Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Determination of plasma concentrations of epirubicin and its metabolites by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy

In order to determine epirubicin and its metabolites at low concentrations (<38 ng/ml) in small plasma samples, a fast reliable method based on a precipitation pre-treatment and sensitive reversed-phase isocratic HPLC has been developed and validated for epirubicin in the range 5–100 ng/ml. The R.S.D. was 5–9% over this concentration range. For human serum containing 25 ng/ml of epirubicin, the inter- and intra-day variation was <10%. Recoveries of the metabolites epirubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone at 20 ng/ml ranged from 94–104%. The assay has been used to study human plasma samples taken during a 96-h infusion of epirubicin in a patient with multiple myeloma. The combined levels of the unseparated metabolites, epirubicin glucuronide and epirubicinol glucuronide, were semiquantitatively determined after treatment with β-glucuronidase. The metabolites epirubicinol and 7-deoxydoxorubicinolone, but not 7-deoxydoxorubicinone, were also detected and measured.     

Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography

A method is described for the determination of the two enantiomers of mirtazapine in human blood plasma by high-performance liquid chromatography. Measurements were performed on drug free plasma spiked with mirtazapine and used to prepare and validate standard curves. Levels of enantiomers of mirtazapine were also measured in patients being treated for depression with racemic mirtazapine. Mirtazapine was separated from plasma by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved using a Chiralpak AD column and pre-column and compounds were detected by their absorption at 290 nm. Imipramine was used as an internal standard. The assay was validated for each analyte in the concentration range 10–100 ng/ml. The coefficient of variance was 16% and 5.5% for(+)-mirtazapine for 10 and 100 ng/ml control specimens respectively and 15% and 7.3% for mirtazapine for 10 and 100 ng/ml control specimens respectively. This assay is appropriate for use in the clinical range. The range of plasma mirtazapine concentrations from eleven patients taking daily doses of 30–45 mg of racemate was <5 to 69 ng/ml for (+)-mirtazapine and 13–88 ng/ml for (−)-mirtazapine for blood specimens collected 10–17.5 h after taking the dose.     

High-performance liquid chromatographic assay for the α-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100–1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.     

Solid-phase extraction–high-perfomance liquid chromatography determination of verapamil and norverapamil enantiomers in urine

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C₈ or C₁₈ bonded phase (C₈ in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5–300 ng/ml. Intra-day and inter-day reproducibilities were 10.5–14.2% at 3 ng/ml, 4.8–9.3% at 138.5 ng/ml and 7.8–10.1% at 280 ng/ml level, respectively, for all the enantiomers.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Liquid chromatographic–electrospray tandem mass spectrometric determination of novel antifungal agents in plasma

A rapid, selective, sensitive and reproducible HPLC–electrospray tandem mass spectrometric method has been developed for the analysis of novel triazole antifungal agents, SYN-2869 and its derivatives (SYN-2836, SYN-2903 and SYN-2921), in rat plasma using SYN-2506 as an internal standard. Isolation of these compounds from plasma and sample desalting were performed by a simple extraction procedure involving protein precipitation, vacuum-drying and reconstitution with acetonitrile. For all the agents, linearity was observed over the range of 10–10 000 ng/ml (r≥0.996) and the limit of quantitation was 10 ng/ml using a 100-μl plasma volume. A measurement rate of 400–500 samples/day/instrument could be achieved using this method.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane—ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile—ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5–500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63–74 and 63–68%, respectively, over a concentration range of 0.5–500 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Sensitive high-performance liquid chromatographic determination of nifedipine in cat plasma following improved sample treatment

A simple, easy and accurate reversed-phase high-performance liquid chromatographic method is described for the determination of nifedipine in cat plasma. The procedure involves extraction of nifedipine from plasma using a Sep-Pak C₁₈ cartridge and ultraviolet detection at 350 nm. The present method provides the required reproducibility and sensitivity for the determination of low concentrations of nifedipine without interference from plasma components or photodegradation products. The method was validated over the range 1–50 ng/ml nifedipine. Accuracy and precision were, respectively, 97% or more and 5% or less over the concentration range examined. The minimum quantifiable concentration of nifedipine was found to be 1 ng/ml.     

Enantioselective analysis of propafenone in plasma using a polysaccharide-based chiral stationary phase under reversed-phase conditions

We present a method for the enantioselective analysis of propafenone in human plasma for application in clinical pharmacokinetic studies. Propafenone enantiomers were resolved on a 10-μm Chiralcel OD-R column (250×4.6 mm I.D.) after solid-phase extraction using disposable solid-phase extraction tubes (RP-18). The mobile phase used for the resolution of propafenone enantiomers and the internal standard propranolol was 0.25 M sodium perchlorate (pH 4.0)–acetonitrile (60:40, v/v), at a flow-rate of 0.7 ml/min. The method showed a mean recovery of 99.9% for (S)-propafenone and 100.5% for (R)-propafenone, and the coefficients of variation obtained in the precision and accuracy study were below 10%. The proposed method presented quantitation limits of 25 ng/ml and was linear up to a concentration of 5000 ng/ml of each enantiomer.     

High-performance liquid chromatographic determination of diclofenac in human plasma using automated column switching

A validated high-performance liquid chromatographic procedure employing ultraviolet detection for the analysis of diclofenac in human plasma is reported. The method is rapid and, coupled with column switching, leads to a sensitive, accurate and reproducible assay. The retention times of diclofenac and the internal standard (4′-methoxydiclofenac, CGP-4287) are 6.4 and 7.6 min, respectively. The peak height versus plasma concentration is linear over the range 5.0–2000 ng/ml with a detection limit below 2.5 ng/ml. The mean absolute recovery of diclofenac using the described assay is 96.5% (n = 24). The inter- and intra-day accuracy and precision are within 8.3% of the actual values for all concentrations investigated. Furthermore, this procedure is applied to assess the pharmacokinetics of a single 75-mg oral dose of diclofenac sodium.     

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 μg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 μg/ml for acrylamide and from 0.5 to 100 μg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration–time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.     

Determination of olopatadine, a new antiallergic agent, and its metabolites in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and specific assay method has been developed to determine plasma concentrations of olopatadine hydrochloride (A) and its metabolites, M1 (B), M2 (C) and M3 (D), using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Olopatadine, its metabolites, and internal standard, KF11796 (E), were separated from plasma using solid-phase extraction (Bond Elut C₁₈ cartridge). The eluate was dried, reconstituted and injected into the LC–ESI-MS–MS system. The calibration curves showed good linearity over the ranges 1–200 ng/ml for olopatadine and M3, and 2–100 ng/ml for M1 and M2, and the method was thoroughly validated and applied to the determination of olopatadine and its metabolites in plasma collected during Phase I clinical trials. Furthermore, the assay values were compared with those determined by the radioimmunoassay method, which has been routinely used to determine olopatadine in plasma.     

Development and validation of a high-performance liquid chromatography assay for the quantitative determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma

A new, simple, reproducible and reliable high-performance liquid chromatography method with ultraviolet absorbance detection at 240 nm was developed and validated for the determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma. The method was based upon solid-phase (C₁₈) extraction of plasma after addition of 17β-hydroxy-3β-methoxyandrost-5-en-7-one as internal standard. Using 1 ml of plasma for extraction, the detection limit of the assay was 3 ng/ml. The standard curve was linear over the concentration range 10–1000 ng/ml. Stored at −20°C for about 4 months at various concentrations in plasma, 7-oxo-dehydroepiandrosterone-3β-sulfate did not reveal any appreciable degradation. Also included herein is a method for the simultaneous detection and determination of 7-oxo-dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone-3β-acetate in plasma.     

High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment

This paper describes the development of a simple and sensitive analytical method for the quantification of melatonin in human plasma and rabbit serum, using standard analytical equipment and on-line column enrichment without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. For human plasma, the accuracy was 101% (range 89–106%), and the mean precision was 5% (range 2–9%) for all concentrations (0, 2, 10, 50 and 200 ng/ml) tested (n=6). The accuracy in rabbit serum was 101% (range 90–112%), and the mean precision was 13% (range 8–19%) for all concentrations (0, 2, 10, 50, 200 and 500 ng/ml) tested (n=6). The retention time of melatonin was about 8 min and the total recoveries were found to be approximately 65 and 85%, respectively, for human plasma and rabbit serum. The limit of detection was found to be lower than 1 ng/ml for human plasma and around 2 ng/ml for rabbit serum. The method is, therefore, found to be suitable for melatonin bioavailability studies in rabbits and presumably also in humans.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.