Quantification of doripenem in human plasma and peritoneal fluid by high-performance liquid chromatography with ultraviolet detection

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and peritoneal fluid protein was developed to determine doripenem concentrations in human plasma and peritoneal fluid. Doripenem was stabilized by immediate mixing of the plasma or peritoneal fluid with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Doripenem and an internal standard were detected by measuring their ultraviolet absorbance at 300 nm. The calibration curves for doripenem in human plasma and peritoneal fluid were linear from 0.05 to 100 microg/mL. For plasma, both the intra- and the interday precision were less than 3.41% (CV), and the accuracy was between 97.4 and 101.7% above 0.05 microg/mL. For peritoneal fluid, the intra- and the interday precision were less than 2.98% (CV), and the accuracy was between 94.4 and 103.9% above 0.05 microg/mL. The limit of detection was 0.02 microg/mL in both plasma and peritoneal fluid. The assay has been applied to the therapeutic drug monitoring of doripenem in both plasma and peritoneal fluid.     

Quantification of paclitaxel metabolites in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic (HPLC) method has been validated for the quantitative determination of the three major paclitaxel metabolites (6α-hydroxypaclitaxel, 3′-p-hydroxypaclitaxel, 6α,3′-p-dihydroxypaclitaxel) in human plasma. The HPLC system consists of an APEX-octyl analytical column and acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5 (AMW; 4:1:5, v/v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The sample pretreatment of the plasma samples involves solid-phase extraction (SPE) on Cyano Bond Elut columns.The concentrations of the metabolic products could be determined by using the paclitaxel standard curve with a correction factor of 1.14 for 6α,3′-p-dihydroxypaxlitaxel. The recoveries of paclitaxel and the metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in human plasma were 89, 78, 91 and 89%, respectively. The accuracy of the assay for the determination of paclitaxel and its metabolites varied between 95 and 97%, at a 50 ng/ml analyte concentration. The lower limit of quantitation was 10 ng/ml for both the parent drug and its metabolites.     

Determination of mitiglinide in rat plasma by high-performance liquid chromatography with UV detection

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and 0.02 mol/l KH(2)PO(4) buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1-20 microg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.     

Quantification of darunavir (TMC114) in human plasma by high-performance liquid chromatography with ultra-violet detection

A precise and accurate high-performance liquid chromatography (HPLC) method with UV detection has been developed and validated for darunavir, a peptidic protease inhibitor. An internal standard, methylclonazepam, was added to 100 microL of plasma before a solid-phase extraction on C18 Bond Elut column. The eluted solutions were evaporated to dryness and reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The separation was performed on a C8 column using an acetonitrile and ultrapure water mixture (40:60, v/v) as mobile phase. All compounds were detected at a wavelength of 266 nm. The method was linear and validated over a concentration range of 0.25-20mg/L. The within-day precision, ranged from 3.0 to 7.9%, while the within-day accuracy ranged from -11.4 to 0.5%. The between day precision and accuracy were respectively less than 13.7 and -11.4%. The mean recovery was 75.7% for darunavir and 66.7% for methylclonazepam. This method provides a useful tool for therapeutic drug monitoring in HIV patients.     

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection

A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi Max RP analytical column (150 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1+/-5.5% (n=6) and 72.4+/-5.9% (n=7), respectively; for OZP at 200 ng the value was 68.2+/-6.8% (n=14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n=7 in all cases) and 6.6% (n=8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n=7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of +/-20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.     

Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection

A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from ?15.0 to ?0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.     

Measurement of ribavirin and evaluation of its stability in human plasma by high-performance liquid chromatography with UV detection

A simple high-performance liquid chromatography method for the determination of the antiviral agent ribavirin in human plasma was developed and validated. The method involved solid-phase extraction on phenyl boronic acid cartridges, a reversed-phase liquid chromatography with a Waters Atlantis dC18 (150 mm x 3.9 mm, 5 microm) column and a mobile phase consisting of 10 mM potassium phosphate buffer (pH 4.0), and ultraviolet detection at 207 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 microg/ml), linear (correlation coefficients >or=0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation 相似文献   

Determination of tadalafil in small volumes of plasma by high-performance liquid chromatography with UV detection

Tadalafil is a potent reversible phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction. This study describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of tadalafil in 50 microl of rat plasma. Tadalafil and the internal standard lamotrigine were extracted with 0.5 ml of tert-butyl methyl ether, after the samples alkalinized with 20 microl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a C18 column with the mobile phase of acetonitrile-water containing 20 mM phosphate buffer (pH 7) (35/65, v/v), at a flow rate of 1 ml/min. The eluant was detected at 290 nm. The retention time was about 4.5 min for lamotrigine and 15 min for tadalafil. No endogenous substances were found to interfere. Calibration curves were linear from 10 to 2000 ng/ml. The recovery of tadalafil from plasma was greater than 77%. The limit of quantitation was 10 ng/ml. The intra- and inter-day imprecision (expressed as coefficient of variation, C.V.) did not exceed 10.7%, and the accuracy was within 5.9% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring tadalafil concentration.     

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection

A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid-liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4-256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.     

Determination of naloxone in human plasma by high-performance liquid chromatography with coulometric detection

Naloxone, the analyte and the internal standard, sumatriptan, are extracted from plasma using solid-phase extraction columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric end-point detection. The standard curve was linear over the range 0–50 ng/ml of naloxone in plasma. The reproducibility, the coefficient of variation (C.V.) of the method over the range of the standard curve was 6.2–11.2%. The recovery averaged 90.4±8.9%. A plasma profile following i.v. administration of naloxone in one normal healthy volunteer is presented.     

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 metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Determination of succinylcholine in human plasma by high-performance liquid chromatography with electrochemical detection

An alternative HPLC method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine in human plasma is described. Drug spiked plasma and patient plasma samples were extracted using a C₁ solid-phase cartridge. Succinylcholine was separated on a Cyano column and quantitated using electrochemical detection at a potential of 450 mV and 750 mV. Mobile phase consisted of a mixture of phosphoric acid–acetonitrile–methanol (45:35:25) adjusted to an apparent pH of 5. Standard curves for the quantitation were linear in the range of 250–8000 ng/ml. Between-day and within-day relative standard deviations were 5.1% and 1.7%, respectively. Mean drug recovery and accuracy was 68% and 104%, respectively.