Determination of isoniazid methanesulphonate and its metabolites in rabbit blood by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method is described for simultaneous determination of isoniazid methanesulphonate (IHMS) and its metabolites, such as isoniazid (INH) and acetylisoniazid (AcINH) in rabbit blood. According to stability studies, IHMS was most stable at pH 3-5. After acidifying the blood to pH 5.0, a suitable amount of acetonitrile was added to the supernatant for extraction and niacinamide served as an internal standard. After evaporation, the residue was reconstituted with phosphate buffer and aliquots of this solution were separated on a reversed-phase phenyl column by a mobile phase consisting of 0.25 mM tetrabutylammonium phosphate as a paired-ion reagent. UV detection was performed at 280 nm. Under these conditions, the between-run coefficients of variation of IHMS, INH and AcINH from 1 to 25 microns/ml were 4.7 +/- 2.5, 5.4 +/- 1.0 and 5.1 +/- 3.1%, respectively. Hence this sensitive, reproducible and accurate method was suitable for pharmacokinetic studies of IHMS.     

Analytical methodology for the determination of aflatoxins in peanut butter: comparison of high-performance thin-layer chromatographic, enzyme-linked immunosorbent assay and high-performance liquid chromatographic methods

High-performance thin-layer chromatography (HPTLC) was applied to the separation and quantification of aflatoxin in 300 jars of "crunchy" peanut butter. A critical evaluation of the proposed HPTLC method has been carried out by statistical comparisons with a commercially available enzyme-linked immunosorbent assay (ELISA) kit and a high-performance liquid chromatographic (HPLC) method. The statistical tests indicated that whilst the distributions of the data sets obtained with each method were similar, the HPLC method was found to be biased. Over-all results indicated that the HPTLC method gave more consistent data, relatively lower standard deviations and lower coefficients of variation. The ELISA kit was found to be less precise than the HPTLC and HPLC methods and prone to some loss of sensitivity caused by matrix interference.     

Comparative titration of ginsenosides by different techniques in commercial ginseng products and callus cultures

The ginsenoside content of different ginseng species (Panax ginseng, P. quinquefolium, and P. vietnamensis) from different sources (roots from field-grown plants or from in vitro cultures, cells from solid calluses or from liquid cultures, commercial powders, and suspensions) is evaluated by means of a new high-performance thin-layer chromatography (HPTLC) technique combining an automatic TLC sampler and scanner. The results are compared with those obtained through more classical gross spectrometric and high-performance liquid chromatography (HPLC) techniques. HPTLC and HPLC allow the separation and estimation of the different ginsenosides. For this, HPTLC is faster and simpler than HPLC. Both techniques determine less amounts of ginsenosides than spectrophotometry, which displays overestimated values caused by light absorption by contaminating osides. In vitro cultured cells and roots contain the same ginsenosides as those produced by their mother plants, although at quite lower levels. The culture media also accumulates ginsenosides.     

Analysis of flavonol aglycones and terpenelactones in Ginkgo biloba extract: A comparison of high-performance thin-layer chromatography and column high-performance liquid chromatography

Advancements in automated high-performance thin-layer chromatography (HPTLC) have made it feasible to assess its use for the quantitative analysis of marker compounds in botanical preparations. We report here the findings of method comparisons for the terpenelactones and flavonol aglycones by column high-performance liquid chromatography (HPLC) with evaporative light scattering and UV detection, and HPTLC with a scanning densitometer. For the HPTLC assay of terpenelactones, total bilobalide, ginkgolide A, and ginkgolide B consistently achieved <70% of the total determined using HPLC, regardless of variations to postchromatographic derivatization time and temperature. Accuracy testing showed the possibility of a matrix interference. In contrast, a good relationship (95%) was determined between HPTLC and HPLC for determination of total flavonol glycosides (calculated from combined quercetin, kaempferol, and isorhamnetin) from an acid-hydrolyzed Ginkgo biloba L. (GBE) sample. The HPTLC flavonol aglycone method also performed well in terms of accuracy (overall average of 96% recovery for the 3 aglycones) and consecutive plate repeatability (overall percent relative standard deviation of 4.4). It is demonstrated that HPTLC can be a time-saving complement to HPLC for routine analysis of the flavonol glycosides in GBE.     

High-performance thin-layer chromatography method for monitoring norfloxacin residues on pharmaceutical equipment surfaces

This paper presents a high-performance thin-layer chromatography (HPTLC) method with direct fluorescence measurement for the determination of norfloxacin. The method was validated for the monitoring of norfloxacin residues on stainless steel surfaces at the allowed limit of 10 mg of norfloxacin per square meter. However, it can be adapted for lower amounts of residues owing to the low detection limit of norfloxacin (about 5 ng) and can also be used for other surface materials. Test solutions were analyzed by the new HPTLC method and the known HPLC method for comparison. Accuracy and precision of the new HPTLC method, with a subsequent quantification by densitometer or video system, are comparable with those of the HPLC method.     

Polarographic determination of isoniazide, N-acetylisoniazide and isonicotinic acid by superimposed sinusoidal tension]

The a.c. polarographic determination of isoniazid (INH), N-acetylisoniazid (AcINH) and isonicotinic acid (INA) is described. Under the optimal conditions of pH, ionic strength and electrical parameters, the limits of detection are 0.5 μg ml^-1 for AcINH, 0.2 μg ml^-1 for INH, and 0.03 μg ml^-1 for INA.     

Assay of tinidazole in human serum by high-performance thin-layer chromatography--comparison with high-performance liquid chromatography

Determination of tinidazole in human serum by high-performance thin-layer chromatography (HPTLC) is presented. It includes use of 10 x 10 cm plates coated with silica gel 60 and chloroform-acetonitrile-acetic acid (60 + 40 + 2) as mobile phase. Quantitation was performed by densitometry at 320 nm. The linearity (1-10 ng), precision (6%), reproducibility (5%), recovery (96%), and detection limit (1 mg/L) of tinidazole determination by HPTLC were comparable with corresponding method parameters by reversed-phase HPLC. A satisfactory correlation was found between the 2 analytical methods. The procedure was used to quantitate tinidazole in patient sera.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

High-performance thin-layer chromatography method for assessment of the quality of combinatorial libraries,and comparison with liquid chromatography-ultraviolet-mass spectrometry

A high-performance thin-layer chromatography (HPTLC) method was developed for fast evaluation of the purity of solid-phase synthesis products. The results obtained were in good agreement with results obtained by the LC-MS method (r(2) = 0.8404) or by the LC-UV method (r(2) = 0.8053), confirming the suitability of HPTLC for purity analysis of combinatorial syntheses. The synthesis products can be quantified and identified by measuring UV densitograms or in situ UV spectra or by ESI-MS after isolation of the zone of interest. A new, simple, and fast method for transferring the zone of the analyte from the plate to the ESI-MS equipment is described. The new HPTLC method enables rapid and efficient analysis of approximately 40 samples in parallel. As such, it offers a cheaper and easier way to analyze the purity of synthesis products than the commonly used LC-UV-MS.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

Characterization of nucleobases in sea buckthorn leaves: An HPTLC approach

The present study aims to establish a high-performance thin layer chromatography (HPTLC)-based comparative analysis, directed toward characterization of nucleobases in aqueous and alcoholic extracts of sea buckthorn leaves from three different varieties: Hippophae salicifolia, Hippophae rhamnoides mongolica, and Hippophae rhamnoides turkestanica. The alcoholic and aqueous leaf extracts from these sea buckthorn varieties were prepared using accelerated solvent extraction technique. A novel HPTLC method for separating and identifying six nucleobases, namely, guanosine, guanine, cytosine, adenine, uracil, and thymine were adopted. HPTLC analysis indicated the presence of one or more of these nucleobases in a total of six leaf extracts evaluated, their quantities varying from 0.23 to 7.76?µg nucleobase per mg of extract. Though a typical trend could not be observed in the values obtained, the extracts were found to be considerably rich with respect to nucleobase contents. The results acquired from HPTLC were subsequently validated by hyphenation with mass spectrometry and also by applying chemometric tools in form of heat maps, hierarchical cluster dendrograms, and principal component analysis. The presence of nucleobases in the leaf extracts was confirmed by HPLC as well but HPTLC proved to be a better approach for characterization of nucleobases in plant extracts, than high performance liquid chromatography (HPLC).     

Synthesis and evaluation of molecularly imprinted polymeric microspheres for highly selective extraction of an anti-AIDS drug emtricitabine

The molecularly imprinted polymeric microspheres (MIPMs, 3～5 μm), used as high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) packing materials for anti-AIDS drug emtricitabine (FTC), were synthesized by precipitation polymerization. The effects of ratio of chloroform to acetonitrile on the morphology and diameter of MIPMs were investigated. The prepared MIPMs were characterized by HPLC. The imprinting factor (2.26) suggests that the resultant MIPMs exhibit good recognition and affinity to FTC. In addition, the MIPMs were used in SPE as packing material for separation and enrichment of FTC. The recovery of FTC on MIPMs cartridge was 97.6 % in standard solution. Finally, the MIPMs cartridge was applied to extract the FTC in human serum samples. Impurities in sample have been mostly removed, and the average recovery of 92.5 % was obtained with a detection limit of 0.005 μg/mL and a linear range of 0.02～4.0 μg/mL. The method established can be used to monitor the FTC in human serum sample with good accuracy and selectivity.     

Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry

Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid chromatography (HPLC)-MS/MS.     

Utilization of a scientifically operated charge-coupled device detector for high-performance thin-layer chromatographic analysis of tetracyclines

A high-performance thin-layer chromatography (HPTLC) method using a scientifically operated charge-coupled device detector is described for the assay of tetracycline pharmaceutical products. Quantitative information can be obtained for all samples on a TLC plate within a few seconds. The separation efficiency and detection limits were determined on both normal phase and reverse phase TLC plates. Fluorescence detection mode offers higher sensitivity than fluorescence quenching mode. The dynamic range, sensitivity, accuracy and precision of the system were evaluated. Detection limits of the impurities are in the range of 0.1 to 0.5 ng or 0.3 to 1% of tetracycline, depending on the compound, with a recovery percentage over 85%. The existing impurities in tetracycline capsules were determined using both HPLC and HPTLC techniques. All of the impurities were below the regulation level.     

Determination of selected β-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS

A new method is presented for the determination of five selected β-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08–7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of β-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.     

Comparison between HPLC and HPTLC densitometry for the determination of icariin from Epimedium koreanum extracts

Dry extracts of the aerial parts of Epimedium koreanum were quantified by HPLC and high performance TLC (HPTLC). A gradient HPLC method was used for the quantification of the prenylflavone glycoside icariin at 270 nm. A direct HPTLC assay was developed for the determination of icariin at 270 nm. The UV detection of both analytical assays were used to examine the purity of icariin peaks and compared with the standards. The assays provide good accuracy, reproducibility, and selectivity for the quantitative analysis of icariin. The icariin contents of five different dry extracts were compared by HPLC and HPTLC densitometry. The quantitative results of both analytical methods did not show any statistically significant differences between them, although a trend to slightly lower mean values could be found for the HPLC method.     

Therapeutic drug monitoring by high performance thin-layer chromatography

Summary Methods for measuring concentrations of therapeutic drugs in blood serum have been devised which involve separation of the drugs by high-performance thin-layer chromatography (HPTLC) on silica gel followed by quantification by reflectance or fluorescence scanning. Aliquots of from 20 to 300 L serum are extracted with organic solvent. Portions of the extract containing approximately 1/4 of the drug present are deposited on a Contact Spotter (Clarke Analytical Systems), concentrated to a pin-point and applied to the HPTLC plate which is then developed and scanned.HPTLC permitted 12 samples and three standard solutions of drugs used to treat cardiac arrhythmias to be assyed for 6 drugs simultaneously, with scanning at a single wavelength. Results of these assays correlated well with those by EMIT^® immunoassays and by HPLC. No interference by other common drugs was observed. The total time required was two hours, making the work output esiily competitive with that of moderately rapid HPLC. Since sample preparation and quantification by scanning required only a small portion of this time, and the chromatography, which was time consuming, could be done on all the samples simultaneously, analysis of twice as many samples requires only an additional 1/2 hour. HPTLC thus offered the possibility of highly efficient mass production with relatively little capital equipment. Assay of chloramphenicol in serum, which in our laboratory requires fairly rapid turnaround of nnly a few samples at one time, required a similar length of time. Since standards and quality assurance samples could be assayed simultaneously, the analytical output was again comparable to that from a single HPLC instrument.Dedicated to Professor A. Zlatkis on the occasion of his 60th birthday.     

Simultaneous estimation of pantoprazole and domperidone in pure powder and a pharmaceutical formulation by high-perfomance liquid chromatography and high-performance thin-layer chromatography methods

This paper describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous estimation of pantoprazole (PANT) and domperidone (DOM) in pure powder and capsule formulations. The HPLC separation was achieved on a Phenomenex C18 column (250 mm id, 4.6 mm, 5 pm) using 0.01 M, 6.5 pH ammonium acetate buffer-methanol-acetonitrile (30 + 40 + 30, v/v/v, pH 7.20) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol (60 + 40, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 287 nm over the concentration range 400-4000 and 300-3000 ng/mL with mean recovery of 99.35+/-0.80 and 99.08+/-0.57% for PANT and DOM, respectively (HPLC method). Quantification was achieved with UV detection at 287 nm over the concentration range 80-240 and 60-180 ng/spot with mean recovery of 98.40+/-0.67 and 98.75+/-0.71% for PANT and DOM, respectively (HPTLC method). These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of PANT and DOM in pure powder and capsule formulations.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Application of new high-performance liquid chromatography and solid-phase extraction materials to the analysis of pesticides in human urine

A method for the simultaneous determination of diuron and linuron pesticides in human urine was developed, using both solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) phases made in our own laboratory. These materials were prepared by sorption of polysiloxanes onto a silica surface, followed by immobilization. The HPLC columns were prepared from poly(methyloctylsiloxane), PMOS, immobilized onto silica with microwave radiation while the SPE cartridges where made with poly(methyloctadecylsiloxane), immobilized thermally. Method validation was performed for diuron and linuron for three fortification levels. The recoveries obtained were 85-103%, the inter- and intra-assay precisions were less than 1.6 and 1.8%, respectively. The limits of quantitation and detection for diuron were 2.4 and 8.0 microg/l and for linuron were 5.0 and 12 microg/l, respectively.