Mobile Phase Preparation Research Articles

Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat

The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling. In order to increase laboratory efficiency, and minimize opportunities for errors, a suite of methods, based on a single acetonitrile (ACN)-aqueous buffer mobile phase combination, has been developed. This mobile phase can be used for hydrophobic interaction liquid chromatography on an amide stationary phase (for polar analytes), reversed-phase (RP) LC analysis on a C8 stationary phase (for moderately polar-non-polar compounds) and RPLC using a CSH phenyl-hexyl bonded column (for lipids). All of these sub 10 minute separations had good throughput and reproducibility with CV's of analyte response <25 % whilst eliminating the need for complex mobile phase preparation and the use of IPA as an organic modifier for lipidomics. Advantages of removing IPA and replacing it with the ACN-based method were a 58 % increase in peak capacity for lipids, with improved resolution for the di- and triglycerides and cholesterol esters compared to current methods. Compared to the IPA-containing solvent system the ACN-based mobile phase also resulted in a 61 % increase in lipid feature detection. The utility of this “universal” mobile phase approach was demonstrated by its application to a rat toxicology study investigating the consequences of methapyrilene administration through on the endogenous metabolite profiles of plasma and urine. Methapyrilene and its metabolites were also profiled in these samples.

Simultaneous analysis of the of levamisole with triclabendazole in pharmaceuticals through developing TLC and HPLC–PDA chromatographic techniques and their greenness assessment using GAPI and AGREE methods

Two simple and rapid chromatographic methods were developed and validated for the analysis of levamisole and triclabendazole simultaneously in pure and pharmaceutical products. The first method is thin-layer chromatography (TLC) with densitometry, and the second method is high-performance liquid chromatography with PDA detection (HPLC–PDA). A Hypersil BDS C18 column with dimensions of 4.6 × 150 mm and a particle size of 5 µm was used in the HPLC–PDA method. An isocratic condition was used to carry out the separation, and the mobile phase was made up of acetonitrile and a 0.03 M potassium dihydrogen phosphate buffer in double-distilled water. The ratio of the mobile phase preparation was 70:30 (v/v), and the flow rate was 1 mL/min. A wavelength of 215 nm was employed for analyte detection. Precoated silica gel 60 F254 aluminium plates were used for the TLC method's separation. Mobile phase was made of ethyl acetate, hexane, methanol, and ammonia (69:15:15:1) for the separation. The detection wavelength selected was 215 nm. According to the International Council for Harmonization (ICH) guidelines, the proposed methods were validated and it was found that the two chromatographic methods are accurate, precise, and linear for both compounds in the range of 3.75–37.5 and 6–60 mg/L for the HPLC method for levamisole and triclabendazole, respectively and in the range of 2–14 µg/spot for the TLC method. The developed methods greenness profile was assessed using AGREE and ComplexGAPI tools.Graphical

Development and validation of an analytical method for the determination of related substances in bisoprolol fumarate in dosage forms by HPLC-UV-DAD

The aim of this work was the development of an efficient analytical method for the determination of bisoprolol and related substances in the finished drug product using HPLC/DAD. Different bonded phases (alkyl and phenyl) with variable pore sizes and column dimensions, temperatures, and mobile phases with variable pH and additives were tested. Experiments that included using Hypersil 3 BDS C18 100 × 4 mm, 3 mm; Zorbax SB C18 150 × 4.6 mm, 3.5 mm; Acquity UPLC BEH C18 50 × 2.1 mm, 1.7 mm; and Xterra MS C18 100 × 4.6 mm, 3.5 mm gave the best results when considering the separation and resolution of the tested analytes within a reasonable run time. The method using Xterra MS C18 100 × 4.6 mm, 3.5 mm was validated as the most suitable when taking into account the mobile phase preparation and versatility, analysis time, and equipment maintenance.Validation parameters (linearity, accuracy, precision, selectivity) confirmed the method as suitable for its intended use.

Glycerol as a novel green mobile phase modifier for reversed phase liquid chromatography

Introducing green organic solvents that can be used as mobile phase modifiers in liquid chromatography is crucial. The currently used modifiers are either expensive, less efficient, toxic or incompatible with UV detection. In this work, glycerol has been tried as a renewable cheap green solvent for mobile phase preparation in reversed phase liquid chromatography. The enthalpy–entropy calculations showed that analyte transfer from the glycerol-containing mobile phase to the stationary phase was favourable and exothermic. The thermodynamic calculation showed that the retention process was governed by partitioning rather adsorption. The high viscosity of glycerol could be manipulated by addition of water as a co-fluidizing solvent. The Van Deemter plot showed high efficiency using 7%glycerol, which could be due to the beneficial effects of glycerol on eddy and longitudinal diffusions. The mechanism of retention was studied by increasing the percentage of glycerol in the mobile phase and monitoring the change in retention. The observed decrease in retention with the increase in the mobile phase strength and the elution order of the more polar analytes first are typical for the reversed phase mode. The glycerol-containing mobile phase was successfully applied for separation of a mixture of four antiviral drugs using C18 column. The chromatographic conditions were optimized using factorial design. The mixture was separated in 13 min with acceptable resolution and peak shapes. The method was validated according to the ICH guidelines. Glycerol was found to be a promising green alternative for conventional mobile phase modifiers.

Characterization of hydrophilic interaction liquid chromatography retention by a linear free energy relationship. Comparison to reversed- and normal-phase retentions

The Abraham solvation parameter model, a linear free energy relationship (LFER) approach, has been used to characterize a polymeric zwitterionic (sulfobetaine) column in HILIC mode. When acetonitrile (MeCN) is used in the preparation of mobile phases the main solute characteristics affecting the chromatographic behavior of analytes are the molecular size and the hydrogen-bonding (both acidity and basicity) interactions. The former property is more favorable in the acetonitrile-rich mobile phase, reducing thus the retention, but the latter reveals a higher affinity for the water layer adsorbed on the stationary phase, enhancing retention. However, if the aprotic acetonitrile is replaced by methanol, a hydrogen-bond acidic solvent, solute hydrogen-bond basicity does not contribute any more to retention, quite the opposite. Thus, a slightly different selectivity is observed in methanol/water than in acetonitrile/water. Normal-phase mode and HILIC-MeCN share the same main factors affecting retention. For reversed-phase and immobilized artificial membrane (IAM) chromatography, the solute molecular size increase retention because of the lower amount of energy required in the formation of a cavity in the solvated stationary phase. On the contrary, the analyte hydrogen-bond basicity favors interactions with the hydroorganic mobile phase and reduces retention. The determined parameters justify the reversed selectivity commonly observed in HILIC in reference to reversed-phase. In most instances, the least retained solutes in reversed-phase are the most retained in HILIC.

Methanol linear gradient counter-current chromatography for the separation of natural products: Sinopodophyllum hexandrum as samples

Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many compounds with a large range of polarities and partition coefficients, which are common features of natural products.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Emtricitabine and Tenofovir Disoproxil Fumarate in Bulk and Tablet Dosage Form

An analytical method was developed for estimation of Emtricitabine and Tenofovir Disoproxil Fumarate in drug product by using high performance liquid chromatography. The chromatographic separation was performed on column Cosmosil C18 (250mm x 4.6ID, Particle size: 5micron) using methanol: water (70:30 v/v) as mobile phase with flow rate of 0.8 ml/min. The detection was carried by using UV-300-M detector at 266 nm wavelength. The retention times of Emtricitabine and Tenofovir Disoproxil Fumarate were found to be 3.561 and 5.478 min respectively. Run time was observed as 5.54 min and 7.05 min respectively for Emtricitabine and Tenofovir Disoproxil Fumarate. Validation was performed as per ICH guidelines which include accuracy, precision, linearity, specificity, LOD, LOQ, ruggedness and robustness all of these were found within standard range as per ICH norms. Preparation of mobile phase is very simple &amp; does not require lengthy procedure. Proposed method was found to be accurate and specific for routine quality control of pharmaceutical dosage form like bulk and tablet dosage form. Keywords: Emtricitabine, Tenofovir Disoproxil Fumarate, HPLC, Validation.

Molecular weight distribution characterization of reactive higher ethyleneamines using size-exclusion chromatography with conventional calibration

Ethyleneamines have been produced and commercialized for decades in the chemical industry for a diverse range of applications. The presence of amine functional groups provides them opportunity to adsorb onto surfaces which can make them a very challenging sample matrix to analyze using separation techniques. In the present report, a new aqueous SEC-RI method, which enables MWD characterization of higher ethyleneamines, is described. The sample preparation was based on the dilute-and-shoot methodology. A surface-modified SEC column with positively charged groups attached to the stationary phase was used. The mobile phase composition (salt concentration, pH) was optimized to suppress interaction between the ethyleneamines and the packing material. Very symmetrical peak shapes were achieved for low MW monodisperse ethyleneamines despite their high primary amine content. MWD calculations were conducted using conventional narrow standard calibration with partial linear extrapolation of the calibration curve. The narrow standards were of the same chemistry as the samples of interest. Consequently, the standard components display a consistent behaviour towards the column packing as the sample components which makes the present method more robust and the interpretation of the quantitative results more convenient. Effect on the measured MW averages and MW distribution due to various experimental parameters (e.g., system variability, mobile phase preparation, sample concentration) were investigated showing good repeatability (RSD < 2%) for Mn, Mw, and Mz.

Method Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Propranolol and Hydralazine in Pharmaceutical Dosage Form

The method developed and validation was carried out by using isocratic mode with simple mobile phase preparation and separation was achieved on Phenomenex Luna C18 column (250 X 4.6 mm, 5 μm) using the mobile phase consisting of Methanol: water in the ratio of 40: 60 (0.01%O-phosphoric acid). The mobile phase was pumped at a flow rate of 1.0 mL/min and detection was done by UV detector at 293 nm. The retention times were 2.76 min for Propranolol and 4.69 min for Hydralazine. The linearity ranges for Propranolol and Hydralazine were 10–60 μg/ml and 6–36 μg/ml respectively with correlation coefficient 0.999. The LOD and LOQ values of propranolol were 0.099μg/ml and 0.300μg/ml obtained and then hydralazine LOD and LOQ values were 0.093μ/ml and 0.281μg/ml respectively. This method can be applicable for routine analysis which shows good reproducible, precise and accurate for simultaneous estimation of propranolol and hydralazine in pharmaceutical dosage forms.

Development of quantitative HPTLC–Densitometry methods for the analysis of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide using a model process developed earlier for the transfer of TLC screening methods

A model process, previously developed in a series of studies, allows for the transfer of thin-layer chromatography (TLC) methods for qualitative screening of counterfeit drug products published in the Global Pharma Health Fund (GPHF) Minilab manual and US Food and Drug Administration (FDA) Compendium of Unofficial Methods for Screening of Pharmaceuticals by TLC to quantitative high-performance TLC (HPTLC)–densitometry methods. This article describes HPTLC–densitometry methods developed and validated according to this model process for pharmaceutical products of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide, for which qualitative screening methods have not been published in the Minilab manual or FDA Compendium. These methods use relatively inexpensive and nontoxic “green solvents” for sample and standard solution and mobile phase preparation, Merck Premium Purity silica gel 60 F254 plates, automated standard and sample solution bandwise application, and automated densitometry for the assessment of peak purity and identity and quantification. Corresponding to the quantitative HPTLC–densitometry methods, qualitative TLC screening methods for these drug products were developed and posted online with open access as supplements to the FDA Compendium.

A review on analytical method development, optimization and validation of combination of Azithromycin and benzoyl peroxide by RP-HPLC using design of experiment as per ICH guideline

Pharmaceutical analysis is one of the most challenging fields of analytical chemistry. Pharmaceutical analysts carry out the qualitative and quantitative control of APIs and drug products and also develop and validate appropriate methods. One of my main goals was to develop modern, rapid, precise and reproducible, but also cost-effective HPLC assay methods which are generally available and applicable for most users. The aim of this work was to develop LC methods for both compounds. The assay of erythromycin by LC offers several advantages, such as high specificity, the possibility of determining and quantifying impurities and degradation products, and improved accuracy. The developed methods were validated. My whole work containing following plan of work as Selection of drug, Review Literature, FITR of both drugs and Mixture, Preparation of standard solutions, Preparation of sample of pure drug in Standard solution, Method development by HPLC (as Selection of solvents to be used as diluents and mobile phase, Selection of wavelength, Selection of mobile phase and Selection of chromatographic conditions) Preparation of Mobile phase, Preparation of standard calibration curve combination of drug, Optimization of HPLC condition using box behnken design. Validation of analytical method following parameters as per ICH guidelines. (i). System suitability (ii). Linearity and range (iii). Specificity (iv).Accuracy and precision (v). Limits of detection (LOD) and Quantitation (LOQ). (vi). Selectivity and (vii).Robustness.

Validation of a straightforward high performance liquid chromatographic method for morphine quantitation

BackgroundMorphine is illegal to use unless it is prescribed by a medical doctor. This compound is commonly abused in the form of illicit heroin.MethodsAn easy-to-use method for determining morphine in illicit heroin using high performance liquid chromatography-photodiode array detector (HPLC-PDA) was developed. With the aid of 50:50 acetonitrile:ammonium formate, this target compound traveling in a pentafluorophenyl (PFP) column was successfully separated from other compounds frequently present in the heroin matrix.ResultsThe method was precise with an intra-day relative standard deviation (RSD) ≤ 0.8% and inter-day RSD < 5%. It was able to detect as low as 0.001 mg/mL morphine. The detector’s response was linear (R2 > 0.999) and reliable for morphine quantitation from 0.005 up to 1 mg/mL. Through recovery studies, accuracy of the method was averagely estimated to be 89.54–101.91%.ConclusionsThis method saves time in terms of mobile phase preparation and cuts cost by excluding additional purchase of expensive chemicals. More importantly, it proves to be able to determine the target analyte with sufficient accuracy and precision.

The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides.

Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase. Graphical Abstract ᅟ.

Protein quantitation using various modes of high performance liquid chromatography

Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% – RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15μg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study.

Aqueous chromatographic system for the quantification of propofol in biological fluids using a temperature-responsive polymer modified stationary phase

A new method for the quantitative analysis of monkey serum propofol, which is widely used as an anaesthetic agent, was developed by utilizing a temperature-responsive polymer of N-isopropylacrylamide (NIPAAm) and butyl methacrylate (BMA) as the stationary phase of HPLC–fluorescence detection. This poly(NIPAAm- co-BMA) copolymer undergoes a reversible phase transition from a hydrophilic to a hydrophobic microstructure when triggered by change in the temperature. Also this chromatographic system is possible to separate the analytes by using only water as a mobile phase. A pretreatment of the serum (80 μL) was only solid-phase extraction, and the recovery rate of propofol and internal standard was more than 77%, respectively. This method covered the calibration range from 0.5 μg/mL to 10 μg/mL and allowed a reproducible quantification of the serum propofol in administrated monkey serum. The intra- and inter-assay relative standard deviations were less than 14.1%. In addition, there was good relationship of the quantification values between the developed method and the widely used reversed-phase HPLC method. Our developed method has proven to be useful for a simple analysis of propofol in clinical practice, because the avoidance of complicated mobile phase preparation was possible, and only temperature changing could regulate the retention time of the analyte. In addition, by using water instead of fossil fuel, it is the ideal analytical method according to green chemistry.

Influence of Solvent Additive Composition on Chromatographic Separation and Sodium Adduct Formation of Peptides in HPLC–ESI MS

Mobile phase additives have a strong influence on sensitivity and spectral quality when HPLC is coupled to mass spectrometry. They may cause ion suppression, as frequently observed with trifluoroacetic acid, or may lead to increased adduct formation, complicating data analysis and interfering with automated LC–MS–MS analysis. When reversed-phase HPLC is performed under neutral conditions, ammonium acetate is the additive of choice because of its volatility. Because of the limited solubility of ammonium acetate in commonly employed organic solvents like acetonitrile usually a small amount of water is added for dissolution, and in addition, acetic acid can be added to obtain a buffered system. We compared six different acetonitrile/ammonium acetate blends, differing in their water and acetic acid content and evaluated their performance for HPLC–MS analysis of model peptides (bradykinins) under neutral conditions. The results demonstrate that mobile phase preparation strongly influences chromatographic separation and adduct formation in electrospray ionization MS, as shown for a series of bradykinin peptides. By varying the solvent composition only slightly to influence the pH curves during gradient elution, three nearly coeluting peptides could be baseline separated. At the same time, the formation of sodiated instead of protonated peptides was found to vary by up to a factor of four, depending on the preparation of the solvent blends.

An automated mobile phase preparation workstation

An automated solvent dispensing workstation capable of delivering volumes ranging from 10 mL to 4.5 L for the preparation of solutions/mobile phases was developed and implemented into the industrial R&D laboratory. The workstation was designed to address business, safety, and compliance needs while meeting or exceeding the precision and accuracy of current manual methods of preparation. The system's performance was optimized with respect to liquid transfer tubing inner diameter, pumping pressure, flow characteristics of the valve, and computer control logic. The automated solvent dispensing workstation was shown to exceed the specifications set by the ASTM for Class A graduated cylinders for all dispense volumes (10 mL–4.5 L).

Enhancement of retention predictions in reversed-phase liquid chromatography using reference compounds

The use of reference compounds to correct errors associated with the preparation of mobile phase is studied, in order to enhance modelling of retention for optimisation purposes in reversed-phase liquid chromatography. The method fits individual retention models for a set of reference compounds, which are present in all injections. In second step, mobile-phase composition is corrected for each chromatogram, according to the fitted models of the reference compounds. The enhanced precision provided by the large number of replicates for the reference compounds is partially propagated to the retention models of the analytes. The approach is assayed in the separation of amino acids, after pre-column derivatisation with o-phthalaldehyde (OPA) and N-acetylcysteine (NAC) to form isoindoles, using acetonitrile–water mobile phases and a C18 column. In this example, the reference compounds were three by-products present in the reagent mixture, which produce peaks usually eluting before the analytes. Detection of isoindoles and reference compounds was performed at 335 and 254 nm, respectively. In other samples, similar procedures can be developed by adding one or more external inert compounds to the sample solution. The approach is especially useful for situations where the experimental work is carried out along a rather long period, and random variations in the instrument, reagents and environmental conditions are expected.

Using ultrapure water in ion chromatography to run analyses at the ng/L level

Thanks to enhanced capabilities, ion chromatography (IC) occupies an increasing position in many types of applications. Achieving ideal performances for an extended life-time can only be reached, however, if the IC system is operated in optimum experimental conditions. Among the various parameters that need to be controlled, water is particularly important, because it is used throughout the analysis, from sample preparation to column rinsing, elution, and mobile phase preparation. More and more, devices are included in IC systems to generate the eluent in situ, and ultrapure water becomes the major reagent. Data of pre-concentration of high purity water show that detection limits at the ng/L level can be expected with water purified using the right combination of technologies.

Using ultrapure water in ion chromatography to run analyses at the ng/L level

Thanks to enhanced capabilities, ion chromatography (IC) occupies an increasing position in many types of applications. Achieving ideal performances for an extended life-time can only be reached, however, if the IC system is operated in optimum experimental conditions. Among the various parameters that need to be controlled, water is particularly important, because it is used throughout the analysis, from sample preparation to column rinsing, elution, and mobile phase preparation. More and more, devices are included in IC systems to generate the eluent in situ, and ultrapure water becomes the major reagent. Data of pre-concentration of high purity water show that detection limits at the ng/L level can be expected with water purified using the right combination of technologies.