Stereospecific high-performance liquid chromatographic assay of ketoprofen in human plasma and urine

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of ketoprofen (KT), a 2-arylpropionic acid (2-APA) non-steroidal antiinflammatory drug (NSAID), in plasma and urine was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), plasma containing the KT enantiomers and I.S. was extracted by liquid-liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in mobile phase and injeted into the HPLC system. The enantiomers were separated at ambient temperature on a commercially available 250 × 4.3 mm amylose carbamate-packed chiral column (Chiralpak AD) column with hexane-isopropyl alcohol-trifluoroacetic acid (80:19.9:0.1, v/v/v) as the mobile phase pumped at 1.0 ml/min. The enantiomers of KT were quantified by ultraviolet detection with the wavelength set at 254 nm. The assay described allows for the direct quantification of KT enantiomers without pre-column derivatization, and is suitable for clinical studies of KT enantiomers in human plasma and urine after administration of therapeutic doses.     

Sensitive high-performance liquid chromatographic assay for endralazine and two of its metabolites in human plasma

Endralazine (I) is a new antihypertensive which is chemically and pharmacologically related to hydralazine and dihydralazine. A sensitive high-performance liquid chromatographic-fluorescence assay for the drug and two of its metabolites [methyltriazoloendralazine (VII) and hydroxymethyltriazoloendralazine (VIII)] in human plasma was developed. After conversion of I and its internal standard to triazolopyridopyridazine derivatives the latter and metabolites were separated by high-performance liquid chromatography and detected using their fluorescence. The limits of detection of the assay were 1 nmol/l for I and VII and 0.1 nmol/l for VIII. Intra-assay coefficients of variation were 2.5–5.1% for I (range 1000–10 nmol/l), 4.2–4.5% for VII (range 100–5 nmol/l) and 3.4–5.7% for VIII (range 100–1 nmol/l). Following oral administration of 5 and 10 mg of I to two normal volunteers (slow acetylators) peak plasma levels of I occurred between 0.75 and 1 h after the dose, and declined in a biexponential fashion. The terminal half-life ranged from 2.8–3.7 h. These results contrast with those obtained for hydralazine in plasma where in vitro and in vivo half-lives were 30 min.     

Enantioselective determination of tramadol and its main phase I metabolites in human plasma by high-performance liquid chromatography

A sensitive and relatively rapid reversed-phase HPLC method was applied to the enantiomeric separation of tramadol and its two main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in plasma samples. Chromatography was performed on an AGP column containing alpha1-acid glycoprotein as chiral selector with a mobile phase of 30 mM diammonium hydrogen phosphate buffer-acetonitrile-triethylamine (98.9:1:0.1, v/v), adjusted to pH 7 by phosphoric acid, and a flow rate of 0.5 ml/min. The fluorescence of analytes was detected at excitation and emission wavelengths of 200 and 301 nm, respectively. The sample preparation was a simple extraction with ethyl acetate using fluconazol as internal standard (IS). The enantiomers of all analytes and IS peaks eluted within 32 min, without any endogenous interference. The calibration curves were linear (r(2) > 0.993) in the concentration range of 2-200, 2.5-100 and 2.5-75 ng/ml for tramadol, M1, and M2 enantiomers, respectively. The within- and between-day variation determined by the measurement of quality control samples at four tested concentrations, showed acceptable values. The lower limit of quantitation was 2 ng/ml for tramadol enantiomers and 2.5 ng/ml for M1 or M2 enantiomers. Mean recoveries of enantiomers from plasma samples were > 81% for all analytes. The procedure was applied to assess the pharmacokinetics of the enantiomers of tramadol and its two main metabolites following oral administration of single 100-mg doses to healthy volunteers.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Achiral and chiral high-performance liquid chromatographic determination of tramadol and its major metabolites in urine after oral administration of racemic tramadol

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tramadol and its major metabolites O-demethyltramadol and N-demethyltramadol in urine has been developed. The determination of the enantiomeric ratios of the three compounds was achieved using a Chiralpak AD column and a Chiralcel OD column, respectively. After oral administration of racemic tramadol to five healthy volunteers, inter-individual differences of the excreted amounts and the enantiomeric ratios of the compounds were observed.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Improved high-performance liquid chromatographic analysis of teniposide in human plasma

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure. VM26 and internal standard (ibuprofen) were extracted from human plasma (0.5 ml) with ethyl acetate. A phenyl μBondapak column eluted with a mobile phase, consisting of acetonitrile–distilled water–acetic acid (30:68:2, v/v/v) was used for separation, and quantitation was achieved with a UV monitor set at 240 nm. Average extraction efficiency was 96.8±6.6% for VM26 between 1 and 25 μg/ml, and 91.4±4.3% for internal standard, with both intra- and inter-day coefficients of variation being less than 10%. The detection limit with a 100-μl injection was estimated at 0.2 μg/ml with a signal-to-noise ratio of 3 for VM26 in human plasma. The stability data of VM26 in plasma, standard and stock solutions were also obtained. The present method was found to be an alternative to the previously reported method with an electrochemical detection, and can be easily applied to routine clinical pharmacokinetic studies of VM26.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid—liquid extraction. Separation was achieved on a C₁₈ reversed-phase column. The limit of detection of ciprofloxacin is 0.05 μg/ml and that of its three metabolites is 0.25 μg/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-a]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-a]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V–VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 μmole/l and the inter-assay variability in the slope of the standard curves ranged from 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37°, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level—time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level—time curve of apparent hydralazine.     

Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma

Quinupristin–dalfopristin (30:70, w/w) is a new streptogramin, which has been developed for intravenous use. A specific and sensitive HPLC method was developed to measure simultaneously quinupristin (RP 57669) and dalfopristin (RP 54476) and their main metabolites in human plasma. The metabolites measured by this method were RP 69012 (glutathione-conjugated) and RPR 100391 (cysteine-conjugated) from quinupristin and RP 12536 (natural pristinamycin IIA), from dalfopristin. Solid-phase extraction with disposable cartridges was combined with reversed-phase HPLC and fluorimetric detection for RP 57669, RP 69012 and RPR 100391 and UV detection for RP 54476 and RP 12536. The method provided good recovery and low limits of quantitation (0.025 mg l⁻¹ for RP 57669, RP 54476 and RP 12536, and of 0.010 mg l⁻¹ for RP 69012 and RPR 100391). The validated range of concentrations of the method was: 0.025–5000 mg l⁻¹ for RP 57669, RP 54476 and RP 12536 and 0.010–0.750 mg l⁻¹ for RP 69012 and RPR 100391.     

Rapid, sensitive high-performance liquid chromatographic method for the determination of cyclosporin A and its metabolites M1, M17 and M21

Cyclosporin A (CyA) and its metabolites seem to have nephro-, hepato- and neurotoxic side effects. Immunosuppressive therapy is a narrow path between the risk of rejection by inderimmunosuppression and toxic organ damage by overdosage. Thus CyA dosage must be calculated to avoid the risks of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In routine monitoring of CyA therapy, it can be important to measure not only the patent drug but also the metabolites. We describe a rapid and isocratic high-performance liquid chromatographic method for measurement of CyA and its metabolites M1, M17 and M21 in whole blood. CyA was detected by ultraviolet absorption at 212 nm with a CN analytical column maintained at 50°C and recycling of hexane-isopropanol as mobile phase for improved long-term column stability and efficiency. The minimum detectable concentration of CyA and the three metabolites was 10 ng/ml blood. Our modified HPLC method for the determination of CyA and its metabolites is a simple (isocratic), rapid (the retention times were 7.1 min for CYD, internal standard, 8.9 min for CyA, 11.0 min for M21, 12.9 min for M17 and 16.3 min for M1) and economical method suitable for measuring the concentration of the major metabolite, M17, and for routine monitoring of CyA-treated patients.     

Determination of the calcium antagonist SIM6080 and its N- and O-demethylated metabolites in plasma,urine and tissues by high-performance liquid chromatography

A sensitive and versatile high-performance liquid chromatographic assay for the determination of the calcium antagonist SIM6080 and its four N- and O-demethylated metabolites in plasma, urine and tissues has been developed and validated. A two-step extraction procedure is employed followed by reversed-phase liquid chromatographic analysis using ultraviolet detection. An isomer of SIM6080 was used as the internal standard. The analysis of spiked plasma, urine and tissues demonstrated the accuracy and precision of the assay with quantitation limits of 5 ng/ml (plasma and urine) or 100 ng/g (tissues). This assay has been used for urinary recovery and tissue distribution studies, as well as for toxicokinetic protocols.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Method development and validation of a high-performance liquid chromatographic method for tramadol in human plasma using liquid-liquid extraction

An HPLC system using a simple liquid-liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma

A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO₃ to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and were baseline-separated. Calibration curves were linear in the concentration range studied (5–500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Assay of moguisteine metabolites in human plasma and urine: conventional and chiral high-performance liquid chromatographic methods

Moguisteine is a novel peripheral non-narcotic antitussive agent. Pharmacokinetic studies in animal and in man showed that no unchanged drug is present in plasma, urine and faeces after oral administration. The main active metabolite, M1, is the free carboxylic acid of moguisteine, which maintains a stereogenic centre and consists of R(+)-M1 and S(−)-M1 enantiomers. M1 is partly metabolized to M2, its sulfoxidation derivative. A conventional HPLC method is described for the simultaneous determination of M1 and M2 in human plasma and urine after administration of therapeutic moguisteine doses. Plasma samples, previously acidified with phosphoric acid, are extracted with dichloromethane; urine samples are analyzed after appropriate dilution with methanol. Chromatography is performed using a Lichrosorb RP2 column and a linear gradient. M1 enantiomers can be determined in plasma extracts and urine samples by a chiral HPLC method using a β-cyclodextrin column. The analytical characteristics of both HPLC procedures proved to be adequate to analyze samples of subjects treated with therapeutic doses of moguisteine during clinical pharmacokinetic studies.     

Stereospecific high-performance liquid chromatographic assay of isosakuranetin in rat urine

A stereospecific method of analysis of racemic isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study pharmacokinetics. A simple high-performance liquid chromatographic method was developed for the determination of isosakuranetin enantiomers. Separation was achieved on a Chiralpak AD-RH column with ultraviolet (UV)-detection at 286 nm. The standard curves in urine were linear ranging from 0.5 to 100.0 microg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV) and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was <15% and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of isosakuranetin enantiomers in rat urine.     

Quantitative high-performance liquid chromatographic determinations of aminopyrine and its metabolites in man

A quantitative high-performance liquid chromatographic method, using a polystyrene—divinyl benzene (Hitachi No. 3010 gel) column and aqueous methanol as the mobile phase, was employed for the determination of aminopyrine and its related compounds, 4-acetylaminoantipyrine, 4-aminoantipyrine and 4-monomethylaminoantipyrine. Baseline separation could be achieved within 25 min. The method was applied to the recovery of these materials from control urine and human urine. Before separation human urine was adjusted to pH 9 and extracted with ethyl acetate, chloroform and diethyl ether.