Indopolycarbocyanine dyes as perspective analytical reagents for spectrophotometric determination of nitrite by radical nitration.

A spectrophotometric method for the quantitative determination of nitrite was developed, based on the radical nitration of indopolycarbocyanine dyes in the presence of 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO). The rate of the reaction of the studied dyes with nitrite increases with the lengthening of the polymethine chain and the presence of hydrophilic sulfo groups in the side chain of the dye. TEMPO acts as a co-reagent, significantly accelerating the reaction rate and increasing the sensitivity of nitrite determination. The proposed reaction mechanism is supported by spectrophotometric and HPLC/MS studies. For Ind2 (tetramethine indocarbocyanine cationic dye), the limit of detection for nitrite is 0.50 µM within a linearity range of 1-13 µM. The developed method is sensitive, with a LOD 130 times lower than the maximum contaminant level (MCL) of nitrite in drinking water (65 µM), as specified by the WHO. The method is of low-toxicity and good selectivity, as the determination of nitrite is not significantly affected by the main components of water. The method was successfully applied for the analysis of nitrite in natural and bottled water.

Optimizing the Vanillin-Acid Sulfuric Method to Total Saponin Content in Leaves of Yerba Mate Clones.

Yerba mate (Ilex paraguariensis) is a forest species consumed in the form of non-alcoholic beverages in South America, with applications in foods, cosmetics, and pharmaceutical industries. The species leaves are globally recognized for their important bioactive compounds, including, saponins. We adjusted the vanillin-acid sulfuric method for determining spectrophotometrically the total saponin in yerba mate leaves. Seeking to maximize the extraction of saponins from leaves, a Doehlert design combined with Response Surface Methodology (RSM) was used, considering ethanol:water ratios and ultrasound times. In addition, the same methodology was used for the analysis of times and temperatures in the vanillin-sulfuric acid reaction heating. The contents of total saponin in mature leaves were compared in four yerba mate clones. The extraction was maximized using 40 % ethanol:60 % water and 60 minutes of ultrasound assisted extraction (UAE) without heating. For the reaction conditions, 70 °C for 10 minutes heating is recommended, and UV/Vis reading from 460 to 680 nm. Using the optimized methodology, total saponin contents ranged from 28.43 to 53.09 mg g-1 in the four yerba mate clones. The significant difference in saponin contents between clones indicate great genetic diversity and potential for clones' selection and extraction of these compounds from yerba mate leaves.

FIA- spectrophotometric method for the determination of amoxicillin in pharmaceuticals; application of AES, GAPI, and AGREE greenness assessment tools.

A new, simple, and sensitive FIA-spectrophotometric method has been developed for evaluating pure amoxicillin and pharmaceutical formulations. The FIA method involves the reaction of dapsone with sodium nitrite and hydrochloric acid. Subsequently, the diazotized dapsone is coupled with amoxicillin in an alkaline medium, resulting in a stable orange dye with a maximum wavelength of 440 nm. The developed method was validated according to the ICH guidelines and found to have a concentration range of 1-150 µg/mL, a correlation coefficient of 0.9994, a molar extinction coefficient of 0.273 × 104 L/mol.cm, and a detection limit of 0.074 µg/mL. The FIA method was then evaluated using AES, GAPI, and AGREES analytical greenness assessment tools. The FIA method uses dapsone as an eco-friendly reagent, in addition to the FIA method's advantages of reduced sample and reagent usage, reduced waste generation, and cheaper equipment. So, it has been proposed as an excellent eco-friendly method for the determination of AMX in pharmaceutical formulations.

Analytical Quality by Design Driven Development and Validation of UV-Visible Spectrophotometric Method for Quantification of Xanthohumol in Bulk and Solid Lipid Nanoparticles.

Objectives: Xanthohumol (XH) is a prenylated chalcone available naturally and has diverse pharmacological activities. It has some limitations in the physiological environment such as biotransformation and less gastrointestinal tract absorption. To overcome the limitations, we prepared nanoformulations [solid lipid nanoparticles (SLNs)] of XH. Therefore, an analytical method is required for the estimation of XH in the bulk nanoformulations, so we developed and validated a quality by design (QbD)-based ultraviolet (UV)-spectrophotometric method as per the International Conference of Harmonization (ICH) Q2 (R1) guidelines. Materials and Methods: The new analytical Qbd based UV-visible spectrophotometric technique is developed and validated for estimation of XH in bulk and SLNs as per ICH guidelines Q2 (R1). Critical method variables are selected on the basis of risk assessment studies. Optimization of method variables was performed using the a central composite design (CCD) model. Results: Multiregression ANOVA analysis showed an R2 value of 0.8698, which is nearer to 1, indicating that the model was best fitted. The optimized method by CCD was validated for its linearity, precision, accuracy, repeatability, limit of detection (LOD), limit of quantification (LOQ), and specificity. All validated parameters were found to be within the acceptable limits [% relative standard deviation (RSD) <2]. The method was linear between 2-12 g/mL concentration with R2 value 0.9981. Method was accurate with percent recovery 99.3-100.1%. LOD and LOQ were found to be 0.77 and 2.36 µg/mL, respectively. The precision investigation confirmed that the method was precise with %RSD <2. Conclusion: The developed and validated method was applied to estimate XH in bulk and SLNs. The developed method was specific to XH, which was confined by the specificity study.

Green-assisted spectrophotometric techniques utilizing mathematical and ratio spectra manipulations to resolve severely overlapped spectra of a cardiovascular pharmaceutical mixture.

Cardiovascular diseases, in particular hypertension and hypercholesterolemia, are two of the main causes of death worldwide. These conditions are silent killer syndromes that need a variety of pharmacological treatments to be effectively controlled. This study introduces novel, environmentally friendly spectrophotometric techniques for the simultaneous determination of telmisartan (TMS) and rosuvastatin calcium (RVS) in their pharmaceutical dosage forms. For the simultaneous determination of the binary mixture, the suggested methods included the dual wavelength method (DWM) which utilizes mainly the absorbance difference at 233 nm and 253 for TMS determination and, the absorbance difference at 274 nm and 310 for RVS determination as the selected wavelengths for each drug is directly proportional to the drug of interest independent on the other interfering component. The Fourier-self deconvolutions method (FSDM) depends on compressing their bandwidth to resolve the overlap. Ratio difference spectrophotometric method (RDSM) that utilizes TMS 35 µg.mL-1 and RVS 20 µg.mL-1, respectively as divisors to produce the ratio spectra for each drug. Further manipulation of the produced ratio spectra was applied for the determination of the two drugs. Mean centering method (MCM) where a suitable wavelength range was chosen to exclusively use the informative portions and prevent experimental spectrum noises. The investigated methods showed good levels of detection and quantification together with excellent linearity. The suggested methods' greenness was evaluated using two different greenness evaluation tools, which showed that the methods were green in terms of several factors, including the safety of the chemicals, instruments, and waste. The validity of the methodologieswas investigated by resolving prepared laboratory mixtureswith varying TMS and RVS ratios. The standard addition method also assured the newly added methods. Finally, statistical analysis using the reported method did not reveal any appreciable differences in terms of accuracy and precision. The developed methods can be employed in quality control laboratories to ascertain the binary mixture due to their high precision and affordability.

A novel spectrophotometric method based on plasmonic nanoparticles for nicotine detection.

In this paper, a spectrophotometric method for nicotine detection based on plasmonic nanoparticles (AuNPs and AgNPs) is proposed. Due to their specific properties, plasmonic nanoparticles have become interesting to use in the development of sensitive analytical methods. The localized surface plasmon resonance (LSPR) absorption bands for AgNPs and AuNPs with a wavelength at 395.5 nm (A395.5) and 543.5 (A543.5) nm, respectively, are used for the detection. Experimental variables such as solvent type and pH were optimized so as to determine the optimum working conditions. The analytical calibration curve for both AgNPs and AuNPs based on spectrophotometric methods was prepared with nicotine concentrations range from 0.10 to 5.00 µM (R2 = 0.9903) and 0.001-0.300 µM (R2 = 0.9960), respectively. The detection limits were found to be 0.001 µM for AuNPs based method and 0.09 µM for AgNPs based method. The proposed nanoparticle-based spectrophotometric methods showed a good stability, selectivity and low detection limit.

An expedient ion chromatography based method for high-throughput analysis of phytic acid in groundnut kernels.

A study was made to expedite ion chromatography method using IonPac analytical column and self-regenerating anion suppressor for phytic acid determination in groundnut seeds and compared with a widely adopted spectrophotometric method based on enzymatic hydrolysis. The Ion Chromatography method equipped with AG11 guard and AS11 analytical columns in isocratic mode using 65 mM NaOH mobile phase at 1 mL min-1 flow rate showed a sharp peak for phytic acid with a retention time of 2.42 ± 0.2 min. The peak area was plotted v/s concentration showed linearity with an R2 value of 0.997, detection limit of 0.028 mg L-1 and recovery of 98% as against R2 value of 0.988 and detection limit of 0.065 mg L-1 in the spectrophotometric method. The study demonstrates that Ion Chromatography method was more accurate with a better detection limit than spectrophotometry. Also, this method provides robust handling with lesser reagent requirements due to combined eluent generation and self-regenerating suppression. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05527-9.

Aloe Vera Functionalized Magnetic Nanoparticles Entrapped Ca Alginate Beads as Novel Adsorbents for Cu(II) Removal from Aqueous Solutions.

CABs (Ca alginate beads), AVCABs (Aloe vera Ca alginate beads), and AVMNCABs (Aloe-vera functionalized magnetic nanoparticles entrapped Ca alginate beads) were developed as adsorbents for the removal of Cu(II) from aqueous solutions. The materials were characterized using Fourier-transform infrared (FTIR) spectroscopy, high-resolution scanning electron microscopic (HR-SEM) analysis, X-ray diffraction (XRD), energy-dispersive X-ray (EDX) spectroscopy, and a vibrating-sample magnetometer (VSM). The effect of several parameters, such as pH, time, temperature, adsorbent dose, etc., were investigated. The adsorption isotherm of Cu(II) was adjusted best to the Langmuir model. The maximum adsorption capacities were 111.11 mg/g, 41.66 mg/g, and 15.38 mg/g for AVMNCABs, AVCABs, and CABs, respectively. The study of the adsorption kinetics for Cu(II) ions on beads followed a pseudo-second-order kinetic model, with a very good correlation in all cases. The adsorption studies used a spectrophotometric method, dealing with the reaction of Cu(II) with KSCN and variamine blue.

In Vitro and in Silico Analysis of Phytochemicals From Fallopia dentatoalata as Dual Functional Cholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

Current studies have found that butyrylcholinesterase (BuChE) replaces the biological function of acetylcholinesterase (AChE) in the late stage of Alzheimer's disease. Species in the genus of Fallopia, rich in polyphenols with diverse chemical structures and significant biological activities, are considered as an important resource for screening natural products to against AD. In this study, thirty-four compounds (1-34) were isolated from Fallopia dentatoalata (Fr. Schm.) Holub, and their inhibitory effects against AChE and BuChE were assessed. Compounds of the phenylpropanoid sucrose ester class emerged as the most promising members of the group, with 31-33 displaying moderate AChE inhibition (IC50 values ranging from 30.6 ± 4.7 to 56.0 ± 2.4 µM) and 30-34 showing potential inhibitory effects against BuChE (IC50 values ranging from 2.7 ± 1.7 to 17.1 ± 3.4 µM). Tacrine was used as a positive control (IC50: 126.7 ± 1.1 in AChE and 5.5 ± 1.7 nM in BuChE). Kinetic analysis highlighted compounds 31 and 32 as non-competitive inhibitors of AChE with Ki values of ∼30.0 and ∼34.4 µM, whilst 30-34 were revealed to competitively inhibit BuChE with Ki values ranging from ∼1.8 to ∼17.5 µM. Molecular binding studies demonstrated that 30-34 bound to the catalytic sites of BuChE with negative binding energies. The strong agreement between both in vitro and in silico studies highlights the phenylpropanoid sucrose esters 30-34 as promising candidates for use in future anti-cholinesterase therapeutics against Alzheimer's disease.

A Simple Spectrophotometric Method for Coccidian Oocysts Counting in Broiler Feces.

PURPOSE: This study aimed to develop a rapid spectrophotometric method for counting coccidian oocysts in broilers feces, based on a standard count method "Malassez cell". METHODS: Therefore, a raw suspension containing purified oocysts of Eimeria known to parasitize broiler chicken was used. Five concentrations of oocyst suspensions were prepared, and three series of counts using the proposed method were compared with the conventional protocol using Malassez cell to quantify oocysts. Calibration curves were drawn to correlate the specific absorbance measurement at 550 nm and the number of coccidian oocysts quantified using Malassez cell counting (oocysts mL-1). RESULTS: The obtained results allowed to establish a formula for an easy estimation of the cell count based on their respective absorbance. The developed method can be used not only for the in vitro evaluation of the anticoccidian activity of natural or synthetic substances, but also for assessing oocysts production and the level of coccidian infection in broilers. CONCLUSION: The estimation formula has been found to be satisfactory and useful for a wide range of Eimeria oocyst suspension, offering a methodology with high potential for automation in anticoccidian tests in vivo and in vitro.

The Chitosan-Based System with Scutellariae baicalensis radix Extract for the Local Treatment of Vaginal Infections.

Scutellarie baicalensis radix, as a flavone-rich source, exhibits antibacterial, antifungal, antioxidant, and anti-inflammatory activity. It may be used as a therapeutic agent to treat various diseases, including vaginal infections. In this study, six binary mixtures of chitosan with stable S. baicalensis radix lyophilized extract were obtained and identified by spectral (ATR-FTIR, XRPD) and thermal (TG and DSC) methods. The changes in dissolution rates of active compounds and the significant increase in the biological properties towards metal chelating activity were observed, as well as the inhibition of hyaluronic acid degradation after mixing plant extract with chitosan. Moreover, the combination of S. baicalensis radix lyophilized extract with a carrier allowed us to obtain the binary systems with a higher antifungal activity than the pure extract, which may be effective in developing new strategies in the vaginal infections treatment, particularly vulvovaginal candidiasis.

Improved spectrophotometric method for nitrite determination in processed foods and dietary exposure assessment for Korean children and adolescents.

A spectrophotometric method based on diazo-coupling reaction for nitrite analysis was established and validated, including inter-laboratory validation, linearity, accuracy, precision, the limit of detection (LOD) and limit of quantification (LOQ). The time-saving and high-recovery method was established by examining the filtration step, colorimetric process and concentration range of the calibration curve. This method showed good linearity (r2 > 0.999) in the range of 0.025-1.0 µg/mL. The three-level recoveries were between 86.7% and 108.6%, with the coefficient of variation (CV) below 5.8%. Mean nitrite concentration ranges in processed foods were ND-33.47 mg/kg. The mean nitrite intake was 0.8% of the Acceptable Daily Intake (ADI, 0.07 mg/kg bw/day) for all children and adolescents and 2.8% for the consumer group. The major contributors for all subjects and consumers were ham, sausage and bacon. These results indicated that the improved method was suitable for analyzing nitrite in processed foods and the nitrite exposure levels were safe.

Low consumption and portable technology for dithionite detection based on potassium ferricyanide differential spectrophotometry method in related advanced oxidation processes.

Carbon neutrality has been received more attention and emerged in wastewater treatment processes. Due to the development of treating technologies with the rising of new-emerging pollutants, the coupled chemical processes also should remain current for the goal of carbon-neutral operation. Among of those updated strategies, several advanced oxidation processes (AOPs) based on dithionite (DTN, S2O42-), a common water treatment agent, have been established for refractory organic contaminations removal. However, in terms of DTN detection, the traditional formol-titration method has several application limits including the low detection sensitivity and high consumption of formaldehyde. In this study, compared with traditional method, a low energy consumption technology has been developed based on the potassium ferricyanide with the carbon consumption decreasing by about 5 times. Moreover, detection limit of DTN (mmol/L level) also was lower than the titration method. The method was established based on the fact that every 1 mol of DTN can react with 2 mol [Fe(CN)6]3- under alkaline condition. According to that potassium ferricyanide (K3 [Fe(CN)6]) has the maximum absorption at 419 nm wavelength, a fitting equation based on the linear relationship between the absorbance variation of K3 [Fe(CN)6] and DTN amount in the ranges of 0-30 µmol with the detection limit of 0.6 µmol was established with the determination coefficient of 0.99935. It was found that there was no obvious influence of the ubiquitous foreign species with the amount lower than 6 mM, 4 mM, 6 mM, 4 mM and 1 mg/L for Cl-, HCO3-, NO3-, SO42- and NOM, respectively. Moreover, methanol and tert-butanol were employed to verify the influence of the presence of organic matters on the determination of DTN and no impact was observed in this study. The proposed method provides a new way for DTN detection with stable and countable performance in the related AOPs with the low electric energy and carbon source consumption and high detection efficiency.

Is there a relationship between dynamic thiol/disulfide homeostasis and osteoarthritis progression?

We aim to determine serum dynamic thiol/disulphide homeostasis with novel methods in early-stage osteoarthritis and late-stage osteoarthritis patients and investigated whether it was associated with the progression of osteoarthritis risk or not. One hundred eighteen patients were included in this prospective study. Osteoarthritis patients were divided into five stages, according to the Kellgren-Lawrence scale. Dynamic thiol/disulphide homeostasis was determined with a novel spectrophotometric method. Late-stage osteoarthritis patients had significantly lower levels of native and total thiol than the patients of early-stage osteoarthritis. Disulphide, index-1, index-2 levels, and WOMAC score of late-stage osteoarthritis patients were significantly higher than the ones belonging to patients of early-stage osteoarthritis. Decreased native thiol and total thiol levels and increased WOMAC score and disulphide levels were independently associated with increased risk of late-stage osteoarthritis. We suggest that both WOMAC score and dynamic thiol/disulphide homeostasis may be implicated in the pathogenesis and progression of osteoarthritis. We also recommend that dynamic thiol/disulphide homeostasis may have clinical utility as possible markers of differential diagnosis of early-stage and late-stage osteoarthritis.

Development of a simplified spectrophotometric method for nitrite determination in water samples.

A new eco-friendly, rapid, and sensitive spectrophotometric method was developed to determine small quantities of nitrite, based on a diazotization mechanism. In an acidic solution, sulfathiazole was first diazotized with sodium nitrite, followed by adding phosphate buffer to form a yellow-colored compound, which showed maximum absorption at 450 nm, without the need for the addition of coupling agents such as N-(1-naphthyl) ethylenediamine. The effects of reagents amount and the optimal experimental conditions were examined by Central composite design. The simplified method presented a wide linear range of nitrite between 0.091 µg mL-1 and 1.47 µg mL-1, a sensitivity of 0.447 Abs mL µg-1, a determination coefficient of 0.998, and a low limit of detection of 0.053 µg mL-1. The simplified method was found to be comparable to the Griess method. It was evaluated for the measurements of nitrite using the accuracy profile approach. The validation procedure results established that 80% of the future results would be within the acceptability limit of 10% over the validation domain ranging from 0.174 µg mL-1 to 1.37 µg mL-1. The developed method was furtherly applied in the determination of nitrite using a developed paper-based analytical device that detected a nitrite concentration of 3 µg mL-1 which is considered by the World Health Organization to be the maximal permissible limit of nitrite in drinking water.

The Direct Spectrophotometric Method Is Reliable for Initial Assessment of Total Bilirubin in Neonatal Venous Plasma.

OBJECTIVE: To determine the consistency of bilirubin values between the direct spectrophotometric (DS) and colorimetric diazo (diazo) methods in neonatal venous plasma specimens. METHODS: We measured the total bilirubin via the DS and diazo methods in 255 neonatal venous plasma specimens and compared the overall and subgroup results. RESULTS: Slight underestimation of total bilirubin values in most specimens using the DS method was observed, with higher mean biases found in higher concentrations. Significantly high positive correlations were found in all groups in which most of the different values were within the limits of agreement. DS cutoff of > 12 mg/dL showed 100% for all predictive indices in comparison with the diazo cutoff > 15 mg/dL. CONCLUSIONS: Measurement of total bilirubin in neonatal venous plasma using the DS method had favorable agreement and high correlation with the diazo method. Therefore, the direct spectrophotometric method can be used as a reliable screening method.

A rapid spectrophotometric method to identify inhibitors of human erythropoiesis.

Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min). Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3', 5,5'- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 µl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H2SO4. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation. Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.

A Simple and Cost-Effective Method for Measuring Hemolysis in Biobank Serum Specimens.

Background: During sampling and processing, blood samples can be affected by hemolysis. Information is lacking regarding hemolysis for biobank samples. There is a need for a method that can easily measure hemoglobin as an indicator of hemolysis in stored samples before they are included in research projects. In this study we present a simple method for estimating hemolysis and investigate the effect of centrifugation speeds and temperatures on sample turbidity that commonly interferes with measurements. Methods: Using a variation of the Beer-Lambert law, we quantified the hemoglobin concentration in 75 long-term stored samples at a wavelength of 414 nm with a NanoDrop™ 8000 spectrophotometer. Owing to interference from turbidity, the samples underwent different treatments post-thawing: centrifugation at 10,000 and 20,000 g at two different temperatures (4°C and 19°C) for 15 minutes. In addition, freshly collected serum samples (n = 20) underwent a single freeze-thaw cycle, with hemoglobin measured prefreeze, post-thaw, and postcentrifugation. Kruskal-Wallis rank sum test groups and pairwise Wilcoxon rank test were used for statistical analysis. Results: A strong effect of centrifugation on the turbidity was shown for the long-term stored samples, however, this effect was independent of the temperature or centrifugation speeds. Centrifugation at 20,000 g for 15 minutes at 19°C reduced the turbidity up to 50%. A single freeze-thaw cycle in the fresh samples increased the optical density at 414 nm slightly, indicating a false increase of hemoglobin concentration. The following centrifugation reduced the concentration to less than the initial sample measurements, suggesting the presence of interference immediately after sampling. Conclusion: We describe here a simple and cost-effective NanoDrop-based method for measuring hemolysis levels intended for use in biobank facilities. We found that centrifugation, but not temperature, is a crucial step to reduce interference from turbidity.

Charge-transfer complex-based spectrophotometric method for the determination of mesotrione in environmental samples.

A simple, accurate, sensitive, and selective spectrophotometric method has been developed for the determination of mesotrione. This method is based on the reaction of mesotrione with Fe(III) to form a charge transfer metal complex having λmax at 348 nm. Beer's law was obeyed in the concentration range of 0.2-10.0 µg mL-1 with limit of detection (LOD) and limit of quantification (LOQ) equal to 0.053 and 0.162 µg mL-1, respectively. The percent recovery of mesotrione from different environmental and agricultural samples was found to be 95.00-106.50% at various levels. Notably, the developed method was successfully employed for the determination of mesotrione in environmental (pond water, canal water, tap water, and soil) and agricultural (maize grains) samples.

Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase.

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A-ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 µM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.