Post-column Reaction Detection Research Articles

Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.

Liquid Chromatography Coupled On-Line to Flow Cytometry for Postcolumn Homogeneous Biochemical Detection

The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a postcolumn continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.

Liquid chromatography—Post-column photochemical conversion and electrochemical detection for determination of peroxide-based explosives

A quantitative trace analysis method for peroxide-based explosives is described. This method uses reversed-phase high-performance liquid chromatography with post-column UV irradiation and electrochemical detection for analysis of triacetonetriperoxide (TATP) and hexamethylenetriperoxide diamine (HMTD). The analytes are initially separated and subsequently decomposed photochemically to hydrogen peroxide, which is finally determined by electrochemical detection. Parameters of the method were optimized for lowest detection limits and fast separation. The limits of detection were 3×10−6 M for both TATP and HMTD, respectively. This detection scheme is significantly easier to use than earlier methods applying photochemical decomposition, enzymatic post-column reaction and fluorescence detection for analysis of peroxide-based explosives.

High-performance liquid chromatographic separation and indirect fluorescence detection of thiols

A fluorescent post-column reaction detection scheme has been devised for selective determination of thiols. The post-column reagent is 40 μ M Cd 2+ and 100 μ M 8-hydroxyquinoline-5-sulfonic acid (HQS) in non-complexing buffer at pH 10. HQS complexes Cd 2+ to form a fluorescent product. Thiols in the HPLC effluent compete for complexation of the Cd 2+, resulting in a decrease in the fluorescence response. Detection limits of 0.2 μ M (0.04 ppm) are achieved for cysteine, homocysteine and glutathione in a 5 min separation. Recoveries from spiked synthetic urine samples are 87–120%.

Comments on “Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection”

Comments on “Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection”

Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines.

An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 microl injection volume) for interleukin 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukins 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules.

Comparison of microbiological and HPLC – fluorescence detection methods for determination of niacin in fortified food products

A comparison has been made between results obtained by a recently published HPLC method, involving post-column reaction and fluorescence detection, with a microbiological assay using Lactobacillus plantarum. The HPLC method includes a modified acid hydrolysis extraction step (0.1 M HCl) and yields niacin values from fortified foods somewhat lower than by the microbiological assay. The most significant differences were observed for the cereal-based products. These differences arise principally from the lack of specificity of L. plantarum and from the stronger acid hydrolysis extraction conditions (1 M HCl) of the microbiological assay, which probably releases a part of the non-bioavailable niacin. Moreover by HPLC, excellent recoveries of added nicotinic acid and nicotinamide (95–100%) were obtained and better precision (RSDr=0.3–0.8%) was observed than from the micobiological assay.

Improved measurement of formaldehyde in water-soluble polymers by high-performance liquid chromatography coupled with post-column reaction detection

An improved methodology for the analysis of free formaldehyde in water-soluble polymers used for industrial water treatment is reported. Previously, derivatization prior to HPLC or colorimetric techniques has been used. The data generated by these approaches are suspect in that the derivatizing agent can react with the polymer or other sample components to produce high results. Post-column reaction derivatization is applied after separation of the free formaldehyde from the product interferences. The type of polymer product analyzed influences the choice of column(s). The degree of high bias of the commonly used 2,4-dinitrophenylhydrazine pre-column derivatization is reported and the results are compared to those with the post-column reaction for two polymer products. This method, being more selective, should be applicable to any polymer containing formaldehyde.

Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection

A simple ion-exchange high-performance liquid chromatographic method was developed and employed for the analysis of bisphosphonate compounds in dosage formulations using on-line post-column photochemical reactions. The method used molybdate as the post-column reagent to react with the photolyzed bisphosphonate to form phosphomolybdate for enhanced spectrophotometric detection. A bisphosphonate compound, 2-thioethane-1,1-bisphophonic acid, was selected to evaluate the separation using both isocratic and gradient elution methods, along with the effects of other experimental parameters including mobile phase composition, flow-rate and post-column reagent concentration. The gradient elution method showed improved resolution and detection sensitivity compared to the isocratic elution method. The optimized gradient method was simple, reproducible, and specific to bisphosphonate compounds. It was successfully employed for the stability study of the bisphosphonate compound in pharmaceutical dosage formulations.

SELECTIVE CHEMILUMINESCENCE ANALYSIS OF AMADORI FORM OF GLYCATED HUMAN SERUM ALBUMIN

Human serum albumin was purified by size-exclusion liquid chromatography. The glycated albumin in the effluent was selectively measured by a post-column reaction detector. The selective reaction mechanism is to measure the amount of Amadori form of glycated albumin. According to the Maillard reaction, glycosylated amines are rearranged to Amadori form ketoamines which can produce superoxide in strong alkaline solution, and this superoxide produces chemiluminescence with lucigenin. The chemiluminescence intensity was related to the concentration of glycated albumin in albumin while the fluorescence intensity was the same.

Effect of food and gender on the pharmacokinetics of RP 73401, a phosphodiesterase IV inhibitor.

Early exploratory clinical pharmacokinetic studies can provide valuable information for the design and control of subsequent phase 2 studies. This philosophy was instituted for the compound RP 73401, a specific phosphodiesterase IV inhibitor, that was being developed simultaneously for delivery by both oral and pulmonary routes of administration. The objective of these studies was to separately evaluate the effect of food and gender on the pharmacokinetics of RP 73401 using small-scale focused pilot studies. In the first study, 400 mcg of inhaled RP 73401 were administered to male and female healthy volunteers (n = 8 f, 8 m. In the second study, 400 mcg oral RP 73401 were administered to healthy male volunteers (n = 8) in the fed and fasted state in a crossover fashion. Serial plasma samples were collected for 24 hours and analyzed for RP 73401 using an HPLC method with post-column photochemical reaction and fluorescence detection that had a minimum quantifiable limit of 10 pg/ml. Pharmacokinetic parameters were calculated using non-compartmental techniques. Comparison of male and female pharmacokinetics following inhalation administration showed no statistically significant differences in the absorption and disposition of RP 73401 with respect to AUC, Cmax, tmax, and t1,2 values. Conversely, RP 73401 administered subsequently to a high fat meal showed a 51% reduction in Cmax and a 5-fold prolongation in tmax as compared to the fasted state. However, there was no statistically significant difference in the systemic availability of RP 73401 as assessed through AUC0-infinity comparisons. These results successfully allowed the uncomplicated inclusion of females in oral and inhalation studies with RP 73401 and indicated the need to address oral drug dosing conditions in order to minimize sources of pharmacokinetic variability in subsequent phase 2 studies.

Multiresidue Method for the Determination of Residues of 251 Pesticides in Fruits and Vegetables by Gas Chromatography/Mass Spectrometry and Liquid Chromatography with Fluorescence Detection

A method is described for the determination of 251 pesticide and degradation product residues in fruit and vegetable samples. Extraction of the sample with acetonitrile is followed by a salting-out step. Co-extractives are removed by passing a portion of the acetonitrile extract through an octadecyl (C18) solid-phase extraction cleanup cartridge and then, in a second cleanup, through a carbon cartridge coupled to an amino propyl cartridge. Determination is by gas chromatography with mass-selective detection in the selected-ion monitoring mode, and by liquid chromatography with post-column reaction and fluorescence detection for N-methyl carbamates. The method has been used for analysis of various fruits and vegetables, such as apple, banana, cabbage, carrot, cucumber, lettuce, orange, pear, pepper, and pineapple. Limits of detection range between 0.02 and 1.0 mg/kg for most compounds. Over 80% of the compounds have a limit of detection of < or = 0.04 mg/kg.

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method: II. Methods of detection and quantification and overall long-term performance

By separating the first six linear and cyclic oligomers of polyamide-6 on a reversed-phase high-performance liquid chromatographic system after sandwich injection, quantitative determination of these oligomers becomes feasible. Low-wavelength UV detection of the different oligomers and selective post-column reaction detection of the linear oligomers with o-phthalic dicarboxaldehyde (OPA) and 3-mercaptopropionic acid (3-MPA) are discussed. A general methodology for quantification of oligomers in polymers was developed. It is demonstrated that the empirically determined group-equivalent absorption coefficients and quench factors are a convenient way of quantifying linear and cyclic oligomers of nylon-6. The overall long-term performance of the method was studied by monitoring a reference sample and the calibration factors of the linear and cyclic oligomers.

Post-column photochemical reaction detection in liquid chromatography based on photosensitized generation of singlet molecular oxygen: Study of reaction parameters and application to the determination of polychlorinated biphenyls

The parameters effecting the sensitivity and selectivity of a photochemical reaction detection scheme based on the reaction of 3-substituted pyrroles with singlet molecular oxygen (1O2) in HPLC are reported. Polychlorinated biphenyls (PCBs) were chosen as model compounds for the detection scheme. Following separation by reverse-phase chromatography, PCBs are excited by a Hg pen-ray lamp in a crocheted PTFE photochemical reactor. PCBs that are efficient1O2-sensitizers promote ground state O2 (3Σ-3) to an excited state (1Σg+ or1Δg) which rapidly oxidizes 3-substituted pyrroles which are added to the mobile phase. Detection is based on the loss of pyrrole. The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of1O2. Detection limits for 4,4′-dichlorobiphenyl and Aroclors 1242, 1248 and 1254 were improved by 1–2 orders of magnitude over optimized UV-absorbance detection. Configuration of the photochemical reactor and judicious choice of the wavelength used to follow the loss of pyrrole were determined to be the most important factors in terms of sensitivity of the detection scheme. Comparison of three reagents (i.e. 3-substituted-pyrroles) used for trapping1O2 demonstrates the effect of substitution on sensitivity and to some extent selectivity of PCB determination.

Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region.

High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates.

Major sensitivity improvements in ion chromatography determinations involving post-column spectrophotometric reaction detectors through elimination of pump noise using a dual wavelength monitoring procedure

A novel dual wavelength procedure is described for the elimination of baseline pump noise in spectrophotometric post-column reaction detection systems in high-performance liquid chromatography. The method is applicable if there is some overlap between the spectrum of the analyte reaction product and the spectrum of the excess reagent. One wavelength is chosen to monitor the analyte reaction product and the other wavelength is set close to the isosbestic point. After simple arithmetic manipulation of the data using commonly available computer software, the pump noise is subtracted from the analyte baseline, allowing much higher sensitivity settings to be used. The two examples shown are taken from the ion chromatography of trace metals using colorimetric post-column reaction detectors, where close to a 20-fold increase in signal to noise ratio was obtained when using the dual wavelength procedure.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection: a biomarker of oxidative DNA damage upon kidney transplantation.

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35+/-0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Post-column reaction detection based on fluorescence energy transfer in the far red spectral region

Post-column reaction detection may result in enhanced analytical sensitivity and selectivity. This paper describes an on-line HPLC with post-column fluorescence energy transfer assay using biotin as a model analyte. Biotin labeled with R-phycoerythrin was used as the donor labeled ligand and streptavidin labeled with an indodicarbocyanine dye (Cy5), the acceptor labeled binder protein. The use of these labels provided a detection wavelength in the far red spectral region which is more selective for biological samples. In the on-line system, biotin was injected into the HPLC system followed by Cy5 labeled streptavidin and R-phycoerythrin labeled biotin, post-column. The mixture was incubated on-line in an open tubular reactor coil maintained at 37°C. The measured response was the sensitized emission of Cy5 due to fluorescence energy transfer from R-phycoerythrin labeled biotin measured at 670 nm. Excitation was at 488 nm, which provided a large Stokes shift for reduction of scatter interference. The system was optimized with regard to the post-column reagents to obtain the minimum detectable concentration while maintaining appropriate dynamic range for the analysis of biotin. Biotin spiked in 0.01 M phosphate buffer, pH 7.4, showed a dynamic range of 304.0 pg/ml–122.20 ng/ml with a correlation coefficient of 0.993. The limit of detection for this assay was 304.0 pg/ml. The precision calculated at the blank ( n=6) was 4.14%.

New strategies for determination of transition metals by complexation ion-exchange chromatography and post column reaction

A method for the simultaneous determination of metals (Fe 3+, Fe 2+, Cu, Ni, Zn, Co, Cd, Mn and Pb) at μg/l levels in environmental matrices is demonstrated. The metals are separated by ion chromatography on a mixed-bead ion-exchange column (IonPac CS5A) and detected by post-column reaction and spectrophotometric detection. The use of a gradient program (based on oxalic acid–hydrochloric acid–sodium chloride and sodium nitrate eluents) allows the separation of nine metals in a single run. The direct injection of a large volume of samples lowers the detection limits to below and/or few μg/l. The chromatographic conditions and colour-development reaction were optimised. Two post-column reagents, 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) and 4-(2-pyridylazo) resorcinol, were tested and detection limits were compared. The use of 5-Br-PADAP increased the sensibility for Cu, Cd, Zn and Mn. For the proposed method, linearity, reproducibility, detection limits and interference due to alkaline-earth metals (noticeably Mg and Ca) were discussed. Under the suggested chromatographic conditions Mg 2+ and Ca 2+ up to 0.2 g/l do not interfere with separation and quantitative analysis of the metals. The calibration graphs were linear ( r 2>0.986) in the range 5–5000 μg/l. The application of the method to the determination of metals in coastal marine sediments is shown.

セミミクロカラムを用いた高速液体クロマトグラフィー カラムスイッチングシステムによるヒト血清および血清限外ろ過液中のワルファリンの定量

A simple and sensitive reversed-phase high-performance liquid chromatographic method was developed for the analysis of warfarin (WF) in both human serum and serum ultrafiltrate. WF and internal standard (I.S.) were extracted from the serum by simple deproteinization and filtration with a membrane filter. The Column-switching system utilized the short polymer-coated mixed-function (PCMF) phase deproteinization column coupled with a C18 semi-micro analytical column using a mobile phase of acetonitrile, water and NH4H2PO4, followed by a post-column photochemical reaction and fluorimetric detection. The extraction recovery was greater than 80% and the WF and I. S. were separated from the endogenous components in the serum and serumultrafiltrate. The calibration curve was linear from 5 to 1000 ng/mL of serum and 1 to 150 ng/ mL of the serum ultrafiltrate. The limit of quantification was 5 and 1 ng/mL for found to be the serum and serum ultrafiltrate, respectively. The inter and intra-day coefficients of variation for the serum and serum ultrafiltrate ranged less than 10%, respectively. WF was found to be the stable in the serum for a 4-month period during storage at -20°C.