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A novel, environmentally friendly, and sensitive HPTLC method has been developed and validated for simultaneous quantification of duloxetine (DLX) and tadalafil (TDL) in their pure state, laboratory-prepared mixtures, and spiked human plasma. This method is particularly important for patients dealing with depression and sexual issues, as it allows for the measurement of these co-administered antidepressant and sexual stimulant drugs in biological fluids. The separation process employed a stationary phase of pre-coated silica gel 60 F254 and a mobile phase consisting of ethyl acetate, acetonitrile, and 33% ammonia (8:1:1, v/v). The optimized mobile phase resulted in well-defined bands for DLX and TDL, with Rf values of 0.3 and 0.8, respectively with dual-wavelength detection at 232 nm for DLX and 222 nm for TDL. Polynomial regression analysis revealed exceptional linearity for both drugs, with correlation coefficients of 0.9999 over concentration ranges of 10-900 ng/band for DLX and 10-1200 ng/band for TDL. The quantitation limits were 8.2 ng/band for DLX and 8.6 ng/band for TDL, while the detection limits (LOD) were 2.7 ng/band for DLX and 2.8 ng/band for TDL. The validation of this method followed the guidelines set by the International Council for Harmonization (ICH). Additionally, the suggested method's greenness was assessed by means of four up-to-date ecological tools, namely the Eco-Scale, the National Environmental Method Index (NEMI), the Green Analytical Procedure Index (GAPI), and the Analytical Greenness metric approach (AGREE). The proposed method was also assessed using the Blue Applicability Grade Index (BAGI), a recently developed metric for assessing the practicality (blueness) of procedures.
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Clorhidrato de Duloxetina , Tadalafilo , Humanos , Tadalafilo/sangre , Clorhidrato de Duloxetina/sangre , Cromatografía en Capa Delgada/métodos , Tecnología Química Verde/métodos , Límite de Detección , Cromatografía Líquida de Alta Presión/métodos , Reproducibilidad de los ResultadosRESUMEN
Regular monitoring of blood oxygenation is important for disease prevention and treatment. Image photoplethysmography (IPPG) technology is a non-contact physiological parameter detection technology, which has been widely used in blood oxygenation detection. However, traditional imaging devices still have issues such as low detection accuracy, narrower receiving spectral range. In this paper, we proposed two improved detection methods based on the dual-wavelength measurement principle, that is, dual-band IPPG signal ratio method and dual-band IPPG signal AC/DC method. To verify the effectiveness of the two methods, we used different heartbeat period IPPG signals as sample data sets, and combined PLS and RF algorithms for model training, thus obtaining the best data processing method. The experimental results showed that the dual-band IPPG signal AC/DC method can effectively reduce the model training time. This method meets the strong demand for non-contact blood oxygen measurement and provides a new measurement idea.
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Tetracyclines family is considered as the first-line antibiotic drugs for food animals. Formulating bromhexine (BR) with oxytetracycline (OTC) improved the antibacterial activity of OTC, besides it is considered as a mucolytic agent. Four precise, rapid, and simple spectrophotometric methods were successfully developed for resolution of the overlapped spectra of OTC and BR in their pure form and in their pharmaceutical formulation. The proposed methods are absorption correction (AC), dual wavelength (DW), induced dual wavelength (IDW), and spectrum subtraction (SS) spectrophotometric methods. The developed methods were used for the determination of OTC and BR in the ranges of 2-50 µg/mL and 1-30 µg/mL, respectively for all methods. For (AC) and (SS) methods, OTC and BR were determined at 380 nm and 245.6 nm, respectively after spectral resolution steps. While for (DW) method, the absorbance difference between λ (271.8 -287.6 nm) and λ (245.6 -283.2 nm) were used for the determination of OTC and BR, in order. For IDW, it depended on using the absorbance difference between 271.8 nm and 245.6 nm as well as the equality factor (F) calculated for each drug at the selected wavelengths. In all methods, HCl was used as a solvent and they are validated according to ICH guidelines. Several green metric tools have been developed to evaluate the greenness of the analytical methods like National Environmental Methods Index (NEMI), Modified NEMI, Analytical Eco-scale, Green Analytical Procedure Index (GAPI) and Analytical GREEnness calculator (AGREE), and all ensured the low impact of the suggested approaches on health and environment. The proposed methods are highly selective, robust and precise. Additionally they are time and money effective and can be used in any analytical laboratory.
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This study analyzed the optical and laser spot acquisition capabilities of a newly developed dual-wavelength-band camera. The camera performance was evaluated using a 3.0 m × 1.8 m Styrofoam target and a 70-mJ laser target designator; mid-infrared images were acquired based on the target distance, and a laser beam was irradiated onto the target to detect laser spots. The dual-wavelength-band camera demonstrated target recognition and spot detection ranges of 3 and 2 km, respectively. The results demonstrated that laser spot images could be obtained with a laser reception power of 65 µW or higher.
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In the fields of agriculture and forestry, the Normalized Difference Vegetation Index (NDVI) is a critical indicator for assessing the physiological state of plants. Traditional imaging sensors can only collect two-dimensional vegetation distribution data, while dual-wavelength LiDAR technology offers the capability to capture vertical distribution information, which is essential for forest structure recovery and precision agriculture management. However, existing LiDAR systems face challenges in detecting echoes at two wavelengths, typically relying on multiple detectors or array sensors, leading to high costs, bulky systems, and slow detection rates. This study introduces a time-stretched method to separate two laser wavelengths in the time dimension, enabling a more cost-effective and efficient dual-spectral (600 nm and 800 nm) LiDAR system. Utilizing a supercontinuum laser and a single-pixel detector, the system incorporates specifically designed time-stretched transmission optics, enhancing the efficiency of NDVI data collection. We validated the ranging performance of the system, achieving an accuracy of approximately 3 mm by collecting data with a high sampling rate oscilloscope. Furthermore, by detecting branches, soil, and leaves in various health conditions, we evaluated the system's performance. The dual-wavelength LiDAR can detect variations in NDVI due to differences in chlorophyll concentration and water content. Additionally, we used the radar equation to analyze the actual scene, clarifying the impact of the incidence angle on reflectance and NDVI. Scanning the Red Sumach, we obtained its NDVI distribution, demonstrating its physical characteristics. In conclusion, the proposed dual-wavelength LiDAR based on the time-stretched method has proven effective in agricultural and forestry applications, offering a new technological approach for future precision agriculture and forest management.
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An ultraviolet-infrared (UV-IR) dual-wavelength photodetector (PD) based on a monolayer (ML) graphene/GaN heterostructure has been successfully fabricated in this work. The ML graphene was synthesized by chemical vapor deposition (CVD) and subsequently transferred onto GaN substrate using polymethylmethacrylate (PMMA). The morphological and optical properties of the as-prepared graphene and GaN were presented. The fabricated PD based on the graphene/GaN heterostructure exhibited excellent rectify behavior by measuring the current-voltage (I-V) characteristics under dark conditions, and the spectral response demonstrated that the device revealed an UV-IR dual-wavelength photoresponse. In addition, the energy band structure and absorption properties of the ML graphene/GaN heterostructure were theoretically investigated based on density functional theory (DFT) to explore the underlying physical mechanism of the two-dimensional (2D)/three-dimensional (3D) hybrid heterostructure PD device. This work paves the way for the development of innovative GaN-based dual-wavelength optoelectronic devices, offering a potential strategy for future applications in the field of advanced photodetection technology.
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The simultaneous assay of duloxetine hydrochloride (DLX) and avanafil (AVN) in their pure forms, synthetic mixtures, and spiked human plasma was achieved using a novel, eco-friendly, sensitive, and specific HPTLC methodology that have been established and validated. Measuring the levels of co-administered antidepressants and sexual stimulants in biological fluids is an important step for individuals with depression and sexual problems. Separation was performed successfully using pre-coated silica gel 60-F254 as a stationary phase and a mobile phase composed of methanol, acetone, and 33% ammonia (8:2:0.05, v/v/v). Compact bands were produced by the optimized mobile phase that was chosen for development (Rf values were 0.23 and 0.75 for DLX and AVN, individually) after dual-wavelength detection for DLX and AVN at 232 and 253 nm, respectively. The results of polynomial regression analysis were exceptional (r = 0.9999 for both medicines) over concentration ranges of 5-800 and 10-800ng/spot for DLX and AVN, respectively. The quantitation limits were 4.69 and 9.53 ng/spot (0.31 and 0.94 µg/mL), whereas the detection limits were 1.55 and 3.15 ng/spot (0.63 and 1.91 µg/mL), for DLX and AVN, respectively. The International Council for Harmonization (ICH) criteria served as the basis for validating the established approach. Moreover, the proposed technique was evaluated in terms of greenness using four contemporary ecological metrics: The Analytical Greenness software (AGREE), the Green Analytical Procedure Index (GAPI), Eco-Scale, and the National Environmental Method Index (NEMI). Additionally, the Blue Applicability Grade Index (BAGI), a newly developed tool for evaluating the practicality (blueness) of procedures, was taken into consideration when evaluating the sustainability levels of the established approach.
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Developing a reliable and effective quality evaluation system for traditional Chinese medicine (TCM) is both challenging and crucial for its advancement. This study employs fingerprinting techniques to establish precise and comprehensive quality control for TCM, taking Xuezhikang capsules as an example and aiming to facilitate the internationalization of TCM. The "double wavelength absorption coefficient ratio fingerprint" and "Reliability theory" are developed to determine the fingerprint peak purity and fingerprint reliability respectively. Subsequently, the dual-wavelength fusion fingerprint was obtained to avoid the limitations of a single wavelength. In addition, an electrochemical fingerprint (ECFP) was obtained to assess the similarity of electroactive components in the sample, and the Differential Scanning Calorimetry quantized fingerprint (DSC QFP) was introduced for thermal analysis. Fingerprint-efficacy correlations between PL-EC* and dual-wavelength fusion fingerprint (DWFFP) provided valuable insights that there are 76.6 % of the fingerprint compounds exhibited electroactivity. Finally, samples were classified into grades 1â¼3 by combining DWFFP, ECFP and DSC QFP through the mean method, meeting the evaluation standard (SL-M > 0.9, PL-M between 80 % and 120 %). This study provides valuable information for ensuring the quality of TCM products, which represents a significant step forward in enhancing the reliability and authenticity of TCM products.
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Rastreo Diferencial de Calorimetría , Medicamentos Herbarios Chinos , Técnicas Electroquímicas , Medicina Tradicional China , Control de Calidad , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/análisis , Técnicas Electroquímicas/métodos , Reproducibilidad de los Resultados , Cromatografía Líquida de Alta Presión/métodosRESUMEN
4D microstructured actuators are micro-objects made of stimuli-responsive materials capable of induced shape deformations, with applications ranging from microrobotics to smart micropatterned haptic surfaces. The novel technology dual-wavelength volumetric microlithography (DWVML) realizes rapid printing of high-resolution 3D microstructures and so has the potential to pave the way to feasible manufacturing of 4D microdevices. In this work, DWVML is applied for the first time to printing stimuli-responsive materials, namely, liquid crystal networks (LCNs). An LCN photoresist is developed and characterized, and large arrays of up to 5625 LCN micropillars with programmable shape changes are produced by means of DWVML in the time span of seconds, over areas as large as â¼5.4 mm2. The production rate of 0.24 mm3 h-1 is achieved, exceeding speeds previously reported for additive manufacturing of LCNs by 2 orders of magnitude. Finally, a membrane with tunable, micrometer-sized pores is fabricated to illustrate the potential DWVML holds for real-world applications.
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An optical thermometry strategy based on Mn2+ -doped dual-wavelength emission phosphor has been reported. Samples with different doping content were synthesized through a high-temperature solid-phase method under an air atmosphere. The electronic structure of Li4 Zn(PO4 )2 was calculated using density functional theory, revealing it to be a direct band gap material with an energy gap of 4.708 eV. Moreover, the emitting bands of Mn2+ at 530 and 640 nm can be simultaneously observed when using 417 nm as the exciting wavelength. This is due to the occupation of Mn2+ at the Zn2+ site and the interstitial site. Further analysis was conducted on the temperature-dependent emission characteristics of the sample in the range 293-483 K. Mn2+ has different responses to temperature at different doping sites in Li4 Zn(PO4 )2 . Based on the calculations using the fluorescence intensity ratio technique, the maximum relative sensitivity at a temperature of 483 K was determined to be 1.69% K-1 , while the absolute sensitivity was found to be 0.12% K-1 . The results showed that the Li4 Zn(PO4 )2 :Mn2+ phosphor has potential application in optical thermometry.
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Termometría , Temperatura , Iones , Litio , ZincRESUMEN
The study on structure-property relationship has been a significant focus in the field of organic molecular luminescence. In the present work, three chiral binaphthyl-based triphenylethylene (HTPE) derivatives were prepared through condensation reactions. Despite their similar structures, these compounds exhibited distinct luminescent properties. Diphenylmethane-derived HTPE displayed dual-state emissions, characterized by dual-wavelength emissions which were insensitive to the polarity of solvents. The dual emissions in solution state could be attributed to the different locally excited (LE) excitons. However, upon aggregation, two stable conformers were generated, probably leading to different emission peaks. In contrast, dibenzocycloheptadiene-derived HTPE aggregates showed only a single emission peak. Surprisingly, fluorene-derived HTPE exhibited obvious luminescence in neither solution nor aggregate states due to inherent π-π interactions. These conclusions were substantiated by X-ray analysis, spectroscopic analysis, and theory calculations. Application studies demonstrated that fluorescence on/off switches could be achieved through exposure to acetone. More importantly, trace amounts of acetone could be detected using luminescent materials in both organic and aqueous phases with a detection limit of 0.08 %. Thus, this work not only presents a strategy for designing chiral triphenylethylene fluorophores but also provides valuable information for dual wavelength emissions resulting from two stable conformations.
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976 nm + 1976 nm dual-wavelength pumped Er-doped ZBLAN fiber lasers are generally accepted as the preferred solution for achieving 3.5 µm lasing. However, the 2 µm band excited state absorption from the upper lasing level (4F9/2 â 4F7/2) depletes the Er ions population inversion, reducing the pump quantum efficiency and limiting the power scaling. In this work, we demonstrate that the pump quantum efficiency can be effectively improved by using a long-wavelength pump with lower excited state absorption rate. A 3.5 µm Er-doped ZBLAN fiber laser was built and its performances at different pump wavelengths were experimentally investigated in detail. A maximum output power at 3.46 µm of ~ 7.2 W with slope efficiency (with respect to absorbed 1990 nm pump power) of 41.2% was obtained with an optimized pump wavelength of 1990 nm, and the pump quantum efficiency was increased to 0.957 compared with the 0.819 for the conventional 1976 nm pumping scheme. Further power scaling was only limited by the available 1990 nm pump power. A numerical simulation was implemented to evaluate the cross section of excited state absorption via a theoretical fitting of experimental results. The potential of further power scaling was also discussed, based on the developed model.
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Partial unilateral lentiginosis (PUL) is a pigmentation disorder characterized by multiple lentigines restricted to the normal skin in a unilateral and segmental pattern. Various lasers have been used for the treatment of this pigmented disorders, one of which is the copper bromide (CuBr) laser, which emits a dual-wavelength beam at 511 nm and 578 nm. A 35-year-old woman presented with multiple brown macules with a sharp demarcation from the midline of the forehead to the right cheek in a dermatomal distribution. Histopathological results showed increased melanin pigmentation in the basal layer with elongated rete ridges, supporting the diagnosis of PUL. Following photoprotection and skin priming, CuBr laser treatment was performed in two steps using 511 nm and 578 nm wavelength as the first and second steps, respectively, with the second step delivered immediately after. Clinical improvement was achieved after two sessions of treatment at a one-month interval, as shown by an increasing lightness value (L*) in spectrophotometry. No side effects, such as hyperpigmented macules, was observed. The patient also expressed satisfaction at her brighter facial skin. A sustainable favorable result persisted after six months of laser treatment. Interactions between cutaneous vasculature and melanocytes can affect the development of pigmented lesions. Melanocytes express the functional vascular endothelial growth factor receptor (VEGF) and are responsive to angiogenic factors. Ideally, these conditions should be treated with a dual-wavelength CuBr laser. The combination of 511 nm and 578 nm CuBr laser showed favorable results, supporting its potential as an effective treatment choice for PUL.
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The present work was developed to create three rapid, simple, eco-friendly, cheap spectrophotometric methods for concurrent assay of Sofosbuvir (SOF) and Simeprevir (SMV) in their pure, laboratory prepared mixture and pharmaceutical dosage form with high degree of accuracy and precision. Three methods were developed including iso-absorptive point, ratio subtraction and dual wavelength. The linear range of the proposed methods was 3.0-50.0 and 2.0-50.0 µg mL-1 for SMV and SOF, respectively. The proposed methods were validated according to ICH guidelines in terms of linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed approach is highly simple and the procedure is environmentally green making it suitable for the drug analysis in routine works.
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Background: To investigate the application value of 980-nm/1470-nm dual-wavelength fiber laser in thyroidectomy. Methods: The clinical data of 130 patients undergoing thyroid surgery from March 2017 to December 2018 were retrospectively analyzed. According to the use types of energy devices, the patients were divided into laser group and ultrasonic scalpel group, with 65 patients in each group. The baseline data, operation-related indicators, operation complications, postoperative pathological conditions, and follow-up results of the two groups were compared. Results: The operations were successfully completed in both groups. The median operative time of total thyroidectomy (TT), lobectomy+central lymph node dissection (CLND), TT+CLND in the laser group were longer than that in the harmonic scalpel group, and the difference was statistically significant (p < 0.05). The incidence of parathyroid gland congestion in the laser group (10.3%) was lower than that in the harmonic scalpel group (19.2%), and the difference was statistically significant (p < 0.05). No significant differences were found in operative type, intraoperative blood loss, postoperative drainage volume, operative complications, postoperative hospital stay, and lymph node metastasis rate between the two groups (p > 0.05). No incidence was noted of recurrence, metastasis, or death in both groups. Conclusions: The 980-nm/1470-nm dual-wavelength fiber laser had an efficacy in open thyroidectomy similar to that of the harmonic scalpel, was safe and feasible, and less damage to the parathyroid gland blood supply than a scalpel. It can be used as a new option for thyroid surgery.
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Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
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Colorantes Fluorescentes , Fagosomas , Microscopía Fluorescente/métodos , Ionóforos , Concentración de Iones de Hidrógeno , Espectrometría de FluorescenciaRESUMEN
Speckle Plethysmography (SPG) and Photoplethysmography (PPG) are different biophotonics technologies that allow for measurement of haemodynamics. As the difference between SPG and PPG under low perfusion conditions is not fully understood, a Cold Pressor Test (CPT-60 s full hand immersion in ice water), was used to modulate blood pressure and peripheral circulation. A custom-built setup simultaneously derived SPG and PPG from the same video streams at two wavelengths (639 nm and 850 nm). SPG and PPG were measured at the right index finger location before and during the CPT using finger Arterial Pressure (fiAP) as a reference. The effect of the CPT on the Alternating Component amplitude (AC) and Signal-to-Noise Ratio (SNR) of dual-wavelength SPG and PPG signals was analysed across participants. Furthermore, waveform differences between SPG, PPG, and fiAP based on frequency harmonic ratios were analysed for each subject (n = 10). Both PPG and SPG at 850 nm show a significant reduction during the CPT in both AC and SNR. However, SPG showed significantly higher and more stable SNR than PPG in both study phases. Harmonic ratios were found substantially higher in SPG than PPG. Therefore, in low perfusion conditions, SPG seems to offer a more robust pulse wave monitoring with higher harmonic ratios than PPG.
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Presión Arterial , Fotopletismografía , Humanos , Dedos , Presión Sanguínea/fisiología , ManoRESUMEN
In recent years, traditional analytical methods have failed to meet the widespread use of multi-component Chinese pharmaceutical formulations. To solve this problem, this study proposed a comprehensive analytical strategy using compound liquorice tablets (CLTs) as an example, both in terms of chemical quality and dissolution curve consistency. Firstly, the peak purity of the two wavelengths was checked using dual-wavelength absorbance coefficient ratio spectra (DARS) to avoid the fingerprint bias caused by peak purity. Secondly, liquid-phase dual-wavelength tandem fingerprint (DWTF) of 38 batches of CLTs was established for the first time. The two analytical methods were also evaluated using the systematically quantified fingerprint method (SQFM), and the 38 batches of samples were classified into two grades with good quality consistency. Quantitative analysis of the five markers of CLTs was performed simultaneously using the standard curve method (SCM) and the quantitative analysis of multiple components by single marker (QAMS). The results showed no significant differences between the two analytical methods (p > 0.5). In addition, the in vitro dissolution of CLTs in two media (pure water and pH = 4.5 medium) was determined by the total UV fingerprint dissolution assay. The similarity of the dissolution curves was also analyzed by combining the f2 factor and the dissolution-systematically quantified fingerprint method (DSQFM). The result showed that most of the samples had f2 > 50 and Pm satisfied the range of 70-130%. Finally, a principal component analysis (PCA) model was developed to combine the evaluation parameters of chemical fingerprint and dissolution curves for comprehensive analysis of the samples. In this study, a chromatographic and dissolution-based quality analysis method was proposed, which effectively overcomes the shortcomings of previous analytical methods and provides a scientific analytical method for the quality control of natural drugs.
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Medicamentos Herbarios Chinos , Glycyrrhiza , Medicamentos Herbarios Chinos/química , Glycyrrhiza/química , Solubilidad , Cromatografía Líquida de Alta Presión/métodos , Comprimidos/químicaRESUMEN
The UV spectra of Diclofenac Potassium DIC and Methocarbamol MET are superimposed making their analysis using direct or derivative spectrophotometric methods quite complicated. This study presents four effective spectrophotometric methods that enable simultaneous determination of both drugs without interference. The first method is based on application of simultaneous equation method on their zero order spectra where DIC has shown absorbance maxima at 276 nm and MET displays two absorbances maxima at 273 nm and 222 nm in distilled water. The second method relies on dual wavelength method, the two wavelengths (232 and 285 nm) were chosen for determination of DIC where the absorbance differences at these wavelengths are proportional to DIC concentration while the absorbance differences of MET are equal to zero. For the determination of MET, the two wavelengths (212 and 228 nm) were selected. The third method of first-derivative ratio has been applied where the derivative ratio absorbances of DIC and MET were measured at 286.1 and 282.4 nm, respectively. The fourth method utilizing ratio difference spectrophotometric (RD) method was eventually performed on the binary mixture. The amplitude difference between the two wavelengths (291and 305 nm) was calculated for DIC estimation while the amplitude difference between the two wavelengths (227and 273 nm) for MET determination. All methods show linearity range from 2.0-25 µg. mL-1 and 6.0-40 µg. mL-1 for DIC and MET respectively. The developed methods have been statistically compared with a reported method based on first derivative method and the results of statistical comparison confirm the accuracy and precision of the proposed methods therefore they can be effectively applied for determination of MET and DIC in pharmaceutical dosage form.
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Metocarbamol , Metocarbamol/análisis , Diclofenaco , Espectrofotometría/métodosRESUMEN
Pulse oximetry is a non-invasive method for measuring blood oxygen saturation. However, its detection scheme heavily relies on single-point measurements. If the oxygen saturation is measured at a single location, the measurements are influenced by the profile of illumination, spatial variations in blood flow, and skin pigment. To overcome these issues, imaging systems that measure the distribution of oxygen saturation have been demonstrated. However, previous imaging systems have relied on red and near-infrared illuminations with different profiles, resulting in inconsistent ratios between transmitted red and near-infrared light over space. Such inconsistent ratios can introduce fundamental errors when calculating the spatial distribution of oxygen saturation. In this study, we developed a novel illumination system specifically designed for a pulse oximetry imaging system. For the illumination system, we customized the integrating sphere by coating a mixture of barium sulfate and white paint inside it and by coupling eight red and eight near-infrared LEDs. The illumination system created identical patterns of red and near-infrared illuminations that were spatially uniform. This allowed the ratio between transmitted red and near-infrared light to be consistent over space, enabling the calculation of the spatial distribution of oxygen saturation. We believe our developed pulse oximetry imaging system can be used to obtain spatial information on blood oxygen saturation that provides insight into the oxygenation of the blood contained within the peripheral region of the tissue.