High-performance liquid chromatographic determination of the enantiomers of the benzoquinolinone LY191704, a human type 1 5 alpha-reductase inhibitor, in plasma

A stereoselective reversed-phase liquid chromatographic method for the determination of compounds LY300502 and LY300503 (enantiomers of LY191704) in rat and dog plasma was developed. The assay involved extraction of the compounds using a strong cation-exchange solid-phase extraction column, from which the compounds are eluted with 1% of 1 M HCI in methanol. The enantiomers were separated on a Daicel Chiralcel OD-R column. The mobile phase consisted of water-acetonitrile-methanol (50:40:10, v/v) at a flow-rate of 0.3 ml/min. UV detection was achieved at 220 nm. The disposition of the enantiomers of LY191704 in rats and dogs was found to be stereoselective and species specific.     

Simple high-performance liquid chromatographic method for determination of pentoxifylline in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of pentoxifylline in human plasma. Prior to analysis, pentoxifylline and the internal standard (chloramphenicol) were extracted from plasma sample using dichloromethane. The mobile phase comprised 0.02 M phosphoric acid adjusted to pH 4, methanol and tetrahydrofuran (55:45:1, v/v). Analysis was run at a flow-rate of 1.4 ml/min with the detector operated at a wavelength of 273 nm. The method was specific and sensitive with a detection limit of approximately 3.0 ng/ml at a signal to noise ratio of 3:1, while the limit of quantification was 12.5 ng/ml. Mean recovery value of the extraction procedure was about 99.9%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 10.0%. The calibration curve was linear over a concentration range of 12.5-400.0 ng/ml.     

Determination of dapoxetine, an investigational agent with the potential for treating depression, and its mono- and di-desmethyl metabolites in human plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of dapoxetine and its mono- and di-desmethyl metabolites in human plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane-ethyl acetate. The organic extract was evaporated to dryness and the residue reconstituted with acetonitrile. The analytes were separated from late-eluting endogenous substances on a Zorbax RX-C8 pre-column. The front-cut fraction containing the analytes was further separated on a second RX-C8 column. The analytes were detected by their native fluorescence, using excitation and emission wavelengths of 230 and 330 nm, respectively. The limit of quantitation was determined to be 20 ng/ml, and the response was linear from 20 to 200 ng/ml. The method has been successfully applied to human plasma samples in a Phase I study.     

Simultaneous determination of nerisopam, a novel anxiolytic agent showing polymorphic metabolism, and its N-acetyl metabolite from human plasma by a validated high performance liquid chromatographic method

OBJECTIVE: Our goal was to determine the effect of chronic and acute umbilical-placental embolization on placental hemodynamic and fetal heart rate patterns in relation to fetal oxygenation in the near-term ovine fetus. STUDY DESIGN: Daily fetal placental embolization was performed during 10 days in 9 sheep fetuses until fetal arterial oxygen content decreased by approximately 30%. Nine control fetuses received saline solution. Mean and pulsatile umbilical blood flow, perfusion pressure, placental vascular resistance, fundamental impedance, pressure pulsatility index, and umbilical artery resistance index corrected to a fetal heart rate of 160 beats/min were measured. On day 10 both groups were acutely embolized until fetal arterial pH decreased to approximately 7.00. Fetal heart rate was measured with the Sonicaid System 8000 (Oxford Sonicaid, Oxford, United Kingdom). RESULTS: Chronic fetal placental embolization was associated with a progressive reduction in umbilical blood flow (p < 0.00001) and fetal arterial oxygen content (p < 0.001) whereas fetal heart rate patterns remained unaltered. A chronic increase in umbilical artery resistance index corrected to a fetal heart rate of 160 beats/min could be entirely explained only if the changes in umbilical artery pressure pulsatility index and the fundamental impedance were taken into account, in addition to the changes observed in placental vascular resistance. During acute embolization leading to a 50% reduction in umbilical blood flow (p < 0.0002) and a three times increase in placental vascular resistance (p < 0.0001), the most consistent change in fetal heart rate patterns related to progressive metabolic acidosis was an 84% decrease in absolute acceleration frequency (p < 0.0001) whereas short-term fetal heart rate variability remained unaltered. CONCLUSION: Changes in umbilical artery resistance index induced by chronic umbilical-placental embolization resulting in fetal hypoxemia occurred before any changes in fetal heart rate patterns were detectable. A decrease in the absolute acceleration frequency was the only component of fetal heart rate patterns related to progressive metabolic acidosis in the near-term ovine fetus.     

Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection

Two high-performance liquid chromatography (HPLC) methods were developed for the determination of trecetilide in plasma samples. Differing only in the addition of a derivatization step and different detection wavelengths, the two methods encompassed a wide concentration range. In both methods, plasma samples (0.1 ml) with added internal standard were applied to solid-phase extraction discs containing a non-polar/strong cation mixed-phase, washed and eluted with an acetone-acetonitrile triethylamine mixture. The eluate was evaporated to dryness, and either reconstituted and directly injected onto an HPLC column or first derivatized with 1-naphthyl isocyanate before HPLC analysis. In both methods, the separation was performed isocratically on a cyano analytical column utilizing a mobile phase composed of acetonitrile-pH 7.9 phosphate buffer (70:30, v/v). The column effluent was monitored by fluorescence detection at 290/345 nm (with derivatization) or 235/320 nm (without derivatization). The limits of detection and quantitation of the assay were 0.57 and 1.9 ng/ml, respectively, when derivatization was used, or 4.3 and 14 ng/ml, respectively, without derivatization.     

High-performance liquid chromatographic method for the determination of fenofibric acid and reduced fenofibric acid in human blood, plasma and urine

In this study, a very reliable HPLC method was developed for the determination of fenofibric acid and reduced fenofibric acid in the biological samples described as follows. After addition of the internal standard solution and 0.5 M HCl to the biological sample, fenofibric acid, reduced fenofibric acid and the internal standard were extracted with a mixed solvent of n-hexane and ethyl acetate (90:10) from the mixture. The acids were back-extracted from the organic phase with 0.1 M Na2HPO4 and then re-extracted from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5) after addition of 0.5 M HCl. The organic phase was evaporated to dryness under the vacuum. The residue was dissolved in MeOH and diluted with distilled water. An aliquot of the resulting solution was injected on the HPLC. High reproducibility was observed in this HPLC method (C.V.% less than 4%). Moreover it was confirmed that the conjugates in the urine could be hydrolyzed by incubation at 37 degrees C for 18 h after addition of 400 IU of beta-glucuronidase.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 microl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Sensitive high-performance liquid chromatographic method with fluorometric detection for the simultaneous determination of gabapentin and vigabatrin in serum and urine

Twenty-seven cases of Kala-azar were treated with sodium stibogluconate at a dose of 20 mg/kg/day for 20 days (group A) and an equal number of cases were treated with the same dose but for a longer duration of 30 days (group B). Clinical and laboratory evaluation of these cases were carried out before and after therapy, during a follow up of cases every month, upto 6 months. Renal and liver function tests and electrocardiography were carried out of monitor any toxic effect of the drug during therapy. The cure rates of patients were 77.78% and 92.59% in group A and B cases respectively. Six and two patients in group A and B respectively were unresponsive to the treatment and showed relapse. Results of the study show that treatment of cases of Kala-azar with sodium stibogluconate in a dosage of 20 mg/kg/day for a longer period of 30 days is effective with a higher cure rate and minimum side effects, for treatment of cases of Kala-azar in this eastern part of Nepal, endemic for the disease.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C4 reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent gadobenate ion in plasma, urine, faeces, bile and tissues

The gadobenate ion is an intravascular paramagnetic contrast agent for magnetic resonance imaging. An HPLC method for assaying gadobenate ion in plasma, urine, faeces, bile and tissue samples is described. The analysis is based on the reversed-phase chromatographic separation of gadobenate ion from the endogenous components of biological matrices and detection by UV absorption at 210 nm. The selectivity of the method was satisfactory. The mean absolute recovery was greater than 95%. The precision and accuracy of the analytical methods were in the range 0.1-6.5% and -12 to +9.3%, respectively. The detection limits in plasma (0.1 ml), urine (0.05 ml), dried faeces (200 mg suspended in 4 ml water), bile (0.5 ml), and dried liver tissue (100 mg suspended in 1 ml water) were, respectively, 0.24, 0.47, 2.6, 0.63 and 2.8 nmol ml(-1) (corresponding to 0.16, 0.31, 1.7, 0.42 and 1.9 microg ml(-1)).     

High-performance liquid chromatographic determination and stability of 5-(3-methyltriazen-1-yl)-imidazo-4-carboximide, the biologically active product of the antitumor agent temozolomide, in human plasma

5-(3-Methyltriazen-1-yl)-imidazo-4-carboximide (MTIC) is a highly unstable compound which is believed to be the biologically active degradation product of the antitumor agent temozolomide. An HPLC method has been developed and validated for the analysis of MTIC in human plasma. Because of the instability of MTIC, sample processing was kept to minimal. The method involved precipitation of plasma protein with methanol followed by analysis of the supernatant using reversed-phase column and UV detection at 316 nm. The linearity (r>0.99), precision (C.V.<9%) and accuracy (bias<5%) were satisfactory. The lower limit of quantitation (LOQ) was 10 ng/ml. The recovery of MTIC and internal standard was > or = 86.7%. MTIC was stable in plasma though three freeze-thaw cycles, and was stable at 4 degrees C for 1 h and at -80 degrees C for at least 70 days. MTIC may be unstable at 10 degrees C in processed samples; therefore, samples were placed in the autosampler (10 degrees C) immediately prior to injection. By using this analytical method, MTIC was quantified in plasma of cancer patients (n=12) within 0.25-12 h after oral administration of temozolomide at 150 mg/m2. The mean maximum plasma concentration (Cmax) was 211 ng/ml which was observed at a mean Tmax of 1.88 h post dose. MTIC disappeared rapidly from plasma with an apparent in vivo half-life (t1/2) of 1.9 h similar to that of temozolomide. Following in vitro incubation of MTIC in human plasma at 25 degrees C, MTIC disappearance was bioexponential with estimated t1/2 values of 25 and 60 min for the first and second phases, respectively. Therefore, the elimination t1/2 of MTIC in human in vivo (1.9 h) was controlled by the rate of its formation from temozolomide.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C18 column (75 x 4.6 mm ID, 3 mu, Altex, USA) as clean-up column and a CSC-nucleosil C8 column (150 x 4.6 mm ID, 5 mu, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH = 3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min-1 for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH = 5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml-1. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml-1). It is applicable in clinical and pharmacokinetic studies.     

Validated high-performance liquid chromatographic assay for the determination of promazine in human plasma. Application to pharmacokinetic studies

A high-performance liquid chromatographic method for the determination of promazine in human plasma is described. The assay involves a single-step liquid-liquid extraction using pentane-2-propanol (98:2, v/v). The analyte of interest and the internal standard chlorpromazine were separated on a Spherisorb CN column using a mobile phase of acetonitrile-50 mM ammonium acetate (9:1, v/v). Electrochemical detection was achieved using an applied potential of +750 mV. The assay was validated according to international requirements prior to application to a pharmacokinetic study and was found to be specific, accurate and precise with a linear range of 0.25-25 ng ml(-1).     

Improved column-switching liquid chromatographic method for the determination of the enantiomers of mefloquine

A liquid chromatographic method for the determination of the enantiomers of mefloquine has been improved. The chromatography involved two columns: an achiral cyanopropyl stationary phase for the quantification of (+/-)-mefloquine and a chiral naphthyl-urea stationary phase for the determination of the enantiomeric ratio. Compared with the previous method, which needed two detectors, this one used one detector-integrator to which the two columns are connected alternately by an automated column-switching system. The method is suitable for the quantification (0.05 microgram/ml) of mefloquine and the determination of enantiomeric ratios from 500-microliters plasma samples with ultraviolet detection.     

Validated method for the determination of idazoxan in human plasma by liquid chromatography with tandem mass spectrometric detection

The effect of perchlorate anion as a mobile phase modifier on the retention of dansyl norvaline and dansyl tryptophan enantiomers on a human serum albumin (HSA) column was studied by varying the chaotropic agent concentrations. The thermodynamic parameters for the transfer of a solute from the mobile to the HSA stationary phases were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction between the solute and HSA was independent of the molecular structure. The parabolic variations observed with the enthalpic and entropic terms of dansyl amino acid transfer in relation to the concentration of perchlorate anion were considered to be the result of the change from reversed to normal-phase conditions for this chromatographic system.     

High performance liquid chromatographic method for the determination of lobenzarit disodium in a sustained release tablet formulation

A rapid and simple high performance liquid chromatographic method is described and validated for the determination of lobenzarit disodium (CAS 64808-48-6) in a sustained release tablet formulation. The calibration graph was linear over the range 20-105 micrograms/ml. The sensitivity (discriminator capacity) was 2.079 micrograms/ml. The coefficient of variations for repeatability and reproducibility were less than 1.60% and 1.30%, respectively. The accuracy of the method did not depend on lobenzarit concentration in tablets. The mean recovery was found to be 100.62%. The method was selective, even when degradation products were present.     

Brain proton magnetic resonance spectroscopy (1H-MRS) in Alzheimer's disease: changes after treatment with xanomeline, an M1 selective cholinergic agonist

OBJECTIVE: Higher than normal cellular levels of the phospholipid catabolic intermediate glycerophosphocholine have been found in postmortem brain tissue of persons with Alzheimer's disease. Proton magnetic resonance spectroscopy (1H-MRS) can detect a choline resonance that is largely due to glycerophosphocholine. The authors tested the hypothesis that treatment with xanomeline, an M1 selective muscarinic cholinergic agonist, would be associated with a decrease in the 1H-MRS choline resonance. METHOD: Patients with mild to moderate Alzheimer's disease received placebo or xanomeline for 6 months. 1H-MRS spectra were collected at baseline and after treatment discontinuation for 12 patients, two taking placebo and 10 taking xanomeline at a dose of 25 mg t.i.d. (N = 4), 50 mg t.i.d. (N = 3), or 75 mg t.i.d. (N = 3). RESULTS: For the combined group of patients taking xanomeline, there was a significant decrease in the choline/creatine ratio from baseline to endpoint. CONCLUSIONS: Treatment of Alzheimer's disease with a cholinergic agonist is associated with a decrease in the MRS choline resonance. Xanomeline may reduce breakdown of cholinergic neuron membranes by reducing the cellular requirement for free choline for acetylcholine synthesis.