An isocratic high-performance liquid chromatography method for the simultaneous analysis of plasma retinol, alpha-tocopherol, and various carotenoids

An isocratic high-performance liquid chromatography method for the simultaneous determination of various fat-soluble vitamins and carotenoids is reported. The method utilizes a Radial-Pak C-18, 5-microns column and an elution solvent composed of methanol:acetonitrile:chloroform (25:60:15). Only 100 microliters of plasma sample is required for one determination. Retinol, alpha-tocopherol, alpha-carotene, beta-carotene, lycopene, zeaxanthin, and two other unidentified carotenoids can be clearly separated and quantified in one HPLC run using alpha-tocopheryl acetate or tocol as the internal standard. The eluted peaks are quantified by either a photodiode-array detector at preprogrammed wavelengths at the absorption maxima of the compounds or a dual-wavelength detector at 280 and 436 nm. The total run time is 16 min. With an automatic injector and a programmable detector, the system allows unattended operation. The within-run and day-to-day coefficients of variation range from 1 to 8%. The lower limits of determination are 2, 40, and 2 ng for retinol, alpha-tocopherol, and carotenes, respectively. In addition, the system monitors the absorption spectra of the eluant during the HPLC run; this allows the spectral identification of various compounds separated in the same run.     

Reference ranges of retinol, tocopherols, lycopene and alpha- and beta-carotene in plasma by simultaneous high-performance liquid chromatographic analysis

Using a high-performance liquid chromatographic procedure, age- and gender-based reference ranges for plasma retinol, alpha- and gamma-tocopherol, lycopene, and alpha- and beta-carotene have been established. In addition to confirming higher retinol levels in men than in women (p less than 0.001), this study demonstrates significantly higher plasma levels of both carotenes in women than in men (p less than 0.001). The plasma levels of retinol and beta-carotene showed a positive relationship with age after adjusting for plasma lipids (p less than 0.001 and p less than 0.02, respectively). In contrast to beta-carotene, there was no gender difference for plasma lycopene and a strong (p less than 0.001) inverse relationship of lycopene with age. These results suggest that differences may exist between beta-carotene and lycopene in their intestinal absorption, plasma transport, or tissue metabolism.     

Rapid method for the determination of piroxicam in rat plasma using high-performance liquid chromatography

A previously published method was used for the determination of piroxicam in plasma samples obtained from rat. The sample preparation involved liquid extraction, centrifugation and evaporation. Separation of piroxicam from internal standard occurred on a reversed-phase C₁₈ column with a mobile phase consisting of methanol-phosphate buffer pH 2 (45:55). The detection limit of the assay was 0.02–20 μg/ml. The assay linearity was good (typically r = 0.9992). The method was applied for determination of piroxicam in rats after administration of an oral dose of 2 mg/kg piroxicam.     

Reversed-phase high-performance liquid chromatography method for the analysis of nitro-arginine in rat plasma and urine

Nitro--arginine (-NNA) is an inhibitor of the enzyme nitric oxide synthase (NOS). We developed a simple, sensitive and reproducible reversed-phase high-performance liquid chromatographic method for detection of nitro-arginine (- and -enantiomer) in rat plasma and urine. Samples were treated with perchloric acid, neutralized and eluted through a C₈ reversed-phase column with a mobile phase of 18.5 mM heptanesulfonic acid-10% methanok in water using theophylline as an internal standard. Plasma recovery for both isomers was complete, and the sensitivity limit was 0.5 μg/ml. This method may be used for disposition studies of -NNA in small animals.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

High-performance liquid chromatography method for quantifying sphingomyelin in rat brain

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Influence of dietary vitamins A and E on serum alpha- and gamma-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti)

Serum retinol, retinyl palmitate, beta-carotene, cryptoxanthin, lutein, alpha-tocopherol and gamma-tocopherol were measured in 18 captive Humboldt penguins (Spheniscus humboldti) prior to and following the removal of Columbia River (CR) smelt (Thaleichthys pacificus) from the diet. Dietary vitamin A was reduced from 59.8 to 13.5 IU g-1 (dry matter basis) when CR smelt was removed from the diet. Minimal changes were noted in dietary vitamin E. Serum samples Without-CR smelt had significantly lower circulating retinol (1.19 +/- 0.09 vs. 1.94 +/- 0.08 micrograms ml-1) and retinyl palmitate (0.033 +/- 0.012 vs. 0.105 +/- 0.004 microgram ml-1) compared to samples With-CR. The Without-CR smelt diet resulted in increased serum alpha-tocopherol from 26.4 +/- 0.94 to 39.1 +/- 3.72 micrograms ml-1. More serum samples taken Without-CR smelt had detectable levels of gamma-tocopherol than those With-CR smelt. Serum lutein was higher for the samples taken Without versus With-CR smelt. Serum cryptoxanthin did not differ. beta-Carotene was not detected. Data indicate that high levels of dietary vitamin A can affect circulating levels of retinol, retinyl palmitate and vitamin E. Thus, dietary vitamin A and the interrelationship between vitamins A and E should be considered when assessing captive penguins.     

Direct plasma injection method for the analysis of tryptophan metabolites by high-performance liquid chromatography coupled with precolumn deproteinization

Reversed-phase HPLC method by direct plasma injection has been developed for the analysis of major tryptophan metabolites (both metabolites in kynurenine pathways and in indole pathways). Two columns were used: one was a short precolumn of protein-coated octadecylsilane (ODS) for deproteinization and also for trapping of tryptophan metabolites, and the other was an analytical column of the usual ODS. By a column-switching method, the metabolites trapped in the precolumn were allowed to be eluted through the analytical column. The recovery of the spiked metabolites in plasma by the present method was almost quantitative (98-102%) with good reproducibility (CV less than 3%, within-run), and the method is determined to be simple and reproducible for the analysis of total (free + protein-bound) tryptophan metabolites in plasma. The analysis of rabbit plasma showed several peaks corresponding to kynurenine, kynurenic acid, 5-hydroxyindole-3-acetic acid, indole-3-lactic acid, indole-3-acetic acid, indole-3-propionic acid, and 5-hydroxy-tryptamine in addition to tryptophan.     

Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography

An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 microm, 150 mm x 2 mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 microg/ml for plasma and amniotic fluid and 0.2-50 microg/ml for the placental and fetal tissues.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Alternative method for determination of ceftazidime in plasma by high-performance liquid chromatography

A high-performance liquid chromatography procedure was developed to analyze ceftazidime concentrations in plasma. The procedure consisted of solid phase extraction followed by ion-pairing reverse-phase chromatography. An excellent linear relationship between ceftazidime peak height measurements and concentrations was demonstrated over the concentration range of 1–200 μg ml⁻¹. The advantage of this assay is the elimination of interference at the ceftazidime elution time that has been noted in previous studies and in our experience. Thus, this study describes an alternative, simple methodology that is clinically useful for analyzing ceftazidime in the research setting.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography spectrometric analysis of trans-resveratrol in rat plasma

A HPLC method for determination of trans-resveratrol concentrations in rat plasma was developed. Plasma samples were treated with acetonitrile to deposit proteins. The analysis used a Hypersil ODS(2) C(18) column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow-rate=1 mL/min). The UV detection wavelength was 303 nm, and chlorzoxazone was used as the internal standard. The calibration curve was linear over the range of 0.02-40 microg/mL with a correlation coefficient of 0.9997. This concentration range corresponds well with the plasma concentrations of resveratrol in pharmacokinetic studies. There was 98.7%, 91.3% and 84.4% recovery from 0.02, 0.4 and 40 microg/mL plasma samples respectively. The R.S.D. of intra- and inter-day assay variations were all less than 12%. This HPLC assay is a quick, precise and reliable method for the analysis of resveratrol in pharmacokinetic studies.     

High-performance liquid chromatography spectrometric analysis of tripterin in rat plasma

A quick, precise and reliable HPLC method has been developed to determine tripterin in rat plasma. After liquid-liquid extraction, the analytes was analyzed on a Discovery ODS C(18) column (5microm, 4.6mmx250mm) with an isocratic elution consisting of methanol-water-phosphoric acid (87:13:0.2, v/v/v). Ultraviolet detection was at 425nm. Using trioxymethylanthraquinone as an internal standard, the assay was linear over the concentration range of 0.025-1.60microg/mL (r(2)=0.9988). The extraction recovery of tripterin in rat plasma was more than 62%. The intra- and inter-day precision was less than 13% (CV). This validated method was successfully applied to the pharmacokinetics of tripterin in rats.     

Sensitive and rapid isocratic liquid chromatography method for the quantitation of curcumin in plasma

An HPLC assay was developed using three methods of plasma sample preparation in order to quantitate curcumin, the main constituent in the herbal dietary supplement turmeric. Each method involves simple and rapid processing of samples (either an ethyl acetate or chloroform extraction) with resulting different quantitation limits for curcumin. The assay was developed in an effort to quantify extremely low curcumin plasma concentrations observed in preliminary in vivo studies. The most sensitive assay can reliably detect concentrations down to 2.5 ng/ml. Plasma quantitation was precise and accurate based on both intra- and inter-day validations as indicated by low values for coefficients of variation and bias, respectively (< or =15%). The analytical validation was reproducible between different analysts. The resulting analytical method couples desired sensitivity with the ease of an isocratic system.     

Sensitive column-switching high-performance liquid chromatography method for determination of propiverine in human plasma

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2-200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Improved method for the analysis of furosemide in plasma by high-performance liquid chromatography

Modifications of existing rapid high-performance liquid chromatographic procedures for the determination of furosemide in plasma were made in order to achieve greater sensitivity. To a small volume of plasma was added an internal standard structurally related to furosemide. Then, following previously described procedures, acetonitrile was added to precipitate the proteins and the clear supernatant was separated. However prior to injection of the supernatant the pH and composition of the sample were adjusted. This modification of the sample enabled an injection volume of up to 300 μl of the supernatant to be injected onto the chromatographic column. The effluent was monitored spectrofluorimetrically. A standard linear calibration curve with a mean precision of ± 4.4% was obtained for plasma samples containing 20–900 ng/ml of furosemide. Two structurally related compounds were used as internal standards in the furosemide assay.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C₁₈ column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).