A Novel and Simple Method for a Differentiation of Alcohol Types.

BACKGROUND: Alcohol poisoning is a significant global problem that has become an epidemic. The determination of the alcohol type is hereby essential as it may affect the course of the treatment; however, there is no routine laboratory diagnostic method for alcohol types other than for ethanol. In this study, we aimed to define a simple method for alcohol type differentiation by utilizing a combination of breathalyzer and spectrophotometrically measured serum ethanol results. METHODS: A breathalyzer and spectrophotometry were used to measure four different types of alcohol: ethanol, isopropanol, methanol, and ethylene glycol. To conduct serum alcohol analysis, four serum pools were created, each containing a different type of alcohol. The pools were analyzed using the spectrophotometric method with an enzymatic ethanol test kit. An experiment was conducted to measure the different types of alcohol using impreg-nated cotton and a balloon, simulating a breathalyzer test. An algorithm was created based on the measurements. RESULTS: Based on the results, the substance consumed could be methanol or isopropanol if the breathalyzer test indicates a positive reading and if the blood ethanol measurement is negative. If both the breathalyzer and the blood measurements are negative, the substance in question may be ethylene glycol. CONCLUSIONS: This simple method may determine methanol or isopropanol intake. This straightforward and innovative approach could assist healthcare professionals in different fields with diagnosing alcohol intoxication and, more precisely, help reducing related morbidity and mortality.

Spectrophotometric determination of celecoxib and tramadol in the new approved formulated dosage form using principle component regression assistive model.

Celecoxib and tramadol have been combined in a novel FDA-approved medication to address acute pain disorders requiring opioid treatment when other analgesics proved either intolerable or ineffective. The absorbance spectra of celecoxib and tramadol exhibit significant overlap, posing challenges for their individual quantification. This study introduces a spectrophotometric quantification approach for celecoxib and tramadol using a principle component regression assistive model to assist resolving the overlapped spectra and quantifying both drugs in their binary mixture. The model was constructed by establishing calibration and validation sets for the celecoxib and tramadol mixture, employing a five-level, two-factor experimental design, resulting in 25 samples. Spectral data from these mixtures were measured and preprocessed to eliminate noise in the 200-210 nm range and zero absorbance values in the 290-400 nm range. Consequently, the dataset was streamlined to 81 variables. The predicted concentrations were compared with the known concentrations of celecoxib and tramadol, and the errors in the predictions were evidenced calculating root mean square error of cross-validation and root mean square error of prediction. Validation results demonstrate the efficacy of the models in predicting outcomes; recovery rates approaching 100 % are demonstrated with relative root mean square error of prediction (RRMSEP) values of 0.052 and 0.164 for tramadol and celecoxib, respectively. The selectivity was further evaluated by quantifying celecoxib and tramadol in the presence of potentially interfering drugs. The model demonstrated success in quantifying celecoxib and tramadol in laboratory-prepared tablets, producing metrics consistent with those reported in previously established spectrophotometric methods.

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Determination of Phosphoglycolate Phosphatase Activity via a Coupled Reaction Using Recombinant Glycolate Oxidase.

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.

Spectrophotometric Assays for Measuring Hydroxypyruvate Reductase Activity.

Hydroxypyruvate reductase (HPR; EC 1.1.1.81) activity is integral to the photorespiratory pathway. Within photorespiration, HPR catalyzes the reduction of hydroxypyruvate, a product of the serine:glyoxylate aminotransferase reaction to glycerate, a substrate for glycerate kinase, using NADH as cofactor. Here we detail a spectrophotometric assay for measuring HPR activity in vitro by following the consumption of NADH at 340 nm.

Facile green spectrophotometric approaches for the determination of three natural edible antioxidant polyphenols in different matrices.

The combination of Curcumin (CRN), resveratrol (RSV), and quercetin (QRN) has significant antioxidant effects and is found to be more effective than a single polyphenol. Spectrophotometric methods are considered one of the most common analytical techniques for the determination of the drugs due to their sensitivity, rapidness, low cost, and reproducibility. Therefore, the presence of new, and simple methods for the determination of such compounds will be highly valuable, specially in the presence of spectral overlap. In this research, five different facile spectrophotometric methods were investigated for the simultaneous determination of that ternary mixture for the first time, including zero order (I), first derivative (II), ratio difference double divisor (III), first derivative ratio spectra (IV), and mean centering (V) methods. The designed approaches were linear over the concentration ranges of (1.0-10.0), (0.5-8.0), and (1.0-14.0) µg/mL, respectively for curcumin, resveratrol, and quercetin. The different methods were then validated as stated by the International Council of Harmonization. The accuracy and precision have been evaluated by statistical analysis including student t-test, variance ratio F-test, and ANOVA. Moreover, the greenness and whiteness of the proposed methods were assessed to ensure the adherence to the greenness characters.

Novel sample double dilution calibration method for determination of lithium in biological samples using automatic flow system with in-syringe reaction.

A novel sample double dilution calibration method (SDDCM) and an automatic flow system with in-syringe reaction and spectrophotometric detection were developed for determining lithium in biological samples. The method is based on the reaction of lithium with Thorin in an alkaline medium and the signal was measured at 480 nm. The reaction was performed simultaneously for both standards and samples in three syringes of the automatic flow system. The method was validated and successfully applied to the determination of lithium in synthetic and pharmaceutical samples, with results consistent with the ICP OES method. The novel calibration method, developed for the determination of lithium in biological samples, uses a sample with two dilution degrees. Using the method, the concentration of the analyte is determined by relating the signal for a less diluted sample to the calibration plot for a more diluted sample and vice versa. The implementation of the calibration method was facilitated by preparing solutions directly in the flow system. The use of two sample dilutions makes it possible to determine the analyte in the sample without preliminary preparation. Moreover, obtaining two results based on signals for a sample diluted to different degrees allows them to be verified for accuracy. The proposed approach was successfully verified by the determination of lithium in certified reference materials of blood serum and urine. Using the developed method lithium was determined within the concentration range of 0.06-1.5 mg L-1, with precision (CV, %) less than 6.7, and accuracy (RE, %) better than 6.9. The detection limit was 0.03 mg L-1.

Non-invasive prediction of maca powder adulteration using a pocket-sized spectrophotometer and machine learning techniques.

Discriminating different cultivars of maca powder (MP) and detecting their authenticity after adulteration with potent adulterants such as maize and soy flour is a challenge that has not been studied with non-invasive techniques such as near infrared spectroscopy (NIRS). This study developed models to rapidly classify and predict 0, 10, 20, 30, 40, and 50% w/w of soybean and maize flour in red, black and yellow maca cultivars using a handheld spectrophotometer and chemometrics. Soy and maize adulteration of yellow MP was classified with better accuracy than in red MP, suggesting that red MP may be a more susceptible target for adulteration. Soy flour was discovered to be a more potent adulterant compared to maize flour. Using 18 different pretreatments, MP could be authenticated with R2CV in the range 0.91-0.95, RMSECV 6.81-9.16 g/,100 g and RPD 3.45-4.60. The results show the potential of NIRS for monitoring Maca quality.

Digital Photocolorimetric Analysis of In Vitro Tooth Color Changes.

OBJECTIVE: Tooth color matching is challenging, and digital photocolorimetry using eLABor_aid (eLAB) provides objective evaluation through polarized photographs. However, its comparability with spectrophotometry remains unclear. METHODS AND MATERIALS: Bovine incisor root canals (n=30) were prepared to simulate an incomplete root apex. The teeth were randomly assigned to three groups based on intracanal medication: control (without medication); calcium hydroxide/propylene glycol; and triple-antibiotic paste (n=10 each). Tooth color was assessed using both eLAB and spectrophotometry. Measurements were taken at the crown medio-cervical region on five-time intervals (baseline, 1, 3, 7, and 14 days). Statistical analysis included two-way repeated-measures ANOVA, Sidak post hoc and Pearson's correlation test (α=0.05). RESULTS: No significant differences were observed between the two methods for either medication or follow-ups (p>0.05). Triple-antibiotic paste exhibited higher color variation (p<0.05). After 7 days, all groups presented significant color changes (p<0.05). Moderate to high correlations (R2 from 0.51 to 0.84, p<0.0001) were found between both methods for all groups at all intervals. CONCLUSION: The eLAB is a reliable method for detecting tooth color changes, and its results are comparable to spectrophotometry analysis.

Reliability of the color measurement of resin composites using images obtained using a stereoscopic loupe.

This study assessed the reliability of a color measurement method using images obtained from a charge-coupled device (CCD) camera and a stereoscopic loupe. Disc-shaped specimens were created using the composite Filtek Z350 XT (shades DA1, DA2, DA3, and DA4) (n = 3). CIELAB color coordinates of the specimens were measured using the spectrophotometer SP60 over white and black backgrounds. Images of the same specimens were taken using a CCD camera attached to a stereoscopic loupe. The color of the image was measured (red-green-blue [RGB]) using an image processing software and converted to CIELAB coordinates. For each color coordinate, data from images were adjusted using linear regressions predicting those values from SP60. The whiteness index for dentistry (WID) and translucency parameter (TP00) of the specimens as well as the color differences (ΔE00) among pairwise shades were calculated. Data were analyzed via repeated-measures analysis of variance and Tukey's post hoc test (α = 0.05). Images obtained using the loupe tended to be darker and redder than the actual color. Data adjustment resulted in similar WID, ΔE00, and TP00 values to those observed for the spectrophotometer. Differences were observed only for the WID of shade DA3 and ΔE00 for comparing DA1 and DA3 over the black background. However, these differences were not clinically relevant. The use of adjusted data from images taken using a stereoscopic loupe is considered a feasible method for color measurement.

Investigating the Z-scan technique for quantifying circulating cell-free DNA (ccfDNA) extracted from blood plasma as a potential biomarker for various cancers.

The Z-scan technique is a nonlinear optical method that has found applications in characterizing various materials, particularly those exhibiting nonlinear optical response (NLOR). This study applies the continuous wave (CW) Z-scan technique to examine the NLOR in terms of the nonlinear optical phase shifts(ΔΦ0) exhibited by the ccfDNA extracted from blood plasma samples collected from a group constituting 30 cancer-diagnosed patients and another group constituting 30 non-diagnosed individuals. The cancer group exhibited significantly higherΔΦ0versus incident power slopes compared to the non-cancer group (0.34 versus 0.12) providing a clear distinction between the two groups. The receiver operating characteristic (ROC) curve analysis of the results indicates a clear separation between cancer and non-cancer groups, along with a 94% accuracy rate of the data. The Z-scan results are corroborated by spectrophotometric analysis, revealing a consistent trend in the concentration values of ccfDNA samples extracted from both cancerous and non-cancerous samples, measuring 3.24 and 1.41 respectively. Additionally, more sensitive fluorometric analyses of the respective samples demonstrate significantly higher concentrations of ccfDNA in the cancer group, further affirming the correlation with the Z-scan results. The study suggests that the Z-scan technique holds promise as an effective method for cancer detection, potentially contributing to improved oncology diagnosis and prognosis in the future.

Novel assaying method for the accurate and rapid analysis of antioxidant total capacity based on hexachloroiridate(IV).

We introduce a novel method, namely IrRAC, for assessing total antioxidant capacity utilizing the single electron oxidant hexachloroiridate(IV). This method leverages the 488 nm absorption band of [IrCl6]2- largely reducing interferences from antioxidants and their oxidation products. [IrCl6]2- is stable 6 h in phosphate-buffered saline (PBS) ensuring consistent and reproducible absorbance readings and rendering spectrophotometric determinations under physiological neutrality. Individual assessments of 23 antioxidants reveal a linear correlation between decreasing absorbance and increasing antioxidant concentration. When the IrRAC assay was compared with several established water-based methods, strong correlations were found. Importantly, [IrCl6]2- shows a minimal oxidation of non-antioxidative substances. Moreover, IrRAC performs well with synthetic antioxidant mixtures and real samples, highlighting that the nature of antioxidants dominates the assay without much disturbance. Commercial availability of K2[IrCl6] eliminates the need of pretreatment of the oxidant. Undoubtedly, the new method confers a compelling and cost-effective alternative to the existing electron transfer-based methodologies.

Development of Two Eco-Friendly and High-Throughput Microwell Spectrophotometric Methods for Analysis of an Antibacterial Drug Tulathromycin in Pharmaceutical Bulk Form.

BACKGROUND: Tulathromycin (TUL) is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The existing methods for determination of TUL in its pharmaceutical bulk form are very limited and suffer from major drawbacks. OBJECTIVES: The aim of this study was the development of two innovative microwell spectrophotometric methods (MW-SPMs) for determination of TUL in its pharmaceutical bulk form. METHODS: The formation of charge-transfer complexes (CTCs) of TUL, as an electron donor, was investigated with 2,5-dihydroxy-3,6-dichlorocyclohexa-2,5-diene-1,4-dione (HCD) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (CBQ), as π-electron acceptors. The CTCs were characterized using UV-Vis spectrophotometry and computational calculations. The reactions were employed for the development of two MW-SPMs with one step for the quantitative analysis of TUL. RESULTS: The formation of CTCs was confirmed via the formation of characteristic absorption bands with maximum absorption at 520 and 460 nm for CTCs with HCD and CBQ, respectively. The stoichiometry of both CTCs was found to be 1:1, and the values of different spectroscopic and electronic constants confirmed the stability of the CTCs. The mechanisms of the reactions were postulated. The linear range of both MW-SPMs was 10-500 µg/mL. The LOQs were 13.5 and 26.4 µg/mL for methods involving reactions with HCD and CBQ, respectively. Both methods were successfully applied to the quantitation of TUL in pharmaceutical bulk form with acceptable accuracy and precision. The results of eco-friendliness/greenness assessment proved that both MW-SPMs fulfill the requirements of green analytical approaches. In addition, the one-step reactions and simultaneous handling of a large number of samples with micro-volumes in the proposed methods gave them the advantage of high-throughput analysis. CONCLUSION: This study described two new MW-SPMs as valuable analytical tools for the determination of TUL. HIGHLIGHT: The proposed methods are valuable analytical tool for the analysis of bulk form of TUL.

Protocol for assessing single-cell persister recovery kinetics and physiology in Escherichia coli using spectrophotometry.

Bacterial persisters constitute a small fraction of cells that transiently display multidrug tolerance, allowing them to survive antibiotic treatment and to establish a new population upon recovery from the persistent state. Here, we present a protocol to quantify post-antibiotic persister recovery kinetics and physiological states at the single-cell level. We describe steps for sample preparation, technical setup, and data acquisition using spectrophotometry. Our assay allows for the elucidation of genes and mechanisms involved in persister survival. For complete details on the use and execution of this protocol, please refer to Wilmaerts et al.1.

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.

A comparative evaluation of the variations on the shades of pressable ceramic system with the layering technique after different number of firing cycles: An in vitro study.

AIM: The main aim of the present study was to compare and evaluate the effect of repetitive firings on different shades of a pressable all ceramic system layered with veneering porcelain. SETTING AND DESIGN: In-vitro comparative study. MATERIALS AND METHODS: An in vitro comparative study was conducted, and a total of 60 disc shaped specimens (15 mm in diameter and 0.8 mm in thickness) were made of heat pressed ceramic of shades A2, A3, and B2 (20 discs of each shade) grouped as Group I, II, and III, respectively, using the lost wax technique. The discs were subsequently layered with veneering porcelain followed by glazing and overglazing and underwent a firing cycle at each step until six times combined. CIE L*a*b* measurements were noted on each sample after the third, fourth, fifth, and sixth firing using VITA Easyshade Advance 4.0 spectrophotometer. STATISTICAL ANALYSIS USED: Statistical Analysis was done by SPSS 17.0 software. One way analysis of variance, multiple comparisons using the Tukey test, and descriptive statistical analysis were done for all the groups in the study. P <0.05 was statistically significant. RESULTS: The mean color differences for the repeated firings were imperceptible (ΔE <1.67) to the human eye for all ceramic samples tested except between the fourth and fifth firing of Group II (shade A3). CONCLUSION: The analysis revealed that although repeated firings lead to changes in L*, a*, and b* values, the mean color difference was below the clinically acceptable color change (ΔE <3.7).

Field evaluation of a novel semi-quantitative point-of-care diagnostic for G6PD deficiency in Indonesia.

BACKGROUND: The WHO recommends routine testing of G6PD activity to guide radical cure in patients with Plasmodium vivax malaria. Females may have intermediate G6PD enzyme activity and to date, only complex diagnostics are able to reliably identify them. The semi-quantitative G6PD diagnostic "One Step G6PD Test" (Humasis, RoK; "RDT") is a lateral flow assay that can distinguish deficient, intermediate, and normal G6PD status and offers a simpler diagnostic alternative. METHODS: G6PD status of participants enrolled in Malinau and Nunukan Regencies and the capital Jakarta was assessed with the RDT, and G6PD activity was measured in duplicate by reference spectrophotometry. The adjusted male median (AMM) of the spectrophotometry measurements was defined as 100% activity; 70% and 30% of the AMM were defined as thresholds for intermediate and deficient G6PD status, respectively. Results were compared to those derived from spectrophotometry at the clinically relevant G6PD activity thresholds of 30% and 70%. RESULTS: Of the 161 participants enrolled, 10 (6.2%) were G6PD deficient and 12 (7.5%) had intermediate G6PD activity by spectrophotometry. At the 30% threshold, the sensitivity of the RDT was 10.0% (95%CI: 0.3-44.5%) with a specificity of 99.3% (95%CI: 96.4-100.0%); the positive predictive value was 50.0% (95%CI: 1.3-98.7%) and the negative predictive value 94.3% (95%CI: 89.5-97.4%). The corresponding figures at the 70% threshold were 22.7% (95%CI: 7.8-45.4%), 100.0% (95%CI: 97.4-100.0%), 100.0% (95%CI: 47.8-100.0%) and 89.1% (95%CI: 83.1-93.5%), respectively. CONCLUSION: While there is a dire need for an easy-to-use, economical, semi-quantitative diagnostic for the point of care, the observed performance of the "One Step G6PD Test" in its current form was insufficient to guide antimalarial treatment.

Development of Green and High-Throughput Microwell Spectrophotometric Methods for Determination of Galidesivir in Bulk Drug and Dosage Forms Based on Simple Oxidimetric Reactions With Inorganic Agents.

BACKGROUND: Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk or dosage form. OBJECTIVE: The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules). METHODS: The reactions were carried out in 96-well plates, and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized. RESULTS: Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry (GAC) approach. CONCLUSIONS: The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness. HIGHLIGHTS: Overall, the proposed MW-SPMs are versatile, valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Spider chart, greenness and whiteness assessment of experimentally designed multivariate models for simultaneous determination of three drugs used as a combinatory antibiotic regimen in critical care units: Comparative study.

In this study, five earth-friendly spectrophotometric methods using multivariate techniques were developed to analyze levofloxacin, linezolid, and meropenem, which are utilized in critical care units as combination therapies. These techniques were used to determine the mentioned medications in laboratory-prepared mixtures, pharmaceutical products and spiked human plasma that had not been separated before handling. These methods were named classical least squares (CLS), principal component regression (PCR), partial least squares (PLS), genetic algorithm partial least squares (GA-PLS), and artificial neural network (ANN). The methods used a five-level, three-factor experimental design to make different concentrations of the antibiotics mentioned (based on how much of them are found in the plasma of critical care patients and their linearity ranges). The approaches used for levofloxacin, linezolid, and meropenem were in the ranges of 3-15, 8-20, and 5-25 µg/mL, respectively. Several analytical tools were used to test the proposed methods' performance. These included the root mean square error of prediction, the root mean square error of cross-validation, percentage recoveries, standard deviations, and correlation coefficients. The outcome was highly satisfactory. The study found that the root mean square errors of prediction for levofloxacin were 0.090, 0.079, 0.065, 0.027, and 0.001 for the CLS, PCR, PLS, GA-PLS, and ANN models, respectively. The corresponding values for linezolid were 0.127, 0.122, 0.108, 0.05, and 0.114, respectively. For meropenem, the values were 0.230, 0.222, 0.179, 0.097, and 0.099 for the same models, respectively. These results indicate that the developed models were highly accurate and precise. This study compared the efficiency of artificial neural networks and classical chemometric models in enhancing spectral data selectivity for quickly identifying three antimicrobials. The results from these five models were subjected to statistical analysis and compared with each other and with the previously published ones. Finally, the whiteness of the methods was assessed by the recently published white analytical chemistry (WAC) RGB 12, and the greenness of the proposed methods was assessed using AGREE, GAPI, NEMI, Raynie and Driver, and eco-scale, which showed that the suggested approaches had the least negative environmental impact. Furthermore, to demonstrate solvent sustainability, a greenness index using a spider chart methodology was employed.