Post-column Reaction Detection Research Articles

Determination of auranofin, a chrysotherapy agent, in urine by HPLC with a postcolumn reaction and visible detection.

Auranofin [(2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S) (triethylphosphine)gold(I): AF] is a unique orally active chrysotherapy agent. A HPLC method has been developed for determining AF in urine. The proposed method comprises initial chromatographic separation of AF followed by on-line decomposition by potassium iodide with a released mercapto group undergoing a color-developing reaction with 5,5'-dithiobis(2-nitrobenzoic acid). An aliquot (100 microliters) of a urine sample was chromatographed on a YMC AM-302 octadecylsilica column (4.6 mm i.d. x 15 cm, ambient) with a water-methanol (35:65) eluent delivered at a flow rate of 1 ml/min. A reagent solution for a postcolumn reaction comprised of 50 microM 5,5'-dithiobis(2-nitrobenzoic acid), 0.3 M potassium iodide and a 50 mM phosphate buffer (pH 7.4), was delivered at a flow rate of 0.5 ml/min. The postcolumn reactor consisted of a poly(tetrafluoroethylene) tube (0.5 mm i.d. x 5 m) at 60 degrees C. Detection wavelength was 412 nm. The identity of the AF peak was confirmed by a 3-dimensional chromatogram as well as by atomic absorption spectrophotometric analysis of gold in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and AF concentration in the range 0.1 to 10 microM, with a correlation coefficient of 0.999. The detection limit was 50 nM (S/N = 3 at 0.005 AUFS) and the reproducibility was within 4% for 5 determinations. The AF concentrations in the urine of a rabbit given AF intraperitoneally were determined.

Optimisation of post-column reaction detector for HPLC of explosives

Improvements in selectivity and sensitivity in the analysis of common explosives, like nitrate esters, nitramines and nitroaromatic compounds can be achieved by post-column derivatisation in a two step reaction detector. The first step in derivatisation is the photolysis of the analytes with UV at 254 nm. The photo reactor consists of a crocheted 20 m Tefzel capillary, which is coiled around a low pressure mercury lamp. In second step the nitrite ion generated is subsequently detected by a colourimetric reaction. The azo dye formed can be selectively detected at 540 nm.

Highly sensitive detection of non-reducing carbohydrates by liquid chromatography

A highly sensitive post-column reaction detection system was developed for the liquid chromatographic determination of non-reducing carbohydrates. Non-reducing carbohydrates were oxidized by periodate prior to forming fluorescent compounds by reaction with guanidine in alkaline solution. The calibration graphs were linear between 50 pmol and 10 nmol.

Residues of glyphosate and its metabolite AMPA in strawberry fruit following spot and wiper applications

In a 3-yr study, residues of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) were determined, using high-pressure liquid chromatography with post-column reaction and fluorescence detection, in strawberry fruit following spot and wiper applications of glyphosate to established strawberries. Residues in the fruit were not significantly different for the wiper and spot treatments. Maximum residues of glyphosate detected for either treatment were of the order of 0.04 mg kg−1; residues of AMPA were non-detectable in all samples. Recoveries of glyphosate and AMPA from fortified strawberry fruit tissue were 76 ± 8% and 79 ± 13%, respectively, at 0.1 mg kg−1.Key words: Glyphosate, AMPA, strawberry, wiper application, spot application, residues, HPLC

Low ng/1-level determination of twenty N-methylcarbamate pesticides and twelve of their polar metabolites in surface water via off-line solid-phase extraction and high-performance liquid chromatography with post-column reaction and fluorescence detection

Reversed-phase high-performance liquid chromatography (HPLC) was used for the determination of twenty parent N-methylcarbamate pesticides and twelve major metabolites in surface water. A 50-ml water sample was passed through a disposable solid-phase extraction cartridge, packed with 500 mg of low-carbon C 18-bonded silica (C 18/OH, 40 μm particle size), which selectively retained polar compounds. The preconcentrated analytes were eluted with acetonitrile, reconstituted in 1 ml of water and 100 μl were injected into the HPLC system. The carbamates were separated via a water-methanol-acetonitrile gradient. Detection was performed via postcolumn hydrolysis on a solid-phase (anion-exchange) catalyst, derivatization of the methylamine formed with o-phthalaldehyde-2-mercaptoethanol and fluorescence detection of the isoindole derivative. The detection limits for surface water were between 20 and 30 ng/l. Recoveries were determined for thirteen carbamates and ten metabolites at the 0.1 and 1 μg/1 level and generally ranged from 76 to 106% with relative standard deviations (R.S.D.s) of 0.5–8.5%. Only the sulphoxide metabolite of ethiofencarb and thiofanox had lower recoveries and correspondingly higher R.S.D.s.

On-line dialysis, liquid chromatography and post-column reaction detection of oxytetracycline in salmon muscle extracts

The development of a sensitive automated method for residue control of oxytetracycline (OTC) in salmon muscle is described. Tissue homogenate is dialysed and the dialysate enriched on a small on-line polystyrene column. OTC and the internal standard (tetracycline) are separated by HPLC on a polystyrene column using an ion-pair eluent system. The column effluent is mixed with sodium hydroxide and irradiated at 366 nm and the resulting derivatives monitored by means of a fluorescence detector (excitation: 358 nm, emission: 460 nm). By the method OTC is detected down to 5 ng g −1. The standard curve was linear ( r = 0.9999) over the range 50–1000 ng g −1. Within-day and between-day relative standard deviations ( n = 6) at 50 and 200 ng g −1 ranged from 1.0 to 1.7%.

Determination of alkaline phosphatase isoenzymes in serum by high-performance liquid chromatography with post-column reaction detection

A weak anion-exchange high-performance liquid chromatographic procedure with post-column reaction detection for simultaneous determination of alkaline phosphatase (EC 3.1.3.1, ALP) isoenzymes is described. We identified six peaks with ALP isoenzyme activity in normal serum. The peaks were, in order of elution, one intestinal/bone, two bone and three liver ALP isoenzymes. This new assay with automatic injection, on-line post-column reaction detection and powerful integration data system could be of significant value in the routine clinical biochemistry laboratory. The advantages include improved sensitivity and selectivity over previous methods for the determination of ALP isoenzymes.

Determination of National Survey of Pesticides analytes in groundwater by liquid chromatography with postcolumn reaction detection

Multi-residue methods for pesticides in water were developed using liquid chromatography (LC) with postcolumn reaction detection. Over 100 analytes from the US Environmental Protection Agency's National Survey of Pesticides in Drinking Water Wells were screened for response using postcolumn photolysis followed by fluorescence (PFD), electrochemical (PED) or conductivity (PCD) detection. LC-PED and LC-PFD are suitable for multi-residue pesticide determinations in groundwater. These two detection methods are complementary as PED responds to several sulfur-containing pesticides whereas PFD responds to many nitrogenous pesticides. Approximately half of these analytes could be determined in low nanograms amounts using these two detection systems and multi-residue separations with gradient reversed-phase LC are demonstrated. The LC-PCD system tested was not suitable for sensitive, multi-residue determinations and further examination of this technique is recommended using the commercial PCD instrument.

High-performance liquid chromatographic determination of monohydroxy compounds by a combination of pre-column derivatization and post-column reaction detection

A novel high-performance liquid chromatographic method was developed for the determination of monohydroxy compounds using the combined approach of pre-column derivatization and post-column reaction with fluorescence detection. Monohydroxy-containing drugs were modified by pre-column derivatization with propyl isocyanate (in pyridine, 50°C, 1 h) to form the corresponding n-propyl carbamate esters. Excess of reagent, reagent impurities and solvent were then removed by evaporation. Following chromatographic separation on a reversed-phase octadecylsilica column, the n-propyl carbamate ester derivative was subjected to post-column reaction and detection using alkaline hydrolysis to generate free propylamine, which was subsequently derivatized in-line using o-phthaladehyde and 3-mercaptopropionic acid to form a fluorescent isoindole. The fluorescence emission of the isoindole product was measured at 455 nm following excitation at 340 nm. As propyl isocyanate is highly volatile and physico-chemically different to many drug molecules, problems associated with reagent impurities and reaction by-prodycts were substantially minimized. The method was highly sensitive, allowing detection of sub-nanogram amount of monohydroxy-containing drugs, hydroxy steroids and hydroxamic acids. The utility of this derivatization scheme was demonstrated by the highly sensitive measurement in human plasma at oxiracetam (4-hydroxy-2-oxo-1-pyrrolidineacetamide), an investigational drug inteded for use in dementia and other memory disorders.

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyl-oxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l-1 potassium acetate (pH 3.20)-50 mmol l-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 + 4.85 + 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate (40 degrees C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol l-1 ED and 175 mmol l-1 imidazole in acetonitrile-ethanol (90 + 10 v/v) and flow rate, 0.25 ml min-1. Reaction coil (15 m x 0.5 mm i.d.) heated at 80 degrees C. Chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-1 hydrogen peroxide and 110 mmol l-1 trifluoroacetic acid in dioxane-ethyl acetate (50:50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of metal complexes of ethylenediaminetetraacetic acid in environmental water samples by ion chromatography with UV and potentiometric detection

An ion chromatographic method is described for the analysis of several metal complexes of ethylenediaminetetraacetic acid (EDTA) in water samples. Separations are achieved on C 18 bonded silica, typically using a mobile phase comprising 1% (w/v) cetrimide-1.2 m M phosphate buffer (pH 7)-25% (v/v) acetonitrile-15% (v/v) methanol, as well as on a polymer-based anion exchanger using 2 m M phosphate buffer (pH 7) as eluent. Direct UV detection at 250 nm is employed for EDTA complexes of Fe(III), Cu(II), Pb(II) and Ni(II), whilst UV detection at 250 nm after post-column reaction with copper ions is utilized for EDTA complexes of Zn(II), Cd(II), Co(II), Pb(II), Ni(II) and Cu(II). Detection limits are in the range 1.5–4.0 ng for direct UV detection and 30–50 ng for post-column reaction detection. Indirect potentiometric detection after post-column reaction with copper ions is utilized with metallic copper as the indicator electrode, giving detection limits in the range 1.0–1.5 μg. These separations are applied to the determination of metal-EDTA complexes in river water at the ppb b level and to remobilization studies of metal ions in sediment.

Determination of Nabam Fungicide in Crops by Liquid Chromatography with Postcolumn Reaction Detection

The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.

Liquid Chromatographic Determination of Glyphosate and Aminomethylphosphonic Acid (AMPA) in Environmental Water: Collaborative Study

A new method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in environmental water was collaboratively studied by 6 laboratories. The method is simpler and shorter than previous methods. A filtered volume of water is evaporated to dryness and the residue is dissolved in a buffered EDTA solution. Glyphosate and AMPA are determined by liquid chromatography with postcolumn reaction detection. The method was validated over the range 0.50-5000 ppb, although one of the collaborating laboratories could not reliably quantitate below 1.0 ppb. Statistical analysis of the results showed that typical reproducibility relative standard deviations (RSDR) ranged from 11 to 20% for both glyphosate and AMPA, which compares very well with predicted values for this concentration range. Total variability (as measured by sR) increased with increasing fortification level. The method has been adopted official first action by AOAC.

Analysis of Aldehydes in Fogwater by HPLC with Postcolumn Reaction Detector

An analytical procedure that couples HPLC with the postcolumn reaction detector of flow injection method was developed to analyze aldehydes in fogwater samples. The aldehydes were derivatized with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the reactor and detected at 640 nm. The optimal condition was established and formaldehyde, glyoxal, and glycolaldehyde concentrations were determined. The data obtained by this method were consistent with those obtained by 2,4-dinitrophenylhydrazine (DNPH) method.

Determination of Traces of Scandium in Geological Materials by Preconcentration - Ion-Interaction Chromatography

Preconcentration and ion-interaction chromatography of scandium have been worked out for the determination of scandium in geological materials. Scandium can be enriched by cation-exchange in thiocyanate - hydrochloric acid medium after wet decomposition of rock sample with hydrofluoric - perchloric acids. Subsequent ion-interaction chromatography in lactate medium allows scandium to be determined selectively and sensitively with post-column reaction detection with Arsenazo III. About 5 to 50 µg Sc/g of silicate rocks has been determined with good precision.

Determination of non-ionic surfactants with esters groups by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic (HPLC) method with a post-column reaction detector was developed for the determination of non-ionic surfactants with esters groups (NSEG) such as sorbitan fatty acid esters and sucrose fatty acid esters. NSEG from the mono- to pentaester are separated by reversed-phase HPLC with gradient elution. NSEG eluted from a chromatographic column are hydrolysed with potassium hydroxide, and the resulting fatty acids are reacted with 2-nitrophenylhydrazine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form 2-nitrophenylhydrazides. The reaction mixture is then made strongly alkaline in order to reduce the blank absorbance and develop a violet colour of the hydrazide, which is detected at 550 nm. The post-column reaction detector, for example, can detect 1 μg of monomyristin. With the proposed method, NSEG are well separated and determined with high sensitivity and selectivity.

Determination of arsenate, germanate, phosphate and silicate by ion chromatography using a post-column reaction (molybdenum blue) detector

A selective post-column detection system was developed for the determination of As(V), Ge(IV), P(V) and Si(IV) oxoanions after separation by ion chromatography. Detection is based on the molybdenum blue reaction which, by careful choice of reaction conditions, will react with all four oxoanions with good sensitivity. Quantitative studies revealed slightly curved, though acceptable, calibration functions, with detection limits ranging from 0.06 to 2.0 mg l −1.

Promotion of hepatocellular foci in female rats by chenodeoxycholic acid

Chenodeoxycholic acid (CDC), a dihydroxylated primary bile acid, was evaluated for promotional activity in the liver of rats using a two-stage initiation-promotion model. CDC is a primary bile acid that can attain high concentrations in serum and liver during induced or naturally occurring hepatocellular disorders. Female Sprague-Dawley rats were injected once (i.p.) with diethylnitrosamine (DEN, 150 mg/kg) or sterile physiologic saline (SAL, 0.85% NaCl). Two weeks later, rats in each group were placed into one of two subgroups and fed either NIH-31 mash (Control) or NIH-31 mash containing 0.5% CDC for a 10 week period. At the end of the feeding period, blood and liver samples were collected for determination of bile acid profiles and quantitation of hepatocellular foci respectively. Serum samples were analyzed for concentrations of individual bile acids using a HPLC method that utilizes a post-column enzymatic reaction and fluorescence detection. Liver slices from the left hepatic lobe were stained for foci positive for placental glutathione S-transferase. In serum, significant increases occurred in concentrations of all forms of CDC and were accompanied by mild, insignificant increases in lithocholic acid. Decreased serum concentrations occurred in all forms of cholic and deoxycholic acids. Analysis of liver sections revealed that rats treated with DEN-CDC had significant increases in numbers and volume of foci compared to those treated with DEN-Control. For rats in groups DEN-CDC and DEN-Control, the numbers of foci per square centimeter were 32 and 12; per cubic centimeter, 2221 and 937; and the per cent volume of foci, 1.487 and 0.385 respectively. In this study, CDC was a promoter of hepatocellular foci. Because concentrations of CDC in liver and serum increase in a variety of hepatobiliary disorders, the possibility that increases in endogenous concentrations can enhance the formation of hepatocellular foci is being explored.

Determination of organophosphorus and carbamate pesticide standards by liquid chromatography with detection by inhibition of immobilized acetylcholinesterase

A sensitive method for post-column reaction detection of carbamate and organophosphorus pesticides is described, based on the inhibition of immobilized acetylcholinesterase. The compounds are separated by reversed-phase liquid chromatography with tetrahydrofuran—water as the mobile phase in a isocratic system. The reactor used for detection comprises a conventional flow injection assembly for monitoring activity of acetylcholinesterase immobilized on controlled pore glass in a mini-column with spectrophotometric detection. The detection limits and linear calibration ranges are 2.6 and 5–40 ng for paraoxon, 0.04 and 0.1–1.6 ng for diisopropylfluorophosphate, 18 and 20–100 ng for isopropyl N-phenyl-carbamate and 29 and 40–400 ng for isopropyl N-(3-chlorophenyl)carbamate. Only 16 min is required for the determination of a mixture of the above pesticides, and each enzyme column can be used for 90 determinations.

Post-column reaction detection and flow injection analysis

Post-column derivatization with chemical reaction detection (CRD) and flow injection analysis (FIA) use the same principle, viz. reaction in a flow system. For CRD, dispersion must be minimized so as to maintain the separation achieved. In FIA, dispersion is required to mix the reagent and solute. It has been demonstrated that by using low-dispersion knitted capillaries and various modifications of the FIA principle (forced mixing in a branched system; inverse FIA; plug injection of solute and reagent in an inert carrier), the detection limit can be improved. In plug injection, one can also use coloured reagents and solutes to reduce reagent consumption.