Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H₃PO₄/CH₃CN (96:4) to 0.15 M H₃PO₄/CH₃CN (85:15) over 30 min followed by isocratic elution with 0.15 M H₃PO₄/CH₃CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g^-1) and cystine (0.62 μmol g^-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g^-1) was observed in liver, spleen, heart and testicle.     

Simultaneous determination of five fat-soluble vitamins in feed by high-performance liquid chromatography following solid-phase extraction

A high-performance liquid chromatography method is developed for the simultaneous determination of menadione, retinyl acetate, cholecalciferol, alpha-tocopherol, and alpha-tocopherol acetate in feed. The present study uses an enzyme to destroy the coating film, ethanol to extract free vitamins, and Oasis HLB cartridges to purify. Vitamins are separated using an Atlantis dC18 column. The mobile phase is methanol-water (98:2 v/v). Detection is performed with a UV-vis detector at 230 and 265 nm. The linearity, accuracy, and repeatability of this method are all satisfactory. Application of the method is suitable for the determination of the fat-soluble vitamins in general feed.     

Simultaneous determination of some antibacterial drugs in isosensitest broth using high-performance liquid chromatography with solid-phase extraction.

A method is described for the simultaneous determination of combinations of some antibacterial drugs in a matrix of isosensitest broth. A double solid-phase extraction procedure is described in which trimethoprim and dibromopropamidine isethionate together with 4-chlorophenylbiguanide as internal standard are freed from endogenous components by a cation-exchange extraction cartridge and subsequently removed and individually separated by reversed-phase ion-pair chromatography. Sulfadiazine, sulfamerazine and p-aminobenzoic acid, unretained by ion exchange, are similarly isolated for chromatography by adsorption on a CH-bonded phase cartridge and individually assayed using the same chromatographic system. The rationale of the pre-treatment and chromatography is described and the quantitative aspects of the analyses of selected combinations of these drugs are reported.     

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.     

Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection.

High-performance liquid chromatography has become an important analytical tool for the quantitation of opioid drugs. Using solid-phase extraction and coulometric electrochemical detection, we have developed a chromatographic method for the simultaneous measurement of morphine and hydromorphone which is both sensitive and specific. Using 1 ml of plasma, intra-assay and inter-assay data show that the detection limit for accurate quantitation of these compounds is about 1.2 ng/ml (coefficient of variation 11.6%) for morphine and 2.5 ng/ml (coefficient of variation 10.5%) for hydromorphone. The method is simple and readily adaptable to most pharmacokinetic studies and toxic screens involving these drugs.     

Matrix solid-phase dispersion extraction and determination by high-performance liquid chromatography with fluorescence detection of residues of glyphosate and aminomethylphosphonic acid in tomato fruit

A method based on matrix solid-phase dispersion (MSPD) is described for the quantitative extraction of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) from tomato fruit. After application of 120 microL of HNO3 1M to the sample, the dispersion column was packed with 0.5 g of sample blended into 1 g of NH2-silica. Two aqueous fractions were obtained. First, AMPA was eluted from the column using deionized water (F1), and then a NaH2PO4 0.005 M solution was used for the elution of glyphosate (F2). Cleanup of F1 and F2 was made by ion exchange chromatography on a SAX anion exchange silica. Determination was done by HPLC with fluorescence detection after precolumn derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). Mean recoveries calculated at fortification levels of 0.5 microg/g for glyphosate and 0.4 microg/g for AMPA were 87% and 78%, respectively. The relative standard deviations (n=7) for the total procedure were 10% and 16%. Detection limits were 0.05 microg/g for glyphosate and 0.03 microg/g for AMPA.     

Determination of aflatoxin M1 in milk using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

Solid-phase extraction of aflatoxin Ml from milk using Diapak S16M and Diapak S solid-phase extraction cartridges followed by HPLC determination with a Flyuorat-02 fluorescence detector was studied.     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

Simultaneous determination of 15 marker constituents in various radix Astragali preparations by solid-phase extraction and high-performance liquid chromatography

An improved quality control method was developed to simultaneously determine 15 major constituents (eight flavonoids and seven saponins) in various radix Astragali preparations, using SPE for pretreatment of samples, HPLC with diode-array and evaporative light scattering detectors (DAD-ELSD) for quantification in one run, and HPLC-ESI-TOF/MS for definite identification of compounds in preparations. Optimum separations were obtained with a ZORBAX C(18) column, using a gradient elution with 0.3% aqueous formic acid and ACN. This established method was fully validated with respect to linearity, precision, repeatability, and accuracy, and was successfully applied to quantify the 15 compounds in 19 commercial samples, including 3 dosage forms, i. e., oral solution, injection, concentrated granule, and its processed products of radix Astragali. The results demonstrated that many factors might result in significant differences in quality of the final preparations, including crude drugs, pretreatment processes, manufacturing procedure, storage conditions, etc. Then the developed method provided a reasonable and powerful manner to ensure the efficacy, safety, and batch-to-batch uniformity of radix Astragali products by standardizing each procedure, and thus should be proposed as quality control for the clinical use and modernization of herbal preparations.     

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 x 4.6 mm, 5 microns 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 micrograms/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.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

Determination of terfenadine and terfenadine acid metabolite in plasma using solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

This work describes the methodology for the analysis of terfenadine and the acid metabolite of terfenadine in plasma using high-performance liquid chromatography. The use of solid-phase extraction allows the use of robotic or manual sample preparation for the efficient clean-up of terfenadine and terfenadine acid metabolite from plasma. Additional selectivity is obtained through the use of fluorescence detection. For terfenadine, the validated quantitation range of this method is 10.0-84.2 ng/ml with coefficients of variation of 5.7-30%. For terfenadine acid metabolite, the validated quantitation range of this method is 8.2-500 ng/ml with coefficients of variation of 4.1-24%.     

Analysis of tetrahydro-beta-carboline-3-carboxylic acids in foods by solid-phase extraction and reversed-phase high-performance liquid chromatography combined with fluorescence detection

The presence and analysis of two tetrahydro-beta-carboline-3-carboxylic acids in foods are studied. Sample preparation with benzenesulfonic acid strong cation-exchange columns followed by RP-HPLC-fluorescence allowed a reliable analysis and spectral characterization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA). Experimental data showed that upon oxidation tetrahydro-beta-carboline-3-carboxylic acids gave rise to beta-carbolines (norharman and harman) that were also chromatographically separated and their fluorescent profile monitored. This approach was useful to confirm identification of tetrahydro-beta-carboline-3-carboxylic acids in foods. Several foods and beverages contained THCA and MTCA in varying proportions. Their occurrence in foods implies that diet is a source of these compounds in humans.     

On-line solid-phase extraction of piroxicam prior to its determination by high-performance liquid chromatography

A direct method for the determination of piroxicam in plasma is described. Plasma is directly injected onto the extraction column (10 mm x 2 mm I.D., packed with 40-microns Bond Elut C2) where proxicam is separated from the plasma concomitants using a solid-phase extraction procedure. Using a laboratory-made on-line column-switching system, the drug is quantitatively transferred and separated on the analytical column (15 cm x 4.6 mm I.D., Supelcosil LC18 DB, 5 microns) followed by determination using ultraviolet absorption at 331 nm. Validation of the method demonstrated a good recovery (100%), sensitivity (limit of determination 0.2 microgram/ml, based on a 20-microliters sample volume), accuracy and precision (better than 5%). The developed method has been adopted for studying the steady-state pharmacokinetics of the drug.     

Sensitive determination of buspirone in serum by solid-phase extraction and two-dimensional high-performance liquid chromatography

A selective and sensitive determination of buspirone in serum by high-performance liquid chromatography is described. The procedure is based on separation on a C18 column. A solid-phase extraction procedure is used for sample clean-up. The retention on the first column is based on the hydrophobic interaction of buspirone with the stationary phase, and the retention on the second column is based on ionic interactions due to the presence of sodium lauryl sulphate in the mobile phase as well as hydrophobic interaction. This allows for good separation of buspirone from impurities and consequently allows lower detection limits than previously reported for liquid chromatographic methods. Detection by ultraviolet absorbance gives a detection limit of 0.2 ng/ml.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 micrograms/ml of succinylcholine. In a pilot study the plasma concentration-time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Quantitative determination of Closantel residues in milk by high-performance liquid chromatography with fluorescence detection.

A HPLC method with fluorescence detection for quantitative determination of Closantel residues in milk has been developed and validated. The proposed cleaning procedure with acetonitrile and acetone extraction, and solid-phase clean-up with Florisil enables concentrations of Closantel below 50 micrograms/l to be determined. The method was shown to be sufficient, precise, accurate, selective and rugged. The method was applied in the regular monitoring of Closantel residues in milk and of the pharmacokinetic behavior of Closantel in sheep.     

Simultaneous analysis of three avermectins in soils by high-performance liquid chromatography with fluorescence detection

A simple and sensitive method has been developed and validated for the determination of abamectin B_1a (ABA B_1a), emamectin B_1a (EMA B_1a) benzoate and ivermectin H₂B_1a (IVM H₂B_1a) in soils. The avermectins (AVMs) residues were extracted from soils with acetonitrile/water (9?:?1, v/v) and then were purified on C₁₈ solid-phase extraction (SPE) cartridge. After being derivatised by N-methylimidazole (N-MIM) and trifluoroacetic anhydride (TFAA), the residues of three AVMs were analysed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method was validated in terms of system suitability, linearity, selectivity, precision, recovery, specificity and stability. There was a good linear relationship (R ^2?>?0.99) for three AVMs ranged from 0.01 to 5?µg?mL^?1. The LOD and LOQs of ABA B_1a, EMA B_1a benzoate and IVM H₂B_1a for standard solutions were 1.1–1.7 and 3.6–5.7?µg?L^?1 respectively. The accuracy of AVMs in soils was from 83.7 to 115.5% with precision less than or equal to 12.4%. Using the developed method, 9 soil samples with 9.3–12806.3?µg?kg^?1 of AVMs residues had been detected.