Determination of risperidone and 9-hydroxyrisperidone in plasma, urine and animal tissues by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of the new anti-psychotic risperidone and its major metabolite 9-hydroxyrisperidone in plasma, urine and animal tissues. The alkalinized plasma samples were extracted with ethyl acetate and further purified prior to reversed-phase chromatography with ultraviolet detection at 280 nm. The method could also be applied to urine samples and animal tissue homogenates. Quantification limits were 2 ng/ml for plasma and urine and 10 ng/g for animal tissue. The method was applied to pharmacokinetic studies in experimental animals, human volunteers and patients.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

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.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Assay of paclitaxel (Taxol) in plasma and urine by high-performance liquid chromatography

A new, rapid and sensitive high-performance liquid chromatographic method for the analysis of paclitaxel (Taxol) in human plasma and urine was developed and validated. After addition of an internal standard, paclitaxel was extracted from plasma or urine by a liquid–liquid extraction using diethyl ether. Extraction efficiency averaged 90%. Chromatography was performed isocratically on a reversed-phase column monitored at 227 nm. Retention times were 7.7 and 6.7 min for paclitaxel and docetaxel, respectively, and the assay was linear in the range 25–1000 ng/ml. The limits of quantification for paclitaxel were 25 and 40 ng/ml in plasma and urine, respectively. The assay was shown to be suitable for pharmacokinetic studies of children involved in a phase I clinical trial.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues.The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method.Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.     

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C₁₈ columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.     

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.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025–25 and 2–150 μg/ml in plasma and urine, respectively. An aliquot of 200 μl of plasma was extracted with solid-phase extraction using Oasis^® cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 μl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

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.     

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane—2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5–100 ng/ml and 25–1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.     

Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography

A coupled achiral-chiral liquid chromatographic assay has been developed to determine the concentrations of metyrapone and the enantiomers of its chiral metabolite metyrapol in plasma and urine. The chromatographic system consisted of a silica precolumn (75 × 4.6 mm I.D.) coupled in-line to a 250 × 4.6 mm I.D. column containing cellulose tris(4-methylbenzoate) coated on silica gel (Chiralcel OJ-CSP). When plasma samples were analyzed, the mobile phase was hexane-ethanol (92:8, v/v) modified with 0.1% diethylamine and when urine samples were analyzed the mobile phase was hexane-ethanol (94:6, v/v) modified with 0.2% diethylamine. Under these chromatographic conditions the chromatographic retentions [expressed as capacity factors (k′)] for metyrapone were k′ = 2.35 (plasma) and 2.52 (urine); for (−)-metyrapol k′ = 4.22 (plasma) and 4.62 (urine); for (+)-metyrapone k′ = 5.16 (plasma) and 5.86 (urine); enantioselectivities (α) were 1.09 (plasma) and 1.13 (urine). The assay has been validated for use in metabolic studies. The analyses of plasma and urine samples from one subject following oral administration of 750 mg of metyrapone indicated that the enzymatic reduction of myterapone by aldo-keto reductase was enantiospecific.     

High-performance liquid chromatographic assay for the measurement of the novel microtubule inhibitor 1069C85 in biological tissues and fluids

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation <5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 ± 2.2 h. The drug remained detectable in plasma, at 1 ± 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.     

Liquid chromatographic analysis of penem antibiotic FCE22101 in biological fluids with a liquid-liquid extraction procedure

A sensitive high-performance liquid chromatographic method has been developed for the determination of penem antibiotic FCE22101 in plasma and urine. FCE22101 was extracted from plasma and urine with ethyl acetate. After evaporating, the sample solutions were analyzed by a reversed-phase high-performance liquid chromatographic system using a two-sided bracketing injection technique. The determination limit of FCE22101 was 5 ng/ml in plasma. Analysis of the spiked plasma samples demonstrated the good accuracy and precision of the method. The proposed method improved from five- to ten-fold on the analytical sensitivity in comparison with the most commonly ultrafiltration method.     

High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm x 150 mm, 4 microm) protected by a Nova-Pak C18 guard column (3.9 mm x 20 mm, 4 microm), where 10 mM KH(2)PO(4)-methanol-triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 microl mouse plasma. The assay was shown to be linear over a concentration range of 1-500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT.