Determination and identification of isoflavonoids in Radix astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection

The isoflavonoids in Radix astragali were determined and identified by HPLC-photodiode array detection-MS after extraction employing matrix solid-phase dispersion (MSPD). As a new sample preparation method for R. astragali, the MSPD procedure was optimized, validated and compared with conventional methods including ultrasonic and Soxhlet extraction. The amounts of two major components in this herb, formononetin (6) and ononin (2), were determined based on their authentic standards. Four major isoflavonoids, formononetin (6), ononin (2), calycosin (5) and its glycoside (1), and three minor isoflavonoids, (6aR,11aR)-3-hydroxy-9, 10-dimethoxypterocarpan (7), its glycoside (3), and (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavone-7-O-beta-D-glycoside (4), were identified based on their characteristic two-band UV spectra and [M + H](+), [aglycone + H](+) and [A1 + H](+) ions, etc. The combined MSPD and HPLC-DAD-MS method was suitable for quantitative and qualitative determination of the isoflavonoids in R. astragali. (C) 2003 Elsevier B.V. All rights reserved.

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MSn. In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol.

Drug-protein interaction studied by technique of microdialysis combined with high performance liquid chromatography

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines the method of microdialysis combined with high-performance liquid chromatography (HPLC) that has been developed;by ours to study such interactions, in which the microdialysis was applied to sample the free drug in the mixed solution of drug with protein, and HPLC to quantify the concentration of free drug in the microdialysate. This technique has successfully been used for determining various types of binding interactions between the low affinity drugs, high affinity drugs and enantiomers to HSA. For the case of competitive binding of two drugs to a protein in solution, a displacement equation has been derived and examined with four nonsteroidal anti-inflammatory drugs and HSA as model drugs and protein, respectively. Microdialysis with HPLC was adopted to determine simultaneously the free solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Analysis of estrogens in environmental waters using polymer monolith in-polyether ether ketone tube solid-phase microextraction combined with high-performance liquid chromatography

A simple, rapid and sensitive on-line method for simultaneous determination of four endocrine disruptors (17 beta-estradiol, estriol, bisphenol A and 17 alpha-ethinylestradiol) in environmental waters was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). A poly(acrylamide-vinylpyridine-NAP-methylene bisacrylamide) monolith, synthesized inside a polyether ether ketone (PEEK) tube, was selected as the extraction medium. To achieve optimum extraction performance, several parameters were investigated, including extraction flow-rate, extraction time, and pH value, inorganic salt and organic solvent content of the sample matrix. By simply filtered with nylon membrane filter and adjusting the pH of samples to 6.0 with phosphoric acid, the sample solution then could be directly injected into the device for extraction. Low detection limits (S/N = 3) and quantification limits (S/N = 10) of the proposed method were achieved in the range of 0.006-0.10 ng/mL and 0.02-0.35 ng/mL from spiked lake waters, respectively. The calibration curves of four endocrine disruptors showed good linearity ranging from quantification limits to 50 ng/mL with a linear coefficient R-2 value above 0.9913. Good method reproducibility was also found by intra- and inter-day precisions, yielding the RSDs less than 12 and 9.8%, respectively. Finally, the proposed method was successfully applied to the determination of these compounds in several environmental waters. (c) 2006 Elsevier B.V. All rights reserved.

Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data. (C) 2002 Elsevier Science B.V. All rights reserved.

Studies on principal components and antioxidant activity of different Radix Astragali samples using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

The principal components, isoflavonoids and astragalosides, in the extract of Radix Astragali were detected by a high-performance liquid chromatography Couple to electrospray ionization ion trap multiple-stage tandem mass spectrometry (HPLC-ESI-IT-MSn) method. By comparing the retention time (t(R)) of HPLC, the ESI-MSn data and the structures of analyzed Compounds with the data of reference compounds and in the literature, 17 isoflavonoids and 12 astragalosides have been identified or tentatively deduced. By Virtue of the extracted ion chromatogram (EIC) mode, simultaneous determination of isoflavonoids and astragalosides could be achieved when the different components formed overlapped peaks. And this method has been utilized to analyze the constituents in extracts of Radix Astragali from Helong City and of different growth years. Then the antioxidant activity of different samples has been Successfully investigated by HPLC-ESI-MS method in multiple selected ion monitoring(MIM) mode, applying the spin trapping technology, and the Ferric Reducing Antioxidant Power (FRAP) assay was applied to support the result.

Investigation of the hydrolysis of ginsenosides by high performance liquid chromatography-electrospray ionization mass spectrometry

The hydrolysis of ginsenoside standards and the crude extracts of ginseng has been investigated at different pH values (2.4 - 11.2) using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). The experimental results indicated that the pH value of aqueous solutions is an important factor in changing the composition of ginsenosides. For (20S)-protopanaxadiol ginsenosides, ginsenosides with a large mass hydrolyzed to form hydrolysates (20S)-Rg(3) and (20R)-Rg(3) at pH 4.3. There were more hydrolyzed products observed at pH 3.3: (20S)-F-2, C-25,26 hydrated ginsenoside "C-Y-1" and "C-Y-2" (MW = 802 Da) accompanied with (20S)-Rg(3), (20R)-Rg(3). At pH 2.4, only (20R)-Rg(3), (20S)-F-2, a small quantity of (20S)-Rg(3) and three C-25,26 hydrated ginsenosides were obtained. For (20S)protopanaxatriol Re, no hydrolysates were observed at pH 4.3; it was hydrolyzed at pH 3.3 to form hydrolysates (20S)-Rg, (20R)Rg(2) and hydrated C-25,26 (MW = 802 Da) and at pH 2.4 only C-25,26 hydrated ginsenosides "C-Y-1" and "C-Y-2" (MW = 802 Da) were left in the solution. Similar hydrolysis reactions could be also observed for the crude extracts of ginseng. It showed that HPLC/ESI-MS is a fast and convenient method to study the hydrolysis of ginseng.

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation.

Studies on lignan constituents from Schisandra chinensis (Turcz.) Baill. fruits using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

A reversed-phase high-performance liquid chromatography-diode array detector-electrospray ionization multiple-stage tandem mass spectrometry (RP-HPLC-DAD-ESl-MSn) method has been developed for the detection and analysis of lignan constituents in the methanol extract from the fruits of Schisandra chinensis (Turcz.) Baill. RP-HPLC-DAD-ESI-MSn and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-TCR-MSn) have been applied to investigate the characteristic product ions of four lignan reference compounds. Then, the logical fragmentation pathways of the lignans have been proposed. By comparing the retention time (t(R)) of HPLC, the ESI-MSn data and the structures of analyzed compounds with the data of reference compounds and in the literature, 11 peaks in HPLC have been unambiguously identified and another 5 peaks have been tentatively identified or deduced. Also, in the present paper, the extracted ion chromatograms (EIC) have been used to analyze the lignan isomers. The experimental results demonstrate that RP-HPLC-DAD-ESI-MSn is a specific and useful method for the identification of the lignan constituents and their isomers.

Simultaneous Determination of Multi-Organotin Compounds in Seawater by Liquid-Liquid Extraction-High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds (trimethyltin, dibutyltin, tributyltin, diphenyltin and triphenyltin) in seawater samples. Agilent TC-C18 column was used for the separation, the mobile phase of HPLC was CH3CN : H2O: CH3COOH=65 : 23 : 12 (phi), 0.05% TEA, and pH value was adjusted to 3.0 by diluent ammonia. The flow rate was 0.6 mL . min(-1). Five mixed organotin compounds in a mix standard solution from 100 to 0.5 mu g . L-1 were applied for the method assessment. The experimental results indicate that the correlation coefficient of calibration curves (R-2) for each organotin compound was over 0.998 and the detection limits of the five organotin compounds were lower than 3 ng . L-1. Different mixed organic solvents including dichloromethane or toluene were used for extraction of organotin and the extraction condition of organotin from seawater was optimized. The 100 mL seawater acidized by hydrochloric acid was extracted by 10 mL carbon dichloride (CH2Cl2) with 2% tropolone for 10 min twice. Extracted organic solvents were mixed And blown to one drop by nitrogen with the rate of 1.7 mL . min(-1), then 1 mL acetonitrile was added to the drop for redissolving the organotin compounds. Finally, the mixed redissolution was filtered by 0.22 mu m organic filter membrane before analysis. it was found that the only organotin compound in seawater was triphenyltin (TPHT) and the content was 53.2 ng . L-1. The recoveries test from the standard addition for diphenyltin (DPHT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPHT) were over 80%. However, the recovery for trimethyltin (TMT) was relatively low and the value was 50%. The reason might be attributed to the decomposition or adsorption of those compounds during the extraction procedure. Further study on this subject is in progress.

Speciation Analysis of Organotin Compounds in Shellfish by Hyphenated Technique of High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry(HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds in the shellfish samples. Agilent TC-C-18 column was selected, mobile phase of the HPLC was CH3CN:H2O: CH3COOH = 65:23:12 (V/V), 0. 05% TEA, pH = 3.0 at flow rate 0.4 mL/min. Five mixed organotin standards from 100 mu g/L to 0. 5 mu g/L was used for the method evaluation. The experimental results indicate that the linearity (R-2) for each compound was over 0.998. The shellfish samples were treated by supersonic extraction with mobile phase for 30min. Four organotin compounds including dibutyltin (DBT), tributyltin (TBT), diphenyltin (DphT) and triphenyltin (TPhT) in shellfish samples were detected with method mentioned above. It was found that the domain compounds in the samples were tributyltin (TBT) and triphenyltin (TPhT). The recoveries test from the standard addition for trimethyltin (TMT tributyltin (TBT), and triphenyltin (TPhT) were, over 80%. However, the recoveries for diphenyltin (DPhT) and dibutyltin (DBT) were relatively low, 37.3% and 75.2% respectively. The reason might be attributed to the decomposition of those compounds during the extraction procedure. The further study on this subject is under the progress.

Fingerprinting quality control of Qianghuo by high-performance liquid chromatography-photodiode array detection

A novel, simple and accurate fingerprint method was developed using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) for the quality control of Qianghuo, a Tibetan folk and Chinese herbal medicine used as a diaphoretic, an antifebrile and an anodyne. For the first time, the feasibility and advantages of employing chromatographic fingerprint were investigated and demonstrated for the evaluation of Qianghuo by systematically comparing chromatograms of aqueous extracts with the professional analytical software recommended by State Food and Drug Administration (SFDA). Our results revealed that the chromatographic fingerprint combing similarity evaluation could efficiently identify and distinguish raw herbs of Qianghno from different sources and different species. The effects on Notopterygium forbesii Boiss (Apiaceae) chromatographic fingerprints resulted from collecting locations, harvesting time were also examined. (c) 2006 Elsevier lrelanc Ltd. All rights reserved.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.