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1.
Analysis of hormonal steroids in fish plasma and bile by coupling solid-phase extraction to GC/MS 总被引:1,自引:0,他引:1
Budzinski H Devier MH Labadie P Togola A 《Analytical and bioanalytical chemistry》2006,386(5):1429-1439
An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, androstenedione, 17β-estradiol,
estrone, pregnenolone, progesterone, dihydroandrostenedione, dihydrotestosterone, 11α-ketotestosterone, 17α-hydroxyprogesterone,
17α-hydroxypregnenolone, 17α,20β-dihydroxy-4-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and
gas chromatography/mass spectrometry (GC–MS) has been developed. After enzymatic hydrolysis for bile samples only, samples
were concentrated and purified using two successive SPE (C18 and NH2) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) / mercaptoethanol / ammonium iodide (NH4I) and determined by GC–MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range
90–120% in plasma and in the range 60–70% in bile, and the reproducibility was below 10% for the complete procedure. Limits
of detection obtained ranged from 0.1 to 0.4 ng/g in fish plasma and from 1.6 to 14 ng/g in fish bile. The developed method
was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries. 相似文献
2.
Vanesa Guillén-Casla María Eugenia León-González Luis Vicente Pérez-Arribas Luis María Polo-Díez 《Analytical and bioanalytical chemistry》2010,397(1):63-75
Changes in free amino acids content and its potential racemization in ready-to-eat foods treated with E-beam irradiation between
1 and 8 kGy for sanitation purposes were studied. A simple heart cut two-dimensional high performance liquid chromatographic
method (LC–LC) for the simultaneous enantiomeric determination of three pairs of amino acids used as markers (tyrosine, phenylalanine,
and tryptophan) is presented. The proposed method involves the use of two chromatographs in an LC–LC achiral–chiral coupling.
Amino acids and their decomposition products were firstly separated in a primary column (C18) using a mixture of ammonium acetate buffer (20 mM, pH 6) (94%) and methanol (6%) as the mobile phase. Then, a portion of
each peak was transferred by heart cutting through a switching valve to a teicoplanin-chiral column. Methanol (90%)/water
(10%) was used as the mobile phase. Ultraviolet detection was at 260 nm. Detection limits were between 0.16 and 3 mg L−1 for each enantiomer. Recoveries were in the range 79–98%. The LC–LC method combined with the proposed sample extraction procedure
is suitable for complex samples; it involves an online cleanup, and it prevents degradation of protein, racemization of L-enantiomers,
and degradation of tryptophan. Under these conditions, D-amino acids were not found in any of the analyzed samples at detection
levels of the proposed method. 相似文献
3.
Determination of individual bile acids in serum by high performance liquid chromatography 总被引:2,自引:0,他引:2
A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4-bromomethyl-7-methoxycoumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep-Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18-crown-6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm. Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free-bile acids. Recoveries of individual bile acids varied from 83-96% for free and glycine conjugates and 72-83% for taurine conjugates. Coefficients of variation were in the range of 5.1-12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods. 相似文献
4.
A fluorimetric liquid chromatography for highly sensitive analysis of very long chain fatty acids as naphthoxyethyl derivatives 总被引:3,自引:0,他引:3
Summary A simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important
very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based
on the derivatization of the fatty acids with 2-(2-naphtoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the
presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction
temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were
analyzed by HPLC with fluorimetric detection (λex=235 nm; λem=366 nm). The linear range for the determination of docosanoic,
tetracosanoic and hexacosanoic acids was 0.028–1.4 μM with a detection limit of about 5.6 nM (S/N=3) (56 fmol per 10 μL injection).
Application of the method to the analysis the non-esterified (free) very long chain fatty acids spiked in plasma proved feasible. 相似文献
5.
Summary A capillary zone electrophoretic method for the analysis of phenolic acids in soil and plant extracts was developed with direct
UV detection using a phosphate electrolyte solution. The electrophoretic separation required the phenolic acids to be charged
at a pH above their pKa in order to achieve their migration towards the anode. Electroosmotic flow (EOF) was reversed in direction by adding tetradecyltrimethylammonium
bromide (TTAB). Factors affecting the separation selectivity, including the buffer pH and EOF modifiers, were investigated
systematically. Eight phenolic acids were separated and detected in 10 min using an electrolyte containing 25 mM phosphate,
0.5 mM TTAB and 15% acetonitrile (v/v) at pH of 7.20. Linear plots for the test phenolic acids were obtained in a concentration range of 0.01–1 mM with detection
limits in the range of 1.0–7.0 μM. The recoveries ranged from 92.8 to 102.3% in soil and plant tissues samples spiked at 100
μM and the relative standard deviation based on the peak area were ranged 2.0 to 4.5%. The proposed method was used for the
determination of phenolic acids in plant tissue and soil extracts with direct injection. 相似文献
6.
The mobilities of several free and conjugated 5 beta-bile acids, cholesterol and analogues, and alpha, beta-unsaturated sterols and steroidal acids have been investigated with a microbore reversed-phase high-performance liquid chromatographic column (50 cm X 1 mm I.D., 12% C18) with appropriate solvent mixtures at flow-rates of 50-100 microliter/min and a UV monitor set at 193, 198, 212, or 243 nm. With a solvent mixture of 2-propanol-10 mM phosphate buffer, pH 7.0 (160:340) bile acids or their conjugates were separated in a manner similar to those by microBondapak columns (10% C18). The lower detection limit of the conjugates was 20 pmoles with the UV detector set at 193 nm, whereas the lower limit for alpha, beta-unsaturated keto sterols or steroidal acids was 5 pmoles at 243 nm. The detection limit for cholesterol with the UV monitor at 198 nm was 10 pmoles. Contributions of substituent groups of sterols to their time of elution (capacity factor) were calculated for several substituted 4-cholesten-3-ones. 相似文献
7.
Fathalla Belal Saadia M. El-Ashry Ihsan M. Shehata Magda A. El-Sherbeny Dina T. El-Sherbeny 《Mikrochimica acta》2000,135(3-4):147-154
A highly sensitive differential-pulse (DPP) polarographic method is described for the determination of three N-substituted
phenothiazine derivatives, chlorpromazine (CZ), promazine (PZ) and promethazine (PMZ). The method involves the use of nitrous
acid as an oxidant. Polarographically-active sulphoxides with diffusion-current constants (Id) of 2.53, 3.05 and 3.37 were
obtained for CZ, PZ and PMZ, respectively. The polarographic waves were characterized as being diffusion-controlled, irreversible
and partly affected by adsorption phenomena. All parameters affecting the oxidation process and polarographic behaviour were
optimized and incorporated into the procedure. The limiting current-concentration plots in the DPP mode were rectilinear over
the range: 0.006–0.1 mM, 0.005–0.08 mM and 0.008–0.1 mM for CZ, PZ and PMZ, respectively, with minimum detectability (S/N=3)
of 3 × 10−7 M for CZ and PZ, and 4 × 10−7 M for PMZ, respectively. The kinetic parameters of the electrode reaction, including rate constant, free energy of activation
ΔG and effect of temperature on both parameters were studied. The proposed method was successfully applied to the determination
of phenothiazines in dosage forms; the results obtained were in agreement with those given with the official methods. The
method was further applied to the determination of promazine in spiked human urine. The percentage recovery was 96.86 ± 0.30.
The advantages of the proposed method over other reported methods were discussed. A proposal of the electrode reaction was
made.
Received June 1, 1999. Revision March 10, 2000. 相似文献
8.
This paper dealt with a simple and efficient method for separating a mixture of different series of ionic, high polar, and hydrophilic conjugates of bile acids by high-performance ion-pair chromatography (HPIPC) with a new volatile ion-pair chromatographic reagent, di-n-butylamine acetate (DBAA), as a mobile phase additive. The substrates examined included eleven different classes of C-24 glycine- or taurine-amidated, 3-sulfated, 3-glucosylated, 3-N-acetylglucosaminidated, and 3-glucuronidated conjugates of cholic, chenodeoxycholic, urosodeoxycholic, and deoxycholic acids, as well as their double-conjugated forms. The anionic conjugated bile acids were chromatographed on a C18, reversed-phase ion-pair column, eluting with methanol-water (65:35, v/v) containing 5 mM of DBAA as a counter ion. Satisfactory chromatographic separation and column performance were attained by DBAA, compared with conventionally used non-volatile tetra-n-butylammonium phosphate. The present HPIPC method with DBAA provides an insight into the separation and structural elucidation of these biologically important bile acid conjugates and may be proved to be applied to HPLC-mass spectrometric analysis. 相似文献
9.
An enzymatic fluorimetric method is described for the determination of chenodeoxycholic acid and its conjugates and of cholic acid and its conjugates in aqueous solutions and serum. The method is based on the oxidation of 7 α-hydroxy bile acids by β-NAD+ in the presence of 7 α-hydroxysteroid dehydrogenase; the NADH produced is monitored fluorimetrically. Chenodeoxycholic acid is determined in the presence of cholic acid by a differential kinetic procedure; the sum of the two acids (primary bile acids) is determined by an equilibrium procedure, and cholic acid is calculated by difference. The r.s.d. was ca. 3% and 10% for aqueous solutions and sera, respectively. Recoveries of chenodeoxycholic acid, cholic acid and primary bile acids added to serum samples averaged 100.5, 105.1, and 102.9%, respectively. Ten samples can be analyzed per working day. 相似文献
10.
Isolation and Identification of Peptides from Soy 11S-Globulin with Hypocholesterolemic Activity 总被引:2,自引:0,他引:2
A peptide with hypocholesterolemic activity was isolated by HPLC from the pepsin hydrolysate of 11S-globulin. Its molecular
weight (755.2 Da) and amino-acid sequence (Ile-Ala-Val-Pro-Gly-Glu-Val-Ala) were established. The hypocholesterolemic effect
was determined by analysis of bile-acid binding and the percent inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase
in vitro. The lowering of the cholesterol content is explained by bile acids bound to hydrolysate peptides shielding them
from reabsorption and stimulating the transformation of cholesterol in blood plasma.
__________
Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 585–588, November–December, 2005. 相似文献
11.
Using synthetic substrates, an uncomplicated and sensitive procedure for the identification and determination of extracellular
aminopeptidase was developed. The β-naphthylamides of the amino acids were applied for the identification of extracellular
aminopeptidase, whereas the 4-(phenylazo) phenylamides of the amino acids were used for the determination of intra-and extracellular
aminopeptidase activity. The results show a 81.8–88.9% intracellular and 11.1–18.2% extracellular distribution of the studied
enzyme activity.
Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 167–169, March–April, 2007. 相似文献
12.
Mitamura K Sakai T Nakai R Wakamiya T Iida T Hofmann AF Ikegawa S 《Analytical and bioanalytical chemistry》2011,400(7):2061-2072
Previous work from this laboratory has reported the chemical synthesis of N-acetylcysteine (NAC) conjugates of natural bile acids (BAs) and shown that such novel conjugates can be formed in vivo in
rats to which NAC has been administered. The subsequent fate of such novel conjugates is not known. One possible biotransformation
is sulfation, a major pathway for BAs N-acylamidates in patients with cholestatic liver disease. Here, we report the chemical synthesis of the 3-sulfates of the
S-acyl NAC conjugates of five natural BAs (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic). We also
measured the sulfation of N-acetylcysteine–natural bile acid (BA-NAC) conjugates when they were incubated with a rat liver cytosolic fraction. The chemical
structures of the BA-NAC 3-sulfates were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray
ionization-linear ion trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation of singly and
doubly charged deprotonated molecules, structurally informative product ions were observed. Using a triple-stage quadrupole
instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of
a highly sensitive and specific assay. When BA-NACs were incubated with a rat liver cytosolic fraction to which 3’-phosphoadenosine
5’-phosphosulfate was added, sulfation occurred, but the dominant reaction was hydrolysis of the S-acyl linkage to form the unconjugated BAs. Subsequent sulfation occurred at C-3 on the unconjugated BAs that had been formed
from the BA-NACs. Such sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus, NAC conjugates
of BAs as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes. 相似文献
13.
Bierla K Dernovics M Vacchina V Szpunar J Bertin G Lobinski R 《Analytical and bioanalytical chemistry》2008,390(7):1789-1798
A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken
and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction
with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide
(IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid
chromatography (LC) and analysed by ion-paring reversed-phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS).
The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination
of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues
analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing
SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect
and tolerance). 相似文献
14.
Animal biles and gallstones are popularly used in traditional Chinese medicines, and bile acids are their major bioactive constituents. Some of these medicines, like cow-bezoar, are very expensive, and may be adulterated or even replaced by less expensive but similar species. Due to poor ultraviolet absorbance and structural similarity of bile acids, effective technology for species differentiation and quality control of bile-based Chinese medicines is still lacking. In this study, a rapid and reliable method was established for the simultaneous qualitative and quantitative analysis of 18 bile acids, including 6 free steroids (cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid) and their corresponding glycine conjugates and taurine conjugates, by using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This method was used to analyze six bile-based Chinese medicines: bear bile, cattle bile, pig bile, snake bile, cow-bezoar, and artificial cow-bezoar. Samples were separated on an Atlantis dC18 column and were eluted with methanol–acetonitrile–water containing ammonium acetate. The mass spectrometer was monitored in the negative electrospray ionization mode. Total ion currents of the samples were compared for species differentiation, and the contents of bile acids were determined by monitoring specific ion pairs in a selected reaction monitoring program. All 18 bile acids showed good linearity (r2 > 0.993) in a wide dynamic range of up to 2000-fold, using dehydrocholic acid as the internal standard. Different animal biles could be explicitly distinguished by their major characteristic bile acids: tauroursodeoxycholic acid and taurochenodeoxycholic acid for bear bile, glycocholic acid, cholic acid and taurocholic acid for cattle bile, glycohyodeoxycholic acid and glycochenodeoxycholic acid for pig bile, and taurocholic acid for snake bile. Furthermore, cattle bile, cow-bezoar, and artificial cow-bezoar could be differentiated by the existence of hyodeoxycholic acid and the ratio of cholic acid to deoxycholic acid. This study provided bile acid profiles of bile-based Chinese medicines for the first time, which could be used for their quality control. 相似文献
15.
A procedure for the quantification of 9 organic acids, acetic, formic, citric, tartaric, lactic, malic, succinic, oxalic,
and fumaric, in alcoholic and alcohol-free beverages by reversed-phase HPLC on a Pronto-SIL C18 AQ (300 × 3 mm) column (3
μm) with the mobile phase 5 mM Li2SO4 (pH 3.00, H2SO4) at the rate 0.5 mL/min and conductometry detection. The analytical ranges made 5–200 mg/L for tartaric, malic, lactic and
acetic acids, 2–200 mg/L for the citric and fumaric, 10–400 mg/L for succinic, 15–400 for oxalic, and 20–200 for the formic
acids, and so the detection limits: 1 mg/L for tartaric, formic, malic and fumaric, 2 mg/L for lactic, acetic and citric,
5 mg/L for succinic, and 10 mg/L for oxalic acids. The analysis of alcoholic beverages takes 30–40 min, and of non-alcoholic
ones, 20–30 min; the standard deviation of the results of analyses does not exceed 5%. 相似文献
16.
The composition is given of the fatty acids of the red marine algaPalmaria stenogona. The amount of eicosapentaenoic acid (EPA) in the alga was 69.9–77.0% of the total fatty acids. The procedure for isolating
EPA of 80–90% purity from the alga is described which includes the saponification of the algal lipids, the selective extraction
of the unsaponifiable lipids, the isolation of the free fatty acids, and their separation by the method of crystallizing inclusion
complexes of the fatty acids with urea.
Institute of Marine Biology, Far Eastern Branch, USSR Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh
Soedinenii, No. 3, pp. 330–333, May–June, 1990. 相似文献
17.
Summary Capillary electrophoresis with indirect UV detection at 254 nm was applied to simultaneous determination of ∼20% of azelaic
acid and ∼0.1% of sorbic acid in AKNOREN cream. The acids were separated in fused silica capillary (45 cm × 50 μm) at 30 kV.
Optimised back-ground electrolyte was 30 mM benzoate buffer (pH∼6, adjusted with TRIS) containing 7 mM β-cyclodextrin and
5% of methanol; internal standard was 2-hydroxysobutyric acid (HIBA). Rectilinear calibration ranges were 0.4–4 mg mL−1 for azelaic acid and 2–20 μg mL−1 for sorbic acid and the recoveries were 97.2–100.5%. A single analysis took <15 min. 相似文献
18.
We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile. 相似文献
19.
A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile. 相似文献
20.
Liu Z Zhang B Liu Z Li S Li G Geng L Zhao X Bi K Tang X Chen X 《Analytical and bioanalytical chemistry》2012,403(1):323-330
A UPLC-ESI-MS/MS method has been developed and validated for the determination of larotaxel in beagle dog plasma. After addition
of the internal standard, plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether and separated
on a 50 × 2.1 mm ACQUITY 1.7 μm C18 column (Waters, USA), with acetonitrile and 5 mM ammonium acetate as mobile phase, within a runtime of 3.0 min. The analytes
were detected without interference in Multiple Reaction Monitoring mode with positive electrospray ionization. The linear
range was 2.5–5,000 ng/mL. The intra-day and inter-day precisions (relative standard deviation, RSD, %) were within 9.3% and
10.2%, respectively, and the accuracy (relative error, RE, %) was less than 11.5%. The validated method was successfully applied
to a pharmacokinetic study of larotaxel in beagle dogs after intravenous administration of larotaxel-loaded lipid microsphere
with different doses of 0.4, 0.8, and 1.6 mg/kg. The area under the concentration–time curve and the peak concentration of
larotaxel seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics. 相似文献