Sensitive determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in human plasma by HPLC with electrochemical detection

Summary This study deals with the development of a new HPLC method for the determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), the main noradrenaline metabolite in human plasma. A Varian reversed-phase column (C₈; 250 mm×4.6 mm i.d.; 5 μm particles) was used as the stationary phase and an aqueous solution of citric acid, 1-octanesulfonic acid, EDTA, and methanol was used as the mobile phase. Coulometric electrochemical detection (ED) was used to obtain the highest sensitivity. Isolation of MHPG from plasma was accomplished by means of a new solid-phase extraction procedure after a protein precipitation step. The extraction yield of MHPG from plasma was very high (>97%). Linearity was observed in the 0.5–25 ng mL⁻¹ concentration range; the limit of detection was 0.2 ng mL⁻¹ and the limit of quantitation was 0.5 ng mL⁻¹. Repeatability (RSD,%) for plasma samples was found to be <3.2% and intermediate precision was <4.3%. The method was applied to the determination of MHPG in the plasma of healthy subjects under experimentally-induced psychological stress.     

Determination of sulbutiamine and its disulfide derivatives in human plasma by HPLC using on-line post-column reactors and fluorimetric detection

Summary A new direct HPLC procedure for the simultaneous determination of sulbutiamine (Arcalion) and other thiamine disulfides in human plasma has been developed. The method involves an automated solidphase extraction on octadecylsilyl (C18) cartridges and chromatographic separation of the compounds on an RP Select B column with gradient elution using methanol and phosphate buffer. Detection was by fluorescence of the resulting thiochromes obtained from two on-line post-column reactors. Optimization of post-column reaction parameters has been achieved. This method has been proved to be highly selective for the determination of the thiamine disulfide derivatives and quantitation limits of 5 ng·ml^–1 were obtained for each compound in human plasma. Linearity was in the range 5–200 ng·ml^–1. Precision and accuracy were also demonstrated by within-day and between-day assays, and showed the good reliability of the method.     

Simultaneous determination of cefoxitin,cefuroxime, cephalexin and cephaloridine in plasma using HPLC and a column-switching technique

Summary A new high performance liquid chromatographic method was developed using a column-switching technique for the simultaneous determination of cephalexin, cefuroxime, cefoxitin and cephaloridine in plasma. The plasma samples were injected onto a precolumn packed with Corasil RP C₁₈ (37–50 m) after simple dilution with an internal standard solution in 0.01 M acetate buffer (pH 3.5). Polar plasma components were washed out using 0.01 M acetate buffer (pH 3.5). After valve switching, the concentrated drugs were desorbed in back-flush mode and separated on a Partisil ODS-3 column using acetonitrile in 0.02 M acetate buffer (pH 4.3) (1585, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed with a detection limit of 0.5 g/ml. The total analysis time per sample was less than 25 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9 %.This method has been successfully applied to plasma from rats after subcutaneous injection of cefuroxime.     

Enhanced sensitivity by laser-induced fluorescence for the determination of calcitriol and other vitamin D3 metabolites in plasma

Summary A sensitive method for the determination of hydroxymetabolites of vitamin D₃, parficularly calcitriol (1,25-(OH)₂-D₃), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system. The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature in a strong-acid medium. A LOD of 0.01 pg mL⁻¹ (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients (≥0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n=5) were acceptable (between 2.6 and 4.7%). The method was also checked by applying it to human plasma spiked with the target analytes and excellent recoveries were obtained. This is the first time that these species have been determined at the sub-pg mL⁻¹ level.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 °C for 30 min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5 mL min⁻¹ for 1 min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250 mm × 4.6 mm, 5 μm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0 mL min⁻¹. The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470 nm and an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9995, n = 5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500 ng mL⁻¹, with limits of detection and quantification of 0.5 and 1.7 ng mL⁻¹, respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11 ± 2.24% and 100.99 ± 2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12 min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.     

High-Performance Liquid Chromatographic Assay of Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in Human Plasma using a Solid-Phase Extraction Technique

A selective and sensitive liquid chromatographic method was developed for the determination of zonisamide in small volumes of plasma. Zonisamide and the internal standard methyl 4-hydroxybenzoate were extracted from 0.2 mL of plasma with solid-phase extraction columns and eluted with methanol. Analysis of the extracts was performed on a Symmetry C₁₈ column with ultra-violet spectrophotometric detection. The calibration curve was linear over the concentration range of 0.05–5 g mL^–1 in plasma. Recoveries were reasonable for routine analyses; the limit of quantification was 0.05 g mL^–1 with a signal-to-noise ratio of 5. This method could be useful for the pharmacokinetic study of zonisamide in a limited volume of human plasma and for therapeutic drug monitoring.     

Determination of vitamins D2 and D3 in pharmaceuticals by supercritical-fluid extraction and HPLC separation with UV detection

Summary The supercritical-fluid extraction of vitamins D₂ and D₃ with carbon dioxide is reported for the first time. The extraction recovery was enhanced by direct addition of diethyl ether to sample contained in the extraction cell. Separation and detection of the analytes was performed off-line by reversed-phase liquid chromatography with UV-detection. The quantification limit of the method is 4.1 μg for both analytes, with precision, expressed as relative standard deviation, of 3.8 and 6.3% for vitamins D₂ and D₃, respectively (η=7). The proposed method has been applied to the determination of vitamin D in different pharmaceutical products; recoveries were between 85 and 105%.     

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

Summary An HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C₈ column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL⁻¹, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL⁻¹ for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.     

Simultaneous sample preparation for high-performance liquid chromatographic determination of Vitamin A and β-carotene in emulsified nutritional supplements after solid-phase extraction

Determination of small amounts of the fat-soluble species Vitamin A (VA) (2.5 μg/g) and β-carotene (9 μg/g) from emulsified nutritional supplements containing 50 kinds of co-existing compounds and a fat content between 2000 and 8000 times higher was performed by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with fluorescence detection set at ex. 350 nm and em. 480 nm, and visible detection at 450 nm using an Inertsil ODS 80A (5 μm) analytical column. Mobile phases of methanol-ethanol (50:50) and acetonitrile-ethanol (70:30) were used for the both vitamins. A Bond Elut C₁₈ cartridge was chosen for SPE after comparison with eight other types of SPE cartridge. Retention time of VA and β-carotene was 7 and 8 min, respectively, giving a limit of detection of ca. 0.1 ng per injection at a signal-to-noise ratio 3:1. Recoveries of VA and β-carotene were over 90% by the standard addition method. Relative standard deviation of VA and β-carotene were ca. 2.9 (n=5) and 2.3% (n=5), respectively.     

Determination of adapalene (CD271/differin®) and retinol in plasma and tissue by on-line solid-phase extraction and HPLC analysis

Summary Adapalene, the active constituent of Differin^?, is a novel potent retinoid (vitamin A analogue) for the topical treatment of acne vulgaris. The clinical usefulness of retinoids is limited by a number of side effects, such as teratogenicity and skin irritation. A method has been developed for simultaneous determination of adapalene and retinol in plasma and tissue in in vivo and in vitro studies for the determination of the pharmacokinetic profile and the influence of adapalene on the endogenous retinol level. The new method was developed by coupling an autosampler to an automated solid-phase extraction unit on-line with a gradient HPLC system using UV and fluorescence detection. The low detection limit (0.25 ng mL⁻¹ for adapalene), the small sample weight (50 mg) and the high degree of automation make this method convenient for analysis of biological samples in animal and human studies. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Simultaneous determination of CGP 22 848 and its major metabolite (free carboxylic acid) CGP 35 652 in plasma by HPLC

Summary A specific method for the quantitative determination of CGP 22 848 (a basic compound) and its major metabolite CGP 35 652 (a free carboxylic acid metabolite) in plasma using CGP 17 582 as internal standard is described. Separation of components is by reversed-phase chromatography using a mobile phase consisting of pH 3 phosphate buffer-acetonitrile (76:24, v/v) at a flow-rate of 1 ml/min in conjunction with a Radialpak C18 cartridge combined with a radial compression separation system or by using a mobile phase consisting of pH 3 phosphate buffer-acetonitrile (80:20, v/v) at a flow-rate of 0.8 ml/min in conjunction with a Novapak C18 column. Rapid extraction of CGP 22 848 and CGP 35 652 from plasma is achieved using reversed-phase C18 columns (Bond-Elut or Supelco). With 250 μl of plasma, concentrations down to 0.078 μM (25 ng/ml) of CGP 22 848 B and 0.326 μM (100 ng/ml) of CGP 35 652 A can be determined. A wavelength of 221 nm was used to monitor CGP 22 848, 280 nm for CGP 35 652 and 320 nm for the internal standard.     

Determination of clozapine in human plasma by solid-phase extraction and liquid chromatography with amperometric detection

Summary A sensitive and selective high-performance liquid chromatographic method has been developed for monitoring clozapine levels in human plasma. Chromatography was performed on a reversed-phase column (C₈, 150 mm×4.6 mm i.d., 5 μm) with acetonitrile-aqueous sodium acetate solution, 88∶12 (v/v), as mobile phase; the flow rate was 1 mL min⁻¹. Clozapine oxidation at +800 mV was detected amperometrically. Response was linearly dependent on concentration over the range 50–1500 ng mL⁻¹ clozapine in plasma. Sample preparation by solid-phase extraction before HPLC analysis gave high extraction yield (94%). The accuracy and precision of the method were both very good (recovery: 97%;RSD<3.3%).     

Direct measurement of pentoxifylline and its hydroxymetabolite from plasma using HPLC with column switching techniques

Summary Using column switching techniques a rapid and precise HPLC method for the determination of pentoxifylline was developed. Shorter precolumns with RP 2 filling material and longer purging times at neutral pH reduce the amount of interfering peaks while retaining a 100% extraction yield. Of all stationary phases of the analytical column tested only Nucleosil Phenyl yielded base line separation and acceptable life time of the column. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05-5.0 μg mL⁻¹. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2-6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL⁻¹ for andrographolide and 0.022 μg mL⁻¹ for dehydroandrographolide. This method was successfully applied to the plasma concentration-time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.     

Determination of paraquat in human blood plasma using reversed-phase ion-pair high-performance liquid chromatography with direct sample injection

This report describes the determination of paraquat (PQ) in human blood plasma samples by a direct-injection reversed-phase ion-pair chromatographic method. Blood plasma filtrate was injected directly into the LiChrospher® RP-18 alkyl-diol silica (ADS) precolumn integrated in a column switching system using a mixture of 3% 2-propanol and 10 mM sodium octane sulfonate (SOS) in a 0.05 M phosphate buffer (pH 2.8). After washing with this phase, the ADS precolumn was back-flushed with the analytical mobile phase consisting of 40% of methanol and 10 mM SOS in a 0.05 M phosphate buffer (pH 2.8) at a flow rate of 1.0 ml min⁻¹, in order to carry the analyte to a conventional reversed-phase analytical column, where the separation of PQ was achieved and finally detected by UV at 258 nm. The recoveries of PQ from human blood plasma samples ranged between 95.0 and 99.5% at nine different concentrations (from 0.05 to 3.00 μg of PQ ml⁻¹) with coefficients of variation <2.5% (n=3). The precision expressed as relative standard deviation was below 3.5% for between-day and below 4.3% for within-day measurements (n=5). The detection limit (signal-to-noise ratio, S/N>3) was 0.005 μg ml⁻¹ with an injection volume of 200 μl. The proposed method is promising for the identification and quantification of PQ at low concentration levels and is suitable for its analysis in human blood plasma samples from intentional or accidental poisonings cases with a sample throughput of 5 samples per hour.     

Vitamin D analysis in plasma by high performance liquid chromatography (HPLC) with C(30) reversed phase column and UV detection--easy and acetonitrile-free

Two physiologically important forms of vitamin D exist: vitamin D₂ and vitamin D₃, which by liver based hydroxylase enzymes are converted to 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃, respectively. These hydroxylated metabolites of vitamin D are measured in plasma to assess the vitamin D status of animals and humans. Therefore cheap and reliable analytical methods are very much in demand in nutritional and physiological research. After saponification and extraction of plasma or serum samples the current method uses reverse phase high performance liquid chromatography on a C₃₀ column and with UV detection at 265 nm for quantifying vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, and 25-hydroxyvitamin D₃. The method proved versatile with respect to plasma lipid content, sample amount, and plasma concentration of the vitamin D metabolites as it was tested using plasma from six different species: cattle, pigs, poultry, mink, horses, and humans. In cattle plasma recoveries were between 86.6 and 101.0%, within day error between 0.9 and 5.9%, and between day error between 0.2 and 1.7%. However, depending on species and sample amount error percentages varied. When running the method on standard reference material^® 972 “Vitamin D in human serum” from the National Institute of Standards and Technology (NIST) (Gaithersburg, USA) the results for 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ concentrations were within the boundaries provided by NIST, reflected by Z-scores between 0.1 and 0.9.     

Determination of cyclosporin A in blood and plasma by column switching-HPLC after rapid sample preparation

A method for the determination of cyclosporin A in human whole blood and plasma is described which uses liquid chromatography with step gradient elution and a column switching technique. The chromatographic conditions chosen allow simple and rapid sample preparation, so that a result can be obtained within one hour. Blood and plasma are deproteinized with diluted methanol and an aliquot of the clear supernatant is directly injected. The detection limit for cyclosporin A is about 20 ng/ml starting from a 0.5 ml sample. The method is sensitive enough for monitoring the drug in the therapeutic range.     

Selective quantification of doxycycline in human plasma and urine with optimised chromatography

Summary A method for the selective quantification of doxycycline in human plasma and urine has been developed using HPLC with UV-detection at 350 nm. Analyte and internal standard were extracted from plasma by extraction columns filled with octadecylsilica. Urines were only mixed with diluted acid prio to injection. The influence of pH on peak shapes is discussed as well as comparative investigations on selectivity and peak symmetry on commerical octadecylsilica. The method was successfully applied to the samples of a clinical study with an oral single dose of 100 mg. Precision and accuracydata of the assay and calculated pharmacokinetic parameters are presented.     

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

Summary Dobutamine is one of the synthetic catecholamines acting directly onβ ₁-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.