Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9 -anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The organic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The eluate was reacted on-line with a mixture of tris(2,2'-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 10 ng/ml, respectively. This method has been used for the determination of 1 in samples from clinical trials.     

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.     

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.     

Determination of a new proton pump inhibitor, YH1885, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new proton pump inhibitor, YH1885 (I), in human plasma and urine, and rat blood and tissue homogenate using fenticonazole as an internal standard. The sample preparation was simple: a 2.5 volume of acetonitrile was added to the biological sample to deproteinize it. A 50-microliter aliquot of the supernatant was injected onto a C8 reversed-phase column. The mobile phase employed was methanol-0.005 M tetrabutylammonium dihydrogenphosphate (77:23, v/v), and it was run at a flow-rate of 1.0 ml/min. The column effluent was monitored using an ultraviolet detector at 270 nm. The retention times for I and the internal standard were 9.0 and 10.3 min, respectively. The detection limits for I in human plasma and urine, and in rat tissue homogenate (including blood) were 50, 100 and 100 ng/ml, respectively. The coefficients of variation of the assay (within- day and between-day) were generally low (below 8.84%) for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

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.     

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.     

Personality disorder in patients infected with HIV: a controlled study with implications for clinical care

A reversed-phase isocratic high-performance liquid chromatographic method is described for the simultaneous determination of EO9, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta- en-alpha-ol (I), and its ring-opened aziridine analogue EO5A (II), employing ultraviolet detection. Solid-phase sample extraction was used without addition of an internal standard. Plots of peak heights and areas of I and II were linear in the range 5-10,000 ng/ml. The lower limit of detection of both I and II in plasma was 2 ng/ml. The between-day variation of I was 13.9% at 5 ng/ml and lower than 6.2% for concentrations > or = 10 ng/ml. The between-day variation of II at 5 ng/ml was 13.8% and lower than 4.5% for concentrations > or = 10 ng/ml. The assay was developed to enable pharmacological guiding of a phase I study of I in solid tumour cancer patients.     

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).     

High-performance liquid chromatographic determination of angiotensin II receptor antagonists in human plasma and urine. I. DuP 532 (L-694,492)

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet detection was developed for the analysis of a new angiotensin II receptor antagonist, DuP 532 (L-694,492), in human plasma and urine. The analyte and internal standard are extracted from plasma and urine at a pH between 3.3 to 3.6 by liquid-liquid extraction and analyzed on a C6 column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limits of quantification are 6 and 7.5 ng/ml for plasma and urine, respectively.     

Methamphetamine-stimulated striatal dopamine release declines rapidly over time following microdialysis probe insertion

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.     

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.     

A simple method for measurement of angiotensin II in plasma

A simple radioimmunological (RIA) method for the determination of angiotensin II in 0.5-1.0 ml samples of plasma is described and carefully evaluated. Before RIA was performed, the interfering plasma proteins were eliminated by ion exchange chromatography, and recovery from every column was checked with a small amount of [125I]angiotensin II. The sensitivity of the method was 4.0 ng/l; the coefficient of intra-assay variation was 10.0% and that of inter-assay variation 12.1%. Accuracy was studied both by adding various amounts of angiotensin II to plasma samples and by diluting plasma containing angiotensin II with the RIA buffer. Both studies gave very good correlations between found and expected values (r=0.998 and r=0.987). In a normal material (n=36), the mean angiotensin II concentration at 8 a.m. after 2 h ambulation 42.4 +/- 12.8 (S.D.) ng/l. Because the present method is accurate, precise, and practical, and allows measurement of angiotensin II in small samples, it seems useful for routine as well as for research work.     

The natural interleukin-1 receptor antagonist in the fetal, maternal, and amniotic fluid compartments: the effect of gestational age, fetal gender, and intrauterine infection

OBJECTIVES: The interleukin-1 receptor antagonist is a newly discovered cytokine that blocks the biologic effects of interleukin-1 in vitro and in vivo. This cytokine is a physiologic component of amniotic fluid and is considered to be of critical importance in the homeostasis of the cytokine network. This study was undertaken to systematically examine the bioavailability of interleukin-1 receptor antagonist in the maternal, fetal, and amniotic fluid compartments during term and preterm parturition in women with and without microbial invasion of the amniotic cavity. STUDY DESIGN: The patient population consisted of (1) pregnant women in the midtrimester (n = 42), (2) patients who underwent cordocentesis for diagnostic purposes (n = 39), (3) patients with preterm labor (n = 126), (4) women with term gestation (n = 102), and (5) healthy nonpregnant women (n = 8). Amniotic fluid was cultured for aerobic and anaerobic bacteria, as well as Mycoplasma sp. Interleukin-1 receptor antagonist concentrations were determined by enzyme-linked immunoassay in maternal and fetal plasma, amniotic fluid, and neonatal urine. Microbial invasion of the amniotic cavity was defined as the presence of a positive amniotic fluid culture for microorganisms. RESULTS: (1) Interleukin-1 receptor antagonist was normally present in fetal plasma samples obtained by cordocentesis, and its concentration increased with advancing gestational age (n = 39; r = 0.61, p < 0.001). (2) Patients at term not in labor had higher amniotic fluid interleukin-1 receptor antagonist concentrations than patients in the midtrimester (median 40.1 ng/ml, range 5.7 to 213.1 vs median 16.2 ng/ml, range 3.2 to 62.2, respectively, p < 0.001). (3) Amniotic fluid and cord plasma interleukin-1 receptor antagonist concentrations were significantly higher in patients with preterm labor and microbial invasion of the amniotic cavity than in those without microbial invasion of the amniotic cavity (amniotic fluid: median 219.9 ng/ml, range 35.4 to 504 vs median 80.6 ng/ml, range 24.3 to 399, respectively, p < 0.001; umbilical cord plasma: median 4.8 ng/ml, range 0.3 to 167.0 vs median 1.0 ng/ml, range 0 to 276.0, respectively, p < 0.05). In contrast, these differences were not found in patients with term labor either with or without microbial invasion of the amniotic cavity. (4) In both term and preterm patients the amniotic fluid and neonatal urine concentrations of interleukin-1 receptor antagonist were significantly higher in female fetuses than in male fetuses (amniotic fluid, preterm: median 191.9 ng/ml, range 51.6 to 504.0 vs median 61.1 ng/ml, range 11.5 to 284.9, respectively, p < 0.001; amniotic fluid, term: median 58.7 ng/ml, range 25.5 to 264.0 vs median 33.9 ng/ml, range 3.4 to 132.4, respectively, p < 0.001; neonatal urine: median 317 ng/ml, range 59.0 to 440.8 vs median 12.2 ng/ml, range 2.5 to 61.6, respectively, p < 0.005). CONCLUSIONS: (1) Interleukin-1 receptor antagonist is physiologically present in the fetal, maternal, and amniotic fluid compartments; (2) microbial invasion of the amniotic cavity in the preterm gestation is associated with a significant increase in the concentrations of this cytokine in the fetal and amniotic fluid compartments but not in maternal plasma; (3) fetal urine is a source of amniotic fluid interleukin-1 receptor antagonist; (4) fetal plasma interleukin-1 receptor antagonist concentrations increase with gestational age; (5) there is a significant effect of fetal gender in amniotic fluid and neonatal urine concentrations of interleukin-1 receptor antagonist.     

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.     

Combined pharmacological and traction treatment in cases of painful syndromes of the cervical spine with degenerative and deformative changes

A simple, specific, and sensitive radioimmunoassay was developed for the determination of the diuretic bumetanide in plasma and urine. Antiserum to bumetanide was obtained from rabbits immunized with an immunogen prepared by covalently coupling the glycine conjugate of bumetanide to bovine serum albumin. Following extraction of the sample at pH 5.5 with ether, radioimmunoassay of the residue from the ether extract allows for the determination of bumetanide with a limit of sensitivity of about 1 ng/ml using 0.1 ml of plasma or urine. The specificity of the radioimmunoassay was established by comparison with specific radiometric and spectrofluorometric techniques. The pharmacokinetic profile of bumetanide in eight human subjects receiving single 2-mg oral doses of the drug was elucidated using the radioimmunoassay. The peak plasma levels ranged from 39 to 50 ng/ml at 1-4 hr after administration and declined with a mean apparent half-life of 1.17 hr. The mean plasma clearance rate was calculated to be 255 ml/min. During the first 24 hr, a mean of 43% of the bumetanide dose was excreted in the urine as intact drug.     

Screening for the detection of angiotensin-converting enzyme inhibitors, their metabolites, and AT II receptor antagonists

A gas chromatography-mass spectrometry (GC-MS) screening procedure was developed for the detection of angiotensin-converting enzyme (ACE) inhibitors, their metabolites, and angiotensin (AT) II receptor antagonists in urine as part of a systematic toxicologic analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 157, 160, 172, 192, 204, 220, 234, 248, 249, and 262, the possible presence of ACE inhibitors, their metabolites, and AT II antagonists could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed detection of therapeutic concentrations of ACE inhibitors (benazepril, enalapril, perindopril, quinapril, ramipril, trandolapril, their metabolites, or both) and therapeutic concentrations of the AT II antagonist, valsartan, in human urine samples. Human urine samples were not available for testing cilazapril, moexipril, and losartan; they were detected only in rat urine. The overall recoveries of ACE inhibitors ranged between 80% and 88%, with a coefficient of variation (CV) of less than 10% and the limit of detection of at least 10 ng/ml (signal to noise ratio 3) in the full-scan mode. The overall recovery of the valsartan was 68%, with a CV of less than 10%; the limit of detection was at least 10 ng/ml (S/N 3) in the full scan mode.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography-mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography-mass spectrometry (GC-MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC-MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one-five days) in all samples analysed.     

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.     

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.