Corona CAD Research Articles

Simultaneous determination of six main components in Bushen Huoxue prescription by HPLC-CAD

BackgroundThe Bushen Huoxue prescription is a traditional Chinese medicine formula treating diabetic retinopathy, which was developed by our research group. Catalpol, puerarin, salvianolic acid B, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd are six of main effective components, which could be partly representative of this prescription. The corona charged aerosol detector (corona CAD) is one kind of universal detectors equipped with the high performance liquid chromatography (HPLC). The CAD has many advantages for the analysis of complex mixtures, but too few applications in traditional Chinese medicine compounds. ObjectiveThe aims of this study are to establish a method for the determination of six components in Bushen Huoxue prescription, and to increase the use of the CAD in traditional Chinese medicine compounds. MethodsHPLC-CAD analysis was performed on an Inertsil ODS-SP (4.6 mm × 250 mm, 5 μm) with a mobile phase consisting of 0.5 % formic acid solution(A)-acetonitrile(B) at a flow rate of 1 mL/min (0−7 min, 1 % B; 7−12 min, 1 %–12 % B; 12−22 min, 12 %–19 % B; 22-40 min, 19 %–28 % B; 40−43 min, 28 %–33 % B; 43−50 min, 33 % B; 50−65 min, 33 %–42 % B). The column temperature maintained at 30 ℃, the injection volume was 20 μL, the atomization temperature mode was LOW, the filtration constant (filter) was 3.6 and data collection rate was 10 Hz. The methodology was examined and the linearity of regression of different functions was compared. Sixteen batches of samples were prepared and their contents were determined. ResultsThe six compounds showed a better linearity (R2 > 0.9990) in their concentration ranges when using the linear function. The average recoveries were 99.18 %–101.30 %. Although the RSD value of puerarin and ginsenoside Rg1 was slightly out of 3 % during the average recovery investigation, all the other methodological investigations of the six components were within 3 %. The precision, stability and repeatability of the method were good. In sixteen batches of Bushen Huoxue prescription samples, the contents of six components were 0.3138 %–0.6042 % for catalpol, 0.8095 %–1.2917 % for puerarin, 0.7416 %–1.1189 % for salvianolic acid B, 0.0231 %–0.0418 % for ginsenoside Rg1, 0.0702 %–0.1724 % for ginsenoside Rb1, 0.0384 %–0.1196 % for ginsenoside Rd. ConclusionIn this experiment, a method for the determination of six components in Bushen Huoxue prescription based on HPLC-CAD was established with high accuracy, good repeatability and simple operation, and it can provide references for the improvement of quality standard of the Bushen Huoxue prescription. It is reasonable and accessible for the CAD application in the determination of traditional Chinese medicine compound prescriptions.

Quantitative analysis of underivatized amino acids in the sub- to several-nanomolar range by ion-pair HPLC using a corona-charged aerosol detector (HPLC–CAD)

The separation and quantification of underivatized naturally occurring amino acids (AAs) by high-performance liquid chromatography (HPLC) is advantageous for reducing preparation time, running costs, and analytical errors, and is compatible with the isolation of intact AAs. This study establishes and validates an analytical method for the separation and quantification of underivatized AAs and taurine (Tau) in the sub- to several-nanomolar range (ca. 0.1–1.6 nmol) by optimizing ion-pair HPLC coupled to a corona charged aerosol detector (corona CAD). Chromatographic separation of 19 AAs and Tau was achieved using a porous graphite carbon (PGC) column and nonafluoropentanoic acid (NFPA) as a volatile ion-pair reagent. The response of the corona CAD to the AAs was highly dependent on the eluate composition, whereas these response factors were similar for AAs in eluate with similar compositions. Regression curves and coefficients (r2) >0.998 were obtained for plots of injection amount versus peak area, except for Arg which co-eluted with a background peak. On the other hand, all plots of injection amount versus peak height regressed to curves with r2 > 0.997. Repeat quantification based on peak area showed lower relative standard deviations (RSDs) (typically better than 5%) than those based on peak height. The present method is useful for quantifying AAs from proteins and Tau in the sub- to several-nanomolar range without derivatization, and constant repeatability can be maintained by quantification using peak areas.

Effects of densities of brominated flame retardants on the detection response for HPLC analysis with a corona-charged aerosol detector.

Dependence of the response of a corona-charged aerosol detector (corona CAD) on the concentration and densities of brominated flame retardants and some related substrates was studied. The calibration curves of the substrates did not show linearity and the substrate with a lower density exhibited the stronger response. Regardless of the solvents (chloroform or toluene), and the injected volume of the substrate solution, the signal intensity of the substrate observed by a corona CAD was substantially proportional to 2/3 power law of concentration and proportional to (-2/3) power law of the density of the substrates. These results suggest that the responses should be proportional to the surface area of the particles generated through the drying process in corona CAD. Contrary to the former reports that the detector response of a corona CAD was independent of chemical species, it was proved that the response varies with the density of a substrate.

The relationship between plasma concentration of metoprolol and CYP2D6 genotype in patients with ischemic heart disease

BackgroundMetoprolol is the one of the most commonly used β-blockers in the treatment of ischemic heart disease and it is extensively metabolized in the liver undergoing oxidation by CYP2D6 isoenzyme of cytochrome P450. Gene encoding the CYP2D6 enzyme is characterized by genetic polymorphism. The CYP2D6 oxidation polymorphism has a major impact on the effectiveness and safety of the treatment. The aim of the study was to evaluate the relationship between plasma concentration of metoprolol and the CYP2D6 genotype in patients with ischemic heart disease. MethodsFifty patients were interviewed and subsequently enrolled into the study. The patients received metoprolol twice daily at a dose of 50mg. The blood samples were analyzed for two major defective alleles for CYP2D6 – CYP2D6*4 and CYP2D6*3 – by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Metoprolol concentration in plasma was determined by using the new and unique high-performance liquid chromatography (HPLC) method in the author's own modification with Corona CAD detector (Charged Aerosol Detection). ResultsIn the test group, genotypes conditioning poor oxidation (PM) occurred in 3 patients (6%), while 47 patients (94%) had genotypes coding for extensive metabolism (EM). Patients with PM genotypes had significantly higher plasma concentrations of metoprolol than the patients with EM genotype (mean 92.25±SD 36.78ng/ml vs. mean 168.22±SD 5.61ng/ml, respectively). Established relationships were statistically significant (NIR test, p=0.0009). ConclusionsThis study demonstrated that the CYP2D6 genotype remains a major determinant of the metoprolol plasma concentrations. The pharmacogenetic effect is likely to have consequences on both, the clinical benefit of metoprolol treatment and adverse drug reactions. The use of Corona CAD detector seems to be a very good alternative method for the determination of metoprolol concentration in plasma.

Simultaneous determination of aspartame, acesulfame-K, saccharin, citric acid and sodium benzoate in various food products using HPLC–CAD–UV/DAD

A newly developed method for simultaneous determination of aspartame, acesulfame-K, saccharin, citric acid and sodium benzoate in various diet supplements and non-alcoholic beverages in a single run is presented. The analytes were analysed by high-performance liquid chromatography coupled to a charged aerosol (Corona CAD) and ultraviolet–diode array detectors simultaneously connected in series. Mass spectrometer MicrOTOF-QII from Bruker Daltonik (Bremen, Germany) was used to obtain the mass spectra for peak identifications. The method was validated using a Thermo Hypersil Gold-C18 column packed with 5 μm shell particles (150 × 4.6 mm) and methanol–water with 0.05 % TFA gradient mobile phase at a flow rate of 0.80 mL/min. The elaborated method was validated for linearity, precision and accuracy. The analytical results obtained in the validation study were highly satisfying; the recoveries for the analytes studied ranged between 98.1 and 101 % and the precision values from 0.11 to 1.73 %. By these procedures, the three sweeteners (aspartame, acesulfame-K and saccharin), citric acid and sodium benzoate could be well separated and quantitatively determined in varied food products. Hundred millilitres of soft drinks contained on average 5.50 mg of aspartame, 6.38 mg of saccharin, 8.94 mg of acesulfame-K, 9.05 mg of sodium benzoate and 111 mg of citric acid. Citric acid was the most abundant additive in all the samples analysed except for table sweeteners, and its highest concentration was determined in diet supplements, i.e. 347 mg/g. The percentage of adequate daily intake realisation in case of all additives is lower than 10 %, except for sodium benzoate in isotonic drinks (10.1 %).

Investigation of polar organic solvents compatible with Corona Charged Aerosol Detection and their use for the determination of sugars by hydrophilic interaction liquid chromatography

A range of organic solvents (ethanol, isopropanol and acetone) has been investigated as alternatives to acetonitrile and methanol when used in conjunction with Corona Charged Aerosol Detection (Corona CAD). These solvents have been evaluated with regard to their effect on the response of the Corona CAD. Three dimensional response surfaces were constructed using raw data showing the relationship between detector response, analyte concentration and percentage of organic solvent in the mobile phase, using sucralose or quinine as the test analyte. The detector response was non-linear in terms of analyte concentration for all solvents tested. However, detector response varied in an approximately linear manner with percentage of organic solvent over the range 0–40% for ethanol or isopropanol and 0–80% for acetone and methanol. The chromatographic performance of the various solvents when used as aqueous–organic mobile phases was evaluated for isocratic and gradient separations of sugars and sugar alcohols by hydrophilic interaction liquid chromatography (HILIC) using an Asahipak NH2P-504E column coupled with Corona CAD detection. It was found that whilst acetonitrile provided the highest column efficiencies and lowest detection limits of the solvents studied, acetone also performed well and could be used to resolve the same number of analytes as was possible with acetonitrile. Typical efficiencies and detection limits of 5330platesm−1 and 1.25μgmL−1, respectively, were achieved when acetone was used as the organic modifier. Acetone was utilised successfully as an organic modifier in the HILIC separation of carbohydrates in a beer sample and also for a partially digested dextran sample.

Simultaneous Determination of Caffeine and Aspartame in Diet Supplements and Non-Alcoholic Beverages Using Liquid-Chromatography Coupled to Corona CAD and UV-DAD Detectors

A method for the simultaneous determination of caffeine and aspartame in various diet supplements and non-alcoholic beverages is presented. The analytes were analysed by high-performance liquid chromatography coupled to a charged aerosol detector (Corona CAD) and ultraviolet-diode array detector (UV-DAD) simultaneously connected in series. The method was validated using a Thermo Hypersil Gold-C18 column packed with 5 μm shell particles (150 × 4.6 mm) and acetonitrile–water (15/85% v/v) mobile phase at a flow rate of 1.00 mL/min. The elaborated method was validated for linearity, precision and accuracy. The rapid HPLC–CAD–UV-DAD technique is suitable for quantifying caffeine and aspartame in the range of 0.25–75 μg/mL in diet supplements and non-alcoholic beverages. The limit of detection for caffeine and aspartame was 62 and 43 ng/mL for Corona CAD and 31 and 30 ng/mL for UV-DAD detector, respectively. Each analyte calibration curve had a correlation coefficient of at least 0.999 and was linear in the defined range. The accuracies of CAD and UV-DAD detection were all acceptable, with the mean value of 100% for aspartame and 98.3% for caffeine. Precision values ranged from 0.09% to 1.12%. The work has demonstrated that charged aerosol detector can be successfully used in a rapid screening technique for biologically active substances in non-alcoholic beverages and diet supplements.

Sensitive Analysis of Commonly Used Artificial and Natural Sweeteners Including Stevia and Their Impurities and Degradation Products

Many recently commercialized sweeteners tend to have increased potency, reducing the amount of active ingredient added to beverages and other food products and often providing cost savings to the manufacturer. Using increased potency has also contributed to a need for sensitive analytical methods to quantify the active product and to detect low levels of breakdown products and impurities, which are required for quality and safety issues. Because compounds typically do not possess any chromophore, traditional HPLC-UV approaches are inappropriate. The work in this study describes several methods using HPLC with the Thermo Scientific Dionex CoronaTM Charged Aerosol Detector (CADTM) for the study of common natural sugars (fructose, glucose, turanose, saccharose, trehalose, maltose, melezitose, and raffinose); artificial sweeteners (sucralose, aspartame, saccharin, and acesulfame K); and newly introduced products containing stevia extracts (rebaudioside A and stevioside). These HPLC-CAD methods provide sensitivity at low levels (ng) with good reproducibility and accuracy, and correlation to the component concentrations. Stevia products were analyzed by both charged aerosol detection and UV. Charged aerosol detection showed a greater than fivefold improvement in sensitivity over UV for all major components. Finally, the UHPLC methods developed showed a decreased run time and an increased sensitivity for glucose, lactose, and sucrose. Typical limits of detection (LOD) were found to be <500 pg (on column) for glucose and other monoand disaccharides. HPLC-CAD is a very flexible approach to measuring sweeteners and overcomes many of the limitations of UV, refractive index (RI), LCMS, evaporative light scattering detection (ELSD), and HPLC-pulsed amperometric approaches. The HPLC-CAD platform can be used throughout the manufacturing process to ensure finished quality and batch-to-batch uniformity. Introduction The Dionex Corona CAD is a mass-sensitive universal detector for nonvolatile and some semivolatile analytes. Unlike absorbance, fluorescence, RI, or electrochemical detection, analyte response is independent of chemical Sensitive Analysis of Commonly Used Artificial and Natural Sweeteners Including Stevia and Their Impurities and Degradation Products Ian Acworth, Chris Crafts, and Bruce Bailey, Thermo Fisher Scientific, Chelmsford, MA, USA structure. Carbohydrates lack chromophores and the typical approaches used for their analysis may lack sensitivity, require derivatization, or cannot be used with gradient. The work in this study examines different HPLC-CAD methods for the analysis of honey sugars, artificial sweeteners, and other natural products including stevia extracts. Finally, a new UHPLC method for fast analysis of simple carbohydrates is presented. The Corona CAD system is easy to use and offers a simple, sensitive, reproducible, and direct approach for the routine analysis of natural and artificial sweeteners. Artificial Sweeteners Global Method HPLC Parameters Column: ACE®, C18 4.6 × 250 mm, 5 μm Mobile Phase: A) Deionized water B) Acetonitrile + 0.1% trifluoroacetic acid Gradient: 2 to 40% B over 25 min, 40 to 60% 25–30 min Flow Rate: 1.0 mL/min Inj. Volume: 50 μL Column Temp.: 30 °C Samples: 1.2 to 20 μg on column FIGURE 1. Chromatogram of artificial sweeteners. 26831 5 10 15 20 25 30 35 400 A ce su lfa m e K C yc la m at e S ac ch ar in S uc ra lo se A sp ar ta m e

The Corona Charged Aerosol Detector – A „Universal“ Detector for the Measurement of Non-volatile Components in Food, Natural Products and Supplements

The Corona® Charged Aerosol Detector® is a „universal“ mass-sensitive detector capable of measuring any non-volatile and many semi-volatile analytes. Its response is independent of chemical structure and does not require the analyte to possess a chromophore (a prerequisite for UV and fluorescence detection) or to be ionized (essential for MS detection). The Corona CAD is compatible with gradient elution, has a wide dynamic range (4+ orders of magnitude), high sensitivity (sub-nanogram), good reproducibility, and a predictable response independent of chemical structure. As discussed in this presentation, charged aerosol detection overcomes many of the limitations seen with other „universal“ detectors including evaporative light scattering, condensation nucleation light scattering, refractive index, and UV. Charged aerosol detection has great applicability to the measurement of foods, supplements, botanicals and natural products. Some of the topics I will discuss include the measurement of carbohydrates, profiling lipids, measurement of fat-soluble vitamins, analysis of antibiotics, examination of potential bioactives in foods and supplements, and the evaluation of the composition of supplements and botanicals.

Study of Xanthophyll Concentration in Spinach Leaves by Means of HPLC Coupled with UV–VIS and Corona CAD Detectors

In this study, the determination of lutein in fresh and frozen spinach leaves has been done. Changes of pigment concentration in spinach leaves were observed in various heating time periods (1–20 min). An improved and simplified analytical method which offers rapid, accurate determination and identification of lutein in spinach samples is reported. During experiments, two detection systems, ultraviolet–visible (UV–vis) spectroscopy and Corona CAD coupled with high-performance liquid chromatography (HPLC), were applied. The determination of lutein in plant extracts was carried out by HPLC–UV–vis. Concentrations of lutein ranged between 0.43 and 0.88 mg/g xfor three frozen spinach samples and 0.83 mg/g for fresh spinach, respectively. Changes of lutein concentration after cooking of frozen spinach were also determined. Primary concentration of lutein in examined frozen spinach sample was 0.43 mg/g. After a 1-min heating time, the concentration of lutein was 0.46 mg/g. After 20 min, it was only 0.11 mg/g. The decreasing of lutein concentration in spinach during the cooking from 2 to 20 min was observed. Over a 20-min heating time of spinach leaves, a loss of primary concentration of lutein was observed. Short cooking time (1–2 min) of spinach has an effect in the increasing of contents of lutein and other biologically active compounds. Long cooking time of spinach induces lutein degradation process.

Development of a Reversed-Phase HPLC Impurity Method for a UV Variable Isomeric Mixture of a CRF Drug Substance Intermediate with the Assistance of Corona CAD

The compound studied, an antagonist of corticotropin-releasing factor (CRF) receptor 1, was under development for the treatment of major depressive disorder and generalized anxiety disorder. To control the quality of intermediate compounds in the synthetic pathway, the preference is to develop a single HPLC method that could determine impurities of the sequential and structurally similar intermediates. However, the chemical nature of one of those intermediates, Compound C, posed a number of challenges to the development of a suitably rugged impurity method for this isolated intermediate. In the solid state, Compound C is a stable mixture of its trans-isomer and semicarbazone forms. In most solution states, Compound C interchanges between the trans and semicarbazone forms as well as degrades rapidly to impurity X, resulting in sample solution instability. Furthermore, the trans-isomers and semicarbazone exhibited different UV response factors, but reference standards of the two forms were not available to determine the relative response factor. Consequently, irreproducibility was observed during the HPLC analysis when conventional UV detection was applied directly. This article describes the following approaches that were employed to meet these challenges: (1) diluent screening was performed to select a \solvent that afforded adequate sample solution stability; (2) appropriate chromatographic conditions were selected to eliminate or minimize any on-column degradation; (3) alternate HPLC detection, Corona charged aerosol detection, in conjunction with UV analysis, was used to determine relative amounts of the isomeric forms which subsequently permitted the use of conventional UV detection for routine analysis. The validation data of this reversed-phase liquid chromatographic method are also discussed.

Comparison of the response of four aerosol detectors used with ultra high pressure liquid chromatography

The responses of four different types of aerosol detectors have been evaluated and compared to establish their potential use as a universal detector in conjunction with ultra high pressure liquid chromatography (UHPLC). Two charged-aerosol detectors, namely Corona CAD and Corona Ultra, and also two different types of light-scattering detectors (an evaporative light scattering detector, and a nano-quantity analyte detector [NQAD]) were evaluated. The responses of these detectors were systematically investigated under changing experimental and instrumental parameters, such as the mobile phase flow-rate, analyte concentration, mobile phase composition, nebulizer temperature, evaporator temperature, evaporator gas flow-rate and instrumental signal filtering after detection. It was found that these parameters exerted non-linear effects on the responses of the aerosol detectors and must therefore be considered when designing analytical separation conditions, particularly when gradient elution is performed. Identical reversed-phase gradient separations were compared on all four aerosol detectors and further compared with UV detection at 200 nm. The aerosol detectors were able to detect all 11 analytes in a test set comprising species having a variety of physicochemical properties, whilst UV detection was applicable only to those analytes containing chromophores. The reproducibility of the detector response for 11 analytes over 10 consecutive separations was found to be approximately 5% for the charged-aerosol detectors and approximately 11% for the light-scattering detectors. The tested analytes included semi-volatile species which exhibited a more variable response on the aerosol detectors. Peak efficiencies were generally better on the aerosol detectors in comparison to UV detection and particularly so for the light-scattering detectors which exhibited efficiencies of around 110,000 plates per metre. Limits of detection were calculated using different mobile phase compositions and the NQAD detector was found to be the most sensitive (LOD of 10 ng/mL), followed by the Corona CAD (76 ng/mL), then UV detection at 200 nm (178 ng/mL) using an injection volume of 25 μL.

Protein, Peptide, and Amino Acid Analysis with Corona CAD: A New Universal HPLC Detector

Protein, Peptide, and Amino Acid Analysis with Corona CAD: A New Universal HPLC Detector

Protein, Peptide, and Amino Acid Analysis with Corona CAD: A New Universal HPLC Detector

Protein, Peptide, and Amino Acid Analysis with Corona CAD: A New Universal HPLC Detector