Simultaneous determination of 11 saponins in Panax notoginseng using HPLC-ELSD and pressurized liquid extraction

A new HPLC coupled with evaporative light scattering detection (ELSD) method was developed for simultaneous determination of 11 major triterpene saponins, namely notoginsenoside R1 (1), ginsenosides Rg1 (2), Re (3), Rf(4), Rb1 (5), Rg2 (6), Rc (7), Rb2 (8), Rb3 (9), Rd (10), and Rg3 (11) in Panax notoginseng, a commonly used traditional Chinese medicine (TCM). Pressurized liquid extraction (PLE) was employed for sample preparation, and the analysis was achieved using a Zorbax ODS C18 column eluted with gradient water-ACN in 60 min. The drift tube temperature of ELSD was set at 60 degrees C, and nitrogen flowrate was at 1.4 L/min. The method provided good repeatability and sensitivity for quantification of 11 saponins with overall precision (including intra- and interday) and LOD of less than 2.9% (RSD) and 98 ng, respectively. The validated method was successfully applied to quantify 11 saponins in 28 samples of P. notoginseng collected in different places, which is helpful to control the quality of P. notoginseng and its related products.     

Qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps by pressurized liquid extraction and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS)

A simple and rapid pressurized liquid extraction (PLE) and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps. The samples were extracted using PLE. The separation was achieved on a ZORBAX Eclipse XDB-C₁₈ column with gradient elution of methanol and 5 mM aqueous ammonium acetate as mobile phase. Target compounds were identified by characterizing their product ions, precursor ions and retention times. Quantitative analysis of investigated compounds were performed using time programmed selective ion monitoring (SIM) or selective reaction monitoring (SRM) with 10 segments in positive (negative for uridine) ion mode. The results showed that 43 bases, nucleosides and their analogues were detected in Cordyceps, of these 16 compounds were identified. The simultaneous determination of seven nucleosides and six bases in Cordyceps was achieved using PLE and HPLC-ESI-MS/MS method described above, which afforded good linearity, selectivity, precision, recovery, short analysis time as well as LOD and LOQ in the ng/ml range.     

Determination of flavonoids and saponins in Gynostemma pentaphyllum (Thunb.) Makino by liquid chromatography-mass spectrometry

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chinese herb possessing antitumor and antioxidant activities, has been shown to contain several functional components like saponins and flavonoids. However, their identities remain uncertain. The objectives of this study were to develop an appropriate extraction, purification and HPLC-MS method to determine saponins and flavonoids in G. pentaphyllum. Both flavonoids and saponins were extracted with methanol, followed by purification with a C18 cartridge to elute the former with 50% methanol and the latter with 100% methanol. A total of 34 saponins were separated within 40 min by a Gemini C18 column and a gradient mobile phase of acetonitrile and 0.1% formic acid in water, in which 18 saponins were identified by LC-MS with ESI mode and Q-TOF (LC/MS/MS). Similarly, a total of eight flavonoids were separated within 45 min by the same column and a gradient solvent system of methanol and 0.1% formic acid in water, with identification being carried out by a post-column derivatization method and LC-MS with ESI mode. The amounts of flavonoids in G. pentaphyllum ranged from 170.7 to 2416.5 μg g⁻¹, whereas saponins were from 491.0 to 89,888.9 μg g⁻¹.     

Chemical investigation of saponins in different parts of Panax notoginseng by pressurized liquid extraction and liquid chromatography-electrospray ionization-tandem mass spectrometry

A pressurized liquid extraction (PLE) and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for the qualitative determination of saponins in different parts of P. notoginseng, including rhizome, root, fibre root, seed, stem, leaf and flower. The samples were extracted using PLE. The analysis was achieved on a Zorbax SB-C18 column with gradient elution of acetonitrile and 8 mM aqueous ammonium acetate as mobile phase. The mass spectrometer was operated in the negative ion mode using the electrospray ionization, and a collision induced dissociation (CID) experiment was also carried out to aid the identification of compounds. Forty one saponins were identified in different parts of P. notoginseng according to the fragmentation patterns and literature reports, among them, 21 saponins were confirmed by comparing the retention time and ESI-MS data with those of standard compounds. The results showed that the chemical characteristics were obviously diverse in different parts of P. notoginseng, which is helpful for pharmacological evaluation and quality control of P. notoginseng.     

Evaluation of surfactant assisted pressurized liquid extraction for the determination of glycyrrhizin and ephedrine in medicinal plants

Surfactant assisted pressurized liquid extraction (PLE) with a laboratory made system was applied for the extraction of glycyrrhizin in Radix glycyrrhizae/liquorice and ephedrine in Ephedra sinica. The proposed system set-up for this current work was simpler as no heating and back pressure regulator was required. Extraction with surfactant assisted PLE was carried out dynamically at a flow of 1.5 mL min⁻¹, at room temperature, under an applied pressure of 10-20 bar with an extraction time of 45-50 min. The extraction efficiencies of the proposed method using surfactants such as sodium dodecyl sulfate (SDS) and Triton X-100 were compared with sonication using organic solvent for different batches of medicinal plants materials. For the determination of glycyrrhizin in R. glycyrrhizae, the extraction efficiencies of surfactant assisted PLE with SDS and Triton X-100 was observed to be comparable with sonication. The method precision was found to vary from 1.6 to 2.6% (R.S.D., n = 6) on different days. For ephedrine in E. sinica, surfactant assisted PLE with SDS was found to give higher extraction efficiencies compared to Triton X-100. The overall method precision for surfactant assisted PLE with SDS for ephedrine in E. sinica was found to vary from 1.5 to 4.1% (R.S.D., n = 6) on different days. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC. Our data showed the possibility of PLE at room temperature and the advantages of eliminating the use of organic solvents in the extraction process.     

Qualitative and quantitative analysis of multiple components for quality control of Deng‐Zhan‐Sheng‐Mai capsules by ultra high performance liquid chromatography tandem mass spectrometry method coupled with chemometrics

Deng‐Zhan‐Sheng‐Mai capsules are a well‐known traditional Chinese patent medicine that was developed in China for the treatment of ischemic stroke. Its quality control focuses on Erigerontis Herba but ignores the contributions of Ginseng Radix et Rhizoma, Schisandrae Chinensis Fructus, and Ophiopogonis Radix. To improve the quality standards for this medicine, this work reports the application of a systematic ultra high performance liquid chromatography tandem mass spectrometric method coupled with chemometrics. Three qualitative and quantitative parameters are established for the evaluation of quality: chemical profiling, the relationship between the contents of 18 compounds and the antioxidant activity, and chemometric analysis. A total of 55 compounds, including 20 phenolic acids, 10 flavonoids, 15 saponins, and 10 lignans, were identified. The method for the quantitative determination of the aforementioned 18 compounds was validated. The limit of quantification ranged from 0.13 to 9.60 ng/mL. The overall recoveries ranged from 95.31 to 103.54%. Hierarchical cluster analysis and principal component analysis were applied to the data of 18 components in ten batches of samples. Nine compounds, including scutellarin, 3,5‐O‐dicaffeoylquinic acid, 4,5‐O‐dicaffeoylquinic acid, ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, ophiopogonin D, schisandrin, and schisandrol B, are suggested as chemical markers for evaluating the quality.     

改性松香键合二氧化硅高效液相色谱固定相的制备及其对三七总皂苷的分离

三七中发挥药效的主要成分为三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1和人参皂苷Rd,用于贫血、冠心病、高血压、脑卒中后遗症等疾病的治疗,但其化学成分多且难分离.将氢化松香丙烯酸羟乙酯(HRHA)通过巯基-烯点击化学反应键合到烷基化硅胶表面,制备出一种新型的改性松香键合二氧化硅高效液相色谱固定相(SiO2...     

Comparative pharmacokinetics of the main active components in normal and ulcerative colitis rats after oral administration of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair and its single herb extracts by LC–MS/MS

Zingiberis Rhizoma and Ginseng Radix et Rhizoma are usually used together for the treatment of ulcerative colitis in clinical practices. However, their compatibility mechanism remains unclear. In this study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed for simultaneous quantification of ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, and 6-gingerol in rat plasma after oral administration of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair and its single herb extracts. The calibration curves exhibited good linearity, with correlation coefficients of more than 0.993. The precision deviations of intra- and interday analysis were within 10.66%, and accuracy error ranged from −12.74 to 11.56%. The average recoveries of analytes were higher than 76.60% and the matrix effects were minimal. Thus, the validated method was successfully applied to a pharmacokinetic study of four ingredients in normal and ulcerative colitis rat plasma. The results indicated that the pharmacokinetic parameters of four analytes in normal and model groups showed significant differences. The larger exposure (the mean AUC_0-t of ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, and 6-gingerol were increased by 50.93, 141.90, 3.68, and 37.25%, respectively) and slower elimination (the CLz/F of ginsenoside Re, ginsenoside Rg1, and 6-gingerol were decreased by 52.94, 83.64, and 32.18%, respectively) were observed in ulcerative colitis rats. Furthermore, compared with single herbs, the analytes in rat plasma after oral administration of combined extracts presented relatively high systemic exposure levels with AUC_0-t > 2000 h·ng/mL and C_max > 200 ng/mL. Collectively, the differences of pharmacokinetic characteristics revealed the synergistic effect of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair, which provided a valuable and reliable basis for its clinical application in the treatment of ulcerative colitis.     

Pressurized liquid extraction of isoflavones from soybeans

Isoflavone derivatives from freeze-dried soybeans were extracted by pressurized liquid extraction (PLE) and determined by reverse-phase high performance liquid chromatography (HPLC) with both photo diode array and mass spectrometry (MS) detection. Both real and spiked samples were used in the development of the method.Several extraction solvents (methanol (MeOH) and ethanol (EtOH), 30-80% in water and water), temperatures (60-200 °C), pressures (100-200 atm), as well as the sample size (0.5-0.05 g) and cycle length (5-10 min) were studied for the optimization of the extraction protocol. The optimized extraction conditions for quantitative recoveries were: 0.1 g of sample, 100 °C, three (7 min) static extraction cycles and ethanol 70% as extracting solvent. The stability of the isoflavones during the PLE was also determined. Under PLE conditions, degradation of malonyl glucoside forms of the isoflavones takes place using temperatures higher than 100 °C whereas degradation of glucosides takes place above 150 °C. Using the optimized protocol, isoflavones can be extracted from freeze-dried soybeans without degradation.     

亲水/反相二维色谱法制备桔梗中的三萜皂苷

建立了亲水/反相二维色谱用于制备桔梗中三萜皂苷单体的方法。桔梗经水煮醇沉、反相和亲水两种模式的固相萃取后得到三萜皂苷类组分。选定XAmide色谱柱（150 mm×20 mm,5 μm）,以乙腈和水为流动相,在亲水色谱模式下进行组分制备。选择时间触发模式,以1 min为单位进行馏分收集,得到6~25 min之间的20个三萜皂苷精细组分。以第18个馏分（JG23）为例,在反相色谱模式下采用Atlantis Prep T3色谱柱（100 mm×30 mm,5 μm）制备,得到两个单体化合物。通过质谱和核磁共振对其进行定性,确定分别为deapi-platycoside E和platycoside E。实验结果表明,该制备方法具有好的正交选择性,对于复杂样品中三萜皂苷类化合物的分离纯化有一定的借鉴意义。     

Pressurized liquid extraction and liquid chromatography–tandem mass spectrometry applied to determine iodinated X-ray contrast media in sewage sludge

Pressurized liquid extraction (PLE) has been successfully applied for the first time to the extraction of five iodinated X-ray contrast media from sludge. Once optimized all PLE parameters, the extract has been analyzed by liquid chromatography–tandem mass spectrometry, being the method developed sensible enough to reach limit of quantifications (LOQs) of 25 μg kg⁻¹ (d.w.). The developed method has been applied to the analysis of sludge from urban sewage treatment plants and although some compounds such as iopromide, diatrizoic acid and iopamidol have been identified, their concentrations have been lower than their LOQs.     

Determination of four major saponins in the seeds of Aesculus chinensis Bunge using accelerated solvent extraction followed by high-performance liquid chromatography and electrospray-time of flight mass spectrometry

A new method based on accelerated solvent extraction (ASE) followed by a reliable high-performance liquid chromatography-diode array detector (HPLC-DAD) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) analysis has been developed for the characterization and quantification of four major saponins in extracts of the seeds of Aesculus chinensis Bunge (Semen Aesculi). The saponins escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted from seeds of A. chinesis Bunge via ASE, and the operational parameters of ASE were optimized, such as extraction solvent, extraction temperature, static extraction time and extraction cycles. The optimized procedure employed 70% MeOH as extraction solvent, 120 °C of extraction temperature, 7 min of static extraction time, 60% flush volume and the extraction recoveries of the four compounds were nearly to 100% for two cycles. The HPLC conditions are as follows: SinoChrom ODS BP C₁₈ (4.6 mm × 200 mm, 5 μm) column, acetonitrile and 0.10% phosphoric acid solution as mobile phase, flow rate is 1.0 mL min⁻¹, detection length of UV is 203 nm, injection volume is 10 μL. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of four major saponins in seeds of A. chinesis Bunge with a good linearity (r² > 0.9994), precision (relative standard deviation (R.S.D.) <1.5%) and the recovery ranges of 95.2-97.3%. The limits of detection (LOD) of the four compounds were in the range of 0.40-0.75 μg mL⁻¹. This assay can be readily utilized as a quality control method for Semen Aesculi and other related medicinal plants.     

Qualitative and quantitative determination of polyacetylenes in different Bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry

Polyacetylenes are main toxic ingredients in Bupleurum longiradiatum, a poisonous plant that has ever been misused as substitutes for Chaihu (Bupleuri Radix). For the first time, a high-performance liquid chromatography method coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for qualitative and quantitative analysis of nine polyacetylenes in Bupleurum species. All references, including two new polyacetylenes, were isolated from B. longiradiatum and purified using a semi-preparation HPLC chromatography. The analysis was performed on a TSKgel ODS-100V C18 column (3 μm, 150 mm x 4.6 mm i.d.) using a gradient system of acetonitrile and water, with diode array detection (254 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. A total of 27 Bupleurum samples were examined with this method, which showed a great variety in the distribution and contents of the polyacetylenes. It was found that polyacetylenes (1-8) were the main ingredients in B. longiradiatum, while a few kinds of polyacetylenes (5-8) were also identified in B. smithii, B. smithii var. parvifolium, B. bicaule and B. angustissimum. However, no polyacetylenes (1-9) were detected in the authentic Chaihu samples and the other Bupleurum species. The results indicated that the toxic B. longiradiatum could readily be distinguished from other medicinal Bupleurum species based on the polyacetylene profiles, and the guidelines and quality control of polyacetylenes for Chaihu are useful. The acute toxicity of the ethanol extract of B. longiradiatum and its fractions was also investigated.     

Suitability of selective pressurized liquid extraction combined with gas chromatography-ion-trap tandem mass spectrometry for the analysis of polybrominated diphenyl ethers

A one-step extraction and clean-up method using pressurized liquid extraction (PLE) (selective PLE) combined with gas chromatography-ion-trap tandem mass spectrometry (GC-ITMS-MS) was evaluated for the analysis of polybrominated diphenyl ethers (from tri- to hepta-PBDEs) at low concentrations in fish and shellfish samples. To this end, the performance of an on-line PLE extraction/clean-up method and of a classical Soxhlet extraction and clean-up method using a multi-layer modified silica column were compared. The two sample treatment methods provided similar results, although an important reduction in the sample treatment time (40 min per sample) was achieved using the selective PLE method. In addition, the suitability of the PLE combined with GC-ITMS-MS method was evaluated by comparing the results obtained in the analysis of fish samples with those obtained by gas chromatography-high resolution mass spectrometry (GC-HRMS). Good agreement between both techniques was obtained with differences between the mean values of less than 16%. The selective PLE method coupled to GC-ITMS-MS produced accurate results for PBDE determination with low limits of detection (1.0-16.8 pg g⁻¹ wet weight) and quantification (3.1-51 pg g⁻¹ wet weight) as well as good precision (RSD < 16%). This method has been applied to the analysis of PBDEs in fish and shellfish samples collected at fish markets in Catalonia (NE Spain).     

Optimization of a pressurized liquid extraction method by experimental design methodologies for the determination of fluoroquinolone residues in infant foods by liquid chromatography

In the present study, we have developed a method based on pressurized liquid extraction (PLE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in infant food products. PLE extraction has been optimized by the application of experimental design methodologies. Initially, a fractional factorial design (FFD) was used to screen the significance of four extraction parameters: solvent composition, temperature, pressure and number of cycles. The most significant factors, identified by ANOVA analysis, were the solvent composition, temperature and pressure, which were further optimized with the aid of a face centred design (FCD) and the desirability function. The optimized operating PLE conditions were as follows: ACN/o-phosphoric acid 50 mM pH 3.0 (80:20, v/v), 80 °C, 2000 psi and three extraction cycles of 5 min. Under these conditions, recoveries of the target FQs varied between 69% and 107% with RSDs below 9%. The whole method was validated according to the Commission Decision 2002/657/EC guidelines. The proposed method has been successfully applied to the analysis of different infant food products bought in local supermarkets and pharmacies. The results showed the presence of residues of enrofloxacin in a non-compliant baby food sample corresponding to a chicken-based formulation, which were also confirmed and quantified by LC–MS/MS analysis.     

Quality assessment of a formulated Chinese herbal decoction, Kaixinsan, by using rapid resolution liquid chromatography coupled with mass spectrometry: A chemical evaluation of different historical formulae

Kaixinsan is an ancient Chinese herbal decoction mainly prescribed for patients suffering from mental depression. This decoction was created by Sun Si-miao of Tang Dynasty (A.D. 600) in ancient China, and was composed of four herbs: Radix and Rhizome Ginseng, Radix Polygalae, Rhizoma Acori Tatarinowii and Poria. Historically, this decoction has three different formulations, each recorded at a different point in time. In this study, the chemical compositions of all three Kaixinsan formulae were analyzed. By using rapid resolution LC coupled with a diode-array detector and an ESI triple quadrupole tandem MS (QQQ-MS/MS), the Radix and Rhizome Ginseng-derived ginsenosides including Rb(1), Rd, Re, Rg(1), the Radix Polygalae-derived 3,6'-disinapoyl sucrose, the Rhizoma Acori Tatarinowii-derived α- and β-asarone and the Poria-derived pachymic acid were compared among the three different formulations. The results showed variations in the solubility of different chemicals between one formula and the others. This systematic method developed could be used for the quality assessment of this herbal decoction.     

Selective pressurized liquid extraction of polybrominated diphenyl ethers in fish

A fast and simple method for the analysis of polybrominated diphenyl ethers (PBDEs) in fish samples was developed using a one-step extraction and clean-up by means of pressurized liquid extraction (PLE) combined with gas chromatography-ion trap tandem mass spectrometry (GC-ITMS-MS). The selective PLE method provided to obtain ready-to-analyse extracts without any additional clean-up step, using a sorbent as fat retainer inside the PLE cell. Several PLE operating conditions, such as solvent type, extraction temperature and time, number of cycles and type of fat retainer, were studied. Using Florisil as fat retainer, maximum recoveries of PBDEs (83-108%) with minimum presence of matrix-interfering compounds were obtained using a mixture of n-hexane:dichloromethane 90:10 (v/v) as solvent, an extraction temperature of 100 °C and a static extraction time of 5 min in combination with three static cycles. Quality parameters of the method were established using standards and fish samples. Limits of detection and quantification ranged from 10 to 34 pg g⁻¹ wet weight and between 34 and 68 pg g⁻¹ wet weight, respectively. In addition, good linearity (between 1 and 500 ng ml⁻¹) and high precision (RSD % < 15%) were achieved. The method was validated using the standard reference material SRM-1945 (whale blubber) and was then applied to the analysis of PBDEs in fish samples.     

Determination of fluoroquinolones in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

We developed a sensitive and useful method for the determination of five fluoroquinolones (FQs), enoxacin, ofloxacin, ciprofloxacin, norfloxacin, and lomefloxacin in environmental waters, using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These compounds were analysed within 7 min by high-performance liquid chromatography (HPLC) using a CAPCELL PAK C8 column and aqueous ammonium formate (pH 3.0, 5 mM)/acetonitrile (85/15, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection. In order to optimize the extraction of FQs, several in-tube SPME parameters were examined. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample at a flow-rate of 150 μL/min, using a Carboxen 1010 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r ≥ 0.997) was obtained in the concentration range from 0.1 to 10 ng/mL for all compounds examined. The limits of detection (S/N = 3) of the five FQs ranged from 7 to 29 pg/mL. The in-tube SPME method showed 60-94-fold higher sensitivity than the direct injection method (5 μL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface waters and wastewaters were collected from the area around Asahi River, and ofloxacin was detected in wastewater samples of a sewage treatment plant and other two hospitals at 17.5-186.2 pg/mL. The recoveries of FQs spiked into river water were above 81% for a 0.1 or 0.2 ng/mL spiking concentration, and the relative standard deviations were below 1.9-8.6%.     

Determination of Cinchona alkaloids and Vitamin B6 by high-performance liquid chromatography with fluorescence detection

A simple and specific method has been developed for the simultaneous determination of the four major Cinchona alkaloids and their dihydroderivatives and pyridoxine hydrochloride (Vitamin B₆) by high-performance liquid chromatography (HPLC) with fluorescence detection (λ_em=420 nm with λ_ex=330 nm). The chromatographic separation was performed on a Phenomenex Prodigy ODS column (5 μm,  mm i.d.), recommended for basic compounds, under isocratic reversed-phase conditions. The method allowed a good peak shape and an effective resolution of the tested compounds. The extraction of alkaloids from the Cinchona succirubra bark was carried out in mild and fast conditions (ambient temperature, 20 min) by ultrasonication. The procedure showed to be advantageous respect to a reference method, which involved Soxhlet extraction. The results were compared statistically by means of the Student’s t-test and the variance ratio F-test; no significant difference was found. The method was reproducible (relative standard deviations in the range of 1.0-5.0% for the different alkaloids) and gave quantitative recovery of alkaloids added to bark samples (97.8-105%). For additional informations a photoreactor was arranged between the analytical column and the detector and the online post-column photochemical conversion (irradiation=254 nm) was investigated. Vitamin B₆ was shown to be highly photosensitive, giving significantly different fluorescence spectra with and without UV irradiation. The proposed method was successfully applied to the quality control of Cinchona bark, liquid extract and cosmetics.