Quantification of Colorimetric Data for Paper-Based Analytical Devices.

Colorimetric measurements by image analysis, giving RGB or HSV data, have become commonplace with optical indicator-based assays and as a readout for paper-based analytical devices (PADs). Yet, most works on PADs tend to ignore the quantitative relationship between color data and concentration, which may hamper their establishment as analytical devices and make it difficult to properly understand chemical or biological reactions on the paper substrate. This Perspective Article discusses how image color data are computed into colorimetric absorbance values that correlate linearly to dye concentration and compare well to traditional spectrophotometry. Thioflavin T (ThT), Neutral Red (NR), and Orange IV are used here as model systems. Absorbance measurements in solution correlate well to image data (and Beer's law) from the color channel of relevance if the gamma correction normally used to render the picture more natural to the human eye is removed. This approach also allows one to correct for color cast and variable background color, which may otherwise limit quantitation in field measurements. Reflectance measurements on paper color spots are equally found to correlate quantitatively between spectroscopy and imaging devices. In this way, deviations from Beer's law are identified that are explained with dye interactions on the paper substrate.

Deep Kernel for Genomic and Near Infrared Predictions in Multi-environment Breeding Trials.

Kernel methods are flexible and easy to interpret and have been successfully used in genomic-enabled prediction of various plant species. Kernel methods used in genomic prediction comprise the linear genomic best linear unbiased predictor (GBLUP or GB) kernel, and the Gaussian kernel (GK). In general, these kernels have been used with two statistical models: single-environment and genomic × environment (GE) models. Recently near infrared spectroscopy (NIR) has been used as an inexpensive and non-destructive high-throughput phenotyping method for predicting unobserved line performance in plant breeding trials. In this study, we used a non-linear arc-cosine kernel (AK) that emulates deep learning artificial neural networks. We compared AK prediction accuracy with the prediction accuracy of GB and GK kernel methods in four genomic data sets, one of which also includes pedigree and NIR information. Results show that for all four data sets, AK and GK kernels achieved higher prediction accuracy than the linear GB kernel for the single-environment and GE multi-environment models. In addition, AK achieved similar or slightly higher prediction accuracy than the GK kernel. For all data sets, the GE model achieved higher prediction accuracy than the single-environment model. For the data set that includes pedigree, markers and NIR, results show that the NIR wavelength alone achieved lower prediction accuracy than the genomic information alone; however, the pedigree plus NIR information achieved only slightly lower prediction accuracy than the marker plus the NIR high-throughput data.

Novel spectrophotometric methods for the determination of Leflunomide and Diacerein in binary mixtures.

Two novel spectrophotometric methods were presented in this work using ethanol as a solvent. The first method was the ratio difference spectrophotometric method [RDSM], in which the amplitude difference between two selected wavelengths on the ratio spectra were recorded and used for estimation of each of Leflunomide LEF in mixture with its alkaline induced degradate DEG and also for Diacerein DIA determination in mixture with Aceclofenac ACEC without interference from the other component in the mixture. The second method is the ratio subtraction coupled with constant multiplication [RS-CM], where LEF was determined in its mixture with its alkaline degradate DEG at 261 nm which is considered as a stability indicating assay. In addition to simultaneous determination of Diacerein DIA and Aceclofenac ACEC in their mixtures at 257 and 277 nm, respectively, by the second method without previous separation. Linearity was shown over the concentration range of [1.5-15 µg/ml] for LEF, [1-11 µg/ml] for DIA and [2.5-25 µg/ml] for ACEC, by both proposed methods. Leflunomide was found to be completely degraded when subjected to alkaline degradation producing one alkaline product. Validation of the suggested methods was conducted according to ICH guidelines, concerning precision, accuracy, repeatability. The suggested spectrophotometric methods were statistically compared to reference methods showing no significant difference. The suggested spectrophotometric methods are considered to be simple, sensitive and could be easily applied in quality control laboratories instead of LC methods.

New spectrophotometric/chemometric assisted methods for the simultaneous determination of imatinib, gemifloxacin, nalbuphine and naproxen in pharmaceutical formulations and human urine.

In this paper, novel univariate and multivariate regression methods along with model-updating technique were developed and validated for the simultaneous determination of quaternary mixture of imatinib (IMB), gemifloxacin (GMI), nalbuphine (NLP) and naproxen (NAP). The univariate method is extended derivative ratio (EDR) which depends on measuring every drug in the quaternary mixture by using a ternary mixture of the other three drugs as divisor. Peak amplitudes were measured at 294nm, 250nm, 283nm and 239nm within linear concentration ranges of 4.0-17.0, 3.0-15.0, 4.0-80.0 and 1.0-6.0µgmL-1 for IMB, GMI, NLP and NAB, respectively. Multivariate methods adopted are partial least squares (PLS) in original and derivative mode. These models were constructed for simultaneous determination of the studied drugs in the ranges of 4.0-8.0, 3.0-11.0, 10.0-18.0 and 1.0-3.0µgmL-1 for IMB, GMI, NLP and NAB, respectively, by using eighteen mixtures as a calibration set and seven mixtures as a validation set. The root mean square error of predication (RMSEP) were 0.09 and 0.06 for IMB, 0.14 and 0.13 for GMI, 0.07 and 0.02 for NLP and 0.64 and 0.27 for NAP by PLS in original and derivative mode, respectively. Both models were successfully applied for analysis of IMB, GMI, NLP and NAP in their dosage forms. Updated PLS in derivative mode and EDR were applied for determination of the studied drugs in spiked human urine. The obtained results were statistically compared with those obtained by the reported methods giving a conclusion that there is no significant difference regarding accuracy and precision.

Evaluation of reliability and validity of three dental color-matching devices.

PURPOSE: To assess the repeatability and accuracy of three dental color-matching devices under standardized and freehand measurement conditions. MATERIALS AND METHODS: Two shade guides (Vita Classical A1-D4, Vita; and Vita Toothguide 3D-Master, Vita), and three color-matching devices (Easyshade, Vita; SpectroShade, MHT Optic Research; and ShadeVision, X-Rite) were used. Five shade tabs were selected from the Vita Classical A1-D4 (A2, A3.5, B1, C4, D3), and five from the Vita Toothguide 3D-Master (1M1, 2R1.5, 3M2, 4L2.5, 5M3) shade guides. Each shade tab was recorded 15 continuous, repeated times with each device under two different measurement conditions (standardized, and freehand). Both qualitative (color shade) and quantitative (L, a, and b) color characteristics were recorded. The color difference (ΔE) of each recorded value with the known values of the shade tab was calculated. The repeatability of each device was evaluated by the coefficient of variance. The accuracy of each device was determined by comparing the recorded values with the known values of the reference shade tab (one sample t test; α = 0.05). The agreement between the recorded shade and the reference shade tab was calculated. The influence of the parameters (devices and conditions) on the parameter ΔE was investigated (two-way ANOVA). Comparison of the devices was performed with Bonferroni pairwise post-hoc analysis. RESULTS: Under standardized conditions, repeatability of all three devices was very good, except for ShadeVision with Vita Classical A1-D4. Accuracy ranged from good to fair, depending on the device and the shade guide. Under freehand conditions, repeatability and accuracy for Easyshade and ShadeVision were negatively influenced, but not for SpectroShade, regardless of the shade guide. CONCLUSION: Based on the total of the color parameters assessed per device, SpectroShade was the most reliable of the three color-matching devices studied.

Factors involved in the spectrophotometric measurement of soft tissue: A clinical study of interrater and intrarater reliability.

STATEMENT OF PROBLEM: The reliability of spectrophotometric measurements of gingival color has not been tested. PURPOSE: The purpose of this study was to evaluate the repeatability and reproducibility of gingival color measurements with a digital spectrophotometer. Measurement error was estimated by determining the interrater agreement and by repeating measurements in different illumination environments with and without contact of the device with the gingiva. MATERIAL AND METHODS: Two trained examiners measured the gingival shade around 30 central incisors with a spectrophotometer with and without external illumination and with and without contact of the device with the gingiva. Color data obtained (CIELab color coordinates; L*, c*, h*, a*, b*) were analyzed with the intraclass correlation coefficient (ICC) and the Student t test for paired samples. RESULTS: Mean L*, c*, a*, and b* values differed significantly between measurements made with and without contact of the device with the tissue, but no difference was found in h* values. An ICC of >0.9 was obtained for interrater and intrarater agreements in all cases. Shade measurements did not differ between the presence and absence of stable ambient light. CONCLUSIONS: The repeatability and reproducibility of soft tissue shade measurements were almost perfect (ICC >0.9) under the examination conditions tested. The measurements were affected by pressure but not by ambient light.

Quantification of hepatic iron concentration in chronic viral hepatitis: usefulness of T2-weighted single-shot spin-echo echo-planar MR imaging.

OBJECTIVE: To investigate the usefulness of single-shot spin-echo echo-planar imaging (SSEPI) sequence for quantifying mild degree of hepatic iron stores in patients with viral hepatitis. METHODS: This retrospective study included 34 patients with chronic viral hepatitis/cirrhosis who had undergone histological investigation and magnetic resonance imaging with T2-weighted gradient-recalled echo sequence (T2-GRE) and diffusion-weighted SSEPI sequence with b-factors of 0 s/mm(2) (T2-EPI), 500 s/mm(2) (DW-EPI-500), and 1000 s/mm(2) (DW-EPI-1000). The correlation between the liver-to-muscle signal intensity ratio, which was generated by regions of interest placed in the liver and paraspinous muscles of each sequence image, and the hepatic iron concentration (µmol/g dry liver), which was assessed by spectrophotometry, was analyzed by linear regression using a spline model. Akaike information criterion (AIC) was used to select the optimal model. RESULTS: Mean ± standard deviation of the hepatic iron concentration quantified by spectrophotometry was 24.6 ± 16.4 (range, 5.5 to 83.2) µmol/g dry liver. DW-EPI correlated more closely with hepatic iron concentration than T2-GRE (R square values: 0.75 for T2-EPI, 0.69 for DW-EPI-500, 0.62 for DW-EPI-1000, and 0.61 for T2-GRE, respectively, all P<0.0001). Using the AIC, the regression model for T2-EPI generated by spline model was optimal because of lowest cross validation error. CONCLUSION: T2-EPI was sensitive to hepatic iron, and might be a more useful sequence for quantifying mild degree of hepatic iron stores in patients with chronic viral hepatitis.

Data comparison between two dental spectrophotometers.

OBJECTIVES: The objective of this study was to clinically test whether the data from two different spectrophotometers, based on spot and surface measurements, can be compared. METHODS: Under standardized clinical conditions two devices (Vita Easyshade and Spectro Shade-Micro) were used to record the color of three areas (cervical, middle, and incisal) per tooth for three upper maxillary anterior teeth in 102 participants. Each position was measured three times to attain an average for the CIE L*a*b* coordinates and to attain the corresponding Vita Classical shade tab integrated in the software of both devices. Vita tabs were also described as L*a*b* values using earlier published translations so that color differences (ΔE) could be calculated between them. RESULTS: The regression analysis between the two devices showed that the independent correlation coefficients of the L*a*b* values are low. Yet when the suggested shade codes are compared with Vita colors instead of L*a*b*, 40% of the cases were equal and 51% were clinically acceptable. SIGNIFICANCE: According to this study the two devices do not give a comparable shade selection output, and thus the exchange of L*a*b* values between the two spectrophotometers cannot be recommended.

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and isoconazole nitrate in creams.

A new RP-LC method and two new spectrophotometric methods, principal component regression (PCR) and first derivative spectrophotometry, are proposed for simultaneous determination of diflucortolone valerate (DIF) and isoconazole nitrate (ISO) in cream formulations. An isocratic system consisting of an ACE C18 column and a mobile phase composed of methanol-water (95 + 5, v/v) was used for the optimal chromatographic separation. In PCR, the concentration data matrix was prepared by using synthetic mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 29 wavelengths in the range of 242-298 nm for DIF and ISO in the zero-order spectra of their combinations. In first derivative spectrophotometry, dA/dlambda values were measured at 247.8 nm for DIF and at 240.2 nm for ISO in first derivative spectra of the solution of DIF and ISO in methanol-water (3 + 1, v/v). The linear ranges were 4.00-48.0 microg/mL for DIF and 50.0-400 microg/mL for ISO in the LC method, and 2.40-40.0 microg/mL for DIF and 60.0-260 microg/mL for ISO in the PCR and first derivative spectrophotometric methods. These methods were validated by analyzing synthetic mixtures. These three methods were successfully applied to two pharmaceutical cream preparations.

Measuring the color of maxillofacial prosthetic material.

Color information from different color-measuring systems varies during color matching in maxillofacial prosthetics. We studied the hypothesis that a non-contact measuring system and 4 contact color-measuring instruments perform comparably in accuracy and precision on measurements of pigmented maxillofacial elastomer specimens having human skin colors. Measurement comparisons in accuracy on opaque standard color patches were made in Phase I. In Phase II, the system with the best accuracy was used as the reference instrument, and comparisons in accuracy and precision on elastomer specimens were made. The CIEDE2000 color difference formula was used. Repeated-measures ANOVA with Tukey testing and linear regression analysis for CIELAB and color differences among the instruments were performed. The contact measuring systems perform differently in accuracy, possibly due to edge loss and other factors, but performed comparably in precision with the non-contact measuring instrument. This non-contact system is recommended for color measurement of maxillofacial prosthetic materials.

Simultaneous spectrophotometric determination of 2-thiouracil and 2-mercaptobenzimidazole in animal tissue using multivariate calibration methods: concerns and rapid methods for detection.

Two multivariate calibration methods, partial least squares (PLS) and principal component regression (PCR), were applied to the spectrophotometric simultaneous determination of 2-mercaptobenzimidazole (MB) and 2-thiouracil (TU). A genetic algorithm (GA) using partial least squares was successfully utilized as a variable selection method. The concentration model was based on the absorption spectra in the range of 200 to 350 nm for 25 different mixtures of MB and TU. The calibration curve was linear across the concentration range of 1 to 10 microg mL(-1) and 1.5 to 15 microg mL(-1) for MB and TU, respectively. The values of the root mean squares error of prediction (RMSEP) were 0.3984, 0.1066, and 0.0713 for MB and 0.2010, 0.1667, and 0.1115 for TU, which were obtained using PCR, PLS, and GA-PLS, respectively. Finally, the practical applicability of the GA-PLS method was effectively evaluated by the concurrent detection of both analytes in animal tissues. It should also be mentioned that the proposed method is a simple and rapid way that requires no preliminary separation steps and can be used easily for the analysis of these compounds, especially in quality control laboratories.

Improvement of direct calibration in spectroscopy.

Several linear calibration methods have been proposed for predicting the concentration of a particular compound from a spectrum. Some methods are based on experimental data, such as Partial Least Square Regression. Other methods are based on expert data, e.g. Direct Calibration. This article proposes a new method, called Improved Direct Calibration, which uses expert and experimental information. It performs a projection onto the pure interest spectrum, after correcting it from influence factors. No calibration dataset is necessary to build this model. This method has been successfully applied to the quantification of ethanol in musts during fermentation, using near infrared spectrometry.

Rapid and accurate estimation of blood saturation, melanin content, and epidermis thickness from spectral diffuse reflectance.

We present a method to determine chromophore concentrations, blood saturation, and epidermal thickness of human skin from diffuse reflectance spectra. Human skin was approximated as a plane-parallel slab of variable thickness supported by a semi-infinite layer corresponding to the epidermis and dermis, respectively. The absorption coefficient was modeled as a function of melanin content for the epidermis and blood content and oxygen saturation for the dermis. The scattering coefficient and refractive index of each layer were found in the literature. Diffuse reflectance spectra between 490 and 620 nm were generated using Monte Carlo simulations for a wide range of melanosome volume fraction, epidermal thickness, blood volume, and oxygen saturation. Then, an inverse method was developed to retrieve these physiologically meaningful parameters from the simulated diffuse reflectance spectra of skin. A previously developed accurate and efficient semiempirical model for diffuse reflectance of two layered media was used instead of time-consuming Monte Carlo simulations. All parameters could be estimated with relative root-mean-squared error of less than 5% for (i) melanosome volume fraction ranging from 1% to 8%, (ii) epidermal thickness from 20 to 150 mum, (iii) oxygen saturation from 25% to 100%, (iv) blood volume from 1.2% to 10%, and (v) tissue scattering coefficient typical of human skin in the visible part of the spectrum. A similar approach could be extended to other two-layer absorbing and scattering systems.

Direct spectrophotometric determination of serum fructose in pancreatic cancer patients.

OBJECTIVES: We sought to develop an assay to measure circulating fructose concentrations in pancreatic cancer patients. METHODS: Using fructose dehydrogenase-catalyzed conversion of d-fructose to 5-ketofructose, followed by quantitation of MTT [3-(4,5-dimethylthiaze-syl)-2,5-diphenyltetrazolium bromide] formazan production by direct spectrophotometry, an assay to measure serum fructose concentration was developed, and assay sensitivity and specificity were tested. Validity of the assay was confirmed by gas chromatography-mass spectroscopy, and the assay was tested in healthy subjects and pancreatic cancer patients. RESULTS: The assay was highly specific, exhibiting no cross-reactivity with other sugars. Mean serum fructose concentration in fasting healthy volunteers was 1.9+/-0.4 mM and after ingestion of a fructose and glucose-containing drink rose to 16.3+/-1.2 mM at 15 minutes and peaked at 30 minutes when serum fructose was 17.2+/-1.1 mM. Mean fasting serum fructose level was significantly higher at 5.7+/-2.5 mM in patients with pancreatic cancer than those with no pancreatic cancer. The fructose dehydrogenase-based enzymatic assay correlated highly with gas chromatography-mass spectroscopic analysis of serum fructose with a correlation coefficient of 0.94. CONCLUSION: This spectrophotometric assay for fructose is easy to perform and well suited to determination of serum fructose. Measurement of serum fructose concentration may provide insight into the relationship between refined fructose intake and diseases including pancreatic cancer.

A comparison of human raters and an intra-oral spectrophotometer.

Consistently choosing an accurate shade match is far more difficult than it appears. Recently, several electronic shade-matching devices have been marketed. One device is an intraoral spectrophotometer, Easyshade. The current study compared the accuracy and consistency of the Easyshade (ES) device to three clinicians experienced in tooth whitening trials and trained in the use of the Vitapan 3D Master shade. The maxillary anteriors of 16 participants were matched on three separate occasions one month apart. At each appointment, the three clinicians (R1, R2 & R3) and ES independently chose a single 3D Master tab. A trained research assistant used the Easyshade device to record CIE L*, C* and H* and a shade tab. In addition, color differences between shade tabs were calculated using the Delta E 2000 (delta e 00) formula. The CIE L*C*H* data were also used to establish standards for the five lightness groups of the 3D Master. An intrarater agreement was evaluated using an intraclass correlation statistic, and an inter-rater agreement was evaluated using a weighted Kappa statistic. The percentages of exact matches were: ES = 41%; R1 = 27%; R2 = 22% and R3 = 17%. Matches within a half-shade were also calculated. This represents a mismatch that is perceptible but acceptable. The percentages of matches within a half-tab were: ES = 91%; R1 = 69%; R2 = 85% and R3 = 79%. In terms of lightness, the intra-rater agreement was considered to be very good for ES and R2 and good for R1 and R3. For chroma, agreement for ES was considered good, and for the three clinicians, it was considered moderate. The mean color difference for the L*, C*, H* data recorded at each evaluation was 1.5, or only slightly greater than the color difference between the same tab on different guides (1.2). The delta e 00 data were the most accurate data collected, and they were used to establish a standard to which the tab choices of the four raters were compared. A weighted Kappa statistic was performed and, in terms of lightness, agreement was found to be good for all raters. For chroma, agreement was very good for ES and it was good for the clinicians. In terms of the number of exact matches and matches within a half-shade, the performance of ES was at least comparable to, if not better than, the dentists. Statistically, the same was true in terms of consistency and accuracy when making repeated matches of lightness and chroma using the 3D Master shade guide.

Interexaminer reliability in the clinical measurement of L*C*h* values using a laminar spectrophotometer.

This in vivo study investigated interexaminer reliability in the clinical measurement of L*C*h* values using a spectrophotometer (Shadepilot, DeguDent), which provides laminar measurement. Thirty incisors were measured by 3 trained clinicians. Intraclass correlation coefficient was used to assess reliability. Additionally, the range of differences between the measurements by all examiners for each subject was calculated to assess the clinical impact of the differences. Agreement was acceptable to excellent for all measurements. Additionally, the reliability of the measurement of L* and C* was excellent (all measurements < 5). Laminar spectrophotometric measurement seems to be superior to punctiform measurement.

Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arthrospira) supplements and raw materials by spectrophotometry.

A single-laboratory validation study was conducted for a 2-wavelength spectrophotometric method for the determination of c-phycocyanin (cPC) and allophycocyanin (aPC) in Spirulina supplements and raw materials. The absorption maxima of cPC and aPC are at 620 and 650 nm, respectively. The concentrations of the analytes were calculated from measurement of absorbance at 2 wavelengths. This method provided linear responses for cPC and aPC in the range of 25-250 microg/mL. Duplicate determinations of cPC and aPC in 4 different test materials on 5 different days resulted in relative standard deviations of 0.3-1.0 and 1.0-1.5% for cPC and aPC, respectively. Recoveries were 99.2-102.4 and 100.0-104.0% for cPC and aPC, respectively. The results were satisfactory for the determination of cPC and aPC in Spirulina supplements.

Simultaneous spectrophotometric determination of mercury and palladium with Thio-Michler's Ketone using partial least squares regression and orthogonal signal correction.

A simple, novel and sensitive spectrophotometric method was described for simultaneous determination of mercury and palladium. The method is based on the complex formation of mercury and palladium with Thio-Michler's Ketone (TMK) at pH 3.5. All factors affecting on the sensitivity were optimized and the linear dynamic range for determination of mercury and palladium found. The simultaneous determination of mercury and palladium mixtures by using spectrophotometric method is a difficult problem, due to spectral interferences. By multivariate calibration methods such as partial least squares (PLS), it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. Orthogonal signal correction (OSC) is a preprocessing technique used for removing the information unrelated to the target variables based on constrained principal component analysis. OSC is a suitable preprocessing method for PLS calibration of mixtures without loss of prediction capacity using spectrophotometric method. In this study, the calibration model is based on absorption spectra in the 360-660 nm range for 25 different mixtures of mercury and palladium. Calibration matrices were containing 0.025-1.60 and 0.05-0.50 microg mL(-1) of mercury and palladium, respectively. The RMSEP for mercury and palladium with OSC and without OSC were 0.013, 0.006 and 0.048, 0.030, respectively. This procedure allows the simultaneous determination of mercury and palladium in synthetic and real matrix samples good reliability of the determination.

Kinetic spectrophotometric determination of trace amounts of palladium by whole kinetic curve and a fixed time method using resazurine sulfide reaction.

The univariate and multivariate calibration methods were applied for the determination of trace amounts of palladium based on the catalytic effect on the reaction between resazurine and sulfide. The decrease in absorbance of resazurine at 602 nm over a fixed time is proportional to the concentration of palladium over the range of 10.0-160.0 ng mL(-1). The calibration matrix for partial least squares (PLS) regression was designed with 14 samples. Orthogonal signal correction (OSC) is a preprocessing technique used for removing the information unrelated to the target variables based on constrained principal component analysis. OSC is a suitable preprocessing method for PLS calibration without loss of prediction ability using spectrophotometric method. The root mean square error of prediction (RMSEP) for palladium determination with fixed-time, PLS and OSC-PLS were 3.71, 2.84 and 0.68, respectively. This procedure allows the determination of palladium in synthetic and real samples with good reliability of the determination.

Evaluation of visual and spectrophotometric shade analyses: a clinical comparison of 3758 teeth.

This study aimed to evaluate the performance of visual and spectrophotometric tooth shade analysis. Two operators independently selected the best match of 3758 anterior teeth of 106 patients at 3 different dates, using the Chromascop-Complete shade guide. Additionally, tooth color was analyzed 3 consecutive times using a reflectance spectrophotometer. Spectrophotometry showed high agreement values (89.6%); both examiners agreed in 49.7% of the measurements. Visual assessment resulted in significantly darker ratings than spectrophotometry (P < .0005). However, a positive association was observed for both procedures (P = .548). Spectrophotometric shade determination seems to be significantly more reproducible than the visual procedure.