Quantification of midodrine and its active metabolite in plasma using a high performance liquid chromatography column switching technique

An automated column-switching HPLC system is described for the simultaneous determination of midodrine, an alpha-adrenergic stimulating drug, and its active metabolite, ST-1059. Serum or plasma (850 microliters) is directly injected onto a RP18 (30 micrograms particle size) pre-column (9 x 4 mm ID) which acts as an on-line liquid-solid extractor and analyte enrichment system. The injection is followed by washing steps. The fraction containing the analytes is transferred onto an analytical RP18 column via step gradient elution where the final analysis is performed. Fluorescence detection is used (lambda ex 290 nm and lambda em 322 nm), and method detection limits of 0.8 ng/mL plasma were reached. These were sufficiently low to determine the plasma concentration-time profiles for both compounds following oral administration of 2.5 mg and 5 mg midodrine hydrochloride. The assay in serum or plasma was linear in the range of 1 to 15 ng analyte/mL, the recovery was greater than 95%, and the reproducibility was sufficient. The assay was rugged and was maintained by routinely changing the home-made, dry packed pre-column every 20th serum injection.     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

A simple determination of mizoribine in human plasma by liquid chromatography with UV detection

A simple liquid chromatography (LC) method was developed for determination of the therapeutic level of mizoribine in human plasma. After precipitation of plasma proteins with 6% perchloric acid, mizoribine was determined by LC with spectophotometric detection. The peak height for mizoribine was linearly related to its concentrations, which ranged from 0.09 to 3.13 microg/mL. Therefore, the limit of quantitation was considered to be 0.09 microg/mL. The accuracy was 104.96-107.37%. The intra- and interday relative standard deviation values were in the range of 1.10-3.25%. The detection limit was 0.025 microg/mL, defined as a signal-to-noise ratio of 3. The plasma concentrations of mizoribine were not related to the dosage. Because mizoribine was mainly excreted in the urine, the plasma concentrations of mizoribine might be affected by a change in renal function. Therefore, the mizoribine concentration in blood should be monitored and the dosage adjusted, depending on the condition of renal function. It was suggested that the present method may be applied well in the therapeutic drug monitoring for mizoribine.     

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.     

Simultaneous determination of rosiglitazone and metformin in plasma by gradient liquid chromatography with UV detection

A novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in human plasma. The analysis was performed on a phenyl column (250 mm × 4.6 mm i.d., 5 μm) using a gradient method starting with mobile phase composed of acetonitrile:5 mM acetate buffer pH 5.5 (75:25, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The total run time was less than 10 min. Sample preparation included a simple protein precipitation step with acetonitrile. Validation experiments were performed to demonstrate stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 100 ng mL⁻¹ for rosiglitazone and 250 ng mL⁻¹ for metformin. The extraction recoveries were 100.02-105.0% for rosiglitazone and 105.64-103.88% for metformin. The method was applied with success to plasma samples obtained from diabetic patients undergoing treatment with rosiglitazone and metformin.     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.     

Rapid quantitative determination of epirubicin and its metabolites in plasma using high performance liquid chromatography and fluorescence detection

A rapid sensitive and selective isocratic technique was developed for the analysis of plasma epirubicin and three of its known fluorescent metabolites epirubicinol, 4'-O-beta-D-glucuronyl-4'-epidoxorubicin and 4'-O-beta-D-glucuronyl 1,3-dihydro-4'-epidoxorubicin, with daunorubicin as an internal standard, by using high performance liquid chromatography (HPLC) with fluorescence detection and a 'Hypersil ODS' column. The drugs were easily and efficiently extracted with a Sep-Pak C18 cartridge and the mean recoveries were greater than 85%. Intraassay and Interassay coefficients of variation (plasma samples) were better than 8.25%. An example of pharmacokinetic study obtained in a cancer patient after intravenous injection of epirubicine is described.     

Determination of naphazoline in rat plasma using column liquid chromatography with ultraviolet detection.

A sensitive (1 ng/mL) and rapid method for the determination of naphazoline in rat plasma is described. Following extraction, the compound is analysed by reversed phase high performance liquid chromatography and ultraviolet detection at 214 nm.     

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection

A simple high‐performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed‐phase Kromasil KR 100 C₁₈ (5 µm particle size) column using a mobile phase of acetonitrile–potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10–1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

High performance liquid chromatography method for the determination of meropenem in human plasma

Summary This paper describes an HPLC method for the determination of meropenem in human plasma. The method uses solid phase extraction (SPE) of the samples and has good sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.02 μg mL⁻¹. Calibration curves were linear over a large dynamic range, namely within 0.02–50 μg mL⁻¹. The method was applied to the determination of meropenem levels in patients receiving meropenem, as a single dose or at steady state.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.     

Full automation of catecholamine metabolite determination by column switching and high-performance liquid chromatography

The urinary catecholamine metabolites, vanimandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid, were extracted on a silica-bonded strong-anion-exchanger cartridge (SAX) and then injected into an high-performance liquid chromatographic (HPLC) system by column switching. Chromatography was performed on a reversed-phase analytical column with electrochemical detection. Full automation was obtained by coupling two devices: a solid-phase automatic sampler and intelligent autosampler. For each substance the recovery was greater than 95% and the coefficient of variation was ca. 3%; the analysis takes 11 min. Substance instability problems are overcome, because the samples are extracted and injected in rapid succession. The normal values and correlation with manual HPLC were established for a large number of samples.     

Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

Sensitive determination of cinnarizine in human plasma by high performance liquid chromatography and fluorescence detection

Summary A sensitive high performance liquid chromatographic method has been developed for the determination of cinnarizine in human plasma. Cinnarizine and clocinizine (internal standard) were extracted from acidified plasma (pH 4.7) into carbon tetrachloride and the organic layer was evaporated. The products were separated on a Microspher C18 (3 m) column, using a mixture of 0.04 % triethylamine in 0.01 M ammonium dihydrogen phosphate (NH₄H₂PO₄), pH adjusted to 4.2 with orthophosphoric acid (H₃PO₄), and acetonitrile (2080, v/v) as mobile phase, at a flow rate of 1 ml/min at 40°C. Fluorescence detection (_ex = 245 nm, _em = 310 nm) was used; the detection limit was 0.5 ng/ml under the conditions used, and the calibration curve linear in the concentration range evaluated (1–60 ng/ml). The assay has been used to measure cinnarizine concentrations in plasma after oral administration to volunteers.