ABSTRACT
BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.
Subject(s)
Amino Acids, Diamino/analysis , Lathyrus/chemistry , Neurotoxins/analysis , Chromatography, Liquid/economics , Chromatography, Liquid/methods , Costs and Cost Analysis , Isotope Labeling , Lathyrus/genetics , Mass Spectrometry/economics , Mass Spectrometry/methods , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry/economics , Spectrophotometry/methods , Time FactorsABSTRACT
Spectrophotometry is a fundamental technique in many areas of science, with many applications and uses. The cost of spectrophotometers has acted as a barrier on the teaching and use of the technique. Here, we provide open-source plans to a 3D-printed cuvette holder with an interchangeable narrow-spectral bandwidth light-emitting diode (LED) block that can be used in conjunction with a smartphone's ambient light sensor (ALS) to perform spectrophotometry. A Lego version with an interchangeable LED block is also presented. Results from the smartphone spectrophotometer in comparison with commercially available spectrophotometers demonstrated functionality, and the model may have many applications, especially in indirect spectrophotometry, such as in the protein assay shown here. The plans for the 3D-printed model are freely available on GitHub, as are editable files to allow customisation by users. We would encourage users to share adaptations with the scientific community.
Subject(s)
Spectrophotometry/instrumentation , Information Dissemination/methods , Printing, Three-Dimensional , Smartphone , Spectrophotometry/economicsABSTRACT
With the rapid development and proliferation of mobile devices with powerful computing power and the ability of integrating sensors into mobile devices, the potential impact of mobile health (mHealth) diagnostics on the public health is drawing researchers' attention. We developed a Smartphone Octo-channel Spectrometer (SOS) as a mHealth diagnostic tool. The SOS has nanoscale wavelength resolution, is self-illuminated from the smartphone itself, and is ultra-low cost (less than $20). A user interface controls the optical sensing parameters and precise alignment. After calibrating and testing the SOS by quantifying protein concentrations, we clinically validated the SOS by comparing the diagnostic performance of our device with that of a clinical spectrophotometer. About 180 serum samples from de-identified patients with 4 types of autoantibodies were blindly read the ELISA results. The accuracy of the SOS achieved 100% across the clinical reportable range compared with the FDA-approved instrument. Furthermore, the self-illuminated SOS only requires about half of the light intensity of the FDA-approved instrument to achieve clinical-level sensitivity. The low-energy-consumption and low-cost SOS enables point-of-care spectrophotometric sensing in low-resource areas, and can be integrated into point-of-care diagnostic systems for rapid multiplex readout and analysis at patient bedside or at home.
Subject(s)
Costs and Cost Analysis , Point-of-Care Systems , Smartphone/instrumentation , Spectrophotometry/economics , Spectrophotometry/instrumentation , Telemedicine/instrumentation , Animals , Cattle , Equipment Design , Humans , Serum Albumin, Bovine/analysisABSTRACT
We report a miniature, visible to near infrared G-Fresnel spectrometer that contains a complete spectrograph system, including the detection hardware and connects with a smartphone through a microUSB port for operational control. The smartphone spectrometer is able to achieve a resolution of ~5 nm in a wavelength range from 400 nm to 1000 nm. We further developed a diffuse reflectance spectroscopy system using the smartphone spectrometer and demonstrated the capability of hemoglobin measurement. Proof of concept studies of tissue phantoms yielded a mean error of 9.2% on hemoglobin concentration measurement, comparable to that obtained with a commercial benchtop spectrometer. The smartphone G-Fresnel spectrometer and the diffuse reflectance spectroscopy system can potentially enable new point-of-care opportunities, such as cancer screening.
Subject(s)
Hemoglobins/analysis , Point-of-Care Systems , Smartphone , Humans , Phantoms, Imaging , Spectrophotometry/economics , Spectrophotometry/instrumentation , Spectrophotometry/methodsABSTRACT
Current reported smartphone spectrometers are only used to monitor or measure one sample at a time. For the first time, we demonstrate a multichannel smartphone spectrometer (MSS) as an optical biosensor that can simultaneously optical sense multiple samples. In this work, we developed a novel method to achieve the multichannel optical spectral sensing with nanometer resolution on a smartphone. A 3D printed cradle held the smartphone integrated with optical components. This optical sensor performed accurate and reliable spectral measurements by optical intensity changes at specific wavelength or optical spectral shifts. A custom smartphone multi-view App was developed to control the optical sensing parameters and to align each sample to the corresponding channel. The captured images were converted to the transmission spectra in the visible wavelength range from 400nm to 700nm with the high resolution of 0.2521nm per pixel. We validated the performance of this MSS via measuring the concentrations of protein and immunoassaying a type of human cancer biomarker. Compared to the standard laboratory instrument, the results sufficiently showed that this MSS can achieve the comparative analysis detection limits, accuracy and sensitivity. We envision that this multichannel smartphone optical biosensor will be useful in high-throughput point-of-care diagnostics with its minimizing size, light weight, low cost and data transmission function.
Subject(s)
Biosensing Techniques/instrumentation , High-Throughput Screening Assays/instrumentation , Interleukin-6/blood , Point-of-Care Systems , Serum Albumin, Bovine/analysis , Smartphone/instrumentation , Animals , Biosensing Techniques/economics , Cattle , Equipment Design , High-Throughput Screening Assays/economics , Humans , Immunoassay/economics , Immunoassay/instrumentation , Limit of Detection , Smartphone/economics , Spectrophotometry/economics , Spectrophotometry/instrumentationABSTRACT
A sensor was developed for simultaneous measurements of carbon monoxide (CO) and carbon dioxide (CO2) fluctuations in internal combustion engine exhaust gases. This sensor utilizes low-cost and compact light-emitting diodes (LEDs) that emit in the 3-5 µm wavelength range. An affordable, fast response sensor that can measure these gases has a broad application that can lead to more efficient, fuel-flexible engines and regulation of harmful emissions. Light emission from LEDs is spectrally broader and more spatially divergent when compared to that of lasers, which presented many design challenges. Optical design studies addressed some of the non-ideal characteristics of the LED emissions. Measurements of CO and CO2 were conducted using their fundamental absorption bands centered at 4.7 µm and 4.3 µm, respectively, while a 3.6 µm reference LED was used to account for scattering losses (due to soot, window deposits, etc.) common to the three measurement LEDs. Instrument validation and calibration was performed using a laboratory flow cell and bottled-gas mixtures. The sensor was able to detect CO2 and CO concentration changes as small as 30 ppm and 400 ppm, respectively. Because of the many control and monitor species with infra-red absorption features, which can be measured using the strategy described, this work demonstrates proof of concept for a wider range of fast (250 Hz) and low-cost sensors for gas measurement and process monitoring.
Subject(s)
Air Pollutants/analysis , Carbon Dioxide/analysis , Carbon Monoxide/analysis , Environmental Monitoring/instrumentation , Spectrophotometry/instrumentation , Environmental Monitoring/economics , Equipment Design , Lasers , Light , Limit of Detection , Spectrophotometry/economicsABSTRACT
Four rapid, simple, accurate and precise spectrophotometric methods were used for the determination of ciprofloxacin in the presence of its acidic degradation product. The methods under study are ratio derivative, ratio difference, mean centering and dual wavelength. All the methods were validated according to the ICH guidelines and the obtained accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can be used for the routine analysis of ciprofloxacin in quality-control laboratories.
Subject(s)
Anti-Bacterial Agents/analysis , Ciprofloxacin/analysis , Spectrophotometry/methods , Acids/analysis , Analysis of Variance , Drug Stability , Limit of Detection , Reproducibility of Results , Spectrophotometry/economicsABSTRACT
Four rapid, simple, accurate and precise spectrophotometric methods were used for the determination of ciprofloxacin in the presence of metronidazole as interference. The methods under study are area under the curve, simultaneous equation in addition to smart signal processing techniques of manipulating ratio spectra namely Savitsky-Golay filters and continuous wavelet transform. All the methods were validated according to the ICH guidelines where accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can therefore be used for the routine analysis of ciprofloxacin in quality-control laboratories.
Subject(s)
Anti-Infective Agents/analysis , Ciprofloxacin/analysis , Metronidazole/analysis , Spectrophotometry/methods , Anti-Bacterial Agents/analysis , Limit of Detection , Spectrophotometry/economics , Wavelet AnalysisABSTRACT
In this paper, a simple and cost effective method was developed for extraction and pre-concentration of carmine in food samples by using cloud point extraction (CPE) prior to its spectrophotometric determination. Carmine was extracted from aqueous solution using Triton X-100 as extracting solvent. The effects of main parameters such as solution pH, surfactant and salt concentrations, incubation time and temperature were investigated and optimized. Calibration graph was linear in the range of 0.04-5.0 µg mL(-1) of carmine in the initial solution with regression coefficient of 0.9995. The limit of detection (LOD) and limit of quantification were 0.012 and 0.04 µg mL(-1), respectively. Relative standard deviation (RSD) at low concentration level (0.05 µg mL(-1)) of carmine was 4.8% (n=7). Recovery values in different concentration levels were in the range of 93.7-105.8%. The obtained results demonstrate the proposed method can be applied satisfactory to determine the carmine in food samples.
Subject(s)
Carmine/analysis , Chemical Fractionation/methods , Food Analysis/methods , Food Coloring Agents/analysis , Spectrophotometry/methods , Carmine/isolation & purification , Food Coloring Agents/isolation & purification , Octoxynol/chemistry , Spectrophotometry/economics , Surface-Active Agents/chemistryABSTRACT
Four simple, sensitive, accurate and precise spectrophotometric methods were developed for the simultaneous determination of a binary mixture containing Pantoprazole Sodium Sesquihydrate (PAN) and Itopride Hydrochloride (ITH). Method (A) is the derivative ratio method ((1)DD), method (B) is the mean centering of ratio spectra method (MCR), method (C) is the ratio difference method (RD) and method (D) is the isoabsorptive point coupled with third derivative method ((3)D). Linear correlation was obtained in range 8-44 µg/mL for PAN by the four proposed methods, 8-40 µg/mL for ITH by methods A, B and C and 10-40 µg/mL for ITH by method D. The suggested methods were validated according to ICH guidelines. The obtained results were statistically compared with those obtained by the official and a reported method for PAN and ITH, respectively, showing no significant difference with respect to accuracy and precision.
Subject(s)
2-Pyridinylmethylsulfinylbenzimidazoles/analysis , Benzamides/analysis , Benzyl Compounds/analysis , Spectrophotometry/methods , Calibration , Capsules/analysis , Chemistry, Pharmaceutical/methods , Drug Stability , Green Chemistry Technology , Pantoprazole , Reproducibility of Results , Spectrophotometry/economicsABSTRACT
A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 µL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.
Subject(s)
Glutathione/blood , Spectrophotometry/methods , Adult , Chromatography, High Pressure Liquid , Colorimetry/economics , Colorimetry/methods , Dithionitrobenzoic Acid/chemistry , Female , Humans , Indicators and Reagents , Limit of Detection , Male , Oxidation-Reduction , Oxidative Stress , Sample Size , Spectrophotometry/economicsABSTRACT
Simple, reliable, and sensitive kinetic spectrophotometric method has been developed for the simultaneous determination of diloxanide furoate and metronidazole using H-point standard addition method (HPSAM). The method is based on the oxidation rate difference of diloxanide and metronidazole by potassium permanganate in basic medium. A green color has been developed and measured at 610 nm. Different experimental parameters were carefully optimized. The limiting logarithmic and the initial-rate methods were adopted for the construction of the calibration curve of each individual reaction with potassium permanganate. Under the optimum conditions, Beer's law was obeyed in the range of 1.0-20.0 and 5.0-25.0 µg ml(-1) for diloxanide furoate and metronidazole, respectively. The detection limits were 0.22 µg ml(-1) for diloxanide furoate and 0.83 µg ml(-1) for metronidazole. Correlation coefficients of the regression equations were greater than 0.9970 in all cases. The precision of the method was satisfactory; the maximum value of relative standard deviation did not exceed 1.06% (n=5). The accuracy, expressed as recovery was between 99.4% and 101.4% with relative error of 0.12 and 0.14 for diloxanide furoate and metronidazole, respectively. The proposed method was successfully applied for the simultaneous determination of both drugs in pharmaceutical dosage forms and human urine samples and compared with alternative HPLC method.
Subject(s)
Anti-Infective Agents/urine , Furans/urine , Metronidazole/urine , Spectrophotometry/methods , Anti-Infective Agents/analysis , Furans/analysis , Humans , Limit of Detection , Metronidazole/analysis , Oxidation-Reduction , Pharmaceutical Preparations/chemistry , Potassium Permanganate/chemistry , Spectrophotometry/economicsABSTRACT
OBJECTIVES: Pigmented skin lesions are commonly presented in primary care. Appropriate diagnosis and management is challenging because the vast majority are benign. The MoleMate system is a handheld SIAscopy scanner integrated with a primary care diagnostic algorithm aimed at improving the management of pigmented skin lesions in primary care. METHODS: This decision-model-based economic evaluation draws on the results of a randomized controlled trial of the MoleMate system versus best practice (ISRCTN79932379) to estimate the expected long-term cost and health gain of diagnosis with the MoleMate system versus best practice in an English primary care setting. The model combines trial results with data from the wider literature to inform long-term prognosis, health state utilities, and cost. RESULTS: Results are reported as mean and incremental cost and quality-adjusted life-years (QALYs) gained, incremental cost-effectiveness ratio with probabilistic sensitivity analysis, and value of information analysis. Over a lifetime horizon, the MoleMate system is expected to cost an extra £18 over best practice alone, and yield an extra 0.01 QALYs per patient examined. The incremental cost-effectiveness ratio is £1,896 per QALY gained, with a 66.1% probability of being below £30,000 per QALY gained. The expected value of perfect information is £43.1 million. CONCLUSIONS: Given typical thresholds in the United Kingdom (£20,000-£30,000 per QALY), the MoleMate system may be cost-effective compared with best practice diagnosis alone in a primary care setting. However, there is considerable decision uncertainty, driven particularly by the sensitivity and specificity of MoleMate versus best practice, and the risk of disease progression in undiagnosed melanoma; future research should focus on reducing uncertainty in these parameters.
Subject(s)
Dermoscopy/economics , Melanoma/diagnosis , Melanoma/economics , Primary Health Care/economics , Computer Simulation , Cost-Benefit Analysis , Decision Support Techniques , Decision Trees , Dermoscopy/instrumentation , Dermoscopy/methods , Diagnosis, Computer-Assisted , Diagnosis, Differential , England , Humans , Markov Chains , Monte Carlo Method , Nevus, Pigmented/diagnosis , Nevus, Pigmented/economics , Primary Health Care/methods , Quality-Adjusted Life Years , Randomized Controlled Trials as Topic , Referral and Consultation/economics , Referral and Consultation/statistics & numerical data , Skin Neoplasms/diagnosis , Skin Neoplasms/economics , Spectrophotometry/economics , Spectrophotometry/instrumentation , Spectrophotometry/methodsABSTRACT
New, simple, specific, accurate and precise spectrophotometric technique utilizing ratio spectra is developed for simultaneous determination of two different binary mixtures. The developed ratio H-point standard addition method (RHPSAM) was managed successfully to resolve the spectral overlap in itopride hydrochloride (ITO) and pantoprazole sodium (PAN) binary mixture, as well as, mosapride citrate (MOS) and PAN binary mixture. The theoretical background and advantages of the newly proposed method are presented. The calibration curves are linear over the concentration range of 5-60 µg/mL, 5-40 µg/mL and 4-24 µg/mL for ITO, MOS and PAN, respectively. Specificity of the method was investigated and relative standard deviations were less than 1.5. The accuracy, precision and repeatability were also investigated for the proposed method according to ICH guidelines.
Subject(s)
2-Pyridinylmethylsulfinylbenzimidazoles/analysis , Benzamides/analysis , Benzyl Compounds/analysis , Gastrointestinal Agents/analysis , Morpholines/analysis , Spectrophotometry/methods , Pantoprazole , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry/economicsABSTRACT
A simple and fast on line spectrophotometric method combined with a hybrid hard-soft modeling multivariate curve resolution (HS-MCR) was proposed for the monitoring of photodegradation reaction of ciprofloxacin under UV radiation. The studied conditions attempt to emulate the effect of sunlight on these antibiotics that could be eventually present in the environment. The continuous flow system made it possible to study the ciprofloxacin degradation at different pH values almost at real time, avoiding errors that could arise from typical batch monitoring of the reaction. On the base of a concentration profiles obtained by previous pure soft-modeling approach, reaction pathways have been proposed for the parent compound and its photoproducts at different pH values. These kinetic models were used as a constraint in the HS-MCR analysis. The kinetic profiles and the corresponding pure response profile (UV-Vis spectra) of ciprofloxacin and its main degradation products were recovered after the application of HS-MCR analysis to the spectra recorded throughout the reaction. The observed behavior showed a good agreement with the photodegradation studies reported in the bibliography. Accordingly, the photodegradation reaction was studied by high performance liquid chromatography coupled with UV-Vis diode array detector (HPLC-DAD). The spectra recorded during the chromatographic analysis present a good correlation with the ones recovered by UV-Vis/HS-MCR method.
Subject(s)
Anti-Infective Agents/chemistry , Ciprofloxacin/chemistry , Photolysis , Spectrophotometry/methods , Drug Stability , Equipment Design , Hydrogen-Ion Concentration , Kinetics , Models, Chemical , Multivariate Analysis , Spectrophotometry/economics , Spectrophotometry/instrumentation , Ultraviolet RaysABSTRACT
Granisetron is a selective 5-HT3 receptor antagonist used in prevention and treatment of chemotherapy-induced nausea and vomiting. The drug is available in tablet dosage form and parenteral dosage form containing benzyl alcohol as a preservative. The main route of degradation of granisetron is through hydrolysis. The present work describes the development of a simple, rapid, and reliable first derivative spectrophotometric method for the determination of granisetron in presence of its hydrolytic products as well as the formulations adjuvant and benzyl alcohol. The method is based on the measurement of the first derivative response of granisetron at 290 nm where the interference of the hydrolytic products, the co-formulated adjuvant and benzyl alcohol is completely eliminated. The proposed method was validated with respect to specificity, linearity, selectivity, accuracy, precision, robustness, detection, and quantification limits. Regression analysis showed good correlation between the first derivative response and the concentration of granisetron over a range of 8-16 µg ml(-1) . Statistical analysis proved the accuracy of the proposed method compared with a reference stability indicating high performance liquid chromatography method. The described method was successfully applied to the determination of granisetron in different batches of tablets and ampoules. The assay results obtained in this study strongly encourage us to apply the validated method for the quality control and routine analysis of tablets and parenteral preparations containing granisetron.
Subject(s)
Granisetron/analysis , Serotonin 5-HT3 Receptor Antagonists/analysis , Spectrophotometry/methods , Benzyl Alcohol/chemistry , Hydrolysis , Limit of Detection , Pharmaceutical Preparations/chemistry , Preservatives, Pharmaceutical/chemistry , Spectrophotometry/economics , Time FactorsABSTRACT
New, simple and rapid spectrophotometric method has been developed and validated for the assay of two macrolide drugs, azithromycin (AZT) and erythromycin (ERY) in pure and pharmaceutical formulations. The proposed method was based on the reaction of AZT and ERY with sodium 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline medium at 25 °C to form an orange-colored product of maximum absorption peak at 452 nm. All variables were studied to optimize the reaction conditions and the reaction mechanism was postulated. Beer's law was obeyed in the concentration range 1.5-33.0 and 0.92-8.0 µg mL(-1) with limit of detection values of 0.026 and 0.063 µg mL(-1) for AZT and ERY, respectively. The calculated molar absorptivity values are 4.3 × 10(4) and 12.3 × 10(4) L mol(-1) cm(-1) for AZT and ERY, respectively. The proposed methods were successfully applied to the determination of AZT and ERY in formulations and the results tallied well with the label claim. The results were statistically compared with those of an official method by applying the Student's t-test and F-test. No interference was observed from the concomitant substances normally added to preparations.
Subject(s)
Anti-Bacterial Agents/analysis , Azithromycin/analysis , Erythromycin/analysis , Naphthoquinones/chemistry , Pharmaceutical Preparations/chemistry , Spectrophotometry/methods , Coloring Agents/chemistry , Limit of Detection , Spectrophotometry/economics , Sulfonic Acids/chemistryABSTRACT
There is limited and inconclusive information regarding detectable limits and linear dynamic ranges of various quantitative protein assays. We thus performed systematic comparisons of seven commonly used methods, including direct spectrophotometric quantitation at λ205 and λ280 nm (A205 and A280, respectively), bicinchoninic acid (BCA), Biuret, Bradford, Lowry and Ninhydrin methods. Purified BSA, porcine kidney extract, tryptic digested peptides derived from purified BSA, and glycine, were used as representative purified protein, complex protein mixture, peptide and amino acid, respectively. Bradford method was the most sensitive assay (LOD=0.006 mg/ml) and had the widest range of detectability (LOD-UOD=0.006-100mg/ml) for purified protein and complex protein mixture. For peptide, A205, A280, Lowry and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but Ninhydrin method had the widest detectability range (LOD-UOD=0.006-100mg/ml). For amino acid, A205 and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but A205 had a wider detectability range (LOD-UOD=0.006-6.250 mg/ml). Biuret method offered the widest linear dynamic range for purified protein and complex protein mixture (0.391-100mg/ml), A280 offered the widest linear dynamic range for peptide (0.024-6.250 mg/ml), and Ninhydrin method offered the widest linear dynamic range for amino acid (0.024-0.195 mg/ml). Both Laemmli's and 2-D lysis buffers had dramatic interfering effects on all assays. Concerning the practicality and unit costs, A205 and A280 were the most favorable. Among the colorimetric methods, Bradford method consumed the least amount of samples and shortest analytical time with the lowest unit cost. These are the most extensive comparative data of commonly used quantitative protein assays that will be useful for selecting the most suitable method for each study.
Subject(s)
Colorimetry/standards , Glycine/analysis , Peptides/analysis , Spectrophotometry/standards , Animals , Cattle , Colorimetry/economics , Colorimetry/methods , Kidney/chemistry , Limit of Detection , Serum Albumin, Bovine/analysis , Spectrophotometry/economics , Spectrophotometry/methods , Swine , Tissue Extracts/chemistryABSTRACT
A flow-injection indirect spectrophotometric method for the determination of ascorbic acid (AA) in pharmaceutical preparations is proposed. The method is based on the reduction of iron(III) to iron(II) by the AA, and by the subsequent reaction of the produced iron(II) with 2,4,6-tripyridyl-s-triazine (TPTZ) in buffered medium (pH=3.6) to form a coloured complex (λ(max)=593nm). The three-line manifold with one reaction coil was used. The linear range of the method is from 0.08 to 10µM of ascorbic acid, with the detection limit 24nM of AA. The proposed method is simple, rapid (sampling rate of 180 samples per hour), sensitive and reproducible (RSD 0.8%, n=100). The proposed method is very selective, because only the reducing substances with standard (formal) potentials lower than 0.6V would have the thermodynamic predisposition to interfere in the proposed method. Tested reducing substances (thiol compounds) did not give serious errors when present at the same concentrations as the ascorbic acid. The proposed method can be applied for the determination of AA in pharmaceutical preparations, down to picomolar quantity.
Subject(s)
Ascorbic Acid/analysis , Flow Injection Analysis/methods , Spectrophotometry/methods , Triazines/chemistry , Calibration , Color , Drug Compounding , Flow Injection Analysis/economics , Reproducibility of Results , Spectrophotometry/economicsABSTRACT
The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The "gold standard" method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-L-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-L-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents.