Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography–tandem mass spectrometry

A method was developed and validated for the quantification of (±)-trans-[2-morpholino-1-(1-naphthaleneethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5–3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

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.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C₁₈ column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

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.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Improved high-performance liquid chromatography analysis of P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification

An improved HPLC-based ³²P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C₁₈ precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10⁷ bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10⁴ to ≈ 2±1 adducts per 10⁹ bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3′-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.     

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).     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

Determination of 2-n-propylquinoline in mouse plasma and liver by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the specific determination of 2-n-propylquinoline, a new anti-leishmaniasis drug, in plasma and liver homogenates of mice. 2-n-Propylquinoline was extracted with methyl-tert.-butyl ether with quinoline as internal standard. Separation was carried out using a Nucleosil C₁₈ column. The mobile phase consisted of methanol–0.005 M ammonium acetate buffer (60:40) at pH 5.5 and 8 for plasma and liver homogenates, respectively. Detection was monitored at 233 nm. The method was validated and shown to be accurate and precise for plasma and liver homogenates. Extraction yield was 96% in plasma and 81% in liver homogenates. This method was used to determine the pharmacokinetic profile of 2-n-propylquinoline following oral administration to mice.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

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.     

Measurement of synthetic phenolic antioxidants in human tissues by high-performance liquid chromatography with coulometric electrochemical detection

The antioxidants, 2-tert.-butyl-4-methoxyphenol (BHA) and its oxidative peroxidation product 2,2′-dihydroxy-3,3′-di-tert.-butyl-5,5′-dimethoxybiphenyl (di-BHA), 3,5-di-tert.-butyl-4-hydroxytoluene (BHT) and propyl gallate, were measured in plasma and tissue homogenates by HPLC and electrochemical detection, with a sensitivity down to 0.2 (BHA), 0.1 (di-BHA), 0.4 (BHT) and 1 (propyl gallate) ng ml⁻¹ of plasma or tissue homogenate. The data demonstrate that in man, at the current level of exposure to dietary antioxidants, significant amounts of BHA, BHT and propyl gallate are accumulated in the omentum. Furthermore, they provide the first evidence that the peroxidase-catalysed oxidation of BHA is operative in man.     

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4′-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4′-hydrazino-2-stilbazole (4′H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis–trans isomerism at the proline–pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4′H2S is performed in a water–acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2–100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4′H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.     

The beneficial role of different auxins and polyamines at successive stages of somatic embryo formation and development of Panax ginseng in vitro

The production of viable plantlets via somatic embryogenesis in Panax ginseng requires different culture media corresponding to successive developmental stages. The effects of several auxins and polyamines have been tested at various steps. Multiplication of the embryogenic root-derived callus has been optimized on half-strength MS medium supplemented with 3-(benzo[b]selenyl)acetic acid (BSAA, a synthetic auxin) and kinetin; exogenously applied polyamines were deleterious at this stage, causing browning of the callus, diminished capacity of embryo initiation, and an increased tendency to hyperhydricity. BSAA again appeared to be the most favourable auxin at the initiation stage, but here its action was reinforced by the presence of polyamines, spermidine being the most efficient. Among the auxin needed at the next step, i.e., for the regeneration of embryos, the two seleniated auxins BSAA and seleniated 2,4-dichlorophenoxyacetic acid (2,4-D-Se) were the more efficient. For the harmonious development of plantlets, i.e., the simultaneous outgrowth of shoots and roots, the polyamines were favourable, with a greater efficiency for spermine.     

Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1–85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2±7.9, 74.9±8.5, 81.7±6.9, 73.1±7.8, 74.3±8.3, 80.8±6.6, 81.6±7.3 and 81.4±6.5, and from urine 79.4±6.9, 77.8±8.4, 83.3±6.6, 72.8±9.0, 76.3±7.7, 83.4±7.9, 81.6±7.9 and 81.8±6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Analysis of N,N-diethyl-m-toluamide in porcine skin perfusates using solid-phase extraction disks and reversed-phase high-performance liquid chromatography

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5-1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 micro g/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET-ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55 x 10(-5)+/-0.54 x 10(-5) cm/h.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.