High-performance liquid chromatographic assay for amiloride in plasma and urine

A sensitive and simplified high-performance liquid chromatographic procedure has been developed for quantification of amiloride in rabbit plasma, as well as human plasma and urine. Following protein precipitation with perchloric acid, the supernatant was directly injected into a C₁₈ Nucleosil column. The mobile phase consisted of methanol—water (45:55) containing 0.1 M perchloric acid, and the compound was quantitated using a fluorescence detector at excitation and emission wavelengths of 286 and 418 nm, respectively. The average recovery was 97.6%. The calibration curve was linear over the range 2.0–20.0 ng/ml. The limit of detection was 0.5 ng/ml.     

High-performance liquid chromatographic validated assay of doxorubicin in rat plasma and tissues

A specific and selective high-performance liquid chromatography (HPLC) technique that requires few manipulations, and is readily adaptable to analysis for a large series of samples, has been developed for the determination of the concentration of the anticancer drug doxorubicin (DXR) in rat serum and tissues. The biological samples were efficiently deproteinised and resolved from a reversed-phase nucleosil C₁₈ column with fluorescence detection. The validation study of the proposed method was successfully carried out in an assay range of between 5 and 5000 ng/ml and was subsequently implemented in a pharmacokinetic study of DXR in Wistar rats that were treated by intravenous administration of the drug.     

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H(3)PO(4) with 0.2% triethylamine mobile phase was employed and separation was achieved with a C(18) analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9+/-0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 microg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 microg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 microg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 microg/ml. Results of the stability study indicated that when frozen at -80 degrees C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.     

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis™ HLB cartridge and were injected into a C₈ Nucleosil column. The HPLC eluent contained acetonitrile–methanol–distilled water–0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1–25 μg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2–17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4°C, rapid degradation of the drug occurred. Frozen at −30°C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The method’s ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.     

High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma

An isocratic high-performance liquid chromatographic method for the determination of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. The method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C₁₈ cartridges. Separation was achieved with an ODS column using acetonitrile and phosphate buffer supplemented with octanesulfonic acid (an ion-pairing agent). The pH of the mobile phase (2.5) was optimal with respect to the mode of detection (fluorescence). The method was validated in the range of 5-MTHF concentrations from 0.0625 μmol/l to 4.0 μmol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exceed 8.7%. The method is specific, accurate and sensitive enough to be used in pharmacokinetic studies for the assessment of the systemic availability of 5-MTHF after leucovorin administration to patients as a rescue after high-dose therapy with methotrexate. The limit of detection was 0.17 pmol which corresponds to a plasma concentration of 1.7 nmol/l. Thus, the assay could potentially be used for the measurement of 5-MTHF in the range of physiological concentrations in plasma (5–20 nmol/l).     

High-performance liquid chromatographic assay of formycin A in plasma after solid-phase extraction

A new, simple and accurate high-performance liquid chromatography (HPLC) method for the determination of formycin A in plasma is presented. The samples were chromatographed on a LiChrosorb RP-18 column after purification using a Bakerbond SPE column. The mobile phase was methanol–0.067 M phosphate buffer, pH 4.20 (1:4, v/v) containing 0.005 M sodium hexanesulfonate. Azathioprine was applied as an internal standard. UV detection was carried out at 293 nm. The method was tested for linearity (over the range 0.1–9.0 μg/ml). The recovery was 91.89% (mean). The described method has been successfully applied to the quantitative determination of formycin A in plasma and should be useful for clinical and bioavailability investigations.     

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.     

High-performance liquid chromatographic assay of cefotaxime,desacetylcefotaxime and ceftriaxone in rat plasma

A simple and selective high-performance liquid chromatographic method is described for the analysis of the cephalosporins cefotexime (CXM), desacetylcefotaxime (DACXM) and ceftriaxone (CFX) in rat plasma. Plasma was deproteinized with methanol, and the supernatant was directly injected into the chromatograph and monitored at 254 nm. For determination of the unbound drugs, a centrifugal ultrafiltration method was employed. The calibration curves were linear (r=0.999) from 2.5 to 500 μg/ml; the detection limits were 100 ng/ml for DACXM and 250 ng/ml for CXM and CFX. The method was not interfered with by other plasma components, nor by barbital sodium or caffeine, and has been applied to study the pharmacokinetics of the cephalosporins in rats.     

High-performance liquid chromatographic assay for cefepime in serum

A simple, rapid, specific and sensitive high-performance liquid chromatographic method was developed for the determination of cefepime 1-[[6R, 7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo-[4.2.0] oct-2-en-3-yl]methyl]-1-methylpyrrolidinium hydroxide, inner salt, 7²-(Z)-(O-methyloxime) in human serum. Separation was achieved on a reversed-phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4). Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV absorbance at 254 nm. The lower limit of quantitation of cefepime in plasma was 0.5 μg/ml. The average absolute recovery was 106.2±2.1%. The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient greater than 0.999. The within-day C.V.s for human samples were 4.9 and 2.3% for 1 and 50 μg/ml, respectively. The between-day C.V.s for human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50 μg/ml, respectively. Cefepime was found to be unstable in serum at room temperature. For delayed assay, samples must be stored at −80°C.     

High-performance liquid chromatographic assay for melphalan in human plasma Application to pharmacokinetic studies

This paper describes a simple, rapid and reproducible high-performance liquid chromatographic method (HPLC) with ultraviolet absorbance detection for the analysis of melphalan in plasma. The HPLC column was an Ultrasphere ODS (5 μm) and the eluent was composed of methanol, purified water and acetic acid (49.5:49.5:1, v/v). The detection was performed at 261 nm. The method involved a simple treatment of the samples with methanol. The propylparaben was used as internnal standard. Linear detection response was obtained for concentrations ranging from 50 to 2500 ng/ml. Recovery from plasma proved to be more than 90%. Precision, expressed as C.V., was in the 0.5 to 9% range. Accuracy ranged from 95 to 102%. This method was used to determine the pharmacokinetic parameters of melphalan following high-dose (140 mg/m²) intravenous administration in patients with advanced malignancies undergoing peripheral blood hematopoietic progenitor-cell transplantation.     

High-performance liquid chromatographic assay for meropenem in serum

High-performance liquid chromatographic procedures have been developed for the measurement of meropenem in serum. The separation was performed on an Ultrasphere XL-ODS analytical column (75×4.6 mm I.D.). The mobile phase consisted of 10.53 mmol/l ammonium acetate-acetonitrile (95:5, v/v) (pH 4). The UV detection was at 298 nm. The quantitation limit both in serum and water was 0.25 μg/ml. The method was validated in serum and aqueous solution over the concentration range 0.25–50 μg/ml. The extraction recovery from serum spiked with meropenem was 99.7±3.4%. The intra- and inter-assay coefficients of variation were below 6%. Stored at −80°C for three months at various concentrations in serum and in aqueous solution, meropenem did not reveal any appreciable degradation. After 24 h, it was also stable at 4°C in serum, aqueous solution and supernatant of extraction but not at room temperature. The stability of the drug was also confirmed in serum after repeated freezing-thawing cycles at −80°C on four consecutive days.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.     

High-performance liquid chromatographic determination of ganciclovir in plasma

A rapid, selective and sensitive isocratic reversed-phase high-performance liquid chromatographic method for the determination of ganciclovir in plasma samples was developed. This method, which was applied to the analysis of plasma ganciclovir from heart transplant patients under ganciclovir therapy for cytomegalovirus infections, represents a suitable analytical tool for drug monitoring and pharmacokinetic investigations.