Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Liquid chromatographic determination of manidipine in serum with electrochemical detection

A highly sensitive and selective method for determining a dihydropyridine calcium antagonist, manidipine, by liquid chromatography using column switching with electrochemical detection was developed. Manidipine in serum was extracted by a rapid and simple procedure based on C₈ bonded-phase extraction and then silica extraction. Manidipine and nilvadipine as an internal standard were separated on a C₈ bonded-phase HPLC column and detected by high conversion efficiency amperometric detection at +0.7 V. Manidipine and nilvadipine (I.S.) were separated from an endogenous interference peak in serum and concentrated on a pre-column (C₁₈) by column switching using an isocratic mobile phase, and then the corresponding fractions were introduced to an analytical column with a C₈ stationary phase. Determination of manidipine was possible over the concentration range 0.5–10 ng/ml: the limit of detection was 0.3 ng/ml. The recovery of manidipine added to serum was 93.1–98.4% with coefficients of variation of less than 7.1%. The method is applicable to drug level monitoring in the serum of healthy volunteers treated with manidipine and to the analysis of pharmacokinetics.     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C₁₈ solid-phase extraction cartridge, elution from a Spherisorb C₈ reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8±3.5% and 89.1±6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 μg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

Validated assay for the determination of celiprolol in plasma using high-performance liquid chromatography and a silanol deactivated reversed-phase support

Previously reported methods for the determination of celiprolol in plasma could not be satisfactorily employed due to interference from plasma components. Thus, an improved, convenient and efficient method for the determination of the plasma concentration of celiprolol was developed using a simple solvent extraction step followed by high-performance liquid chromatography on a silanol deactivated C₁₈ column with fluorescence detection. The plasma interference was resolved from celiprolol and the typical trailing of basic compounds on reversed-phase HPLC was eliminated. The peak-area ratio versus plasma concentration was linear over the range of 5–1000 ng/ml and the detection limit was 5 ng/ml.     

Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C₁₈-, C₈- or C₄-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C₈-Alkyl-Diol Silica, 25 μm, 25 × 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5–101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2–3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproductivity. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).     

Stereoselective determination of apomorphine enantiomers in serum with a cellulose-based high-performance liquid chromatographic chiral column using solid-phase extraction and ultraviolet detection

A novel and rapid method for the separation and determination of R-(−)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(−)- and S-(+)-enantiomers of apomorphine and the internal standard R-(−)-propylnorapomorphine from serum using a C₈ Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250×4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0.05 M sodium perchlorate (pH 2.0, adjusted with 60–62% perchloric acid) at a flow-rate of 0.5 ml/min with UV detection at 273 nm. The detection and quantitation limits were 10 ng/ml for each enantiomer using 1 ml of serum. Linear calibration curves from 10 to 1000 ng/ml for both R-(−)- and S-(+)-enantiomers show coefficient of determination of more than 0.9995. Precision calculated as %R.S.D. and accuracy calculated as % error were 0.2–4.7 and 3.1–6.9%, respectively, for the R-(−)-enantiomer and 1.3–4.2 and 0.3–6.8%, respectively, for the S-(+)-enantiomer.     

Determination of celiprolol and oxprenolol in human plasma by high-performance liquid chromatography and the analytical error function

Two reversed-phase HPLC methods with UV detection to quantify celiprolol and oxprenolol in human plasma are described. The analytical methods for the determination of both drugs used the same reversed-phase HPLC column, mobile phase and extraction procedure. Linearity was obtained in the ranges 15.63–1000 and 25–800 ng/ml for celiprolol and oxprenolol, respectively. Intra-day and inter-day variation was lower than 14%. After validation of the methods, analytical error functions were established as S.D. (ng/ml)=3.096+0.041C for celiprolol and S.D. (ng/ml)=8.906+8.075·10⁻⁸C³ for oxprenolol.     

Gas chromatography with surface ionization detection: a highly sensitive method for determining underivatized codeine and dihydrocodeine in body fluids

Underivatized codeine and dihydrocodeine in human plasma and urine have been determined with a high degree of accuracy by capillary gas chromatography (GC) with surface ionization detection (SID). The drugs were extracted with the aid of Sep-Pak C₁₈ cartridges. Recovery of both drugs was 90%. The calibration curves obtained with dimemorfan as an internal standard showed linearity in the range 4.5–72.3 and 3.0–75.5 ng/ml of plasma for codeine and dihydrocodeine, respectively. The detection limit was about 100 pg on column (2.5 ng/ml sample). Codeine was determined quantitatively in plasma and urine obtained from a volunteer who had received 10 mg codeine phosphate orally 3 h before the sampling: the levels were found to be 14.1 and 142 ng/ml, respectively. The present GC-SID method has been compared carefully with GC-NPD (nitrogen-phosphorus detection) using the same extracts; the sensitivity of GC-SID was more than ten times greater than that of GC-NPD, with background noise correspondingly lower.     

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.     

Simple method for the analysis of tenoxicam in human plasma using high-performance liquid chromatography

A simple, rapid and cost-effective method for the determination of tenoxicam in human plasma is described, using ketorolac as the internal standard. The extraction procedure utilised 5% zinc sulphate and methanol. A nucleosil C₁₈ column and 35:65 acetonitrile-water phosphate buffered mobile phase (pH 2.8) were used, with ultraviolet detection at 355 nm. The assay was linear in the range 40 ng/ml-10 μg/ml, with recovery of extraction ranging from 87 to 102%. The intra- and inter-assay reproducibility had coefficients of variation of 3.9–7.7 and 1.6% respectively. The limit of detection for this method was 40 ng/ml.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

High-performance liquid chromatographic analysis for the determination of miconazole in human plasma using solid-phase extraction

A high-performance liquid chromatographic method for the determination of miconazole in human plasma is described. A solid-phase extraction was performed on an octadecyl (C₁₈) cartridge. Miconazole was eluted with methanol, separated on a reversed-phase column and was measured by ultraviolet detection at 230 nm. The absolute extraction recovery from plasma samples was 85%. The limit of detection was established as 5 ng/ml. The coefficient of variation of the determination of plasma levels by this method over the standard curve concentration range was less than 10%, except with the concentration of 10 ng/ml. The plasma levels of miconazole in twelve healthy volunteers given a 250-mg oral dose of two tablet forms were determined by this method.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C₈ (150×4.6 mm) 5 μm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5–800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3–4.5% and 2.2–4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.