Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam

Citicoline and piracetam were subjected separately to different stress conditions as recommended by the international conference on harmonization. In addition, new stability indicating thin layer chromatographic and ultra high performance liquid chromatographic methods have been developed and validated for simultaneous determination of citicoline and piracetam in presence of their degradation products. Separation on the proposed thin layer chromatographic method was carried out using a developing system containing methanol:chloroform:ammonium chloride buffer (9:1:2, v/v/v) on silica gel plates at 230 nm. On the other hand, the mobile phase in the ultra high performance liquid chromatographic method was composed of water (containing 0.1% triethylamine):ethanol (92:8, v/v). The flow rate was 1 mL/min and ultraviolet detection was at 230 nm. Moreover, results of the developed methods were statistically compared to those obtained by the reported high‐performance liquid chromatography method and no significant difference between them was found. The greenness profile of ultra high performance liquid chromatographic method was assessed and compared with those of the previously published high‐performance liquid chromatography methods, it was noticed that the proposed ultra high performance liquid chromatographic method more environmentally friendly and greener than other methods.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

Novel strategy for quality consistency evaluation of Chinese medicine “YIQING” tablet that combines the simultaneous quantification and screening of ten bioactive constituents

The UV characteristics for different categories compounds in complex traditional Chinese medicines and herbal preparations usually vary. Thus, to achieve the integral analysis of multiwavelength fingerprint characteristics, we introduced a novel strategy of multiwavelength total fusion profiling. The simultaneous separation and quantification of multiple components by reversed‐phase high‐performance liquid chromatography coupled with diode array detection was developed in an effective, accurate, and reliable way. Furthermore, a 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH) scavenging assay was set up to detect and screen the bioactivity of similar‐structure constituents (aloe‐emodin, rhein, emodin, chrysophanol, baicalein, wogonin, baicalin, wogonoside, berberine hydrochloride, and jatrorrhizine hydrochloride). Moreover, the high‐performance liquid chromatography DPPH assay was developed to monitor the relationship between the biological activity and the spatial structure, the number of hydroxyl groups, the concentration of the analytes in samples. The result of qualitative classification for 15 batches of “YIQING” tablets using principle component analysis was consistent with the quantitative fingerprint assessment using the average linear quantitative fingerprint method. Therefore, chemometrics, multiwavelength total fusion profiling in conjunction with average linear quantitative fingerprint method and antioxidant activity can control the quality of traditional Chinese medicines/herbal preparations comprehensively and practically.     

Rapid simultaneous quantification of fructooligosaccharides in Morinda officianalis by ultra‐high performance liquid chromatography

Many Chinese herbal medicines with tonifying effects contain high levels of inulin fructooligosaccharides. These herbal medicines have high development and utilization value because of their effects against dementia, depression, and oxidative stress; on improving learning and memory ability; and on enhancing immunity. In this study, a method was developed for the separation and simultaneous quantitation of fructose, glucose, sucrose, and ten inulin fructooligosaccharides by ultra‐high‐performance liquid chromatography with evaporative light scattering detection within 10 min. Separation was performed on an Amide column with gradient elution. The calibration curves for the 13 constituents showed good linearity (R² > 0.9991). The limits of detection and quantification were 10.78–33.44 and 35.94–124.81 μg/mL, respectively, and the recoveries ranged from 98.90 to 103.67%. This method was successfully used to quantify the 13 constituents in the Chinese herbal medicine Morinda officinalis. The contents of the ten inulin fructooligosaccharides ranged from 56.28 to 60.71%. This method is accurate, rapid and simple and can be used for quantitative analysis in the quality control of herbal medicines and functional foods.     

Quality assessment of Cinnamomi Ramulus by the simultaneous analysis of multiple active components using high‐performance thin‐layer chromatography and high‐performance liquid chromatography

A novel and improved method for the quality assessment of Cinnamomi Ramulus was developed and completely validated. The method was established using fingerprint technology and simultaneous quantitative determination of six main marker compounds including coumarin, cinnamic alcohol, cinnamic acid, 2‐methoxy cinnamic acid, cinnamaldehyde, and 2‐methoxy cinnamaldehyde in the herbal medicine for the first time. A newly developed high‐performance thin‐layer chromatography method, which achieved simultaneous definition of five marker components by comparing the colors and retardation factor values of the bands in high‐performance thin‐layer chromatography, was first used for the authentication of Cinnamomi Ramulus. The fingerprints of 26 batches of herbal samples from different regions of China showed very similar chromatographic patterns that were evaluated by similarity analysis and hierarchical clustering analysis. In addition, six marker compounds were simultaneously determined using single standard to determine multiple components by the relative response factors. Compared with the external standard method, the new quantitative method was validated to determine multiple compounds in 26 batches of Cinnamomi Ramulus samples. All results demonstrated that the simple and rapid method could be effectively utilized for the quality control of Cinnamomi Ramulus.     

Development, optimization and validation of a fingerprint of Ginkgo biloba extracts by high-performance liquid chromatography

A four-step development, optimization and validation strategy for high-performance liquid chromatography (HPLC) fingerprints of Ginkgo biloba extract is described. A suitable chromatographic system was selected first. The following step was performing a screening design to select important parameters. After selecting some controllable parameters and their range to further optimize, gradient optimization with uniform design was done. At last, method validation including determination of injection precision, repeatability, and a sample stability test, was performed. Through this effective and integrated four-step method, a feasible and reliable HPLC fingerprint to identify and assess the Ginkgo biloba quality can easily be established using a linear gradient elution with acetonitrile/0.1% phosphoric acid (from 14/86 to 30/70, v/v, in 40 min) as mobile phase, a column temperature of 30 degrees C and a detection wavelength of 350 nm. The strategy can also be applied for the development of fingerprints in the quality control of other herbal medicines.     

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high‐performance liquid chromatography with diode array detection and tandem mass spectrometry

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry and high‐performance liquid chromatography with electrospray multistage tandem ion‐trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p‐coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high‐performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.     

Simultaneous determination of chlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and protocatechuic aldehyde in Chinese herbal preparation by RP-HPLC

In the present study, a reversed phase high performance liquid chromatographic (RP-HPLC) method was established for simultaneous determination of chlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and protocatechuic aldehyde in a Chinese herbal preparation (Fufang-Pugongying-Mixture). The separation was performed on a Hypersil ODS-2 column by isocratic elution with methanol and 0.2 M acetate buffer (pH 3.6) (15 : 85, v/v) as the mobile phase at the flow-rate of 1.0 ml/min with operating temperature of 30 degrees C, and detection wavelength of 300 nm. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 2-200 microg/ml for the five marker compounds mentioned above. The spike recoveries were within 96.72-104.07%. The variation coefficient (CV) values of the precision were in the range of 0.89-4.50%. Moreover the developed method has reference value for quantitative analysis of Taraxacum, Lonicera and Angelica.     

Chemical interaction between Paeonia lactiflora and Glycyrrhiza uralensis,the components of Jakyakgamcho‐tang,using a validated high‐performance liquid chromatography method: Herbal combination and chemical interaction in a decoction

The herbal combination is the basic unit of a herbal formula that affects the chemical characteristics of individual herbs. In the present study, a method of simultaneous determination of the 11 marker compounds in Jakyakgamcho‐tang was developed using high‐performance liquid chromatography with photodiode array detection. The validated analytical method was successfully applied to approach the chemical interaction between Paeonia lactiflora and Glycyrrhiza uralensis in co‐decoction. In P. lactiflora, the contents of gallic acid, oxypaeoniflorin, (+)‐catechin, paeoniflorin, and benzoylpaeoniflorin were decreased, while those of albiflorin and benzoic acid were increased; in G. uralensis, the contents of liquiritin, isoliquiritin, ononin, and glycyrrhizin were decreased, when decocting two herbs together. Moreover, as the ratio between P. lactiflora and G. uralensis was increased, the contents of chemical contents from each herb were proportionally increased. However, each content of marker compound per the gram of herbal medicine was decreased as the ratio of combinative herbs increased. The results showed that P. lactiflora and G. uralensis affect the extraction efficiency of chemical compounds in a Jakyakgamcho‐tang decoction. Overall, the method established in this study was simple, rapid, and accurate, and would be useful for the determination of marker compounds and for the investigation of the chemical interaction between herbal medicines.     

A simple and sensitive HPLC method for the simultaneous determination of eight bioactive components and fingerprint analysis of Schisandra sphenanthera

A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi-capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250 mm × 4.6 mm i.d., 5 μm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mL min⁻¹, an operating temperature of 35 °C, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera.     

Rapid and sensitive determination of major polyphenolic components in Euphoria longana Lam. seeds using matrix solid‐phase dispersion extraction and UHPLC with hybrid linear ion trap triple quadrupole mass spectrometry

A rapid and sensitive method for the extraction and determination of four major polyphenolic components in Euphoria longana Lam. seeds is presented for the first time based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry. Matrix solid‐phase dispersion method was designed for the extraction of Euphoria longana seed constituents and compared with microwave‐assisted extraction and ultrasonic‐assisted extraction methods. An Ultra high performance liquid chromatography with hybrid triple quadrupole linear ion‐trap mass spectrometry method was developed for quantitative analysis in multiple‐reaction monitoring mode in negative electrospray ionization. The chromatographic separation was accomplished using an ACQUITY UPLC BEH C₁₈ (2.1 mm × 50 mm, 1.7 μm) column with gradient elution of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. The developed method was validated with acceptable linearity (r² > 0.999), precision (RSD ≤ 2.22%) and recovery (RSD ≤ 2.35%). The results indicated that matrix solid‐phase dispersion produced comparable extraction efficiency compared with other methods nevertheless was more convenient and time‐saving with reduced requirements on sample and solvent volumes. The proposed method is rapid and sensitive in providing a promising alternative for extraction and comprehensive determination of active components for quality control of Euphoria longana products.     

Fast and simultaneous determination of 12 polyphenols in apple peel and pulp by using chemometrics‐assisted high‐performance liquid chromatography with diode array detection

In this work, a smart chemometrics‐enhanced strategy, high‐performance liquid chromatography, and diode array detection coupled with second‐order calibration method based on alternating trilinear decomposition algorithm was proposed to simultaneously quantify 12 polyphenols in different kinds of apple peel and pulp samples. The proposed strategy proved to be a powerful tool to solve the problems of coelution, unknown interferences, and chromatographic shifts in the process of high‐performance liquid chromatography analysis, making it possible for the determination of 12 polyphenols in complex apple matrices within 10 min under simple conditions of elution. The average recoveries with standard deviations, and figures of merit including sensitivity, selectivity, limit of detection, and limit of quantitation were calculated to validate the accuracy of the proposed method. Compared to the quantitative analysis results from the classic high‐performance liquid chromatography method, the statistical and graphical analysis showed that our proposed strategy obtained more reliable results. All results indicated that our proposed method used in the quantitative analysis of apple polyphenols was an accurate, fast, universal, simple, and green one, and it was expected to be developed as an attractive alternative method for simultaneous determination of multitargeted analytes in complex matrices.     

Ionic liquids as mobile phase additives for determination of thiocyanate and iodide by liquid chromatography

An analytical method was developed for the simultaneous determination of thiocyanate and iodide by reversed‐phase liquid chromatography with UV detection using imidazolium ionic liquids as mobile phase additives. The chromatographic behaviors of the two anions on a C₁₈ column were studied and compared with four types of reagents including imidazolium ionic liquids, pyridinium ionic liquids, 4‐aminophenol hydrochloride and tetrabutylammonium as mobile phase additives. The effects of the concentrations of imidazolium ionic liquids, organic solvents and detection wavelength on separation and detection of the anions were investigated. The role of ionic liquids, retention rules and mechanisms were discussed. The separation of the anions was performed on the C₁₈ reserved‐phase column using acetonitrile‐0.3 mmol/L 1‐amyl‐3‐methylimidazolium tetrafluoroborate (10:90, v/v) as mobile phase, with column temperature of 35°C, flow rate of 1 mL/min and detection wavelength of 210 nm. Under these conditions, the two anions can be completely separated within 6 min. The limits of detection were 0.05 mg/L. The method was applied for the determination of thiocyanate and iodide in ionic liquid samples and iodide drugs, and the spiked recoveries ranged from 97 to 101%. The method is simple, accurate and meets the requirements of quantitative analysis for thiocyanate and iodide.     

Evaluation of the influence of sulfur fumigation on the pharmacokinetics of four active ingredients in Si Wu Tang

Sulfur fumigation may induce the decrease or the chemical transformation of some active ingredients of traditional Chinese medicines in vitro. Whether sulfur fumigation can cause the pharmacokinetic changes of the active ingredients in vivo is related to the efficacy and the safety of Chinese medicines’ application clinically. A sensitive, specific, and accurate method for the simultaneous determination of paeoniflorin, ferulic acid, senkyunolide A, and senkyunolide I in rat plasma by ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry was developed to evaluate the influence of sulfur fumigation to Si Wu Tang for the first time. Each compound was extracted from plasma samples by liquid–liquid extraction with ethyl acetate, and the chromatographic separation was accomplished on an Agilent Extend C₁₈ column with a linear gradient elution. The mass spectrometric detection and analysis were performed by using an AB Sciex triple quadrupole 5500 mass spectrometer in multiple reaction monitoring mode. The validated method was successfully applied to a pharmacokinetic study of four compounds in rats after oral administration of sun‐dried and sulfur‐fumigated Si Wu Tang. The results provided a meaningful basis for evaluating the affection of sulfur fumigation to the clinical application and the efficacy of Si Wu Tang.     

Postcolumn determination of polyphenolic antioxidants in Cirsium vulgare (Savi) Ten. extracts

Novel methods for the determination of polyphenolic antioxidants present in extracts from inflorescences of Cirsium vulgare (Savi) Ten. based on ultra‐high performance liquid chromatography with photodiode array and chemiluminescence detection have been developed. Under the optimized conditions of chromatographic separation the analytical characteristic of the method was performed. The proposed method was successfully applied to the determination of ten polyphenols present in inflorescences of Cirsium vulgare . A comparison of the contents of analytes in extracts prepared by using various extraction media (methanol, ethanol, 70% methanol, 70% ethanol, and water) was carried out for the first time. For the postcolumn detection of scavenging activity of polyphenolic antioxidants against reactive oxygen species (H₂O₂, ^•OH, O₂^{• −}) three systems based on chemiluminescence of luminol were used. A review of the current scientific literature shows that this is the first report on the application of luminol‐based postcolumn detection for the on‐line investigation of ^•OH scavenging activity. The main compound determined in extracts from inflorescences of Cirsium vulgare was apigenin 7‐O‐glucuronide, whereas the highest antioxidant activity was observed for chlorogenic acid, luteolin 7‐O‐glucoside, and apigenin.     

Quantitative Determination of Nadolol in Tablets by High‐Performance Liquid Chromatography and UV‐Derivative Spectrophotometry

Abstract

High‐performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of nadolol in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 230 and 300 nm, respectively. A mobile phase composed by acetonitrile‐water containing 0.1% triethylamine (15∶85 v/v) and pH adjusted to 4.6 with formic acid was used. The UVDS method was performed taken a signal at 279.5 nm. The correlation coefficient (r) obtained for both methods was 0.9999. The proposed methods are simple, precise, accurate, and can be used in routine analysis.     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

Phenolic acids analysis in ligusticum chuanxiong using HPLC

A reversed-phase high-performance liquid chromatographic method with diode array UV detection is developed for the determination of five kinds of phenolic acids common in herbal medicines. Based on this method, ferulic acid and caffeic acid are found to be two main phenolic acids in Chuanxiong (one of the important crude drugs in traditional Chinese medicine). More important, ferulic acid is found to exist in free form, and caffeic acid--a previously unreported component--is found to exist in esterified or insoluble-bound form.     

Quality assessment of crude and processed ginger by high‐performance liquid chromatography with diode array detection and mass spectrometry combined with chemometrics

A sensitive, simple, and validated high‐performance liquid chromatography with diode array detection and mass spectrometry detection method was developed for three ginger‐based traditional Chinese herbal drugs, Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata. Chemometrics methods, such as principal component analysis, hierarchical cluster analysis, and analysis of variance, were also employed in the data analysis. The results clearly revealed significant differences among Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata, indicating variations in their chemical compositions during the processing, which may elucidate the relationship of the thermal treatment with the change of the constituents and interpret their different clinical uses. Furthermore, the sample consistency of Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata can also be visualized by high‐performance liquid chromatography with diode array detection and mass spectrometry analysis followed by principal component analysis/hierarchical cluster analysis. The comprehensive strategy of liquid chromatography with mass spectrometry analysis coupled with chemometrics should be useful in quality assurance for ginger‐based herbal drugs and other herbal medicines.     

Fast quantitative analysis of four tyrosine kinase inhibitors in different human plasma samples using three‐way calibration‐ assisted liquid chromatography with diode array detection

A simple method has been developed by combining high‐performance liquid chromatography with diode array detection with the alternating trilinear decomposition method for simultaneous determination of four tyrosine kinase inhibitors in different human plasma samples. Chromatographic separation of the analytes was performed on a reversed‐phase column with methanol (65%, v/v, A) and 0.1% aqueous solution of formic acid (35%, v/v, B). Analysis time was 5.0 min per run and analytes could be completely eluted within 2.8??3.8 min. The calibration concentration ranges of vandetanib, pazopanib, afatinib and dasatinib were designed as 0.50–6.10, 0.50–6.10, 0.70–7.00 and 0.70–7.00 μg·mL^?1, respectively. The intra‐ and inter‐day RSDs ranged between 0.1 and 8.9%. Quantitative information could be extracted from the unsegregated interferences of different human plasma samples with the aid of the “second‐order advantage” of three‐way (second‐order) calibration methods. All results demonstrated that the proposed method for direct quantitative analysis of four tyrosine kinase inhibitors in different complex systems possessed good characteristics of rapidity, sensitivity and efficiency, and it is expected to be an attractive choice in the fast analysis of clinical samples.