Method For Routine Analysis Research Articles

Classification of Fresh and Frozen-Thawed Beef Using a Hyperspectral Imaging Sensor and Machine Learning

The demand for safe and edible meat has led to the advancement of freeze-storage techniques, but falsely labeled thawed meat remains an issue. Many methods have been proposed for this purpose, but they all destroy the sample and can only be performed in the laboratory by skilled personnel. In this study, hyperspectral image data were used to construct a machine learning (ML) model to discriminate between freshly refrigerated, long-term refrigerated, and thawed beef meat samples. With four pre-processing methods, a total of five datasets were prepared to construct an ML model. The PLS-DA and SVM techniques were used to construct the models, and the performance was highest for the SVM model applying scatter correction and the RBF kernel function. These results suggest that it is possible to construct a prediction model to distinguish between fresh and non-fresh meat using the spectra obtained by purifying hyperspectral image data cubes, which can be a rapid and non-invasive method for routine analyses of the meat storage state.

Evaluation of the Novel CE-IVD-Marked Multiplex PCR QIAstat-Dx Meningitis/Encephalitis Panel.

Central nervous system (CNS) infections such as meningitis and encephalitis are life-threatening conditions that demand hospital care and prompt identification of the causative agent. Since 2015, there has been only one CE-IVD-marked rapid multiplexed diagnostic assay in cassette format for bacterial and viral detection from cerebrospinal fluid (CSF): the BioFire FilmArray meningitis/encephalitis (ME) panel. In the beginning of 2022, Qiagen introduced the QIAstat-Dx meningitis/encephalitis panel. It is a CE-IVD-marked multiplex PCR cassette test intended for the identification of suspected infectious meningitis, encephalitis, or meningoencephalitis caused by bacterial, viral, or fungal pathogens. In this study, we evaluated patient and quality control samples using the QIAstat-Dx meningitis/encephalitis panel and compared the results to those of the BioFire FilmArray meningitis/encephalitis panel and reference methods (current routine analysis methods in our laboratory, PCR, or cultivation). The combined positive percent agreement between the two panel assays was 100%, and the negative percent agreement was 94%. We further compared specifically herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) dilution series using six commercial herpesvirus assays, including the two cassette tests. The results suggested that real-time PCR methods (with separate extraction) were the most sensitive methods. When comparing the cassette tests, the BioFire FilmArray meningitis/encephalitis panel produced more positive results than the QIAstat-Dx meningitis/encephalitis panel in the herpesvirus analyses. IMPORTANCE The diagnosis of infectious meningitis and encephalitis relies mostly on specific PCR and culturing methods, but commercial syndromic panel assays are bringing a change in diagnostics. With multiplexed analysis, the identification of the pathogen is potentially faster, and less sample material is needed. The novel QIAstat-Dx meningitis/encephalitis panel assay is intended for the rapid identification of pathogens from cerebrospinal fluid for suspected central nervous system (CNS) infection, which is a life-threatening condition and difficult to diagnose. We studied the performance of this panel assay using patient samples and dilution series of selected viruses. The evaluation data for this novel meningitis/encephalitis panel assay are useful for other clinical laboratories and organizations using or considering using this test.

Lab-made 3D-printed electrochemical sensors for tetracycline determination

Antibiotics such as tetracycline (TC) are widely prescribed to treat humans or dairy animals. Therefore, it is important to establish affordable devices in laboratories with minimal infrastructure. 3D printing has proven to be a powerful and cost-effective tool that revolutionizes many applications in electrochemical sensing. In this work, we employ a conductive filament based on graphite (Gr) and polylactic acid (PLA) (40:60; w/w; synthesized in our lab) to manufacture 3D-printed electrodes. This electrode was used “as printed” and coupled to batch injection analysis with amperometric detection (BIA-AD) for TC sensing. Preliminary studies by cyclic voltammetry and differential pulse voltammetry revealed a mass transport governed by adsorption of the species and consequent fouling of the redox products on the 3D printed surface. Thus, a simple strategy (solution stirring and application of successive potentials, +0.95 V followed by +1.2 V) was associated with the BIA-AD system to solve this effect. The proposed electrode showed analytical performance comparable to costly conventional electrodes with linear response ranging from 0.5 to 50 μmol L−1 and a detection limit of 0.19 μmol L−1. Additionally, the developed method was applied to pharmaceutical, tap water, and milk samples, which required minimal sample preparation (simple dilution). Recovery values of 92–117% were obtained for tap water and milk samples, while the content found of TC in the capsule was close to the value reported by the manufacturer. These results indicate the feasibility of the method for routine analysis involving environmental, pharmaceutical, and food samples.

Exploration of HPTLC Technology for Rapid Chemical Fingerprinting and Simultaneous Determination of Bioactive Constituents from Clitoria ternatea Linn.

AbstractThe analytical methods used for herbal analysis are need to be economic, fast and also produce minimum quantities of hazardous chemical waste. Presently analytical community put interest in the research area of non‐hazardous and eco‐friendly practices to develop various green chromatographic methods for routine quality analysis. High cost of phytochemical analysis and uses of hazardous chemicals with high‐end sophisticated instrument, the attempt made to develop a simple analytical method for multiple samples with short time and less uses of solvents. A HPTLC method was developed for simultaneous determination of biological important constituents like β‐sitosterol, taraxerol, clitorienolactone B and β‐sitosterol glycoside from Clitoria ternatea Linn. The proposed method was validated and satisfied the ICH guidelines to demonstrate that the method is adaptable for its intended purpose. The method is simple, sensitive and economic it therefore embraces potential for detection, monitoring, and simultaneous quantification of the four bioactive compounds for C. ternatea and could also be apply to other species.

Quantification and stability assessment of urinary phenolic and acidic biomarkers of non-persistent chemicals using the SPE-GC/MS/MS method

Nowadays, people are exposed to numerous man-made chemicals, many of which are ubiquitously present in our daily lives, and some of which can be hazardous to human health. Human biomonitoring plays an important role in exposure assessment, but complex exposure evaluation requires suitable tools. Therefore, routine analytical methods are needed to determine several biomarkers simultaneously. The aim of this study was to develop an analytical method for quantification and stability testing of 26 phenolic and acidic biomarkers of selected environmental pollutants (e.g., bisphenols, parabens, pesticide metabolites) in human urine. For this purpose, a solid-phase extraction coupled with gas chromatography and tandem mass spectrometry (SPE-GC/MS/MS) method was developed and validated. After enzymatic hydrolysis, urine samples were extracted using Bond Elut Plexa sorbent, and prior to GC, the analytes were derivatized with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA). Matrix-matched calibration curves were linear in the range of 0.1–1000 ng mL−1 with R > 0.985. Satisfactory accuracy (78–118%), precision (< 17%), and limits of quantification (0.1–0.5 ng mL−1) were obtained for 22 biomarkers. The stability of the biomarkers in urine was assayed under different temperature and time conditions that included freezing and thawing cycles. All tested biomarkers were stable at room temperature for 24 h, at 4 °C for 7 days, and at −20 °C for 18 months. The total concentration of 1-naphthol decreased by 25% after the first freeze–thaw cycle. The method was successfully used for the quantification of target biomarkers in 38 urine samples.

PathogenDx DetectX Combined Demonstrates Equivalent Performance in Comparison to Four AOAC Certified Methods for the Detection of Aspergillus Species, Salmonella Species, and STEC in Dried Hemp Flower.

The PathogenDx DetectX Combined method is a certified Performance Tested MethodSM (012201) that is enrichment-free and utilizes a DNA microarray-based end point PCR method for the simultaneous detection of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus), Salmonella spp., and a broad range of Shiga toxin-producing Esherichia coli (STEC) from hemp and cannabis flower, edibles, and concentrates. This study aimed to compare the PathogenDx DetectX Combined enrichment-free method to four AOAC INTERNATIONAL certified molecular methods that utilize enrichment prior to quantitative PCR (qPCR) amplification in hemp flower for the detection of Aspergillus (A. flavus), S. enterica, and Escherichia coli 026. In this method comparison study, each method was evaluated according to the AOAC validated instructions for use (IFU) and the AOAC Appendix J validation guidelines. A total of 16 samples at three levels of contamination (0, 0.7, and 2 CFU/10g test portion) were analyzed by each method. The results for all methods were evaluated by using the probability of detection statistical model (POD). Results of the validation study demonstrate that the PathogenDx DetectX Combined enrichment-free method is equivalent in performance to the three proprietary methods evaluated in this study. The method comparison study indicated that the PathogenDx DetectX Combined enrichment-free method provides equivalent detection of the target analytes (A. flavus, Salmonella, and a broad range of STEC) in hemp flower. The performance of The PathogenDx DetectX Combined method is significantly faster and possesses a higher or equivalent degree of sensitivity and specificity. Implementation of this method for routine microbial pathogen analysis in laboratories would save significant time and resources.

Application of quantitative mineralogy to determine sources of airborne particles at a European copper smelter

Copper processing operations, such as smelters and refineries, can produce airborne particles that may impact the health of workers. At these operations, worker exposure to chemicals are regularly monitored to ensure that regulatory compliance with occupational exposure limit values (OELVs) are maintained. Determining the type of airborne particles present is important for characterizing the composition of dust exposures and better understanding the relationship between worker exposure and health. Routine methods of analysis (e.g., chemical assay) are unable to differentiate between phases containing the same elements and may result in ambiguity. A novel approach of a combination of Quantitative Evaluation of Materials by Scanning Electron Microscope (QEMSCAN) and chemical characterization was used here to evaluate airborne and settled dust collected at key locations throughout a copper smelter in Europe. The copper (Cu) phases present in the airborne dust are indicative of the activities performed at specific locations. In the batch preparation area where Cu concentrate is received, significant amounts of Cu were carried in sulfidic minerals (chalcocite, chalcopyrite/bornite, >40%), whereas near the anode and electric furnace, the majority of Cu in dust was carried in metallic and oxidic phases (60–70%). Particle size analysis of the settled dust indicates that the sulfidic and oxidic Cu minerals are more likely to become airborne over metallic Cu. Furthermore, overall Cu concentrations decreased with particle size where metallic and oxidic Cu dominate, which suggests that differences in the proportion of Cu forms present in the dust will impact how much Cu ends up in the respirable fraction. These results highlight the need to understand the characterization of Cu in dust in order to set better OELVs.

Impact of Crystal Nuclei on Dissolution of Amorphous Drugs.

Amorphous drugs are used to improve bioavailability of poorly water-soluble drugs. Crystallization must be managed to take full advantage of this formulation strategy. Crystallization of amorphous drugs proceeds in a sequence of crystal nucleation and growth, with different kinetics. At low temperatures, crystal nucleation is fast, but crystal growth is slow. Therefore, amorphous drugs may generate dense but nanoscale crystal nuclei. Such tiny nuclei cannot be detected using routine powder X-ray diffraction (PXRD) and polarized light microscopy (PLM). However, they may negate the dissolution advantage of amorphous drugs. In this work, for the first time, the impact of crystal nuclei on dissolution of amorphous drugs was studied by monitoring the real-time dissolution from amorphous drug films, with and without crystal nuclei, and the evolving crystallinity in the films. Three model drugs (ritonavir/RTV, posaconazole/POS, and nifedipine/NIF) were chosen to represent different crystallization tendencies in the supercooled liquid state, namely, slow-nucleation-and-slow-growth (SN-SG), fast-nucleation-and-slow-growth (FN-SG), and fast-nucleation-and-fast-growth (FN-FG), respectively. We find that although the amorphous films containing nuclei do not show obvious differences from the nuclei-free films under PLM and PXRD before dissolution, they have inferior dissolution performance relative to the nuclei-free amorphous films. For SN-SG drug RTV, crystal nuclei have negligible impact on the crystallization of amorphous films, dissolution rate, and supersaturation achieved. However, they cause earlier de-supersaturation by inducing crystallization in solution as heterogeneous seeds. For FN-SG drug POS and FN-FG drug NIF, crystal nuclei accelerate crystallization in the amorphous films leading to lower supersaturation achieved with POS, and elimination of any supersaturation with NIF. Dissolution profiles of amorphous films can be further analyzed using a derivative function of the apparent dissolution rate, which yields amorphous solubility, initial intrinsic dissolution rate, and onset of crystallization in the amorphous films. This study highlights that although crystal nuclei are undetectable with routine analytical methods, they can significantly negate, or even eliminate, the dissolution advantage of amorphous drugs. Hence, understanding crystal nucleation process and developing approaches to prevent it are necessary to fully realize the benefits of amorphous solids.

In vitro/in vivo identification of zein degraded peptides using HPLC-MS/MS and their safety evaluation

The identification of degraded products of implanted scaffolds is desirable to avoid regulatory concerns. In vivo identification of products produced by the degradation of natural protein-based scaffolds is complex and demands the establishment of a routine analytical method. In this study, we developed a method for the identification of peptides produced by the degradation of zein both in vitro and in vivo using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). For in vitro experiments, zein was degraded enzymatically and analyzed produced peptides. In vitro study showed cytocompatibility of peptides present in the hydrolysate of zein with no induction of apoptosis and cell senescence. For in vivo experiment, zein gels were prepared and subcutaneously implanted in rats. Peptides produced by the degradation of zein were identified and few were selected as targeted (unique peptides) and two peptides were synthesized as the reference sequence of these peptides. Further, peptide analysis using HPLC-MS/MS of different organs was performed after 2 and 8 weeks of implantation of zein gel in rats. It was found that zein-originated peptides were accumulated in different organs. QQHIIGGALF or peptides with same fractions were identified as unique peptides. These peptides were also found in control rats with regular rat feed, which means the degradation of implanted zein biomaterial produced food related peptides of non-toxic nature. Furthermore, hemotoxylin and eosin (H&E) staining exhibited normal features. Overall, zein degraded products showed cytocompatibility and did not induce organ toxicity, and QQHIIGGALF can act as a standard peptide for tracing and determining zein degradation. The study also provides the feasibility of complex analysis on identification and quantification of degradation products of protein-based scaffolds.

Carbon dots as fluorescent nanoprobes for assay of some non-fluorophoric nitrogenous compounds of high pharmaceutical interest

BackgroundCarbon dots, CDs, have excellent photoluminescence properties, good biocompatibility, low toxicity and good light stability. The optical, magnetic and electronic properties of CDs make them a hugely relevant tool to be used in pharmaceutical analysis, bioimaging, drug delivery, and other fields. The fluorescence of carbon nanodots makes it suitable for assay of some nitrogenous compounds of high pharmaceutical interest. In this work, we develop simple, fast and green spectrophotometric methods for quantification of Azithromycin and Rasagiline mesilate using synthesized fluorescent CDs from garlic peels.ResultsThe spectrometric methods depend on stoichiometric reactions of both drugs with fluorescent CDs. Carbon dots exhibit a declared absorption peak λmax at 238 nm and potent fluorimetric emission at λem 528 nm, upon excitation at λex 376 nm. Drugs’ concentrations in ppm are efficiently calculated using Stern–Volmer Equation. Decrease in fluorescence (ΔF = Fo − F) and the F-ratio values are linearly correlated to molar concentration of each quencher (drug). A significant linear diminish in the dots’ measured absorbance and fluorimetric emission values was observed. Validation of all the developed methods was according to the ICH guidelines.ConclusionsIn a new way, this work successfully indicates, spectrometric methods for rapid detection of two non-fluorophoric nitrogenous compounds using potent carbon nanodots. Consequently, these green developed methods offer several benefits as simplicity, ease of quantification, accuracy and precision that encourage the application of the developed methods in routine analysis of Azithromycin and Rasagiline mesilate in quality control laboratories as analytical tool.

Fast Raman imaging through the combination of context-aware matrix completion and low spectral resolution.

Raman hyperspectral imaging is an effective method for label-free imaging with chemical specificity, yet the weak signals and correspondingly long integration times have hindered its wide adoption as a routine analytical method. Recently, low resolution Raman imaging has been proposed to improve the spectral signal-to-noise ratio, which significantly improves the speed of Raman imaging. In this paper, low resolution Raman spectroscopy is combined with "context-aware" matrix completion, where regions of the sample that are not of interest are skipped, and the regions that are measured are under-sampled, then reconstructed with a low-rank constraint. Both simulations and experiment show that low-resolution Raman boosts the speed and image quality of the computationally-reconstructed Raman images, allowing deeper sub-sampling, reduced exposure time, and an overall >10-fold improvement in imaging speed, without sacrificing chemical specificity or spatial image quality. As the method utilizes traditional point-scan imaging, it retains full confocality and is "backwards-compatible" with pre-existing traditional Raman instruments, broadening the potential scope of Raman imaging applications.

Advanced Non-small Cell Lung Cancer: EGFR Mutation Analysis Using Pyrosequencing and the Fully Automated qPCR-Based IdyllaTM System.

Epidermal growth factor receptor (EGFR) mutation status is of a major clinical significance in non-small cell lung cancer (NSCLC) management, as it guides therapeutic decision making to target patients for a better response to therapy. This implicates the introduction of EGFR mutation analysis as the standard of care for Moroccan NSCLC patients, which in itself entails the implementation of targeted methods for routine EGFR mutation analysis in our laboratories. In this study, we aimed to present 2 targeted methods for EGFR mutation identification and to determine the prevalence and spectrum of EGFR mutations in NSCLC Moroccan patients. A retrospective investigation of a cohort of 340 patients was undertaken to analyze somatic EGFR mutations in exons 18 to 21 using pyrosequencing and the IdyllaTM system. Of the enrolled patients, 70.9% were males and 29.1% were females. Predominately, 92% of cases had adenocarcinoma, and 53.7% of patients self-reported a history of smoking. Overall, 73 patients (21.7%) harbored an EGFR mutation, the most prevalent of which were the exon 19 deletions (53.4%) followed by exon 21 substitutions (31%). Exon 18 mutations and exon 20 alterations occurred in 8.1% and 6.7% of the positive EGFR mutation cases, respectively. Of the analyzed cases, all of the EGFR-mutated patients had adenocarcinoma. EGFR mutation prevalence was significantly higher in females (females vs males: 38.4% vs 14.5%, P < .001) and non-smokers (non-smokers vs non-smokers: 36% vs 10.3%, P < .001). The featured pyrosequencing and the IdyllaTM system are targeted methods endowed with high sensitivity and specificity as well as other compelling characteristics which make them great options for routine EGFR mutation testing for advanced NSCLC patients. These findings underline the imperious need for implementing quick and efficient targeted methods for routine EGFR mutation testing among NSCLC patients, which is particularly useful in determining patients who are more likely to benefit from targeted therapy.

Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects.

Large- and small-headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. Six sperm staining methods, including Papanicolaou, Diff-Quik, Shorr, Hematoxylin-eosin (HE), Wright, and Wright-Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer-aided sperm morphological analysis system. The highest sperm head length and width were observed with the Wright-Giemsa and Wright staining, followed by the Diff-Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff-Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff-Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright-Giemsa staining. Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff-Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.

Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage

Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs—procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin—for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.

Methods of Analyses for Biodegradable Polymers: A Review.

Biodegradable polymers are materials that can decompose through the action of various environmental microorganisms, such as bacteria and fungi, to form water and carbon dioxide. The biodegradability characteristics have led to a growing demand for the accurate and precise determination of the degraded polymer composition. With the advancements in analytical product development, various analytical methods are available and touted as practical and preferable methods of bioanalytical techniques, which enable the understanding of the complex composition of biopolymers such as polyhydroxyalkanoates and poly(lactic acid). The former part of this review discusses the definition and examples of biopolymers, followed by the theory and instrumentation of analytical methods applicable to the analysis of biopolymers, such as physical methods (SEM, TEM, weighing analytical balance, etc.), chromatographic methods (GC, THM-GC, SEC/GPC), spectroscopic methods (NMR, FTIR, XRD, XRF), respirometric methods, thermal methods (DSC, DTA, TGA), and meta-analysis. Special focus is given to the chromatographic methods, because this is the routine method of polymer analysis. The aim of this review is to focus on the recent developments in the field of biopolymer analysis and instrument application to analyse the various types of biopolymers.

Four Greenness Evaluations of Two Chromatographic Methods: Application to Fluphenazine HCl and Nortriptyline HCl Pharmaceutical Combination in Presence of Their Potential Impurities Perphenazine and Dibenzosuberone

Nowadays, when analysts develop a new method, they are taking into consideration the green aspects of the developed method. Two chromatographic methods were developed for the determination of antidepressant pharmaceutical combination nortriptyline hydrochloride and fluphenazine hydrochloride in presence of their potential impurities and the methods were assessed using four tools; eco-scale, analytical greenness profile (AGP), analytical greenness metric approach (AGREE), and green analytical procedure index (GAPI). Method (A) was RP-HPLC, in which the separation was carried out on C18 column (250 × 4.5 mm, 5 µm) by gradient elution using mobile phase consisting of a mixture of water containing 0.1% H3PO4 (pH 2.25) and methanol, at a flow rate of 2 mL/min with DAD detection at 254 nm. Method (B) was TLC, in which the separation was carried out on silica gel TLC F254 plates. The mobile phase used was a mixture of methanol and acetone (9:1, v/v) with UV detection at 245 nm. The proposed methods agreed with ICH guidelines of method validation and were successfully applied for determination of the proposed components in their dosage form and the results were compared statistically to those obtained by the reported RP-HPLC method with no significant difference; which suggests the application of the developed methods for routine quality control analysis of these drugs.

Optimisation and validation of a modified QuEChERS method for the determination of 222 pesticides in edible oils using GC-MS/MS: a case study on corn oil

ABSTRACT Recently, various pesticides have been employed at different stages of cultivation to protect the oil crop from insect, disease, and weed. Those pesticides, especially lipophilic ones, bio-accumulate in the oilseeds and get co-extracted into edible oils during the refining process. Hence, this study aimed to optimise an extraction method for routine pesticide multi-residue analysis in edible vegetable oil samples using gas chromatography coupled to tandem mass spectrometry (GC – MS/MS). Two parameters were optimised using a factorial design of experiment (solvent type and solvent/sample ratio). In general, the number of pesticides that conformed to our criteria (mean recovery 70–120%) increased as the solvent/sample ratio increased. Each of the three tested solvents had an advantage over the other, ethyl acetate showed the highest capability for non-polar pesticides extraction, acetonitrile had the highest sensitivity, and the ethyl acetate/acetonitrile mixture showed the highest number of pesticides with acceptable criteria. The three solvents were tested using two different cleanup sorbents (EMR, and Z-Sep), the best solvent/cleanup combination was found to be ethyl acetate/acetonitrile mixture in addition to Z-Sep. The final method was validated in accordance with the European guidelines for Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed (SANTE/11312/2021). The precision and trueness of the method were determined from recovery experiments on six replicates of spiked blank corn oil samples at 0.01, 0.025, and 0.05 mg/kg. The number of pesticides that showed accepted criteria were 130, 166, and 182 out of 222 for each spiking level respectively.

CycleFlow simultaneously quantifies cell-cycle phase lengths and quiescence in vivo

SummaryPopulations of stem, progenitor, or cancer cells show proliferative heterogeneity in vivo, comprising proliferating and quiescent cells. Consistent quantification of the quiescent subpopulation and progression of the proliferating cells through the individual phases of the cell cycle has not been achieved. Here, we describe CycleFlow, a method that robustly infers this comprehensive information from standard pulse-chase experiments with thymidine analogs. Inference is based on a mathematical model of the cell cycle, with realistic waiting time distributions for the G1, S, and G2/M phases and a long-term quiescent G0 state. We validate CycleFlow with an exponentially growing cancer cell line in vitro. Applying it to T cell progenitors in steady state in vivo, we uncover strong proliferative heterogeneity, with a minority of CD4+CD8+ T cell progenitors cycling very rapidly and then entering quiescence. CycleFlow is suitable as a routine method for quantitative cell-cycle analysis.

Estimation of Nutraceutical Astaxanthin by RP-HPLC Method: Development and Validation

Background: Nutraceuticals have always been considered natural and safe supplements that may prevent disease, may substitute prescription drugs, compensate for a poor diet or promote health. Several nutraceutical products in the market today play roles as antioxidants and probiotics. One such nutraceutical is Astaxanthin, a red-pigmented keto-carotenoid, which offers a plethora of uses and health benefits. Astaxanthin, like many carotenoids, is a lipid-soluble pigment which faces solubility and bioavailability issues owing to its lipophilicity, due to which its estimation is challenging. And the analysis of a simple solvent extract of Astaxanthin would produce many ester-astaxanthin “peaks” and utilize costly organic solvents. Additionally, the literature review indicates the use of very complex mobile phases and tedious solvent extraction procedures involved in the estimation of Astaxanthin. &#x0D; Objective: The manuscript describes the development and validation of a rapid, sensitive and specific Reverse Phase-High Performance Liquid Chromatographic (RP-HPLC) method involving Ultraviolet (UV) detection for the determination and quantification of nutraceutical Astaxanthin in bulk and capsule dosage forms. &#x0D; Methods: Chromatography was carried out on an Inertsil ODS C18 (250 x 4.6 mm), 5µ, using a filtered and degassed mixture of Water: Acetonitrile (30:70% v/v) as the mobile phase at a flow rate of 1 mL /min with isocratic elution method and effluent was monitored at 478 nm. &#x0D; Results: The method was linear in the range of 5 – 15 μg mL-1 of Astaxanthin. The method was validated with respect to precision, accuracy, specificity and robustness. The run time of the analyte was 8.45 min. &#x0D; Conclusion: The developed method is simple, specific, precise, accurate, and robust and shows no interference with diluents which proves the adaptability of the method for routine analysis of the nutraceutical Astaxanthin in bulk and marketed formulations.

Screening method for the characterization of anabolic steroids seized in Brazil using paper spray mass spectrometry and chemometric tools.

This paper reports the use of paper spray mass spectrometry (PS-MS) combined with chemometric models to analyze seized samples of anabolic steroids. Because many forensic laboratories typically demand high-throughput analysis for this type of sample, we developed a quicker and simpler alternative analytical method for routine analysis with minimal sample preparation. Oily samples (n = 39) resulting from seizures carried out by Brazilian Federal and State Police units were selected for this study. These samples were analyzed by PS-MS in the positive ion mode and full scan (50-1000 m/z), providing spectra containing patterns of the respective active ingredients present in each product. A principal component analysis (PCA) model was built, which discriminated samples mainly according to their active ingredients and allowed to detect and characterize some cases of product counterfeiting. The variable selection method ordered predictors selection was employed jointly with PCA to improve sample cluster separation and to provide model simplification. The final PCA model was built with three principal components and using only 28 spectral variables. This model accounted for 69.82% of the variance and discriminated samples according to their specific active ingredients.