Use of Sequential Injection technique and robotics for the automation of rhFXIII fluorometric activity assay. case study

This paper evaluates the feasibility of two systems—the Sequential Injection (SI) analyzer and the Zymark Benchmate (ZB) robot—for the automation of a recombinant human Factor Thirteen (rhFXIII) fluorometric assay. The goal was to develop a routine analytical procedure suitable for the quality control lab environment. The experimental efforts focused on monitoring of the product formation for the condensation reaction between monodansyl cadaverin and dimethyl casein. The acquired kinetic data demonstrated that both systems are capable of automating the solution handling operations associated with the assay. Using a method developed with the SI system, samples containing 0–410 μg/ml of rhFXIII were analyzed, with a throughput of one sample per 8 min, and a total solution consumption of 0.8 ml. The relative standard deviation for 10 injections of 100 μg/ml rhFXIII sample was 0.91%. With the ZB robot, samples containing 0–2500 μg/ml of rhFXIII were analyzed, and the linear response was obtained for a concentration range between 0 and 1250 μg/ml of rhFXIII. The method had a sample throughput of one sample per 11 min and a total solution consumption of 6.3 ml for each analysis. Due to its commercial availability, the ZB system was preferred over the experimental SI system for the purpose of routine automated analysis of a large number of samples in the quality control lab environment.     

顺序注射停流分光光度法测定痕量碘

食物中碘的含量一直是人们关注的问题,研究食物中微量碘的分析方法具有重要的现实意义.目前测定碘的方法主要有色谱法[1,2]、荧光法[3,4]、分光光度法[5,6]等.     

Sequential injection technique for automated titration: spectrophotometric assay of vitamin C in pharmaceutical products using cerium(IV) in sulfuric acid

For the first time sequential injection analysis (SIA) technique has been employed for titrimetry. A new SI titrimetric spectrophotometric method for the assay of vitamin C in drug formulations was explored. The method is based on the oxidation reaction of vitamin C with cerium(IV) in sulfuric acid media using a spectrophotometer as a detector with the wavelength monitored at 410 nm. A 2(3) factorial design chemometric approach was employed to study the interaction effect of the chemical and system variables, mainly cerium(IV), sulfuric acid concentrations and the flow rate. The results of the chemometric optimization revealed that the optimum operating conditions for the SI titrimetric analysis of vitamin C were 7.0 x 10(-3) M cerium(IV), 0.455 M sulfuric acid and 28.9 microL s-1 flow rate. A linear calibration plot for the determination of vitamin C was obtained in the concentration range between 30 to 200 ppm. The method was applied to the determination of vitamin C in pharmaceutical preparations and no excipient was found to pose any interference, thus rendering the method suitable for the determination of the drug in pharmaceutical preparations. The SIA method is found to be accurate when the results were statistically compared with the results obtained by the BP standard method. The SIA method is superior when compared to the conventional titration method, the BP standard method and previous methods with respect to precision and automation in solution handling.     

Sequential injection analysis for automation of the Winkler methodology, with real-time SIMPLEX optimization and shipboard application

A multipurpose analyzer system based on sequential injection analysis (SIA) for the determination of dissolved oxygen (DO) in seawater is presented. Three operation modes were established and successfully applied onboard during a research cruise in the Southern ocean: 1st, in-line execution of the entire Winkler method including precipitation of manganese (II) hydroxide, fixation of DO, precipitate dissolution by confluent acidification, and spectrophotometric quantification of the generated iodine/tri-iodide (I₂/I₃⁻), 2nd, spectrophotometric quantification of I₂/I₃⁻ in samples prepared according the classical Winkler protocol, and 3rd, accurate batch-wise titration of I₂/I₃⁻ with thiosulfate using one syringe pump of the analyzer as automatic burette.In the first mode, the zone stacking principle was applied to achieve high dispersion of the reagent solutions in the sample zone. Spectrophotometric detection was done at the isobestic wavelength 466 nm of I₂/I₃⁻. Highly reduced consumption of reagents and sample compared to the classical Winkler protocol, linear response up to 16 mg L⁻¹ DO, and an injection frequency of 30 per hour were achieved. It is noteworthy that for the offline protocol, sample metering and quantification with a potentiometric titrator lasts in general over 5 min without counting sample fixation, incubation, and glassware cleaning. The modified SIMPLEX methodology was used for the simultaneous optimization of four volumetric and two chemical variables. Vertex calculation and consequent application including in-line preparation of one reagent was carried out in real-time using the software AutoAnalysis. The analytical system featured high signal stability, robustness, and a repeatability of 3% RSD (1st mode) and 0.8% (2nd mode) during shipboard application.     

Sequential injection technique for determination of phenoxybenzamine hydrochloride and metoclopramide in pharmaceutical formulations

A sequential injection analysis (SIA) system is described for the determination of phenoxybenzamine hydrochloride and metoclopramide using spectrophotometer as detector. The method is based on the detection of an unstable red intermediate compound resulting from the reaction of phenoxybenzamine hydrochloride or metoclopramide with the diazotizating product of p-phenylenediamine with sodium nitrite in hydrochloric acid medium. The sampling frequency is 69 h⁻¹ and 75 h⁻¹ for phenoxybenzamine hydrochloride and metoclopramide, respectively. The linear range is 10–400 μg/mL for phenoxybenzamine hydrochloride with a detection limit of 0.081 μg/mL and 20–250 μg/mL for metoclopramide with a detection limit of 0.034 μg/mL. The RSD is 1.01 and 0.45% for phenoxybenzamine hydrochloride and metoclopramide, respectively. The proposed methods were used to determine phenoxybenzamine hydrochloride and metoclopramide in pharmaceuticals. The results are compared with those obtained by pharmacopoeia method. The article is published in the original.     

Sequential injection titration with spectrophotometric detection for the assay of acidity in fruit juices.

A simple sequential injection analysis (SIA) with spectrophotometric detection for an assay of acidity in fruit juice was investigated. An alkaline reagent (sodium hydroxide), a sample and an indicator (phenolphthalein) were first aspirated and stacked as adjacent zones in a holding coil. With flow reversal through a reaction coil to the detector, zone penetration occurred, leading to a neutralization reaction that caused a decrease in the color intensity of the indicator being monitored for absorbance at 552 nm. The effects of various parameters were studied. Linear calibration graphs for acidities of 0.2 - 1.0 and 0.5 - 2.5% w/v citric acid as a standard, with a relative standard deviation of 1% (acidity of 0.3 - 0.6% w/v as citric acid, n=11) and a sample throughput of 30 samples h(-1), were achieved. The developed method was validated by a standard titrimetric method for assaying the acidity of fruit juice samples.     

顺序注射停留分光光度法测定磺胺乙酰钠

在HCl浓度为5.0×10-4mol/L时,磺胺乙酰钠-NaNO2-1-萘胺的重氮化偶合反应,生成不稳定的红色产物,利用顺序注射停留技术在473nm波长处检测该不稳定产物,建立了快速自动测定磺胺乙酰钠的新方法。该方法线性范围为5～2500μg/mL,检出限为0.018μg/mL,进样频率为62个/h。应用于药物中磺胺乙酰钠的测定,并与药典规定方法进行对照。     

Sequential optimization of a flow injection spectrophotometric method for the assay of chlorpromazine in pharmaceutical preparations

A new simple flow injection spectrophotometric method for the assay of chlorpromazine using cerium(IV) in sulfuric acid media was developed. The oxidized form of the drug was monitored at the maximum absorbance of 526 nm. The optimum conditions were 0.035M sulfuric acid, 3.80 x 10(-3)M cerium(IV), flow rate 4.85 ml/min, coil length 45 cm and sample size 110 mm(3). Optimization was carried out by the modified simplex method. Response surface methodology was employed to investigate the ruggedness of the method. A sampling frequency of 120 hr(-1) was attained. Relative standard deviations for standard sample were usually less than 0.75. The method was applied to the determination of chlorpromazine in proprietary drugs and results were statistically compared with the official British Pharmacopoeia (BP) method.     

Sequential injection technique for the determination of chlorpromazine hydrochloride in pure form and pharmaceutical formulations

A simple, economical, and automated spectrophotometric method for the determination of chlorpromazine hydrochloride by sequential injection analysis using ammonium metavanadate as colorimetric reagent is proposed. The various chemical and physical conditions that affected the reaction have been thoroughly investigated. The calibration curve was linear within the range 10–100 μg/mL. The detection limit (S/N = 3) was 0.7 μg/mL and the limit of quantification (S/N = 10) was 2.3 μg/mL. The sampling frequency was 22 h⁻¹. The method has been used for the determination of chlorpromazine hydrochloride in pure form and pharmaceutical formulations. The t-test has revealed that there is no evidence of significant differences between the obtained results at the 95% confidence level. The method can be applied to the quantitative determination of chlorpromazine hydrochloride. It is also applicable in the quality control of chlorpromazine hydrochloride preparations. The text was submitted by the authors in English.     

Notes on analytical procedures : Determination of carbon dioxide

For the determination of carbon dioxide, methods suitable for general applications are described. a. Macro-titration method. The CO2 is absorbed in sodium hydroxide in a bubble column filled with glass beads. The excess of NaOH is titrated after the addition of BaCI2. b. Scmi-micro-titration method. The CO2 is absorbed in barium hydroxide, the excess of which is determined by titration with 0.1 n hydrochloric acid on cresolphthalein as the indicator. The absorption and titration are performed in a special vessel in which the liquid is kept in circulation by the injected stream of air. c. Gravimetrical method.Method b. is specially recommended.     

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

Micro flow injection analysis combined with a separation technique for the urinary glucose assay.

A urinary glucose assay has been investigated, employing a micro flow injection analysis (microFIA) combined with a separation technique of glucose from the analyte. The adsorption part using activated alumina for the glucose in the analyte can be successively integrated onto a microFI chip. The selective adsorption-desorption of glucose in the artificial urine can progress on the adsorption part. Along with this selective preconcentration of glucose, the typical FI peak of glucose can be obtained just by feeding the sample and deionized water as an elutant sandwiched with the reagent on the carrier stream. The glucose concentration in artificial urine can be quantitatively determined with the present microFIA system, while the interference of other components coexisting in urine occurs in the case of the conventional FIA system without any separation part. The described method serves as a template for improving the selectivity for the analyte in the multi-component system.     

分子信标探针荧光法测定慢性粒细胞性白血病相关基因

根据慢性粒细胞性白血病(CML)相关基因b3a2序列设计了一种带有荧光基团和淬灭基团的凸环结构探针(分子信标,MB),研究其与互补目标DNA杂交前后的荧光变化行为,建立了b3a2基因检测的新方法.在最适条件下,得到杂交后溶液荧光强度与本底荧光强度的比值(S/B)和目标DNA 的浓度呈线性关系,r=0.9973,线性范围4.0×10-9～3.2×10-8 mol/L.该方法为实际样品的检测奠定了基础.     

Modifications to increase the efficiency of the fluorometric cycling assay for cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is an intracellular messenger that triggers the release of calcium ions from intracellular stores in a variety of cell types. The fluorometric cycling assay has become the preferred method for measuring cADPR due to its high level of sensitivity (in the sub-nanomolar range) and its use of commercially available reagents. Additionally, the assay is performed in multiwell plates, making it suitable for high throughput screening using a fluorescence plate reader. The findings reported in this paper present several problems that may be encountered during various stages of the assay, and provide solutions to these problems. Modifications to the assay address reduced recovery of sample and cADPR with removal of perchloric acid (PCA) using organic solvent, reduction in diaphorase activity with heat treatment, and effects on resorufin fluorescence by pH range. Using these modifications, we report an increase of approximately 15% in recovery of brain cADPR, and show that between-subject variability is greatly reduced. We hope that these observations will encourage more widespread application of this valuable assay.     

Flow injection analysis: A useful alternative for solving analytical problems

Summary In addition to its well-known advantages (e.g. ease of automation, high sample throughout and versatility, low sample and reagent consumption and analytical costs), flow injection analysis (FIA) offers the possibility of solving a number of specific analytical problems where poor sensitivity or selectivity, time-consuming preliminary steps, the use of HPLC in routine control for the analysis of a large number of samples, the determination of various analytes (multidetermination) or process monitoring in near-real time is involved.     

Enzyme-linked immunosorbent assay for okadaic acid: investigation of analytical conditions and sample matrix on assay performance

Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28 +/- 0.38 ng/mL, corresponding to 12.8 +/- 3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y = 1.0064x - 10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.