The new ultra-high performance liquid chromatography method with tandem mass spectrometry and fluorescence detection allowing fast, selective, and high-throughput analysis of neopterin, kynurenine, tryptophan, and creatinine in gingival crevicular fluid (GCF) has been optimized. Defining the pathophysiology of periodontal disease and identification of potential diagnostic test for active periodontitis remains a significant challenge in the field of oral disease diagnosis. Analysis of GCF provides a non-invasive means of evaluating the role of the host response in periodontal disease. In addition, the analysis of GCF provides an information about current inflammation level of sampled site/tooth. Determination of GCF inflammatory biomarkers such as neopterin, kynurenine, and tryptophan can contribute to diagnosis, evaluation of treatment, and progression of periodontal diseases such as gingivitis and periodontitis. The separation of target analytes was carried out using a column KinetexTM Polar C18 100 Å, (100 × 3.0 mm) packed with 2.6 μm core-shell particles applying an elution with a gradient formed from 0.2% aqueous formic acid and 90% aqueous acetonitrile. Kynurenine, tryptophan, and creatinine were detected using mass spectrometry with electrospray ionization to improve the sensitivity while neopterin was detected using fluorescence detection. The separation of these four substances was achieved after using a very simple sample preparation technique convenient for small amount of biological sample. Only less than 20 μL sample was needed and the separation was completed in 4 min. MS/MS analysis was performed using multiple reaction monitoring (MRM) under a positive ionization mode. Deuterium labeled internal standard was used for the more precise quantification. The lower limits of quantification (LLOQ) for target analytes were 0.50 × 10-3 μmol/L for neopterin, 0.10 μmol/L for kynurenine, and 0.20 μmol/L for tryptophan and creatinine. The within-run and between-run accuracy were in a range of 96.67-114.77% for all quality controls and LLOQ of all analytes. Matrix effect, extraction recovery, and stability testing have all been investigated. The method was tested with real-life samples using GCF collected from patients suffering from periodontitis and from healthy controls. Neopterin levels in patients were significantly higher (P = 0.020) than in healthy subjects and indicate good potential of this method for using in evaluation of periodontal pathogenesis and healing outcomes following a treatment.
Nintedanib (NIN) is a tyrosine kinase inhibitor recently approved for the treatment of idiopathic pulmonary fibrosis. As a new drug, no monograph is available so far in official compendia. A liquid chromatography-tandem mass spectrometry method is presented for the simultaneous determination of NIN and its seven potential impurities. The risk-based approach of Analytical Quality by Design was applied in method development. The critical method parameters (CMPs) were the type of organic solvent in the mobile phase, formic acid percentage, column flow rate, oven temperature, gradient slope of organic eluent. The critical method attributes (CMAs) were selected as analysis time and selectivity between the main compound NIN and the adjacent peaks. Design of Experiments methodology was effectively employed for establishing the relationship between the CMPs and the CMAs. In the scouting step, a Restek Ultra AQ C18 (100 × 2.1 mm, 2.7 µm) core-shell column was selected, and then the effects of different levels of the five CMPs on the CMAs were evaluated by means of a 35//16 symmetric screening matrix. A Box-Behnken Design made it possible to obtain detailed maps of predicted CMAs throughout the investigated experimental domain, pointing out the presence of interaction and quadratic effects. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations, performing a risk analysis and drawing risk of failure maps, which were used to visualize and define the method operable design region (MODR) with a probability π ≥ 90%. The final working conditions (enclosing the MODR interval) were as follows: methanol as organic solvent; formic acid percentage, 0.15% v/v; flow rate, 0.40 mL min-1 (0.37-0.43 mL min-1); oven temperature, 40 °C (38-40 °C); gradient slope of organic eluent, 14.00% eluent B min-1 (12.85-15.15% eluent B min-1). The resulting analysis time was about 10 min. Validation was carried out according to International Council for Harmonisation guidelines and the optimized method was applied to the analysis of NIN soft capsules for quality control purposes.
Zwitterionic chiral ion-exchange selectors (ZWIX) obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond were immobilized on three different silica particle types, viz. 120 Å 3 μm fully porous particles (FPP), 200 Å 3 μm FPP and 160 Å 2.7 μm superficially porous particles (SPP). Selector densities were determined by elemental analysis and the porosities of packed columns measured by inverse size exclusion chromatography with polystyrene standards. Liquid chromatographic tests with a set of chiral zwitterionic, acidic and basic analytes showed that the surface chemistry was successfully transferred to the distinct particle morphologies. The chromatographic performance of the three columns was evaluated by acquiring van Deemter curves. The results showed that the column packed with the SPP particles gives the best performance and kinetic plots further demonstrated that they represent the most favorable compromise in terms of speed, efficiency and pressure drop. Sub-minute separations could be accomplished at much lower pressure drop on the core-shell column, e.g. 2-amino-2-phenylbutyric acid was baseline separated in less than 15 s on a 5 cm long column. The Maxwell effective medium theory with second order approximation was applied to calculate effective diffusion in the mesoporous zones of SPP and FPP, which allowed eventually to deconvolute the individual peak dispersion contributions (ha, hb, hc,m, hc,s, hc,ads). The efficiency gain of the 160 Å SPP column compared to the 120 Å FPP (benchmark) column was mainly due to lower eddies (ha), smaller c-term accounting for slow adsorption-desorption kinetics in enantioselective chromatography (hc,ads), and also due to lower stationary mass transfer resistance (hc,s). Enhanced effective diffusion (Deff) in the SPP column contributed to a lower longitudinal diffusion (hb). In contrast, the mobile phase mass transfer coefficient was similar in the two columns leading to comparable hc,m contributions. This study discloses some options for improvement of the efficiency of ZWIX-type chiral columns such as replacing narrow pore (120 Å) by wide pore (200 Å) particles, substituting FPP by SPP and reducing the selector density on the surface.
Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 Å) being advantageous (over 90 or 120 Å). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 Å SPPs outperformed their fully porous sub-2 μm 120 Å counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 Å, 200 Å; SPPs: 90 Å, 160 Å), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.
The separation of seven phenolic compounds including gallic acid, chlorogenic acid, epicatechin, quercitrin, rutin, phloridzin, and phloretin present in apple peel and pulp and differing in elution properties has been optimized using high-performance liquid chromatography with diode array detection. Several stationary phases were tested to achieve the efficient separation of phenolic compounds in fruit extracts and C18 was found to be the most efficient. Core-shell and fully porous C18 packings were assessed with respect to the complex composition of the fruit extracts. The developed high-performance liquid chromatography method comprised gradient elution in which mobile phase A was water at pH 2.8 adjusted with acetic acid and B was acetonitrile. The gradient shape was the following: 0 min 95% A/5% B, 2.5 min 85% A/15% B, 12 min 50% A/50% B, 15 min 95% A/5% B. The flow rate was 1 mL/min, injection volume 10 μL, and UV detection at 255, 280, 320, and 365 nm was applied. Our method was validated for both C18 core-shell and fully porous packings. The resolution 6.2-14.8, symmetry 0.99-1.34, peak capacity 18-60, peak area repeatability 0.45-1.00% relative standard deviation, calibration range 0.125-5 mg/mL (0.25-10 mg/mL for chlorogenic acid and rutin), correlation coefficients of calibration curve 0.9976-0.9997, and accuracy evaluated as recovery 95.56-107.54% were determined for the core-shell column.
- MeSH
- Phenols analysis MeSH
- Malus chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
Correct adjustment of the mobile phase is equally important as the selection of the appropriate column for the separation of polar compounds in LC. Both solvophobic and selective polar interactions control the retention in the Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects of the volume fraction of the strong eluent component in binary mobile phases on the sample retention factors apply in a limited mobile phase composition range. We introduced a three-parameter retention model, which provides improved prediction of retention over a broad mobile phase range, under isocratic and gradient elution conditions. The model does not imply any assumptions concerning either adsorption or partition distribution mechanism, but allows estimating retention in pure strong and in pure weak mobile phase components. The experimental retention data for phenolic acids and flavones on several core-shell columns with different types of stationary phases agree with the theory. Many polar columns with important structural hydrophobic moieties show dual retention mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the two modes for separations of samples containing compounds largely differing in polarity. The three-parameter model describes the retention in each mode, with separately determined best-fit parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column.
In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core-shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum, is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour activity. Silymarin is currently being widely used in commercial preparations and herbal teas. Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real samples was performed on the Kinetex 1.7μ F5 100A (150×2.1mm), 1.7μm particle size core-shell column, with a mobile phase methanol/100mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35mLmin(-1) flow rate and 50°C temperature was used for the separation. The detection wavelength was set at 288nm. Under optimal chromatographic conditions, good linearity with a correlation coefficient of R(2) >0.999 for all compounds was achieved. The available commercial samples of herbal teas and food supplements were extracted with methanol using an ultrasonic bath. After dilution with water and centrifugation, a 2μL sample of the filtered supernatant was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with methanol as the organic component of the mobile phase showed new ways to effectively separate isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary phase.
- MeSH
- Teas, Herbal analysis MeSH
- Chemistry, Pharmaceutical methods standards MeSH
- Isomerism MeSH
- Silybum marianum * MeSH
- Dietary Supplements analysis MeSH
- Plant Extracts analysis chemistry MeSH
- Silymarin analogs & derivatives analysis chemistry MeSH
- Chromatography, High Pressure Liquid methods standards MeSH
- Publication type
- Journal Article MeSH
A high-throughput miniaturized liquid-liquid extraction procedure followed by a simple ultra-high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core-shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λex = 325, 295 nm and λem = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 μM, α-tocopherol 0.38 μM, β-tocopherol 0.18 μM, γ-tocopherol 0.14 μM, and δ-tocopherol 0.01 μM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.
In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-shell column (100×4.6mm, 5μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile with gradient elution at a flow-rate of 1mLmin-1. The total time of the whole analytical run, including the extraction step, was 13.25min. The on-line MISPE-HPLC method was optimized and validated. The results showed good linearity (0.10-100μgmL-1) with correlation coefficients higher than 0.99. The LOD values were from 0.03 to 0.15μgmL-1. The proposed method was successfully applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.
The presented work describes the development and validation of a rapid UHPLC-UV method using a core-shell particle column with a pentafluorophenyl stationary phase for the separation and quantitative analysis of the six anthocyanins in acai berry and dry blueberry extracts. The anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutenoside, delphinidin-3-galactoside, delphinidin-3-glucoside, delphinidin-3-rutenoside, and peonidin-3-glucoside) were separated and analyzed in 5min. The chromatographic separation was performed on a Kinetex PFP (150×2.1mm) core-shell column with a particle size of 1.7μm at a temperature of 50°C. Acetonitrile was used as mobile phase B and 5% formic acid, filtrated through a 0.22μm filter, as mobile phase A. They were delivered at a flow rate of 0.55mLmin-1according to the elution gradient program. The detection wavelength was set at 520nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25+75v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of food supplements with a content of acai berry extract and blueberry extract. Under optimal chromatographic conditions, the method was validated. Recoveries for all analyzed anthocyanins were 97.8-102.6% and the relative standard deviation ranged from 0.4% to 3.0% for within-day and from 0.6% to 3.1% for between-day repeatability. The limits of detection were in the range of 0.11-0.14μgmL-1.
- MeSH
- Anthocyanins MeSH
- Euterpe * MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
