The preferential adsorption coefficient, lambda, of poly(methyl methacrylate), PMMA, in solutions formed by an epoxy resin in tetrahydrofuran (THF), was studied by size-exclusion chromatography (SEC). Only PMMA of lowest molar mass was preferentially solvated by epoxy but at low concentrations of epoxy in the mixture. At higher epoxy content PMMA was preferentially solvated by THF. A simultaneous and competitive solvation between the specific interactions PMMA-epoxy and the self association of epoxy at high concentrations would be the responsible of this inversion point. The more compacted coil of PMMA of higher molecular weights in solution could explain the lack of interaction of these polymers with epoxy. The results also indicated that lambda decreased with the molar mass. This variation has been attributed to the influence of the coil segment density on preferential adsorption.
Ethanol weakens the specific interaction between the human red blood cell (RBC) glucose transporter GLUT1 and the inhibitor cytochalasin B (CB). The chromatographic retention volume of cytochalasin B on stationary phases consisting of GLUT1-containing membranes decreased with increasing ethanol concentration in the eluent. The apparent Kd values for the ethanol-GLUT1 interaction were 0.37, 0.45 and 0.64 M for red blood cells, red blood cell membrane vesicles and proteoliposomes, respectively, all much higher than the Kd values for D-glucose or cytochalasin B interaction with GLUT1. Ethanol also decreased the partitioning of cytochalasin B and drugs into phospholipid bilayers.
At present, mass spectrometry (MS) is the most reliable method for identification but there is not yet a quantitative equation describing this fact. In this investigation an approach to the quantitative assessment of the reliability of identification by MS is proposed which is useful for determination of the selectivity and the validation of analytical methods. Mass spectra of the analytes are presented as maps in which the characteristic ions and their intensities are used for identification. A formula for the quantitative expression of the significance of these parameters to the reliability and the identification is given. The contribution of the resolution of MS instruments or their possibilities of a multiple fragmentation to the reliability of the identification is shown. This approach makes it possible to compare the reliability of identification with different MS instruments. Despite the small contribution of the separation of the chromatographic column compared to the MS separation, the role of the column in the identification is very important to distinguish isomers because their MS spectra are similar.
A post-column derivatization method has been developed for the determination of cisplatin and its monohydrated form. Cisplatin was isolated on a strong anion-exchange column, while a strong cation-exchange column was used for the monohydrated complex. Diethyldithiocarbamate was used as reagent and the influence of temperature, pH and methanol content on the yield of derivative was investigated. The reaction was quantitative using a packed-bed reactor with a surrounding temperature of 115 degrees C and a mobile phase consisting of 0.125 M succinic acid-sodium hydroxide buffer pH 5.2 and methanol (2:3, v/v). The resulting complex, Pt(DDTC)2, was monitored photometrically at 344 nm. The precision of the determination was 11.5% (C.V.) at an injected amount of 20 ng (n = 12) for monoaqua and 8.0% (C.V.) at 9 ng (n = 10) for cisplatin. The method was used to evaluate the plasma concentration of cisplatin and its monohydrated form in a patient.
A high-performance liquid chromatographic method for the sensitive determination of 1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-bisphosphonic acid monohydrate (YM529) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is treated by method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and dissolution of the precipitate in EDTA. Plasma obtained in low-dose clinical studies is treated by method B, a more sensitive method using 4 ml of plasma, which is based on direct precipitation of the drug prior to the deproteinization in method A. Urine and bone samples are prepared by solid-phase extraction using a Sep-Pak C18 cartridge coupled with method A. The drug is separated with a reversed-phase column using a mobile phase at pH 7, and detected with a fluorescence detector following postcolumn alkalization of the mobile phase to enhance fluorescence intensity. The limit of determination is 0.2 ng/ml for method A and 0.05 ng/ml for method B in plasma, 0.05 ng/ml in urine, and 5 ng/g in bone.
A direct high-performance liquid chromatographic assay for the determination of labetalol diastereoisomers in plasma without derivatization was developed. Baseline resolution of diastereoisomers was accomplished on a C18 bonded reversed-phase polymeric column with a basic (pH 11.5) mobile phase and isocratic elution. Sample treatment was optimized in order to achieve a complete extraction of labetalol diastereoisomers and to avoid racemization during extraction. Fluorimetric detection improved the selectivity and afforded a detection limit of 3 ng/ml for each diastereoisomer. This method is suitable for routine quantification of labetalol diastereoisomers and has been applied to a pharmacokinetic study in small laboratory animals.
Ligands with an apparent affinity for various structural elements on the surface of synaptic membrane fragments have been bound to the polymers poly(ethylene glycol) and dextran. The ligand-polymer derivatives have been included in aqueous two-phase systems composed of water, poly(ethylene glycol) and dextran. The uneven distribution of the polymers resulted in the concentration of the polymer-bound ligand in one of the two phases. The effect of the ligand-polymer on the partition of membranes was studied by using synaptic membranes from calf brain, obtained by standard centrifugation methods. By using ligand-containing two-phase systems for nine-step counter-current distribution of membranes, it was shown that the distribution behaviour of various parts of the membrane preparation could be affected. The distribution was followed by determination of opiate binding, acetylcholinesterase, and total membrane (using protein and light-scattering measurements).
A method for the analysis of epoxy polyunsaturated fatty acids (EpPUFAs) and epoxyhydroxy polyunsaturated fatty acids (EpHPUFAs) in rat tissue homogenate, with homo-gamma-linolenic acid (20:3, n - 6), arachidonic acid (20:4, n - 6), eicosapentaenoic acid (20:5, n - 3) or docosahexaenoic acid (22:6, n - 3) as a substrate, has been developed. Extraction with dichloromethane at pH 4-5 and concentration in the presence of pyridine were performed. Spectral analysis of chromatograms obtained with high-performance liquid chromatography-thermospray mass spectrometry showed the presence of EpPUFAs, EpHPUFAs and dihydroxy metabolites (DiHPUFAs) of EpPUFAs corresponding to each precursor fatty acid. On a selected-ion monitoring chromatogram, many EpPUFAs, EpHPUFAs and DiHPUFAs in an extract from an incubation mixture of each precursor fatty acid in aged rat tissue homogenate were detected simultaneously within 70 min. EpPUFAs and DiHPUFAs derived from 20:3 (n - 6) or 20:5 (n - 3) were detected in significant amounts. From these results, a highly active cytochrome P450 system or non-enzymic oxidative reactions in aged rat tissue homogenate were suggested.
A rapid and sensitive method for the determination of five different alkylphosphocholines including the antineoplastic phospholipid analogues hexadecylphosphocholine and octadecylphosphocholine is presented. The method is based on the separation of the lipids by high-performance liquid chromatography and quantitation by light-scattering mass detection. The lower limit of detection is approximately 50 pmol for each alkylphosphocholine tested. Quantitation is linear over the range 0.05–75 nmol. Hexane—isopropanol extracts of cultured cells can be applied to the column without further cleanup. The high resolution of separation and the sensitivity of detection render this method useful for pharmacokinetic investigations dealing with the uptake of alkylphosphocholines into different types of cells.
A gas chromatographic—mass spectrometric (GC—MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-(tert.-butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.
A gradient reversed-phase HPLC analysis for the direct measurement of salicylic acid (SA) with the corresponding glycine and glucuronide conjugates in plasma and urine of humans was developed. The glucuronides were isolated by preparative HPLC from human urine samples. The concentration of the glucuronides in the isolated fraction were determined after enzymatic hydrolysis. Salicylic acid acyl glucuronide (SAAG) was not present in plasma. No isoglucuronides were present in acidic and alkaline urine of the volunteer. The limits of quantitation in plasma are: SA 0.2 microgram/ml, salicyluric acid (SU) 0.1 microgram/ml, salicylic acid phenolic glucuronide (SAPG) 0.4 microgram/ml and salicyluric acid phenolic glucuronide (SUPG) 0.2 microgram/ml. The limit of quantitation in urine is for all compounds 5 micrograms/ml. Salicylic acid acyl glucuronide is stable in phosphate buffer pH 4.9 during 8 h at 37 degrees C; thereafter it declines to 80% after 24 h. The subject's urine was therefore acidified by the oral intake of 4 x 1.2 g of ammonium chloride/day. With acidic urine, hardly any salicylic acid is excreted unchanged (0.6%). It is predominantly excreted as salicyluric acid (68.7%).
A high-performance liquid chromatographic method was developed for the simultaneous determination of the enantiomers of flosequinan [(+/-)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone] and its metabolites, flosequinan sulphide and sulphone, in human plasma. These compounds were extracted from plasma with chloroform. The compounds were separated on a chiral stationary phase of cellulose tris-3,5- dimethylphenylcarbamate coated on silica gel, with a mobile phase of ethanol-methanol (22:78, v/v). Flosequinan enantiomers and flosequinan sulphone were determined by UV detection at a wavelength of 320 nm. Flosequinan sulphide was determined using fluorescence detection (excitation at 370 nm, emission at 430 nm). Standard curves were linear over the concentration range 5-10,000 ng/ml for both enantiomers and flosequinan sulphide, and 20-10,000 ng/ml for flosequinan sulphone. This method is adequate for pharmacokinetic studies of the enantiomers of flosequinan and its metabolites.
A sensitive, stereoselective and rapid reversed-phase liquid chromatographic method for the determination of (SR)- and (RS)-mefloquine enantiomers in 100 microliters plasma and capillary blood collected on chromatographic paper is presented. The assay involves protein precipitation from plasma, liquid-liquid extraction of mefloquine from plasma, capillary blood with methyl tert.-butyl ether under alkaline conditions and derivatization of MQ with (-)-1-(9-fluorenyl)ethyl chloroformate. Liquid chromatographic separation of the diastereomers was performed using an C18 reversed-phase column with acetonitrile-water-acetic acid 82:18:0.07 (v/v/v) as the mobile phase, and a flow-rate of 1.0 ml/min. When using 100 microliters of plasma the limit of determination is 250 nmol/l with ultraviolet- and 10 nmol/l with fluorescence detection. The present method offers several advantages over those previously reported; very low limit of determination, small sample volume, sampling onto paper and use of an inexpensive standard achiral HPLC column. No racemization during the derivatization procedure or storage of the MQ enantiomers was found.
A rapid and efficient isocratic high-performance liquid chromatographic assay for the measurement of atovaquone in plasma has been developed and validated. The drug was extracted from plasma with organic solvents, assayed on a C1 column with a mobile phase of methanol-0.1% acetic acid (70:30, v/v), and detected by ultraviolet absorbance at 254 nm. Recovery of atovaquone from plasma was greater than 85%. Intra- and inter-assay variability were less than 8%, and the average accuracy of the assay (expressed as % bias) ranged from -7.4 to + 2.2%. The upper and lower limits of quantitation were 100 and 0.25 microgram/ml, respectively. Measurement of atovaquone in spiked plasma control samples during routine runs of clinical trial samples confirmed the reliability of the assay.
A simple and specific reversed-phase high-performance liquid chromatographic (HPLC) assay for the determination of novobiocin levels in human plasma has been developed. The sample preparation was performed by deproteinization with methanol. Prednisone was used as an internal standard. Both novobiocin and prednisone were separated on a C8 column with a gradient elution of acidic water (pH 3.0)—methanol. The recovery of novobiocin from plasma was nearly complete. The linear range was 5–1000 μM in 0.5 ml of plasma with a minimum limit of determination of 2.25 fmol of novobiocin at 254 nm. The method has been implemented and validated in an ongoing clinical trial.
A sensitive and selective gas chromatographic method for the determination of the calcium antagonist SIM6080 in plasma has been developed and validated. A three-step extraction procedure is employed followed by capillary gas chromatographic analysis using nitrogen-selective detection and the programmed temperature vaporizer injection technique. The defluorinated analogue was used as the internal standard. The analysis of spiked plasma demonstrated the good accuracy and precision of the method with limit of detection of 1 ng/ml. The method has been used for pharmacokinetic studies in laboratory animals.
Determination of amino acids in pig plasma with the classical ninhydrin system is influenced by the excessive amount of protein and lipophilic compounds in the sample, leading to a decline in resolution. This problem was eliminated by using 80 mg of sulphosalicylic acid per ml of plasma, and solid-phase extraction with a C18 cartridge as an additional clean up step. The latter resulted in significantly higher quantities of threonine, asparagine, glutamic acid, glutamine, glycine, alanine, valine and lysine, and lower levels of phenylalanine and tryptophan (P < 0.05). The use of a C18 cartridge had a minor effect on the analytical error.
A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids (ca. 200 μg). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U-14C]glycerol or [U-14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.
In an isotope dilution assay, prostaglandin (PG) E2, 6-keto-PGF1α, thromboxane (Tx) B2 and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF1α, 2,3-dinor-TxB2 and 11-dehydro-TxB2 were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.
An automated technique involving switching valves and a filter assembly has been developed and evaluated for the on-line precipitation of proteins and peptides from plasma samples. In the set-up, the proteins were precipitated on-line by injecting the plasma sample into a stream of organic precipitating agent. The precipitates so formed are filtered on-line by a set of filter assemblies consisting of ordinary in-line HPLC solvent filters. Evaluation of the technique was performed using ibuprofen and a mixture of three estrogens, estradiol, equilin and estrone, spiked in dog plasma. The coefficients of variation (C.V.) for system suitability parameters were below 10%. Absolute recovery of ibuprofen in plasma ranged from 80% for 100 micrograms/ml to 114% for 5 micrograms/ml spiked concentrations, respectively. Resolution for equilin and estrone, two closely eluting peaks, was 1.79 (C.V. = 5.8%, n = 7). The switching-valve--filter assembly had no significant effect on the efficiency of the HPLC system.
