LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative evaluation main of the components in Paeoniae Radix Alba–Atractylodis Macrocephalae Rhizoma herbal pair by high‐performance liquid chromatography

The aim of this study was to investigate the influence of compatibility on the contents of main compounds in Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. Ten compounds were separated on an Inertsil ODS‐SP Extend C₁₈ column (250 mm × 4.6 mm, 5 μm) and detected by a diode array detector with the mobile phase consisting of aqueous phosphoric acid (0.1%, v/v; A) and acetonitrile (B) by linear gradient elution. All analytes showed good linearity over a wide concentration range (r² ≥ 0.9989). The limits of detection and quantification were <8.10 and 10.80 μg/mL, respectively. The intra‐ and interday variations were <4.36%. The average recoveries were observed from 94.90 to 103.38%, with relative standard deviation ranging from 1.23 to 3.15% for the analytes. The established method was reliable enough for global quality evaluation of Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, and their co‐decoctions.     

Determination of cinnamic acid and paeoniflorin in traditional Chinese medicinal preparations by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of cinnamic acid in Cinnamomi ramulus and paeoniflorin in Paeoniae radix was established. The samples were separated by a LiChrospher RP-18 column with water-acetonitrile-methanolacetic acid (61:34:5:0.1 or 80:15:5:0.1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Cinnamic acid and paeoniflorin were determined by UV detection at 280 and 250 nm, respectively. The method was applied to determine the optimum conditions for the extraction of the traditional Chinese medicinal preparation Huang Chi Chien Chung Tong, which contains Cinnamomi ramulus and Paeoniae radix. The results indicate that the best extraction conditions involved the use of an ultrasonic bath at 60 degrees C for 30 min. In this experiment, butyl paraben and methyl paraben were used as the internal standards for cinnamic acid and paeoniflorin, respectively. A good and reproducible separation of cinnamic acid and paeoniflorin was obtained within 15 min. The method was also applicable to other preparations that contain Cinnamomi ramulus and Paeoniae radix such as Guey Chi Chia Long Ku Muu Li Tong, Kuei Chi Chien Chung Tong and Tang Kuei Chien Chung Tong.     

Simultaneous determination of hydroxysafflor yellow A and ferulic acid in rat plasma after oral administration of the co-extractum of Rhizoma chuanxiong and Flos Carthami by HPLC-diode array detector

A simple, rapid and accurate HPLC method has been developed for simultaneous determination of hydroxysafflor yellow A and ferulic acid in rat plasma after oral administration of the co-extractum of Rhizoma chuanxiong and Flos Carthami. Plasma samples were deproteinized with 6% perchloric acid, and riboflavin was used as internal standard. The supernatant after centrifuge was injected into a Shimadzu C(18) (150 x 4.6 mm, i.d. 5 microm) column. Gradient elution for A:B (0 min, 90:10; 25 min, 70:30; 27 min, stop) was applied. The mobile phase was composed of 0.022 mol/L potassium dihydrogen phosphate solutions, adjusted to pH 3.0 with phosphoric acid for pump A, and 90% (v/v) acetonitrile for pump B. The assay was shown to be linear over the range 0.046-4.6 microg/mL (r(2) = 0.9995) for hydroxysafflor yellow A and 0.037-3.7 microg/mL (r(2) = 0.9998) for ferulic acid. Mean recovery was 97.5% for hydroxysafflor yellow A and 83.6% for ferulic acid. Both of the intra-day and inter-day precisions were 相似文献   

Fast profiling of chemical constituents in Yiqing Capsule by ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

An ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC-ESI-MS(n)) has been developed for structural characterization and identification of multi-constituents in Yiqing Capsule, a well-known combined herbal remedy prepared from the extract mixtures of Rhizoma Coptidis, Radix et Rhizoma Rhei and Radix Scutellariae. The UPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6 mm×50 mm, 1.8 μm) and gradient elution of 0.1% formic acid solution and acetonitrile in 16 min. Based on their retention times and mass spectra in comparison with the data from standards or references, a total of 29 compounds including 3 phenolic acids and 4 anthraquinones from Radix et Rhizoma Rhei, 8 alkaloids from Rhizoma Coptidis and 14 flavonoids from Radix Scutellariae were unambiguously identified or tentatively characterized in the complex system. The MS data and fragmentation information of two isomers of feruloylquinic acid were first reported in Radix et Rhizoma Rhei and in Yiqing Capsules. This study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of this commonly used Chinese herbal preparation.     

A simple isocratic HPLC method for the simultaneous determination of bioactive components of Scutellariae Radix extract

Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi, has been widely used in Asian countries for the treatment of dermatitis, diarrhoea, inflammatory disease and hepatic disease. A simple, sensitive and precise reversed-phase liquid chromatographic method with isocratic elution was developed to simultaneously determine four bioactive compounds in Scutellariae Radix: baicalein, baicalin, wogonin and wogonoside. Chromatographic analysis was performed on a YMC Pack Pro C(8) column (150?×?4.6?mm(2), 3?μm), with a mobile phase of 0.1% formic acid?:?acetonitrile (70?:?30, v/v) at a flow rate of 1.0?mL?min(-1), and UV detection at 280?nm. Linear behaviour was observed over the investigated concentration range (0.25-10?μg?mL(-1)) for all analytes, with a correlation coefficient of >0.997. The intra- and inter-day precisions were <8.07%, and accuracies were 92.3-102.9%. This method was successfully applied for the analysis of marker compounds for the quality control of Scutellariae Radix extract.     

Quantitative analysis of different batches of raw,wine‐processed,and vinegar‐processed Paeoniae Alba Radix using ultra‐performance convergence chromatography coupled with photo diode array detection

Supercritical fluid chromatography is a safe and ecofriendly analytical technique that has not been fully applied to the analysis of traditional Chinese medicine. This is the first study on the separation of six quality markers—paeoniflorin, albiflorin, benzoyl paeoniflorin, oxypaeoniflorin, gallic acid and benzoic acid—from raw, wine‐baked and vinegar‐baked Paeoniae Alba Radix (PAR) by Supercritical fluid chromatography. Optimum separation was achieved on an HSS C₁₈ SB column (100 × 3.0 mm, 1.8 μm particles) with a gradient elution of high‐purity carbon dioxide as mobile phase A and methanol–acetonitrile (70:30, v/v) with 0.10% phosphoric acid as mobile phase B. The flow rate was set at 0.7 mL/min for 15.0 min. The method was validated in terms of the overall intraday and interday precision, with relative standard deviations (RSDs) of 0.87–2.87 and 1.47–3.63%, respectively. The recoveries were 98.10–103.60% with an RSD of 1.00–3.40%. The stability of the RSD values was in the range 1.10–3.78%. The developed approach was successfully applied and provides a valuable reference for the quality assessment of PAR and processed PAR. The results also revealed that the standardization of processing technology is of great significance to the fluctuations in quality before and after the processing of traditional Chinese medicine.     

Kinetic distribution of paeoniflorin in cortex of normal and cerebral ischemia-reperfusion rats after intravenous administration of Paeoniae Radix extract

The time course of paeoniflorin in the cortex of normal and cerebral ischemia-reperfusion rats, following intravenous administration of Paeoniae Radix extract at a dose of 60 mg/kg of paeoniflorin, was determined using high-performance liquid chromatographic (HPLC) assay. The results showed that paeoniflorin could penetrate through the blood-brain barrier to reach the cortex, and that the injuries of ischemia-reperfusion could play an important role in pharmacokinetic process of paeoniflorin in the cortex after intravenous administration of Paeoniae Radix extract. The cortex concentrations of paeoniflorin in cerebral ischemia-reperfusion rats were lower 5 min after dosing and declined more slowly than that in normal control.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Detection of albiflorin and paeoniflorin in Paeoniae Radix by reversed-phase high-performance liquid chromatography with pulsed amperometric detection

We have developed a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method for the detection of albiflorin and paeoniflorin in Paeoniae Radix and Wu-ji-san. Albiflorin and paeoniflorin were completely separated using 10% acetonitrile in 5 mM sodium phosphate buffer (pH 3.0) as an eluent and detected by PAD under alkaline conditions after using a post-column delivery system. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) were 0.10 and 0.35 ng for albiflorin, and 0.20 and 0.50 ng for paeoniflorin, respectively. The coefficients of linear regression were 0.9995 and 0.9999 for concentrations between 0.035 and 100 μg/mL. The intra- and inter-day precision (RSDs) was less than 3.56% in Paeoniae Radix and Wu-ji-san. The average recoveries from Paeoniae Radix and Wu-ji-san were 99.01–100.94% and 99.46–100.64%. This method shows higher selectivity than HPLC–UV method for analyzing albiflorin and paeoniflorin in Chinese medicinal preparation.     

A chromatographic method for baicalin quantification in rat thalamus

A rapid reversed-phase high-performance liquid chromatographic (rp-HPLC) assay for the determination of baicalin in rat thalamus was developed. This was carried out on a Hypersil -C(18) column using 4-nitro-benzoic acid as the internal standard with a mobile phase of methanol-water-H(3)PO(4) (45:55:0.2, v/v/v). Detection was by UV at 277 nm. The calibration curve for baicalin was linear (r=0.9992) over the concentration range of 0.05--4.0 microg/mL and the limit of detection was 10 ng/mL. The coefficients of variation of intra- and inter-day assays were 2.64, 5.19 and 3.19% and 3.46, 6.21 and 5.58% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The recoveries of baicalin from rat thalamus were 85.4+/- 5.62, 90.7+/- 2.43 and 89.1+/- 4.75% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The method was applied to determine the time course of baicalin in rat thalamus, following a single dosage of intravenous administration of Scutellariae radix extract at 90 mg/kg of baicalin to male Wistar rats.     

HPLC determination of ferulic acid in rat plasma after oral administration of Rhizoma Chuanxiong and its compound preparation

An HPLC method is described for determination of ferulic acid in rat plasma. The concentration of ferulic acid in rat plasma was determined after deproteinization with acetonitrile using sulfamethoxazole as internal standard. Chromatographic separations were performed on a C(18) stationary phase with a mobile phase composed of acetonitrile-water (16:84, v/v) with 1% glacial acetic acid. The UV detection wavelength was set at 320 nm. The method was successfully applied to the determination of pharmacokinetic parameters in rat plasma after oral administration of Rhizoma Chuanxiong and and its compound preparation Suanzaoren decoctions. The calibration curve was linear over the range 0.0510-4.08 micro g/mL in rat plasma. Within-day and between-day precisions were less than 4.5% RSD. Mean recovery was determined as 96.9%. The limit of quantitation was 0.0510 micro g/mL. The pharmacokinetic parameters of the two preparations were different significantly (p < 0.05), which may attribute to the effects of other ingredients present in Suanzaoren decoction.     

Determination of Baicalin by High Performance Liquid Chromatography Coupling with Flow Injection Chemiluminescence Detection

A new method for the determination of baicalin in Scutellariae Radix extract and its preparations by high performance liquid chromatography (HPLC) coupling with flow injection chemiluminescence detection (FIA‐CL) has been developed. The method was based on the chemiluminescence reaction of potassium permanganate with baicalin in nitric acid medium; the CL intensity can be enhanced by formaldehyde. In this study, the conditions of chemiluminescence and chromatography were examined, and the schematic diagram of the HPLC‐FIA‐CL analyzer was optimized. The analytes were separated on Hypersil RP‐C18 columns (100 × 4.6 mm, I.D., 5 μm) by equality elution with 47:53 (v/v) methanol‐0.3% phosphoric acid as a mobile phase at a flow rate of 0.8 mL·min^?1 and a column temperature of 40 °C. Under the optimum condition, the CL intensity was proportional to the concentration of baicalin over the range of 4.10 × 10^?7 ? 6.15 × 10^?5mol·L^?1. The limit of detection (S/N = 3) was 2.79 × 10^?7mol·L^?1 with the relative standard deviation 2.5% (Cs = 6.15 × 10^?6 mol·L^?1, n = 5). The method has been applied to the determination of baicalin in Scutellariae Radix extract and its preparations, and satisfactory results were obtained.     

黄芩中黄芩苷的亚临界水提取及高效液相色谱分析

建立了黄芩药材中黄芩苷的亚临界水提取 高效液相色谱测定方法。分别考察了温度、压力、提取时间、提取物颗粒度、溶剂比等因素对提取量的影响,并与有机溶剂提取法比较。结果表明,当两者具有相同的提取效果时,亚临界水提取法的提取时间及提取溶剂的消耗量大大减少,避免了使用有机溶剂造成的污染。黄芩苷提取最佳条件为:样品颗粒度80～100目,溶剂比0.2 mL/mg ,5 MPa,130 ℃保持10 min。并通过提取 高效液相色谱联机分析实现了对提取物的实时监测。该技术有望成为从中药材中提取脂溶性成分的有效方法。     

Simultaneous determination of wogonin,oroxylin a,schisandrin, paeoniflorin and emodin in rat serum by HPLC–MS/MS and application to pharmacokinetic studies

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti‐inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high‐performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). The separation was performed on a Symmetry C₁₈ column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple‐reaction monitoring with electrospray ionization source in positive–negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu‐gan‐kan‐kang‐yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.     

Simultaneous determination of ten constituents in herbal formulation Sipjeondaebo-tang using HPLC-PDA

A simple, rapid, and accurate high-performance liquid chromatographic method was applied to the quantitative determination of 10 components, namely, 5-hydroxymethyl-2-furaldehyde, albiflorin, paeoniflorin, liquiritin, ferulic acid, nodakenin, coumarin, cinnamic acid, cinnamaldehyde and glycyrrhizin in the traditional herbal formulation Sipjeondaebo-tang (SJDBT). All components were separated within 40 min using a Gemini C₁₈ column maintained at 40°C. The mobile phase was composed of 1.0% (v/v) aqueous acetic acid and 1.0% (v/v) acetic acid in acetonitrile by gradient elution. The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 230, 254, 280, 320, and 330 nm. The calibration curves showed good linearity (r ² ≥ 0.9993) in different concentration ranges. The recovery of each component was in the range of 92.3–104.8%, with relative standard deviations (RSDs) less than 2.5%. The RSDs for intra- and inter-day precision were 0.09–2.8% and 0.07–3.2%, respectively. The amounts of the 10 components in SJDBT were in the range of 0.18–5.77 mg/g.     

Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography

A method of micellar electrokinetic capillary electrophoresis for determining the six flavonoids in Scutellariae Radix (i.e., baicalin, baicalein, wogonin 7-O-glucuronide, wogonin, oroxylin A 7-O-glucuronide, and oroxylin A) has been developed. The buffer solution (pH 7.24) composed of 20 mM sodium dodecyl sulfate (SDS), 20 mM sodium dihydrogen phosphate, and 25 mM sodium borate was found to be the most suitable electrolyte for the separation. The contents of the six flavonoids in crude Scutellariae Radix could easily be determined within about 15 min. On-column UV (254 nm) monitoring allowed the quantitative determination of baicalin. The effects of pH, surfactant concentration, and applied voltage on the migration behavior of the solutes were studied.     

中药复方小续命汤抗阿尔茨海默病有效成分组中6种有效成分的同时测定

建立了用高效液相色谱分析中药复方小续命汤抗阿尔茨海默病有效成分组中各成分的方法,并将其应用于此有效成分组的质量控制。采用ODS色谱柱,以水-甲酸-乙二胺(流动相A，体积比为100∶0.1∶0.1)和甲醇-甲酸(流动相B，体积比为100∶0.05)为流动相,采用梯度洗脱，流速1 mL/min，检测波长240 nm。在上述色谱条件下,可分离测定有效成分组中的6种成分。6种成分均具有良好的线性；其回收率分别为:芍药苷99.1%,升麻苷99.6%,黄芩苷98.4%,5-O-甲基维斯阿米醇苷99.9%,防己诺林碱99.6%,粉防己碱102.0%;相对标准偏差（RSD）分别为1.3%,1.4%,0.4%,0.8%,0.2%,1.4%。所建立的方法简便、快捷,可用于小续命汤有效成分组的质量控制。     

Simple preparation of baicalin from Scutellariae Radix

A simple, rapid and reproducible method was developed to isolate high-purity baicalin from Scutellariae Radix, the dried roots of Scutellaria baicalensis Georgi. The method involves partition/recrystallization steps without repeated column chromatography or special instruments. Isolated baicalin was characterized by comparisons of TLC, HPLC, IR, MS, and NMR data with an authentic sample. Moreover, beta-glucuronidase hydrolysis of baicalin sequentially yielded glucuronic acid and baicalein as confirmed by co-TLC with authentic samples. The purity of baicalin was more than 97% with yield ca. 8.7% (w/w). The method presented here appears suitable for commercial application.     

Screening the anti-gout traditional herbs from TCM using an in vitro method

The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation and xanthine oxidase(XOD) activity.The anti-inflammation effects of the herbal extracts were detected by an in vitro cell model,which was established by stimulating human umbilical vein endothelial cells(HUVEC) using sodium urate(MSU).In this model,the intercellular adhesion molecule-1(ICAM-1) and interleukin-1 beta(IL-1β) were expressed,and the anti-inflammation effects of herbal extracts were evaluated by detecting the content changes of ICAM-1 and IL-1β in cell lysates and cell culture supernates using an enzyme-linked immunosorbent assay(ELISA).Moreover,an ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC-MS/MS) method was used for the detection of XOD activity and the screening of XOD inhibitors in this research.The amount of uric acid from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid level could be reduced via the addition of an inhibitor.Results indicated that Salviae Miltiorrhizae Radix et Rhizome,Rhei Radix et Rhizoma,Polygoni Cuspidati Rhizoma et Radix,Selaginellae Herba,Paeoniae Radix Rubra,especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD activity.The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated with their bioactive components.And the differences between the herbal extracts were correlated with the amount of flavonoid and anthraquinone components.In our study,we have investigated the potential anti-inflammation bioactivity of 33 herbal extracts in vitro,which could provide a reference for further in vivo research in the prevention and treatment of gout.