Chromatographic Method For Simultaneous Determination Research Articles

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.

Application of a Fast High-Performance Liquid Chromatography Method for Simultaneous Determination of Furanic Compounds and Glucosylisomaltol in Breakfast Cereals

The high-performance liquid chormatography method reported in the present paper provides a fast, low-cost, precise, and simple technique to analyze simultaneously 3 different indicators of nonenzymatic browning, i.e., hydroxymethylfurfural (HMF), furfural, and glucosylisomaltol (GIM), in breakfast cereals. These compounds were extracted in aqueous solution and separated on a reversed-phase C18 column with water-acetonitrile (95 + 5, v/v) mobile phase under isocratic conditions. Average recovery rates for HMF, furfural, and GIM were 99.1, 98.4, and 99.4%, respectively. The coefficients of variation were 3.67, 2.42, and 1.59% for HMF, furfural, and GIM, respectively. The detection limit was 0.01 mg/kg, and the quantitation limit was 0.05 mg/kg for the 3 studied compounds.

High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates

A variety of degradation products are produced upon dilute acid pretreatment of lignocellulosic biomass. Within this larger construct, organic acids, phenols and aromatic aldehydes represent important compound classes to investigate due to increasing evidence of their inhibitory effect on fermentative microorganisms. An analytical extraction procedure is presented, enabling isolation of potential analytes away from alternative products in biomass hydrolysates. Additionally, a reversed-phase high-performance liquid chromatography (HPLC) method has been developed and validated, affording simultaneous separation and quantitative determination of 32 potential analytes in water with UV detection at 210 nm. The method was subsequently employed to quantify a variety of aliphatic acid, aromatic acid, aldehyde and phenolic degradation products in a corn-stover hydrolysate at concentration levels ranging from 0.02 to 41 mM.

A rapid chromatographic method for simultaneous determination of β-sitosterol and tocopherol homologues in vegetable oils

Tocopherols and phytosterols are important components of the unsaponifiable fraction in vegetable fatty foods. Their multiple beneficial effects in nutrition are to some extent well established; nevertheless they continue to be an object of intense research. In this work a simple method for simultaneous determination of both phytochemicals by reversed phase HPLC is proposed. The advantage of this method is a considerable reduction in time with simultaneous information on two types of analytes, whose health effects seem to be complementary. The optimized method is applied to a set of olive and nut oils. The results fill gaps in the literature, and highlight the possible capacity of these phytochemicals to discriminate very similar oils.

High-performance liquid chromatographic method for simultaneous determination of sophoridine and matrine in rat plasma.

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of sophoridine and matrine in rat plasma. Sophoridine and matrine in the resulting supernatant of the plasma deproteinized with acetonitrile containing an internal standard (acetanilide) were directly determined by reversed-phase HPLC and ultraviolet detection. The result of limits of quantitation for matrine and sophoridine were 200 and 350 ng/mL in plasma, respectively, and recovery of both analytes was greater than 98%. The assay was linear from 250 to 4000 ng/mL for matrine and from 500 to 8000 ng/mL for sophoridine. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of matrine and sophoridine in the plasma following oral administration of Kexieling tablets, which is one of the preparations of Kudouzi at a dose equivalent to 30 and 60 mg/kg of matrine and sophoridine, respectively.

Simultaneous separation and determination of four bioactive constituents in traditional chinese medicinal tablet Xinkeshu by HPLC‐DAD

AbstractA reversed‐phase high performance liquid chromatographic method for simultaneous determination of four bioactive constituents was established for the quality control of traditional Chinese medicinal tablet Xinkeshu. Danshensu, protocatechualdehyde, puerarin and daidzein were successfully separated on a Diamonsil C18 column (150×4.6 mm i.d., 5 μm) with a guard column (12.5×2.1 mm i.d., 5 μm) packed with the same material at 25 °C. The mobile phase was a mixture of acetonitrile and 0.02 mol/L potassium dihydrogen phosphate employing gradient elution at a flow rate of 0.8 mL/min. Detection was accomplished with a diode‐array detector and detection wavelength was set at 280 nm before 22 min, then it was varied to 250 nm. The four constituents were identified by comparing their retention time and W spectra in the range of 190–400 nm with those of authentic standards. The linear calibration ranges were 15.4–123.2, 3.11–25.22, 21.6–172.8, and 0.26–3.47 μg/mL for danshensu, protocatechualdehyde, puerarin and daidzein, respectively. The detection limits were 0.03, 0.04, 0.10, 0.03 μg/mL for danshensu, protocatechualdehyde, puerarin, and daidzein, respectively. The contents of these four bioactive constituents in Xinkeshu tablet were successfully determined by the proposed method.

High-performance liquid chromatographic method for simultaneous determination of baicalein and baicalein 7-glucuronide in rat plasma

A simple HPLC method with ultraviolet detection was developed for the simultaneous determination of baicalein and its major metabolite, baicalein 7-glucuronide (baicalin) in rat plasma. Following solid phase extraction with HLB cartridge, the analytes were separated using a gradient mobile phase consisting of methanol–acetonitrile–phosphate buffer. The flow-rate was set at 1 ml/min and the eluent was detected at 320 nm. The method is linear over the studied range of 1–10 and 0.05–1 μg/ml for baicalein and baicalin, respectively. The intra-day and inter-day variations of the analysis were less than 6.38%, with relative error ranging from −3.6 to 6%. The limit of quantification in plasma was 0.05 μg/ml for both baicalein and baicalin. The developed method proves to be an improved and more reliable method because of its capability for determining baicalein and baicalin simultaneously in a single chromatographic run.

Simple and rapid micellar electrokinetic capillary chromatographic method for simultaneous determination of four antiepileptics in human serum.

A very rapid and simple MEKC method was developed for the simultaneous determination of four antiepileptic drugs, ethosuximide (Etho), primidone (Pri), phenytoin (Pht) and carbamazepine (Cbz) in human serum. Sample analysis required only 100 microL of human serum which only needed to be centrifuged, decanted and combined with the running buffer [5.3 mM Na(2)HPO(4)/3.2 mM borax buffer (pH 9.5) containing 55 mM SDS and 3.5% (v/v) acetone]. The analysis was performed in only 10 min into fused-silica capillaries (57 cm total length with 50 microm i.d. and 50 cm to the detector) using the MEKC methodology with diode-array detection at 220 nm. The calibration graphs were established for ethoximide, primidone, phenytoin and carbamazepine between 0 and 20 mg/L. Recoveries were between 85 and 87%. The simplicity of the proposed methodology makes it suitable for routine clinical use, especially for epileptic patients on polytherapy.

Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO 4 and methanol. The analysis was performed on a 3 μm, C 18 Tracer Excel 15 cm×0.4 cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH=3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10–500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 °C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 μl plasma. Detection limits were 0.1 μg/ml for MPA and AcMPAG, and 2.0 μg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid–liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol–acetonitrile–phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5–16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.

Simple and sensitive high-performance liquid chromatography method for simultaneous determination of urinary free cortisol and 6β-hydroxycortisol in routine practice: For CYP 3A4 activity evaluation in basal conditions and after grapefruit juice intake

Cytochrome P450 3A4 activity displays a wide variability. The urinary 6β-hydroxycortisol to cortisol ratio, as a non-invasive assay, can be useful for its pretherapeutic characterization. We developed an HPLC–UV method preceded by liquid–liquid extraction for assessment of this ratio in clinical practice. Urine was collected on second void morning-spot sample. Percentage recoveries were high and reproducible. The 6β-hydroxycortisol to cortisol ratio ranged from 1.6 to 9.9 in 12 Caucasian healthy volunteers. It was reduced by 30 to 70% after ingestion of white grapefruit juice, a CYP3A4 inhibitor. Our method, simple, sensitive and accurate, could be helpful for determination of CYP 3A4 activity before oral chemotherapy, or for the monitoring of the use of grapefruit juice as a pharmacological modulator.

Facile and rapid high-performance liquid chromatography method for simultaneous determination of allopurinol and oxypurinol in human serum.

To develop a rapid and sensitive assay for the simultaneous determination of allopurinol and oxypurinol in serum. High-performance liquid chromatography (HPLC) with UV-detection. Sample preparation consists of protein precipitation by an addition of trichloracetic acid. Percentage recovery and intra-assay coefficient of variation (CV%) for allopurinol were 97.4-101.3 and 0.66-5.13, respectively, in the concentration range 0.5-5.0 microg/mL. For oxypurinol, the percentage recovery and the intra-assay CV% were 93.2-98.1 and 0.88-5.62, respectively, in the concentration ranges 0.4-20 microg/mL. There was no interference of endogenous compounds in this assay. This method is useful for routine therapeutic drug monitoring of allopurinol in a clinical setting.

Liquid chromatographic analysis of phenylpropanoids fromRhodiola rosea extracts

Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. The first liquid chromatographic method for simultaneous determination of eight phenylpropanoids of the plant, including its main bioactive compounds, was developed. The method utilizes Waters Xterra RP182.1×50 mm reverse-phased column, 10 mM aqueous ammonium acetate/acetonitrile gradient elution and photodiode array detection at UV wavelengths 276 nm and 254 nm. Good linearity was obtained over the range 0.2–50 μg mL1 for salidroside and 0.2–100 μg mL1 for rosavin.

Sensitive and rapid high-performance liquid chromatographic method for simultaneous determination of antidepressants in pharmaceutical formulations

An easy, rapid, and sensitive high-performance liquid chromatographic (HPLC) method is proposed for simultaneous determination of five antidepressants (trazodone, citalopram, fluoxetine, fluvoxamine and clomipramine) and norfluoxetine, a major metabolite of fluoxetine. Optimum conditions for the quantitative separation were investigated. The compounds were separated on a Nova Pack C18 column with acetonitrile-pH 2.5 phosphate buffer, 40∶60 (v/v), as mobile phase at a flow rate of 1.5 mL min−1. Stability of solutions, linearity, repeatability and reproducibility, accuracy, specificity, and detection and quantification limits were examined to validate the method. The method is rapid and sensitive and can be applied to the analysis of antidepressants in pharmaceutical formulations and biological fluids; norfluoxetine can also be determined in these matrices. We have tested the method by analysis of several pharmaceutical formations; recoveries obtained were between 102.7% and 98.2% of the labelled values.

High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of honokiol and magnolol in rat plasma. The plasma was deproteinized with acetonitrile which contained an internal standard (diphenyl) and was separated from the aqueous layer by adding sodium chloride. Honokiol and magnolol are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC and ultraviolet detection. The limits of quantitation for honokiol and magnolol were 13 and 25 ng ml −1 in plasma, respectively, and recovery of both analytes was greater than 93%. The assay was linear from 20 to 200 ng ml −1 for honokiol and from 40 to 400 ng ml −1 for magnolol. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration–time profiles of honokiol and magnolol in the plasma following rectal administration of Houpo extract at a dose of 245 mg kg −1, equivalent to 13.5, 24.4 mg kg −1 of honokiol and magnolol, respectively.

A high performance liquid chromatography method for simultaneous determination of albendazole metabolites in human serum

A simple assay for albendazole (ABZ) main metabolites—albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO 2) and albendazole amino sulphone (ABZ-SO 2-NH 2)—in serum using high performance liquid chromatography was developed. The method involves liquid–liquid extraction of the serum by ethyl acetate, clean up with n-hexane and re-extraction with ethyl acetate, followed by separation on RP-C 8 column with a mixture of methanol: acetonitrile: acetic acid: water (40:1:10:49) as the eluting solvent. ABZ-SO and mebendazole—used as internal standard—were detected by UV ( λ=286 nm), and ABZ-SO 2 and ABZ-SO 2-NH 2 with fluorescence spectrophotometer at (Excitation=286 nm, Emission=333 nm) and (Excitation=286 nm, Emission=315 nm), respectively. The assay was accurate and reproducible with a detection limit of 10 ng/ml for ABZ-SO, 2 ng/ml for ABZ-SO 2 and 4 ng/ml for ABZ-SO 2-NH 2. Disregarding ABZ determination, which is not of pharmacokinetic importance as it is not found in human plasma after oral administration, the proposed method is appropriate for further pharmacokinetic and metabolism study of this drug.

Micellar electrokinetic capillary chromatographic method for simultaneous determination of drugs used to treat advanced breast cancer

A micellar electrokinetic capillary chromatography (MEKC) method has been developed for quantification of four drugs-tamoxifen, anastrozole, letrozole, and methotrexate—used to treat advanced breast cancer. Separation was performed at 25°C and 25kV, with 20mm borate buffer (pH 9.2) containing 40mm sodium dodecylsulfate as electrolyte solution. Under these conditions analyses were performed in 12 min. The linearity of the response was investigated for the concentration range 2.0–20.0 mg L−1. The intra-day residual standard deviation (n=4 graphs) between the slopes of the calibration graphs was acceptable for the four drugs studied. Detection limits (signal-to-noise ratio=3) were below 1 mg L−1 for all the compounds. The simplicity, precision, and sensitivity of MEKC proved suitable for quality control of pharmaceutical preparations used to treat advanced breast cancer. Six different pharmaceutical preparations, each containing one of the above-mentioned drugs, were successfully analyzed.

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn ( Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C 18 column with a gradient elution of 5 and 25% acetonitrile in 25 m M phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.

Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, α-tocopherol, 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 in human plasma with photodiode-array ultraviolet detection

A new rapid and sensitive high-performance liquid chromatographic method using 0.5 ml of plasma has been developed for the simultaneous determination of retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3. The eluate was monitored with a photodiode-array detector with two fixed wavelengths (267 nm for vitamin D, 292 nm for α-tocopherol and retinol). For all compounds, including internal standards, the method provides extraction recoveries greater than 81%. Detection limits were equal to or lower than 1.5 μg/l for the 4 vitamins. Linearity of standards was excellent ( r>0.999 in all cases). Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was 3 4 7.7 for all compounds and the inter-day-assay C.V. was <9.2% except for the lower concentrations of 25-hydroxyvitamin D 3, 25-hydroxyvitamin D 2 and α-tocopherol (10.8, 11.8 and 11.9, respectively). The important properties of the present method are its ease of use, its rapidity, since sample preparation was achieved in 15 min and all the compounds were eluted in less than 15 min, and its small sample volume required (=0.5 ml), which enables it to be used in pediatric practice.