Ultra High-pressure Liquid Chromatography Method Research Articles

A Review on UHPLC Method

Ultra-high-pressure liquid chromatography (UHPLC) has emerged as the contemporary HPLC platform of choice in recent years. Because UHPLC requires less time for column equilibration and analysis, it is the perfect tool for developing methods quickly. The present state, advantages, and drawbacks of UHPLC in technique development are critically reviewed in this paper. To explain current developments and best practices, we employ case studies. Converting current HPLC methods to speedier analysis, quick screening of columns and mobile phases, and automated process optimisation are a few examples. Although the development of reversed-phase techniques for small-molecule pharmaceutical assay and impurity analysis is our primary emphasis, our findings and insights are applicable to other sample types and applications as well. When utilised with short, small-particle columns, UHPLC's greater pressure limitations not only produce quicker analysis but also make it possible to use longer columns more effectively for improved routine analysis of complicated materials.

Ceftriaxone dosing in patients admitted from the emergency department with sepsis.

Unbound ceftriaxone pharmacokinetics in adult patients have been poorly characterised. The objective of this study is to determine the ceftriaxone dose that achieves an unbound trough concentration ≥ 0.5 mg/L in > 90% of adult patients receiving once-daily dosing presenting to the emergency department (ED) with sepsis. We performed a prospective single-centre pharmacokinetic study. A single unbound plasma ceftriaxone concentration was obtained from each patient using blood collected as part of routine clinical practice within the first dosing interval. Samples were analysed using a validated ultra-high pressure liquid chromatography method. Population pharmacokinetic analysis and Monte Carlo simulations (n = 1000) were performed using Pmetrics for R. A ceftriaxone concentration obtained throughout the first dosing interval was available for fifty adult patients meeting sepsis criteria. Using this concentration time-curve data, a pharmacokinetic model was developed with acceptable predictive performance per the visual predictive check. Simulations show that a 1-g once-daily dose is unlikely to achieve the minimum therapeutic ceftriaxone exposure in > 90% patients with a creatinine clearance ≥ 60 mL/min. However, a 2-g once-daily dose will provide a therapeutic exposure for target pathogens infecting patients with a creatinine clearance ≤ 140 mL/min. Ceftriaxone administered as a 1-g once-daily dose is unlikely to achieve a therapeutic exposure in > 90% of patients presenting to the ED with sepsis. Increasing the ceftriaxone dose to 2 g once daily will likely achieve the desired exposure against target pathogens. Future clinical trials are required to determine any potential clinical benefit of optimised ceftriaxone dosing.

Improved analysis of olive oils triacylglycerols by UHPLC-charged aerosol detection

In the present study, a novel ultra high-pressure liquid chromatography method using charged aerosol detector (UHPLC-CAD) with an inverse-gradient solvent compensation has been developed for the analysis of triacylglycerols (TAGs) in extra-virgin olive oil. Using a gradient method with a superficially porous particles column, an improved separation of TAGs was obtained, with notably shortening of both solvent consumption and analysis time, in comparison with the IOC official method for ECN 42 analysis. Furthermore, an almost baseline separation of trilinolein was obtained. CAD provides good linearity (R2 > 0.999) in the range of 0.05–10 μg of trilinolein injected on column as well as uniformity of response factor for different TAG molecular species. Finally, the proposed UHPLC-CAD methodology has been successfully applied to the determination of trilinolein in low-linoleic acid extra-virgin olive oil mixed in different proportions with high-oleic sunflower oil, palm olein and a mix of them at different percentages of 2, 4, 6, 8 and 10% (w/w). Good linearity was achieved in all the three blends with correlation coefficients greater than 0.975 in all cases.

A practical approach for predicting retention time shifts due to pressure and temperature gradients in ultra-high-pressure liquid chromatography

Large pressure gradients are generated in ultra-high-pressure liquid chromatography (UHPLC) using sub–2μm particles causing significant temperature gradients over the column due to viscous heating. These pressure and temperature gradients affect retention and ultimately result in important selectivity shifts. In this study, we developed an approach for predicting the retention time shifts due to these gradients. The approach is presented as a step-by-step procedure and it is based on empirical linear relationships describing how retention varies as a function of temperature and pressure and how the average column temperature increases with the flow rate. It requires only four experiments on standard equipment, is based on straightforward calculations, and is therefore easy to use in method development. The approach was rigorously validated against experimental data obtained with a quality control method for the active pharmaceutical ingredient omeprazole. The accuracy of retention time predictions was very good with relative errors always less than 1% and in many cases around 0.5% (n=32). Selectivity shifts observed between omeprazole and the related impurities when changing the flow rate could also be accurately predicted resulting in good estimates of the resolution between critical peak pairs. The approximations which the presented approach are based on were all justified. The retention factor as a function of pressure and temperature was studied in an experimental design while the temperature distribution in the column was obtained by solving the fundamental heat and mass balance equations for the different experimental conditions. We strongly believe that this approach is sufficiently accurate and experimentally feasible for this separation to be a valuable tool when developing a UHPLC method. After further validation with other separation systems, it could become a useful approach in UHPLC method development, especially in the pharmaceutical industry where demands are high for robustness and regulatory oversight.

A new ultra-high pressure liquid chromatography method for the determination of antioxidant flavonol aglycones in six Lysimachia species

UPLC-DAD method was developed and validated for the quantitative determination of free flavonol aglycones (kaempferol, quercetin and myricetin) after acidic hydrolysis in six Lysimachia species. Quantitative analyses showed that the amounts of various flavonol aglycones were significantly different in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, Lysimachia ciliata and Lysimachia clethroides. The L. clethroides sample was found to be the richest in kaempferol (25.77 ± 1.29 μg/mg extract) and quercetin (97.67 ± 4.61 μg/mg extract), while the L. nummularia sample contained the highest amount of myricetin (20.79 ± 1.00 μg/mg extract). The antioxidant capacity of hydrolysed extracts was evaluated using in vitro DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS•+ [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] decolourisation tests. The observed radical scavenging capacities of the extracts showed a relationship with the measured flavonol aglycone content and composition. The acidic treatment resulted in an increased free radical scavenging activity compared to the untreated methanol extract.

Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs

An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41–61% resolution and a decrease of 91–422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds’ retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.

Analysis of Alkylresorcinols in Cereal Grains and Products Using Ultrahigh-Pressure Liquid Chromatography with Fluorescence, Ultraviolet, and CoulArray Electrochemical Detection

Alkylresorcinols are phenolic lipids, with homologues ranging from C17 to C25, found in high concentrations in whole grain wheat and rye, lower concentrations in barley, and negligible concentrations in refined wheat flour. The analysis of alkylresorcinols is of importance due to their potential as biomarkers of whole grain intake and emerging evidence for some biological effects. Present HPLC methods have insufficient resolution for accurately quantitating the mix of alkyl- and alkenylresorcinols found in rye. An ultrahigh-pressure liquid chromatography method was developed, and three detection methods (CoulArray (CAED), ultraviolet (UV), and fluorescence detection (FD)) were compared for cereal alkylresorcinol analysis. The lower limits of quantitation and detection were 50 and 20 pg injected, 5 pg and 2 pg injected, and 500 and 1250 pg injected for FD, CAED, and UV, respectively. FD and CAED provided similar results, with some bias for higher results with FD (<10% difference). UV detection generally resulted in overestimation of alkylresorcinol concentrations. The method was applied to cereal (15) and cereal product (90) samples mainly from the United States with results in the same range as previous methods. The improved resolution with this method allows facile analysis of alkylresorcinols from cereal products, including minor unsaturated homologues such as those found in rye.

Fast simultaneous determination of free and conjugated isoflavones in soy milk by UHPLC–UV

Ultra high pressure liquid chromatography (UHPLC) has become a widely used technique in separation science as it can shorten analysis time while maintaining resolution and increasing sensitivity. Here we developed a UHPLC method with UV detection for the simultaneous determination of isoflavone aglycones (daidzein, glycitein and genistein), their corresponding glycoside forms (daidzin, glycitin and genistin), and acetyl and malonyl conjugate forms of glycosides (malonyldaidzin, malonylglycitin, malonylgenistin, acetyldaidzin, acetylglycitin, acetylgenistin) in soy milk. The method allowed reliable determinations of 12 isoflavones in less than 8min of chromatographic elution. It provided a satisfactory linearity ranging from 0.1 to 5mg/L, except for daidzin and genistin, for which it ranged from 0.1 to 10mg/L. In addition, it showed acceptable chromatographic sensitivity, with a detection limit lower than 0.05mg/L and a determination limit below 0.2mg/L. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was greater than 95%. Total isoflavone content in commercial soy milk samples (n=8) ranged from 1805.22 to 3195.59mg/kg dw. Genistin and Daidzin were the major isoflavones in all samples. The proportion of aglycones, ß-glucosides, and acetyl and malonyl glucosides differed among the samples. ß-Glucosides were identified as the predominant forms.

Product Quality of Parenteral Vancomycin Products in the United States

In response to concerns raised about the quality of parenteral vancomycin products, the U.S. Food and Drug Administration (FDA) is investigating the product quality of all FDA-approved parenteral vancomycin products available in the United States. Product quality was evaluated independently at two FDA Office of Testing and Research (FDA-OTR) sites. In the next phase of the investigation, being done in collaboration with the National Institute of Allergy and Infectious Diseases, the in vivo activity of these products will be evaluated in an appropriate animal model. This paper summarizes results of the FDA investigation completed thus far. One site used a validated ultrahigh-pressure liquid chromatography method (OTR-UPLC), and the second site used the high-performance liquid chromatography (HPLC) method for related substances provided in the British Pharmacopeia (BP) monograph for vancomycin intravenous infusion. Similar results were obtained by the two FDA-OTR laboratories using two different analytical methods. The products tested had 90 to 95% vancomycin B (active component of vancomycin) by the BP-HPLC method and 89 to 94% vancomycin by OTR-UPLC methods. Total impurities were 5 to 10% by BP-HPLC and 6 to 11% by OTR-UPLC methods. No single impurity was >2.0%, and the CDP-1 level was ≤ 2.0% across all products. Some variability in impurity profiles of the various products was observed. No adverse product quality issues were identified with the six U.S. vancomycin parenteral products. The quality parameters of all parenteral vancomycin products tested surpassed the United States Pharmacopeia acceptance criteria. Additional testing will characterize in vivo performance characteristics of these products.

A validated ultra high-pressure liquid chromatography method for separation of candesartan cilexetil impurities and its degradents in drug product

Introduction: A selective, specific, and sensitive “Ultra High-Pressure Liquid Chromatography” (UPLC) method was developed for determination of candesartan cilexetil impurities as well asits degradent in tablet formulation. Materials and Methods: The chromatographic separation was performed on Waters Acquity UPLC system and BEH Shield RP18 column using gradient elution of mobile phase A and B. 0.01M phosphate buffer adjusted pH3.0 with Orthophosphoric acid was used as mobile phase A and 95% acetonitrile with 5% Milli Q Water was used as mobile phase B. Ultraviolet (UV) detection was performed at 254nm and 210nm, where (CDS-6), (CDS-5), (CDS-7), (Ethyl Candesartan), (Desethyl CCX), (N-Ethyl), (CCX-1), (1N Ethyl Oxo CCX), (2N Ethyl Oxo CCX), (2N Ethyl) and any unknown impurity were monitored at 254nm wavelength, and two process-related impurities, trityl alcohol and MTE impurity, were estimated at 210nm. Candesartan cilexetil andimpurities were chromatographed with a total run time of 20min. Results: Calibration showed that the response of impurity was a linear function of concentration over the range limit of quantification to 2 μg/mL (r2≥0.999) and the method was validated over this range for precision, intermediate precision, accuracy, linearity, and specificity. For the precision study, percentage relative standard deviation of each impurity was <15% (n=6). Conclusion: The method was found to be precise, accurate, linear, and specific. The proposed method was successfully employed for estimation of candesartan cilexetil impurities in pharmaceutical preparations.

A High‐Throughput UPLC Method for the Characterization of Chemical Modifications in Monoclonal Antibody Molecules

Development of high-throughput release and characterization assays is critical for the effective support of the rapidly growing biologics pipeline for biotherapeutics. Clipping of polypeptide chains is commonly monitored during process optimization, formulation development, and stability studies. A reduced capillary electrophoresis-sodium dodecyl sulfate (rCE -SDS) method is often used as a purity release assay for monitoring clips in monoclonal antibodies (mAbs); however, it has a cycle time of approximately 40 min, which is not suited for high-throughput screening. Additionally, the characterization of clips and variants from electropherograms is not straightforward and takes significant time. Reduced reversed-phase (RP) chromatography has been a popular assay for the characterization and identification of clips and variants because it can be directly coupled with online mass spectrometric analysis. However, the high-column temperature and low pH required for RP assays can induce on-column cleavage and therefore skew the results. To minimize on-column degradation, we have developed a high-throughput method with a significantly shorter cycle time of 5 min. The short cycle time was achieved using an ultra-high-pressure liquid chromatography (UPLC) system with a 1.7 μm phenyl column. This UPLC method allowed quantitation of hinge clipping in an IgG1 molecule and acid induced aspartic acid/proline (D/P) clip in an IgG2 molecule. The results from the UPLC method were comparable to those obtained with rCE-SDS. Additionally, the phenyl column offered partial resolution of oxidation and other chemical modifications, making this technique an attractive assay for high-throughput process characterization and formulation screens.

A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: Application to analyze isoflavone levels in soybean cultivars

Since only isoflavone aglycones are considered to be bioactive, the determination of total aglycones that are released from conjugated isoflavones after hydrolytic treatment may facilitate an objective alternative for quantifying isoflavone contents in soy products. Given this major benefit, a new ultra-high pressure liquid chromatography (UV-UPLC™) method was developed for the fast and reliable determination of total aglycones in soybeans (daidzein, glycitein, and genistein) after enzymatic hydrolysis applying helix pomatia digestive juice. Capitalizing on the enhanced performance of UPLC™, aglycones were separated within 3min only, with a total runtime of 8min till the next injection. Thus, especially compared to HPLC protocols, UPLC™ proved to be superior due to significantly shorter runtimes and accordingly increasing sample throughput. Additionally, regarding the performed validation (linearity, precision, recovery, selectivity, and robustness), the established method proved to be suitable for quantifying total aglycones in soybeans. Moreover, method applicability was demonstrated by analyzing 23 commercial soybean cultivars for their isoflavone contents. Cumulative aglycone levels ranged from 100 to 255mg per 100g, hence implying an average ratio of 52%, 41%, and 7% of total isoflavones for genistein, daidzein and glycitein, respectively. However, for some soybeans, other distinct aglycone distributions were observed as well.

Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food

Biologically active amines include the so called biogenic amines, such as histamine, tyramine and cadaverine, and polyamines such as spermidine and spermine. Ultra high pressure liquid chromatography (UHPLC) is a new generation of separation techniques that takes full advantage of chromatographic principles to increase speed flow which drastically reduce analysis time. The aim of the present work was to validate a rapid method of UHPLC to detect the presence of biogenic amines and polyamines in food. Different food matrixes (wine, fish, cheese, and dry fermented sausage) were used in order to test the versatility of the method. The UHPLC method described in this article has been demonstrated as a reliable procedure to determine 12 biogenic amines and polyamines in less than 7 min of chromatographic elution. The method provides a satisfactory linearity and chromatographic sensitivity with a detection limit lower than 0.2 mg/L and a determination limit falling below 0.3 mg/L for all amines. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was between 93% and 98%, depending on the food matrix.

Separation and quantification of two diastereomers of a Drug Candidate in rat plasma by ultra-high pressure liquid chromatography/mass spectrometry

Ultra-high pressure liquid chromatography (UHPLC) is a relatively new technology which utilizes chromatographic media with a 1.7 μm particle size. This technology has the potential to offer significant advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometric detection. Drug Candidate A, under development at Merck Research Laboratories, contains two chiral centers which have the absolute configuration R, S. Under in vivo and ex vivo conditions, one of the chiral centers readily epimerizes to produce the R, R diastereomer. Initially, a traditional high performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed to separate and quantify these two diastereomers in rat plasma. The lower limit of quantification (LOQ) of the two analytes was 2 ng/mL, and a chromatographic run time of approximately 11 min was needed to separate R, S-(A) and R, R-(A). In this study, we explored a simple and robust UHPLC–MS/MS method in order to increase sample throughput and productivity. We were able to achieve a two-fold reduction in the lower limit of quantification and a three-fold reduction in retention time utilizing the UHPLC method, while keeping the same sample extraction procedure and similar MS/MS methodology. The new method exhibited good intra- and inter-day accuracy and precision, and was linear over a dynamic range of 1–500 ng/mL for each diastereomer. The method was successfully applied for the determination of R, S-(A) and R, R-(A) concentrations for in vitro and in vivo studies of epimerization of A in Sprague-Dawley rats.