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).     

Development of a perfusion reversed-phase HPLC method for the separation of soybean and cereal (wheat, corn, and rice) proteins in binary mixtures. Application to the detection of soybean proteins in commercial bakery products

A perfusion reversed-phase HPLC method enabling the simultaneous separation of soybean and cereal (wheat, corn, and rice) proteins in commercial bakery products has been proposed for the first time. The method utilises an acetonitrile-water gradient containing an ion-pairing agent. Different ion-pairing agents were tried, 0.3% (v/v) acetic acid being observed to enable the separation of soybean from wheat, rice, and corn proteins while with 0.1% (v/v) trifluoroacetic acid only the separation of soybean and corn proteins was possible. Optimisation of the solubilisation conditions for proteins was achieved by testing different acetonitrile concentrations for the simultaneous extraction of soybean and cereal proteins: best recoveries were found with 25% (v/v) acetonitrile + 0.3% (v/v) acetic acid and with 40% (v/v) acetonitrile + 0.1% (v/v) trifluoroacetic acid. Chromatographic conditions such as gradient, temperature, and wavelength detection were also optimised. The method enabled the separation of soybean and cereal proteins in binary mixtures (soybean and wheat, soybean and corn, or soybean and rice proteins) in less than 5 minutes in a total analysis time of 20 min.     

葛根提取物高效液相色谱分析方法的优化

针对葛根提取物的复杂体系,分别从色谱模式、流动相、添加剂以及梯度洗脱条件几个方面对其高效液相色谱分析 方法进行了优化。其中梯度洗脱条件的优化是根据各组分在5次线性梯度下的保留时间并借助于CSASS软件快速准确地计 算出各组分的液相色谱保留参数a,c值以及峰形参数σ和τ,在此基础上使用CSASS软件对葛根提取物的色谱分离情况进行 高精度仿真预测,从而优化了梯度洗脱条件。考察了优化条件下的分析方法的精密度和重现性,结果表明该方法稳定、可 靠、重现性好。     

Liquid chromatographic-electrospray tandem mass spectrometric determination of bisoprolol in human plasma

An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned up by protein precipitation with acetonitrile, reconstituted in mobile phase and separated by reversed-phase high-performance liquid chromatography (HPLC) using methanol:10 mm ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on an LC-MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5-100 ng/mL with a limit of quantitation (LOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 and 9.95%, respectively, while the accuracy was in the range 89.4-113%. This method has been utilized in a pharmacokinetic study, where healthy volunteers were treated with an oral dose of 5 mg bisoprolol.     

Determination of polyacetylenes and ginsenosides in Panax species using high performance liquid chromatography

A new HPLC method was developed to separate and identify three polyacetylenes (panaxynol, panaxydol and 1,8-heptadecadiene-4,6-diyne-3,10-diol) found in Panax species. The mobile phase was a linear gradient of 2 : 1 : 3 to 2 : 1 : 1 (v/v/v) methanol/acetonitrile/water in 40 min. HPLC analysis was performed at a flow rate of 1.5 ml/min with UV detection at 254 nm. The contents of the polyacetylenes and ginsenosides in Panax ginseng (white ginseng and red ginseng), P. quinquefolium, P. japonicus, and P. noteginseng were determined using these methods. The species containing the highest polyacetylene content (0.080%) was P. quinquefolium cultivated in Nagano, Japan. Meanwhile, the species with the highest ginsenoside content (9.176%) was P. noteginseng cultivated in Yunnan, China.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

High-performance liquid chromatographic analysis of the anticancer drug oxantrazole in rat whole blood and tissues.

A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the antitumor anthrapyrazole analogue, oxantrazole (OX), in rat whole blood and tissues. Blood samples were mixed with equal volumes of a 25% (w/v) aqueous solution of L-ascorbic acid, whereas tissue samples were homogenized with 1.5-3 volumes of an L-ascorbic acid-methanol-water (1:10:1, w/v/v) mixture to prevent oxidative degradation of OX. Samples were then treated with 60% (v/v) perchloric acid (25-30 microliters/ml of stabilized sample) to precipitate proteins, and centrifuged, with the resultant supernatants analyzed on HPLC utilizing a C8 column. The mobile phase for blood and urine samples consisted of 8% (v/v) glacial acetic acid, 13% (v/v) acetonitrile, 79% (v/v) water, 0.16% (w/v) sodium acetate, and 0.05% (w/v) L-ascorbic acid (final pH 2.7), and was pumped at 1.8 ml/min. Tissue samples were eluted at 2 ml/min with a mobile phase consisting of 8% (v/v) glacial acetic acid, 12% (v/v) acetonitrile, 80% (v/v) water, 0.16% (w/v) sodium acetate, and 0.0;5% (w/v) L-ascorbic acid. OX and internal standard were detected at 514 nm and had retention times of 2.3 and 3.1 min, respectively. The limit of quantitation of OX was 25-50 ng/g. Recovery of OX from biological samples ranged from 50 +/- 0.9% in spleen to 102.8 +/- 1.8% in RG-2 glioma. The analytical method was applied to a pharmacokinetic study in rats.     

Rapid determination of ambrisentan enantiomers by enantioselective liquid chromatography using cellulose-based chiral stationary phase in reverse phase mode

A sensitive, specific, and rapid high-performance liquid chromatography (HPLC) method for the determination of ambrisentan enantiomers has been developed and validated. Six chiral columns were tested in a reversed-phase system. Excellent enantioseparation with the resolution more than 2.5 was achieved on Chiralcel OZ-3R (cellulose 3-chloro-4-methylphenylcarbamate) using mixture of 20 mM sodium formate (pH 3.0) with acetonitrile (55:45; v/v). Validation of the HPLC method including linearity, limit of detection, limit of quantification, precision, accuracy, and selectivity was performed according to the International Conference on Harmonisation (ICH) guidelines. The method has an advantage of a very quick chromatographic separation (less than 6 min) and therefore is highly suitable for routine determination of (R)-ambrisentan in enantiopure active pharmaceutical ingredient (S)-ambrisentan.     

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

Determination of pKa values of organic bases in aqueous acetonitrile solutions using capillary electrophoresis

Capillary electrophoresis (CE) was used for the determination of ionisation constants (pKa) of a variety of organic bases in aqueous acetonitrile solutions over the range 0-60% (v/v) acetonitrile. These bases are used as test compounds in HPLC column evaluation, thus knowledge of their pKa in hydro-organic solutions is useful. The base pKa decreased with acetonitrile concentration and significant shifts from the aqueous pKa (up to -0.8) were found using 60% acetonitrile. The CE application was confirmed to be very suitable for fast and accurate pKa measurement in aqueous organic solutions.     

反相高效液相色谱法测定阿德福韦酯及其降解产物

建立了一快速、简单地测定阿德福韦酯及其降解产物阿德福韦单特戊酸甲基酯、阿德福韦的反相高效液相色谱方法。以Inertsil CN-3化学键合硅胶为固定相,以乙腈-25 mmol/L磷酸盐缓冲液(pH 4.0)(体积比为33∶67)为流动相,流速1.0 mL/min,检测波长260 nm。阿德福韦酯、阿德福韦的质量浓度分别为1.861~181.7 mg/L和2.018~197.2 mg/L时与峰面积呈良好的线性关系(r分别为0.9999和0.9998);阿德福韦酯及阿德福韦平均加样回收率分别为99.5%~10     

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.     

Quantitative determination of DRF-1042 in human plasma by HPLC: validation and application in clinical pharmacokinetics

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.     

Separation of natural pyrethrum extracts using micellar electrokinetic chromatography

The separation of the six pyrethrin esters in a technical pyrethrum extract (Riedel-de-Ha?n, Cresent Chemical Co. Inc. Hauppauge, NY, USA) by micellar electrokinetic chromatography (MEKC) using both sodium dodecyl sulfate (SDS) and a polymerized surfactant as pseudo-stationary phases has been investigated and optimized. Parameters such as pH, SDS and polymerized sodium N-undecyl sulfate (poly-SUS) concentration, type and concentration of background electrolyte and organic modifier, as well as the acetonitrile/water ratio in the sample were studied to optimize the resolution, efficiency, and analysis time. An optimized separation of the six pyrethrin esters was achieved in 25 min with 25 mM Tris, buffered at pH 9, containing 30 mM SDS, 25% (v/v) acetonitrile, and an equal volume ratio of acetonitrile/water sample matrix at a voltage of 25 kV. The use of 0.5% (w/v) poly-SUS enhanced resolution of the pyrethrin esters and shortened the total analysis time from 25 to 20 min, compared to the SDS mediated separation. The optimized MEKC results are compared to the HPLC separation of these esters and show an improvement in efficiency and total analysis time.     

High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Gradient HPLC-diode array detector stability-indicating determination of lidocaine hydrochloride and cetylpyridinium chloride in two combined oral gel dosage forms

A simple, rapid, and selective HPLC-diode array detector method was developed for the simultaneous determination of lidocaine hydrochloride (LD) and cetylpyridinium chloride (CPC) in two combined pharmaceutical formulations. Effective chromatographic separation was achieved on a Zorbax SB-C8 (4.6 x 250 mm, 5 microm particle size) column with gradient elution using a mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (v/v) acetonitrile, ramped up linearly to 85% in 5 min, and then was constant until the end of the run. The mobile phase was pumped at a flow rate of 1.2 mL/min. The multiple wavelength detector was set at 214 and 258 nm, and quantification of the analytes was based on measuring their peak areas. The retention times for LD and CPC were about 3.4 and 7.3 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. Calibration curves were linear in the range of 5-200 and 10-400 microg/mL for LD and CPC, respectively, with correlation coefficients > 0.999. The proposed method was proven to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) as well as from forced-degradation products. The validated HPLC method was extended to the analysis of LD and CPC in two combined oral gel preparations for which the two analytes were successfully resolved from the pharmaceutical adjuvants and quantified with recoveries not less than 97.9%.     

A Fast,Simple Method for Analysis of Phenoxy Acid Salt and Ester Formulations by High Performance Liquid Chromatography

Abstract

Salt formulations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid] have been analysed by reversed-phase HPLC using a C18-column with 50:50 (v/v) acetonitrile/2% acetic acid as eluant. Internal and external standard HPLC methods are compared.

Ester formulations of 2,4-D and 2,4,5–T are analysed, without hydrolysis, on the same column using 60:40 (v/v) acetonitrile/2% acetic acid as eluant. The method has been used in this laboratory to determine free phenoxy acid in ester formulations, and for the identification of esters in mixed ester formulations.

The methods are fast and accurate, and offer some advantages over previously-described methods.     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.