固相萃取-高效液相色谱-串联质谱法检测香辛料中罗丹明B

建立了香辛料中罗丹明B的固相萃取-高效液相色谱-串联质谱分析方法。样品经乙腈提取后离心,在提取液中加入10 mL 1%三氯乙酸溶液后,用Bond Elut Plexa PCX强阳离子固相萃取柱净化、富集,Pursuit C18色谱柱(100 mm×2.0 mm, 3 μm)分离,以0.1%甲酸水和甲醇为流动相梯度洗脱,电喷雾电离正离子模式下多反应监测(MRM)模式进行定性、定量检测。结果表明,在0.6～6 μg/L范围内的线性相关系数R2＞0.99;方法的定量限为1.2 μg/kg;添加量分别为1.197、2.992及5.985 μg/L时的加标回收率为80%～121%,相对标准偏差＜15%。同时对流动相的洗脱梯度、提取溶剂、固相萃取柱等条件进行了优化。该方法的专属性较强,基质效应较小,可用于固体香辛料产品中罗丹明B的定性、定量分析。     

Normal-phase high-performance liquid chromatographic determination of spiramycin in eggs and chicken

A precise method is presented for determination of residual spiramycin (SP) in chicken eggs and tissues by high-performance liquid chromatography (HPLC). The sample preparation was performed by homogenizing with a mixture of acetonitrile and n-hexane (5:4, v/v) to minimize the fat amount followed by ultra-filtration using a MolCutII(R). The extracts containing SP were free from interfering compounds when examined by the normal-phase HPLC using a LiChrosorb(R) NH(2) column and a mobile phase of acetonitrile-water (85:15, v/v) with a photo-diode array detector. The average recoveries from spiked SP (0.1, 0.5 and 1.0 ppm) were in excess of 89.0% with coefficients of variation between 1.4 and 2.4%. The limit of detection was 0.1 ppm.     

Comparative study on determination of fumagillin in fish by normal and reversed phase chromatography

Summary Fumagillin is an antibiotic agent mostly used in the veterinary profession. A normal-phase liquid chromatographic method was developed for the determination of this drug in fish matrices. A hexane-dichloromethanedioxan-2-PrOH-acetic acid (43:43:9:5:0.1 v/v %) eluent was used on a Perkin Elmer silica gel column. For validation of the process a reversed-phase HPLC method was also developed (eluent: acetonitrile-water-bicyclohyxylamine 70:30:0.05 v/v %, with Spherisorb ODS, as stationary phase). Recovery is about 100% and the limit of detection is 5 ng g⁻¹ for meat samples. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Determination of halofuginone in bovine plasma by competing-ion high performance liquid chromatography after solid phase extraction

A high performance liquid chromatography (HPLC) method for the determination of the anticoccidial and antitheilerial drug halofuginone in bovine plasma was developed. Samples were diluted with acetic acid (10%, v/v) and cleaned up on a Bond Elut C8 column. The analyte was eluted from the extraction column and chromatographed by reversed-phase HPLC using decylamine as a competing-ion reagent. Detection was by UV at 243 nm. Recovery from plasma was 75%, and within-day and between-day coefficients of variation were 5.23 and 6.35% respectively. The specificity and sensitivity of this method (limit of detection in plasma, 1 ng/mL) were sufficiently high to enable us to characterize the time course of the drug in plasma after oral administration of therapeutic doses to cattle.     

Development of a simple and sensitive high-performance liquid chromatography method for determination of glucosamine in pharmaceutical formulations

A column high-performance liquid chromatography (HPLC) method was developed for the determination of glucosamine in dosage forms. Glucosamine was derivatized by addition of a solution containing orthophthaldialdehyde. The HPLC separation was achieved on a Spherimage 80 ODS2 column (250 x 4 mm id, 5 microm particle size) using an isocratic mobile phase containing phosphate buffer-methanol (90 + 10, v/v, pH 6.50) and methanol-tetrahydrofuran (97 + 3, v/v) in proportions of 85 + 15 at a flow rate of 1 mL/min, followed by fluorescence detection. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for glucosamine HCI was linear over the concentration range of 0.1-20 microg/mL with a correlation coefficient of 0.9980. The accuracy was between 99.4 and 100.8%. The LOD and the LOQ were 0.009 and 0.027 microg/mL, respectively. The method was applied to determination of glucosamine in solid dosage forms.     

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859–4.524 μg/mL was found to be R² = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.     

High-performance liquid chromatographic determination of glyphosate in water and plant material after pre-column derivatisation with 9-fluorenylmethyl chloroformate

This paper describes a robust and sensitive HPLC method for determination of glyphosate in water and plant material after pre-column derivatisation with 9-fluorenylmethyl chloroformate (FMOC-Cl) using single and coupled polymeric amino columns, respectively. New findings about optimisation of the derivatisation reaction and chromatographic behaviour of glyphosate and AMPA on polymeric amino column are also presented.The best HPLC conditions for the separation of glyphosate and AMPA in water samples were achieved using a single polymeric amino column and mobile phase at pH 10 which contained 55% (v/v) acetonitrile and 50 mM phosphate buffer. The method was validated by analysing 10 fortified rainwater samples at a level of 1 μg/l. The average recovery was 94% with relative standard deviation (R.S.D.) of 5.4% and the method detection limit of 0.16 μg/l.The HPLC separation of glyphosate (only) in plant material entailed the coupling of two polymeric amino columns via a switching valve to separate interfering products from the desired analyte. The method was validated using grass samples spiked at the level of 1 mg/kg and gave a method detection limit of 0.3 mg/kg for glyphosate and a recovery of 82.4% with R.S.D. of 10.3%.     

Determination of Benzocaine Using HPLC and FIA with Amperometric Detection on a Carbon Paste Electrode

A new method for benzocaine determination employing FIA and HPLC with electrochemical detection on a carbon paste electrode was developed. The optimum conditions for the determination were found. Carrier solution for FIA consisted of B–R buffer pH 4 (80 % methanol, v/v) and used flow rate was 1.0 mL min^?1. Mobile phase for HPLC consisted of B–R buffer pH 4 (75 % methanol, v/v) with flow rate 0.4 mL min^?1. Working potential of +1.2 V was employed. Practical applicability of the methods was tested on the determination of benzocaine in selected pharmaceuticals. The results were in agreement with results obtained using spectrophotometric detection and with one exception also with the content declared by the manufacturer.     

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory.

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).     

Determination of diethylhexyl phtalate in water by solid phase microextraction coupled to high performance liquid chromatography

Difficulties detected in the determination of the diethylhexylphthalate (DEHP) at trace levels by gas chromatography–mass spectrometry (GC–MS) using SPME, due to its ubiquitous distribution in the environment has been overcome and a new method for the determination of DEHP in drinking water has been proposed. The method is based on solid phase microextraction (SPME) coupled to high-performance liquid chromatography (HPLC). Detection was carried out spectrophotometrically. Calibration graph was linear in the range 10–110 μg/L with a regression coefficient of r² = 0.998 and a detection limit of 0.6 μg/L. The relative standard deviation was 5 and 2% (n = 4) for chromatographic areas and retention times, respectively. The usefulness of the SPME–HPLC technique was confirmed.     

Quantitative determination of haloperidol in tablets by high performance thin-layer chromatography

A densitometric high performance thin-layer chromatography (HPTLC) method was developed and validated for the quantitative analysis of haloperidol in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/acetic acid glacial/water (5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out at a wavelength of 254 nm. The method was linear in the 10-100 ng/microL range, with a determination coefficient of 0.999. The coefficients of variation for precision were not higher than 2.35%. The detection limit was 0.89 ng/microL, and the quantification limit was 2.71 ng/microL. The accuracy ranged from 97.76 to 100.33%, with a CV not higher than 4.50%. This method was successfully applied to quantify haloperidol in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision and accuracy for the quantitative determination of haloperidol in tablets.     

反相高效液相色谱法测定人肌腱中的胶原蛋白

建立了测定人肌腱中胶原蛋白含量的高效液相色谱法。动物肌腱中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用2,4-二硝基氯苯对水解产物衍生化,然后以0.01 mol/L乙酸钠-乙酸缓冲液(pH 6.0)-乙腈(体积比为80∶20)为流动相,经反相C18柱分离,紫外检测器于360 nm波长处检测来测定羟脯氨酸的含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量来计算胶原蛋白的含量。该方法对羟脯氨酸的检出限为3 μg/L,羟脯氨酸为3 μg/L~100 mg/L时与峰面积的线性关系良好;样品测定的相对标准偏差为1.95%,加标回收率为98.4%~110.8%。对60份人肌腱样品中胶原蛋白的含量进行了测定。所建立的方法灵敏、准确、干扰少,适用于肌腱中胶原蛋白含量的测定。     

Hyphenation of reverse-phase HPLC and ICP-MS for metabolite profiling—application to a novel antituberculosis compound as a case study

In this study, a high-performance liquid chromatography (HPLC) inductively coupled plasma (ICP) mass spectrometry (MS) method was developed intended for use in metabolism studies of bromine-containing drugs, administered to test animals (or test persons). As a case study, the method was applied to a new antituberculosis compound, the bromine-containing diarylquinoline R207910. A method has been proposed to overcome the incompatibilities between the high organic solvent content (45%CH₃OH and 45% CH₃CN) used in the reverse-phase liquid chromatography (LC) separation on one hand and the limitations of the ICP on the other hand. Therefore, several instrument modifications had to be made. For the introduction of the column effluent, a combination of a perfluoroalkoxy LC nebulizer with a PC³ Peltier-cooled inlet system (operated at 2 °C) was used. Additionally, the standard injector tube (internal diameter 2 mm) was replaced by an injector tube with an internal diameter of 1 mm and to avoid carbon build-up on the interface cones and the torch, the nebulizer gas was admixed with 6% v/v of oxygen. After optimization of the method, HPLC-ICP-MS was applied for metabolite profiling of faeces samples after dosing of ¹⁴C-radiolabelled R207910 to dogs and rats. To evaluate the method developed, the HPLC-ICP-MS results were compared with those of HPLC with UV spectrophotometric and ¹⁴C radiochemical detection. As the HPLC-ICP-MS method gave rise to a higher selectivity than HPLC with UV detection and to a better detection limit (5 ng R207910) than the method with radiochemical detection (65 ng R207910), it can be concluded that ICP-MS can be used as a good alternative to the more traditional detection methods, even when a mobile phase with high organic solvent content has to be used in the LC separation.     

Residues analysis of post-harvest imidazole fungicides in citrus fruit by HPLC and GLC

The determination of imazalil and prochloraz fungicide residues has been carried out by HPLC with an UV detector at 204 nm and by GLC with an electron capture detector (ECD). In both cases fungicide residues were extracted with hexane/acetone (90:10, v/v) after pH adjustment and purified by a liquid-liquid partitioning process. When HPLC was used for prochloraz and imazalil analysis, it was necessary to eliminate the interfering substances with a further clean-up process. This was also required when samples with low residue levels were analyzed by GLC. Recovery was always higher than 70%. The detection limit was 0.04 ppm for the HPLC method and 0.02 for the GLC method. Imazalil and prochloraz residues in "Washington Navel" oranges and "Hernandina" clementine fruits, dipped in a 1000 ppm fungicide solution, are reported.     

高效液相色谱法同时测定发酵液中赤藓糖醇和L-赤藓酮糖的含量

建立了采用高效液相色谱(HPLC)同时测定发酵液中底物赤藓糖醇和产物L-赤藓酮糖含量的方法。采用Lichrospher 5-NH2色谱柱(250 mm×4.6 mm),柱温30 ℃,以乙腈-水(体积比为9:1)为流动相,流速1.0 mL/min。用示差折光检测器检测赤藓糖醇,检测器温度为35 ℃。用紫外检测器在室温下检测L-赤藓酮糖,检测波长为277 nm。所得赤藓糖醇的线性范围为1.00～100.00 g/L,相关系数为0.9985,检出限为0.10 g/L,定量限为0.45 g/L;所得L-赤藓酮糖的线性范围为1.00～100.00 g/L,相关系数为0.9958,检出限为0.50 g/L,定量限为0.87 g/L;赤藓糖醇的日内和日间相对标准偏差(RSD)分别小于3.28%和5.30%, L-赤藓酮糖的日内和日间RSD分别小于2.16%和2.25%;回收率均大于99%。取不同时间的发酵液样品分别用上述方法测定,结果表明所建立的HPLC法不受发酵液中其他组分的影响,可同时测定底物赤藓糖醇和产物L-赤藓酮糖的含量。     

Determination of captopril in human plasma with precolumn derivatization using solid phase extraction and HPLC

A rapid, accurate and sensitive method for the determination of captopril in human plasma was developed by solid phase extraction and high performance liquid chromatography (HPLC), using precolumn derivatization of captopril with chromophore label o-phethaldialdehyde (OPA). The extraction of captopril from human plasma was carried out by an amino propyl cartridge. A 0.01 M solution of HCl in methanol showed the best recovery and was chosen for elution of captopril in cartridge. This methanolic solution was applied to react with aqueous solution of OPA and glycine as a coderivatization reagent. The process of derivatization was completed within 2 min at room temperature. The derivatized captopril was injected into a reverse phase HPLC system. Mobile phase was consisted of water:acetonitrile:trifluoroacetic acid (85:15:0.1 v/v/v) with a flow rate of 1 ml min^?1 and detector was used at 345 nm. Linear dynamic range and limit of detection were found as 0.1–6 ppm and 0.1 ppm, respectively.     

Quantitative determination of paroxetine and its 4-hydroxy-3-methoxy metabolite in plasma by high-performance liquid chromatography/electrospray ion trap mass spectrometry: application to pharmacokinetic studies

A high-performance liquid chromatography (HPLC) method with tandem mass spectrometric detection is described for the determination of paroxetine, an antidepressant drug, and its metabolite (3S,4R)-4-(4-fluorophenyl)-3-(4-hydroxy-3-methoxyphenoxymethyl)piperidine (HM paroxetine) in human plasma. Plasma samples were hydrolysed with hydrochloric acid and then analytes were extracted with ethyl acetate at alkaline pH. Extracts were analysed by HPLC coupled to an atmospheric pressure ionisation-electrospray (ESI) interface and an ion trap mass spectrometer. Chromatography was performed on a reversed-phase column using acetonitrile/0.02% formic acid (66:34, v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated over concentration ranges of 0.75-100 microg/L and 5-100 microg/L for paroxetine and HM paroxetine, respectively. Mean recoveries of 77% for paroxetine and 76% for HM paroxetine were found, with precision always better than 15%. The limits of detection and quantification were 0.20 and 0.70 microg/L for paroxetine, and 0.70 and 2.20 microg/L for its metabolite. The method was applied to the analysis of plasma samples obtained from nine healthy male volunteers administered with a single oral dose of 20 mg paroxetine. After the 20-mg dose, the mean peak plasma concentration was 8.60 microg/L for paroxetine and 92.40 microg/L for HM paroxetine showing a tenfold ratio between the metabolite and the parent drug along the entire time-concentration curve.     

Residues Analysis of Post-Harvest Imidazole Fungicides in Citrus Fruit by H PLC and GLC

Abstract

The determination of imazalil and prochloraz fungicide residues has been carried out by HPLC with an UV detector at 204 nm and by GLC with an electron capture detector (ECD).

In both cases fungicide residues were extracted with hexane/acetone (90:10, v/v) after pH adjustment and purified by a liquid-liquid partitioning process. When HPLC was used for prochloraz and imazalil analysis, it was necessary to eliminate the interfering substances with a further clean-up process. This was also required when samples with low residue levels were analyzed by GLC.

Recovery was always higher than 70%. The detection limit was 0.04 ppm for the HPLC method and 0.02 for the GLC method.

Imazalil and prochloraz residues in “Washington Navel” oranges and “Hernandina” clementine fruits, dipped in a 1000 ppm fungicide solution, are reported.     

Development of a sensitive liquid chromatography-electrospray ionization mass spectrometry method for the measurement of KW-5139 in rat plasma

A sensitive method for the determination of a prokinetic peptide, KW-5139 (Leu(13)-motilin), in rat plasma has been developed utilizing liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). KW-5139 was separated by reversed-phase HPLC, with a mixture of 75 mM ammonium formate (pH 3.0) and acetonitrile (4:1, v/v), and monitored by single ion recording (SIR)-ESI-MS at m/z 894 ([M+3H](3+)). Simple protein precipitation and the LC-ESI-MS analysis allowed the determination of KW-5139 in rat plasma with the mean precision and accuracy at the lower limit of quantitation (LLOQ, 0.5 ng/mL) of 5.7 and 11.2%, respectively. The method was applied to the monitoring of the plasma time-concentration profile of KW-5139, intravenously administered to rats at a dose of 1 microg/kg.     

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography.

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.