High-performance Liquid Chromatography DAD Research Articles

Camptothecin and its Analogs: High-Yielding Ophiorrhiza Species from Sri Lanka for Sustainable Anticancer Compound Production

Introduction: Camptothecin (CPT) is pivotal in cancer treatment, derived from various CPT-producing plant species, and is a fundamental component in synthesizing valuable cancer drugs like Irnotican and Topotecan. Sourcing from nature poses conservation issues, fostering interest in the herbaceous Ophiorrhiza plant as a more sustainable alternative. Ophiorrhiza species in Sri Lanka lack comprehensive study, warranting exploration for echo-friendly anticancer compound production. Objectives: This study examines CPT and analog content in Ophiorrhiza mungos, O. pectinata, and O. rugosa across diverse Sri Lankan regions. Methods: The study employs Thin Layer Chromatography (TLC), High-Performance Liquid Chromatography-DAD (HPLC-DAD), and Liquid Chromatography-Mass Spectrometry (LC-MS) to quantify and confirm CPT and its analogs. Results: Significant variations in the content of CPT and its analogs were observed among plant parts and regions. O. mungos from Deraniyagala and Bibile regions notably exhibited elevated CPT levels in fruits and roots. O. rugosa var. Angustifolia and O. pectinata also exhibited a considerable content of CPT in their roots, though it was significantly lower (p < 0.005) than O. mungos. Conclusion: The study validates analytical methods for specificity, linearity, precision, accuracy, and sensitivity per ICH guidelines. The results indicate that Ophiorrhiza species, especially O. mungos and O. rugosa var. Angustifolia holds the potential to be a sustainable source of CPT. Optimizing cultivation practices offers an eco-friendly solution for anticancer compound production, alleviating species threats and conserving biodiversity.

Comprehensive and Rapid Quality Evaluation Method for the Ayurvedic Medicine Divya-Swasari-Vati Using Two Analytical Techniques: UPLC/QToF MS and HPLC-DAD.

Divya-Swasari-Vati (DSV) is a calcium-containing herbal medicine formulated for the symptomatic control of respiratory illnesses observed in the current COVID-19 pandemic. DSV is an Ayurvedic medicine used for the treatment of chronic cough and inflammation. The formulation has shown its pharmacological effects against SARS-CoV-2 induced inflammation in the humanized zebrafish model. The present inventive research aimed to establish comprehensive quality parameters of the DSV formulation using validated chromatographic analytical tools. Exhaustive identification of signature marker compounds present in the plant ingredients was carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/QToF MS). This was followed by simultaneous estimation of selected marker components using rapid and reliable high-performance liquid chromatography (HPLC) analysis. Eleven marker components, namely gallic acid, protocatechuic acid, methyl gallate, ellagic acid, coumarin, cinnamic acid, glycyrrhizin, eugenol, 6-gingerol, piperine and glabridin, were selected out of seventy-four identified makers for the quantitative analysis in DSV formulation. Validation of the HPLC method was evaluated by its linearity, precision, and accuracy tests as per the International Council of Harmonization (ICH) guidelines. Calibration curves for the eleven marker compounds showed good linear regression (r2 > 0.999). The relative standard deviation (RSD) value of intraday and interday precision tests were within the prescribed limits. The accuracy test results ranged from 92.75% to 100.13%. Thus, the present inclusive approach is first of its kind employing multi-chromatographic platforms for identification and quantification of the marker components in DSV, which could be applied for routine standardization of DSV and other related formulations.

The Effect of Grapefruit Juice on the Pharmacokinetics of Tadalafil in Rats.

We developed and validated a novel, sensitive, selective, and inexpensive high-performance liquid chromatography (HPLC) method for the determination of tadalafil in rats plasma and to investigate the effect of grapefruit juice on the pharmacokinetics of tadalafil in rats. The ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 μm) chromatography column can be used to separate tadalafil and carbamazepine (internal standard, IS). A mixture of acetonitrile-0.2% trifluoroacetic acid-water (48 : 10 : 42, V/V/V) was used as the mobile phase with a flow rate of 1.0 mL/min. The column temperature was set at 35.0°C. The detection wavelength was set at 286 nm. The tadalafil was extracted by ethyl acetate from plasma at the alkaline condition. 12 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups, Group A (experimental group, received grapefruit juice 5 mL/kg for 7 days) and Group B (control group, received normal saline for 7 days). All the rats were given a single dose of tadalafil (5 mg/kg) after the last administration. The main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the conditions of this experiment, the plasma concentrations of tadalafil in the range of 10–2000 ng/ml had a good linear relationship. The intra- and interday precision for tadalafil in plasma were less than 15%, and the relative recovery rate was good at low, medium, and high QC levels. The Cmax of tadalafil in the control group and the experimental group was (725.89 ± 161.59) ng/mL and (1271.60 ± 179.31) ng/mL, t1/2 was (9.28 ± 2.07) h and (11.70 ± 1.47) h, AUC (0-t) was (7399.61 ± 696.85) ng·h/mL and (9586.52 ± 2048.81) ng·h/mL, and AUC(0-∞) was (7995.50 ± 707.23) ng·h/mL and (10639.43 ± 2235.94) ng·h/mL, respectively. Results show that the Cmax of tadalafil in group A was 75.17% higher than that in group B, the Vz/F was also reduced, and the t1/2 was increased by 2.42 h. The developed HPLC–DAD method for the determination of tadalafil in rats plasma was accurate, reproducible, specific, and it was found to be suitable for the pharmacokinetics of tadalafil and food-drug interactions. Grapefruit juice can inhibit the metabolism of tadalafil and increase the exposure of tadalafil in rats.

Analytical Quality by Design Approach for a Stability-Indicating Method to Determine Apixaban and Its Related Impurities

Quality by Design (QbD) is a systematic approach that takes predefined objectives and emphasizes process understanding and control. Analytical QbD (AQbD) is widely used when it comes to the development and optimization of analytical methods. Since impurities and contaminants may be present in the pharmaceutical product, current regulatory guidelines recommend monitoring these substances. Thus, the aim of this work was to develop and validate a fast and sensitive stability indicating method by high-performance liquid chromatography (HPLC) for the simultaneous determination of apixaban and three synthesis impurities using AQbD approach. Experiments were designed and assessed on MODDE® 11 (Umetrics, Sweden) software and carried out in a Shimadzu® LC-20A Prominence HPLC–DAD at 220 nm. Design of experiments was applied to achieve the optimum conditions through response surface methodology with four quantitative critical method parameters (CMPs): column temperature, flow rate, mobile phase pH and organic percentage. Besides, the chromatographic column was the fifth CMP taken as a qualitative variable. The HPLC method was established using an Inertsil® CN-3 column at the temperature of 30 °C. The mobile phase consisted of methanol and water (50.2:49.8) at a flow rate of 1.015 mL/min with no pH adjustment. In order to confirm method selectivity excipients mixture was evaluated, as well as hydrolytic, photolytic, thermolytic, and oxidative conditions. The method was also validated for sensitivity (LOQ 0.5 μg/mL, LOD 0.01–0.05 μg/mL for impurities), linearity (concentration range 1–35 μg/mL for APX and 0.5–10 μg/mL for impurities, r > 0.999), precision (RSD ≤ 10%) and accuracy between levels (RSD < 3.0 for APX and < 4.0% for impurities). The application of AQbD led to a well-understood and validated analytical method that provided assured separation of APX and all significant impurities, turning it into a powerful decision-making tool for the analytical methodologies development.

Fingerprint Analysis and Multi-Component Determination of Ribonucleic Acid for Injection II Recipe by HPLC-DAD and LC-ESI-MS Methods.

Using high-performance liquid chromatography (HPLC), a quantitative method was developed to control the quality of ribonucleic acid for injection II (RA-II), which is used as an anti-cancer drug clinically in China. Using nuclease enzyme and under optimal hydrolysis conditions, the unstable RNA was hydrolyzed into stable nucleosides and nucleotides, which were easily detected by HPLC with diode array detection. Furthermore, by analyzing HPLC chromatograms of 10 batches of samples, six common peaks, namely, cytosine nucleotide, uracil nucleotide, adenine nucleotide, guanine nucleotide, guanosine and adenine, were identified by a HLPC coupled with electrospray ionization mass spectrometry. The similarity, the relative retention time and the relative peak area of each common peak, relative to the reference peak, were calculated to obtain the HPLC fingerprints, and their values were within the scope of the provisions. More importantly, all six components were simultaneously determined. Overall, the developed method in this investigation proved to be a useful tool for monitoring the quality of RA-II in terms of their batch variations and the raw material sources.

Online extraction–high performance liquid chromatography–diode array detector–quadrupole time-of-flight tandem mass spectrometry for rapid flavonoid profiling of Fructus aurantii immaturus

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC–DAD–QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction for Fructus aurantii immaturus (Zhishi), OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of eighteen flavonoids were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and compound 9, natsudaidain-3-O-glucoside, was discovered in Zhishi for the first time. It is concluded that the developed OLE–HPLC–DAD–QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products.

Use of an online extraction liquid chromatography quadrupole time-of-flight tandem mass spectrometry method for the characterization of polyphenols in Citrus paradisi cv. Changshanhuyu peel

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC–DAD–QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction of Citrus paradisi cv. Changshanhuyu (Changshanhuyu) peel, OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of twenty-two secondary metabolites were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and nine of them were discovered in Changshanhuyu peel for the first time to our knowledge. It is concluded that the developed OLE–HPLC–DAD–QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products.

Determination of vortioxetine in human serum and saliva samples by HPLC–DAD and HPLC–MS

Vortioxetine is a new drug against major depressive disorder with high affinity for a range of different serotonergic targets in the central nervous system. Therapeutic drug monitoring is an important tool for the clinical management of patients receiving a pharmacotherapy, particularly in psychiatry. For this reason, determination of drug concentration in biological fluids is important for a rational dosage of drugs. Rapid and reliable analytical assays are also required to detect and identify drugs of toxicological importance. For analysis of vortioxetine by high-performance liquid chromatography (HPLC), no procedures for its determination in saliva have been reported and there are only a few ones for its determination in serum. A sensitive and selective highperformance liquid chromatography with diode array detector (HPLC-DAD) or mass spectrometer (HPLC-MS) method was developed for the fast quantification of vortioxetine in human saliva and serum. The determination was performed on a Synergi Polar RP column in isocratic mode under the optimal mobile phase containing 70% methanol, 20% acetate buffer at pH 3.5, 10% double distilled water, and 0.025 M L−1 diethylamine.

Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5 mM, pH 5.8)/acetonitrile (both with 1% Et3N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500 nm and quantitative analyses were carried out at 278 nm. The LOQ of the method was 1 μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25 μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of + 25, + 4 and −20 °C in order to evaluate plasma stability profiles.

High-speed counter-current chromatography coupled online to high performance liquid chromatography–diode array detector–mass spectrometry for purification, analysis and identification of target compounds from natural products

A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC–HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC–HPLC–diode array detector–mass spectrometry (HSCCC–HPLC–DAD–MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC–DAD–MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4′,6′-dihydroxyhydrochalcone-2′-O-β-d-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products.

Comparative evaluation of ELISA kit and HPLC DAD for the determination of chlorpyrifos ethyl residues in water and sediments

Enzyme linked immunosorbent assay (ELISA) kit is a versatile, cheap and relatively available tool that can be used in remote areas. In this study, performance of ELISA kit was evaluated in terms of accuracy, recovery, precision, sensitivity, cross reactivity and matrix interference for pesticide residue determination in water and sediment samples. This method was compared with high performance liquid chromatography (HPLC) which is not a commonly available analytical technique for chlorpyrifos ethyl residue analysis in developing countries. The ELISA kit had limits of detection (LOD) of 0.37 µg L(-1) and 0.42 µg Kg(-1) dry weight (dw), for chlorpyrifos ethyl in water and sediment samples, respectively using deionized water and a control sediment sample. Mean percentage recoveries and coefficients of variation (CV) for ELISA kit varied from 96.0±5.8% to 108.0±3.4% for water and sediment samples. Comparison between ELISA and HPLC analysis results using water and sediment samples from Lake Naivasha showed no significant difference in results (p≤0.05). Strong correlations (r2=0.9878 water samples and r1=0.9670, p<0.0001 for sediment samples, n=48) were reported between the methods for the two samples analyzed. Bland-Altman bias plot analysis showed that the two methods were in agreement within 95% confidence interval of limits -2.9 to 3.8 and -2.2 to 3.6 for water and sediment, respectively. Given the high sensitivity reported and the obtained acceptable limits of coefficient of variation and percentage recovery, ELISA appears to be a suitable rapid analytical tool in analysis of chlorpyrifos ethyl in water and sediment samples. Results demonstrate comparability to HPLC and could complement conventional tools in regular monitoring program particularly in developing countries. This will hasten results delivery for ecological risk assessment and timely execution of mitigation measures.

Dispersive liquid–liquid microextraction combined with high performance liquid chromatography-DAD detection for the determination of sulfonylurea herbicides in water samples

A new method for the determination of four sulfonylurea herbicides (metsulfuron-methyl, chlorsulfuron, bensulfuron-methyl and chlorimuron-ethyl) in water samples was developed by dispersive liquid–liquid microextraction coupled with high performance liquid chromatography-diode array detector. Parameters that affect the extraction efficiency, such as the kind and volume of the extraction and disperser solvent, extraction time and salt addition, were investigated and optimised. Under the optimum conditions, the enrichment factors were in the range between 102 and 216. The linearity of the method was obtained in the range of 1.0–100 ng mL−1 with the correlation coefficients (r) ranging from 0.9982 to 0.9995. The method detection limits were 0.2–0.3 ng mL−1. The proposed method has been successfully applied to the analysis of target sulfonylurea herbicides in river, stream and well water samples with satisfactory results.

High performance liquid chromatography-diode array and electrospray-mass spectrometry analysis of vardenafil, sildenafil, tadalafil, testosterone and local anesthetics in cosmetic creams sold on the Internet web sites

A simple high-performance liquid chromatography (HPLC) method with ultraviolet diode array (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) detection has been developed for the determination of vardenafil, sildenafil, tadalafil, testosterone, procaine, lidocaine, prilocaine, and benzocaine in cosmetic creams sold as promising remedies for male erectile dysfunction and female genitals stimulation. The presence of these substances in commercial cosmetic samples is prohibited. Aliquots (1 g) of the cosmetic creams under investigation were diluted 1:100 in methanol, subjected to ultrasonic treatment, added with benzoic acid as internal standard, and analyzed by HPLC–DAD and HPLC–ESI-MS after a further 1:1000 dilution. The compounds were separated by reversed phase chromatography with water (0.02% trifluoroacetic acid) and acetonitrile gradient elution and detected by UV-DAD at 228, 255 and 290 nm and by ESI-MS positive ionisation mode. Benzoic acid was used as internal standard. Linearity was studied with UV-DAD detection from 2.5–7.8 to 250 μg/g range, depending on the different compounds and with ESI-MS in the 3.3–8.9 to 250 ng/g range. Good determination coefficients ( r 2 ≥ 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 μg/g for HPLC–UV-DAD assay and between 3.3 and 8.9 ng/g for HPLC–ESI-MS assay depending on different analyzed substances. At three concentrations spanning the linear dynamic ranges of both UV-DAD and ESI-MS assay, mean recoveries were always higher than 90% for the different analytes and intra-assay and inter-assay precision always better than 15% and 12%. This method was successfully applied to the analysis of substances under investigations present in cosmetic creams, freely sold on the Internet web-sites.

Supercritical fluid CO 2 extraction and simultaneous determination of eight annonaceous acetogenins in Annona genus plant seeds by HPLC–DAD method

Annonaceous acetogenins (ACGs) isolated from Annonaceae plants exhibited a broad range of biological bioactivities such as cytotoxic, antitumoral, antiparasitic, pesticidal and immunosuppresive activities. However, their structures were liable to change at more than 60 °C and their extraction yields were low using traditional organic solvent extraction. In the present study, all samples from Annona genus plant seeds were extracted by supercritical carbon dioxide under optimized conditions and a high-performance liquid chromatography (HPLC) method was established for simultaneously determining eight ACGs. All of the eight compounds were simultaneously separated on reversed-phase C 18 column (250 mm × 4.6 mm, 5 μm) with the column temperature at 30 °C. The mobile phase was composed of (A) methanol and (B) distilled water, the flow rate was 1.0 ml/min and the detection wavelength was set at 220 nm. All calibration curves showed good linear regression ( γ > 0.9995) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.87–2.53% and 1.91–3.42%, respectively, and overall recoveries of 95.81–105.39% for the eight compounds analyzed. The established method can be applied to evaluate the intrinsic quality of Annonaceae plant seeds. The determination results recover the content-variation regularities of various ACGs in different species, which are helpful to choose the good-quality Annonaceae plant seeds for anticancer lead compound discovery.

Improved quality control method for Fufang Danshen preparations through simultaneous determination of phenolic acids, saponins and diterpenoid quinones by HPLC coupled with diode array and evaporative light scattering detectors

A herb-combined prescription, mainly derived from roots of Salvia miltiorrhiza and Panax notoginseng, has been widely used for improving coronary or cerebral circulation in China as well as in Western countries. Multiple commercially available preparations, known as Fufang Danshen preparations (FDPs), produced by various manufacturers with the raw materials from different sources, pose a serious challenge to the quality control of this herb medicine. Previous pharmacological studies identify three types of bioactive components correlated with the clinical effect of those herb preparations. Those mainly include four phenolic acids, four saponins and four diterpenoid quinones. In this report, by using high performance liquid chromatography (HPLC) coupled with diode array and evaporative light scattering detectors (DAD–ELSD), we developed an improved quality control method for those herb medicines. A simultaneous separation and quantification of the 12 components was performed on a C 18 column, in which the mobile phase consisted of (A) 0.1% aqueous formic acid and (B) acetonitrile using a gradient elution. The optimum detection wavelength was set at 281 nm, the drift tube temperature of ELSD was set at 113 °C, the nitrogen flow rate at 3.1 L/min, and the gain = 4. All calibration curves showed good linear regression ( r 2 > 0.9927) within test ranges. The method developed showed good precision and accuracy with overall intra- and inter-day variations of 0.64–4.79% and 0.69–4.96%, respectively, and the overall recoveries of 93.50–107.69% for the 12 compounds analyzed. This method was successfully applied to quantify the twelve components in ten commercial samples from three formulas by seven independent manufacturers. This readily available, low-cost and reliable HPLC–DAD–ELSD method improved the quality control of traditional Chinese medicinal preparations consisting of complex compounds with different structures such as FDPs.

Simultaneous determination of salidroside and tyrosol in extracts of Rhodiola L. by microwave assisted extraction and high-performance liquid chromatography

Rhodiola L. has a long history of use in traditional Chinese medicine (TCM) and has shown great promise in recent clinical trials. Salidroside and tyrosol are two important active compounds present in this TCM. In this work, microwave-assisted extraction (MAE) followed by high performance liquid chromatography (HPLC) with a photodiode array detector (DAD) was developed for quantitative analysis of salidroside and tyrosol in Rhodiola L. samples. After systematical investigation, the optimal experimental parameters of soak time (60 min), extraction solvent volume (1 g sample, 5 mL), extract solvent composition (50% methanol/water), microwave power (400 W) and extraction time (5 min) were investigated. The optimized method provided satisfactory precision (R.S.D. values less than 7.5%), good recovery (from 94.4 to 123%), and good linear relation in the range of 5.0–500 μg/mL for salidroside and 1.0–100 μg/mL for tyrosol ( R 2 > 0.999). The proposed method was applied to quantitative analysis of salidroside and tyrosol in Rhodiola L. samples from five different growing areas. To demonstrate the method feasibility, recirculation was also used to analyze salidroside and tyrosol in Rhodiola L. samples. The proposed MAE–HPLC–DAD is a simple, rapid and low-cost method for quantitative analysis of salidroside and tyrosol, and a potential tool for quality assessment of Rhodiola L. sample.

Chromatographic fingerprint analysis of Cephalotaxus sinensis from various sources by high-performance liquid chromatography–diodearray detection–electrospray ionization-tandem mass spectrometry

Selective and efficient analytical methods are required not only for quality assurance but also for authentication of Chinese herbal medicine. A simple, rapid and valid fingerprint method has been first carried out for the quality control of Cephalotaxus sinensis by using high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) and electrospray ionisation-tandem mass spectrometry (ESI-MS). The characteristic analytical fingerprints of this plant extract showed 18 common peaks, and out of these, 10 compounds involving 2 new compounds were identified by comparing the retention time, UV and ESI-MS/MS spectrum of each standard with those of each peak separated by on-line HPLC–DAD–MS/MS. Moreover, the effects of collecting locations, harvesting time, storage time, drying methods, and medicinal portions on herbal chromatographic fingerprints were examined by similarity analysis and principal component analysis (PCA) along with markers. Using the reference fingerprint along with markers, the best harvesting time, cultivation location and medicinal part were determined. The results obtained suggest that the chromatographic fingerprint combining similarity evaluation and PCA along with markers or pharmacologically active constituents can efficiently identify raw herb of Cephalotaxus sinensis from different sources, which provide helpful clues to the study of plant's secondary metabolites and benefit quality control.

Simultaneous analysis and identification of main bioactive constituents in extract of Zizyphus jujuba var. sapinosa (Zizyphi spinosi semen) by high-performance liquid chromatography–photodiode array detection–electrospray mass spectrometry

A high-performance liquid chromatography (HPLC) coupled with UV photodiode array detection and electrospray mass spectrometry (HPLC–DAD–ESI-MS) was developed for the simultaneous analysis and identification of main bioactive constituents in extract of zizyphi spinosi semen including three flavonoids, three alkaloids and four triterpene saponins. The separation was achieved on a C 18 column with a gradient solvent system of 0.08% aqueous formic acid solution-acetonitrile. The compounds were identified with MS-spectra and authentic standards. The contents of jujuboside A, jujuboside B, spinosin and 6‴-feruloyl spinosin were quantified by using DAD in different extract of zizyphi spinosi semen.

Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography–diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring

Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography–diode array detection (HSCCC–HPLC–DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC–DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum ( St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate–ethanol–water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).

On-line identification of the constituents of Buyang Huanwu decoction in pig serum using combined HPLC–DAD–MS techniques

Buyang Huanwu decoction (BYHWD) is a widely used Chinese traditional compound medicine that has proved effective in treating cerebrovascular illnesses; however, its active substances have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), photodiode-array detection and mass-spectrometry techniques were used to study the constituents of BYHWD from pig serum after oral administration. A total of 45 characteristic HPLC peaks were detected from serum containing drug. The chemical structures of nine of the peaks were tentatively elucidated as 7,3'-dihydroxy-4'-methoxyisoflavone-7-O-glucuronide (P1), 7-hydroxy-4'-methoxyisoflavone-7-O-glucuronide (P2), 7,2',4'-trihydroxy-3'-methoxyisoflavane-7-O-sulphate (P3), 3-hydroxy-9,10-dimethoxypterocarpan-3-O-glucuronide (P4), 7,2'-dihydroxy-3',4'-dimethoxyisoflavane-7-O-glucuronide (P5), 3-hydroxy-9,10-dimethoxypterocarpane-3-O-sulphate (P6), 4(1H)-quinolinone (P7 or P8), 4-hydroxyquinoline (P8 or P7) and oleic acid (P9). All of the identified peaks, with the exception of P9, were metabolites of the constituents of BYHWD in vivo.