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.     

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.     

Evaluation and quantitative analysis of 11 compounds in Morinda officinalis using ultra‐high performance liquid chromatography and photodiode array detection coupled with chemometrics

Morinda officinalis (Rubiaceae) is a traditional Chinese medicine widely used for the treatment of impotence and osteoporosis in clinical therapy. In the present study, a rapid and simple ultra‐high performance liquid chromatography with photodiode array detection method was developed and validated for the simultaneous determination of 11 bioactive compounds in M. officinalis . This assay method was validated with respect to linearity (R ²  > 0.9991), precision, repeatability, limit of detection, limit of quantification, and accuracy (with observed recovery rates between 94.21 and 100.38%). The quantitative results revealed significant differences in the concentrations of the selected compounds. Additionally, chemometric methods, including hierarchical clustering analysis, principal component analysis, and partial least‐squares discriminate analysis, were applied to compare and sort the 25 batches of M. officinalis samples based on the quantitative data of the analytes. All of the samples were clearly divided into two groups: the Hainan samples were successfully discriminated from the samples from other origins. Simultaneous determination of multiple compounds using the proposed method combined with chemometrics could be a viable strategy to compare and evaluate the quality of M. officinalis .     

High‐performance liquid chromatography with fluorescence detection for the rapid analysis of pheophytins and pyropheophytins in virgin olive oil

Pheophytins and pyropheophytin are degradation products of chlorophyll pigments, and their ratios can be used as a sensitive indicator of stress during the manufacturing and storage of olive oil. They increase over time depending on the storage condition and if the oil is exposed to heat treatments during the refining process. The traditional analysis method includes solvent‐ and time‐consuming steps of solid‐phase extraction followed by analysis by high‐performance liquid chromatography with ultraviolet detection. We developed an improved dilute/fluorescence method where multi‐step sample preparation was replaced by a simple isopropanol dilution before the high‐performance liquid chromatography injection. A quaternary solvent gradient method was used to include a fourth strong solvent wash on a quaternary gradient pump, which avoided the need to premix any solvents and greatly reduced the oil residues on the column from previous analysis. This new method not only reduces analysis cost and time but shows reliability, repeatability, and improved sensitivity, especially important for low‐level samples.     

Multi‐component determination and chemometric analysis of Paris polyphylla by ultra high performance liquid chromatography with photodiode array detection

Multi‐source analysis of traditional Chinese medicine is key to ensuring its safety and efficacy. Compared with traditional experimental differentiation, chemometric analysis is a simpler strategy to identify traditional Chinese medicines. Multi‐component analysis plays an increasingly vital role in the quality control of traditional Chinese medicines. A novel strategy, based on chemometric analysis and quantitative analysis of multiple components, was proposed to easily and effectively control the quality of traditional Chinese medicines such as Chonglou. Ultra high performance liquid chromatography was more convenient and efficient. Five species of Chonglou were distinguished by chemometric analysis and nine saponins, including Chonglou saponins I, II, V, VI, VII, D, and H, as well as dioscin and gracillin, were determined in 18 min. The method is feasible and credible, and enables to improve quality control of traditional Chinese medicines and natural products.     

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.     

Online solid‐phase extraction with high‐performance liquid chromatography and mass spectrometry for the determination of five tannins in traditional Chinese medicine injections

A rapid analytical method based on online solid‐phase extraction with high‐performance liquid chromatography and mass spectrometry has been established and applied to the determination of tannin compounds that may cause adverse effects in traditional Chinese medicine injections. Different solid‐phase extraction sorbents have been compared and the elution buffer was optimized. The performance of the method was verified by evaluation of recovery (≥40%), repeatability (RSD ≤ 6%), linearity (r² ≥ 0.993), and limit of quantification (≤0.35 μg/mL). Five tannin compounds, gallic acid, cianidanol, gallocatechin gallate, ellagic acid, and penta‐O‐galloylglucose, were identified with concentrations ranging from 3.1–37.4 μg/mL in the analyzed traditional Chinese medicine injections.     

Partial least squares and principal components analysis of wine vintage by high performance liquid chromatography with chemiluminescence detection

HPLC with acidic potassium permanganate chemiluminescence detection was employed to analyse 17 Cabernet Sauvignon wines across a range of vintages (1971-2003). Partial least squares regression analysis and principal components analysis was used in order to investigate the relationship between wine composition and vintage. Tartaric acid, vanillic acid, catechin, sinapic acid, ethyl gallate, myricetin, procyanadin B and resveratrol were found to be important components in terms of differences between the vintages.     

Rapid quantitative analysis of individual anthocyanin content based on high‐performance liquid chromatography with diode array detection with the pH differential method

A new quantitative technique for the simultaneous quantification of the individual anthocyanins based on the pH differential method and high‐performance liquid chromatography with diode array detection is proposed in this paper. The six individual anthocyanins (cyanidin 3‐glucoside, cyanidin 3‐rutinoside, petunidin 3‐glucoside, petunidin 3‐rutinoside, and malvidin 3‐rutinoside) from mulberry (Morus rubra) and Liriope platyphylla were used for demonstration and validation. The elution of anthocyanins was performed using a C₁₈ column with stepwise gradient elution and individual anthocyanins were identified by high‐performance liquid chromatography with tandem mass spectrometry. Based on the pH differential method, the high‐performance liquid chromatography peak areas of maximum and reference absorption wavelengths of anthocyanin extracts were conducted to quantify individual anthocyanins. The calibration curves for these anthocyanins were linear within the range of 10–5500 mg/L. The correlation coefficients (r²) all exceeded 0.9972, and the limits of detection were in the range of 1–4 mg/L at a signal‐to‐noise ratio ≥5 for these anthocyanins. The proposed quantitative analysis was reproducible with good accuracy of all individual anthocyanins ranging from 96.3 to 104.2% and relative recoveries were in the range 98.4–103.2%. The proposed technique is performed without anthocyanin standards and is a simple, rapid, accurate, and economical method to determine individual anthocyanin contents.     

基于双定性原则的高效液相色谱-二极管阵列检测法分析中药组分

双定性是根据样品的保留时间和吸收光谱的特征峰进行复合定性的一种新方法。该文基于自行设计及组装的二极管阵列检测器,构建了一套液相色谱-二极管阵列检测系统。采用该色谱系统分别对6种中药饮片中的非法添加物金胺O和枣仁天麻胶囊中的有效成分五味子醇甲进行了分离及定性研究。结果显示,在蒲黄饮片和枣仁天麻胶囊样品色谱图中均存在与目标检测物保留时间接近的色谱峰,进一步通过吸收光谱的特征峰比对,均排除了目标物存在的可能。应用结果证明,基于保留时间/吸收光谱的双定性原则,可以有效排除样品中杂质的干扰,避免假阳性结果,为中药组分研究提供了参考方法。     

High‐performance liquid chromatography with diode array detection method for the simultaneous determination of seven selected phosphodiesterase‐5 inhibitors and serotonin reuptake inhibitors used as male sexual enhancers

This work presents a simple, sensitive and generic high‐performance liquid chromatography with diode array detection method for the simultaneous determination of seven drugs prescribed for the treatment of erectile dysfunction and premature ejaculation. Investigated drugs include the phosphodiesterase‐5 inhibitors: sildenafil, tadalafil, and vardenafil, in addition to the selective serotonin reuptake inhibitors: dapoxetine, duloxetine, fluoxetine, and paroxetine. The drugs were separated using a Waters C8 column (4.6 × 250 mm, 5 μm) with the mobile phase consisting of phosphate buffer pH 3, acetonitrile and methanol in the ratio 60:33:7. The flow rate was 1.2 mL/min, and quantification was based on measuring peak areas at 225 nm. Peaks were perfectly resolved with retention times 3.3, 3.9, 6.4, 7.5, 9.5, 10.7, and 13.4 min for vardenafil, sildenafil, paroxetine, duloxetine, dapoxetine, fluoxetine, and tadalafil, respectively. The developed method was validated with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and limits of detection and quantification. The proposed method showed good linearity in the ranges 5–500, 2–200, 2–200, 3–300, 1.5–150, 2–200, and 2–200 μg/mL for sildenafil, tadalafil, vardenafil, dapoxetine, duloxetine fluoxetine, and paroxetine, respectively. The limits of detection were 0.18–0.38 μg/mL for the analyzed compounds. The applicability of the proposed method to real life situations was assessed through the analysis of commercial tablets, and satisfactory results were obtained.     

Application of two‐dimensional liquid chromatography in the separation of traditional Chinese medicine

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two‐dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one‐dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two‐dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two‐dimensional liquid chromatography, heart‐cutting two‐dimensional liquid chromatography both in on‐line, and off‐line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two‐dimensional liquid chromatography.     

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H₂O, CO, and CH₂O₂ losses, together with retro‐Diels–Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C‐ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C₄H₈) and 68 Da (C₅H₈) were yielded from a prenyl chain; neutral losses of 42 (C₃H₆), 54 (C₄H₆), and 70 Da (C₄H₆O) were generated by a pyran ring. Particularly, neutral losses of 18 (H₂O), 16 (CH₄), 112 (C₈H₁₆), and 98 Da (C₇H₁₄) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.     

Reversed‐phase high‐performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors

Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of l ‐[³H]‐citrulline produced from l ‐[³H]‐arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed‐phase high‐performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of l ‐citrulline with o‐phthaldialdehyde/N‐acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N‐[3‐(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well‐established radiometric assay.     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Two‐way and three‐way approaches to ultra high performance liquid chromatography–photodiode array dataset for the quantitative resolution of a two‐component mixture containing ciprofloxacin and ornidazole

Two‐way and three‐way calibration models were applied to ultra high performance liquid chromatography with photodiode array data with coeluted peaks in the same wavelength and time regions for the simultaneous quantitation of ciprofloxacin and ornidazole in tablets. The chromatographic data cube (tensor) was obtained by recording chromatographic spectra of the standard and sample solutions containing ciprofloxacin and ornidazole with sulfadiazine as an internal standard as a function of time and wavelength. Parallel factor analysis and trilinear partial least squares were used as three‐way calibrations for the decomposition of the tensor, whereas three‐way unfolded partial least squares was applied as a two‐way calibration to the unfolded dataset obtained from the data array of ultra high performance liquid chromatography with photodiode array detection. The validity and ability of two‐way and three‐way analysis methods were tested by analyzing validation samples: synthetic mixture, interday and intraday samples, and standard addition samples. Results obtained from two‐way and three‐way calibrations were compared to those provided by traditional ultra high performance liquid chromatography. The proposed methods, parallel factor analysis, trilinear partial least squares, unfolded partial least squares, and traditional ultra high performance liquid chromatography were successfully applied to the quantitative estimation of the solid dosage form containing ciprofloxacin and ornidazole.     

Determination of multiple phytohormones in fruits by high‐performance liquid chromatography with fluorescence detection using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling

Plant hormone determination in food matrices has attracted more and more attention because of their potential risks to human health. However, analytical methods for the analysis of multiple plant hormones remain poorly investigated. In the present study, a convenient, selective, and ultrasensitive high‐performance liquid chromatography method for the simultaneous determination of multiple classes of plant hormones has been developed successfully using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling. Eight plant hormones in fruits including jasmonic acid, 12‐oxo‐phytodienoic acid, indole‐3‐acetic acid, 3‐indolybutyric acid, 3‐indolepropionic acid, gibberellin A₃, 1‐naphthylacetic acid, and 2‐naphthaleneacetic acid were analyzed by this method. The conditions employed for dispersive liquid–liquid microextraction were optimized systematically. The linearity for all plant hormones was found to be >0.9993 (R² values). This method offered low detection limits of 0.19–0.44 ng/mL (at a signal‐to‐noise ratio of 3), and method accuracies were in the range of 92.32–103.10%. The proposed method was applied to determine plant hormones in five kinds of food samples, and this method can achieve a short analysis time, low threshold levels of detection, and a high specificity for the analysis of targeted plant hormones present at trace level concentrations in complex matrices.     

Evaluation of a method for the simultaneous quantification of N‐nitrosamines in water samples based on stir bar sorptive extraction combined with high‐performance liquid chromatography and diode array detection

A simple, sensitive, and reliable procedure based on stir bar sorptive extraction coupled with high‐performance liquid chromatography was applied to simultaneously extract and determine three semipolar nitrosamines including N‐nitrosodibutylamine, N‐nitrosodiphenylamine, and N‐nitrosodicyclohexylamine. To achieve the optimum conditions, the effective parameters on the extraction efficiency including desorption solvent and time, ionic strength of sample, extraction time, and sample volume were systematically investigated. The optimized extraction procedure was carried out by stir bars coated with polydimethylsiloxane. Under optimum extraction conditions, the performance of the proposed method was studied. The linear dynamic range was obtained in the range of 0.95–1000 ng/mL (r = 0.9995), 0.26–1000 ng/mL (r = 0.9988) and both 0.32–100 ng/mL (r = 0.9999) and 100–1000 ng/mL (r = 0.9998) with limits of detection of 0.28, 0.08, and 0.09 ng/mL for N‐nitrosodibutylamine, N‐nitrosodiphenylamine, and N‐nitrosodicyclohexylamine, respectively. The average recoveries were obtained >81%, and the reproducibility of the proposed method presented as intra‐ and interday precision were also found with a relative standard deviation <6%. Finally, the proposed method was successfully applied to the determination of trace amounts of selected nitrosamines in various water and wastewater samples and the obtained results were confirmed using mass spectrometry.