Simultaneous determination of a novel angiotensin II receptor blocking agent, losartan, and its metabolite in human plasma and urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of a new angiotensin II receptor blocking agent, losartan (DuP 753, MK-954, I), and its active metabolite, EXP3174 (II), in human plasma or urine is described. The two analytes and internal standard are extracted from plasma and urine at pH 2.5 by liquid—liquid extraction and analyzed on a cyano column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limit of quantification for both compounds in plasma is 5 ng/ml. The limit in urine is 20 and 10 ng/ml for I and II, respectively. The assay described has been successfully applied to samples from pharmacokinetic studies.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

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 μl of plasma) and 25 ng/ml (50 μl 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.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

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 ortanic 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 elute 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 00 ng/ml, respectively. This method has been used for the determination of I in samples from clinical trials.     

Sensitive high-performance liquid chromatographic fluorescence assay for the quantitation of topotecan (SKF 104864-A) and its lactone ring-opened product (hydroxy acid) in human plasma and urine

A sensitive reversed-phase high-performance liquid chromatographic fluorescence method is described for the simultaneous determination of topotecan (I) and the hydrolysed lactone ring-opened product hydroxy acid (II) in plasma and for the determination of I in urine. To 250 μl of plasma, a 750-μl volume of cold methanol was added to stabilize the pH-dependent conversion of I into II. In plasma, the lower limit of quantitation (LLQ) for both compounds was 0.10 ng/ml. The between-day variation for I at the LLQ was 7.1% and for II was 5.5%. Prior to injection, urine samples were acidified with orthophosphoric acid and diluted with phosphate-buffered saline (PBS). In urine, the calibration curve for I was linear in the range of 10 to 250 ng/ml and the LLQ was 10 ng/ml. The assay was developed to enable pharmacological analysis of I, in on-going phase I and II studies, in patients with solid tumors.     

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.     

Sensitive high-performance liquid chromatographic method for a dopamine receptor agonist,CI-1007, and its metabolite PD 147693 in monkey plasma

A sensitive gradient high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of a dopamine autoreceptor agonist CI-1007 (I) and its metabolite PD 147693 (II) is described. Monkey plasma samples were purified by liquid-liquid extraction using hexane. Liquid chromatographic separation was achieved on two C₁₈ analytical columns (installed in series) using gradient elution. Column effluent was monitored using a fluorescence detector programmed to change wavelengths at specified times. Minimum quantitation limits of I and II were 3.0 and 5.0 ng/ml, respectively, for a plasma sample volume of 0.100 ml. Linearity was demonstrated up to 300 ng/ml. The assay has been applied to the analysis of I and II in plasma from monkeys following intravenous and oral doses of I.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

Simultaneous determination of clofibrate and its active metabolite clofibric acid in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection

A reversed-phase high-performance liquid chromatographic (HPLC) using ultraviolet (UV) absorbance detection method for simultaneous determination of clofibrate (I) and its major metabolite clofibric acid (II) in human plasma has been developed to support a clinical study. I, II and internal standard (I.S., III) are isolated from human plasma by 96-well solid-phase extraction (SPE) C(18)z.ccirf;AR plate and quantified by direct injection of the SPE eluent onto the HPLC with UV detection wavelength at 230 nm. Two chromatographic methods, isocratic and step gradient, have been validated from 1.0 to 100.0 microg/ml and successfully applied to plasma sample analysis for a clinical study. The lower limit of quantitation (LLOQ) is 1.0 microg/ml for both I and II when 500 microl plasma sample is processed. Sample collection and preparation is conducted at 5 degrees C to minimize the hydrolysis of I to II in human plasma.     

Liquid chromatographic method for the determination of rosiglitazone in human plasma

A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid-liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C(18), 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5-1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma

A simplified method for the simultaneous determination of irinotecan (CPT-11, I) and its active metabolite (SN-38, II) in human plasma by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Following the addition of the internal standard (I.S.) camptothecin, the drugs were extracted from plasma using methanol. The average extraction efficiencies were 87% for I, 90% for II and 90% for the I.S. Chromatography was performed using a TSK gel ODS-80Ts column, monitored at 556 nm (excitation wavelength, 380 nm) and the mobile phase was acetonitrile-50 mM disodium hydrogen phosphate (28:72) containing 5 mM heptanesulphonate (pH 3.0). The linear quantitation ranges for I and II were 30–2000 and 1–30 ng/ml, respectively.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

High-performance liquid chromatographic assay for the quantitation of irbesartan (SR 47436/BMS-186295) in human plasma and urine

A selective, accurate, precise and reproducible high-performance liquid chromatographic assay was developed for the quantitation of irbesartan, an angiotensin II antagonist, in human plasma and urine samples. The method involved solid-phase extraction of irbesartan and internal standard (I.S.) using a 100-mg Isolute CN cartridge. A portion of the eluate was injected onto an ODS analytical column connected to a fluorescence detector that was set at an excitation wavelength of 250 nm and an emission wavelength of 371 nm. The mobile phase consisted of 50% acetonitrile and a 50% weak phosphate-triethylamine solution, pH 3.5, at a flow-rate of 0.8 ml/min. The assay was linear from 1 to 1000 ng/ml with both plasma and urine. In either matrix, the lower limit of quantitation was 1 ng/ml. The analyses of quality control samples indicated that the nominal values could be predicted with an accuracy >95%. The inter- and intra-day coefficients of variation for the analyses in both matrices were <8%. Irbesartan was stable in both human plasma and urine for at least seven months at −20°C. The application of the assay to a pharmacokinetic study is described.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Isolation,identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and Rhesus monkey by high-performance liquid chromatography

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by β-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N₄-acetylsulfadiazine (N₄), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 μg/ml (S 0.31, N₄ 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 μg/ml). In urine it varies between 0.6 and 1.1 μg/ml (S 0.75, N₄ 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 μg/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.