On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 microm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5+/-2.8% with a linear range of 0.1-100 microg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Effect of alcohol on elevated aggressive behavior in male transgenic TGF alpha mice

A simple method using ion-pair high-performance liquid chromatography was established for the rapid and precise simultaneous determination of honokiol (3', 5-di-2-propenyl-1, 1'-biphenyl-2,4'-diol) and magnolol (5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol) in oriental pharmaceutical decoctions containing Magnolia bark. An ODS column and a mixture of water involving 10 mM tetra-n-amylammonium bromide (TAA) and acetonitrile (4:6) as a mobile phase were used for the separation. Honokiol and magnolol were eluted without interference of other co-existing components within 12 min.     

Determination of synephrine in Oriental pharmaceutical decoctions containing aurantii nobilis pericarpium by ion-pair high-performance liquid chromatography. II

A simple method using ion-pair high-performance liquid chromatography was established for the rapid and precise determination of synephrine in thirty three species of oriental pharmaceutical decoctions containing Aurantii Nobilis Pericarpium. An ODS column and a mixed solvent system of water, acetonitrile, sodium dodecyl sulfate and phosphoric acid as a mobile phase were used for the separation. Synephrine was eluted without interference of other coexisting components within 15 min.     

Validation of a model predicting enrollment status in a chemoprevention trial for breast cancer

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], alpha-(cystein-S-yl)isovalerylurea [CVU], and alpha-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10 mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10 mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90-97.79% from the spiked liver homogenate and 89.68-96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.     

High-performance liquid chromatographic determination of clindamycin in human plasma or serum: application to the bioequivalency study of clindamycin phosphate injections

This paper presents an assay of clindamycin phosphate injection in human plasma or serum. A 0.5-ml volume of plasma was used with the internal standard, propranolol. The sample was loaded onto a silica extraction column. The column was washed with deionized water and then eluted with methanol. The eluates were evaporated under nitrogen gas. The residue was reconstituted with the mobile phase and injected onto the high-performance liquid chromatographic system: a 5-micron, 25 cm X 4.6 mm I.D. ODS2 column was used with acetonitrile, tetrahydrofuran and 0.05 M phosphate buffer as the mobile phase and with ultraviolet detection at 204 nm. A limit of quantitation of 0.05 microgram/ml was found, with a coefficient of variation of 11.6% (n = 6). The linear range is between 0.05 and 20.00 micrograms/ml and gives a coefficient of determination (r2) or 0.9992. The method has been successfully applied to the bioavailability study of two commercial preparations of clindamycin phosphate injection (300 mg each) in twelve healthy adult male volunteers.     

A new method for allantoin determination and its application in allantoin determination in Agrostemma githago L. Seed

We have established several optimal conditions for qualitative and quantitative allantoin determination by applying Ehrlich's reagent. The limit of detection for allantoin determination amounts to 5 x 10(-6) mM. Allantoin is determined quantitatively by measuring the absorbance at 440 nm (from 300 to 1000 micrograms/ml). The color of the complex becomes stable by standing for 10 min at room temperature. We have used these conditions for allantoin determination in Agrostemma githago seed.     

A subpopulation of reactive astrocytes at the immediate site of cerebral cortical injury

Data presented in this paper show that bromhexine and its pharmacologically active metabolite can easily be determined by capillary zone electrophoresis. The composition of the running buffer had a significant effect on the reproducibility of the migration time for which a carrier solution containing 30 mM phosphate buffer (pH 3.0), 5 M urea and 10% (v/v) acetonitrile was used. The method was validated with respect to its response linearity and reproducibility. The method is suitable for the determination of bromhexine and ambroxol in several samples such as pharmaceuticals, urine and serum. Photodiode-array detection permitted the rapid identification of both drugs in the sample analyzed.     

Determination of gamma-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection

A method was developed for the simultaneous determination of gamma-glutamylglutathione (gamma-GluGSH) and other low-molecular-mass thiol compounds (cysteine, cysteamine, homocysteine, cysteinylglycine, gamma-glutamylcysteine, glutathione and N-acetylcysteine) using high-performance liquid chromatography combined with precolumn fluorescence labeling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). These SBD-labeled thiol compounds were separated within 35 min on a Cosmosil 5C-18AR column with isocratic elution using 75 mM sodium citrate buffer (pH 2.90)-methanol (98:2) and detected fluorimetrically (ex. 386 nm, em. 516 nm). The calibration graphs using 2-mercaptoethanol as an internal standard showed good linearity in the range from 20 pmol to 10 nmol for all thiol compounds examined. The application of this method for the quantitative determination of thiol compounds in the urine from gamma-glutamyl transpeptidase-deficient mice was also demonstrated. This method is sufficiently simple, rapid and sensitive for the determination of gamma-GluGSH and other low-molecular-mass thiol compounds in biological samples.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 x 2.1 mm I.D., 5 microns Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate-acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 microliters sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean +/- S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 +/- 1.2% and 91.4 +/- 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

A simple method for measurement of angiotensin II in plasma

A simple radioimmunological (RIA) method for the determination of angiotensin II in 0.5-1.0 ml samples of plasma is described and carefully evaluated. Before RIA was performed, the interfering plasma proteins were eliminated by ion exchange chromatography, and recovery from every column was checked with a small amount of [125I]angiotensin II. The sensitivity of the method was 4.0 ng/l; the coefficient of intra-assay variation was 10.0% and that of inter-assay variation 12.1%. Accuracy was studied both by adding various amounts of angiotensin II to plasma samples and by diluting plasma containing angiotensin II with the RIA buffer. Both studies gave very good correlations between found and expected values (r=0.998 and r=0.987). In a normal material (n=36), the mean angiotensin II concentration at 8 a.m. after 2 h ambulation 42.4 +/- 12.8 (S.D.) ng/l. Because the present method is accurate, precise, and practical, and allows measurement of angiotensin II in small samples, it seems useful for routine as well as for research work.     

Multiresidue liquid chromatographic method for determining residues of mono- and dibasic penicillins in bovine muscle tissues

A liquid chromatographic method with UV detection at 325 nm was developed for simultaneous determination of amoxicillin, ampicillin, penicillin G, and cloxacillin residues in bovine muscle tissue as their mercaptide derivatives. The penicillins are extracted from bovine tissues with 0.1 M phosphate buffer (pH 8.5), cleaned up on a t-C18 Sep-Pak cartridge, and eluted with 2 mL acetonitrile. After the acetonitrile in the eluate is evaporated to dryness, the residue is dissolved in 200 microL (40 + 60, v/v) acetonitrile-phosphate buffer (pH 6.5) and derivatized with acetic anhydride and mercuric chloride in the presence of 1,2,4-triazole at 65 degrees C for 30 min. Gradient analysis on a Spherisorb 5 microns ODS(2) (octadecyl silane) analytical column using a binary mobile phase consisting of acetonitrile and 0.10 M phosphate buffer (pH 6.5) in the presence of 0.0157 M sodium thiosulfate at 1 mL/min permits determination of each intact penicillin in bovine muscle tissue at > or = 10 ppb with recoveries > or = 72%. This laboratory method provides detection sensitivities equivalent to those of rapid tests used for screening beta-lactam drug residues in bovine tissue samples for regulatory enforcement.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).     

Application of micellar electrokinetic capillary chromatography for the determination of benzoic acid and its esters in liquid formula medicines as preservatives

We described a method for the simultaneous determination of preservatives including benzoic acid, methyl-, ethyl- and propyl-benzoate by micellar electrokinetic capillary chromatography (MECC). The factors affecting the reproducibility in the quantitative analysis of pharmaceuticals by MECC were investigated by varying the running buffer and washing condition in-between runs. Preservatives in liquid formula medicines have been determined by optimum MECC condition using p-hydroxy benzoic acid as an internal standard. The reproducibility of this method was acceptable as a validate method for the quality control of pharmaceuticals (RSD < 2%). Routine quantitative analysis of pharmaceuticals using MECC could be possible with well characterized reproducible procedure.     

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm x 75 microm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.     

Isolation of pathogenic bacteria from induced sputum from hospitalized children with pneumonia in Bangladesh

A high-performance liquid chromatographic system, combining solid-phase extraction and automated precolumn derivatization is described for the routine determination of methotrexate in human plasma. The sample extraction and elution onto the analytical column were performed automatically and concomitantly using the column-switching technique and a protein-coated precolumn. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before the analytical column for the conversion of methotrexate into highly fluorescent products. The oxidative-cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing plasma sample through CTH column with a flow rate of 0.5 mL/min at 40 degrees C. The fluorescent products were transferred to the protein-coated precolumn, which was then flushed with the same buffer for clean-up and enrichment from plasma sample. The trapped substances were desorbed from the precolumn and eluted onto the ODS/TM analytical column by isocratical elution with a mobile phase containing 0.05 M phosphate buffer, pH 6.6 and acetonitrile (90-10, v/v) for subsequent separation. The fluorescent products were detected fluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The complete analysis was achieved within 15 min per sample. The calibration graph was linear in the range of 50-500 ng/mL of methotrexate and there was no interference from endogenous components.     

Determination of nimesulide and hydroxynimesulide in human plasma by high performance liquid chromatography

Two specific methods for the simultaneous determination of nimesulide, a non steroidal anti-inflammatory drug, and its hydroxylated metabolite in human plasma are described. Adopting a high performance liquid chromatographic (HPLC) system with UV detection (230 nm), the compounds, extracted from plasma in acidic medium, were separated on ODS columns under gradient conditions, using a phosphate buffer solution and methanol as mobile phase. For each method column length, gradient rate and composition were appropriately selected. The limit of quantitation was 25 ng/mL for both compounds. The two methods were validated by intra day assays at three concentration levels and applied in kinetic studies in healthy volunteers, during which inter-day assays were carried out confirming their feasibility.     

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-SSA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C18 column (75 x 4.6 mm ID, 3 mu, Altex, USA) as clean-up column and a CSC-nucleosil C8 column (150 x 4.6 mm ID, 5 mu, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH = 3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min-1 for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH = 5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml-1. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml-1). It is applicable in clinical and pharmacokinetic studies.     

Determination of free bile acids in pharmaceuticals by thin layer chromatography and high performance liquid chromatography

High-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and thin layer chromatography with flame ionization detection (TLC-FID) have been applied to the separation of five main free bile acids present in humans: cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), lithocholic (LCA) and ursodeoxycholic (UDCA) acid. HPLC separation was performed on Biospher Si 100 column using a mixture of n-heptane, isopropanol, ethylacetate, methanol and glacial acetic acid as a mobile phase. All the compounds were separated in less than 12 minutes by using a gradient elution mode. TLC-FID separation was performed on S-II Chromarods with a mixture of isooctane, ethylacetate and glacial acetic acid as a mobile phase. HPLC-ELSD method was applied to the determination of CDCA and UDCA in pharmaceuticals and their purity control when LCA, DCA and CA were considered as impurities.     

Development of a simple high-performance capillary electrophoretic method with on-line mode in capillary derivatization for the determination of spermidine

A new high-performance capillary electrophoretic (HPCE) method with an on-line mode in-capillary derivatization (ICD) procedure for determinations of some amines using 20 mmol/L sodium dodecyl sulfate (SDS) - 2 mmol/L o-phthalaldehyde (OPA) - 2 mmol/L N-acetylcysteine (NAC) - 20 mmol/L phosphate-borate buffer [9] has previously been shown. Although this technique offers direct fluorescence detection of free amines without any derivatization procedures before or after HPCE separation, the presence of spermidine (Spd) is difficult to detect due to low fluorescence intensity. The purpose of this study is to improve the detection sensitivity of Spd by reoptimizing this method with regard to the run buffer; the reoptimized method was applied to the determination of Spd in human plasma. To enhance the fluorescence intensity of the Spd signal, it is effective to use the run buffer in the presence of both beta-cyclodextrin (beta-CD: 8.8 mmol/L) and NAC at high concentration (16 mmol/L). By contrast, the intensity was remarkably decreased when SDS was used in the presence of beta-CD. After ultrafiltrating (UF) spiked human plasma with Spd, UF plasma was directly analyzed using the reoptimized method. Spd peak was detected and separated from the other peaks of blank plasma. The present method gave good linearity (r = 0.999), reproducibility (3.85% coefficient of variation at 5 micromol/L level; n = 10) and specificity. The detection limit and lower limit of quantitation is for 0.2 micromol/L and 1 micromol/L, respectively.