Solid acids assisted matrix solid-phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time-of-flight mass spectrometry.

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid-phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time-of-flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32-1.59 ng/mL), good recoveries (86.63-98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid-phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time-of-flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.

Determination and Visualization of Peimine and Peiminine Content in Fritillaria thunbergii Bulbi Treated by Sulfur Fumigation Using Hyperspectral Imaging with Chemometrics.

Rapid, non-destructive, and accurate quantitative determination of the effective components in traditional Chinese medicine (TCM) is required by industries, planters, and regulators. In this study, near-infrared hyperspectral imaging was applied for determining the peimine and peiminine content in Fritillaria thunbergii bulbi under sulfur fumigation. Spectral data were extracted from the hyperspectral images. High-performance liquid chromatography (HPLC) was conducted to determine the reference peimine and peiminine content. The successive projection algorithm (SPA), weighted regression coefficient (Bw), competitive adaptive reweighted sampling (CARS), and random frog (RF) were used to select optimal wavelengths, while the partial least squares (PLS), least-square support vector machine (LS-SVM) and extreme learning machine (ELM) were used to build regression models. Regression models using the full spectra and optimal wavelengths obtained satisfactory results with the correlation coefficient of calibration (rc), cross-validation (rcv) and prediction (rp) of most models being over 0.8. Prediction maps of peimine and peiminine content in Fritillaria thunbergii bulbi were formed by applying regression models to the hyperspectral images. The overall results indicated that hyperspectral imaging combined with regression models and optimal wavelength selection methods were effective in determining peimine and peiminine content in Fritillaria thunbergii bulbi, which will help in the development of an online detection system for real-world quality control of Fritillaria thunbergii bulbi under sulfur fumigation.

A non-derivative method for the quantitative analysis of isosteroidal alkaloids from Fritillaria by high performance liquid chromatography combined with charged aerosol detection.

A non-derivative method was developed for the qualitative and quantitative analysis of isosteroidal alkaloids from Fritillaria thunbergii. During method development the performance of two universal detectors, the charged aerosol detector (CAD) and evaporative light scattering detector (ELSD), were evaluated. The CAD was found to be 30 to 55 times more sensitive than ELSD enabling the measurement of low levels of reference compound impurities that could not be detected by ELSD. The peak area percent of the reference compound, peimisine, obtained by CAD was 50.10%, but 91.66% by ELSD showing that CAD is suitable to estimate the presence of impurities. The CAD showed good reproducibility with overall intra- and inter-day peak area RSD values of less than 1.8% and 2.7%, respectively and had a linear dynamic range of up to 4 orders of magnitude (0.06-44mg/L) for peimine and peiminine. The optimized method was used for the quantitative analysis of peimine and peiminine from F. thunbergii.

[Study on accumulation law of peimine in different Fritillaria thunbergii cultivar].

In order to study the accumulation of Fritillaria thunbergii cultivar, peimine content in Xiaye, Kuanye, Duozi and Xiaosanzi bulbs of different sizes and parts was determined by HPLC-ELSE. The results indicated that the peimine content varied significantly with the cultivar type, the size and part of bulb. The distribution laws of peimine were as follow: Xiaosanzi > Duozi > Xiaye > Kuanye, small-size bulb > big-size bulb, core bud > scale. The peimine yield per plant in Duozi was the highest.

Simultaneous determination of five constituents in Qinpijiegu capsule by high-performance liquid chromatography coupled with tandem mass spectrometry.

A rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed for the simultaneous determination of five constituents in Qinpijiegu capsule (QJC), a classical Tibetan prescription. The separation of five compounds such as aesculin, aesculetin, fraxin, peimine and peiminine was performed on a Purospher STAR LP RP-C18 (250 × 4.6 mm, 5 µm) column with linear gradient elution of acetonitrile-0.3‰ formic acid water in 13 min. Detection was carried out by multiple reaction monitoring mode using electrospray ionization in the positive and negative ion switching mode. The sample was prepared with ultrasound extraction with methanol, which could obtain higher extraction efficiency and shorter extraction time comparing to reflux extraction with alkalized chloroform-methanol. The proposed method was applied to analyze three batches of samples with acceptable linearity (r(2) > 0.9977), precision [relative standard deviation (RSD) < 7.40%], repeatability (RSD < 2.49%), stability [relative error (RE) < 9.15%] and recovery (RSD < 10.76%). This is the first development of a multicomponent quantitation method for the quality control of QJC. Furthermore, the new established method was proven to be highly sensitive and effective in evaluating the quality of QJC.

In vitro and in vivo antitumor activity of Bulbus Fritillariae Cirrhosae and preliminary investigation of its mechanism.

Bulbus Fritillariae Cirrhosae (BFC) is widely used in China both for food and folk medicine because of its powerful biological activities. Firstly, this study was designed to examine the antiproliferative activities of the different fractions from BFC in vitro by MTT assay. The results showed that chloroform extracts (CE) and the purified total alkaloids of BFC (TAF) exhibited stronger antiproliferative activity than the other fractions. We further determined the total alkaloids and 3 main alkaloids monomers content of CE and TAF by UV and HPLC-ELSD methods, respectively. Moreover, we assessed the antitumor activity of TAF in vivo and made preliminary investigation of its antitumor mechanism by histological and immunohistochemical staining technique. These results demonstrate that TAF showed significant antitumor activity and low toxicity in vivo. Meanwhile, TAF significantly inhibited tumor angiogenesis and induced apoptosis by improvement of expression level of caspase-3. These results suggest that alkaloids of BFC could hold a good potential for use as an antitumor drug.

A simple and sensitive LC-MS/MS method for quantitation of peimine and its pharmacokinetic in beagle dogs.

A simple LC-MS/MS method was developed for determination and pharmacokinetic study of peimine in beagle dogs. The chromatographic separation was carried out on a Agilent Zobax SB C18 column with a mobile phase consisting of acetonitrile-10 mmol/L ammonium formate (35:65, v/v) at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by MRM via electro spray ionization source with positive mode. The standard curve for peimine was linear (r>0.999) over the concentration range of 1-200 ng/mL with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (relative standard deviation) values were not higher than 15% and the accuracy (relative error) was <5% at 3 quality control levels. This simple, fast and highly sensitive method was fully validated and successfully applied to a preclinical pharmacokinetic study of peimine in dogs after oral and intravenous administration.

[Simultaneous determination of peimisine and sipeimine in Fritillaria walujewii regel and Fritillaria pallidiflora Schrenk by UPLC-ELSD].

The paper reports the establishment of a method for simultaneous determination of peimisine and sipeimine contents in Fritillaria walujewii Regel and Fritillaria pallidiflora Schrenk. The analyses were performed on an ultra-performance liquid chromatography with evaporative light scattering detection (UPLC-ELSD), equipped with a binary solvent manager, a sampler manager and a column compartment, and connected to Waters Empower 2 software. An Acquity UPLC BEH C18 column (100 mm x 2.1 mm, 1.7 microm) was used for all analysis. The investigated compounds were separated with gradient mobile phase consisting of acetonitrile-0.02% triethylamine-water. The temperature of sample manager was set at 25 degrees C. Drift tube temperature was 40 degrees C, and spray parameter was 40% with injection volume of 1 microL. The investigated compounds including peimisine and sipeimine had good linearity (r > or = 0.9991) over the tested ranges. The average recovery was 94.5% and 98.1% with RSD < or = 2.36%. The UPLC-ELSD method is simple, sensitive and accurate with good repeatability, which is available for quality control of F. walujewii Regel and F. pallidiflora Schrenk.

Sensitive determination of verticine and verticinone in Bulbus Fritillariae by ionic liquid assisted capillary electrophoresis-electrochemiluminescence system.

CE/Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system with the assistance of ionic liquids (ILs) was successfully established for sensitive determination of verticine and verticinone in Bulbus Fritillariae for the first time. Migration behavior of alkaloid largely relies on the hydrogen bonding interactions between alkyl imidazolium cations in ILs and the alkaloids. Running buffer containing 40 mmol/L 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF(4)) IL-8 mmol/L phosphate resulted in significant changes in separation selectivity for alkaloids with similar structures. The highest sensitivity of the detection was obtained by maintaining the detection potential at 1.2V. Under the optimized conditions, relative standard derivations of the ECL intensity and the migration time were 3.27 and 2.84% for verticine and 4.42 and 1.69% for verticinone, respectively. The standard curves were linear between 1x10(-8) and 1x10(-6) mol/L for verticine and between 5x10(-8) and 1x10(-6) mol/L for verticinone, respectively. Detection limits of 1.25x10(-10) mol/L for verticine and 1x10(-10) mol/L for verticinone were obtained (S/N=3). Developed method was successfully applied to determine the amounts of alkaloids in Bulbus Fritillariae.

[Simultaneous determination of peimine, peiminine and zhebeinine in Fritillaria thunbergii from different habitat by HPLC-ELSD].

OBJECTIVE: To determine peimine, peiminine and zhebeinine in Fritillaria thunbergii simultaneously by RP-HPLC-ELSD. METHOD: The HPLC was carried out with a Waters Alliance, Model 2690, equipped with an Xterra RP18 column (3.9 mm x 150 mm, .5 microm), and evaporated light scattering detector. It was eluted gradiently with acetonitrile and 10 mmol x L(-1) NH4HCO3 (adjusted to pH 10. 10 by ammonia solution). RESULT: The average recovery rates (n=5) of peimine, peiminine and zhebeinine were 98.2% (RSD 1.7%), 98.2% (RSD 2.2%), 96.5% (RSD 1.3%) respectively. CONCLUSION: The method is simple, sensitive and reliable. It can be used for quantitative determination of F. thunbergii.

[HPLC fingerprint of Fritillaria hupehensis].

AIM: To establish a fingerprint analysis method of Fritillaria hupehensis. METHODS: fingerprint was performed by HPLC-ELSD. Hypersil ODS column was used; the mobile phase was composed of methanol (with 0.05% triethylamine) and water with gradient elution; flow rate was 1.0 mL x min(-1); recording time was 60 min; drift tube temperature was 75 degrees C; gas flow rate was 1.9 L x min(-1). RESULTS: HPLC fingerprint of Fritillaria hupehensis was obtained. CONCLUSION: A reliable method was provided for controlling the quality of Fritillaria hupehensis.

[Determination of peimine and peiminine in Fritillaria thunbergii by HPLC-ELSD].

AIM: To determine peimine and peiminine in Fritillaria drug simultaneously by RP-HPLC-ELSD. METHODS: HPLC was carried out with a Waters Alliance, Model 2690, equipped with XTerra RP18 column (150 mm x 3.9 mm ID, 5 microm) and evaporated light scattering detector. The mobile phase (acetonitrile-10 mmol.L(-1) NH4HCO3 adjusted to pH 10.10 by ammonia solution) was eluted in gradient mode. RESULTS: The recoveries of peimine and peiminine were 98.96% (n = 4), with RSD 1.01% and 98.40% (n = 4), with RSD 2.63%, respectively. CONCLUSION: The method is simple, sensitive and reliable. It can be used for quantitative determination of Fritillaria drug.

Simultaneous determination of the major isosteroidal alkaloids and their glucosides in the bulbs of Fritillaria by high performance liquid chromatography coupled with evaporative light scattering detection.

AIM: To establish an HPLC-ELSD method for the simultaneous determination of five major bioactive isosteroidal alkaloids and gluco-alkaloids in the bulbs of Fritillaria namely peimissine, imperialine, sinpeinine A, imperialine-3 beta-glucoside and yibeinoside A. METHODS: A Nova-Pak C18 column (150 mm x 3.9 mm ID) was used. The chromatography was carried out with a linear gradient programming. The mobile phase was acetonitrile-water (containing 0.1% diethylamine) and the flow rate was 1.0 mL.min-1. RESULTS: The linear range of peimissine was 13.1-288.2 mg.L-1 (r2 = 0.9975), imperialine-3 beta-glucoside 7.7-169.4 mg.L-1 (r2 = 0.9993), yibeinoside A 7.3-160.6 mg.L-1 (r2 = 0.9997), imperialine 16.5-363.0 mg.L-1 (r2 = 0.9992), sinpeinine A 8.7-191.4 mg.L-1 (r2 = 0.9942). CONCLUSION: The method is accurate with overall intra- and inter-day variation less than 5% and recovery more than 95%. The method was successfully applied to analyze five major bioactive alkaloids and gluco-alkaloids in three Fritillaria bulbs.

[Determination of imperialine and imperialine-3 beta-D-glucoside in bulbs of Fritillaria pallidiflora by HPLC-ELSD].

AIM: To establish an HPLC (high performance liquid chromatography)-ELSD (evaporative light scattering detector) method for the quantification of chromophore-lacking imperialine (I) and imperialine-3 beta-D-glucoside (II) in bulbs of Fritillaria pallidiflora. METHODS: By optimizing the chromatographic conditions of HPLC and the parameters of ELSD to test the methodology. Column: Kromasil C-18 (5 microns, 200 mm x 4.6 mm), mobile phase: acetonitrile-water-triethylamine (71:29:0.03), flow rate: 1.0 mL.min-1, drift tube temperature: 88 degrees C, gas flow-rate: 2.38 L.min-1. RESULTS: The calibration curves were linear in the range of 60.72-161.92 micrograms.mL-1 for I and 48.72-194.88 micrograms.mL-1 for II. The precisions (RSD) of intra-day and inter-day were less than 1% for I and II. The recoveries of the amounts of 24.28, 30.35 and 36.42 micrograms for I were 97.1%, 96.6% and 96.8%, and 24.20, 30.24 and 36.29 micrograms for II were 95.8%, 96.9% and 96.0%. The minimal detectable concentrations of I and II were 2.43 and 1.53 micrograms.mL-1 respectively. CONCLUSION: The established method is simple, rapid, accurate and sensitive, and can be utilized to determine the contents of imperialine and imperialine-3 beta-D-glucoside in crude drug Yibeimu (derived from the bulbs of Fritillaria pallidiflora).

Pre-column derivatization and gas chromatographic determination of alkaloids in bulbs of Fritillaria.

A method of precolumn derivatization GC with FID detection was developed for a simultaneous analysis of five major steroidal alkaloids of Fritillaria species, namely ebeiedine, ebeiedinone, verticine, verticinone and imperialine. Derivatization was carried out by trimethylsilylation of the hydroxyl-containing Fritillaria alkaloids to the corresponding trimethylsilylates with trimethylsilylimidazole. Reaction conditions were optimised and the alkaloids derivatives were characterised by on-line GC-MS. The validated GC method demonstrated a good linearity at the sampling ranges used. This analytical method is simple, convenient and reproducible. The developed assay was successfully applied to the determination of the major pharmacologically active alkaloids in three commonly used antitussive Fritillaria species: F. cirrhosa, F. thunbergii and F. pallidiflora.

[Experimental studies on the quality control of xiaoluo pills].

Microscopic and physico-chemical methods have been used in the qualitative study of Xiaoluo Pills. The contents of peimine, peiminine and calcium carbonate in these pills have been determined. The proposed method can be used to control the quality of Xiaoluo Pills.

Quantitative determination of germines. The rate of transformation of germine-3,16-diacetate in buffered solutions of pH 7.4.

Germine (GER), germine-3-monoacetate (GMA) and germine-3,16-diacetate (GDA) when treated with 9:1 mixture of concentrated sulfuric acid and glacial acetic acid yield a deep purple color with characteristic ultraviolet absorption spectra. Combination of the color reaction with TLC makes possible the quantitative determination of these compounds in buffered solutions of pH 7.4 with a > 90% yield and satisfactory accuracy. It was observed that GDA incubated in such solutions is slowly transformed into GMA. The transformation of GDA to GMA was significantly faster with higher than with lower concentrations of GDA. There was no detectable breakdown of GMA under the same experimental conditions.