C8-diol mixed phases for two-dimensional T.L.C. were prepared by mechanical mixing of silica-based bonded phases. Their properties in terms of retention and mechanisms developed were determined using polynuclear aromatic hydrocarbons as the compounds analyzed. Comparisons carried out with C18-cyano mixed phases revealed both similar behaviors and appreciably different behaviors, which we have explained.
Three major metabolites of rapamycin (M2, M3, and M5) were isolated from pooled plasma of orally dosed rats. Metabolites were extracted from the plasma with ethyl acetate/methanol prior to isolation by HPLC using a Supelcosil SPLC-18, 5 mu m, IO x 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M5 is O-demethylated on the C41 methoxy moiety and the macrolide ring is intact.
High Performance Liquid Chromatography(HPLC) and Capillary Electrophoresis (CE) are valuable analytical techniques for the determination of neuropeptides in biological fluids. A crucial aspect of HPLC and CE analyses is the preparation of samples. This review outlines sample preparation protocols for HPLC and CE methods that have been used for this determination, with a focus on enkephalins. Sample preparation protocols have ranged from simple techniques of extraction and centrifugation to solid phase extraction (SPE) and ultrafiltration. Additionally, this review also discusses the final determinant steps of HPLC and CE using various modes of detection. Detection modes for the HPLC methods included ultraviolet (UV), fluorometry, electrochemical and mass spectrometry (MS). Modes of detection when using CE for this determination have been more limited, focusing on UV and MS detection modes.
The HPLC determinations of cocaine and its main metabolites are critically reviewed with special attention to the matrices analysed. A comparison of the different extraction methods is given and the chromatography, internal standardization and detection modes are discussed.
9-Methylamino-methylanthracene (MAMA) is a secondary amine compound which can be used for derivatization and quantitation of isocyanate compounds by HPLC with detection by fluorescence and ultra-violet light absorbance The compound has potential as a chemical label for determination of airborne total reactive isocyanate group (TRIG) compounds arising from partially polymerized polyurethanes and thermal decomposition products of fully cured polyurethanes Six isocyanate oligomers and polyurethane precursors based on methylene-bis-(phenylisocyanate) (MDI), 1,6-hexamethylene-diisocyanate (HDI), and 2,4- and 2,6-toluenediiosocyanate (TDI) were assayed for isocyanate content using MAMA reagent After reaction of isocyanate with MAMA, the urea derivatives were analyzed by reversed phase HPLC using three detection / quantitation modes: fluorescence with excitation at 245 nm and emission at 414 nm, UV absorbance at 245 nm and UV absorbance at 370 nm. The ratio of absorbance at 245 nm and at 370 nm identified multiple peaks originating from isocyanate-containing compounds in each sample. The total amount of TRIG in each sample was then quantitated by absorbance of these peaks at both of the UV wavelengths. For the test samples, observed recoveries of TRIG by MAMA-HPLC assay ranged from 96 to 105%, in comparison to a reference titration assay. The technique was also evaluated for chemical interference from phosgene gas. A single compound was found after reaction of MAMA with phosgene which responded as two equivalents of TRIG.
A method is described to isolate a mixture of taxanes from cell suspension cultures of Taxus species. The aqueous suspension medium is pre-filtered and centrifuged to remove cellular debris, and then passed through either nylon or PVDF membranes. Contaminants are washed from the membranes and the taxanes are eluted with appropriate solvents. This method provides a rapid, efficient, and inexpensive means of extracting taxanes from cell suspension medium, as well as a significant reduction in the total volume of solvents used.
Macroporous and microparticulate poly(hydroxyethyl methacrylate) gel (HEMA) and HEMA containing pores filled with a dextran network were used as supports for immobilization of reactive dye Cibacron Blue 3G-A. The performance of both dextran-filled and non-filled HEMA sorbents, with various ligand densities, in dye-ligand affinity chromatography of several enzymes and of bovine albumin were studied. The obtained results show the positive effect of dextran filling on enzyme recovery and on binding capacity of the separation media.
Radial flow (RF) column configurations have been developed for larger scale production liquid chromatography, with the primary purpose of increasing throughput rates and decreasing trans-bed pressure drops in comparison to conventional axial flow (AF) columns. The RF columns have been quite successful in attaining these two objectives. In this work, we investigated the nature of protein band dispersion in both axial and radial flow chromatography column configurations, utilizing S-200 Sephacryl gel filtration media with bovine serum albumin as the applied protein. The effects of input feed flow rate as well as input feed albumin concentration on the nature of protein band dispersion were studied. Protein band dispersion was quantified by examining the shape of the eluting protein peak and measuring peak height-to-width (HTW) ratio's for the various flow rates and feed concentrations used in the study. Our results indicate that protein dispersion was larger in RF columns as indicated by smaller peak HTW ratio's than those obtained for AF columns, operated at the same flow rates and feed protein concentrations. We also found that moderately increasing the flow rate increased peak HTW ratio's and resulted in sharper peaks for both AF and RF columns.
Sulfated beta-cyclodextrin (SO3-beta-CD) was utilized as a chiral additive for capillary zone electrophoresis (CZE). Chiral separations of several uhcharged enantiomers, such as phensuximide, indapamide, etc., which are difficult to separate using neutral CDs, were achieved. The effects of SO3-beta-CD concentration and the pH and ionic strength of the supporting electrolyte as well as the presence of an organic modifier, methanol, were discussed.
5,6-Dimethoxy-2-(4-hydrazinocarbonylphenyl)benzothiazole (BHBT-hydrazide) was found to be a highly sensitive and stable fluorescence derivatization reagent for carboxylic acids in liquid chromatography. Its reactivity was investigated for various C-18:0 - C-22:6 saturated and unsaturated fatty acids. The reagent readily reacted with the fatty acids in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37 degrees C to produce the corresponding fluorescent derivatives. The derivatives were separated on a reversed-phase column, TSK-gel ODS 120T, with gradient elution using 40-100% (v/v) aqueous acetonitrile, and were detected spectrofluorimetrically at 447 nm with excitation of 365 nm. Calibration curves were linear over the range 10 fmol - 5.0 pmol per 20-mu l injection (r=0.992 - 0.999). The relative standard deviations for ten replicated determinations did not exceed 2.0% for any of the fatty acids (1.0 nmol/ml). The detection limits (signal-to-noise ratio=3) for the acids were 1 - 2 fmol for an injection volumn of 20 mu l. Further, the BHBT derivative of stearic acid was synthesized to examine the fluorescence properties.
Approximate analytical equations were developed for the products of consecutive reactions occurring on a chromatographic column. The equations were tested both by numerically evaluated chromatograms and the acid catalyzed hydrolysis of phthalic dichloride in a liquid chromatographic reactor.
A simple high-performance liquid chromatographic (HPLC) method for simultaneous determination of proguanil and its active metabolite cycloguanil in human urine has been developed. Quinine sulphate was used as the internal standard. The assay uses a reversed phase C-18 microbore column (2 mm LD. x 10 cm) packed with 3 mu m ODS Hypersil. The chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer (10:90, v/v) containing 200 mM sodium dodecyl sulphate adjusted to pH 2. The mobile phase was pumped at 0.4 ml/min. The eluant was monitored by a UV detector operating at 254 nm. The assay was based on an organic extraction with 1-hexanol/ether (40: 60, % v/v) and then back-extracted into a small volume of acidic aqueous solution before injection onto the HPLC column. With this procedure coefficients of variation were less than 8%. The detection limit was 0.5 mu g/ml of urine. The method is simple, sensitive, selective and allows for routine analysis of urine samples in the genetic drug oxidation phenotyping study in ethnic population.
Liquid chromatographic methods for the determination of chloramphenicol (CAP) and thiamphenicol (TAP) residues in gamebird (pheasant, mallard and quail) meats were developed. The drugs were extracted from the homogenised tissues (muscle and liver) by water and the extracts were purified by subsequent partitions with Chem Elut CE 1020 cartridge - ethyl acetate and water - toluene. The recoveries were 67% for CAP and 72% for TAP. Chromatographic separations were performed on a Hypersil C-18 column and the peak identification and quantitation was made with a photodiode array detector. Using the first derivative of the chromatographic peak the selectivity of the analysis was improved. The elimination kinetics of CAP and TAP from the quail tissues were also evaluated.
A rapid, specific and reliable high performance liquid chromatographic assay of Nalidixic acid in tablets has been developed. Reversed-Phase chromatography was conducted using a mobile phase of 0.05 M Ammonium acetate Methanol and acetonitrile, (65, 5, 30% v/v) pH 5 and detection at lambda 254 nm. The recovery and coefficient of variation from six placebo tablets containing 100 mg of Nalidixic acid were 100.2% and 0.56 respectively. Replicate regression analyses of three standard plots in the concentration range 1 - 20 mcg/ml obtained on three different days Save a correlation coefficient (0.99996) and the coefficient of variation of the slopes 0.089%. The assay was precise within day and between days as indicated by ANOVA test. It is suggested that the proposed HPLC method should be used for routine quality control and dosage form assay of Nalidixic acid.
A relatively simple HPLC procedure used to detect and quantify amphetamine and 4'-hydroxyamphetamine in rat urine has been developed. Following Bond Elut(R) solid phase extraction, the two analytes - amphetamine and 4'-hydroxyamphetamine, and the two internal standards - methamphetamine and 4'-hydroxymethamphetamine were separated by HPLC using a phenyl column and detected by UV at 215 nm. The limit of quantitation for amphetamine and 4'-hydroxyamphetamine was 0.92 and 0.81 mu g/ml, respectively. Based on replicated analysis of controls at 1.6, 6.5, and 16.0 mu g/ml, the method is accurate (94 - 103% of target), and precise (% CVs of 1.3 - 5.2). The linear range of the assay is suitable for the quantitation of both analytes at urine concentrations that commonly result from administration of a single dose of amphetamine to the rat.
The retention behavior of five 17-oxosteroids (dehydroepiandrosterone, epiandrosterone, androsterone, 5 beta-androsterone, and 5 beta-androstane-3 beta ol-17-one) with 5-dimethylamino-1-naphthalenesulfonic hydrazide, 4-(N, N-dimethylaminosulfonyl)- 7-hydrazino-2, 1, 3-benzoxadiazole or p-nitrophenylhydrazine are examined using reversed-phase high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as a mobile phase additive is also used for this purpose and found effective in separating the isomeric derivatized 17-oxosteroids.
A quantitative method for the determination of intact saponins in Lupinus angustifolius seed by high-performance liquid chromatography (HPLC) is described. The seed was extracted under mild conditions with 70% aqueous ethanol containing 0.01% EDTA and quantified using an internal standard of alpha-hederin. Soyasaponin VI, also known as soyasaponin beta g, a DDMP (2,3 -dihydro - 2,5 - dihydroxy - 6 - methyl - 4H - pyran - 4 - one)- conjugated form of soyasaponin I, was the only saponin detected in the lupin seed. The technique is compared with a gas chromatographic method which quantifies the sapogenol resulting from the acid hydrolysis of the saponins.
Tolfenamic acid is a potent prostaglandin synthetase inhibitor used clinically as non-steroidal anti-inflammatory and analgesic-antipyretic agent. A simple, sensitive, accurate, and precise reverse phase high performance Liquid chromatographic method has been developed and validated for the quantitative determination of tolfenamic acid in small volumes of human plasma. The chromatographic separation of tolfenamic acid and the internal standard (phenylbutazone) was performed on a reversed phase, 5-mu m C18 column (250 x 4 mm) using acetonitrile-10 mM phosphoric acid (60:40, v/v) as mobile phase with a flow rate of L1 ml/min and the chromatographic peaks were detected at 280 nm. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. Calibration curves were linear in the range 0.2-5.0 mu g/ml with a squared correlation coefficient (r(2)) of 0.999 or better and the detection limit was 50 ng/ml for 100-mu l plasma samples. The method was not interfered with by other endogenous compounds or metabolites and one assay can be completed in 12 min. The within-day and between-day assay variation for three different concentrations was found to be less than 6% and the accuracy was nearly 100%.
An efficient off-line solid-phase extraction (SPE) of lamotrigine, 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine (LTG), a new antiepileptic drug, from human serum and urine, prior to high-performance liquid chromatographic analysis, was tested and optimized. High extraction recoveries were achieved from C-8 Bond Elut cartridges (200mg/3ml), using acidic acetonitrile for the elution of LTG and the internal standard, 3,5-diamino-6-(2-methoxyphenyl)-1,2,4-triazine. Isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis on octylsilica, using a Lichrosorb RP-8, 5 mu m, 250 x 4.6 mm i.d. column and a mobile phase consisting of 0.05M acetate buffer pH5.6 and acetonitrile (72:28 v/v) proved to be sensitive and rapid. The identification of LTG was performed by UV detection at 306nm. The method detects approximately 0.9 ng of LTG on-column, using a 20-mu l loop, and linearity holds from approximately 0.044 to 7.8 mu g/ml in standard solutions. In plasma and urine, the limits of detection are 1.1 and 1.2ng respectively, while linearity holds from approximately 0.087 to 3.49 mu g/ml. The proposed method was also used for the direct analysis of antiepileptic tablets.
N-dodecoxycarbonyl-(S)-valine was evaluated for the separation of some benzoylated amino acids (alanine, aminobutyric acid and leucine). Successful enantiomeric separation required blocking of the carboxylic function by the formation of the methyl esters in order to avoid repulsion of the charged carboxylate group by the hydrophobic interior of the chiral micelles. A model of chiral recognition is presented, The highest enantioselectivity was obtained for the 3,5-dinitrobenzoyl derivatives (alpha > 1.07). Based on the data, the generally accepted MEKC-resolution equation was experimentally verified and confirmed.
