Qinhua Chen

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.

Identification of volatile compounds of Atractylode lancea rhizoma using supercritical fluid extraction and GC-MS.

Supercritical fluid was used to extract volatile components from the rhizoma of Atractylode lancea (A. lancea). An orthogonal array design (OAD), L(9) (3)(4), was employed as a chemometric method for the optimization of the supercritical fluid extraction (SFE) of volatile compounds from the herbal medicine. Four parameters, namely, pressure, temperature, dynamic extraction time, and flow rate of CO(2), were studied and optimized by a three-level OAD in which the interactions between the parameters were neglected. These compounds were identified according to their retention times and mass spectra by GC-MS. A total of 30 compounds of SFE extracts were identified. Atractylon (8.63%), hinesol (1.44%), beta-eudesmol (6.64%), elemol (0.42%), and atractydin (13.92%) were the major sesquiterpenes identified in A. lancea SFE extracts.

Sensitive determination of four camptothecins by solid-phase microextraction-HPLC based on a boronic acid contained polymer monolithic layer.

Camptothecin (CPT) and its derivative have been revealed to possess special anti-cancer activity, extraction methods are necessary for trace determination of CPTs in complex samples. In this work, we prepared a high efficient boronic acid-based polymer monolithic layer for microextraction of CPTs. A disposable membrane filter-based extraction device was developed, and boronic acid groups were co-polymerized into a polyporous polymer skeleton and served as the monolithic sorbent. The prepared poly(4-VB-MA-TRIM) showed good stability and great extraction efficiency toward four CPTs. After optimization of extraction conditions, poly(4-VB-MA-TRIM)-based solid-phase microextraction was coupled HPLC for determination of CPTs in biological samples. The method exhibited low limits of detection of 0.05-0.2 ng mL(-1), which is significantly more sensitive than reported HPLC methods. The method also showed wide linear range (0.1-100 and 0.5-200 ng mL(-1)), good linearity (R(2)≥0.9981) and good reproducibility (RSD ≤3.76%). The method has been applied in plasma samples, with good selectivity and good recoveries ranging from 85.1 to 104.7%.

Simple and fast determination of reserpine and yohimbine from Rauvolfia yunnanensis by nonaqueous capillary electrophoresis

In this article, we propose very simple procedures to analyze two important indole alkaloids, reserpine and yohimbine, in Rauvolfia yunnanensis. A nonaqueous CE method has been employed using berberine as an internal standard. Optimum separation conditions were obtained when the sample was injected by pressure for 3 s at 50 mbar and then separated with the buffer containing 40 mM ammonium acetate, 2.0% (v/v) acetic acid, and 20% (v/v) acetonitrile in methanol medium at an applied voltage of 25 kV. The analytes were detected at 220 nm. The two alkaloids can be separated within 15 min and quantified with high sensitivity. Good recoveries were obtained with both the procedures, using an internal standard. Finally, these procedures were applied to the analysis of two alkaloids in Rauvolfia yunnanensis.

Comparative Validations of Capillary Electrophoresis and High-Performance Liquid Chromatography Methods for the Simultaneous Determination of Five Anthraquinones in Compound Rhubarb Enema

Compound Rhubarb Enema (CRE) is a classic formula comprised of Rhubarb and Prunus mume in a weight ratio of 5:1 for the treatment of severe hepatitis and chronic kidney disease. High-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC) were compared to simultaneously determine and separate five anthraquinones in CRE. An ultraviolet detector at 254 nm with an isocratic elution of methanol and 10% H3PO4 in water (84:16) at 30°C in HPLC effectively detected this separation in 25 min. In addition, separation is performed by using an electrolyte consisting of 25 mM sodium tetraborate, 4 mM sulfobutylether-β-CD (SBE-β-CD), 20 mM sodium dodecyl sulfonate (SDS), and 2% methanol at pH 10.1 and at a separation voltage of 20 kV in 18 min. The detection limits of anthraquinones were 0.04–0.08 µg mL−1 and 0.4–0.8 µg mL−1 for the HPLC and MEKC methods, respectively. Two methods were evaluated and compared with a number of validation characteristics (repeatability, precision, calibration range, and recovery). The established HPLC and capillary electrophoresis (CE) methods are suitable for quantitative determination of the five components in CRE.

Enzyme-free ultrasensitive fluorescence detection of epithelial cell adhesion molecules based on a toehold-aided DNA recycling amplification strategy

Epithelial cell adhesion molecules (EpCAMs) play a significant role in tumorigenesis and tumor development. EpCAMs are considered to be tumor signaling molecules for cancer diagnosis, prognosis and therapy. Herein, an enzyme-free and highly sensitive fluorescent biosensor, with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy, was developed for sensitive and specific fluorescence detection of EpCAMs. Due to highly specific binding between EpCAMs and corresponding aptamers, strand a, which is released from the complex of aptamer/strand a in the presence of EpCAMs which is bound to the corresponding aptamer, triggered the toehold-mediated strand displacement process. An amplified fluorescent signal was achieved by recycling strand a for ultrasensitive EpCAM detection with a detection limit as low as 0.1 ng mL−1, which was comparable or superior to that of reported immunoassays and biosensor strategies. In addition, high selectivity towards EpCAMs was exhibited when other proteins were selected as control proteins. Finally, this strategy was successfully used for the ultrasensitive fluorescence detection of EpCAMs in human serum samples with satisfactory results. Importantly, the present strategy may be also expanded for the detection of other targets using the corresponding aptamers.

PEEK tube-based online solid-phase microextraction–high-performance liquid chromatography for the determination of yohimbine in rat plasma and its application in pharmacokinetics study

Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R2 of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.

RAPID AND SIMPLE QUANTITATIVE DETERMINATION OF ATRACTYLENOLIDE I AND ATRACTYLENOLIDE III IN ATRACTYLODES MACROCEPHALA AND ITS DIFFERENT PROCESSED PRODUCTS BY CAPILLARY ZONE ELECTROPHORESIS

Atractylenolide I (AO-I) and atractylenolide III (AO-III) are the major sesquiterpenes considered to be bioactive from Atractylodes macrocephala (RAM) and its different processed products, popularly used for the treatment of the various digestive diseases and tumors. A rapid and simple capillary zone electrophoresis (CZE) method to quantitatively determine the AO-I and AO-III is described. Using an optimized method, AO-I and AO-III were separated in less than 10 min with A 50 mM borate buffer and a separated voltage 20 kV at 25°C. The CZE method was validated for linearity, sensitivity, accuracy, and precision and then used to determine the content of AO-I and AO-III. This method was successfully applied to determine AO-I and AO-III in real sample RAM and its different processed products.

Preclinical pharmacokinetic analysis of armillarisin succinate ester in mouse plasma and tissues by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated to determine the concentration of armillarisin succinate ester in mouse plasma and tissues, used for preclinical evaluation. Bavachin was employed as the internal standard. Separation was performed on a 3.5 µm Zorbax SB-C(18) column (30 × 2.1 mm), with a mobile phase consisting of methanol and aqueous 20 mm ammonium acetate. Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via selected ion monitoring in negative scanning mode. Quantification was performed using the transitions m/z 333 → 233 and 323 → 221 for armillarisin succinate ester and internal standard, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic and tissue distribution study after intravenous injection of ASE in mouse in a dose of 10 mg/kg.