acetonitrile content in the mobile phase composition, buffer pH, and flow rate. Here, the applied model was central composite design (CCD) to research the response surface methodology and study the effects of independent factors. The Shimadzu (LC 20 AT VP) HPLC system with Spinchrom software has been used. Zodiac, C18 (250×4.6 ID) 5μm column, phosphate buffers, and acetonitrile were used as mobile phase in the ratio 40:60 v/v with a flow rate 1.15 mL/min. The eluent was monitored at 212 nm using the Prominence UV-Visible detector. The retention time for OLM and ATS was 2.673 and 3.717, respectively. The optimized procedure was validated as per ICH guidelines. The correlation coefficient of OLM and ATS was 0.9869 and 0.9832. The % of recovery was 98.59, 99.68 %. OLM had a LOD of 17.568 μg/mL, while ATS had a LOD of 12.88 μg/mL. OLM had a LOQ of 53.24 μg/mL, while ATS had a LOQ of 39.04 μg/mL. The pH aqueous phase, solvent composition, and flow rate were the most stringent variables affecting the responses, according to the 3D response surface graphs. A new accurate and precise RP-HPLC approach has been developed and validated and used to regularly analyse OLM and ATS.
 HIGHLIGHTS
 
 A new, quick, and cost-effective RP-HPLC method for the simultaneous estimation of Atorvastatin Calcium and Olmesartan Medoxomil in bulk and pharmaceutical dosage forms
 RP-HPLC method developed by experimental design and response surface method
 Developed method was validated as per ICH guidelines
 
 GRAPHICAL ABSTRACT

Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD

Evaporative light scattering detectors (ELSD) are commonly used with high-performance liquid chromatography (HPLC) to separate and quantify lipids, which are typically not easily detectable by more conventional methods such as UV–visible detectors. In many HPLC-ELSD methods to analyze lipids, a volatile buffer is included in the mobile phase to control the pH and facilitate separation between lipid species. Here, we report an unintended effect that buffer choice can have in HPLC-ELSD analysis of lipids – the identity and concentration of the buffer can substantially influence the resulting ELSD peak areas. To isolate this effect, we use a simple isocratic methanol mobile phase supplemented with different concentrations of commonly used buffers for ELSD analysis, and quantify the effect on peak width, peak shape, and peak area for seven different lipids (POPC, DOPE, cholesterol, sphingomyelin, DOTAP, DOPS, and lactose ceramide). We find that the ELSD peak areas for different lipids can change substantially depending on the mobile phase buffer composition, even in cases where the peak width and shape are unchanged. For a subset of analytes which are UV-active, we also demonstrate that the peak area quantified by UV remains unchanged under different buffer conditions, indicating that this effect is particular to ELSD quantification. We speculate that this ELSD-buffer effect may be the result of a variety of physical phenomenon, including: modification of aerosol droplet size, alteration of clustering of analytes during evaporation of the mobile phase, and mass-amplification or ion-pair effects, all of which could lead to differences in observed peak areas. Such effects would be expected to be molecule-specific, consistent with our data. We anticipate that this report will be useful for researchers designing and implementing HPLC-ELSD methods, especially of lipids.

Liquid chromatography method for simultaneous quantification of ATP and its degradation products compatible with both UV–Vis and mass spectrometry

ATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV–Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV–Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV–Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV–Vis and their confirmation and/or identification of unknown peaks via MS.

ASSAY METHOD DEVELOPMENT AND VALIDATION OF CILNIDIPINE AND RAMIPRIL, CHARACTERIZATION OF ITS DEGRADANTS BY USING LC-MS/MS

Objective: The current study focused on the development, validation of Ramipril and Cilnidipine using HPLC and characterization of forced degradation products using LC-MS/MS. Methods: Simple, accurate, and precise method has been developed for the simultaneous estimation of Cilnidipine and Ramipril in tablet type of dosage form using X-bridge phenyl column (150x4.6 mm, 3.5m) with mobile phase of buffer and Acetonitrile in the 30:70 v/v ratio was pumped through a column with a 1 ml/min flow rate. The buffer used in this process was 0.1 percent tri ethyl amine in 1 lt of water and adjust its pH-2.5 with 0.1 percent ortho phosphoric acid. At ambient temperature, chromatography was isocratically performed and run time was 8 min. 240 nm was the optimized wave length. Results: Cilnidipine and Ramipril retention times were 2.79 min, 5.11 min. respectively. By injecting the norm six times, device parameters of suitability have been studied and the results were found to be well under the acceptance criteria. This approach offers strong linearity over a range of 2-30 µg/ml Cilnidipine 1-15 µg/ml Ramipril concentrations. The regression coefficient R2>0.999 from the calibrated curve implies that the linearity of the system was within the range. Conclusion: The system was validated by using HPLC in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, degradation and the degradation products were characterized by using LC-MS/MS.

Physiologically relevant hCys concentrations mobilize MeHg from rabbit serum albumin to form MeHg-hCys complexes.

Methylmercury (MeHg) is one of the most potent neurotoxins to which humans are exposed via the consumption of fish, from which it is effectively absorbed via the gastrointestinal tract into the bloodstream. Its interactions with plasma proteins, small-molecular-weight (SMW) molecules, and red blood cells, however, are incompletely understood, but critical as they determine whether and how much MeHg reaches target organs. To better define the role that SMW thiols play in the delivery of MeHg to known transporters located at the placental and blood-brain barrier, we have employed size exclusion chromatography-inductively coupled plasma-atomic emission spectroscopy to analyze MeHg-spiked rabbit plasma in the absence and presence of SMW thiols dissolved in the phosphate-buffered saline buffer mobile phase. While 300μM methionine did not affect the binding of MeHg to rabbit serum albumin (RSA), cysteine (Cys), homocysteine (hCys), and glutathione resulted in the elution of the main Hg peak in the SMW elution range. In addition, 50μM of hCys or Cys in the mobile phase resulted in the mobilization of MeHg from RSA in rabbit plasma and from pure RSA in solution. The Hg peak that eluted in the SMW elution range (50μM of hCys) was identified by electrospray ionization-mass spectrometry as an MeHg-hCys complex. Since l-type amino acid transporters are present at the blood-brain barrier (BBB), which facilitate the uptake of MeHg-Cys species into the brain, our results contribute to establish the bioinorganic mechanisms that deliver MeHg to the BBB, which is critical to predict organ-based adverse health effects.

Choice of Buffer in Mobile Phase Can Substantially Modulate Peak Areas in Quantification of Lipids by Hplc-Elsd

Choice of Buffer in Mobile Phase Can Substantially Modulate Peak Areas in Quantification of Lipids by Hplc-Elsd

Development and validation of Stability-indicating RP-UPLC Method for the Simultaneous Determination of Ivacaftor, Tezacaftor and Elexacaftor in bulk and their Formulation

A simple reproducible stability indicating RP-UPLC method was developed for the simultaneous determination of Ivacaftor, Tezacaftor and Elexacaftor in their combined dosage forms using HSS C18, 1.8μm, 100mm x2.1 mm i.d. column. A mobile phase of phosphate buffer (10mM) pH-4.8 and acetonitrile in the ratio of 70: 30v/v mixture was used for separation and quantification of ivacaftor, tezacaftor and elexacaftor. The present drug analytes were run at a flow-rate of 0.3ml/ min at 30°C temperature. The injection volume was 2μL and with ultraviolet detection at 270nm. Under these conditions, elexacaftor, ivacaftor and tezacaftor were eluted at 0.72min, 1.4min and 1.9min respectively with a total run time shorter than 5min. The developed method was validated according to International Conference on Harmonization (ICH) guidelines. The developed RP-UPLC method was applied successfully for quality control assay of Ivacaftor, Tezacaftor and Elexacaftor in their combination drug product.

QBD APPROACH TO ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR ESTIMATION OF LENVATINIB IN BULK AND PHARMACEUTICAL FORMULATION

Objective: The objective of this research was to develop a simple, very rapid, sensitive, accurate, precise reverse phase High-Performance Liquid Chromatography (RP-HPLC) technique for the estimation of Lenvatinib in bulk and its dosage form. Methods: To perform this study, we employed a central composite design (CCD) to make method robust and effective to create chromatographic database. The factor screening studies were performed using 2-factor 10-runs. The factors were selected as the mobile phase ratio and buffer pH. Results: The desirability value of the optimized model was found to be 0.869 and The optimized chromatographic condition was achieved on Enable C18 analytical column with 0.01M Ammonium acetate buffer pH 3.84: methanol (33.17:66.83 v/v) as the mobile phase and flow rate of 1 ml min-1 and detection wavelength was set to 240 nm. The retention time of Lenvatinib was found to be 5.122 min. Linearity was established for Lenvatinib in the range of 10-50 µg/ml with a correlation coefficient (r2=0.9995). The accuracy values were found to be in the range of 98–102%. Intraday precision and Interday precision were in prescribed (Less than 0.98% RSD). Robustness was found to be less than 1.22% RSD. Conclusion: The proposed method was useful for best analysis of Lenvatinib in Bulk pharmaceutical dosage forms. Central Composite Design was an effective tool for the proposed RP-HPLC method.

Qbd Based RP-HPLC Method Development and Validation for Simultaneous Estimation of Amlodipine Besylate and Lisinopril Dihydrate in Bulk and Pharmaceutical Dosage Form

The objective of this experiment was to develop and validate a simple, robust, and accurate QbD based Reverse-Phase High-Performance Liquid Chromatography method for Simultaneous estimation of Amlodipine besylate and Lisinopril dihydrate in bulk and Pharmaceutical Dosage form. A box-Behnken design was employed for optimizing the mobile phase, flow rate and pH of buffer, the optimized chromatographic conditions were Phosphate buffer: Methanol (25: 75 v/v), pH of buffer: 6.5 and flow rate: 1mL/min.
 Furthermore formulation injected and observed that the additives do not interfere with the peak of Amlodipine besylate and Lisinopril dehydrate. Both drugs are well resolved and Retention times were found to be 2.332 min and 3.584 min respectively.
 Linearity was observed in the concentration range of 10 μg to 50 μg/mL (r2=0.999). The accuracy range was 99.75 to 100.04%. Intra-day and Inter-day precision was found to be less than 2% RSD. The proposed method was useful for the best analysis of Amlodipine besylate and Lisinopril dihydrate in Bulk, pharmaceutical dosage forms and was successfully applied to routine analysis.

Synthesis and enantioseparation characteristics of a novel β-cyclodextrin chiral stationary phase based on diazotized silica in HPLC

A novel chiral selector polyacrylic acid-β-cyclodextrin (PAA-β-CD) was successfully prepared, then a facile synthesis of PAA-β-CD stationary phase based on diazotized silica was developed and characterized. Monodisperse silica microspheres were synthesized by a modified Stöber method. The obtained silica particles were functionalized by self-assembly positive charged photosensitive diazoresin (DR) and PAA-β-CD on the surface. The corresponding chiral stationary phase (CSP) was used in HPLC and the enantioseparation performance was investigated in both polar organic and reverse phase mode. Better separation abilities and excellent enantio-selectivities were obtained on PAA-β-CD CSP for antihistamine drugs in the acetonitrile and triethylammonium acetate buffer mobile phase. Abbreviations: AIBN: azodiisobutyronitrile; β-CD: b-cyclodextrin; β-CD-NH2: amino-β-CDcyclodextrin; β-CD-OTs: sulfonyl-b-cyclodextrin; CD: cyclodextrin; CSP: chiral stationary phase; DCC: dicyclohexyl carbimide; DR: diazoresin; MeOH: methanol; NMP: N-methyl pyrrolidone; PAA: polyacrylic acid; PAA-β-CD: polyacrylic acid-β-cyclodextrin; TEAA: triethylammonium acetate; TEOS: tetraethyl orthosilicate

Stability Indicating RP-HPLC Method Development and Validation for the Determination of Solifenacin Succinate in Bulk and its Pharmaceutical Formulation

The current work aims to establish a novel and advanced reverse phase isocratic liquid chromatography system followed by validation and to conduct stability analysis in active pharmaceutical ingredients and formulations for the quantification of Solifenacin Succinate. The optimized elution was achieved with column Sunfire C8 (4.6 x 150mm, 5µm), using the mobile phase of Buffer: Methanol: Acetonitrile in the composition ratio of 45:45:10 v/v. The wavelength of detection was selected as 220nm with 1.0ml/ min flow rate and 30μl injection volume. The retention time of Solifenacin Succinate was found 2.94 min respectively. The method developed has been validated for various analytical parameters according to ICH guidelines. The Linearity was attained at 20 to100 μg/ml of concentration range. The established method was proved as reproducible. The Assay was obtained as 100.40%. The degradation studies were carried out at all degradative conditions and the results of degradation studies denote that the current method was specific, reliable, and economical. Hence, the developed method can be applied for the qualitative and quantitative determination of the selected drug and its commercial formulations.

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to „non-proportional” behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved – ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use.

Method Development and Validation of Droxidopa by RP-UPLC

A simple, rapid, accurate and economical method has been developed for the quantification of droxidopa by UPLC. The chromatographic system was equipped with Phenomenex column C18 (50mm x 3.0mm, 3µ) as stationary phase and UV detector at 235nm, in conjunction with a mobile phase of buffer: methanol (25:75 % v/v ratio) at a flow rate of 1.0mL/min. Linearity was observed over the concentration range of 100-300µg/mL for droxidopa. The droxidopa peak eluted at 0.35 min. The recovery of Droxidopa was found to be 100.17% - 100.63% respectively. Statistical techniques were employed for the validation of precision, linearity, accuracy, robustness and ruggedness and can be applied for routine analysis. Validation revealed that the developed method was specific, accurate, precise, reliable, robust, reproducible and suitable for the routine quantitative analysis.