Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.     

Determination of norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

A method for the determination of norfloxacin in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5-ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from +/- 0.37 to +/- 4.14% in plasma (concentration range: 70.3-2109.2 ng/ml) and from +/- 0.51 to +/- 1.56% in urine (concentration range: 7.5-299.4 micrograms/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18% to -9.47% in plasma and from -10.56% to - 5.91% in urine. The assay has been used to support human pharmacokinetic studies.     

Determination of a new podophyllotoxin derivative, TOP-53, and its metabolite in rat plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method was developed for the determination of a new podophyllotoxin derivative, TOP-53 (I), and TOP-53 glucuronide (II) as its major metabolite in rat plasma and urine. For the analysis of I, the sample was chromatographed on a reversed-phase C18 column with electrochemical detection after consecutive two-step liquid-liquid extractions. Compound II was determined as I after enzymatic hydrolysis of II. This method was validated sufficiently with respect to specificity, accuracy, and precision. The limits of quantitation for both I and II were 2 ng/ml in plasma and 10 ng/ml in urine. The method is thus useful for the pharmacokinetic study of I.     

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.     

Simultaneous determination of N,N',N"-triethylenethiophosphoramide, cyclophosphamide and some of their metabolites in plasma using capillary gas chromatography

A sensitive assay for the simultaneous determination of N,N',N"-triethylenethiophosphoramide (thioTEPA), its metabolite N,N',N"-triethylenephosphoramide (TEPA), cyclophosphamide (CP) and its metabolite 2-dechloroethylcyclophosphamide (2-DCE-CP) in plasma has been developed and validated. The analytes were determined using gas chromatography with nitrogen/phosphorus selective detection after liquid-liquid extraction with chloroform using 100 microl of plasma. Diphenylamine (for TEPA, thioTEPA and 2-DCE-CP) and imipramine (for CP) were used as internal standards. The limits of quantitation for thioTEPA, TEPA, CP and 2-DCE-CP were 5, 5, 50 and 250 ng/ml, respectively. Linear calibration curves were observed over two decades of concentration. Accuracy, within-day and between-day precision were less than 13% for all analytes. Stability of the analytes proved to be satisfactory for at least 1 month, stored at -70 degrees C. Analysis of samples obtained from patients receiving cyclophosphamide, thioTEPA and carboplatin in a high-dose regimen demonstrated the applicability of the assay.     

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.     

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.     

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.     

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.     

High-performance liquid chromatographic determination of phenylephrine in human serum using column switching with fluorescence detection

A method for the determination of total phenylephrine (free plus conjugated) in human serum was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. After serum was deproteinized with acetonitrile, the conjugated phenylephrine was hydrolyzed with diluted hydrochloric acid. The solution was evaporated to dryness. The reconstituted residue was analyzed with HPLC using a heart-cut technique. Good recoveries of the analytes from spiked human serum samples were obtained with small coefficients of variation. A good linear response was obtained for the concentration range 5-500 ng/ml. The lower limit of quantitation for phenylephrine in human serum was 5 ng/ml. The method was applied to the determination of phenylephrine in human serum after oral administration of phenylephrine hydrochloride.     

Simultaneous determination of GTS-21 and its metabolite in rat plasma by high-performance liquid chromatography using solid-phase extraction

It has been suggested that GTS-21 can improve the learning deficits and inhibit the neuro-degeneration in patients with Alzheimer's disease. This paper describes a reversed-phase high-performance liquid chromatographic assay with visible detection at 405 nm for determination of GTS-21 and its metabolite, 4-hydroxy-GTS-21 in rat plasma. The method uses solid-phase extraction with a Bond Elut C18 column. A quantitation limit of 1.0 ng/ml was achieved using 0.5 ml of rat plasma. In the validation study, the coefficients of variation and the relative errors of each compound were less than 10%. Also freeze-thaw and storage stability were confirmed. This method has proved to be applicable to the pharmacokinetic study of GTS-21 in rats.     

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.     

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 radioimmunoassay for digoxin in plasma and urine

A reproducible and sensitive radioimmunoassay for digoxin in either serum, plasma or urine is described. Using 0.5 ml of serum or plasma, the assay sensitivity is 0.05 ng of digoxin/ml. The antiserum and tracer solutions employed are available in a kit sold in the United States. All other reagents were prepared in the laboratory. The assay allows measurement of digoxin in plasma or serum for 96 hours after single 0.5 mg doses of digoxin; this is necessary in human bioavailability studies to accurately estimate the total area under the digoxin concentration, time curve from zero to infinite time. In contrast, with the kit assay, employing 0.2 ml of plasma or serum, it has been reported that the 12 hr serum digoxin levels, after single 0.5 mg doses, are, in most subjects, below the sensitivity limit (about 0.5 ng/ml) of the assay.     

Noncanonical conformation of nucleic acids: structure with slipped-loops, occurring in DNA oligonucleotides

A HPLC method was developed and validated for the quantitation of 9-cis-retinoic acid (ALRT1057) and its major metabolite, 4-oxo-9-cis-retinoic acid (LG100182) in human plasma. Samples were buffered and extracted with methyl-tert-butyl-ether. The analytes and an I.S. were separated on a C18 HPLC column using a shallow gradient of 70-89% organic solvent. The analytes were quantitated by UV detection at 348 nm. Selectivity against endogenous compounds and potential metabolites (retinol, all trans-, 13-cis-, and 4-hydroxy-9-cis-retinoic acid) was demonstrated. The run time was 29 min. The standard curve was linear from 2.5 to 450 ng ml-1. Interassay precision for both analytes in quality control samples was less than 5.0% RSD. Accuracy was within 11.0% RE for both compounds. Analyte stability during sample storage, extraction processing, and chromatography was established. Method ruggedness was tested by two analysts and on two HPLC systems. This method has been applied to the quantitation of clinical samples.     

Simultaneous determination of granisetron and its 7-hydroxy metabolite in human plasma by reversed-phase high-performance liquid chromatography utilizing fluorescence and electrochemical detection

A highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of granisetron and its active metabolite, 7-hydroxygranisetron (7OH-G) in human plasma. Granisetron is a selective 5-hydroxytryptamine receptor antagonist used in the treatment of cytotoxic drug-induced emesis. The method involves isolation of granisetron, 7OH-G and the internal standards from plasma by solid-phase extraction prior to reversed-phase ion-pair chromatographic separation on an octyl silica column with subsequent quantification of analytes simultaneously either with electrochemical (7OH-G) or fluorescence (granisetron) detectors which are placed in series. The recovery of granisetron and 7OH-G from human plasma was quantitative. Using 1 ml of plasma, the limits of quantification for granisetron and 7OH-G were 0.1 and 0.25 ng/ml, respectively. Linear responses in analyte/internal standard peak-area ratios were observed for analyte concentrations ranging from 0.1 to 50 ng/ml plasma. Precision and accuracy were within 13% across the calibration range for both granisetron and 7OH-G. The method was sufficiently sensitive, accurate and precise to support pharmacokinetic studies for granisetron and 7OH-G, in both normal and patient populations.     

Reversed-phase high-performance liquid chromatographic determination of enoxacin and 4-oxo-enoxacin in human plasma and prostatic tissue. Application to a pharmacokinetic study

A simple high-performance liquid chromatographic method has been developed for the simultaneous determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue. The work-up procedure involves a liquid-liquid extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet absorbance detection (lambda = 340 nm). Using a mobile phase of 20.9% (v/v) acetonitrile buffer (pH 2.1), adequate retention time and separation among the analytes has been obtained using tetrabutylammonium hydroxide included in the eluent. Retention times are 5.2 min for enoxacin, 6.8 min for pefloxacin and 12 min for 4-oxo-enoxacin. For plasma and prostatic tissue, the precision of the assay was below 9%. The percent recovery from the nominal values for accuracy ranged from 94 to 108%. The limits of quantitation were 20 ng/ml for plasma and 50 ng/g for tissue (precision < 18%). The detection limits were 10 ng/ml and 25 ng/g, respectively. The calibration curves were linear from 20 to 1000 ng/ml for plasma and from 50 to 2500 ng/g for tissue. In plasma, the extraction recoveries averaged 52% for enoxacin and 63% for 4-oxo-enoxacin. In prostatic tissue, they were 57 and 76% for the two analytes, respectively. This method has been employed for the determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue samples from patients following repeated oral administration of enoxacin (400 mg twice a day for four days).     

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.     

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.