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.     

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.     

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography–mass spectrometry

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [²H₄]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C₁₈ cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C₁₈ reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]⁺) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.     

Determination of XR510, a balanced angiotensin II receptor antagonist, in dog and rat plasma by combined liquid-liquid/solid-phase extraction and high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the determination of XR510 (I), a new non-peptide angiotensin II (AII) receptor antagonist with balanced affinity for AT₁ and AT₂ receptor subtypes is described. I and the internal standard, XR513, were extracted from acidified plasma by combined liquid-liquid/solid-phase extraction and chromatographed on a phenyl column with ultraviolet absorbance detection at a wavelength of 272 nm. The mobile phase consisted of a mixture of acetonitrile and sodium phosphate buffer. For both rat and dog plasma, the limit of quantitation was 5 ng/ml. This method has been validated and successfully utilized to investigate the disposition of I.     

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.     

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-μl 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.     

Ultrasensitive determination of NE-100, a novel sigma ligand,in human plasma by liquid chromatography and electrospray ionization tandem mass spectrometry combined with a column-switching technique

For the highly sensitive and selective determination of NE-100, a novel sigma ligand, at levels of low picogram per milliliter of human plasma, a method with excellent reliability employing liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS–MS) combined with a column-switching technique has been developed. The method involves the use of a stable isotope labeled compound as the internal standard (I.S.), liquid–solid extraction of a plasma specimen with a C₈ cartridge, automated on-line clean-up on a short trapping column, subsequent separation on a micro-bore C₁₈ column and detection with ESI-MS–MS using m/z 356 ([M+H]⁺) as a precursor ion and m/z 105 as a product ion in a selected reaction monitoring mode. The detection and the quantification limits of NE-100 in plasma were 0.5 pg/ml with a signal-to-noise ratio (S/N) of 3 and 2.3 pg/ml, respectively, with an S/N of 21. The good linearity of the calibration graph was obtained in the range of 2.3∼907.0 pg/ml with excellent reliability. The developed method was applied to the determination of NE-100 in plasma obtained from the clinical trail.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Determination of olopatadine, a new antiallergic agent, and its metabolites in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and specific assay method has been developed to determine plasma concentrations of olopatadine hydrochloride (A) and its metabolites, M1 (B), M2 (C) and M3 (D), using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Olopatadine, its metabolites, and internal standard, KF11796 (E), were separated from plasma using solid-phase extraction (Bond Elut C₁₈ cartridge). The eluate was dried, reconstituted and injected into the LC–ESI-MS–MS system. The calibration curves showed good linearity over the ranges 1–200 ng/ml for olopatadine and M3, and 2–100 ng/ml for M1 and M2, and the method was thoroughly validated and applied to the determination of olopatadine and its metabolites in plasma collected during Phase I clinical trials. Furthermore, the assay values were compared with those determined by the radioimmunoassay method, which has been routinely used to determine olopatadine in plasma.     

Highly sensitive determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase inhibitor,in rat and rabbit plasma by liquid chromatography and atmospheric pressure chemical ionization-tandem mass spectrometry combined with a column-switching technique

A quantitative bioanalytical method with excellent specificity using liquid chromatography (LC) atmospheric pressure chemical ionization-tandem mass spectrometry (APCI-MS–MS) combined with a column-switching technique has been developed for the highly sensitive and reliable determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rat and rabbit plasma. The method involves protein precipitation of a 25-μl aliquot of plasma sample with eight volumes of methanol containing a deuterium-labeled internal standard, the direct injection of a methanolic supernatant into the analytical instrumentation with no sample evaporation and reconstitution steps, automated on-line clean-up on a C₁₈ short trapping column (10 mm×4.0 mm I.D.) followed by separation on a C₁₈ analytical column (50 mm×4.6 mm I.D.), and detection with APCI-MS–MS using m/z 448 ([M+H]⁺) as a precursor ion and m/z 178 as a product ion in a selected reaction monitoring mode. The lower limit of quantification was 1 ng/ml, and good linearity of the calibration graph was obtained in the range of 1∼490 ng/ml with excellent reliability. The developed method enabled pharmacokinetic profiles to be determined for rats and rabbits with sequential plasma collection from an individual animal.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Simultaneous determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous quantification of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma. The method involved the solid-phase extraction of the five drugs and the internal standard (I.S., verapamil) from 400 μl of human plasma. The HPLC analysis used a reversed-phase C₁₈ analytical column and a mobile phase consisting of a gradient with 15 mM phosphate buffer (pH 5.75)–acetonitrile and UV monitoring. The method was linear over the therapeutic concentration range for the five HIV-protease inhibitors. The accuracy of the method ranged from 98.2 to 106.7% and the precision values ranged from 1.4 to 8.1% for intra-day precision and from 3.1 to 6.4% for the inter-day values.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Determination of Ro 23-7637 in dog plasma by multidimensional ion-exchange—reversed-phase high-performance liquid chromatography with ultraviolet detection

Ro 23-7637 (I) is a new drug under development for the treatment of metabolic diseases. A high-performance liquid chromatographic—ultraviolet detection (HPLC—UV) analytical procedure for its analysis in dog plasma was developed and is reported here. Following C₁₈ solid-phase extraction, the sample is applied to a strong cation-exchange column in the first dimension. The analyte and internal standard, Ro 24-4558 (II), are transferred to a base-deactivated C₁₈ reversed-phase column in the second dimension (orthogonal separation mechanism), with UV detection at 254 nm. The reversed-phase solid-phase extraction provides a gross isolation of the drug, based on hydrophobicity. The first-dimension ion-exchange separation allows neutral species and anions to elute with the column void volume, while separating basic species according to pK_a. The second dimension provides a high-resolution separation dependent upon the hydrophobicity of the sample species. The rationale for using orthogonal multidimensional chromatography was that an exhaustive examination of reversed-phase and normal-phase columns invariably resulted in co-elution of I with endogenous plasma components, which limited the sensitivity of the method. We have utilized C₁₈ solid-phase extraction, followed by multidimensional HPLC—UV, to develop an accurate and precise analytical method whose limit of quantitation, 5 ng/ml using 0.5 ml of plasma, is determined by inherent detector sensitivity. Increased sensitivity can be readily achieved by using more sample or by using microbore HPLC on the second dimension.     

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS) after one single liquid—liquid extraction with chloroform—methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml–5 μg/ml with good accuracy and precision and is used in pharmacokinetic studies.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.