Coupling immunoassays with chromatographic separation techniques

Coupling immunoassays with HPLC separation techniques is becoming increasingly useful in the analysis of biological and nonbiological samples of both large and small molecules. This is because it provides both sensitivity and selectivity for molecular analysis at relatively low cost, low maintenance and with excellent potential for automation. This paper reviews application of this hyphenated approach both in the pre-column immunoextraction and post-column immunodetection modes. Systems in which immunoassays are interfaced to chromatographic separations in order to separate bound and free fractions of the immunoassay will not be included since these systems do not provide the enhanced selectivity common to hyphenated systems. Post-column immunodetection is based on various immunoassay formats such as direct detection, one-site, competitive and sandwich immunoassays. Homogeneous immunodetectors are more convenient than heterogeneous immunodectors since there are no separation and column regeneration steps involved in homogeneous immunoassays. On the other hand, heterogeneous immunoassays are generally more sensitive than homogeneous immunoassays since interfering substances are removed prior to immunodetection. Advantages and limitations for the various approaches will be discussed.     

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.     

Analysis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (bentiromide) metabolites in urine by ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of bentiromide metabolites in urine. The procedure involves no more than direct injection of the diluted urine sample, obviating the need for an extraction step or an internal standard. A mu Bondapak C18 column is used with a mobile phase of 0.01 M tetrabutylammonium chloride (pH 7.4)--methanol (9:1). A flow-rate of 1.4 ml/min, detection at 254 nm and column temperature of 40 degrees C are employed. These conditions were achieved by investigating the effects of mobile phase pH, and concentrations and types of organic modifiers, buffers and ion-pairing agents on the resolution of the metabolites. The analysis time is 18 min per sample and the coefficient of variation on replicate assays is less than 10% for most concentrations studied. Analytical recoveries were between 95 and 100% throughout the appropriate concentration ranges and no interferences were obtained with the exception of p-acetamidobenzoyl glucuronide which could be eliminated by treatment of the samples with beta-glucuronidase. Concentration profiles of the metabolites were studied in normal subjects, and the method was found to be potentially useful for clinical situations in which the existing bentiromide test leads to ambiguous results because of small bowel and hepatic dysfunctions.     

Determination of (3S)-3-hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser-induced fluorescence detection

Capillary electrophoresis (CE) has been used with collinear laser-induced fluorescence detection (LIF) to determine the amount of (3S)-3-hydroxy quinidine (3OHQ) formed on direct injection of microsomal incubation mixtures. 3OHQ is the CYP 3A4 metabolite of quinidine sulfate (QS) and is therefore useful for metabolism screening studies. The method was validated analytically and tested for its capability of screening for a weak inhibitor of the CYP 3A4 isozyme. A linear calibration was found to provide the best fit for the standard curve with a correlation of 0.9950 and all concentration residuals less than 15%. The percentage relative standard deviations (RSDs) of two controls, 175 and 2250 ng/mL, were 9.29 and 5.68% and the percentage differences from normal (DFN) were 6.87 and -4.37%, respectively. The concentration limit of detection (LOD) for 3OHQ in the incubation matrix was 52.11ng/mL and the mass LOD was approximately 521.1 fg (injection volume 10 nL). The effectiveness of the method to screen for the weak inhibitor erythromycin has been shown by calculating percentage inhibition when incubating with different concentrations of QS. Sensitive detection coupled with the convenience of the direct injection technique makes this an attractive approach for metabolism screening. The small sample size capability of CE will further reduce the quantities of probe drug, microsomes and other reagents required for incubation studies.     

Quantitative liquid chromatography tandem mass spectrometry analysis of macromolecules using signature peptides in biological fluids

Targeted protein quantification using peptide surrogates has increasingly become important to the validation of biomarker candidates and development of protein therapeutics. These approaches have been proposed and employed as alternatives to immunoassays in biological fluids. Technological advances over the last 20 years in biochemistry and mass spectrometry have prompted the use of peptides as surrogates to quantify enzyme digested proteins using triple quadrupole mass spectrometers. Multiple sample preparation processes are often incorporated to achieve quantification of target proteins using these signature peptides. This review article focuses on these processes or hyphenated techniques for quantification of proteins with peptide surrogates. The most recent advances and strategies involved with hyphenated techniques are discussed.     

Comparative evaluation of two substrates for urinary determination of p-aminobenzoic acid by room-temperature phosphorimetry

Filter paper (S & S 903) impregnated with diethylenetriaminopentaacetic acid, and Whatman DE-81 anion-exchange paper are evaluated for quantitation of urinary p-aminobenzoic acid. The two substrates are compared with respect to drying characteristics, pH variations, heavy atom effect, and some other variables. Impregnated S & S 903 paper is superior in terms of drying characteristics and background interferences but DE-81 has several advantages including lower detection limit, wider pH range, and ready availability. Although both substrates seem applicable to clinical application, DE-81 is probably more useful.     

Runaway Electron Measurements in the EAST Tokamak

An experimental study of runaway electrons in the EAST tokamak has been performed by a recently developed multi‐channel hard x‐ray diagnostics based on NaI(TL) scintillator detectors. It is found that in the current quench phase, the inductive loop voltage plays an important role in the generation of runaway electrons. And the avalanche mechanism was the main mechanism for runaway electrons after the disruptions. The distribution and transportation of runaway electrons were also investigated by multi‐channel hard x‐ray diagnostics. It is also found that the intensity of runaway electrons emission in the core plasma was much higher than those in the downside of the cross‐section, while the emission intensity of runaway electrons in the core plasma was almost the same. Calculated shrinking coefficient of runaway electrons emission after the plasma disruption was about 26 m/s according to the experimental data (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Magnetic Separation Immunoassay for Digoxin in Plasma with Flow Injection Fluorescence Detection

Immunoassay (IA) is a sensitive and selective approach for low level quantitation of drugs. Magnetic separation immunoassays use magnetic beads to facilitate the separation of bound labeled antigens from free antigens in solution. Digoxin was chosen for this study because low level analysis (ngmL^–1) in biological samples isrequired, antibodies to digoxin were commercially available and derivatization procedures for fluorescence labeling were well established. A competitive immunoassay format was used in this study. Streptavidin coated magnetic beads were attached to biotinylated anti-digoxin antibodies for the separation. The inhibition curve for off-line magnetic separation immunoassay of digoxin in spiked plasma was characterized and the dynamic range of the curve was 0.25–2.5ngmL^–1. A power fit weighted by the inverse of concentration was found to provide the best fit to the data (r=0.9934). The percent RSDs for the two controls, 0.8 and 2.2ngmL^–1, were 9.95% and 20.62% (n=6) and the percent errors were 11.75% and 22.63% (n=6), respectively. The limit of detection (LOD) in plasma is 0.14ngmL^–1. The dynamic range of the inhibition curve for on-line magnetic separation immunoassay of digoxin was 0.5–15ngmL^–1 of digoxin. A quadratic fit was found to provide the best fit to the data (r=0.9937). The percent RSDs for the two controls, 4.0 and 12ngmL^–1, were 14.1% and 10.7% (n=6) and the percent errors were 5.8% and 3.3% (n=6) from the spiked value, respectively. The LOD was estimated to be 0.44ngmL^–1 (determined as two times the standard deviation of the blank, n=6). The on-line method has the advantages of being relatively easy to automate in the continuous flow mode and is adaptable for use in conjunction with HPLC separations.     

A cellular automata model of chromatography

Dynamic models of the behavior of solvent and solute molecules can be made using cellular automata. A chromatographic column was represented by use of a cellular automata grid of 43 x 200 spaces. Solvent (mobile phase), solute and stationary phase cells were designated to simulate the chromatographic situation. The movements of solute and solvent cells down the grid were monitored for different numbers of iterations, different flow rates and different affinities of the solutes for the stationary phase and the solvent for itself. The cellular automata dynamics were successfully able to model expected chromatographic behavior except in a few cases where the number of cells was not large enough to provide an average value reflective of the molecular situation.