Simple Sample Preparation Method Research Articles

Application of a Green Deep Eutectic Solvent for Preconcentration and Determination of Copper and Cadmium in Food, Cosmetic, and Water Samples

Background:: Determination of environmental pollutants is important due to their harmful effects on the health of living organisms. However, direct measurement of many of these pollutants is not possible due to their low concentrations, which necessitates the need for sample preparation methods. One of the rapid and simple sample preparation methods is the Air-Agitated Liquid-Liquid Microextraction (AALLME) method, which requires toxic organic solvents that can lead to environmental pollution. Therefore, finding green solvents like Deep Eutectic Solvents (DESs) to replace them can be investigated. Methods:: A novel Deep Eutectic Solvent (DES) was synthesized from a combination of choline chloride and 2-phenylethanol with a molar ratio of 1:4, and it was examined using FT-IR analysis. This solvent was used in an AALLME method for measuring copper and cadmium heavy metal ions in complex real samples with Flame Atomic Absorption Spectroscopy (FAAS). Results:: The influential parameters of this method, such as solution pH (5.4), extraction cycles (12), and extraction solvent volume (440 μL), were optimized utilizing central composite design (CCD). Underneath the optimized circumstances, the detection limits for Cu2+ and Cd2+ were 0.14 and 0.09 ng mL-1, and the linear dynamic range was 0.47-50.0 and 0.32-22.5 ng mL-1, respectively. The preconcentration factors for these cations were 139.7 and 133.4, respectively. Conclusion:: Combining this novel green solvent and rapid sample preparation method for the preconcentration and determination of the studied heavy metal ions has shown promising results in terms of enrichment factors and detection limit values.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol

In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC–MS/MS and clinical application in kidney injury

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93–5.94 % and 2.30–8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1–3.13 ng/mL and 0.5–6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56–400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Pouring and Curing for Sample Preparation Technology for the Analysis of the Silicon-Aluminum Ratio of Molecular Sieves by Energy Dispersive X-Ray Fluorescence (EDXRF)

The silicon-aluminum ratio of the molecular sieves is a crucial control index for the quality of synthetic molecular sieves. Here a fast, simple and reliable sample preparation method, referred to as pouring and curing method, is described for determining the silicon-aluminum ratio of high silicon molecular sieves by energy dispersive x-ray fluorescence (EDXRF). Resin is used as the carrier with the addition of a curing agent, so that the sample powder is embedded in the resin matrix. By measuring sixteen random positions of the same cured sample, the homogeneity of the sample was evaluated to ensure the stability of the results. A double logarithmic calibration curve of the analysis line ratio and the corresponding concentration ratio of silicon and aluminum was established according to the binary ratio method. The results by EDXRF and inductively coupled plasma-optical emission spectrometer (ICP-OES) were compared with a relative standard error from 2.934 to 6.777%. The good precision shows that the pouring and curing method is a suitable for measuring the silicon-aluminum ratio of molecular sieves.

A simple, low cost and fast sample preparation method for fluorine determination by ISE in leguminous seeds and oilseeds.

This work presents the first report of the application of microwave-induced combustion in disposable vessels (MIC-DV) as a simple and fast sample preparation step for fluoride determination by ion-selective electrode (ISE) in leguminous seeds and oilseeds samples. Several experimental conditions of MIC-DV were evaluated, such as sample mass, absorbing solution, volume of combustion igniter, purging time with oxygen, and the use of successive combustion cycles. The accuracy of MIC-DV/ISE method was evaluated by comparing the results obtained by microwave-induced combustion and determination by ion chromatography, with agreements ranged from 94 to 106%. The method allowed to achieve relatively low limits of quantification (8.3 to 16µgg-1) comparing to AOAC method (40µgg-1), besides the use of small amounts of reagents, less handling of digests, making the method greener and with portable features comparing to other methods.

Routine breast milk monitoring using automated molecular assay system reduced postnatal CMV infection in preterm infants.

Human cytomegalovirus (CMV) transmitted through breast milk poses fatal risks to preterm infants. However, current molecular assay systems often do not accommodate breast milk samples. In this study, we evaluated the analytical and clinical performance of the measurement procedure of CMV load in breast milk utilizing the Cobas CMV test on the Cobas 6,800 system. This was enabled by incorporating a simple independent sample preparation procedure before the application of samples on the automated assay system. Clinical data from electronic medical records were retrospectively analyzed. Breast milk samples from mothers of preterm infants born before 33 weeks of gestation were screened for CMV using the automated assay system. CMV positivity rates in breast milk and neonatal samples and the CMV transmission rate were calculated. Furthermore, to validate the analytical accuracy of the overall measurement procedure with newly obtained residual breast milk samples, the linearity of the measurement procedure was assessed, and a simplified sample preparation method was validated against a conventional method. The CMV positivity rates in maternal breast milk and neonatal samples were 57.8 and 5.2%, respectively. The CMV transmission rate through breast milk was 7.7%. No significant differences in gestational age or birth weight were found between the CMV-negative and CMV-positive neonates. The linearity of the procedure was observed within a range of 1.87-4.73 log IU/mL. The simplified sample preparation method had an equivalent or even improved CMV detection sensitivity than the conventional method. Incorporating a simple independent sample preparation procedure effectively resolved any potential issues regarding the application of breast milk on the automated assay system. Our approach contributed to reduced vertical transmission of CMV by providing a convenient and reliable method for the monitoring of breast milk CMV positivity for clinicians.

Development, Validation, and Two-Year Application of Rapid and Simple LC-MS/MS-Based Method for the Determination of K2MK-7 in Blood Samples.

Biological properties of menaquinone-7, one of the vitamin K2 vitamers (K2MK-7), both those proven and those that remain to be investigated, arouse extensive interest that goes beyond the strictly scientific framework. The most important of them is the prevention of age-related diseases, considering that we live in the times identified as the era of aging societies and many people are exposed to the vitamin K2MK-7 deficiency. Therefore, an effective analytical protocol that can be adopted as a diagnostic and preventive analytics tool is needed. Herein, a simple sample preparation method followed by the liquid chromatography-tandem mass spectrometry-based method (LC-MS/MS), was used for the selective and sensitive determination of K2MK-7 in serum samples. Under the optimized conditions, using 500 µL of serum and the same amount of n-hexane, the reproducibility and the accuracy were obtained in the ranges of 89-97% and 86-110%, respectively, and the limit of detection value was 0.01 ng/mL. This method was used for the routine analysis. Statistical interpretation of the data from 518 samples obtained during 2 years of practice allowed for obtaining information on the content and distribution of K2MK-7 in the Polish population, broken down by the sex and age groups.

Multi-Mycotoxin Analysis in Italian Grains Using Ultra-High-Performance Chromatography Coupled to Quadrupole Orbitrap Mass Spectrometry.

Mycotoxins are a major source of contamination in cereals, posing risks to human health and causing significant economic losses to the industry. A comprehensive strategy for the analysis of 21 mycotoxins in Italian cereal grain samples (n = 200) was developed using a simple and quick sample preparation method combined with ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC Q-Orbitrap HRMS). The proposed method showed some advantages, such as multi-mycotoxin analyses with simple sample preparation, fast determination, and high sensitivity. The analysis of the sample revealed the presence of 11 mycotoxins, with α-zearalenol being the most frequently detected, while deoxynivalenol exhibited the highest contamination level. Furthermore, co-occurrence was identified in 15.5% of the samples under analysis. Among these, 13% of the samples reported the simultaneous presence of two mycotoxins, while 2.5% showed the co-occurrence of three mycotoxins. Currently, there has been a renewed interest in guaranteeing the quality and safety of products intended for human consumption. This study holds significant value due to its ability to simultaneously detect multiple mycotoxins within a complex matrix. Furthermore, it provides findings regarding the occurrence and co-occurrence of emerging mycotoxins that currently lack regulation under the existing European Commission Regulation.

Validation of an Aqueous Normal Phase Chromatography Method for the Analysis of Ergothioneine in Commercial Mushrooms

An analytical HPLC method for the identification and quantification of ergothioneine (EGT) has been developed using a silica hydride-based column. A simplified sample preparation method was developed. Several types of commercially available mushrooms were analyzed for quantification of ergothioneine using a Cogent Diamond Hydride column (100 x 4.6 mm) at a flow rate of 1 mL/min, with a deionized water:acetonitrile:0.1% formic acid mobile phase using an aqueous normal phase (ANP) gradient. EGT was detected at 254 nm and was identified by matching retention times of the verified standard. The standard ergothioneine content ranged from 0.01 mg to 0.1 mg/mL with excellent linearity correlation (R2 = 0.99999). EGT recoveries were within 96.5–100.3%. The developed HPLC method was simple, accurate, reproducible, sensitive and rugged.

Determination of Cl and S in crude oil by ICP-OES after sample digestion by microwave-induced combustion in disposable vessels

A simple, fast and promising sample preparation method based on microwave-induced combustion in disposable vessels (MIC-DV) was developed for Cl and S determination in crude oil by inductively coupled plasma optical emission spectrometry (ICP-OES). The MIC-DV consists of a new approach of conventional microwave-induced combustion (MIC). For the combustion, crude oil was pipetted on a disk of filter paper and placed on a quartz holder, followed by the addition of igniter solution (40 μL of 10 mol L−1 NH4NO3). The quartz holder was inserted into a commercial 50 mL disposable polypropylene vessel containing the absorbing solution, which was then inserted in an aluminium rotor. The combustion occurs under atmospheric pressure in a domestic microwave oven not compromising the operator's safety. The following parameters of combustion were evaluated: type, concentration and volume of absorbing solution, sample mass and the possibility of performing consecutive combustion cycles. Using MIC-DV, up to 10 mg of crude oil were efficiently digested, using 2.5 mL of ultrapure H2O as absorbing solution. Moreover, up to 5 consecutive combustion cycles were possible without analyte losses, reaching a total sample mass of 50 mg. The MIC-DV method was validated according to Eurachem Guide recommendations. Results obtained for Cl and S by MIC-DV were in agreement with those obtained using conventional MIC, as well as those obtained for S in a certified reference material of crude oil (NIST 2721). Analyte spike recovery experiments were performed and recoveries at three concentration levels ranged from 99 to 101% for Cl and from 95 to 97% for S, indicating a good accuracy. The limit of quantification achieved by ICP-OES after MIC-DV were 73 and 50 μg g−1 for Cl and S respectively, applying 5 consecutive combustion cycles.

Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing.

High-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer, but low-risk HPV strains can sometimes also be involved. Although HPV genotyping techniques used in clinical diagnosis cannot detect low-risk HPV, next-generation sequencing (NGS) can detect both types. However, DNA library preparation is complicated and expensive. The aim of this study was to develop a simplified, cost-effective sample preparation procedure for HPV genotyping based on next-generation sequencing (NGS). After DNA extraction, a first round of PCR was performed using modified MY09/11 primers specific for the L1 region of the HPV genome, followed by a second round of PCR to add the indexes and adaptors. Then, the DNA libraries were purified and quantified, and high-throughput sequencing was performed using an Illumina MiSeq platform. The sequencing reads were compared with reference sequences for HPV genotyping. The limit of detection for HPV amplification was 100 copies/µl. Analysis of the correlation of pathological cytology with the HPV genotype in individual clinical samples showed that HPV66 was the most common genotype found in the normal stage, whereas HPV16 was the main genotype found in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer. This NGS method can detect and identify several HPV genotypes with 92% accuracy and 100% reproducibility, and it shows potential as a simplified and cost-effective technique for large-scale HPV genotyping in clinical samples.

Simultaneous determination of multiple mycotoxins in corn and wheat by high efficiency extraction and purification based on polydopamine and ionic liquid bifunctional nanofiber mat

Due to the universality and harmfulness of mycotoxin co-contamination in cereals, it is of great significance to simultaneously monitor various mycotoxins co-polluted to ensure food safety and public health. In this work, a nanofiber mat modified by polydopamine and ionic liquid (PDA-IL-NFsM) was prepared and utilized as a solid-phase extraction (SPE) adsorbent for the simultaneous quantitative detection of multiple mycotoxins in corn and wheat. The PDA-IL-NFsM can form multiple retention mechanisms with the targets through hydrogen bond, π-π interaction, electrostatic or hydrophobic interaction, it shows favorable simultaneous adsorption performance (adsorption efficiency mostly higher than 88.27%) for fifteen mycotoxins in seven classes. Moreover, it can significantly reduce the matrix effect (lower than −13.69%), showing a good purification effect on the sample matrix. Based on the superior performance of PDA-IL-NFsM, a simple sample preparation method was established. The sample extract is simply diluted with water for SPE, and the eluent can be directly collected for ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. The detection limit can reach 0.04–4.21 μg kg−1, the recovery was 80.09%–113.01%, and the relative standard deviations of intra-day and inter-day precision were 2.80%–14.81% and 0.68%–13.80% respectively. The results show that the proposed method has good sensitivity, accuracy and precision, and has practical application potential.

Enterokokların MALDI-TOF MS’de Slanetz ve Bartley ve Kanlı Agar Besiyeri ile Karşılaştırılarak Tanımlanması

Introduction: Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used frequently in the last decade as a fast and reliable method for the identification of microorganisms. The fact that only one gram-negative selective medium is recommended in this method causes problems in the diagnosis. There is no information in the scientific literature that Slanetz and Bartley (SB) agar used in the study can be used in MALDI-TOF MS to identify bacteria. Materials and Methods: In this study, it was aimed to compare the identification performance of enterococci isolated from drinking-use and lake waters in Van and its surroundings with blood agar by using MALDI-TOF MS method and to develop a simple and fast sample preparation method that increases this performance. For this purpose, 60 mains and 30 lake water samples were examined for six months. Results: The samples were studied by the membrane filtration method. The isolated enterococci were identified by MALDI-TOF MS method with the isolates taken directly from both blood agar and SB medium, which is a selective medium. The same results were obtained in the species determination in both media. According to these results, 35% of the mains water isolates were Enterococcus casseliflavus, 30% Enterococcus faecalis, 26.7% Enterococcus faecium, 6.6% Enterococcus hirae, 1.7% Enterococcus columbae, and 20% of lake water isolates E. casseliflavus, 40% E. faecalis, 36.7% E. faecium, 3.3% E. hirae, E. casseliflavus in mains water and E. faecalis in lake water are the most frequently isolated enterococci species. Conclusion: Determining that it can also be obtained from selective media such as SB agar, for which there is no data on its suitability for mass spectrometric identification, will contribute to the more effective and rapid use of MALDI-TOF MS in microbiology laboratories. In addition, it has been concluded that all the waters examined are exposed to a significant level of microbial contamination and pose a potential danger to public health. It has been revealed that the consumption of these waters may pose a potential health risk for immunocompromised patients and the elderly, as well as children.

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera.

Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Abstract Viral contamination may occur at any stage of food processing. The study aim was to develop a two-step reverse transcription (RT)-recombinase polymerase amplification (RPA) assay and evaluate for in-field duplex detection of hepatitis A virus (HAV) and norovirus in oysters. The RNA expression plasmids were generated by amplifying a fragment of the VP1 gene of HAV and norovirus through PCR and cloning it into an expression vector. The RNAs were transcribed in vitro from the plasmids and further used for reverse transcription. The resulting single-stranded cDNAs were used as the purified or spiking templates to determine the sensitivities of simplex and duplex RT-RPA compared with RT-PCR, and RT-qPCR assays. The reproducibility and application of duplex RT-RPA in the field were also evaluated. Our results showed that simplex RT-RPA was at least 100 times more sensitive than RT-PCR and RT-qPCR and even more than duplex detection using purified targets. Unlike RT-PCR, the RT-RPA reaction was unaffected by inhibitors found in food, allowing simple sample preparation methods for detection within a fraction of the time. The duplex assay detected HAV, norovirus, or both in 12/30 (40%) oyster samples tested. Duplex RT-RPA proved to be a rapid, accurate, and reproducible method in a field test for detecting HAV and norovirus. Thus, duplex RT-RPA should be suitable for use in minimally equipped laboratories and field settings. If in-field RT-RPA services are provided to oyster farmers, the technique can minimize the risk of infection to consumers, thereby improving food safety. Keywords: Food safety, Hepatitis A virus, Direct extraction, Norovirus, Nucleic acid amplification

Metabolite Fingerprinting and Profiling of Selected Medicinal Plants Using Nuclear Magnetic Resonance

Background: Medicinal herbs are well known for their therapeutic effects and are traditionally used in the treatment and prevention of numerous diseases. Further plant-derived natural products have also been a valuable source of lead compounds for drug discovery and development. As the bioactivity of natural extracts is due to synergism between hundreds of metabolites present in the plant extract, the complete metabolomic analysis can be used for the quality control of phytomedicine. The 1H-nuclear magnetic resonance (NMR) fingerprint of the herbal extract can be used as a promising approach for comprehensive analysis of secondary metabolites to obtain a holistic view. The 1H-NMR spectroscopy has the advantage that it requires little quantity of samples and simple sample preparation method. Objectives: The study aimed to study the secondary metabolites of seven selected herbs, namely Abies webbiana, Cuminum cyminum, Elettaria cardamomum, Zingiber officinale, Glycyrrhiza glabra, Piper longum, and Terminalia chebula. Materials and Methods: The secondary metabolites of the herbal extracts were studied by recording the 1H-NMR spectra using NMR spectrometer in suitable solvent. Results: The putative metabolites that have been identified were 4-methoxy quercetin, luteolin, cuminaldehyde, 1,8-cineole, elettarins, gingerol, shogaol, glycyrrhizin, liquiritigenin, glabridin, betulinic acid, oleanolic acid, arabinogalactan, chebulagic acid, and gallic acid. Conclusion: The study highlighted the potential of NMR metabolomics in the study of plant extracts and their application for the quality control of phytomedicine.

MALDI-TOF MS Identification of some Clinically-Relevant Filamentous Fungi with the Direct Smear Method, a Simple Sample Preparation Method.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful technique for the identification of microorganisms. The technique normally requires a sample preparation procedure before instrumental analysis, which can be somewhat labor-intensive when numbers of samples are large. The direct smear method, in which samples are directly smeared on the sample plates and subsequently subjected to instrumental analyses, can save time and is less labor-intensive. However, the method has rarely been tested on filamentous fungi, although it has been successfully used in the identification of bacteria and yeasts. In the present study, we examined the method using clinically-collected filamentous fungi. Three hundred forty-eight isolates of filamentous fungi representing 9 species collected from body fluids of patients were analyzed on the VITEK MS version 3.0 system, a popular commercial MALDI-TOF MS system, using the direct smear method. For those misidentified or unidentified, the samples were retested. All fungal species were determined through DNA sequencing. Among 334 isolates that were included in the database of the VITEK system, 286 (85.6%) samples were correctly identified. After retesting, the rate of correct identification increased to 91.0%. Aspergillus fumigatus exhibited a 95.2% rate of correct identification before retesting, whereas Aspergillus niger showed the rate at only 46.5% (58.1% even with retesting). The direct smear method could be used in the identification of filamentous fungi found in body fluids of patients by MALDI-TOF MS at good rates of correct identification. The method is simple and time-saving, and deserves further evaluation.

High-performance Liquid Chromatography based Methods for Determination of Antitubercular Drugs in Urine Samples: A Scoping Review

Introduction: Tuberculosis (TB) is a contagious disease and is one of the leading causes of death worldwide. TB is primarily caused by Mycobacterium Tuberculosis (MTB) and the percentage of Multi-Drug Resistance TB (MDR-TB) and Extensively Drug Resistance TB (XDR-TB) is increasing daily in many developing countries. Aim: To summarise different High-performance Liquid Chromatography (HPLC)-based methods for checking treatment adherence and drug monitoring of patients by estimating the amount of Anti-tubercular (Anti-TB) drugs in urine samples. Materials and Methods: The authors conducted a search and screened various databases (PubMed, Scopus, Web of Science, Google Scholar) using different keywords between April 2023 and June 2023. The authors included original research articles, clinical trials, and observational studies that focused on estimating anti-TB drugs in urine samples using HPLC. The authors excluded articles that employed methods other than HPLC for drug estimation in urine samples. A total of 13 articles were included in this review. Results: The authors identified 296 articles from different electronic databases and four articles from other sources (Google Scholar, ResearchGate, etc.). Ultimately, 13 articles were included describing HPLC-based methods for determining anti-TB drugs in urine samples. Data was extracted focusing on mobile phases and sample preparation/extraction procedures. Two articles of 2004 and 2014 reported simple mobile phases and sample preparation methods for estimating rifampicin and isoniazid. Additionally, articles published in the last five years have employed simple mobile phases with minimal or no extraction procedures. Conclusion: The present review summarises various HPLCbased methods reported in the literature, as it is considered the gold standard method for checking treatment adherence in TB patients. Urine samples were chosen for ease of collection, particularly from patients of different age groups, including the paediatric population. This review highlights the need for more HPLC-based methods with simple mobile phases and extraction procedures for early detection of anti-TB drugs in resource-poor settings.

A Simple High-Throughput Field Sample Preparation Method Based on Matrix-Induced Sugaring-Out for the Simultaneous Determination of 5-Hydroxymethylfurfural and Phenolic Compounds in Honey.

In the present work, a high-throughput field sample preparation method was reported for the simultaneous determination of 5-hydroxymethylfurfural and phenolic compounds in honey. Combining a simple and green homogenous liquid−liquid extraction, matrix-induced sugaring-out, with the use of a 96-deepwell plate and multichannel pipette, the proposed method showed its merits in instrument-free and high-throughput preparation. Due to the high-throughput property, the parameters of the method were rapidly and systematically studied using a constructed 4 × 2 × 4 × 3 array (sample amount × ratio of ACN:H2O × standing time × replicates) in a 96-deepwell plate. Analytical performance was fully validated, and the limits of detection and limits of quantification were in the range of 0.17−1.35 μg/g and 0.51−4.14 μg/g, respectively. Recoveries were between 83.98 and 117.11%, and all the precisions were <5%. Furthermore, the developed method was successfully applied in the outdoor preparation of commercial honey samples and the in-field preparation of raw honey samples in apiary. The current work presented a simple, rapid, and high-throughput method for the field sample preparation of honey and provides a valuable strategy for the design of field and on-site sample preparation.