A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

We discribe the development of a simplified radioimmunoassay for triiodothyronine (T₃) using pre-incubated labelled T₃ and antibody. The assay is carried out by adding 50 l of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction is allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm³ of 22% polyethylene glycol solution. Due to the high dissociation constant of T₃-antibody complex at 37° C (2.83·10^–4 S^–1), the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. With a four hour incubation the sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and compared with the equilibrium assay (Y=0.94x+0.046 ng/cm³, r=0.98).     

A solid phase radioimmunoassay for triiodothyronine using antibody-immobilized serum albumin microspheres

A solid phase radioimmunoassay for triiodothyronine (T₃) has been developed using antibody-immobilized serum albumin microspheres. Antibody albumin microspheres were prepared using a spinning disc aerosol generator. The low density of the antibody-albumin microspheres gives greater mobility for the particles there by ensuring better kinetics to the antigen-antibody reaction. The assay has a single incubation of one hour at 37° C and the separation of the antigen-antibody complex is accomplished by centrifugation. The sensitivity of this assay is 0.3 ng/cm³ and has a range of 0.3–4 ng/cm³.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Determination of mefloquine in blood, filter paper-absorbed blood and urine by 9-fluorenylmethyl chloroformate derivatization followed by liquid chromatography with fluorescence detection

We describe a method for determination of mefloquine (MQ) in 100-microliters samples of urine, whole blood, and capillary blood collected on filter paper; quantification is by liquid chromatography with fluorescence detection at 475 nm of the 9-fluorenylmethyleneoxycarbonyl derivative. Whole blood and urine samples were prepared by extraction of MQ and internal standard from aqueous base with methyl tert.-butyl ether (MTBE), separation and evaporation of the MTBE layer, and derivatization using a solution of 9-fluorenylmethyl chloroformate in acetonitrile. Filter paper spots were immersed for 16 h in 0.1 M hydrochloric acid, followed by extraction with MTBE from aqueous sodium carbonate. The separated and evaporated organic layer was treated with the derivatizing solution. An aliquot was injected onto a high-performance liquid chromatography system using a C18 reversed-phase column and acetonitrile-water (72:28) mobile phase for filter paper spot extracts as for whole blood and urine extracts. The method has a limit of determination in blood, blood spots, and urine of 50 ng/ml with 100 microliters sample size (coefficient of variation = 16%). Linearity and precision (within-day and between-day) for the method are good. The MQ derivative was isolated and characterized spectroscopically. Values for MQ concentrations in filter paper blood spots compared favorably with values found in corresponding whole blood samples analyzed by a published method.     

Enzyme Immunoassay of Thyroid-Stimulating Hormone Using Dried Blood Samples a Simple Technique of Screening for Congenital Hypothyroidism

Abstract

A method for enzyme imnunoassay of thyroid-stimulating hormone (TSH) in dried blood spotted onto filter paper has been developed. TSH was conjugated to horse-radish peroxidase according to Nakane's method. Separation of the bound and free fractions was obtained by a double antibody solid phase method using polyacetal beads which were coated with the purified IgG fraction from goat anti-rabbit IgG serum. p-Hydroxyphenyl propionic acid was used as substrate for the fluorophotometric assay of peroxidase activity. The assay sensitivity is 0.07, μU TSH/assay tube, which is equivalent to μU/ml when five 3 mm discs of dried blood spot are assayed. TSH values in dried blood samples obtained by this method correlate well with those of serum samples obtained by radioimmunoassay (r=0.89). The coefficients of variation were 6.8 to 13.4% (within assay) and 5 to 40% (between assay). The enzyme immunoassay of TSH presented here is applicable to the mass-screening for congenital hypothyroidism of neonate.     

Solid phase radioimmunoassay for testosterone in human serum using antibodies coupled to magnetizable cellulose

Summary A simple user-friendly, solid phase radioimmunoassay for testosterone in human serum based on magnetic particles is described. IgG fractions precipitated from polyclonal testosterone antiserum were coupled to magnetizable cellulose particles using carbonyl diimidazole. The prepared antibody solid phase was stable for one year when stored at 4 °C. The optimized assay involves the incubation of 50 ml of testosterone standards (0.3-10 ng/ml), 100 ml of magnetizable cellulose particle coupled antibody suspension and 100 ml of ¹²⁵I-testosterone derivative for 4 hours at 37 °C. At the end of the incubation, the tubes were placed on a magnetic rack for 10 minutes after the addition of wash buffer and decanted. The sensitivity of the assay is 0.2 ng/ml. The intra-assay variation was <15% throughout the assay range. The recovery varied from 88 to 115%. On dilution of samples having high levels of testosterone, linearity ranged between 87 and 125%. Correlation coefficient of 0.978 was obtained when the developed solid phase assay was compared to the earlier established liquid phase assay.     

An Enzyme Immunoassay for Determining Immunoreactive Trypsinogen (IRT) in Dried Blood Spots on Filter Paper Using an Ultra-Microanalytical System

Cystic fibrosis (CF) is a severe autosomal recessive disorder. It is caused by mutations in the CF transmembrane conductance regulator gene. Early diagnosis of CF can be carried out by determining high immunoreactive trypsinogen (IRT) blood values in newborns. A simple sandwich-type ultramicroELISA assay (UMELISA®) has been developed for the measurement of IRT in dried blood spots on filter paper. Strips coated with a high affinity monoclonal antibody directed against IRT are used as solid phase, to ensure the specificity of the assay. The assay is carried out within 20 h. The useful rank of the curve is 0–500 ng/mL, and the lowest detectable concentration is 4.8 ng/mL. Intra- and inter-assay coefficients of variation were lower than 10%. The recovery mean value was 100.3 ± 11.2%. Cross-reactivity with proteins structurally related to IRT (α2-macroglobulin, α1-antitrypsin, and human chymotrypsin) was lower than the detection limit of the assay. Four thousand four hundred six newborn samples from the Cuban Newborn Screening Program were analyzed, and the mean IRT concentration was 12.8 ng/mL. Higher IRT values were obtained when samples were eluted overnight. Regression analysis showed a good correlation with the commercially available AutoDELFIA® Neonatal IRT kit (n?=?3948, r =?0.885, ??=?0.976, p?<?0.01). The analytical performance characteristics of our UMELISA® TIR Neonatal suggest that it can be used for the neonatal screening of CF.     

A resonance light scattering method for the determination of uranium based on a water-soluble salophen and oxalate

A resonance light scattering (RLS) method for the direct detection of uranium (VI) or uranyl in aqueous solution without separation procedure has been reported in this paper. Sulfo-salophen, a water-soluble tetradentate Schiff base ligand of uranyl, reacted with uranyl to form a complex. The complex reacted further with oxalate to form supramolecular dimer with large molecular volume, resulting in a production of strong RLS signal. The amount of uranium (VI) was detected through measuring the RLS intensity. A linear range was found to be 0.2–30.0 ng/mL under optimal conditions with a detection limit of 0.15 ng/mL. The method has been applied to determine uranium (VI) in environmental water samples with the relative standard deviations of less than 5 % and the recoveries of 98.8–105.8 %. The present technique is suitable for the assay of uranium (VI) in environmental water samples collected from different sources.     

A low blood volume LC-MS/MS assay for the quantification of fentanyl and its major metabolites norfentanyl and despropionyl fentanyl in children

Preterm and term neonates often require surgical procedures and analgesia. However, our knowledge about neonatal pharmacokinetics of fentanyl, the most commonly used drug for these procedures, and its metabolites is still incomplete. To facilitate pharmacokinetic studies of fentanyl and its metabolites in neonates and other children, we developed and validated an LC-MS/MS method based on minimally invasive, low blood volume sampling. LC-MS/MS was used for the simultaneous analysis of fentanyl, despropionyl fentanyl (DPF), and norfentanyl from dried blood samples (DBS) collected on filter paper. Positive ions were monitored using multiple reaction monitoring. Since the standard matrix for measuring fentanyl blood concentrations is plasma, the assay was developed and validated in plasma, whole blood, and then DBS. Our method was able to measure clinically relevant levels of fentanyl and its metabolites. In DBS, the lower limits of quantification were 100 pg/mL for fentanyl with a range of reliable response from 0.1 to 100 ng/mL (r(2)>0.99) and 250 pg/mL for both DPF and norfentanyl with a range of reliable response from 0.25 to 100 ng/mL (r(2)>0.99). In plasma and in DBS inter-day accuracy and precisions of fentanyl met predefined acceptance criteria and also indicated comparable assay performance in both matrices.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

Multi-residue screening of bovine urine on xenobiotic oestrogens with an oestrogen radioreceptor assay

An improved radioreceptor assay (RRA) is used for the screening of urine samples from cattle for the presence of exogenous oestrogenic anabolic compounds, e.g., stilbenes and zeranol. The method includes extraction of the hormones from urine samples with simultaneous purification using reversed-phase C18 cartridges. High-performance liquid chromatography is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently analysed using the RRA with the oestrogen receptor, isolated from immature calf uteri, as a binder and tritiated 17 beta-oestradiol as a tracer. Urine samples from untreated calves and cows and samples from calves treated with zeranol-trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol were analysed with this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng/ml 'apparent' oestradiol in urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.     

HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.     

Effect of Reaction Format on a McAb-Based Heterologous ELISA for the Insecticide Parathion

Abstract

Based on the selected monoclonal antibody, the effect of three reaction formats on sensitivity, specificity, and determination ability for real samples were investigated. The results showed the antibody-coated format was the optimal assay for parathion determination. The sensitivity of the assay was 3.20 ng/ml, with a detection limit of 0.40 ng/ml, and the assay time was 1.5 h. The average recoveries of parathion in river water, rice, cucumber, green soy bean, and cabbage were 98.56%, 89.46%, 99.25%, 118.57%, and 101.39%, respectively. In addition, when rice and cabbage extracts were analyzed by the assay and HPLC, the correlation was greater than 0.9.     

Rapid analysis of ranitidine in biological fluids and determination of its erythrocyte partitioning

A reversed-phase ion-pair high-performance liquid chromatographic assay is described for the rapid and sensitive quantitation of the H2-receptor antagonist ranitidine in human plasma and urine. The method involves a single-step extraction of the alkalinized sample with methylene chloride and analysis of the evaporated extract on a cyano column. Detection was performed by ultraviolet absorbance monitored at 318 nm. The overall run time of the assay was 5 min at a flow-rate of 2.0 ml/min. The limit of sensitivity was 1 ng/ml ranitidine in human plasma. Urine and plasma samples collected from a subject after administration of an oral dose of 150 mg of ranitidine were analyzed by this method. Furthermore, the procedure was applied to determine the red blood cell partition coefficient of ranitidine in a concentration range up to 10 micrograms/ml.     

Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.     

High Sensitive and Selective Spectrophotometric Determination of Aluminium after Collection on a Membrane Filter Using 2,3‐Dichloro‐6‐(3‐carboxy‐2‐hydroxy‐1‐naphthylazo)quinoxaline and Zephiramine

Abstract

A high sensitive and selective spectrophotometric method for the determination of aluminium(III) using 2,3‐dichloro‐6‐(3‐carboxy‐2‐hydroxy‐1‐naphthylazo)quinoxaline (DCHNAQ) and zephiramine (zeph) is described. The formed ion pair precipitate between zephiramine and perchlorate ions is effective for the enrichment of aluminium(III) on a membrane filter as its ternary complex with DCHNAQ and zephiramine. The solid–state absorbance of the complex on the membrane filter is measured at 655 nm against a blank thin layer and the difference is calculated. The colour system obeys Beer's law from 5.0–150 ng ml^?1 of aluminium. The detection and quantification limits were calculated. The relative standard deviation for 60 ng of aluminium(III) in 20‐ml sample volume amounts 0.84% (n=10). A ligand buffer solution, composed of transcyclohexane‐1,2‐diaminetetraacetic acid with an excess of zinc(II), is effective for masking interferences from foreign ions, particularly iron(III). The proposed method was applied successfully to tap and environmental water, biological (human blood, urine, and gallstone), and soil samples.     

Quantitation of Verapamil and Norverapamil in Small Blood Samples From the Rat by High Performance Liquid Chromatography

Abstract

A simple, sensitive HPLC assay using flurescence detection was developed for quantitation of verapamil and its active metabolite, norverapamil in 100-200 μl blood samples from the rat. Baseline separation of verapamil, normverapamil and internal standard, propranolol, was attained within 14 minutes. Standard curves for verapamil and norverapamil were linear from 7 ng/ml to 1000 ng/ml with limit of detection of 4 ng/ml for both Compounds. the intraday and interday coefficients of variation in verapamil and norverapamil concentrations, determined from spiked whole blood samples, were less than 10%.     

HPLC Determination of Ochratoxin A in a Low Volume of Human Blood Serum

Abstract

A high‐performance liquid chromatography (HPLC) method has been developed for the determination of ochratoxin A (OTA) in human blood serum. Samples were purified on a C18 solid phase extraction column. The developed method required a relatively low serum volume (0.5 ml). Significant correlation (r of 0.998) was found over the range from 0.10 to 8 ng/ml, with a detection limit of 0.1 ng/ml and better performance in terms of precision and accuracy. Mean recoveries at 0.5 and 2 ng/ml were respectively 69.7±1.2 and 71.9±2.8%. This method was used as a rapid and noninvasive tool to assess human exposure to OTA. Among 40 analyzed serum samples, 27.5% were found to contain OTA with levels going from 0.1 to 11.98 ng/ml with a mean concentration of 0.73±2.35 ng/ml.     

Ion-Paired Liquid Chromatographic Method for the Analysis of Pyridostigmine in Plasma

Abstract

A high performance Liquid chromatographic (HPLC) procedure for the analysis of pyridostigmine in plasma has been developed. Only 0.5 ml of plasma is required for the analysis. The clean-up procedure involves a protein precipitation step and a column elution step prior to separation by HPLC. The assay is quite sensitive with a detection limit of 1.37 ng/ml for pyridostigmine in plasma. Variability of results ranged from 3 to 14% on evaluations of assay precision. Accuracy of results, evaluated using blind samples in the range of 0–50 ng/ml, differed between 6 and 12% from blind sample values. Stability was also determined for pyridostigmine in plasma at ?20°C and ?80°C. The results showed no degradation for pyridostigmine at ?80°C for up to four months. In a preliminary study with one human volunteer, the drug could be detected up to 12 hours following oral syrup solution doses of between 0.4 and 0.9 mg/kg. This assay is suitable for pharmacokinetic studies involving pyridostigmine in human subjects.     

Subnanomolar Determination of Indium by Adsorptive Stripping Differential Pulse Voltammetry Using Factorial Design for Optimization

Abstract

A highly sensitive method to determine of indium is proposed by adsorption stripping differential pulse cathodic voltammetry (AdSDPCV) method. The complex of indium ions with xylenol orange is analyzed based on the adsorption collection onto a hanging mercury drop electrode (HMDE). After accumulation of the complex at ?0.20 V vs. Ag/AgCl reference electrode, the potential is scanned in a negative direction from ?0.40 to ?0.75 V with the differential pulse method. Then, the reduction peak current of In(III)–XO complex is measured. The influence of chemical and instrumental variables was studied by factorial design analysis. Under optimum conditions and accumulation time of 60 s, linear dynamic range was 0.1–10 ng/ml (8.7 × 10^?10 to 8.7 × 10^?8 M) with a limit of detection of 0.03 ng/ml (2.6 × 10^?10 M); at accumulation time of 5 min, linear dynamic range was 0.04–10 ng/ml (3.4 × 10^?10 to 8.7 × 10^?8 M) with a limit of detection of 0.013 ng/ml (1.1 × 10^?10 M). The applicability of the method to analysis of real samples was assessed by the determination of indium in water, alloy, and jarosite (zinc ore) samples.