High-performance liquid chromatography with ultraviolet detection for real-time therapeutic drug monitoring of meropenem in plasma

A simple, rapid, and precise high-performance liquid chromatography (HPLC) method using ultrafiltration to remove plasma protein was developed to determine meropenem concentrations in human plasma in a clinical setting. Plasma was separated by centrifugation at 4 degrees C from blood collected in heparinized vacuum tubes, and meropenem was stabilized by immediately mixing the plasma with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Ultrafiltration was used for plasma deproteinization. Meropenem was detected by ultraviolet absorbance at 300 nm with no interfering plasma peak. The calibration curve of meropenem in human plasma was linear from 0.05 to 100 microg/mL. Intraday and interday precision was less than 7.17% (CV), and accuracy was between 97.7% and 106.3% over 0.05 microg/mL. The limit of detection was 0.01 microg/mL. The assay has been clinically applied to a real-time therapeutic drug monitoring in pediatric patients and pharmacokinetic studies.     

High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

High-performance liquid chromatography with ultraviolet detection for therapeutic drug monitoring of everolimus

We developed and validated a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for determining everolimus concentrations in human whole blood. Sample preparation involved a solid-phase extraction after protein precipitation. The separation of everolimus from internal standard (IS) and endogenous components was achieved using an isocratic elution on an octyl column. The method showed a linear relationship between peak height ratios and blood concentrations in the range of 1-200 ng/mL (r(2)=0.9997). The observed intra- and inter-day assay imprecision had a coefficient of variation (CV)=12.8%, and inaccuracy was 11.4%. The method was found to be precise, accurate, and sensible making it useful for routine therapeutic monitoring of everolimus.     

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCl were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)–acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.     

High-performance liquid chromatographic assays with fluorometric detection for mivacurium isomers and their metabolites in human plasma

Two high-performance liquid chromatographic assays coupled with fluorometric detection have been developed for the determination of mivacurium isomers (trans-trans, cis-trans and cis-cis) and their monoester and alcohol metabolites in human plasma. A novel solid-phase extraction procedure allowed good recovery of the mivacurium isomers (mean 98%) and their monoester metabolites (mean 83%), whereas the alcohol metabolites were analyzed after direct precipitation of plasma proteins. For all analytes, these assays proved to be sensitive (LOQ 3.9–15.6 ng/ml), reproducible (C.V. < 15%) and accurate (>94%) over the therapeutic range of concentrations of mivacurium and its metabolites. These two methods were applied successfully to a pharmacokinetic study of mivacurium after a bolus dose of 0.15 mg/kg in anesthetized patients.     

High-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol-O-methyltransferase-coupled fluorometric detection

A nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells required only about 2.5 microg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of K(m) and V(max) were 6.1+/-0.3 microM and 135.4+/-1.5 nmol AdoMet formed/mg protein/h, respectively.     

Determination of etoposide in human plasma and leukemic cells by high-performance liquid chromatography with electrochemical detection

This paper describes a high-performance liquid chromatographic method with electrochemical detection for the determination of etoposide levels in plasma, total and non-protein bound concentration, and in leukemic cells. The precision for between-runs (n=6) was 7.0, 4.9, and 9.5%, the accuracy was 3.7, 7.1 and 6.3%, and within-runs precision (n=6) was 3.9, 2.9 and 5.1% for total plasma, non-protein bound plasma fraction and leukemic cells, respectively. The correlation coefficients (R²) were 1.00 for all calibration curves. These assays have been applied to analyze samples from one patient with acute myelogenous leukemia during 24 h after i.v. infusion of etoposide (100 mg/m²).     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

Simultaneous quantitation of etoposide and its catechol metabolite in human plasma using high-performance liquid chromatography with electrochemical detection

Etoposide, a highly active and widely used antineoplastic agent, is O-demethylated to its active catechol metabolite. A high-performance liquid chromatographic assay method for the simultaneous quantitation of etoposide and etoposide catechol in human plasma was established. Etoposide and etoposide catechol were extracted from plasma using chloroform and methanol followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Chromatography was accomplished using a reversed-phase phenyl analytical column (390 mm×3.9 mm I.D.) with a mobile phase of 76.6% 25 mM citric acid–50 mM sodium phosphate (pH 2.4)–23.4% acetonitrile pumped isocratically at 1 ml/min with electrochemical detection. The limit of detection for etoposide was 1.2 nM and for etoposide catechol was 0.2 nM. The precision (CV) for etoposide ranged from 0.7 to 3% and for the catechol metabolite from 1 to 6%; accuracy of predicted values ranged from 97 to 106% and 94 to 103%, respectively. The assay was linear from 0.1 to 10 μM for etoposide and from 0.005 to 0.5 μM for etoposide catechol in plasma. Recovery of etoposide and etoposide catechol ranged from 93 to 95% and 90 to 98%, respectively. Stability of etoposide and etoposide catechol in human plasma containing ascorbic acid stored at −70°C for one year was demonstrated. This assay procedure is suitable for evaluation of etoposide and etoposide catechol pharmacokinetics in plasma following etoposide administration.     

High-performance liquid chromatographic determination of α-keto acids in plasma with fluorometric detection

This paper describes a sensitive high performance liquid chromatographic method for the quantitative determination of α-keto acids in plasma using a fluorescence detector. This method is about ten times more sensitive than that reported in a previous paper. Only 50 μl of plasma are needed for the determination of α-keto acids. However, p-hydroxyphenylpyruvic acid could not be analysed because the quinoxalinol derived from it does not exhibit fluorescence.     

Determination of unbound etoposide concentration in ultrafiltered plasma by high-performance liquid chromatography with fluorimetric detection

Etoposide is a highly protein bound drug, and monitoring the concentration of free drug could help individualize dosage in oncological patients. The cost and difficulty of the standard techniques (equilibration dialysis) has hampered the monitoring of free drugs. We describe a simple HPLC method for the measurement of free etoposide concentration in plasma. Sample preparation involves the ultrafiltration of plasma by a Centrifree device for 30 min at 2000 g and extraction with chloroform. The isocratic separation is performed with a μBondapak phenyl analytical column. Fluorimetric detection is used (288–328 nm excitation and emission wavelengths). Linearity of the calibration curve is excellent between 0.05 and 1 μg/ml. Accuracy and precision are reported at the concentrations 0.06 and 0.4 μg/ml: within-run accuracy is 10% and 6.2%, respectively; between-run accuracy is ⩽1%; within-run coefficients of variation (C.V.) are 10.6 and 5.0%; between-run C.V. are 11.6 and 6.8% respectively. The range of the assay is 0.05 to 1 μg/ml. The feasibility of the technique has been tested in 7 patients treated with oral etoposide for hepatocarcinoma (mean protein binding 91%). We found no interference from endogenous substances, co-administered drugs (alizapride, furosemide, ranitidine) and other antineoplastic agents (doxorubicine, idarubicine, vinblastine, vinorelbine).     

High-performance liquid chromatography measurement of hyperforin and its reduced derivatives in rodent plasma

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm x 4.6 mm, i.d. 3.5 microm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.     

High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma

A validated HPLC method for the simultaneous quantitative analysis of the antidepressant mirtazapine and its demethyl metabolite in human plasma is described. The active constituents including internal standard were extracted from 1 ml of plasma with hexane and separated on a μBondapak Phenyl column with fluorescence detection. The lower limit of quantification was 0.5 ng/ml, without significant interferences with endogenous or exogenous components. Inter- and intra-assay accuracy determined at quality control levels of 2, 10 and 80 ng/ml were, respectively, 104.6–113.7% and 105.1–117.7% for mirtazapine, and 91.7–99.3% and 89.9–103.7% for demethylmirtazapine. In all cases the precision was below 6.8%.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma

A rapid and sensitive high-performance liquid chromatographic method is described for the quantitative analysis of dipotassium clorazepate (CZP) and its major metabolite nordiazepam (ND) in fresh human and dog plasma. The method consists of two separate selective ND extractions from a plasma sample without and with conversion of all the CZP to ND. For quantitation, diazepam (DZP) is used as the internal standard. The chromatographic phase utilized in a reversed-phase Hibar® EC-RT analytical column prepacked with LiChrosolv RP-18 with a solvent system consisting of acetonitrile-0.05 M sodium acetate buffer, pH 5.0 (45:55). The UV absorbance is monitored at 225 nm using a variable-wave-length detector. The mean assay coefficient of variation over a concentration range of 20–400 ng per ml of plasma is less than 3% for the within-day precision. Recoveries of ND, DZP and CZP (as ND) are essentially quantitative at all levels investigated. The calibration curves of ND are rectilinear (r² = 0.99) from the lower limit of sensitivity (2 ng/ml) to at least 2000 ng/ml in plasma. Applicability of the method to CZP and ND disposition studies in the anaesthetized mongrel dog is illustrated. When the two separate selective nordiazepam extractions from plasma cannot be performed immediately after blood sampling, an extrapolation kinetic method is suggested for the estimation of CZP concentration. In all previous in vivo studies, CZP has been determined only with gas-liquid chromatographic methods.     

High-performance liquid chromatography and radioimmunoassay of rat plasma corticosterone

Problems inherent in corticosterone radioimmunoassay (RIA) led to consideration of alternative methods. A high-performance liquid chromatography (HPLC) procedure was evaluated that separated and quantitated dichloromethane-extracted corticosterone by reverse-phase chromatography. The results were correlated (r = 0.92) with an RIA procedure. The HPLC recovered nearly 100% of corticosterone added to rat plasma and had excellent reproducibility. In addition, chromatogram profiles of dichloromethane-soluble components obtained from rat plasma, derived from drug effect studies, could have value for characterizing response patterns. Without automated sample injection equipment, HPLC is more appropriately applied in monitoring RIA results than in processing large numbers of samples.     

High-performance liquid chromatography of flufenamic acid in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for the determination of flufenamic acid in rat plasma is described. After liquid-liquid extraction, the drug is separated by HPLC on a 5-μm octadecylsilica column (Nucleosil C₁₈) with ultraviolet detection at 280 nm. Linear calibration graphs for flufenamic acid were constructed from 0.5 to 15 μg/ml. The method has been applied to a pharmacokinetic study in animals.     

Microdetermination of hyaluronan in human plasma by high-performance liquid chromatography with a graphitized carbon column and postcolumn fluorometric detection

A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the ΔDi-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of ΔDi-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for ΔDi-HA was linear over the range 0.2 ng-1 μg. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 μg/ml).