Validation and interlaboratory comparison of anticoagulant rodenticide analysis in animal livers using ultra-performance liquid chromatography-mass spectrometry.

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.

Fusarium Mycotoxins in Maize Field Soils: Method Validation and Implications for Sampling Strategy.

While mycotoxins are generally regarded as food contamination issues, there is growing interest in mycotoxins as environmental pollutants. The main sources of trichothecene and zearalenone mycotoxins in the environment are mainly attributed to Fusarium infested fields, where mycotoxins can wash off in infested plants or harvest residues. Subsequently, mycotoxins inevitably enter the soil. In this context, investigations into the effects, fate, and transport are still needed. However, there is a lack of analytical methods used to determine Fusarium toxins in soil matrices. We aimed to validate an analytical method capable of determining the toxins nivalenol (NIV), deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-AcDON), and zearalenone (ZEN), at environmentally relevant concentrations, in five contrasting agricultural soils. Soils were spiked at three levels (3, 9 and 15 ng g-1), extracted by solid-liquid extraction assisted with ultrasonication, using a generic solvent composition of acetonitrile:water 84:16 (v:v) and measured by LC-HRMS. Method validation was successful for NIV, DON, and 15-AcDON with mean recoveries > 93% and RSDr < 10%. ZEN failed the validation criteria. The validated method was applied to eight conventionally managed maize field soils during harvest season, to provide a first insight into DON, NIV, and 15-AcDON levels. Mycotoxins were present in two out of eight sampled maize fields. Soil mycotoxin concentrations ranged from 0.53 to 19.4 ng g-1 and 0.8 to 2.2 ng g-1 for DON and NIV, respectively. Additionally, we found indication that "hot-spot" concentrations were restricted to small scales (<5 cm) with implications for field scale soil monitoring strategies.

Effect of ethoxylated sorbitan ester surfactants on the chromatographic efficiency of poly(ethylene glycol)-based monoliths.

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate Mn 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate Mn 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m.

Application of a fast and sensitive method for the determination of contaminants of emerging concern in wastewater using a quick, easy, cheap, effective, rugged and safe-based extraction and liquid chromatography coupled to mass spectrometry.

The inefficiency of wastewater treatment plants (WWTPs) to remove contaminants of emerging concern (CECs) leads to their continuous release to the environment. Consequently, CECs are present at low concentrations in the treated wastewater (TWW), producing unpredicted and unwanted effects on living organisms as they are discharged into water receiving bodies. This work presents a fast and reliable method for the determination of CECs in TWW based on the innovative application of a QuEChERS (quick, easy, cheap, effective, rugged and safe) method for water extraction and determination by sensitive liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry (LC-QqLIT-MS/MS). The scope of the proposed QuEChERS-based method allows the monitoring of 107 CECs, including pharmaceuticals (58), antibiotics (16) and pesticides (33). The proposed method was successfully validated in urban TWW at two concentration levels (50 and 500 ng L-1) and it is a feasible alternative to conventional and time-consuming solid-phase extraction (SPE) methodologies. 89% of the CECs presented mean recovery values in the 70-120% range with relative standard deviations (RSDs) always < 20% (intra and inter-day precision), and limits of quantification (LOQs) in the range 5-500 ng L-1 (89% of the compounds showed a LOQ ≤ 50 ng L-1). The applicability of the method was demonstrated by the analysis of urban TWW samples (7 sampling events). In total, 35 CECs (23 pharmaceuticals, 2 antibiotics and 10 pesticides) were detected in the monitored samples with concentrations ranging from 5 to 677 ng L-1.

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanide in human serum by ultra-high-performance liquid chromatography-tandem mass spectrometry.

A simple and rapid ultra-high-performance liquid chromatography coupled with mass spectrometry method was developed for acyclovir and its metabolite 9-carboxymethoxymethylguanine in human serum. After precipitation of serum samples with 0.1% formic acid in acetonitrile/methanol (40:60, v/v), components were separated on a Luna Omega C18 column (1.6 µm; 2.1 × 150 mm) at 40°C. Mobile phase A (2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and mobile phase B (2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v) were used for gradient elution. A linear calibration curve was obtained over the range of 0.05-50 mg/L, and the correlation coefficients were better than 0.999. The limit of quantitation was 0.05 mg/L for both analytes. The intra- and interday accuracy and precision at three concentration levels ranged between 1.6 and 13.3%, and recoveries were achieved with a range between 92.2 and 114.2%. This method was developed and validated for the therapeutic monitoring of acyclovir in patients.

Evolution of cocoa flavanol analytics: impact on reporting and cross-study comparison.

Cocoa flavanols (CF) are a group of dietary bioactives that have been studied for their potential health benefits for over two decades. In this time, multiple methods for CF testing have evolved, introducing the potential for differences in reported CF content. The reliable characterization of CF content in food and test materials used in clinical studies is critical to comparisons of research studies over time, as well as critical to enabling the systematic reviews and meta-analyses required to support dietary recommendations of bioactives. In this work, we compared two analytical methods that have been widely applied to characterize materials used in clinical research and a method newly recognized by AOAC as the official method for CF analysis. Differences in accuracy of -36% to +20% were observed when comparing CF contents determined with these methods, supporting the notion that CF values determined across methods are not directly comparable. To address differences, a linear regression model was developed to predict CF values. This approach was cross-validated and directly applied to the conversion of CF values published in key scientific papers on the benefits of CF. This work provides a valid tool to compare CF values reported across these different methods and enables comparisons and interpretation of studies investigating the bioactivity of CF.

Standardized production of a homogeneous latex enzyme source overcoming seasonality and microenvironmental variables.

Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the enzymes is reported. The latex was mixed with distilled water and the mixture was cleaned through centrifugation. It was dialyzed with distilled water and centrifuged again to recover the soluble fraction [EP]. The dialyze is a key feature of the process. EP was characterized in terms of protein profile, chemical stability, among other criteria. Wild plants belonging to ten geographic regions and grown in different ecological conditions were used as latex source. Collections were carried out, spaced at three-month, according to the seasons at the site of the study. Proteolytic activity was measured as an internal marker and for determining stability of the samples. EP was also analyzed for metal content and microbiology. EP showed similar magnitude of proteolysis, chromatographic and electrophoretic profiles of proteins. Samples stored at 25 °C exhibited reduced solubility (11%) and proteolytic capacity (11%) after six months. Enzyme autolysis was negligible. Microbiological and metal analyses revealed standard quality of all the samples tested. EP induced milk clotting and hide dehairing after storage for up to six months.

Practical application of Westgard Sigma rules with run size in analytical biochemistry processes in clinical settings.

BACKGROUND: The performance of 18 routine chemical detection methods was evaluated by the sigma (σ) metric, and Westgard Sigma rules with run size were used to establish internal quality control (IQC) standards to reduce patient risks. MATERIALS AND METHODS: External quality assessment (EQA) and internal quality control data from 18 assays in a biochemical laboratory were collected from January to June 2020. The sigma values of each assay were calculated, based on the bias, total error allowable, and coefficient of variation, appropriate quality control rules were selected. According to the quality goal index, the main causes of poor performance were determined to guide quality improvement. RESULTS: At IQC material level 1, seven of the 18 assays achieved five sigma (excellent), and five assays (UA, Crea, AMY, TC and Na) showed world-class performance. At IQC material level 2, 14 of the 18 assays achieved 5 sigma (excellent), and thirteen assays (UA, ALT, CK, Crea, AMY, K, AST, ALP, Na, LDH, Mg, TC and GGT) showed world-class performance. The quality goal index (QGI) was calculated for items with analysis performance <5 sigma, and the main causes of poor performance were determined to guide quality improvement. CONCLUSIONS: Westgard sigma rules with run size are an effective tool for evaluating the performance of biochemical assays. These rules can be used to more simply and intuitively select the quality control strategy of related items and reduce the risk to patients.

Revision of the calibration experiment in asymmetrical flow field-flow fractionation.

Asymmetrical flow field-flow fractionation is a versatile chromatographic fractionation method. In combination with at least one detection technique it is used for size-based separation of colloids, biomolecules and polymers. Although often used as pure separation method, a well-elaborated theory is available that allows precise quantification of the translational diffusion coefficient D. Still, current literature suggests different ways to transform this theory into applicable experimental procedures and no "gold standard" for correct data processing exists. While some sources report a direct way to extract diffusion information from the fractogram, others suggest the necessity of an external calibration measurement to obtain the channel width w. In this work, we compare the different approaches and calibration algorithms based on original and literature data using our own open-source AF4 evaluation software. Based on the results, we conclude that available AF4 setups do not fulfill the requirements for absolute measurements of D. We show that the best way to conduct is to consider the area of the channel and D of the calibrant while neglecting the small peak which occurs in the void peak region.

Application of Chiral and Achiral Supercritical Fluid Chromatography in Pesticide Analysis: A Review.

Supercritical fluid chromatography (SFC), the most common mode of which employs pressurized carbon dioxide as the mobile phase, is enjoying resuscitation. It is once again reconsidered as a fast developing chromatographic technique for the separation and identification of compounds in mixtures. In recent years, significant improvements in instrumentation, and its proficiency in specialized applications, have rekindled interest in the technique. SFC applicability in various fields, such as pharmaceutical analysis, bioanalysis, forensic science, environmental analysis, food science, has continued to expand. The present article delineates a comprehensive up-to-date overview of the applications of SFC in pesticide analysis, including the monitoring of their residues in different matrices and the investigation of their environmental behaviors such as dissipation and bioaccumulation. Since ~30% of currently registered pesticides are chiral compounds, attention is also paid to the analysis of such pesticides due to their enantioselective biological activities. Thus, both achiral and chiral SFC in pesticide analysis is reviewed. The article covers discussions on chromatographic conditions, method validation, and sample preparation as well as comparisons with gas chromatographic and liquid chromatographic approaches.

Comparison of internal energy distributions generated by supercritical fluid chromatography versus liquid chromatography hyphenated with electrospray high resolution mass spectrometry.

The internal energy distributions of thermometer ions dissolved in supercritical and liquid solvents and produced in the gas phase by electrospray (ESI) were measured and compared by the survival yield method. The influence of different chromatographic conditions such as the nature of solvents, the composition of the mobile phase, the pressure of the back pressure regulator was studied for supercritical fluid chromatography (SFC) whereas the influence of the composition and of the flow rate of the mobile phase was investigated for liquid chromatography (LC). The MS instrumental parameters were studied in parallel for SFC and LC showing that the drying gas temperature and the fragmentor voltage affected the internal energy distribution, whereas the capillary voltage did not modify the internal energy distribution. A comparison of the internal energy distributions generated by SFC and LC was carried out leading to conclude that SFC led to higher internal energy, i.e. more in-source fragment ions, than LC.

Comparison of three different analytical protocols for 2019 updated D2425 method for renewable jet fuel product certification analysis.

ASTM standard specification D7566 covers the manufacture of synthetic aviation turbine fuel components and their blends with conventional Jet fuel (Jet A or Jet A-1). One of the components is renewable jet fuel (RJF) which is synthetic paraffinic kerosene (SPK) made from hydroprocessed esters and fatty acids (HEFA). The specification D7566 dictates property requirements for the SPK-HEFA, including concentration limits for selected hydrocarbon types (paraffins, cycloparaffins, and aromatics), which are analyzed by using the mass spectrometry (MS) based standard method D2425. The most recent update for D2425 released in 2019 includes the synthetic hydrocarbon sample type (e.g., SPK-HEFA) and defines various analytical procedures for the analysis. Notably, the procedures differ considerably from each other, and the experimental conditions are not defined in details. This leads to laboratories setting up analytical schemes for D2425 that are likely to differ from each other, which may result in variation in the quality of the results obtained in different laboratories. In the present study, the performances of D2425 analytical protocols set up by three laboratories were tested in certification analysis (D7566) of SPK-HEFA type RJF. The tested analytical protocols were proven to comply with the requirements of the 2019 version of the D2425 standard. Furthermore, the precisions of the protocols did not differ significantly from each other. However, a significant bias was found for the results obtained for cycloparaffins and aromatics. Further, considerable differences were found in the bias values between the laboratories. Based on the results of this study, the guidelines of the 2019 updated D2425 standard may result in setting up an analytical protocol for D2425 which may not be optimal for RJF certification.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

The new IVD Regulation 2017/746: a case study at a large university hospital laboratory in Belgium demonstrates the need for clarification on the degrees of freedom laboratories have to use lab-developed tests to improve patient care.

Objectives: The new European In Vitro Diagnostic (IVD) Regulation 2017/746 (IVDR) restricts the use of lab-developed tests (LDT) after 26th May 2022. There are no data on the impact of the IVDR on laboratories in the European Union. Methods: Laboratory tests performed in UZ Leuven were divided in four groups: core laboratory, immunology, special chemistry, and molecular microbiology testing. Each test was classified as Conformité Européenne (CE)-IVD, modified/off-label CE-IVD, commercial Research Use Only (RUO) or LDT. Each matrix was considered a separate test. Results: We found that 97.6% of the more than 11.5 million results/year were generated with a CE-IVD method. Of the 922 different laboratory tests, however, only 41.8% were CE-IVD, 10.8% modified/off-label CE-IVD, 0.3% RUO, and 47.1% LDT. Off-label CE-IVD was mainly used to test alternative matrices not covered by the claim of the manufacturer (e.g., pleural or peritoneal fluid). LDTs were mainly used for special chemistry, flow cytometry, and molecular testing. Excluding flow cytometry, the main reasons for the use of 377 LDTs were lack of a CE-IVD method (71.9%), analytical requirements (14.3%), and the fact the LDT was in use before CE-IVD available (11.9%). Conclusions: While the large majority of results (97.6%) were generated with a CE-IVD method, only 41.8% of laboratory tests were CE-IVD. There is currently no alternative on the market for 71.5% of the 537 LDTs performed in our laboratory which do not fall within the scope of the current IVD directive (IVDD). Compliance with the IVDR will require a major investment of time and effort.

Determination of prescribed and designer benzodiazepines and metabolites in influent wastewater.

Benzodiazepines are important prescription pharmaceuticals used to help in the treatment of anxiety and sleep disorders. However, they also have a strong potential for abuse. In this respect, illicit benzodiazepines, i.e. not prescribed in Australia and designer benzodiazepines, which are new compounds that are not legally prescribed in any jurisdiction, have emerged in the illicit Australian market in recent years. Designer benzodiazepines are a new class of new psychoactive substances (NPS) and are particularly dangerous due to limited toxicity information and propensity to be mistaken for conventional benzodiazepines, leading to severe side effects and potentially death. It is therefore important to assess the prevalence of the use of these compounds in the community. The current work presents a validated liquid chromatography-mass spectrometry method for 20 prescribed and designer benzodiazepines and metabolites: 7-amino nimetazepam, alpha-hydroxy alprazolam, alprazolam, clonazepam, delorazepam, deschloroetizolam, diazepam, diclazepam, etizolam, flubromazepam, flunitrazepam, lorazepam, lormetazepam, meclonazepam, midazolam, nimetazepam, nitrazepam, oxazepam, pyrazolam and temazepam. Quetiapine, a prescription sedative drug that has been diverted for non-medical use, was also validated. Limits of quantification were predominantly below 10 ng L-1, except for the ubiquitous oxazepam, quetiapine and temazepam, which were between 75-300 ng L-1. Stability, recovery and matrix effects were also examined. Finally, this method was applied to influent wastewater from South Australia, which showed the presence of many benzodiazepines including the NPS etizolam.

Desalting and Concentration of Common Hydraulic Fracturing Fluid Additives and their Metabolites with Solid-Phase Extraction.

This work describes the development of a solid-phase extraction method capable of detecting common fracturing fluid additives in flowback and produced water with mass spectrometry. Dissolved organic carbon (DOC) was used as a bulk measurement to investigate the retentive capacity of seven sorbents and to determine a loading volume. Conductivity was used to determine rinse volume. Based on this, four sorbents (HLB, PPL, Carbon, and C-18) were selected for further investigation of their ability to recover common fluid additives. Enrichment factors were calculated for poly(ethylene) glycols (PEGs), PEG-amines, and their metabolites PEG-carboxylates and PEG-carboxylate-amines, poly(propylene) glycols (PPGs), and linear alkyl ethoxylates (LAEs). The sorbent HLB gave the greatest enrichment for all of these compounds, with an average of 8.0× for PEGs, 11.9× for PEG-amines, 4.9× for PEG-carboxylates, and 21.6× for LAEs, though enrichment was highly dependent on sample composition. The effect was more pronounced for higher molecular weight compounds and enabled detection of some compounds in saltier samples. Then, HLB was used to recover these additives from 1:200 and 1:1000 dilutions in groundwater, illustrating the ability of solid-phase extraction to detect these compounds at low levels (<100 ppb) and highlighting the utility of desalting. This method was used to identify ethoxylated amines in flowback and produced waters from across the country.

The Automation Technique Lab-In-Syringe: A Practical Guide.

About eight years ago, a new automation approach and flow technique called "Lab-In-Syringe" was proposed. It was derived from previous flow techniques, all based on handling reagent and sample solutions in a flow manifold. To date Lab-In-Syringe has evidently gained the interest of researchers in many countries, with new modifications, operation modes, and technical improvements still popping up. It has proven to be a versatile tool for the automation of sample preparation, particularly, liquid-phase microextraction approaches. This article aims to assist newcomers to this technique in system planning and setup by overviewing the different options for configurations, limitations, and feasible operations. This includes syringe orientation, in-syringe stirring modes, in-syringe detection, additional inlets, and addable features. The authors give also a chronological overview of technical milestones and a critical explanation on the potentials and shortcomings of this technique, calculations of characteristics, and tips and tricks on method development. Moreover, a comprehensive overview of the different operation modes of Lab-In-Syringe automated sample pretreatment is given focusing on the technical aspects and challenges of the related operations. We further deal with possibilities on how to fabricate required or useful system components, in particular by 3D printing technology, with over 20 different elements exemplarily shown. Finally, a short discussion on shortcomings and required improvements is given.

Are 2D fingerprints still valuable for drug discovery?

Recently, molecular fingerprints extracted from three-dimensional (3D) structures using advanced mathematics, such as algebraic topology, differential geometry, and graph theory have been paired with efficient machine learning, especially deep learning algorithms to outperform other methods in drug discovery applications and competitions. This raises the question of whether classical 2D fingerprints are still valuable in computer-aided drug discovery. This work considers 23 datasets associated with four typical problems, namely protein-ligand binding, toxicity, solubility and partition coefficient to assess the performance of eight 2D fingerprints. Advanced machine learning algorithms including random forest, gradient boosted decision tree, single-task deep neural network and multitask deep neural network are employed to construct efficient 2D-fingerprint based models. Additionally, appropriate consensus models are built to further enhance the performance of 2D-fingerprint-based methods. It is demonstrated that 2D-fingerprint-based models perform as well as the state-of-the-art 3D structure-based models for the predictions of toxicity, solubility, partition coefficient and protein-ligand binding affinity based on only ligand information. However, 3D structure-based models outperform 2D fingerprint-based methods in complex-based protein-ligand binding affinity predictions.

Update and Revalidation of Ghose's Cellulase Assay Methodology.

New studies on cellulolytic enzymes aiming to improve biofuels production lead to a concern over the assaying methods commonly applied to measure their activity. One of the most used methods is Ghose's cellulase and endoglucanase assay, developed by the International Union of Pure and Applied Chemistry in 1987. Carrying out this method demands high volumes of reagents and generation of high amounts of chemical residues. This work aimed to adapt Ghose's methodology to reduce its application cost and residue generation and validate the adjustments. To do so, International and Brazilian laws were applied to validate methodologies. Method's modifications were successfully validated according to all institutions and were considered linear, accurate, precise, and reproducible. It was possible to reduce the volume of reagents and residues in 12 times. Considering the routine work of most laboratories, it is a great reduction on material costs and residue treatment, which reflects in sustainability and environmental impacts.