Development of standardized method for the quantification of azo dyes by UV-Vis in binary mixtures.

The azo dyes, Yellow 5 (Y5), Red 2 (R2) and Blue 1 (B1), quantified in solutions and in mixtures of binary dyes, were studied by means of UV-Vis spectroscopy. In this work was used a CIE algorithm developed in Visual Basic for Applications (VBA). The CIE algorithm is based on the tristimulus chromaticity diagram, as an alternative to the shielding effect that arises in dye mixtures, and it can also be applied to complex quantification methods such as HPLC (High Performance Liquid Chromatography). The results obtained through of the algorithm, showed a higher accuracy from 97 to 99% in relation with similar UV-Vis quantification methods. In contrast, linear methods only managed to reach an accuracy from 78 to 98%. Additionally, the algorithm yielded significant similar values to the UHPLC reference method. The results showed that the method CIE algorithm was accessible and reliable to quantify binary mixtures of the dyes used which suggests the possibility to apply this method on other dyes, within the limits of quantification obtained in this study (0.076-24.56 mg/L) and the pH values from 2 to 10.

Application of Fusarium sp. immobilized on multi-walled carbon nanotubes for solid-phase extraction and trace analysis of heavy metal cations.

In the present work, for the first time, the filamentous fungus Fusarium sp. was utilized for devising a novel method for pre-concentration and determination of trace amounts of Pb(II), Cu(II), Cd(II), and Zn(II) ions, using a mini-column packed with Fusarium-coated multi-walled carbon nanotubes and inductively coupled plasma-optical emission spectrometry. Optimal analytical conditions including pH, ionic strength, elution solution, sample and eluent flow rates, and sample volume were determined. The detection limits were 0.39, 0.060, 0.021, and 0.025 ng mL-1 for Pb(II), Cu(II), Cd(II), and Zn(II) cations, respectively. This new method demonstrated a high performance for the analytes, and their adsorption was not affected by the different co-existing ions. The present procedure was validated by the analysis of standard reference materials, since the obtained data were in close agreement with reference values. Finally, this new procedure was successfully applied to analysis of heavy metal cations in natural food and water samples.

Detection and quantification of adulterants in honey by LIBS.

The present work proposes methods for detection and quantification of honey adulterants using laser-induced breakdown spectroscopy (LIBS). The sample set consisted of 6 pure honey from different botanical sources, 2 sweetener syrups and 228 fortified samples. The spectra acquired using a spark discharge coupled to the LIBS system were used for the development of the PLS-DA (classification) and PLS (calibration) models. Several data preprocessing and variable selection methods were evaluated to obtain the best fit. The detection of adulterants was performed with 100% of accuracy. The quantification of adulterants was possible through a PLS model with the variables selected by iPLS. The PLS model was validated with external samples and presented good accuracy, selectivity, sensitivity, and linearity. The proposed methods highlighted the potential of the LIBS technique for honey authenticity certification, providing fast, simple, and clean determinations since no sample pretreatment was required.

An environmentally friendly method based on micro-cloud point extraction for determination of trace amount of quercetin in food and fruit juice samples.

This study was conducted for quantitative determination of quercetin in food and fruit juice samples based on a green, fast, and accurate method namely, micro-cloud point extraction (MCPE). The proposed MCPE is essentially a miniaturized form of traditional cloud point extraction (CPE) in which only a few microliters of micellar extracting phase is sufficient for determination. Factors influencing the extraction efficiency, such as amount of Triton X-114 (5%, v/v), effect of pH, amount of Na2SO4 salt (5%, w/v) and time of centrifugation were investigated and optimized by experimental design. Under the optimized condition, the calibration range was found to be linear over 10-100 ng mL-1 with the limit of detection of 2.2 ng mL-1. The relative standard deviation of 2.8%, was obtained on a 30 ng mL-1quercetin standard solution (n = 3). Finally, the proposed method was successfully applied for the determination of quercetin in onion, tomato, apple and orange juice samples.

Development of a first order derivative spectrophotometry method to rapidly quantify protein in the presence of chitosan and its application in protein encapsulation systems.

A new protein quantification method based on first order derivative spectrophotometry was established to eliminate various interferences, mainly chitosan, to the utmost using bovine serum albumin (BSA) as a model food protein. Absorbance spectra of BSA solutions were recorded and their first order derivative calculated. The values of derivative absorbance at 288 nm were used to generate linear calibration curve of BSA. The new method was applied in entrapping BSA into chitosan-tripolyphosphate beads. A general calibration curve was established with a CI (width of 95% confidence interval of three repeat measurements of unknown samples) less than 0.0262 g/L and a LOQ (limit of quantification) of 0.11 g/L, showing excellent tolerance to various interferences, which was further verified by the good mass balance of BSA during encapsulation. Overall, the method successfully eliminated the interferences from chitosan and other factors to facilitate the measurement of protein in complicated environments.

Spectrophotometry assays to determine G6PD activity from Trinity Biotech and Pointe Scientific G6PD show good correlation.

OBJECTIVES: Spectrophotometry kits from Pointe Scientific (PS; USA) were compared to kits from Trinity Biotech (Trinity; Ireland) in 50 venous blood samples from purposively selected individuals in Bangladesh. Repeatability and inter-assay variability were assessed by Students t-test, Bland-Altman plot and Pearson correlation coefficient (r). The median glucose-6-phosphate dehydrogenase (G6PD) activity of all G6PD normal participants was calculated per assay and defined as 100% activity. Performance was calculated considering 30% and 70% cut off activities and Trinity as reference. RESULTS: The intra-assay correlation of Trinity (r = 0.9841, p < 0.001) and PS (r = 0.9833, p < 0.001) did not differ significantly (p = 0.904). Both assays were closely correlated (r = 0.9799, p < 0.001), with a mean difference of 0.1 U/gHb (95% limit of agreement: - 1.32 to 1.57). At 30% cut off PS had a sensitivity of 100% (95% confidence interval (95 CI) 59.0-100.0) and specificity of 100% (95% CI 91.8 to 100.0), at 70% cut-off of 100% (95% CI 79.4-100.0) and 97.1% (95% CI 84.7-99.9) respectively. The G6PD assay from PS is a reliable alternative to the assay from Trinity.

A Continuous and Sensitive Spectrophotometric Assay for Lipase and Phospholipase A Activities Using α-Eleostearic Acid-Containing Substrates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

A Generalized Approach to Forensic Dye Identification: Development and Utility of Reference Libraries.

While color is arguably the most important optical property of evidential fibers, the actual dyestuffs responsible for its expression in them are, in forensic trace evidence examinations, rarely analyzed and still less often identified. This is due, primarily, to the exceedingly small quantities of dye present in a single fiber as well as to the fact that dye identification is a challenging analytical problem, even when large quantities are available for analysis. Among the practical reasons for this are the wide range of dyestuffs available (and the even larger number of trade names), the low total concentration of dyes in the finished product, the limited amount of sample typically available for analysis in forensic cases, and the complexity of the dye mixtures that may exist within a single fiber. Literature on the topic of dye analysis is often limited to a specific method, subset of dyestuffs, or an approach that is not applicable given the constraints of a forensic analysis. Here, we present a generalized approach to dye identification that (1) combines several robust analytical methods, (2) is broadly applicable to a wide range of dye chemistries, application classes, and fiber types, and (3) can be scaled down to forensic casework-sized samples. The approach is based on the development of a reference collection of 300 commercially relevant textile dyes that have been characterized by a variety of microanalytical methods (HPTLC, Raman microspectroscopy, infrared microspectroscopy, UV-Vis spectroscopy, and visible microspectrophotometry). Although there is no single approach that is applicable to all dyes on every type of fiber, a combination of these analytical methods has been applied using a reproducible approach that permits the use of reference libraries to constrain the identity of and, in many cases, identify the dye (or dyes) present in a textile fiber sample.

Label-Free, Direct Measurement of Protein Concentrations in Turbid Solutions with a UV-Visible Integrating Cavity Absorbance Spectrometer.

Protein-particle conjugates and mixtures have been investigated extensively for their diverse applications in biotechnology. However, general methods to measure protein concentration of protein-particle solutions are lacking. Typically, proteins in turbid solutions require separation or staining with another chromophore to quantitate their concentration. Here we demonstrate a label-free, direct approach to measure protein concentrations in turbid solutions using a UV-vis integrating cavity absorbance spectrometer. Three systems are used to test the ability to measure accurate protein concentrations: proteins adsorbed to Alhydrogel, proteins in solution with gold nanoparticles, and proteins encapsulated within polymeric microspheres. Protein concentrations in each of the three protein-particle systems were successfully quantified using a calibration curve created from the absorbance at 280 nm.

A pilot study demonstrating the efficacy of transcutaneous bilirubin meters to quantitatively differentiate contusions from Congenital Dermal Melanocytosis.

OBJECTIVE: Congenital Dermal Melanocytosis (CDM) can be difficult to differentiate from contusions. The need for a prompt and accurate diagnosis is best illustrated in cases where child abuse and maltreatment is of concern. Transcutaneous bilirubin (TCB) spectrophotometry has been well established to measure bilirubin under the skin for jaundice in infants. The use of TCB spectrometry has not been used to identify or differentiate contusions from CDM. We hypothesized that bilirubin, a degradation product of hemoglobin, would be elevated in contusions but not in CDM thus demonstrating the efficacy of a novel diagnostic technique to compliment or improve on physical assessment alone. METHODS: Pilot study with thirty-seven infants and children noted to have CDM and fifty-six infants, children and adults with contusions underwent measurement of their lesion with TCB spectrometry. In each patient, the affected skin was scanned along with the adjacent unaffected native skin allowing an internal control for individual pigment variation. RESULTS: TCB measurements of CDM resulted in lower transcutaneous bilirubin values that were not significantly different from adjacent native skin pigmentation. This was in contrast to cutaneous contusions, which resulted in a higher measured value (mean 5.01 mg/dL) compared to adjacent native tissue (1.24 mg/dL) demonstrating a four-fold increase in measurement at the lesion site (P < 0.001). Direct comparison of a ΔTCB value (lesion measurement minus the adjacent tissue) demonstrated a significantly higher value in contusions compared to CDM with a mean value of 3.77 and 0.12 mg/dL, respectively (P < 0.001). CONCLUSIONS: TCB Spectrometry as a novel diagnostic technique has the potential to discern contusions from CDM and may therefore have the ability to compliment the use of physical assessment alone.

Reliability of maximal mitochondrial oxidative phosphorylation in permeabilized fibers from the vastus lateralis employing high-resolution respirometry.

The purpose was to assess the impact of various factors on methodological errors associated with measurement of maximal oxidative phosphorylation (OXPHOS) in human skeletal muscle determined by high-resolution respirometry in saponin-permeabilized fibers. Biopsies were collected from 25 men to assess differences in OXPHOS between two muscle bundles and to assess the correlation between OXPHOS and the wet weight of the muscle bundle. Biopsies from left and right thighs of another five subjects were collected on two occasions to compare limbs and time-points. A single muscle specimen was used to assess effects of the anesthetic carbocaine and the influence of technician. The difference in OXPHOS between two fiber-bundles from the same biopsy exhibited a standard error of measurement (SEM) of 10.5 pmol · s-1  · mg-1 and a coefficient of variation (CV) of 15.2%. The differences between left and right thighs and between two different time-points had SEMs of 9.4 and 15.2 pmol · s-1  · mg-1 and CVs of 23.9% and 33.1%, respectively. The average (±SD) values obtained by two technicians monitoring different bundles of fibers from the same biopsy were 31.3 ± 7.1 and 26.3 ± 8.1 pmol · s-1  · mg-1 . The time that elapsed after collection of the biopsy (up to a least 5 h in preservation medium), wet weight of the bundle (from 0.5 to 4.5 mg) and presence of an anesthetic did not influence OXPHOS. The major source of variation in OXPHOS measurements is the sample preparation. The thigh involved, time-point of collection, size of fiber bundles, and time that elapsed after biopsy had minor or no effect.

Noninvasive Measurement of Hemoglobin Using Spectrophotometry: Is it Useful for the Critically Ill Child?

This study compared the accuracy of noninvasively measuring hemoglobin using spectrophotometry (SpHb) with a pulse CO-oximeter and laboratory hemoglobin (Hb) measurements. A total of 345 critically ill children were included prospectively. Age, sex, and factors influencing the reliabilityof SpHb such as SpO2, heart rate, perfusion index (PI), and vasoactive inotropic score were recorded. SpHb measurements were recorded during the blood draw and compared with the Hb measurement. Thirteen patients (low PI in 9 patients and no available Hb in 4 patients) were excluded and 332 children were eligible for final analysis. The mean Hb was 8.71±1.49 g/dL (range, 5.9 to 12 g/dL) and the mean SpHb level was 9.55±1.53 g/dL (range, 6 to 14.2 g/dL). The SpHb bias was 0.84±0.86,with the limits of agreement ranging from -2.5 to 0.9 g/dL. The difference between Hb and SpHb was >1.5 g/dL for only 47 patients. Of these, 24 patients had laboratory Hb levels <7 g/dL. There was a weak positive correlation between differences and PI (r=0.349; P= 0.032). The pulse CO-oximeter is a promising tool for measuring SpHb and monitoring critically ill children. However, PI may affect these results. Additional studies investigating the reliability of the trend of continuous SpHb values compared with simultaneously measured laboratory Hb values in the same patient are warranted.

Pseudohypophosphatemia associated with high-dose liposomal amphotericin B therapy.

BACKGROUND: Hypophosphatemia is commonly observed in critically ill patients. Inorganic phosphorus is quantified by spectrophotometric measurement of a phosphomolybdate complex, a method with multiple documented interferents. Our clinical laboratory was contacted to investigate a case of asymptomatic hypophosphatemia in a patient receiving high-dose liposomal amphotericin B therapy (L-AMB). METHODS: In vitro experiments were performed by spiking L-AMB into residual plasma specimens. Phosphate was measured on the Beckman Coulter AU and Ortho Diagnostics Vitros instruments. RESULTS: When measured on the AU, phosphate in plasma with approximately 250mcg/mL of L-AMB demonstrated a median negative bias of 3.45mg/dL relative to unspiked samples. In contrast, Vitros phosphate measurements demonstrated excellent agreement for specimens with and without L-AMB (median bias -0.2mg/dL). CONCLUSIONS: High L-AMB concentrations induced a significant negative bias on phosphate measured by the AU assay, but did not affect the Vitros assay. Laboratorians and clinicians should be aware of this phenomenon in patients receiving L-AMB who develop unexplained hypophosphatemia.

Comparison of 3 options for choosing control limits in biochemistry testing.

BACKGROUND: The purpose of statistical quality control (QC) is to provide peace of mind with regard to the production of results that are suitable for analytically sound clinical interpretation and making reliable decisions about patient diagnosis, monitoring, and prognosis. OBJECTIVES: In this study, we compared 3 options for choosing control limits for biochemistry testing. They focus on the probability of error detection (Ped) and probability of false rejection (Pfr) achievable for a veterinary biochemical analyzer using the following 3 combinations: the quality control material (QCM) manufacturer's acceptable ranges; a standard 12s rule customized for the instrument's observed performance; and candidate rules selected for the instrument's observed performance using a computerized program (EZrules). METHODS: For assessing customized QC, we used mean, SD, CV, bias, total error, and sigma metrics calculated from 3 months of control measurements on a laboratory biochemical analyzer, for 24 commonly used analytes, on 2 QCM levels. RESULTS: Given the desirable combination of high Ped (> 90%) and low Pfr (≤ 5%), the candidate rules selected by the computerized program-related EZrules provided the best performance combinations. CONCLUSIONS: The present work shows acceptable QC performance basing the QC on customization of the acceptable ranges of results from the achievable performance of an individual instrument. The QC performance is maximized by the application of candidate rules based on customized ranges obtained from a computerized QC tool, providing the ability to achieve the highest Ped and acceptably low Pfr values.

Comparison between titrimetric and spectrophotometric methods for quantification of vitamin C.

Vitamin C is usually quantified by titrimetric or chromatographic methods. However, these methods have limitations: food color interferes with the titrimetric method and the chromatographic method is costly. The aim of this study was to compare a spectrophotometric method, based on reduction of cupric ions in the presence of cuproine complex, with a titrimetric method, based on reduction of 2,6-dicholorophenolindophenol. Linearity, precision, accuracy, and limits of detection (LOD) and quantification (LOQ) were evaluated using a standard vitamin C solution. Both methods were also applied for AA quantification in industrialized orange and pineapple juices. The methods were precise and accurate when applied to the standard solution. The spectrophotometric method was more sensitive, with lower values for LOD (0.002mgmL-1) and LOQ (0.010mgmL-1), and more accurate with error less than 5% while results from the titrimetric method were affected by the juice color, which generated errors in excess of 15%.

Urinary iodine: comparison of a simple method for its determination in microplates with measurement by inductively-coupled plasma mass spectrometry.

The aim of our study was to develop and validate an inexpensive, rapid, easy to use quantitative method to determine urinary iodine without major procurement costs for equipment. The rationale behind introducing this method is the increasing demand for urinary iodine assessments. Our study included 103 patients (76 female, 27 male), age (arithmetic mean) 52 ± 17.3 years. Urinary iodine was determined in microplates by a modification of the Sandell-Kolthoff reaction. The results were compared with inductively-coupled plasma mass spectrometry (ICP-MS) for iodine, considered as reference method. Geometric mean of urinary iodine determined by the Sandell-Kolthoff reaction method was 62.69 µg/l (95% confidence interval 53.16-73.92) whereas by the ICP-MS method it was 65.53 µg/l (95% confidence interval 54.77-78.41). Passing-Bablok regression equations for both methods gave y = 3.374 + 0.873x (y: Sandell-Kolthoff method, x: ICP-MS). Spearman´s correlation coefficient was 0.981, indicating a very high degree of agreement between the two methods. Bland-Altman plots showed no significant systematic difference between the two methods. The modified Sandell-Kolthoff method using microtiter plate technique presented here is a simple, inexpensive semi-automated method to determine urinary iodine with very little toxic waste. Comparison with the ICP-MS-technique yielded a good agreement between the two methods.

Protein Quantitation and Analysis of Purity.

The accurate quantitation of proteins and an analysis of their purity are essential in numerous areas of scientific research, and is a critical factor in many clinical applications. The large number and variety of techniques employed for this purpose is therefore not surprising. The selection of a suitable assay is dependent on such factors as the level of sensitivity required, the presence of interfering agents, and the composition of the protein itself. Here, protocols for the most commonly used protein determination methodologies are outlined, as well as for the more recently adapted technique of quantitative immuno-Polymerase Chain Reaction.

Perspective: The first ten years of broadband chirped pulse Fourier transform microwave spectroscopy.

Since its invention in 2006, the broadband chirped pulse Fourier transform spectrometer has transformed the field of microwave spectroscopy. The technique enables the collection of a ≥10 GHz bandwidth spectrum in a single shot of the spectrometer, which allows broadband, high-resolution microwave spectra to be acquired several orders of magnitude faster than what was previously possible. We discuss the advantages and challenges associated with the technique and look back on the first ten years of chirped pulse Fourier transform spectroscopy. In addition to enabling faster-than-ever structure determination of increasingly complex species, the technique has given rise to an assortment of entirely new classes of experiments, ranging from chiral sensing by three-wave mixing to microwave detection of multichannel reaction kinetics. However, this is only the beginning. Future generations of microwave experiments will make increasingly creative use of frequency-agile pulse sequences for the coherent manipulation and interrogation of molecular dynamics.