Academic literature on the topic 'RP-HPLC: reversed-phase HPLC'

Chromatography, a separation technique, is mostly used in chemical analysis in which High-performance liquid chromatography (HPLC) is an extremely versatile technique where analytes are separated by passage through a column packed with micrometer-sized particles. Theses day reversed-phase chromatography is commonly used separation technique in HPLC. The reasons for this include the simplicity, versatility, and scope of the reversed-phase method as it is able to handle compounds of a diverse polarity and molecular mass. Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution. This review covers the importance of RP-HPLC in analytical method development and their strategies along with brief knowledge of critical chromatographic parameters need to be optimized for an efficient method development. Key Words- HPLC, RP-HPLC

Chromatographic conditions were optimized and three commercially available columns were evaluated for separation of alcohol-soluble storage proteins of Neepawa wheat using reversed-phase high-performance liquid chromatography (RP-HPLC). Optimal separation was achieved using an extracting solution of 50% 1-propanol, 1% acetic acid, and 4% dithiothreitol and an HPLC elution time of 105 min at a flow rate of 1.0 mL/min. HPLC columns evaluated (SynChropak RP-P, Ultrapore RPSC and Aquapore RP-300) varied in selectivity and resolution. The column providing the greatest versatility was Aquapore RP-300 available in cartridge form. Sodium dodecyl sulfate gradient-gel electrophoresis analysis of protein peaks resolved by RP-HPLC indicated that many of the eluted peaks contained more than one protein species. Chromatographic protein patterns obtained for Neepawa wheat grown at different locations and in different years were qualitatively the same.Key words: Protein, high-performance liquid chromatography, wheat

ZusammenfassungHeparinoide mit Galaktosaminoglykanstruktur können mit chromatographischen Methoden auf ihre Eigenschaften untersucht werden. Mit Hilfe der Hochdruck-flüssigkeits-Gelpermeationschromatographie (GPC-HPLC) wurden daher die durchschnittliche molekulare Masse und die Polydispersität (Q) bestimmt. Die Stoffe zeigten eine unterschiedliche durchschnittliche molekulare Masse Mw von 19077 bis 25193 Da. Die Polydispersität streute von 1,04 bis 1,13 für unfraktionierte Galaktosaminoglykane. Die Reversed-Phase-Hochdruckflüssig-keitschromatographie (RP-HPLC) läßt Unterschiede in der Lipophilie-Hydrophilie erkennen. Die Galaktosaminoglykane lassen sich mit der RP-HPLC detektieren. Aufgrund sehr hydrophiler Eigenschaften zeigten sie ähnliche Elutionsprofile. Durch die Hochdruckflüssigkeits-Affinitätschromatographie (HPLAC) werden mit einer Concavalin-A-Säule die Galaktosaminoglykane analysiert. Sie unterschieden sich in ihrer Retentionszeit (1,00 – 1,40 min).

Cows' and donkey milks (raw and thermally processed) and respective whey were analysed for quantification of major proteins. Two different chromatographic approaches, size exclusion (SE-HPLC) and reversed-phase high performance liquid chromatography (RP-HPLC) both coupled to UV detection were used. Usefulness of these methods for routine control of the effect of thermal processing was evaluated. The external standard method was used to calibrate the SE-HPLC and RP-HPLC systems. Concerning quantification of β-lactoglobulin (β-lg), α-lactalbumin (α-la), lysozyme (lys), and total casein (cn), no significant differences between results obtained by SE-HPLC and by RP-HPLC (t-test, P>0·05) were observed for raw milks and whey. Heating of cows' milk promoted aggregation of denatured proteins as observed by SE-HPLC, whereas α-la and β-lg from donkey milk were stable to thermal processing at 100°C (5 min). Lys was quantified in donkey raw milk and whey however, in thermally processed donkey milk lys was denatured and could not be quantified by HPLC.

RP-HPLC (reversed-phase high-performance liquid chromatography) is widely used to determine the amounts of the different gluten protein types. However, this method is time-consuming, especially at early stages of wheat breeding, when large number of samples needs to be analyzed. On the other hand, LoaC (Lab-on-a-Chip) technique has the potential for a fast, reliable, and automatable analysis of proteins. In the present study, benefits and limitations of Lab-on-a-Chip method over RP-HPLC method in gluten proteins evaluation were explored in order to determine in which way LoaC method should be improved in order to make its results more compliant with the results of RP-HPLC method. Strong correlation (P≤0.001) was found between numbers of HMW glutenin peaks determined by LoaC and RP-HPLC methods. Significant correlations (P≤0.05) were obtained between percentages of HMW and LMW glutenin subunits calculated with regard to total HMW + LMW area. Even more significant correlation (P≤0.001) was found when percentages of individual HMW areas were calculated with regard to total HMW. RP-HPLC method showed superiority in determination of gliadins since larger number and better resolution of gliadin peaks were obtained by this method.

Phenoxy acid herbicides are used worldwide and are potential contaminants of drinking water. Reversed phase high-performance liquid chromatography (RP-HPLC) is commonly used to monitor phenoxy acid herbicides in water samples. RP-HPLC retention of phenoxy acids is affected by both mobile phase composition and pH, but the synergic effect of these two factors, which is also dependent on the structure and pKa of solutes, cannot be easily predicted. In this paper, to support the setup of RP-HPLC analysis of phenoxy acids under application of linear mobile phase gradients we modelled the simultaneous effect of the molecular structure and the elution conditions (pH, initial acetonitrile content in the eluent and gradient slope) on the retention of the solutes. In particular, the chromatographic conditions and the molecular descriptors collected on the analyzed compounds were used to estimate the retention factor k by Partial Least Squares (PLS) regression. Eventually, a variable selection approach, Genetic Algorithms, was used to reduce the model complexity and allow an easier interpretation. The PLS model calibrated on the retention data of 15 solutes and successively tested on three external analytes provided satisfying and reliable results.

Abstract A reversed-phase HPLC method (RP-HPLC) with UV detection was developed and validated for the quantitative determination of cefprozil, a second-generation cephalosporin. Due to β-lactam ring instability under alkaline conditions, this RP-HPLC method was applied for the determination of cefprozil in the presence of its possible degradation product. The interactions that govern the separation process with stationary phase were investigated at both molecular and quantum mechanical levels. Moreover, electrostatic potential maps were generated to determine the sites of interaction with mobile phase. The suggested method was validated in compliance with International Conference on Harmonization guidelines and successfully applied for the determination of cefprozil in its commercial pharmaceutical formulation.

ABSTRACTObjective: To development and validation of a reversed-phase high-performance liquid chromatography (RP-HPLC) for the determination of modafinilin bulk and pharmaceutical dosage forms.Methods: A simple, precise, rapid, and accurate RP-HPLC method was developed for the estimation of modafinil in bulk and pharmaceutical dosageforms. Xterra RP 18 (250 mm × 4.6 mm, 5 µ particle size) with a mobile phase consisting of methanol:water 70:30 V/V was used. The flow rate1.0 ml/min and the effluents were monitored at 260 nm. The retention time and recovery time was 12 minutes. The detector response was linear inthe concentration of 10-50 µg/ml. The respective linear regression equation being Y=452.1x+65237. The limit of detection and limit of quantificationwere 4.547 and 1.377 mcg, respectively. The method was validated by determining its accuracy, precision, and system suitability.Result: The objective of the present work is to develop simple, precise, and reliable HPLC method for the analysis of modafinil in bulk andpharmaceutical dosage forms. This is achieved using the most commonly employed Xterra RP 18 (250 mm × 4.6 mm, 5 μ particle size) columndetection at 260 nm. The present method was validated according to ICH guidelines.Conclusion: In this study, a simple, fast and reliable HPLC method was developed and validated for the determination of modafinil in pharmaceuticalformulations.Keywords: Modafinil, Reversed-phase high-performance liquid chromatography, Estimation, ICH guidelines, Tablets.

A novel RP-HPLC validated method for determination of Exemestane is developed. The chromatographic separation was done on Phenomenex Luna reversed phase C18 (150 mm x 4.6 mm, 5 μm) column in isocratic mode, using Acetonitrile: HPLC grade water (50:50, v/v) as mobile phase at 254 nm wavelength. Exemestane chromatographic peak is eluted with retention time 4.656 min. The linearity range was 5-30 μg/ml with correlation coefficient 0.999. The method was validated as per ICH Guidelines. The quantification and detection limit for estimation of Exemestane was found to be 0.365 and 1.926 μg/ml respectively. Recovery of Exemestane was found in the range of 98.0-102.0%. Keywords: RP-HPLC; Exemestane; Validation; Estimation

Abstract Background: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion. Methods: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0–1 month, 1–12 months, 1–3 years, 3–8 years, 8–16 years, and >16 years) and in two groups for 24-h urine collections (8–16 years and >16 years). Results: Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9–17% and between-run CVs were 7.1–46% for the same period. Amino acid recoveries were 78–122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections. Conclusions: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.

In this study, a reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed to separate and determine Pt and Pd after formation of their chelates with N,N-diethyl-N'
-benzoylthiourea (DEBT). With the aim of reducing the number of steps in treating the samples, the method developed does not require the elimination of excess chelating reagent before the analysis of metal chelates. The different physical and chemical parameters affecting separation were examined in details. The whole analysis was completed on a C18 column in 16 min at 280 nm, with the mobile phase of acetonitrile-methanol-water (80:10:10, v:v:v) containing 0.20 mol l-1 pH 5.0 acetate buffer at a flow rate of 0.8 ml min-1. Detection limits of the method, based on 3s, were found as 14.2 ug l-1 for Pd and 0.77 mg l-1 for Pt using a 20-ul sample loop. Reproducibility of the method for ten repeated measurements was found as 2.36 % for 0.60 mg l-1 Pd and 2.58 % for 10.0 mg l-1 Pt as % RSD. The proposed method is a rapid, simple and highly selective method for the simultaneous determination of Pt and Pd by HPLC without the need for any interference elimination process.

Includes bibliographical references.
The complexes were successfully separated using conventional Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) and a UV detector. A simple method of complexation of platinum(II) and palladium(II) in acidic, aqueous samples was developed. This method involves the mixing of excess ligand (HL") in acetonitrile with aqueous solutions containing traces of platinum(II) and palladium(II) at room temperature followed by salt-induced phase separation prior to RP-HPLC analysis. In the case of rhodium(III), however, complexation was extremely slow and it has not been possible to include this metal in the complexation procedure for platinum(ll) and palladium(II).

Therapeutic proteins are often lyophilized with excipients such as sucrose or trehalose to protect them during manufacturing and achieve a longer shelf-life. Formulation design for therapeutic proteins has been a trial-and-error process, and the mechanisms responsible for the stabilizing effects of excipients are not fully understood. Two proposed theories have been widely accepted: the water replacement theory and the vitrification theory.^1,2The water replacement theory suggests that excipients stabilize protein molecules in the solid state by forming hydrogen bonds that “replace” the hydrogen bonds to water that stabilize the protein in solution, while the vitrification theory asserts that proteins are stabilized by a glassy solid matrix of low mobility and does not require direct interactions between excipient and protein. A better understanding of the interactions between proteins and other components of the lyophilized matrix can facilitate rational formulation design and shorten the time in development. However, most of the analytical methods available can only provide information on the bulk properties of the lyophilized matrix such as moisture content and glass transition temperature (T_g); it has been difficult to measure the interactions between protein and excipient directly, if they exist. In order to characterize the interactions between protein and excipients in a lyophilized matrix with high resolution, a photolytic labeling method was developed in this dissertation, building on previous work in our research group. Photolytic labeling has long been used to identify protein-protein interactions in vivo.^3,4Common types of photo-reaction reagents and their applications are summarized in Chapter 1. The research described in this dissertation utilizes the diazirine functional group, which is activated after UV exposure and undergoes a free radical reaction to form covalent bonds with nearby molecules. The reaction can be used to identify the interactions between excipients and protein or peptide in a solid formulation. Previous studies in our lab have shown that photo-reaction can be applied to lyophilized solids to study protein-matrix properties and interactions in the solid.^5,6This dissertation seeks to further identify photo-reaction products and analyze them in a more quantitative way.

Chapter 2 describes a quantitative analysis of photo-reaction products in solution and lyophilized solids using a model peptide, KLQ (Ac-QELHKLQ-NHCH₃). The purpose of the work in this chapter is to establish a quantitative analytical method for photo-reaction products, enabling studies of peptide-excipient interactions in lyophilized solids. KLQ was derivatized with a bifunctional probe NHS-diazirine (succinimidyl 4,4’-azipentanoate; SDA) at Lys5 to be photo-reactive. The SDA derivatized KLQ (KLQ-SDA) was used to study the photo-reaction products and examine excipient interactions. Identification and quantitation of photo-reaction products of KLQ-SDA was achieved with liquid chromatography mass spectrometry (LC-MS) and reversed phase HPLC (rp-HPLC). Important reaction products such as peptide-excipient adducts and peptide water adducts varied in different formulations. Unexpected reaction products such as unproductive “dead-end” products and peptide-phosphate adducts from buffer salt were also detected and quantified. Together, the photo-reaction products reflected the local environment near Lys5 of the peptide in the solid state. This study has provided a better understanding of photo-reaction with diazirine in the lyophilized solids together with a quantitative description of the local environment near Lys5.

In Chapter 3, the photo-reaction products in lyophilized solids exposed to increasing moisture were analyzed, and the effect of increasing moisture on the local environment near the peptide was examined. Using the analytical method developed in Chapter 2, these studies explored whether peptide-water interactions, as measured by the formation of water adducts formed by photolytic labeling, are linearly correlated with an increase in solid bulk moisture content. Formulations containing the KLQ-SDA peptide were exposed to various relative humidity conditions and photolytic labeling was induced. Solids containing disaccharide excipients behaved differently from those containing amino acids when exposed to the same relative humidity condition, showing different levels of peptide-excipient and peptide-water adducts. With increasing moisture content in the solids, the formation of photo-reaction products did not mimic the pattern of solutions with same composition, indicating differences in the local environment.

An alternative approach to studying lyophilized formulations using photolytic labeling is to incorporate photo-reactive excipients into the solid matrix. In Chapter 4, a new diazirine-labeled photo-excipient, photo-glucosamine (pGlcN), was chemically synthesized and incorporated into formulations of the therapeutic peptide salmon calcitonin (sCT) and compared with the commercially available diazirine-labeled amino acid, photo-leucine (pLeu). The studies in Chapter 4 further compared peptide-excipient interactions at the molecular level with two different photo-excipients, ionizable pLeu and unionizable pGlcN. Changing solution pH prior to lyophilization was expected to change ionic interactions between sCT and pLeu in the solid samples, resulting in different distributions of photo-reactions products; pH-dependent differences were not expected for pGlcN. The results demonstrated that the distribution of photo-reaction products varied with the composition of the formulation and the pH of the solution prior to lyophilization. The photo-reaction products in the pGlcN-containing formulation differed from those pLeu, showing a difference in the interactions of unionizable (pGlcN) and ionizable (pLeu) excipients with sCT in solid samples.

The work in this dissertation has developed photolytic labeling as a tool to study lyophilized peptide formulations, and has provided a more quantitative understanding of the photo-reaction products that are produced from diazirine-labeled peptides or excipients in the solid state. A new photo-reactive excipient has also been presented (pGlcN), which showed different photo-reaction products than a commercially available photo-excipient (pLeu) and is promising for future study. Photolytic labeling for formulation development is still in its early stages, and additional research regarding reaction mechanism and complementary stability studies is needed. Nevertheless, the results presented in this dissertation support continued development of photolytic labeling as a practical method for formulation design and development.