Compounds In Human Plasma Research Articles

Synthesis and biological evaluation of novel A-ring modified hexacyclic camptothecin analogues.

Eleven A-ring modified hexacyclic analogues of camptothecin (CPT) containing a 1,4-oxazine ring were synthesized from 10-hydroxycamptothecin (11a) and 7-ethyl-10-hydroxycamptothecin (3) (SN-38) in four to five steps and were subjected to the biological tests such as cytotoxicity, topoisomerase I (Topo I) inhibitory activity, acetylcholinesterase (AChE) inhibition, and stability in human plasma. Four compounds 15a, 15b, 16a, and 16c were about 2-fold more potent than topotecan and as potent as CPT toward human cancer cell lines A549, H128, WiDr, MKN45, SK-OV-3, and SK-BR-3 in vitro, even though the most active compound 15b was slightly less potent than SN-38. The potency of Topo I inhibition of these compounds showed relatively good correlation with their cytotoxicity. Most of the compounds exhibited AChE inhibitory activity weaker (9 +/- 2 to 20 +/- 3%) than CPT (23 +/- 5%) or topotecan (20 +/- 4%) and similar to SN-38 (13 +/- 2%), indicating that they might have little effect on causing early diarrhea. The stability of lactone forms of these compounds in human plasma seemed to be much higher than that of CPT and similar to that of topotecan but lower than that of SN-38. Among the new hexacyclic CPT analogues, compound 15b showed higher antitumor activity against human tumor xenograft, WiDr, in nude mice compared to that of SN-38. The most promising compound 15b has been selected for further development.

Determination of a sulfur-containing drug in human plasma by an improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography.

An improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography was established. The method was applied to the analysis of a sulfur-containing antiasthma drug, S-1452, and its nine metabolities in human plasma. The high selectivity and sensitivity of the method allowed accurate quantitation of trace levels of these compounds in human plasma with no interferences from the biological components present. To enable stable measurements and maintain reproducibility over a long period, the performance characteristics of a commercially available instrument were investigated. The gas seal in the injection port was found to easily corrode through interaction of the sulfur analyte with the metal gas seal. To prevent this, a disk made from an alloy of platinum and gold (60:40) was mounted on the gas seal. The reproducibility of the measurement was improved remarkably by using the disk. The use of compressed air of high purity significantly lowered the signal-to-noise ratio. The optical filter was kept clean by using a nickel catalyst to trap ozone in place of copper manganese oxide (CuMn2O4). These improvements raised the sensitivity and selectivity with the lower quantitation limits of 0.5-1.0 ng/mL in human plasma.

Development of a sensitive and quantitative analytical method for 1 H-4-substituted imidazole histamine H 3-receptor antagonists utilizing high-performance liquid chromatography and dabsyl derivatization

A sensitive and versatile analytical method utilizing high-performance liquid chromatography (HPLC) and precolumn derivatization of 1 H-4-substituted imidazole compounds is described. A HPLC method using 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) and ultraviolet (UV) detection was developed for the analysis of histamine (HA) H 3-selective compounds in human plasma, rat plasma, or homogenized rat cortical tissue. The average intra- and inter-assay variability, over a range of 10 to 0.01 μg/ml, was determined to be acceptable. The lower limit of detection for the dabsylated ligands was estimated to be <1.0 ng/ml while the lower limit of quantitation (LLOQ) was determined to be 10 ng/ml of conjugate. This assay has demonstrated it's suitability for the sensitive quantitation of several structurally diverse 1 H-4-substituted imidazole HA H 3-receptor antagonists in biological matrices for pharmacokinetic and biodistribution studies.

Quantitative electrospray LC–MS and LC–MS/MS in biomedicine

The use of electrospray LC–MS and LC–MS/MS for the quantitative determination of two low molecular weight (<500 Da) organic compounds in human plasma (Lovastatin) and cell supernatants (Arachidonic acid) and medium molecular weight (>2000 Da) endogenous peptides (Endothelins) in supernatants of human umbilical vein endothelial cell cultures is reported. These methods make use either of deuterium labelled or structurally similar molecules as internal standards for quantitation and one or more pre-purification steps previous the LC–MS analysis. Linear calibration curves and detection limits around 50 pg ml −1 were obtained in all three cases.

High performance liquid chromatography mobile phase composition optimization for the quantitative determination of a carboxylic acid compound in human plasma by negative ion electrospray high performance liquid chromatography tandem mass spectrometry.

A systematic investigation was undertaken to study the effects of varying concentrations of additives in the acetonitrile/water high performance liquid chromatography mobile phase, especially formic acid and ammonium formate, on the negative ion electrospray response of a carboxylic acid compound. The study showed that the response progressively decreased with increase in the formic acid concentration. While such a decrease in the response could be qualitatively explained by the decrease in the concentration of the ionized form of the carboxylic acid compound due to the lower pH of the mobile phase, the change in response was not as large as expected from the change of the concentration of the ionized form. The response also progressively decreased with increase in the ammonium formate concentration but the decrease cannot be explained by the change in the pH of the mobile phase. Although the best negative ion electrospray response was obtained with a water/acetonitrile mobile phase that contained no additives at all, the retention time of the analyte was not found to be adequately reproducible on repeated injections. Thus, this mobile phase was deemed unacceptable for practical, routine use. Comparing formic acid against ammonium formate, the former was preferable since it caused a smaller attenuation of the negative ion response. Equally important was the fact that addition of formic acid had the desirable effect of maintaining a reasonably high capacity factor (k') for the analyte even at a relatively high acetonitrile concentration. A concentration of 1 mM formic acid in the mobile phase was large enough to achieve the reproducible elongated retention time for the analyte, with a loss in the analyte response of about 60% only. It should be noted that the sensitivity achieved with the 1 mM formic acid mobile phase, in which the carboxylic acid is expected to be about 10% in the ionized form, is about 9 times better than the sensitivity achieved in the 1 mM ammonium formate mobile phase, in which the carboxylic acid is expected to be about 99% in the ionized form.

Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive-minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)-and has a working range (between-assay CV < 10%) of 180-10000 pmol/L (54-3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55-168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.

Automated high-performance liquid chromatographic method for the analysis of two novel ergoline compounds in human plasma.

A rapid and sensitive high-performance liquid chromatographic method for the determination of the novel ergoline derivatives sergolexole (compound I), its acid metabolite (compound II) and cis-n-(2-hydroxycyclopentyl)-6-methyl-1-(1-methylethyl)ergoline-8- carboxamide (LY215840, compound III) in human plasma is reported. The compounds were extracted from plasma by automated solid-phase extraction and analysed on a reversed-phase C8 column with fluorescence detection. The limit of quantification for all compounds was 10 ng/ml and the response was linear over the range 10-1000 ng/ml. Validation studies showed the method to be both repeatable and reproducible with no interference from human plasma. The method has been used to support pharmacokinetic studies and has proved to be robust and effective.

Determination of the renin inhibitor Ro 42-5892 in human plasma by automated pre-column derivatization, reversed-phase high-performance liquid chromatographic separation and electrochemical detection after post-column irradiation.

The renin inhibitor Ro 42-5892 has been found to be very potent, thereby necessitating a sensitive assay method for the evaluation of its pharmacokinetics in man. We report here the development of a very sensitive and selective HPLC assay for the analysis of this compound in human plasma. Ro 42-5892 was extracted from plasma with dichloromethane, derivatized with 2,4-dinitrofluorobenzene and then chromatographed on a Novapak C18 column (150 x 3.9 mm I.D.) with acetic acid buffer (pH 7)-acetonitrile (100:85). Detection was performed by irradiation at 254 nm, followed by electrochemical oxidation at 550 mV. The extraction recovery of Ro 42-5892 from human plasma (mean 102%) was quantitative. With this method a limit of quantitation of 0.3 ng/ml was achieved. The assay was linear up to 5 ng/ml, had acceptable inter-assay precision (12.2%) and accuracy (9.3%) and was successfully tested for selectivity. This assay was successfully applied to over 250 samples from a pharmacokinetic study in hypertensive patients.

Synthesis and anti-human immunodeficiency virus type 1 activities of new peptido-nucleoside analogues

In order to investigate whether antiproteasic peptides coupled to anti-reverse transcriptase nucleosides can act as inhibitors at the different stages of the HIV life cycle, various peptido-nucleosides were synthesized using methodologies involving (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as a coupling reagent between the N4-cytosinyl moiety and the peptide carboxy terminus. The anti-HIV-1 activity in MT4 cells of this new class of compounds and their anti-HIV protease activities were determined. Fourteen peptido-nucleosides have been synthesized and six act against both the HIV-protease and viral replication in vitro. Although the activity of the most potent compounds against HIV was found to be one order of magnitude lower than that of the parent nucleoside drug 2′,3′-dideoxy-3′-thiacytidine, this new class of compound could be of biological interest. Indeed, since the in vitro half-lives (t1/2) of the hydrolysis of the most potent compounds in human plasma were found to be longer than 2.5 h, these analogues could reach the infected cells in their structural integrity. This observation does not exclude that these compounds may exert their antiviral effects as combined prodrugs through extracellular or intracellular hydrolysis.

Determination of sulbutiamine and its disulfide derivatives in human plasma by HPLC using on-line post-column reactors and fluorimetric detection

A new direct HPLC procedure for the simultaneous determination of sulbutiamine (Arcalion) and other thiamine disulfides in human plasma has been developed. The method involves an automated solidphase extraction on octadecylsilyl (C18) cartridges and chromatographic separation of the compounds on an RP Select B column with gradient elution using methanol and phosphate buffer. Detection was by fluorescence of the resulting thiochromes obtained from two on-line post-column reactors. Optimization of post-column reaction parameters has been achieved. This method has been proved to be highly selective for the determination of the thiamine disulfide derivatives and quantitation limits of 5 ng·ml−1 were obtained for each compound in human plasma. Linearity was in the range 5–200 ng·ml−1. Precision and accuracy were also demonstrated by within-day and between-day assays, and showed the good reliability of the method.

Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland

P-glycroprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [ 3H]vincristine in MDR cells. Two majo compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5β-metabolic pathway but none in 5α-metabolic pathway were able to enhance the accumulation of [ 3H]vincristine in MDR cells and to inhibit [ 3H]azidopien photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to recognized by P-gp.

Determination of S-1006 and related compounds in human plasma and urine by high-performance liquid chromatography

Determination of S-1006 and related compounds in human plasma and urine by high-performance liquid chromatography

Acyclic Adenine Nucleoside Phosphonates in Plasma Determined by High-Performance Liquid Chromatography with Fluorescence Detection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and (R,S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) are selective antiviral agents with activity against a broad spectrum of DNA viruses and retroviruses. A highly sensitive method has been developed to determine concentrations of these compounds in human plasma. Plasma samples containing PMEA, HPMPA, or FPMPA were treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivatives. Chromatographic separation combined with excitation at 254 nm and fluorescence detection at 425 nm allowed quantification of PMEA, HPMPA, and FPMPA within a concentration range of 0.25-4000 mumol/L. This method proved useful in accurately determining low PMEA concentrations in sera from PMEA-treated monkeys and cats. The assay should be applicable to the quantification of PMEA, HPMPA, and FPMPA in plasma and urine of humans treated with any of these drugs.

Determination of pentoxifylline and its metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction

A simple and reliable method for the determination of pentoxifylline and its main metabolites in human plasma has been developed using high-performance liquid chromatography. After selective solid-phase extraction, pentoxifylline, its metabolites and an internal standard, 7-(2'-chloroethyl)theophylline, were separated on a 5-μm LiChrospher 100 RP-18 column using water—dioxan—acetonitrile (87:6.5:6.5, v/v/v) acidified with acetic acid (0.5%, v/v) as the mobile phase. The analytes were detected at 275 nm. The lowest detectable concentration for all analytes was 25 ng/ml; the recovery was 85%. The assay has been successfully applied to analysis of these compounds in human plasma after administration of an oral dose of 400 mg of pentoxifylline to healthy volunteers.

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH 4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 μ m dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH 4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (<2 pmol) to detect the various forms of the four thiol compounds in human plasma. plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.

Dialysis as an on-line sample-pretreatment technique for column liquid chromatography: influence of experimental variables upon the determination of benzodiazepines in human plasma.

An evaluation is provided of dialysis, coupled on-line to column liquid chromatography, as a sample pretreatment procedure for macromolecule-containing biological samples. The influence of parameters such as acceptor phase flow rate, temperature, hydrophobicity of the analytes, pH, ionic strength and viscosity of the sample on the recovery and rate of dialysis is studied. In addition, methods to reduce the degree of drug-protein binding and thereby improve the recovery are reported. Diazepam, nitrazepam and oxazepam are used as model compounds. A method is reported for the fully automated determination of these compounds in human plasma using only 100 microliters of sample. Data on repeatability, linearity and detectability are given.

Vitamin A supplementation increases levels of retinoic acid compounds in human plasma: possible implications for teratogenesis

The concentrations of retinoic acid compounds were monitored by a newly developed highly sensitive HPLC procedure in plasma of six volunteers who received 833 IU vitamin A per kg body weight per day during a 20-day period. There was a significant increase of all-trans-retinoic acid (two-fold), 13-cis-retinoic acid (7-fold) and 13-cis-4-oxoretinoic acid (5-fold) over endogenous plasma levels of these retinoids. The same compounds had previously been found after treatment with the teratogenic drug isotretinoin (Roaccutan, Accutane). Our results raise the possibility that high vitamin A intake may carry a teratogenic risk attributable to increased levels of retinoic acid compounds generated from retinol by metabolic processes.

Fluorogenic reaction between adenine derivatives and chloroacetaldehyde and its application to the determination of 9-(2-chloro-6-fluorobenzyl)adenine in human plasma

A sensitive (1 ng ml −1) liquid chromatographic method with fluorescence detection is described for the determination of arprinocid and analogous compounds in human plasma. The method is based on chemical derivatization with chloroacetaldehyde to form highly fluorescent derivatives. Extraction of the drug from plasma and separation of the derivative from the reaction mixture are accomplished by solid-phase extraction with two silica cartridges. The effects of pH, solvent, and concentration of the reagents on the efficiency of derivatization were studied. The assay in plasma was validated in the 1–50 ng ml −1 range. The fluorescent derivatives were synthesized and fully characterized. The derivatives were also found to be efficient as energy acceptors in the oxalate ester/hydrogen peroxide chemiluminescent system.

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm×4.6 mm, 5 μm particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5–200 ng/ml for plasma and 0.1–10 μg/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.

Analysis of vinca alkaloids in plasma and urine using high-performance liquid chromatography with electrochemical detection

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.