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 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.

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.

Study on the variation of chemical constituents during combination of ginseng with trogopteroum feces and semen raphani by high performance liquid chromatography-mass spectrometry

A high performance liquid chroatography-electrospray ionization-mass spectrometric method was developed for analysis and identification of ginsenosides from the decoction of ginseng, ginseng with trogopteroum feces and ginseng with semen raphani. Ten ginsenosides were separated and detected. The content variation of these ginsenosides was researched. The experimental results showed, that ginsenosides were less in compatible decoction than in separate one expect Ro. the stripping of ginsenosides were restrained by semen raphani and during combination of ginseng with trogopteroum feces, the precipitates were produced by ginsenosides.

The research of cellulose derivatives as chiral stationary phases and high performance liquid chromatography with polarimeter as on-line detector

Three cellulose derivatives were synthesized and used as chiral stationary phases based on silica gel. The effects of adsorbances on the column numbers and stabilities have been investigated. These stationary phases exhibited high chiral recognition for various racemates. At the same time, the on-line curves of polarimeter were obtained by high performance liquid chromatography with polarimeter as on-line detector.

Detection of menadione sodium bisulfite (vitamin K-3) by reversed-phase high performance liquid chromatography with series dual-electrode amperometric detector

A reversed-phase high-performance liquid chromatography with series dual glassy carbon electrodes for the amperometric detection of water-soluble menadione is described. The complex post-column derivatization reaction and the high background currents were avoided. The menadione sodium bisulfite was reduced at -0.3 V vs. SCE at the upstream (generator) electrode and oxidized at +0.2V vs. SCE at the downstream (collector) electrode. The mobile phase was 0.2moll(-1) HAc-NaAc aqueous buffer (pH 5.50) and 40% (v/v) methanol. The linear response was in the range of 35 ng to 15 mu g, with a detection Limit of 15 ng (S/N=3). The correlation coefficient was 0.9997 (n=6). The electrochemical detection with series dual electrodes has a higher selectivity for menadione (vitamin K-3) compound than with UV detection.

SIMULTANEOUS DETERMINATION OF GUANINE, URIC-ACID, HYPOXANTHINE AND XANTHINE IN HUMAN PLASMA BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH AMPEROMETRIC DETECTION

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C-18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm(-3) pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid; 1-5000 mu mol dm(-3); guanine, 0.5-2000 mu mol dm(-3); hypoxanthine, 0.1-500 mu mol dm(-3) and xanthine, 0.5-5000 mu mol dm(-3). The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.

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.

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.