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 celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO₂ (150×4.6 mm, 5 μm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25–2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.     

Specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea in plasma

A facile, sensitive and highly specific HPLC method for assaying 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extraction with tert.-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I.S.). Following evaporation of the organic solvent, the extract was reconstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded onto a 4 μm Nova-Pak C₁₈ column (15 cm×3.9 mm), which was preceded by a 7 μm Brownlee RP-18 precolumn (1.5 cm×3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol-0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of the effluent was monitored at 240 nm. A flow-rate of 1.0 ml/min was used for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. An automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mobile phase to the analytical column, thereby preventing the apparent build-up of extractable, strongly retained, UV-absorbing components present in mouse and dog plasma. Operating in this manner, more than 100 samples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determination of SarCNU in human plasma by simply decreasing the eluent flow-rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximately 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-μl sample volume, the lowest concentration of SarCNU included in the plasma standard curves, 0.10 μg/ml, was quantified with a 7.8% R.S.D. (n=27) over a 2 month period. Plasma standards, with concentrations of 0.26 to 5.1 μg/ml, exhibited R.S.D. values ranging from 1.3 to 4.7%. Thermospray-ionization MS detection was used to definitively establish the specificity of the method. The sensitivity of the assay was shown by application to be more than adequate for characterizing the plasma pharmacokinetics of SarCNU in mice.     

Fully automated analytical method for codeine quantification in human plasma using on-line solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

A simple, sensitive and fully automated analytical method for the analysis of codeine in human plasma is presented. Samples are added with oxycodone, used as internal standard (I.S.), and directly loaded in the autosampler tray. An on-line sample clean-up system based on solid-phase extraction (SPE) cartridges (Bond-Elut C₂, 20 mg) and valve switching (Prospekt) is used. Isocratic elution improved reproducibility and allowed the recirculation of the mobile phase. A Hypersil BDS C₁₈, 3 μm, 10×0.46 cm column was used and detection was done by UV monitoring at 212 nm. Retention times of norcodeine (codeine metabolite), codeine and oxycodone (I.S.) were 5.5, 6.4 and 9.1 min, respectively. Morphine was left to elute in the chromatographic front. Detection limit for codeine was 0.5 μg l⁻¹ and inter-assay precision (expressed as relative standard deviation) and accuracy (expressed as relative error) measured at 2 μg l⁻¹ were 5.03% and 1.82%. Calibration range was 2–140 μg l⁻¹.     

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.     

Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using three retention mechanisms

An automated two-dimensional HPLC method for the determination of neopterin in serum is described. Neopterin is separated from proteins on a short octadecylsilica column by size exllusion and from the majority of the other serum components by adsorption. The fraction containing neopterin is transferred by column switching to a solvent-generated cation-exchange column using dodecylsulfonic acid as surface activator. Parameters influencing the separation performance and senstivity of the fluorescence detection are discussed. The efficiency of the cleaning of the first column was optimized. The method was validated. It achieves a precision of 1% (R.S.D.) and a detection limit of about 0.3 nmol/1. The accuracy is nearly 100%. The method allow a high sample throughput, requiring 15 min per sample.     

Determination of zopiclone in plasma using column liquid chromatography with ultraviolet detection

A reversed-phase liquid chromatographic method with ultraviolet detection for the determination of zopiclone in plasma is described. It is rapid, sensitive, reproducible and linear over a wide range. The method was used to study plasma zopiclone concentrations in a case of acute intoxication after oral ingestion of 300 mg of the drug. The plasma level was 1600 ng/ml 4.5 h after the dose and the elimination half-life was 3.5 h.     

Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography with ultraviolet detection

A combination of two stereoselective assays was developed using column-switching HPLC with electrochemical detection for the determination of free (unconjugated) silibinin and RP-HPLC with UV detection for the measurement of total (free and conjugated) silibinin in human plasma. After extraction of free silibinin and the internal standard hesperetin with diethyl ether the compounds were pre-separated on a RP-CN column. A cut fraction of eluate containing the analytes was then transferred to the RP-18 main column by means of a switching valve for final separation of the compounds. The limit of quantification with electrochemical detection for free silibinin was 0.25 ng/ml per diastereomer. For the determination of total silibinin diastereomers all conjugates were cleaved enzymatically using β-glucuronidase/arylsulfatase at pH 5.6 followed by extraction with diethyl ether of the pH 8.5 alkalized solution. Separation of the diastereomers and of the internal standard naringenin was achieved on a RP-18 column. The limit of quantification with UV detection at 288 nm for total silibinin was 5 ng/ml per diastereomer. Both assays were successfully applied to the stereospecific analysis of silibinin in plasma samples from a pharmacokinetic study of silymarin in human volunteers.     

Rapid and selective analysis of secnidazole in human plasma using high-performance liquid chromatography with ultraviolet detection

A rapid, reproducible high-performance liquid chromatographic method for the determination of secnidazole, 5-nitroimidazole class of antiprotozoals from blood is described. Metronidazole was used as an internal standard. A simple extraction step with dichloromethane was done before chromatography on a C₁₈ column with the wavelength fixed at 276 nm on the UV detector. Blood levels up to 500 ng/ml have been measured with good precision in the healthy volunteers after 1 g of secnidazole was administered. The present described method can readily be utilized for routine pharmacokinetic studies.     

Determination of hyperforin in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Rapid quantification of indinavir in human plasma by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatography (HPLC) procedure for the quantification of indinavir, a potent human immunodeficiency virus (HIV) protease inhibitor, in human plasma is described. Following C₁₈ solid-phase extraction, indinavir was chromatographed on a reversed-phase C₈ column using a simple binary mobile phase of phosphate buffer–acetonitrile (60:40, v/v). UV detection at 210 nm led to an adequate sensitivity without interference from endogenous matrix components. The limit of quantification was 25 ng/ml with a 0.1 ml plasma sample. The standard curve was linear across the range from 25 to 2500 ng/ml with an average recovery of 91.4%. The mean relative standard deviations for concentrations within the standard curve ranged between 1.4 and 9.7%. Quality control standards gave satisfactory intra- and inter-assay precision (R.S.D. from 3.5 to 15.8%) and accuracy within 15% of the nominal concentration. Sample handling experiments, including HIV heat inactivation, demonstrated analyte stability under expected handling processes. The assay is suitable for the analysis of samples from adult and pediatric patients infected with HIV.     

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.     

Oral platelet aggregation inhibitor Ro 48-3657: Determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 μl of plasma) and 25 ng/ml (50 μl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Determination of opipramol in human plasma by high-performance liquid chromatography with photometric detection using a cyanopropyl column

A high-performance liquid chromatographic method is described for the determination of opipramol in human plasma. Opipramol was extracted into tert.-butylmethyl ether, separated on a cyanopropyl silica column and detected at 254 nm. Imipramine was used as internal standard. The limit of quantitation was 250 pg/ml using 1.5 ml plasma. Precision was better than 9%, inaccuracy less than 8%. The assay is more sensitive than previously published methods, and it has been applied to the analysis of plasma samples from a pharmacokinetic study.