Determination of alkylamines by high-performance liquid chromatography with post-column fluorescence derivatization

A styrene/divinylbenzene polymer column and an amino column are compared for the non-aqueous separation of primary, secondary and tertiary alkylamines. Post-column derivatization with o-phthalaldehyde/2-mercaptoethanol is selective for primary amines and derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is selective for secondary amines after on-line masking of primary amines. This procedure can tolerate 0.4 M butylamine. The limit of detection is 18.5 mM for dioctylamine (with NBD-Cl) and 0.18 mM for decylamine and tetraethylenepentamine (with o-phthalaldehyde/2-mercaptoethanol).     

Detection of zearalenone in cereal extracts using high-performance liquid chromatography with post-column derivatization.

Post-column derivatization has been used to enhance the fluorescence response of the Fusarium mycotoxins zearalenone and zearalenol when determined by reversed-phase high-performance liquid chromatography. Derivatization is based on reaction with aluminum chloride and this results in a more selective response for these toxins. The method was tested on a number of cereals and animal feeds.     

Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method was developed for the determination of citrulline and homocitrulline using a post-column colorimetric reaction with o-phthaladehyde and N-(1-naphthyl)-ethylenediamine. Citrulline and homocitrulline were determined with no interferences from protein amino acids. The results show that the level of citrulline in the plasma of patients with uremia on intermittent hemodialysis is higher than that in healthy human plasma, and that homocitrulline is excreted into the urine of healthy adults.     

Analysis of digoxin at therapeutic concentrations using high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic (HPLC) procedure has been developed for the analysis of digoxin in plasma at therapeutic concentrations. The assay method provides resolution of digoxin from its metabolites using a 15 cm X 4.6 mm HPLC column containing 3-micron octadecylsilane-bonded stationary phase. The effluent of the column is passed through a post-column reactor in which a fluorescent derivative is formed by the co-addition of hydrochloric acid and dehydroascorbic acid. Detection of the derivative is accomplished in a fluorometer with excitation at 336 nm and emission at 425 nm. The extraction efficiency for recovery of digoxin from plasma samples was 70% using chloroform-isopropanol (9:1) following a pre-wash with isooctane to remove endogenous substances. The calibration curve was linear (r = 0.9999) over the range 0.5-4 ng/ml digoxin in plasma using digitoxigenin as internal standard. The minimum detectable quantity of digoxin in plasma was 0.5 ng/ml at a signal-to-noise ratio of 4:1. Split-samples of digoxin control sera were assayed by the HPLC procedure and by the prescribed radioimmunoassay procedure. Excellent correlation was observed between the two methods (r = 0.999). No interference was noted when a selection of commonly co-prescribed drugs were evaluated for chromatographic co-elution or interference in detection with that of digoxin or the internal standard.     

The rapid determination of artemisinin by post-column derivatization high-performance liquid chromatography using matrix solid-phase dispersion method

Artemisinin (an antimalaric compound) is isolated as the active compound of the medicinal plant Artemisia annua L. A simple, rapid, and high-efficient method of extraction is developed, in which it is extracted by matrix solid-phase dispersion (MSPD) and directly analyzed by post-column derivatization high-performance liquid chromatography (HPLC). The quantitation results from the MSPD method are compared with two conventional liquid solvent extraction processes, Soxhlet and ultrasonic wave by F-test, and the result indicates no significant difference. The production rate of Artemisinin during wild plant growth (tested over two years, acquired from Yunnan, provincial Yuan Yan country) is determined. The recovery range of determination is 88.1-91.2%. The relative standard deviation (RSD) is 4.55-6.43%. The limits of detection are 0.1 microg/mL, and the limits of quantitation are 0.5 microg/mL.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization.

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25 +/- 10 pmol per 10(9) cells for inositol 1,4,5-trisphosphate to 6380 +/- 355 pmol per 10(9) cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665 +/- 10 pmol per 10(9) cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and DL-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas DL-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.     

Determination of zeolite-a in detergent powders as acid-soluble aluminum by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method is described for the rapid determination of zeolite-A in detergents as acid-soluble aluminum. Detergent samples are dissolved by digestion with nitric acid. Chromatographic separation on a strong cation exchanger in acidic ammonium sulfate media is combined with the highly selective color reaction of acid-soluble aluminum with pyrocatechol violet. Interferences of the chelating agents in detergents are thus eliminated. After the digestion, determinations take 8 min per sample. The relative standard deviation is less than 1% and quantitative recoveries are obtained. Several commercial detergents were analyzed successfully. The results obtained by the proposed method and by compleximetric titration agreed very closely for zeolite-A contents in the range 2–22%.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Simultaneous determination of 2-deoxy-D-glucose and D-glucose in rat serum by high-performance liquid chromatography with post-column fluorescence derivatization

A simple high-performance liquid chromatographic method for the determination of 2-deoxy-D-glucose and D-glucose in rat serum is described; this method is based on a post-column fluorescence derivatization. The sugars are automatically converted into fluorescent derivatives by reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium after their separation on a strong anion exchanger column (TSK gel Sugar AXG). The detection limits (S/N = 3) for 2-deoxy-D-glucose and D-glucose in rat serum are 0.52 and 0.56 nmol/ml, respectively.     

Determination of streptomycin residues in food by solid-phase extraction and liquid chromatography with post-column derivatization and fluorometric detection

A reliable and sensitive procedure is presented for the analysis of streptomycin (STP) in food of animal origin, like meat, milk and honey. The method is based on a separation by ion-pair liquid chromatography with β-naphthoquinone-4-sulfonate (NQS) postderivatization and fluorescence detection. The clean-up of the extract is done by solid-phase extraction, firstly with a cation-exchange cartridge and secondly with an octadecyl cartridge. The selectivity is very good and not many interfering peaks are observed for various food matrices. The streptomycin recovery of the total procedure is superior to 80%. The procedure is quantitatively characterized and repeatability, linearity, detection and quantification limits are very satisfactory. A special focus is given to STP residues in honeys and a survey on 64 commercial honeys is presented. For honey analysis, the HPLC method is compared with an immunoassay test (ELISA), and the possibility of using this test for screening with and without solid-phase extraction clean-up is also discussed.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Fast simultaneous determination of 23 veterinary drug residues in fish,poultry, and red meat by liquid chromatography/tandem mass spectrometry

The misuses of veterinary drugs can result in the accumulation of residues in food of animal origin that can make its way to the final consumer. Herein we describe a simple method for the accurate determination of beta-lactams, quinolones, sulphonamides, and tetracyclines in fish, poultry, and red meat. No extraction cartridges were used; instead, the extraction process consisted of the addition of an organic solvents, shaking, centrifugation, and dilution. An extensive validation process demonstrated an excellent linearity (R² ≥ 0.99) for 23-drug residues. The recovery of drugs in different matrices at two concentration levels (n = 6) was in the range of 82–119%. The method was proved to be repeatable and reproducible with intra/inter-day measurements (RSDs lower than 20%). The quantification limits of drug residues were in the range of 0.8 to 45.3 ug/kg, which is well below the maximum residue limits set by most regulatory authorities. This method was successfully applied to the routine analysis of 20 fish, poultry, and red meat samples (n = 60).