Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography

The separation and identification possibilities of 27 PTC-amino acids (with particular attention to those present in apples in free forms), are reported on seven RP columns such as, Nucleosil, 3 and 5 microns: 150(+20 guard) x 4.0 mm; Gromsil 3 microns; 150(+10 guard) x 4.0 mm; Hypersil 5 microns: 130(+20 guard) x 4.0 mm, 150(+20 guard) x 4.0 mm and 200(+20 guard) x 4.0 mm, as well as, Hypersil 3 microns: 150(+20 guard) x 4.0 mm: a UV range photodiode array (PDA) detection was employed. Optimization studies carried out in model solutions, as a function of the temperature (30-55 degrees C) and flow-rate (0.8-2.5 ml/min) of eluents proved that optimum resolutions are associated with the highest flow-rate applicable, (remaining on the safe side with a column pressure of < 3500 p.s.i., 1 p.s.i. = 6894.76 Pa), in the temperature range of 30-50 degrees C. Twenty-seven amino acids, characteristic in apples in free forms, have been separated and determined on all seven columns, performing the same gradient program, (the main component asparagine, present in overwhelming excess, and the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, homoarginine and 1-aminocyclopropane-1-carboxylic acid). Optimum conditions, at 2.1 ml/min, at 50 degrees C, with 40 min run time, including equilibration, have been obtained with the Hypersil, 150(+20 guard) x 4 mm column, performing elutions. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range of 15-1500 pmol was < 4.0% R.S.D. (relative standard deviation). Detailed study of the PDA spectra revealed that in addition to the identification/peak purity possibilities further characteristics can be obtained taking advantage of the difference in maximum values and of those of their special ratio values, respectively. The utility of the protocol was shown in the quantitation of the free amino acid content of three apple varieties.     

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C(18) column (150 mm x 3.9 mm, 4 microm) at 35 degrees C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.     

Analysis of methyl tert.-butyl ether and its degradation products by direct aqueous injection onto gas chromatography with mass spectrometry or flame ionization detection systems.

A method was developed to analyze methyl tert.-butyl ether (MTBE) and its degradation products by gas chromatography with mass spectrometry (GC-MS) or flame ionization detection (FID) with direct aqueous injection. The column had dimensions of 30 m x 0.25 mm with film thickness 0.25 microm and a stationary phase of FFAP (nitroterephthalic acid-modified polyethylene glycol). The optimized GC conditions for non-acid components were as follows: carrier gas flow-rate,l mL/min; oven temperature, 35 degrees C for 5.5 min, ramped to 90 degrees C at 25 degrees C/min, then ramped to 200 degrees C at 40 degrees C/min and held at 200 degrees C for 8 min. The conditions for the acid components were: carrier gas flow-rate, 1 mL/min; oven temperature, 110 degrees C for 2 min, ramped to 150 degrees C at 10 degrees C/min, then ramped to 200 degrees C at 40 degrees C/min. The injection port contained a silanized-glass reverse-cup liner filled with Carbofrit. The minimum concentrations for the linear range for the selective ion monitoring mode were 30 to 100 microg/L, depending on the analytes. The minimum detection limit was 1 mg/L for MTBE and tert.-butanol when using FID. More components could be analyzed with the FFAP-type column than with the cyanopropylphenyl-dimethyl polysiloxane-type column.     

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.     

对一种分离测定氨基酸方法的改进

 对Waters公司采用 6 氨基喹啉 N 羟基琥珀酰亚胺基 氨基甲酸酯 (AccQ Tag)柱前衍生化测定氨基酸的方法进行了改进。将流动相流速由原来的 1 0mL/min改变为 2 0mL/min ,用AccQ Tag专用柱 (3 9mmi.d .× 15 0mm ,4μm)在 17 5min(原为 35min) (运行周期为 2 2 5min ,原为 45min)内快速分离测定了 18种氨基酸和牛磺酸。用Nova PakC18柱 (3 9mmi.d .× 15 0mm ,4μm) ,Nova PakC18柱 (4 6mmi.d .× 15 0mm ,4μm) ,SymmetryC18柱 (3 9mmi.d .× 15 0mm ,4μm)和WatersXterraRP 18柱等反相C18柱代替AccQ Tag专用柱 ,均可对氨基酸进行快速分离。     

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%.     

Advantages of pentafluorophenylpropyl stationary phase over conventional C18 stationary phase--application to analysis of triamcinolone acetonide

A pentafluorophenylpropyl (PFPP) stationary phase was for the first time tested for the simultaneous determination of triamcinolone acetonide, its degradation product triamcinolone and two preservatives, methylparaben, and propylparaben. A new simple isocratic reversed phase HPLC method with UV detection, using estradiol hemihydrate as an internal standard, has been developed and validated. Chromatography was performed on a Discovery HS F5 column (150 mm x 4.6 mm, 5 microm) using a binary mobile phase composed of acetonitrile and water 45:55 (v:v). The flow-rate was 0.6 mL/min, the column temperature 25 degrees C and the UV detection was accomplished at 240 nm. The chromatography results using PFPP stationary phase were compared with those obtained using conventional C18 columns.     

Fast capillary GC using a low thermal mass column oven for the determination of residual solvents in pharmaceuticals

A low thermal mass column oven was used for fast capillary GC analysis (high throughput) of residual solvents in pharmaceutical products. A dedicated capillary column, 20 m L x 180 microm ID x 1 microm DB-624 was programmed from 35 degrees C (30 s) to 150 degrees C at 100 degrees C/min and to 250 degrees C (30 s) at 200 degrees C/min, resulting in a total GC cycle time of less than 4 min. Complete separation of a target 20-component mixture was achieved, while method performance in terms of repeatability, sensitivity, and linearity was maintained in comparison to the generic method currently applied in our laboratories.     

Lipophilicity of a series of 1,2-benzisothiazol-3(2H)-ones determined by reversed-phase thin-layer chromatography

A reversed-phase high-performance liquid chromatographic method with detection at 220 nm was developed and validated for the determination of ethyl-3-(N-n-butyl-N-acetyl)aminopropionate, IR 3535, in an insect repellent semi-solid product. A Hypersil ODS RP-C18 column (250 x 4.6 mm), 5 microm particle size, was equilibrated with a mobile phase consisted of water-acetonitrile (60:40, v/v). Its flow-rate was 1.0 ml/min. Excipients did not interfere with the determination of IR 3535 (Rs = 8.663). Intra- and inter-day relative standard deviations for samples were not higher than 0.61 and 1.2%, respectively. Mean recovery was found not lower than 98.5% and not higher than 100.3%. The method of external standard was adopted. Calibration curves were linear in the concentration range between 1.0 x 10(-6) and 5.0 x 10(-4) M. Limits of detection and quantitation were 65 and 196 ng/ml, respectively.     

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.     

Determination of didanosine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography-tandem mass spectrometry

A rapid and efficient high-performance liquid chromatography (HPLC)-tandem mass spectrometry method for the determination of didanosine concentrations in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in optima water and centrifuged. The supernatant was subjected to solid-phase extraction (SPE) prior to analysis. Plasma and amniotic fluid samples were extracted without pretreatment. An Agilent 1100 Series HPLC coupled with a Micromass Quattro II triple quadrupole mass spectrometer was used for all analyses. Chromatographic resolution was achieved on a Nova-Pak phenyl analytical column (2.0 x 150 mm, 4 microm particle size) equipped with a Phenomenex Security-guard phenyl guard cartridge (2.0 x 4.0 mm) using 60% methanol in 10 mm ammonium acetate buffer mobile phase for all matrices at a flow rate of 0.15 mL/min. The method yields retention times of 2.9 min for didanosine and 3.0 min for the internal standard, stavudine. Limits of detection were 1 ng/mL for all matrices. Recoveries were 70% or greater for both compounds in the different matrices. Within- and between-run precision (%RSD) and accuracy (%error) was less than 15% for all matrices.     

Derivatization and chromatographic behavior of the o-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives

An overview is presented of HPLC methods currently in use to determine amino acids as their o-phthaldialdyde derivatives in the presence of various SH-group-containing additives. Crucial points that proved to influence the stability of the amino acid OPA derivatives have been discussed in detail: (i) the mol ratios of the OPA-SH-group-containing additive amino acid; (ii) the preparation and storage conditions of the OPA reagents; (iii) the optimum pH conditions for the interactions and elutions; (iv) the behavior of the, believed to be, less stable amino acids, such as glycine, beta-alanine, gamma-aminobutyric acid, histidine, ornithine and lysine.     

牡励中脂肪酸在储藏过程中的稳定性

用超临界流体萃取及ＧＣ－ＭＳ分析了新冷冻干燥及保存１５ｄ,３０ｄ,４５ｄ,６０ｄ,７５ｄ,９０ｄ后的鲜牡蛎粉中的２３种脂肪酸组分的质量分数。发现在存放过程中牡蛎脂肪酸的稳定性与其不饱和度有关;不饱和度越高,脂肪酸越易被氧化,其中多不饱和记酸的氧化是逐渐进行的,没有特定的稳定期。     

Optimization of parameters of high-performance displacement chromatography for separation of soybean phosphatidylcholine and phosphatidylethanolamine

Hundred milligrams of soybean phospholipids were successfully separated by using high-performance displacement chromatography (HPDC) on a 150mm x 4.6mm analytical silica column (3-5 microm packings) with dichloromethane-methanol (9:1, v/v) as carrier and ethanolamine as displacer. From the viewpoint of preparative separation, the effects of loading amount, concentration and flow-rate of displacer on separation efficiency were investigated using throughput and recovery as indices. The parameters were optimized by orthogonal test design and statistical analysis method. Under the optimum conditions, namely displacer concentration being 167 mM, the flow-rate of displacer at 0.2 ml/min and concentration of sample being 211 mg/ml (factual loading amount 211 mg/ml x 0.7 ml = 148 mg), the purity, throughput and recovery of obtained soybean phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were 80.2%, 65.7 mg/h, 70.9% and 90.5%, 272.6 mg/h, 88.3%, respectively. In addition, selections of regenerant and appropriate regeneration condition were also studied.     

Analysis of carbohydrates in drinks by high-performance liquid chromatography with a dynamically modified amino column and evaporative light scattering detection

A high-performance liquid chromatographic method with a dynamically modified amino column and evaporative light-scattering detector (ELSD) was established for the direct analysis of the carbohydrates in some drinks. A separation column (Zorbax Rx-SIL, 250 mm x 4.6 mm I.D., 5 microm, Hewlett-Packard, USA) which was modified by ethylenediamine and a guard column (Zorbax Rx-SIL, 12.5 mm x 4.6 mm I.D., 5 microm) were used. The mobile phase was a mixture of water-acetonitrile (1:2.6, v/v) containing 0.03% (v/v) ethylenediamine. Regression equations revealed linear relationship (correlation coefficients=0.996-0.999) between the mass of carbohydrates injected and the carbohydrates peak areas detected by ELSD. The detection limits of ELSD (S/N=3) were between 0.2 and 1.2 microg for different carbohydrates. This method is simple and sensitive.     

1-(2-萘基)-3-甲基-5-吡唑啉酮衍生试剂的制备及其在高效液相色谱-质谱法测定糖类化合物中的应用

以1-(2-萘基)-3-甲基-5-吡唑啉酮(NMP)作为柱前衍生试剂,建立了简单、灵敏的糖类组分的反相高效液相色谱测定方法。NMP与糖在氨为催化剂的条件下,于70 ℃下反应可获得稳定的衍生产物。在Hypersil ODS 2反相色谱柱上,实现了8种单糖的基线分离。衍生物线性相关系数均大于0.9985,检出限为0.58～1.1 pmol。利用柱后在线串联质谱的电喷雾电离正离子模式监测,获得了各组分的质谱定性及裂解规律,特别是m/z 473的特征碎片离子可作为单糖NMP衍生物的判定依据。与1-苯基-3-甲基-5-吡唑啉酮(PMP)相比,NMP对糖的衍生化具有灵敏、简单、质谱裂解规律性强、重现性好等优点。该方法用于测定油菜花粉多糖中的单糖组成,结果令人满意。     

Quantitative LC/MS/MS method and pharmacokinetic studies of columbin, an anti-inflammation furanoditerpen isolated from Radix Tinosporae

Columbin is an important component isolated from Radix Tinosporae. It has been demonstrated to possess many pharmacological activities, including anti-inflammation, antitumor and inhibition of enzyme activity in vivo. The purpose of the present study was to examine in vivo pharmacokinetics and bioavailability of columbin in rats using a high-performance liquid chromatography coupled with tandem mass spectrometry quantitative detection method. The columbin was extracted from rat plasma samples by methyl tert-butyl ether, evaporated and reconstituted in 100 microL methanol prior to analysis. The separation was performed using a Luna reversed-phase analytical column (5 microm, 100 x 2.0 mm) and an SB-C18 guard column (5 microm, 20 x 4.0 mm). The mobile phase was a mixture of methanol and water containing 25 mmoL/L NH(4)Ac (80:20, v/v). The method was validated within the concentration range of 5-5000 ng/mL, and the calibration curves were linear with correlation coefficients (r) >0.999. It was further applied to assess pharmacokinetics and oral bioavailability of columbin after i.v. and oral administration to rats. The oral bioavailability of columbin was only 3.18%, which indicated that columbin had poor absorption or underwent extensive first-pass metabolism.     

高效液相色谱法测定饲料中的喹乙醇

报道饲料中喹乙醇的高效液相色谱分析方法。以乙酰苯胺为内标,样品用二甲替甲酰胺（ＤＭＦ）提取,在Ｃ８柱上进行色谱分析,紫外检测波长２６０ｎｍ,流动相为甲醇－水（２０８０,Ｖ／Ｖ）,喹乙醇的回收率为９８．５８％～１０１．６３％,相对标准偏差为２．６７％～４．２５％。     

Radio-detection high-performance liquid chromatographic enzyme assay for inhibitors of fungal sterol delta 14-reductase

An enzyme assay for inhibitors of fungal sterol delta 14-reductase employing isocratic reversed-phase high-performance liquid chromatography is described. A Hypersil 5-microns octadecylsilyl (ODS) column (250 mm x 4.6 mm I.D.) was used and a mobile phase consisting of methanol-water-ethanol (86:4:10, v/v) was pumped at a flow-rate of 1.5 ml/min. Typical analysis times were 15 min. Using [4-14C]ignosterol as a substrate and an enzyme preparation from Saccharomyces cerevisiae, this method was used to compare the inhibition of sterol delta 14-reductase by the fungicides fenpropidin and fenpropimorph with three N-substituted 8-azadecaline compounds.     

超高效液相色谱-串联质谱法测定大鼠血浆中的磷酸西他列汀

建立了大鼠血浆中磷酸西他列汀含量检测的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。以大鼠空白血浆为基质,通过添加标准品的方法配制含磷酸西他列汀和内标物氟西汀的样品,选用甲醇为沉淀剂,经离心除去血浆中的蛋白质,上清液用于目标物的检测。采用Thermo Hypersil Gold C18柱(50 mm×2.1 mm, 1.9 μm)为分析柱,Phenomenex Security Guard C18(4 mm×3.0 mm)为预柱,以乙腈和0.05%(v/v)甲酸水溶液为流动相进行梯度洗脱,流速为200 μL/min, 5 min内实现了快速分离。采用电喷雾正离子(ESI+)模式电离,选择反应监测(SRM)模式检测,确定了磷酸西他列汀和氟西汀的监测离子对分别为m/z 408.0→235.0和m/z 310.0→148.0,用基质匹配标准溶液法进行定量。结果表明: 大鼠血浆中磷酸西他列汀的质量浓度在1～1000 μg/L范围内时线性关系良好(r=0.9991),检出限(信噪比为3)为0.2 μg/L;其平均回收率为85%～115%;日内及日间的相对标准偏差(RSDs)均小于15%,满足生物样品检测的要求。将该方法初步用于大鼠静脉注射后的血浆样品中磷酸西他列汀的检测。该方法快速、灵敏度高、操作简便、重现性好,能够用于磷酸西他列汀药代动力学等方面的研究。