Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

Determination of low levels of 4-fluorophenylalanine incorporation into proteins

This paper describes a rapid method for the determination of low levels of 4-fluorophenylalanine biosynthetically incorporated into proteins. Precolumn derivatization with phenylisothiocyanate followed by reverse-phase high-performance liquid chromatography enables determination of the amino acid composition of the protein as well as accurate detection of a 1-3% substitution of phenylalanine by fluorophenylalanine.     

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry

This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.     

Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample

A flow injection analysis (FIA) system combined with Rayleigh light scattering (RLS) detection is developed for the sensitive and rapid determination of protein concentration in human serum sample. This method is based on the weak intensity of RLS of Eriochrome Black T (EBT, 2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitronaphthalene-4-sulfonic acid sodium salt), which can be enhanced by the addition of protein in weakly acidic solution. The effects of pH and interfering species on the determination of protein were examined. Calibrations for protein, based on RLS intensity, were linear in the concentration ranges of 7-36 microg/ml for human serum album (HSA) and 8-44 microg/ml for bovine serum album (BSA). The detection limits of the method were found to be 0.882 and 2.507 microg/ml for HSA and BSA, respectively. A relative standard deviation of 0.76% (n=5) was obtained with 20 microg/ml HSA standard solution. The FIA-RLS method was more stable than the general RLS method, and the average RSD value of FIA-RLS was less than that of the general RLS. The sample rate was determined to be 90 samples per hour.     

A rapid and direct method for the quantitative determination of tryptophan in the intact protein

1. A method is given for the quantitative determination of free tryptophan or tryptophan in the intact protein by treating with ninhydrin in a mixture of formic acid and hydrochloric acid (reagent b), for 10min at 100 degrees C. Glycyltryptophan was used as a standard for the determination of tryptophan in the intact protein. The extinction at 390nm was linear in the range 0.05-0.5mumol for free tryptophan (in7120) and 0.05-0.30mumol for glycyltryptophan (in15400). 2. Free tryptophan in the presence of protein may be determined by treating with ninhydrin in a mixture of acetic acid and 0.6m-phosphoric acid (reagent a) for 10min at 100 degrees C, the extinction being linear for tryptophan in the range 0.05-0.9mumol. N-Terminal tryptophan peptides also give the typical yellow product on treatment with reagent a. 3. Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others. A mean tryptophan content of 11.25 (s.e.m. +/-0.08) mumol/100mg of protein was found in rat brain during development from 1 to 82 days after birth.     

Armadillo: domain boundary prediction by amino acid composition

The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.     

Simultaneous determination of mycophenolic acid and its phenolic glucuronide in human plasma using an isocratic high-performance liquid chromatography procedure

Simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in plasma was accomplished by isocratic HPLC with UV detection. After protein precipitation and phase separation with saturated sodium dihydrogenphosphate, chromatographic separation was achieved on a monolithic column "Chromolith Performance RP-18e", with acetonitrile/0.01 M phosphate buffer, pH 3, (25:75, v/v), as the mobile phase; flow rate 3.3 ml/min and measurement at 214 nm. Linearity was verified up to 40 mg/l for MPA and up to 400 mg/l for MPAG. Detection limits based on the analysis of 50 microl plasma were 0.05 and 0.5 mg/l for MPA and MPAG, respectively. Accuracy was 99.6-104% for MPA and 95.6-105% for MPAG and total imprecision (CV) was <7% for both compounds. Analytical recovery was >95% for MPA and MPAG. The method is simple, rapid, accurate and suitable for routine determination of MPA and MPAG in plasma.     

Calculation of protein extinction coefficients from amino acid sequence data

Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.     

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Sch?gger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.     

Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species

Recent studies have demonstrated that plasma nitrite (NO2-) reflects endothelial nitric oxide synthase activity and it has been proposed as a prognostic marker for cardiovascular disease. In addition, NO2- itself has been shown to have biological activities thought to be triggered by reduction back to NO in blood and tissues. The development of sensitive and reproducible methods for the quantitative determination of plasma NO2- is, therefore, of great importance. Ozone-based chemiluminescence assays have been shown to be highly sensitive for the determination of nanomolar quantities of NO and NO-related species in biological fluids. We report here an improved direct chemiluminescence method for the determination of plasma NO2- without interference of other nitric oxide-related species such as nitrate, S-nitrosothiols, N-nitrosamines, nitrated proteins, and nitrated lipids. The method involves a reaction system consisting of glacial acetic acid and ascorbic acid in the purge vessel of the NO analyzer. Under these acidic conditions NO2- is stoichiometrically reduced to NO by ascorbic acid. Fasting human plasma NO2- values were found in the range of 56-210 nM (mean=110+/-36 nM). This method has high sensitivity with an accuracy of 97% and high precision (CV<10%) for determination of plasma nitrite. The present method is simple and highly specific for plasma NO2-. It is particularly suited for evaluating vasculature endothelial NO production that predicts the risks for cardiovascular disease.     

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.     

Spectrophotometric determination of acid mucopolysaccharides

The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.     

Determination of iohexol clearance by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

We have developed a simple, rapid, and accurate HPLC-MS/MS method for the determination of iohexol in serum. The column used was a Zorbax Eclipse XDB-C8 (100 mm x 2.1 mm i.d., 3.5 microm). Mobile phases consisted of water containing 2mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2 mM ammonium acetate and 0.1% formic acid (B). After simple protein precipitation with ZnSO4, serum samples were mixed with I.S. (bromperidol) and centrifuged for 3 min. The obtained extraction recovery at three levels was 94.6-107.4%. Quantitative analysis was performed in the multiple reaction-monitoring mode (m/z 822.0-->804.0 for iohexol, 420.1-->122.7 for I.S.) with the total running time of 3 min for each sample. The assay was linear between 0.5 and 1500 microg/mL (r2 > 0.997). The intra- and inter-assay coefficient of variations were 2.4-6.2% and 5.5-6.5%, respectively. Our method provided sufficient analytical range and specificity for the 210 clinical samples analyzed.     

Simultaneous determination of icariin, icariside II and osthole in rat plasma after oral administration of the extract of Gushudan (a Chinese compound formulation) by LC-MS/MS

A sensitive, specific and accurate LC-MS/MS method was developed for simultaneous determination of icariin, icariside II and osthole in rat plasma. With carbamazepine as the internal standard, plasma samples were prepared by protein precipitation with acetonitrile. Analysis was carried out on an ACQUITY UPLC BEH C(18) column with a linear gradient and 0.1% aqueous acetic acid and acetonitrile were used as mobile phase. Detection was performed by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring. Linear calibration curves of icariin, icariside II and osthole were obtained over the concentration ranges of 2.00-200, 2.00-200 and 2.00-500 ng/ml, respectively. The intra- and inter-day precisions were within 8.0% and 14%, and the accuracy was from -6.0% to 9.0%. The method was successfully applied to pharmacokinetic studies of icariin, icariside II and osthole in rats after oral administration of Gushudan extract.     

Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods

The validation of a pre-column derivatization procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to the determination of the amino acid content by RP-HPLC with fluorescence detection (lambda excitation 250 nm, lambda emission 395 nm) in milk-cereal based infant foods was carried out. The analytical parameters: linearity (0.0025-0.2mM), precision of the method (0.2-3.5% variation coefficients), accuracy (derivatization: 86-106% average recovery and method: 88.3-118.2% average recovery) and the limits of detection (0.016-0.367 microM) and quantification (0.044-1.073 microM) were determined. Glutamic acid, proline and leucine were the most abundant amino acid whereas the lowest contents corresponded to tyrosine and cysteine.     

Size exclusion chromatography for extraction of serum bile acids

A major problem in the measurement of serum bile acids is their quantitative extraction from the high molecular protein matrix. In our hands, the standard techniques of adsorption and reversed-phase chromatography have yielded incomplete recovery for different bile acids (33-93%) and poor reproducibility. In contrast, with the novel extraction procedure of size exclusion chromatography, recovery was nearly quantitative (75-104%) and reproducibility was satisfactory. The described method allowed for a reliable determination of serum bile acids in healthy subjects and patients with liver cirrhosis. We conclude that size exclusion chromatography for serum bile acid extraction is more reliable than alternative techniques, because the separation by size is independent of solubility, charge, and polarity.     

Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.     

Measurement of bile acid kinetics in human serum using stable isotope labeled chenodeoxycholic acid and capillary gas chromatography electron impact mass spectrometry

A non invasive method for measurement of bile acid kinetics in serum using (24-13C)chenodeoxycholic acid has been developed. After oral administration of 50 mg (24-13C)chenodeoxycholic acid, the exponential decay of the 13C atom percent excess was measured in serum using capillary gas chromatography mass spectrometry. This required that isotope ratios were measured with high accuracy and coefficients of variation less than 1% by means of selected ion monitoring and scan averaging. The clinical applicability was tested by repeated determination of pool size, fractional turnover and synthesis rate of chenodeoxycholic acid in one healthy volunteer. This method permitted the determination of pool size, synthesis and conversion of chenodeoxycholic acid into lithocholic acid in man without the use of radioactive tracers and without repeated duodenal intubation.     

Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma

A sensitive and specific HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-(13)C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6+/-5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.     

梯度洗脱高效液相色谱法测定桑叶中3种活性成分的含量

建立同时测定桑叶中绿原酸、芦丁和槲皮素含量的分析方法。色谱柱为NUCLEODUR C18 RP(250 mm×4.6 mm,5μm),光电二极管检测器,流动相为甲醇-质量分数0.5%磷酸水溶液,梯度洗脱程序为0 min(30:70)-15min(30:70)-25min(50:50)-35min(85:15)-40min(30:70);流速0.8 ml.min-1,检测波长为350 nm。绿原酸、芦丁和槲皮素的线性范围分别为0.1144~1.0296μg(r=0.9996)、0.0436~0.3924μg(r=0.9999)和0.0452~0.4068μg(r=0.9997),平均回收率分别为97.7%(RSD=1.7%)、98.4%(RSD=2.2%)和100.6%(RSD=1.5%)。方法快速简便,专属性强,重复性好,可作为桑叶中绿原酸、芦丁和槲皮素的定量分析方法。