Genotoxicity of ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine: induction of SCE in human lymphocytes and mutagenicity in Salmonella typhimurium TA 100

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and -10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and +/-2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at -80 degrees C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross-reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.     

Determination of N,N',N"-triethylenthiophosphoramide in biological samples using capillary gas chromatography

A sensitive assay for the determination of N,N',N"-triethylenthiophosphoramide (thioTEPA) in microvolumes of human plasma and urine has been developed. ThioTEPA was analysed using gas chromatography with selective nitrogen-phosphorus detection, after extraction with ethyl acetate from the biological matrix. Diphenylamine is the internal standard. The limit of quantitation was 0.1 ng/ml, using only 100 microl of sample; recoveries ranged between 85 and 100% and both accuracy and precision were less than 10%. Using a flame ionisation nitrogen-phosphorus detector, the assay was not linear over the concentration range of 2-1000 ng/ml for plasma and 10-1000 ng/ml for urine. Linearity was accomplished in the range of 1-1000 ng/ml for plasma and urine when a thermionic nitrogen/phosphorous detector was used. The stability of thioTEPA in plasma proved to be satisfactory over a period of 3 months, when kept at -20 degrees C, whereas it was stable in urine for at least 1 month at -80 degrees C. ThioTEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay.     

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins

A reversed-phase isocratic high-performance liquid chromatographic method has been developed for the determination of AG-331, a novel thymidylate synthase inhibitor, in human serum and urine. The method involves a solid-phase extraction from C18 cartridges without addition of an internal standard. The methanol eluent is evaporated under nitrogen at 40 degrees C, and reconstituted in mobile phase, acetonitrile-water (35:65, v/v) containing 25 mM ammonium phosphate. Separation of AG-331 was obtained on a C18 column at a flow-rate of 1 ml/min. Chromatographic signals were monitored by a photodiode-array detector at a primary wavelength of 457 nm with a bandwidth of 4.8 nm. Standard curves are linear in the range of 22-2175 ng/ml in plasma and 44-2175 ng/ml in urine, respectively. The extraction recovery ranged from 92.9-102.4%. Intra-day coefficient of variation was less than 9.5%, and inter-day coefficient of variation was less than 14.3% for an AG-331 concentration of 44 ng/ml. This method has been used to characterize the pharmacokinetics of AG-331 in cancer patients as part of ongoing Phase I trials.     

Is plaque formation in the common carotid artery representative for plaque formation and luminal stenosis in other atherosclerotic peripheral arteries? A post mortem study

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate-acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15-2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Delivery of salbutamol to nonventilated preterm infants by metered-dose inhaler, jet nebulizer, and ultrasonic nebulizer

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid-liquid extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water-methanol-tetrahydrofuran-ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10-500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6+/-8.52 and 93.7+/-5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.     

Rapid quantitation of (-)-2'-deoxy-3'-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (-)-2'-deoxy-3'-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C18 column using a mixture of phosphate buffer and methanol (88.3:11.7. v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 microl of serum. The standard curve was linear within the range of 20-10,000 ng/ml. Replicate analysis of three quality control samples (40-1500 ng/ml) led to satisfactory intra- and inter-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from 6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Relationship between polypeptide transcytosis and lymphoid tissue in the rabbit nasal mucosa

Two simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) methods were developed and validated for the quantitative determination of a novel hypertension drug CGS 25462 and its major metabolites CGS 24592 and CGS 25659 in human plasma. CGS 25462 and CGS 25798 (internal standard) were purified by one-step liquid-liquid extraction with methylene chloride. The metabolites were analyzed on HPLC after plasma protein precipitation with 10% trichloroacetic acid (TCA). Separations were achieved on a Zorbax RX C18 column. All compounds were detected by using a fluorescence detector. The excitation wavelength was 254 nm, and emission was monitored at 325+/-12.5 nm. Assessment of recovery and reproducibility indicated good accuracy and precision. Over the validation concentration range of 10 to 1000 ng/ml for CGS 25462 and 25 to 5000 ng/ml for both metabolites, overall mean relative recoveries were 96% for CGS 25462, 101% for CGS 25659 and 107% for CGS 24592, and the coefficients of variation were 4.6 to 13% for CGS 25462, 9.5 to 13% for CGS 25659 and 7.7 to 15% for CGS 24592. The limits of quantification (LOQs) were 10 ng/ml for CGS 25462 and 25 ng/ml for CGS 24592 and CGS 25659, which were of sufficient sensitivity to measure the concentrations of CGS 25462, CGS 25659 and CGS 24592 in plasma samples from normal volunteers following a single 800 mg oral dose.     

Osmolarity, ionic flux, and changes in brain excitability

A quantitative capillary electrophoresis-mass spectrometry method has been demonstrated for the determination of methylphenidate in human urine over a dynamic range of 640. The samples were prepared by liquid-liquid extraction using 4 ml of human urine. The extracts were analyzed using a Finnigan LCQ ion-trap mass spectrometer in a two-event, positive-ion full-scan MS and tandem MS selected reaction monitoring mode. The lower level of quantitation was determined to be 1.5 ng/ml methylphenidate in human urine. The intra-assay precision had a relative standard deviation less than 6.8%. The intra-assay accuracy was less than +/-14.7% bias from the nominal concentration for the full-scan MS analysis, and less than +/-26.5% bias for the tandem MS analysis. Six incurred human urine samples containing methylphenidate were analyzed and a simple pharmacokinetic curve is presented.     

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.     

Neuroendocrine responses of flight cadets during midterm tests and of fighter pilots during tail chase sorties

Following detailed study, a rapid and sensitive assay for ethmozine in human plasma has been developed using reversed phase high performance liquid chromatography (HPLC). Plasma samples were prepared for analysis by addition of internal standard (5-chloro-2-amino-benzophenone) followed by protein precipitation using acetonitrile. Analytical column was a C18 Spherisorb. The mobile phase consisted of mathanol-water-triethylamine (70:30:0.4, v/v/v, pH 6.5). The column effluent was monitored at 268 nm. The calibration curve was linear in the range from 20 ng/ml to 4000 ng/ml with r = 0.9994. The detection limit of this method was 3 ng/ml. The method showed good precision and the analytical recovery of ethmozine from plasma was 90-105%. The relative standard deviations for within-day and between-day were 2.4-6.3% and 4.5-10.2% respectively. The plasma drug concentration-time course in man after oral administration of 400 mg after conformed to a 1-compartment open model with a first order absorption phase. Mean T1/2 value was 1.75 +/- 0.45 h.     

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 assay for a new fasciolicide agent, alphaBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (alphaBIOF10) -- a new fasciolicide agent -- and its sulphoxide (SOalphaBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a microBondapak C18 (10 microm) column, using methanol-acetonitrile-water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 7.2 ng/ml for alphaBIOF10 and SOalphaBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 microg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both alphaBIOF10 and SOalphaBIOF10. In urine, recovery was 99.6% and 93.1% for alphaBIOF10 and SOalphaBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Determination of ivabradine and its N-demethylated metabolite in human plasma and urine, and in rat and dog plasma by a validated high-performance liquid chromatographic method with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method with native detection of fluorescence was developed and validated for the quantitation of ivabradine and its N-demethylated metabolite in plasma (rat, dog, human) and human urine. The procedure involves the use of an analogue as internal standard, solid-phase extraction on cyano cartridges, separation on a Nova-Pak C8 column and fluorescence detection. Calibration curves are linear in the concentration ranges from 0.5 to 100 ng/ml in plasma and 2.0 to 500 ng/ml in urine with a limit of quantitation set at 0.5 and 2.0 ng/ml in plasma and urine, respectively. The analysis of plasma and urine samples (spiked with the analytes at low, medium and high concentrations of the calibration range) demonstrates that both analytes can be measured with precision and accuracy within acceptable limits. Quality controls spiked with analyte concentrations up to 10000 ng/ml can also be analysed with excellent precision and accuracy after dilution of the samples. The parent drug and its metabolite are stable in plasma and urine after short-term storage (24 h at room temperature and after three freeze-thaw cycles) as well as after long-term storage at -20 degrees C (at least 6 months in animal plasma and 12 months in human plasma and urine). The method has been used to quantify both compounds in plasma and urine samples from clinical and non-clinical studies with ivabradine.     

High-performance liquid chromatographic determination of diclofenac in human plasma using automated column switching

A validated high-performance liquid chromatographic procedure employing ultraviolet detection for the analysis of diclofenac in human plasma is reported. The method is rapid and, coupled with column switching, leads to a sensitive, accurate and reproducible assay. The retention times of diclofenac and the internal standard (4'-methoxydiclofenac, CGP-4287) are 6.4 and 7.6 min, respectively. The peak height versus plasma concentration is linear over the range 5.0-2000 ng/ml with a detection limit below 2.5 ng/ml. The mean absolute recovery of diclofenac using the described assay is 96.5% (n = 24). The inter- and intra-day accuracy and precision are within 8.3% of the actual values for all concentrations investigated. Furthermore, this procedure is applied to assess the pharmacokinetics of a single 75-mg oral dose of diclofenac sodium.     

Requirement of CD4 T cells for skin graft rejection against thymus leukemia (TL) antigen and multiple epitopes on the TL molecule recognized by CD4 T cells

Two HPLC assays were developed and validated for simultaneous quantitation of two sulfate metabolites, PD 163637 (VI) and PD 163639 (VIII), of an investigational antipsychotic drug CI-1007 (I) in monkey plasma and urine. VI and VIII were identified as major metabolites in monkey plasma, and both were excreted in urine. Monkey plasma samples were directly injected after deproteinization, and urine samples were analyzed after a clean-up procedure using methyl-tert.-butyl ether. Liquid chromatographic separation was achieved on a Zorbax RX C8 analytical column using gradient elution. Column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 254 and 330 nm, respectively. Minimum quantitation limit was 50 ng/ml in plasma and 100 ng/ml in urine. Linearity was demonstrated up to 3000 ng/ml in plasma and urine. Recoveries of the analytes from plasma and urine were greater than 85%. The assay has been applied to the determination of VI and VIII in plasma and urine samples from monkeys receiving oral administration of I.     

Plasma pyrimethamine concentrations during long-term treatment for cerebral toxoplasmosis in patients with AIDS

Steady-state plasma pyrimethamine levels were measured by gas chromatography. The specimens were taken from 74 adults with advanced human immunodeficiency virus infection receiving pyrimethamine-containing drugs for prophylaxis or curative therapy of reactivated cerebral toxoplasmosis. During an overall treatment period of 1,049 months, 1,012 plasma samples were investigated. Pyrimethamine concentrations could be evaluated in 904 plasma samples. The weekly dosage of pyrimethamine ranged from 25 to 1,400 mg; one patient with severe diarrhea received 2,100 mg/week. Steady-state plasma pyrimethamine concentrations were achieved after 12 to 20 days. Pyrimethamine concentrations evidently increased with the weekly dosage given. Mean concentrations were 253 +/- 151 ng/ml with 50 mg of pyrimethamine per week, 471 +/- 214 ng/ml with 100 mg of pyrimethamine per week, 1,893 +/- 1,182 ng/ml with 350 mg of pyrimethamine per week and 3,369 +/- 1,726 ng/ml with 1,050 mg of pyrimethamine per week. A widespread interpatient range was found for every dosage. With the simultaneous use of enzyme-inducing comedication, the plasma pyrimethamine levels decreased in several patients. Mild chronic liver disease did not influence plasma pyrimethamine concentrations. To avoid ineffective therapy or severe side effects, monitoring of pyrimethamine could be useful in patients receiving enzyme-inducing comedications and in patients with severe diarrhea or poor compliance.     

Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined alpha- and beta-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83+/-5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5-1.8 ng ml(-1). Intra-day and inter-day variation at 25 ng ml(-1) were < or = 2.7% and < or = 5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Simultaneous quantification of an anti-inflammatory compound (DuP 697) and a potential metabolite (X6882) in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using fluorescence detection has been developed for the simultaneous analysis of low nanogram concentrations of an anti-inflammatory drug, 5-Bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl]thiophene (DuP 697), and a potential metabolite (X6882) in human plasma and of DuP 697 in human urine. This assay method used an EM Separations Lichrospher C18 endcapped column. The mobile phase was acetonitrile-water (75:25, v/v). The detection of DuP 697 and X6882 was by fluorescence at excitation and emission wavelengths of 256 and 419 nm, respectively. The chromatographic system could separate DuP 697 from X6882, the external standard (anthracene), and other endogenous substances present in human plasma. In human plasma the limits of quantification for DuP 697 and X6882 were 3 and 20 ng/ml, respectively; the limit of quantification for DuP 697 in human urine was 5 ng/ml. These compounds were shown to be stable in frozen (-20 degrees C) human plasma and urine for at least 9 weeks. The assay described has been used to characterize DuP 697 pharmacokinetics after oral administration in humans.     

A comparison of analytic procedures for measurement of fractional dextran clearances

Fractional dextran clearances have been extensively used to study glomerular size selectivity. We report on an analysis of different laboratory procedures involved in measuring fractional dextran clearances. The deproteinization of plasma samples by 20% trichloroacetic acid (TCA) revealed a protein contamination of 0.2% +/- 0.3%, whereas both 5% TCA and zinc sulfate deproteinization revealed a significantly higher remaining sample protein content (2.5% +/- 0.4% and 3.4% +/- 0.1%, respectively). Only zinc sulfate revealed incomplete deproteinization of urine samples (0.6% +/- 0.2%). Dextran recovery in plasma and urine supernatants was significantly lower after 5% TCA and zinc sulfate deproteinization when compared with 20% TCA deproteinization. Gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) showed a variance of calibration smaller than 5% over 1 year. The use of 3 different sets of standard dextrans revealed significant differences in calibration. GPC and HPLC followed by anthrone assay showed a comparable variance in dextran concentration in plasma, from 3 to 6 nm (14% to 25%), whereas the variance in urine was lower for the GPC and anthrone assay, especially from 5.4 to 6 nm (23% to 43% versus 50% to 78%). HPLC and online refractometry showed the lowest variance of dextran concentration in plasma, from 3 to 6 nm (<4%), and in urine, from 3 to 5.2 nm (<7%), whereas it showed a higher variance in urine, from 5.4 to 6 nm, in comparison with GPC and HPLC with the anthrone assay. The GPC and anthrone assay revealed higher fractional dextran clearances in comparison with the HPLC and anthrone assay in healthy subjects (3 to 5.4 nm) as well as in patients with nondiabetic proteinuria (4.2 to 5.8 nm), and lower clearances in patients from 3 to 3.4 nm. The HPLC and anthrone assay revealed higher clearances in comparison with HPLC and online refractometry in healthy subjects (3.6 to 5.4 nm) and in patients (3.6 to 5.2 nm). The GPC and anthrone assay revealed characteristic differences in fractional dextran clearances between healthy subjects and patients. The HPLC and anthrone assay showed no significant differences between both groups, whereas HPLC and online refractometry showed only an increased clearance of dextrans from 4.6 to 5.2 nm in patients. Fractional clearances of dextran 5.6 nm as estimated by all 3 dextran assays were not significantly related to the fractional immunoglobulin G clearance or the immunoglobulin-to-albumin clearance index in our patients. Quantitative and qualitative differences in fractional dextran clearances may be induced by differences in laboratory procedures. We recommend sample preparation by 20% TCA deproteinization, frequent calibration with 1 set of dextran standards with low polydispersity, size-exclusion chromatography by GPC, and dextran detection by anthrone assay for optimal measurement of fractional dextran clearances. Even with such an approach, however, the variability in the measurement remains extremely high in the important range of dextrans greater than 5 nm.