Quantitative determination of imatinib in human plasma with high-performance liquid chromatography and ultraviolet detection

A simple and sensitive high-performance liquid chromatography (HPLC) method was developed to quantitate imatinib in human plasma. Imatinib and the internal standard dasatinib were separated using a mobile phase of 0.5% KH(2)PO(4) (pH3.5)-acetonitrile-methanol (55:25:20, v/v/v) on a CAPCELL PAK C18 MG II column (250 mm × 4.6 mm) at a flow rate of 0.5 mL/min and measurement at UV 265 nm. Analysis required 100 μL of plasma and involved a solid phase extraction with an Oasis HLB cartridge, which gave recoveries of imatinib from 73% to 76%. The lower limit of quantification for imatinib was 10 ng/mL. The linear range of this assay was between 10 and 5000 ng/mL (regression line r(2) > 0.9992). Inter- and intra-day coefficients of variation were less than 11.9% and accuracies were within 8.3% over the linear range. The plasma concentrations of imatinib obtained by our present method were almost the same as those assayed by an LC-MS-MS method at the Toray Research Center, Inc. This method can be applied effectively to measure imatinib concentrations in clinical samples.     

Urinary mesna and total mesna measurement by high performance liquid chromatography with ultraviolet detection

We describe in this report a method for determination of mesna and total mesna in urine by high performance liquid chromatography with ultraviolet detection. The method involves a treatment of the urine sample with tri-n-butylphosphine in order to convert mesna disulfides to its reduced counterpart mesna, ultraviolet labelling with 2-chloro-1-methylquinoluinium tetrafluoroborate, reversed-phase HPLC separation, and detection and quantitation at 350 nm. The result corresponds to total mesna that is sum of mesna, dimesna and its mixed disulfides with endogenous thiols. For determination of mesna the reduction step is omitted. Content of disulfide forms of mesna can be calculated by subtracting the concentration of mesna from the total mesna concentration. The separation of 2-S-quinolinium derivatives of mesna from those of endogenous urinary thiols and internal standard was achieved on an analytical Waters Nova-Pak C18 (150 mm × 3.9 mm, 5 μm) column. A mixture of an aqueous solution of pH 2.3, 0.05 M trichloroacetic acid and acetonitrile (88:12, v/v) was used as a mobile phase at flow rate of 1.2 ml/min and ambient temperature.The assay for mesna and total mesna in urine was proved to be linear over the studied ranges of 0.2-30 and 0.2-800 nmol/ml urine, respectively. The mean recoveries over the calibration ranges were 95.4% for mesna and 99.7% for total mesna. The lower limits of detection and quantitation were 0.1 and 0.2 nmol/ml for both the procedures, respectively. The imprecision did not exceed 8.5%. No interference from endogenous substances was observed.     

Simple and sensitive method for determination of glycoalkaloids in potato tubers by high-performance liquid chromatography with chemiluminescence detection

A novel, simple and sensitive high-performance liquid chromatographic method for the determination of the potato glycoalkaloids, alpha-solanine and alpha-chaconine, based on the chemiluminescent reaction of tris(2,2'-bipyridine)ruthenium(III) has been developed. The calibration graph was linear in the range of 5 ng/ml-10 microg/ml for both alpha-solanine and alpha-chaconine. The detection limits of alpha-solanine and alpha-chaconine were 1.2 and 1.3 ng/ml, respectively. This method was successfully applied to a potato tuber sample without cleanup, pre-concentration, and derivatization steps. The recoveries (mean +/- standard deviation, %) of alpha-solanine and alpha-chaconine spiked in tuber pith at 10 microg/g (n = 6) were 101.0 +/- 4.4% and 103.6 +/- 7.1%, respectively.     

Determination of clindamycin in plasma or serum by high-performance liquid chromatography with ultraviolet detection

A simple, sensitive high-performance liquid chromatographic assay for the determination of clindamycin in human plasma or serum has long been hampered by inability to separate it from endogenous compounds. We describe here such an assay. Proteins from a 200-microliters sample were precipitated by addition of acetonitrile containing the internal standard, triazolam. The sample was then vortex-mixed and centrifuged at approximately 3000 g for 10 min. The supernatant was evaporated to about 250 microliters under nitrogen, and 10-30 microliters were analyzed using an autoinjector. An octadecylsilane column with acetonitrile-water-phosphoric acid-tetramethylammonium chloride as mobile phase and ultraviolet detection at 198 nm provided a reproducibly quantifiable peak corresponding to 0.17 micrograms/ml. Retention times for clindamycin and triazolam averaged 8 and 11.8 min, respectively. Replicate standard curves run over a 36-h period showed no loss of integrity; r2 values generally exceeded 0.999. The method has successfully been applied to the analysis of samples taken up to 12 h after administration of intravenous infusions of 600-1200 mg in healthy volunteers.     

Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.     

Determination of pindolol in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

This paper presents a rapid, simple and economical method for assaying pindolol concentrations in plasma and urine by high-performance liquid chromatography using ultraviolet detection. It is sensitive enough for use in single-dose pharmacokinetic studies and may also be used to determine pindolol concentrations in the plasma from patients taking the drug, provided that the patient is not taking any of the drugs which interfere with the method. Drugs which were found to interfere with the pindolol peak are quinidine, n-acetylprocainamide and lidocaine. Disopyramide, oxprenolol and levobunolol interfered with the internal standard peak.     

A reliable high-performance liquid chromatography with ultraviolet detection for the determination of sulfonamides in honey

The sulfonamides are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value is a potential hazard to human health. Brazilian authorities have included in the National regulatory monitoring program, the control of the three most widely used sulfonamides in honey production, i.e., sulfathiazole, sulfamethazine and sulfadimethoxine. A method for the determination of residual sulfonamides in honey, using sulfapyridine as an internal standard has been developed, optimized and validated. Some changes were implemented on current available methodologies for the analysis of sulfonamides in honey in order to adopt such procedures to Brazilian honey samples. Sulfonamides were extracted from honey with dichloromethane after dissolution with 30% sodium chloride, and cleaned up with solid phase extraction on Florisil columns. The eluate was analyzed by high-performance liquid chromatography with ultraviolet detection. The limit of detection was determined at 3 μg kg⁻¹, 4 μg kg⁻¹ and 5 μg kg⁻¹ for sulfathiazole, sulfamethazine and sulfadimethoxine, respectively with average recoveries of 61.0% for sulfathiazole; 94.5% for sulfamethazine and 86.0% for sulfadimethoxine at the 100 μg kg⁻¹ level. As the final step of validation procedure, the analysts were submitted to a blind spiked sample prepared by the quality assurance officer which results were successfully obtained regarding recovery and deviations.     

Sensitive method for the determination of organophosphorus pesticides in fruits and surface waters by high-performance liquid chromatography with ultraviolet detection.

A sensitive method for the high-performance liquid chromatographic determination of five organophosphorus pesticides (paraoxon, methyl-parathion, ethyl-parathion, guthion and fenitrothion) in fruits and tap and river water samples is described. For the determination of pesticides in fruits a simple and rapid sample preparation procedure was developed that allowed pesticides to be determined at 50-100 micrograms/kg levels with recoveries ranging from 83 to 118% and relative standard deviations below 6%. The determination of pesticide residues in surface water samples was also successfully accomplished. Concentrations at sub-ppb levels can be measured by using a solid-phase concentration step, the recoveries being over 80%. In analyses of both fruits and surface waters, the sensitivity levels achieved were 2-10 times lower than legal limits admitted in the European Economic Community.     

A high-performance liquid chromatography method using ultraviolet detection for the quantitation of flavopiridol from human plasma

Flavopiridol is an inhibitor of cyclin-dependent kinase, a key regulator of cell cycle, and is currently under clinical trials. We developed and validated an HPLC assay method for the quantitation of flavopiridol in human plasma samples. The sample preparation consisted of protein precipitation with acetonitrile. Separation was accomplished on a C(18) column and a C(18) precolumn insert utilizing a gradient profile consisting of ammonium acetate and methanol. Ultraviolet detection was set at 268 nm for flavopiridol and 323 nm for umbelliferone, the internal standard. The method was validated over flavopiridol concentration range of 0.025-3.0 microg/mL using 250 microL of plasma. The assay was linear over this concentration range with a coefficient of variation less than 10% for inter- and intra-assay. The retention times were around 6.2 min for umbelliferone and 9.8 min for flavopiridol. The recoveries of flavopiridol and umbelliferone were 88.6 +/- 1.0% and 97.1 +/- 3.7%, respectively. This method is suitable for quantifying flavopiridol in plasma samples and further characterizing the clinical pharmacology of this compound.     

Determination of morphine 3-esters in rabbit plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of the morphine 3-esters 1[3-(2, 2-dimethylvaleroyl)-morphine (A), 3-(2-phenylbenzoyl)-morphine (B) and 3-(2,2-diphenylpropionyl)-morphine (C)] in rabbit plasma is described. Sample preparation was based on reversed-phase solid-phase extraction. The compounds were separated on C(18) reversed-phase analytical columns and then determined by ultraviolet detection. The recovery from plasma was 78.7 +/- 7.4%, 69.1 +/- 6.9% and 75 +/- 7.2% (mean +/- SD) for A, B, and C, respectively. The present method enabled the detection limit of 0.2, 0.2 and 0.1 ng and quantification limit of 20, 10 and 10 ng/ml for A, B and C, respectively. The developed method was used for determination of the plasmakinetics of these morphine 3-esters in rabbits.