Pt,P-codoped carbon nitride nanoenzymes for fluorescence and colorimetric dual-mode detection of cholesterol.

Cholesterol is an important lipid compound found in a variety of foods, and its level in human blood is closely related to human health. Therefore, development of rapid and accurate POCT (point-of-care testing) methods for cholesterol detection is crucial for assessing food quality and early diagnosis of diseases, in particular, in a resource-limited environment. In this study, a smartphone-assisted colorimetric biosensor is constructed based on platinum,phosphorus-codoped carbon nitride (PtCNP2) for the rapid detection of cholesterol. Phosphorus-doped carbon nitride is prepared by thermal annealing of urea and NH4PF6, into which platinum is atomically dispersed by thermal refluxing. The obtained PtCNP2 exhibits an excellent peroxidase-like activity under physiological pH, whereby colorless o-phenylenediamine (OPD) is oxidized to colored 2,3-diaminophenazine (DAP) in the presence of hydrogen peroxide (H2O2), which can be produced during the oxidation of cholesterol by cholesterol oxidase. A smartphone-assisted visual sensing system is then constructed based on the color recognition software, and rapid on-site detection of cholesterol is achieved by reading the RGB values. Meanwhile, the generated DAP shows an apparent fluorescence signal and can realize highly sensitive detection of cholesterol by the change of the fluorescence signal intensity. Such a cholesterol sensor exhibits a wide linear detection range of 0.5-600 µg mL-1 and a low detection limit of 59 ng mL-1. The practicality of the sensor is successfully demonstrated in the rapid detection of cholesterol in serum and food.

Identification and quantification of nine compounds in Fangwen Jiuwei decoction by liquid chromatography-mass spectrometry.

Fangwen Jiuwei Decoction is a traditional Chinese medicine preparation for the treatment of pneumonia developed by Shenzhen Bao'an Chinese Medicine Hospital, which shows remarkable clinical responses. Qualitative and quantitative analyses of the main active compounds are crucial for the quality control of traditional Chinese medicine prescription in clinical application. In this study, we identified nine active compounds essential for the pharmacological effects of Fangwen Jiuwei Decoction based on the analysis of the Network Pharmacology and relevant literature. Moreover, these compounds can interact with several crucial drug targets in pneumonia based on molecular docking. We applied high-performance liquid chromatography-tandem mass spectrometry method was established these nine active ingredients' qualitative and quantitative detections. The possible cleavage pathways of nine active components were determined based on secondary ions mass spectrometry. The results of high-performance liquid chromatography-tandem mass spectrometry were further validated, which show a satisfactory correlation coefficient (r > 0.99), recovery rate (≥93.31%), repeatability rate (≤5.62%), stability (≤7.95%), intra-day precision (≤6.68%), and inter-day precision (≤9.78%). The limit of detection was as low as 0.01 ng/ml. In this study, we established a high-performance liquid chromatography-tandem mass spectrometry method to qualitatively and quantitatively analyze the chemical components in the Fangwen Jiuwei Decoction extract.

Comprehensive quality evaluation of Aconiti Lateralis Radix Praeparata based on pseudotargeted metabolomics and simultaneous determination of fifteen components, and development of new processed products of black slices with less toxicity.

Aconiti Lateralis Radix Praeparata is one of the most famous traditional Chinese medicines possessing a variety of pharmacological activities on top of the toxicities. Due to the heterogeneity and non-standardization of the processing procedures, the subtypes and contents of the differential compounds between different processed products still remained indistinct, causing great risk in their proper use. In order to achieve the comparison and quality evaluation of different processed products of Aconiti Lateralis Radix Praeparata and develop new processed products with less toxicity, a quantification and pseudotargeted metabolomics method was developed based on the dynamic MRM mode of triple quadrupole (QqQ) mass spectrometry, and multivariate statistical analysis methods were applied to compare different processed products. Method validation results indicated good specificity, linearity, repeatability, precision, stability and recovery of the established quantification method and good linearity, precision and stability of the pseudotargeted metabolomics method. Differential compounds of different processed products were screened out and further confirmed by the quantification results. At last, the processing procedures were optimized to obtain new processed products of "Heishunpian" (black slices) with less toxicity, in which the contents of the toxic diester-type diterpenoid alkaloids were reduced from 106.98 µg/g to 0.85-12.96 µg/g. This study provided a valuable reference for the establishment of comprehensive quality evaluation methods of herbal medicines and a scientific basis for the optimization of processing procedures of Aconiti Lateralis Radix Praeparata.

UGTs-mediated metabolic interactions contribute to enhanced anti-inflammation activity of Jinhongtang.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinhongtang, a traditional Chinese medicine (TCM) formula consisting of dry stems of Rheum palmatum L. (Polygonaceae) and Sargentodoxa cuneata (Oliv.) Rehder & E.H.Wilson (Lardizabalaceae) and whole plant of Taraxacum mongolicum Hand.-Mazz. (Asteraceae), is widely used for the treatment of infection diseases including severe sepsis and COVID-19. AIM OF THE STUDY: The present study aimed to explore the compatibility mechanism in the prescription of Jinhongtang based on the pharmacokinetic interaction. MATERIALS AND METHODS: CLP-induced sepsis mice and LPS-induced RAW264.7 cells were used to explore the anti-inflammatory effect of Jinhongtang and herbs in this clinical prescription. Pharmacokinetics of active components in Jinhongtang (Rhein, Emodin and Aloe emodin) was studied in rats. In vitro analysis of metabolic pathways and interactions mediated by metabolic enzymes were conducted using human liver microsomes (HLMs) and recombinant UGT isoforms. RESULTS: Jinhongtang exhibited much more potent anti-inflammatory effect than its single herbs on CLP-induced sepsis mice and LPS-induced RAW264.7 cells. Next, the bioavailability of active ingredients (Rhein, Emodin and Aloe emodin) in R. palmatum was significantly improved through reduced metabolic clearance when co-administered with S. cuneata and T. mongolicum as Jinhongtang during the in vivo pharmacokinetic study, which presented the rational herbal compatibility mechanism. In detailed, the components in S. cuneata and T. mongolicum including Sargentodoxoside A, Chanitracin Ia, Quercetin and Luteolin inhibited the UGT1A9-mediated glucuronidation of active ingredients in R. palmatum, with Ki values of 2.72 µM, 1.25 µM, 2.84 µM and 0.83 µM, respectively. CONCLUSION: T. mongolicum and S. cuneata, the adjuvant herbs of Jinhongtang, could reduce the metabolic clearance of key active components of R. palmatum, prolong their action time and further enhance their anti-inflammatory activity via inhibition of UGTs. Our findings provided deep insight for the rational compatibility of TCMs and useful guidance for the development of TCM formula.

Integration of offline two-dimensional chromatography and mass defect filtering-based precursor ion list data acquisition for targeted characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii.

The hyphenated technique of offline two-dimensional (2D) chromatography with high resolution mass spectrometry (MS) was an efficient tool for separation and characterization of components in complex systems such as herbal medicines, especially those co-eluting components or isomers. In this study, we constructed the ultra-performance convergence chromatography (UPC2) × reversed phase (RP) chromatographic separation system and developed a mass defect filtering (MDF)-based precursor ion list (PIL) acquisition method to improve the selectivity and sensitivity of this technique, and the systematic characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii (namely "Fuzi" in Chinese) was used as an example. The constructed offline 2D separation system showed a good orthogonality of 0.77. Besides, the in-house databases for known and predicted C19- and C20-diterpenoid alkaloids were established by molecular design in Compound Discoverer software for MS data matching and filtering, and two MDF windows were further constructed to screen out more potential diterpenoid alkaloids with novel structures and to obtain the PIL (mass range: even values between 298 and 1020 Da, parent mass width: ±100 mDa) for data acquisition by calculating the m/z values of potential ions using mass range and corresponding mass defect in the MDF windows. In addition, an integrative structure interpretation strategy was developed by integrating elemental composition analysis, ring double bond analysis, neutral loss filtering, diagnostic ion filtering and database matching, etc. As a result, a total of 659 components in the lateral roots of A. carmichaelii were exposed and characterized, including 526 potential new compounds. This strategy showed significant advantages in improving the coverage and selectivity of screening, and could also be applied in systematic characterization of components in other herbal medicines.

Determination of six core components from Mahuang Xuanfei Zhike syrup in rat plasma and tissues by UPLC-MS/MS: Application to a pharmacokinetics and tissue distribution study.

Mahuang Xuanfei Zhike (MXZ) syrup, a Chinese patent medicine, has been widely used in the clinical treatment of cough. However, there is no reported method for the quantitative analysis of the effective components of MXZ syrup in biological samples. In this study, the effective components of MXZ syrup were screened by network pharmacology and molecular docking technology. A sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to test the active components of MXZ syrup in rat plasma and tissue homogenates, including ephedrine, amygdalin, chlorogenic acid, harpagoside, forsythin and forsythoside A. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) and the mass analysis was conducted using a Waters Xevo TQ mass spectrometer using multiple reaction positive and negative ion simultaneous monitoring mode. The results showed that the linearity ranged from 0.3 to 409.4 ng/ml. The extraction recoveries were all <8.33%, and the matrix effects were all <8.45, which met the requirements. The pharmacokinetic and tissue distribution results indicated that the main active components of MXZ syrup were absorbed quickly and eliminated slowly in vivo, and there may be a reabsorption process.

Systematic Qualitative and Quantitative Analyses of Wenxin Granule via Ultra-High Performance Liquid Chromatography Coupled with Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry and Triple Quadrupole-Linear Ion Trap Mass Spectrometry.

Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography-mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.

Sorafenib blocks the activation of the HIF-2α/VEGFA/EphA2 pathway, and inhibits the rapid growth of residual liver cancer following high-intensity focused ultrasound therapy in vivo.

BACKGROUND: Insufficient high-intensity focused ultrasound (HIFU) can promote the rapid progression of the residual tumor through the hypoxia inducible factor-2α +(HIF-2α)/vascular endothelial growth factor A (VEGFA)/ephrin type-A receptor 2 (EphA2) pathway. Although sorafenib has been shown to significantly improve the survival of patients with advanced liver cancer, the use of sorafenib in residual tumor tissues following HIFU has rarely been elucidated. Thus, this study aimed to investigate the potential adjuvant therapeutic effects of sorafenib following HIFU in order to reduce the relapse rate following insufficient HIFU. METHODS: Xenograft tumors were established using nude mice injected with liver cancer cells. At approximately 4 weeks after the inoculation of the tumor cells (tumors reached 1.3-1.5 cm), all mice were randomly divided into 3 groups as follows: i) The control group (no treatment); ii) the HIFU-alone group, and iii) the combination group (HIFU + sorafenib), with 6 mice per group. The residual tumor volume was determined among the different treatment groups. The protein expression levels of HIF-2α, VEGFA and EphA2 were determined by immunohistochemistry and western blotting, and the mRNA levels were detected by RT-qPCR. The microvessel density (MVD) was calculated by CD31 immunohistochemistry staining. RESULTS: The results revealed that by comparing the control group, insufficient HIFU promoted HIF-2α, VEGFA and EphA2 expression (P < 0.05). Compared with the HIFU-alone group, the protein and mRNA levels of HIF-2α, VEGFA and EphA2 were markedly decreased in the group that received combined treatment with HIFU and sorafenib (P < 0.05). Similar results were obtained for MVD expression. Synergistic tumor growth inhibitory effects were also observed between the control group and HIFU group (P < 0.05). CONCLUSIONS: The findings of this study demonstrate that the expression of HIF-2α, VEGFA and EphA2 can be inhibited by sorafenib, and that sorafenib is likely to provide an effective adjunct treatment for patients with HCC following HIFU ablation.

Assembly and Annotation of a Draft Genome of the Medicinal Plant Polygonum cuspidatum.

Polygonum cuspidatum (Japanese knotweed, also known as Huzhang in Chinese), a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance.

Identification and Quantification of Four Anthraquinones in Rhubarb and its Preparations by Gas Chromatography-Mass Spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the determination of four anthraquinones found in rhubarb. Chrysophanol, physcion, aloe-emodin and emodin were confirmed by GC-MS and the possible main cleavage pathways of fragment ions are discussed in this study. Rhubarb is a traditional Chinese medicinal herb which required an effective evaluation method to quantitate the four major active anthraquinone compounds described. The determinations of analytes were accomplished by GC-MS using osthole as an internal standard. MS detection was performed in selected ion monitoring mode to increase the sensitivity. The method was evaluated by a number of validation characteristics (precision, limit of detection, calibration range and recovery). The calibration ranges were all 3.2-30.0 µg/mL. This method was fully validated and showed good performances in terms of recovery (96.9-102.9%) and precision (1.4-2.9%). Finally, the method was applied to the analysis of four anthraquinones in rhubarb and its preparations in the first time.

Simultaneous determination of camptothecin and 10-hydroxycamptothecine in the Camptotheca acuminate, its medicinal preparation and in rat plasma by liquid chromatography with fluorescence detection.

Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10-hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC-FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL-Cl8 column (4.6 × 150 mm, 5 µm), with the mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10 mm) using an gradient elution at the flow rate of 0.6 mL/min. The LC-FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1 ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0-98.6%. The LC-FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma.

The inhibitory effect of piperine from Fructus piperis extract on the degranulation of RBL-2H3 cells.

Allergy is an abnormal immune response to an allergen. Type I hypersensitivity is an immunoglobulin (Ig) E-mediated allergic disorder. Fructus piperis is derived from the ripe fruit of the pepper, which is widely used as a spice in human diets and is also administered as a medicine in many countries. Piperine has been shown to have anti-oxidant, anti-depressant, anti-tumor, and anti-inflammatory activities. However, the effect of piperine on IgE-mediated allergic responses has not been reported. Here, the rat basophilic leukemia cells by membrane chromatography (RBL-2H3/CMC) coupled to high performance liquid chromatography/mass spectrometry (HPLC/MS) to discover and identify piperine can bind to RBL-2H3 cell membranes. Piperine inhibited the expression of cytokines, and the release of both ß-hexosaminidase and histamine, which could be stimulated by antigen in RBL-2H3 mast cells. We found that the levels of intracellular Ca(2+) also decreased. Furthermore, RT-PCR showed that the mRNA expression levels of IL-4, IL-13, and TNF-α were significantly suppressed by piperine. The inhibitory effect of piperine on IgE-mediated degranulation and cytokine production by RBL-2H3 cells may be caused by the inhibition of IgE-mediated signaling pathways, including the phosphorylation of Lyn, p38, Erk, and Ras. In summary, piperine can inhibit antigen-induced allergic reactions that control degranulation.

Characterization and quantification of 10-hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography-ion trap mass spectrometry.

A rapid and sensitive method for the identification and quantification of 10-hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MS(n) spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI-MS(n) fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MS(n) in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative-ion mode at a product ion at m/z 363 → 319 by LC-MS. The LC-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC-MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time.

Identification and quantification of active alkaloids in Catharanthus roseus by liquid chromatography-ion trap mass spectrometry.

Catharanthus roseus is an important dicotyledonous medicinal plant that produces anticancer compounds. The active alkaloids vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine were identified by direct-injection ion trap-mass spectrometry (IT-MS) for collecting MS(1-2) spectra. The determinations of five alkaloids were accomplished by liquid chromatography (LC) with UV and MS detections. The analytes provided good signals corresponding to the protonated molecular ions [M+H](+) and product ions. The precursor ions and product ions for quantification of vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine were m/z 825→807, 457→397, 353→144, 337→144 and 809→748 by LC-IT-MS, respectively. Two methods were used to evaluate a number of validation characteristics (repeatability, LOD, calibration range, and recovery). MS provided a high selectivity and sensitivity for determination of five alkaloids in positive mode. After optimisation of the methods, separation, identification and quantification of the five components in C. roseus were comprehensively accomplished by HPLC with UV and MS detection.

Screening active anti-breast cancer compounds from Cortex Magnolia officinalis by 2D LC-MS.

Most of the anti-breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA-MB-231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA-MB-231 cells membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 × 2.0 mm, 5 µm). The retention fractions were enriched using an online C(18) column (10 × 1.0 mm, 5 µm) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA-MB-231 cell growth were 23 and 64 µM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.

Identification and quantification of atractylenolide I and atractylenolide III in Rhizoma Atractylodes Macrocephala by liquid chromatography-ion trap mass spectrometry.

Rhizoma Atractylodes Macrocephala (RAM) is an important traditional Chinese medicinal herb that is used for treatment of dyspepsia and anorexia. The active ingredients, atractylenolide I (AO-I) and atractylenolide III (AO-III), were identified by direct-injection ion trap-mass spectrometry (IT-MS) for collecting MS(n) spectra. The major fragment ions of AO-I and AO-III were confirmed by MS(n) both in negative ion mode and in positive ion mode. The possible main cleavage pathway of fragment ions was studied. The determinations of AO-I and AO-III were accomplished by liquid chromatography (LC) with UV and MS. The analytes provided good signals corresponding to the protonated molecular ions [M + H](+) and product ions. The precursor ions and product ions for quantification of AO-III and AO-I were m/z 249 → 231 and m/z 233 → 215, respectively, using selected ion monitoring by LC-IT-MS. Two methods were evaluated for a number of validation characteristics (repeatability, limit of detection, calibration range, and recovery). MS provides a high selectivity and sensitivity for determination of AO-III and AO-I in positive mode. After optimization of the methods, separation, identification and quantification of the two components in RAM were comprehensively tested by HPLC with UV and MS.

Analysis of active alkaloids in the Menispermaceae family by nonaqueous capillary electrophoresis-ion trap mass spectrometry.

A nonaqueous CE-IT MS with a nanospray ionization interface method was developed for the identification and quantification of tetrandrine (TET), fangchinoline (FAN), and sinomenine (SIN) using berberine as internal standard. The TET, FAN, and SIN standard solutions were directly infused into IT-MS for collecting MS(1-3) spectra. The major fragment ions of analytes were confirmed and possible main cleavage pathways of fragment ions were studied. A bare fused-silica capillary was used for separation of the analytes. A sheath liquid (50% aqueous methanol containing 0.2% acetic acid) to the capillary effluent with a nanoelectrospray ionization interface was added. Separation buffer comprised 80 mM solution of ammonium acetate, in a mixture of 70% methanol, 20% ACN, and 10% water, which also contained 1% acetic acid. The CE-MS method was validated for linearity, sensitivity, accuracy, and precision, and then used to determine the content of the above components. The detection limits of TET, FAN, and SIN are 0.05, 0.08, and 0.15 µg/mL, respectively. The precision was no more than 4.67% and the mean recovery of the analytes were 95.36-99.24%. This method was successfully applied to determine TET, FAN, and SIN in real samples radix Stephaniae tetrandrae and rhizomes of Menispermum dauricum.

Simultaneous determination of vinblastine and its monomeric precursors vindoline and catharanthine in Catharanthus roseus by capillary electrophoresis-mass spectrometry.

Catharanthus roseus is an important dicotyledonous medicinal plant that contains various anticancer components, such as vinblastine (VLB) and its monomeric precursors (vindoline and catharanthine). A capillary electrophoresis-mass spectrometry (CE-MS) approach for the simultaneous determination of three components was developed in this work. Baseline separation for three components was achieved by using a running buffer consisting of 20 mM ammonium acetate and 1.5% acetic acid in <20 min. Quantification of three components was assigned in positive-ion mode at a protonated molecular ion [M+H](+). The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The detection limits of VLB, catharanthine and vindoline are 0.8, 0.1 and 0.1 µg/mL, respectively. The precision was not more than 4.54% and the mean recovery of the analytes was 95.04-97.04%. The CE-MS method was successfully applied to determine VLB and its monomeric precursors in real sample C. roseus.

Identification and quantification of oleanolic acid and ursolic acid in Chinese herbs by liquid chromatography-ion trap mass spectrometry.

A rapid and sensitive method for the identification and quantification of ursolic acid (UA) and oleanolic acid (OA) in Chinese herbs is described. The method combines liquid chromatography (LC) with ion trap-mass spectrometry (IT-MS) detection. The UA and OA standard solution were directly infused into IT-MS for collecting MS(n) spectra. The major fragment ions of UA and OA were confirmed by MS(n) at m/z 455, 407, 391, 377 and 363 in negative ion mode, and m/z 457, 439, 411 and 393 in positive mode, respectively. The possible main cleavage pathway of fragment ions was studied. UA and OA provided good signals corresponding to the deprotonated molecular ion [M - H](-). The method is reliable and reproducible, and the detection limit is 5 ng/mL. The method was validated in the concentration range of 0.04-40 µg/mL; intra- and inter-day precisions ranged from 0.78 to 2.15%, and the accuracy was 96.5-108.2% for UA and OA. The mean recovery of UA and OA was 97.1-106.2% with RSD less than 1.86%. An LC-IT-MS method was successfully applied to determine the UA and OA in nine Chinese herbs.

Carboxymethylchitosan covalently modified capillary column for open tubular capillary electrochromatography of basic proteins and opium alkaloids.

A novel open tubular (OT) column covalently modified with hydrophilic polysaccharide, carboxymethylchitosan (CMC) as stationary phase has been developed, and employed for the separations of basic proteins and opium alkaloids by capillary electrochromatography (CEC). With the procedures including the silanization of 3-aminopropyltrimethoxysilane (APTS) and the combination of glutaraldehyde with amino-silylated silica surface and CMC, CMC was covalently bonded on the capillary inner wall and exhibited a remarkable tolerance and chemical stability against 0.1 mol/L HCl, 0.1 mol/L NaOH or some organic solvents. By varying the pH values of running buffer, a cathodic or anodic EOF could be gained in CMC modified column. With anodic EOF mode (pH<4.3), favorable separations of basic proteins (trypsin, ribonuclease A, lysozyme and cytochrome C) were successfully achieved with high column efficiencies ranging from 97,000 to 182,000 plates/m, and the undesired adsorptions of basic proteins on the inter-wall of capillary could be avoided. Good repeatability was gained with RSD of the migration time less than 1.3% for run-to-run (n=5) and less than 3.2% for day-to-day (n=3), RSD of peak area was less than 5.6% for run-to-run (n=5) and less than 8.8% for day-to-day (n=3). With cathodic EOF mode (pH>4.3), four opium alkaloids were also baseline separated in phosphate buffer (50 mmol/L, pH 6.0) with column efficiencies ranging from 92,000 to 132,000 plates/m. CMC-bonded OT capillary column might be used as an alternative medium for the further analysis of basic proteins and alkaline analytes.