Zongtao Lin

Identification of antioxidants in Fructus aurantii and its quality evaluation using a new on-line combination of analytical techniques.

A new on-line method for simultaneous identification and monitoring of antioxidants in Fructus aurantii was established by coupling high performance liquid chromatography-diode array detector-electrospray ionisation-ion trap-time of flight-mass spectrometry with post-column derivatisation and luminol-potassium ferricyanide chemiluminescence (HPLC-DAD-ESI-IT-TOF-MS-PCD-LPFCL). While the HPLC fingerprint, structural identification and radical scavenging profile were rapidly obtained by an on-line assay using ultraviolet (UV) absorption, MS and LPFCL, details of the precise substitution patterns of various structures were achieved through UV absorption using PCD addition of shift reagents. Twenty-five flavonoids were identified by either their PCD and MS data or comparison with reference substances. Data collected both from chromatograms and activity profiles of 12 samples revealed significant differences among samples from different habitats. The results showed that this method was rapid and precise, and therefore would be an effective and sensitive method for biocompounds analysis and quality evaluation for complex food and medicinal samples.

Comprehensive identification of 125 multifarious constituents in Shuang–huang–lian powder injection by HPLC-DAD-ESI-IT-TOF-MS

A high-performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method was established for excellent separation and structural identification of constituents in Shuang-huang-lian powder injection (SHLPI). The typical ultraviolet absorptions, accurate empirical molecular formula and reasonable fragmentation mechanisms of these ingredients were used for their structural elucidation. In consequence, 125 constituents (33 phenolic acids, 29 flavonoids, 32 phenylethanoid glycosides, 15 iridoid glycosides, 8 lignans, 3 amino acids and 2 purines nucleosides, 2 quinoid glycosides and 1 alkylbenzene glycoside) were either unequivocally identified or tentatively characterized by comparing authentic standards or published data. The result showed that this study could provide valuable information for the quality control and further investigation of SHLPI formula.

Simultaneous separation, identification and activity evaluation of three butyrylcholinesterase inhibitors from Plumula nelumbinis using on-line HPLC-UV coupled with ESI-IT-TOF-MS and BChE biochemical detection

We have firstly established a method of high-performance liquid chromatography-ultraviolet analysis coupled with electrospray ionization-ion trap-time-of-flight mass spectrometry and butyrylcholinesterase biochemical detection (HPLC-UV-ESI-IT-TOF-MS-BChEBCD). Applying this on-line method to the identification of BChE inhibitors in a Plumula nelumbinis sample, three alkaloids, namely liensinine, isoliensinine, and neferine, have been detected as having a strong BChE inhibition activity for the first time; in addition, norisoliensinine and 6-hydroxynorisoliensinine were proposed as two new compounds identified by their UV and MS data. The HPLC fingerprint, the MS fragments of the components, and the BChE activity profile could be simultaneously recorded during real-time analysis of complex samples using this on-line approach. Tacrine, a BChE inhibitor, was used as a positive reference compound, and its detection limit in the biochemical detection system was 1 nmol. The BChE activity of 1g of P. nelumbinis sample was equal to that of 127.88 μmol tacrine. The proposed on-line method has been validated as having good precision and reproducibility, and could be used to rapidly identify BChE inhibitors and to screen potential drugs for the treatment of Alzheimers disease in complicated samples.

An on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection system applying two antioxidant methods for activity evaluation of the edible flowers from Prunus mume.

An on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection (HPLC-DAD-ESI-IT-TOF-MS-TACD) system was created for identification and evaluation of antioxidants in Prunus (P.) mume flowers. Applying this system, the HPLC fingerprint, ultraviolet (UV) spectra, mass fragmentations, active profiles against 1,1-diphenylpicryl-2-hydrazyl radical (DPPH•) scavenging activity and ferric reducing antioxidant power (FRAP) of each complex sample were obtained simultaneously after one injection. Synchronous structure identification and activities screening of complex samples were thus accomplished. In this study, 78 compounds were identified from P. mume flowers by their chromatographic behaviors, UV spectra and MS data with the assistance of standard compounds and literature reports. The DPPH and FRAP activity of 24 samples (23 different P. mume varieties and 1 related herbal medicine) were then quantified by their detailed activity profiles from the on-line system, and by the total activity of each sample extract from off-line 96-well plate method. As a result, 21 and 32 compounds in the on-line system showed anti-oxidative effects against DPPH and FRAP, respectively. The established on-line system is efficient, sensitive and reliable to tell the DPPH and FRAP antioxidant activities of individual compound in complex samples, and therefore would be a useful and promising technique for antioxidant screening from different food and medicinal matrices.

Rapid screening and identification of compounds with DNA-binding activity from Folium Citri Reticulatae using on-line HPLC–DAD–MSn coupled with a post column fluorescence detection system

To study the interactions between natural compounds and deoxyribonucleic acid (DNA), a method has been established combining a high-performance liquid chromatography-diode array detector-multi-stage mass spectrometer with a fluorescence detector (HPLC-DAD-MS(n)-FLD). The FLD was used to monitor fluorescence intensity of the ethidium bromide-DNA (EB-DNA) complex when a compound separated by HPLC was introduced. This novel method was used to simultaneously obtain the HPLC fingerprint, UV spectra, MS(n) fragments and DNA-binding activity profile of various components in Folium Citri Reticulatae. As a result, 35 compounds were identified, of which 25 were found in the extract of Folium Citri Reticulatae for the first time, and 33 compounds showed DNA-binding activities, with the most active being feruloylhexaric and p-coumaroylhexaric acids. In addition, the precision, stability and reproducibility of this method were validated by two positive controls, quercetin and hesperidin. This new on-line method is accurate, precise and reliable for further high-throughput screening of DNA-binding compounds from food samples and other complex matrices.

Rapid screening of transferrin-binders in the flowers of Bauhinia blakeana Dunn by on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-transferrin-fluorescence detection system.

Transferrin (Transferrin, TRF, TF) has drawn increasing attention in cancer therapy due to its potential applications in drug delivery. TF receptor, highly expressed in tumor cells, recognizes and transports Fe(3+)-TF into cells to release iron into cytoplasm. Thus, discovering TF-binding compounds has become an active research area and is of great importance for target therapy. In this study, an on-line analysis method was established for screening TF-binding compounds from the flowers of Bauhinia blakeana Dunn using a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-transferrin-fluorescence detector (HPLC-DAD-MS(n)-TF-FLD) method. As a result, 33 of 80 identified or tentatively characterized compounds in the sample were TF-binding active. Twenty-five flavonol glycosides and eight phenolic acids were identified as TF-binders. Twelve of these active compounds together with six standard compounds were used to study the dose-response effects and structure-activity relationships of flavonoids and phenolic acids. The method was validated by vitexin with a good linearity in the range of concentrations used in the study. The limit of detection for vitexin was 0.1596 nmol. Our study indicated that the established method is simple, rapid and sensitive for screening TF-binding active compounds in the extract of Bauhinia blakeana Dunn, and therefore is important for discovering potential anti-cancer ingredients from complex samples for TF related drug delivery.

An analysis method for simultaneous screening of deoxyribonucleic acid-binding active compounds and investigating their mechanisms by ultra-fast liquid chromatography tandem mass spectrometry coupled with fluorescence detection technology

DNA has been known as the cellular target for many cytotoxic anticancer agents over the years. Discovering DNA-binding compounds has become an active research area, while various DNA-binding mechanisms make the drug discovery even more difficult. In this article, we present a novel analysis method to rapidly identify specific DNA-binding compounds from Pyrrosia lingua (Thunb.) using DNA-dual-fluorescent probes, ethidium bromide and Hoechst 33258, with the technology of ultra-fast liquid chromatography-diode array detector-tandem mass spectrometry and dual-wavelength fluorescence detector (UFLC-DAD-MS(n)-DFLD). Sixty-two compounds were identified, of which 22 were found to be active in DNA-binding. After investigation of their dose-response behaviors and structure-activity relationships, chlorogenic acids and flavonoid glycosides were found to be DNA-binders via both minor groove-binding and intercalation modes. The precision, reproducibility and stability of this method were validated by vitexin. The established system was sensitive, precise, and reliable to be used for both screening of DNA-binding compounds and investigating of their mechanisms.

Rapid Identification and Assignation of the Active Ingredients in Fufang Banbianlian Injection Using HPLC-DAD-ESI-IT-TOF-MS.

Fufang Banbianlian Injection (FBI) is a well-known traditional Chinese medicine formula composed of three herbal medicines. However, the systematic investigation on its chemical components has not been reported yet. In this study, a high-performance liquid chromatography combined with diode-array detector, and coupled to an electrospray ionization with ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method, was established for the identification of chemical profile in FBI. Sixty-six major constituents (14 phenolic acids, 14 iridoids, 20 flavonoids, 2 benzylideneacetone compounds, 3 phenylethanoid glycosides, 1 coumarin, 1 lignan, 3 nucleosides, 1 amino acids, 1 monosaccharides, 2 oligosaccharides, 3 alduronic acids and citric acid) were identified or tentatively characterized by comparing their retention times and MS spectra with those of standards or literature data. Finally, all constituents were further assigned in the individual herbs (InHs), although some of them were from multiple InHs. As a result, 11 compounds were from Lobelia chinensis Lour, 33 compounds were from Scutellaria barbata D. Don and 38 compounds were from Hedyotis diffusa Willd. In conclusion, the developed HPLC-DAD-ESI-IT-TOF-MS method is a rapid and efficient technique for analysis of FBI sample, and could be a valuable method for the further study on the quality control of the FBI.

Pharmacokinetics of honokiol after intravenous guttae in beagle dogs assessed using ultra-performance liquid chromatography–tandem mass spectrometry

A simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of honokiol in beagle dog plasma after intravenous guttae. With addition of the internal standard magnolol, plasma samples were precipitated with methanol and separated on a Shim-pack XR-ODS II (2.0 × 100 mm, 2.2 µm) with isocratic elution of methanol and water (80:20) solution at a flow rate of 0.2 mL/min. A good separation of honokiol was achieved within 3.5 min. Quantification was performed on a Waters Quattro Premier XE triple quadrupole mass spectrometer with electrospray ionization inlet in the negative multiple reaction monitoring mode. Good linearity was obtained over the concentration range of 5.12-15580 ng/mL (r(2) > 0.998). Intra- and inter-day precisions were <13.10%, and accuracy ranged from 89.21 to 99.92%. The lower limit of quantification for honokiol was 5.12 ng/mL, and honokiol was stable under various conditions (three freeze-thaw cycles, short-term temperature, post-preparative and long-term temperature conditions.). This validated method was successfully applied to the pharmacokinetic study of honokiol in dogs by intravenous guttae.

An on-line analytical approach for detecting haptens in Shuang-huang-lian powder injection

Shuang-huang-lian powder injection (SHLPI) is a traditional Chinese medicine injection (TCMI) frequently used in the clinical treatment of faucitis, bronchitis, and other viral and bacterial infections of upper respiratory tract. However, its allergenic reactions, being the main adverse effects (AEs) of SHLPI, have been a serious problem of its clinical safety. This problem has not been solved due to short of methods for detecting haptens in complex TCMIs. In this study, an on-line high-performance liquid chromatography-diode-array detector-mass spectrometry combined with bovine serum albumin-fluorescence detector (HPLC-DAD-MS-BSA-FLD) system was established for the first time, validated and applied for identification of haptens in SHLPI. Fourteen of 35 identified compounds showed BSA binding activity, and they were six flavonoids, six caffeoylquinic acids (CQAs), and two phenylethanoid glycosides. The structure-activity relationships of 10 active components were studied, and their ability of sensitization together with that of two CQAs were further verified by ELISA assay. It was found that 10 compounds had sensitization, and flavonoids showed stronger sensitizability than CQAs while the diCQAs were slightly stronger than caffeoylquinic acids. The system was validated using 3-CQA as a positive control, and was proved to have good reproducibility, stability, precision (RSD<0.1%) and linearity (R2>0.9993). This online system is fast, sensitive and efficient for screening haptens in traditional Chinese medicine injection (TCMI), provides a new approach to reveal the chemical basis of haptens in TCMIs.