超高效液相色谱-串联质谱法定性和高效液相色谱法定量分析天南星中氨基酸成分

为建立中药材中氨基类极性非紫外活性成分的定性与定量分析方法,以中药材天南星为研究对象,采用柱前衍生化技术,以异硫氰酸苯酯(PITC)为衍生化试剂,经C₁₈色谱柱(100 mm×2.1 mm, 3.5 μm)分离和超高效液相色谱-串联质谱(UHPLC-MS/MS)分析,共解析了天南星中20个成分,包括18个氨基酸和2个胺类化合物。经优化衍生化条件,应用高效液相色谱法(HPLC),以Diamonsil C₁₈色谱柱(250 mm×4.6 mm, 5 μm)分离,以乙腈和0.05 mol/L醋酸铵-醋酸缓冲液(pH 6.5)为流动相,梯度洗脱,在254 nm下检测,建立了同时测定15种氨基酸含量的方法,经方法学考察符合含量测定要求。谷氨酸、色氨酸在2~100 mg/L范围内、精氨酸在6~300 mg/L范围内、其余各氨基酸在0.8~40 mg/L范围内均呈良好的线性关系,相关系数均不小于0.9995;平均回收率在95%~105%之间,RSD均小于3%;并成功应用于12批中药材的测定。本方法简便、灵敏、准确,具有可操作性,可用于快速鉴定中药中的氨基类成分以及进行含量测定。     

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high‐throughput amino acid analysis, using precolumn derivatization high‐performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI‐MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home‐built auto‐sampler system. Amino acids were derivatized with 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate, and a 3 μm Wakosil‐II 3C8‐100HG column (100 × 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra‐ and inter‐precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%. Copyright © 2009 John Wiley & Sons, Ltd.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

Micro-scale sequence analysis from the N-terminus of peptides using the fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate

The fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate (DNTC) was reacted with amino acids and peptides, cyclized by acid and the liberated 4-N,N-dimethylamino-1-naphthyl thiohydantoin (DNTH) amino acids were then separated and detected by HPLC. The fluorescence intensities of DNTH-amino acids except DNTH-proline and -serine were dramatically increased in the alkaline solution and organic solvent. Thus, the postcolumn reaction with alkaline acetonitrile solution was adopted in HPLC. The polar and aromatic amino acids afforded two DNTH-amino acids on derivatization with DNTC and cyclization with acids. These were suggested to be stereoisomers of DNTH-amino acids. The sequence analysis of 0.5 nmol Leu-enkephalin was achieved by the double coupling method with DNTC and phenyl isothiocyanate followed by the proposed HPLC system.     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸

建立了液相色谱-电喷雾离子阱串联质谱(LC-ESI-IT-MS/MS)同时分析烟草中20种游离氨基酸的方法。烟草样品经萃取后过滤直接进样,无需进行衍生和固相萃取等其他前处理步骤。液相色谱采用HyPURITY C18反相色谱柱(200 mm×2.1 mm, 5 μm),采用1%(体积分数,下同)乙腈水溶液(含0.1%九氟戊酸)和90%乙腈水溶液(含0.1%九氟戊酸)为流动相进行梯度洗脱。结果表明,20种氨基酸的检出限(LOD)为0.01～0.05 μmol/L (S/N=3),线性相关系数均大于0.9977,峰面积测定的相对标准偏差(RSD)为0.78%～4.93%。该方法分析效率、灵敏度和选择性高,已成功应用于多种烟草样品中氨基酸的分析测定。     

Enantiomeric separation of unusual secondary aromatic amino acids

Summary High-performance liquid chromatographic and gas chromatographic methods were developed for the separation of unusual secondary aromatic amino acids. Amino acids containing 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydronorharmane-1-carboxylic acid and 1,2,3,4-tetrahydro-3-carboxy-2-carboline moieties were synthetized in racemic or chiral forms. The high-performance liquid chromatography was carried out either on a teicoplanin-containing chiral stationary phase or on an achiral C₁₈ column. In the latter case the diastereomers of the amino acids formed by precolumn derivatization with the chiral reagents 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate or 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide were separated. The gas chromatographic analyses were based on separation on a Chirasil-L-Val column. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.     

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.     

Determination of free amino acids in biological samples: problems of quantitation

An automatic on-line high-performance liquid chromatographic method was developed to study the effects of various precipitating agents and delayed deproteinization procedures on the estimation of plasma levels of amino acids. The optimized method for analysis is based on pre-column derivatization with o-phthalaldehyde in the presence of 2-mercaptoethanol. The separation of 25 amino acids is accomplished within 45 min on a 5-microns C18 column, using a multi-step gradient with two solvents. The method is sensitive and reproducible, and the relationship between the fluorescence intensity and concentration is linear for each amino acid over a wide concentration range.     

Precolumn derivatization reagents for high‐speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate (APDS), followed by reversed‐phase high‐performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI‐MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra‐ and inter‐day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra‐day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter‐day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra‐day and inter‐day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of plasma amino acids by gas chromatography

A complete methodology, incorporating a novel clean-up technique, for quantitative determination of amino acids in plasma by gas chromatography is described. Glucose, a component causing major analytical interference, is removed by an enzymic reaction included in the pre-chromatographic clean-up. The procedure for derivatisation of amino acid standards is shown to be reproducible down to a level of 2.5 micrograms for each amino acid, relative standard deviations for all amino acids except arginine and histidine being 3% or lower. For the entire procedure applied to plasma, relative standard deviations for most amino acids are below 5% with recoveries ranging from 90 to 120%. Normal values, obtained using eighteen plasma samples, are in reasonable agreement with published data. Plasma amino acid values were determined simultaneously by gas chromatographic and ion-exchange chromatographic techniques. Statistical evaluation shows there to be no significant difference between corresponding values for eleven amino acids. Values for tyrosine, histidine and particularly phenylalanine show significant differences (p less than 0.001).     

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.     

Reversed-phase chromatography of phenylthiocarbamyl amino acid derivatives of physiological amino acids: an evaluation and a comparison with analysis by ion-exchange chromatography.

Reversed-phase chromatography of phenylthiocarbamyl (PTC) amino acid derivatives of physiological amino acids was evaluated and compared with the traditional method of ion exchange. The PTC amino acid derivatives were stable for at least 32 h at ambient temperature before injection. The relationship of detector response to concentration for the PTC derivatives was linear from 39 to 1250 pmol. With few exceptions, the within- and between-run precisions of plasma amino acid retention times were less than 0.2 and 0.3%, respectively; the within- and between-run precisions of their concentrations were less than 4.0 and 5.0%, respectively. Twenty-four plasma samples were quantitated by both reversed-phase and ion-exchange chromatography; fifteen of the twenty amino acids determined had correlation coefficients in the range 0.81-1.00. Nine non-standard amino acids and ten therapeutic drugs were added to plasma; D-glucosaminic acid and alpha-amino-beta-guanidinopropionic acid co-eluted with alpha-aminoadipic acid and threonine, respectively. Of the ten drugs added, only metronidazole and theophylline co-eluted with beta-alanine and histidine, respectively. The precision, stability, and sensitivity of the method render it ideal for the quantitation of plasma amino acids.     

Direct determination of amino acids and carbohydrates by high-performance capillary electrophoresis with refractometric detection

This is an initial report to propose a novel approach in high-performance capillary electrophoresis (HPCE) for the direct detection of compounds without natural absorbance in the UV and visible spectral range, such as amino acids and carbohydrates. A refractometry detector with the 2 nl cell (Applied Systems, Minsk, Belarus) was employed to identify amino acids and carbohydrates without derivatization. The first results are provided on separation of seven free amino acids in the phosphate running buffer and three free carbohydrates in the borate-sodium dodecyl sulfate running buffer and detection by refractometer. Fused capillaries of 50 or 75 microm internal diameter and separation voltage (10-23 kV) were applied. Detection limits ranged typically from 10 to 100 fmol and the response was linear over two orders of magnitude for most of the amino acids and carbohydrates. The HPCE system demonstrated good long-term stability and reproducibility with a relative standard deviation, less than 5% for the migration time (n=10).     

Pharmacokinetic study of metoprolol in rabbit plasma by capillary electrophoresis with laser-induced Fluorescence detection

This work described a sensitive method for determination of metoprolol in rabbit plasma. The method involved purification by ultrafiltration, derivatization with Fluorescein isothiocyanate, separation by capillary electrophoresis and determination by laser-induced fluorescence detector. Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol under experimental conditions. The assay had a wide range (2.0?C500 ng/mL) of linearity and a detection limit of 0.8 ng/mL. The intra- and inter-day precisions of the QC samples were satisfactory with RSD less than 10% and accuracy within 10%. This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

Measurement of plasma and urine amino acids by high-performance liquid chromatography with electrochemical detection using phenylisothiocyanate derivatization

The use of reversed-phase liquid chromatography (LC) with pre-column derivatization for the analysis of amino acid mixtures is becoming established as a possible cheaper alternative to commercial amino acid analysers. The available derivatization procedures all have disadvantages when applied to clinical samples, partly due to the interferences found with body fluids when ultraviolet or fluorescence detection is used. An LC method is described for the separation of amino acids in blood or urine, using pre-column derivatization with phenylisothiocyanate (PITC), gradient elution and electrochemical detection. The use of electrochemical detection of PITC derivatives virtually eliminates interferences and enables the secondary amino acids to be measured. Examples are shown of normal urine and plasma and samples from patients with cystinuria and maple syrup urine disease.     

Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-(13)C amino acids as internal standards.

A method to quantitate blood and plasma amino acids by isotope dilution gas chromatography/mass spectrometry (GC/mS) is described. Samples were spiked with U-(13)C amino acids as internal standards and the tert-butyldimethylsilyl derivatives (tBDMS) separated by capillary column gas chromatography. Linear regression curves, generated for individual amino acids, gave correlation coefficients of 0.9999. The reproducibility of the method was assessed from the analysis of 10 replicate blood and plasma samples. For most amino acids a coefficient of variance (CV) of 相似文献