Matrix Interference Effects Research Articles

Smartphone-based digital image colorimetry for the determination of total capsaicinoid content in chili pepper extracts

A simple smartphone-based digital image colorimetry was proposed for the determination of total capsaicinoid content and the assessment of chili pepper pungency. The biobased solvent D-limonene was used for the first time to isolate analytes. Capsaicinoids were efficiently separated from chili pepper by solid–liquid extraction with D-limonene followed by partitioning of the analytes into the ammonium hydroxide solution to eliminate the matrix interference effect. For colorimetric detection of total capsaicinoid content, a selective chromogenic reaction was performed using Gibbs reagent (2,6-dichloroquinone-4-chloroimide). Measurements were performed using a smartphone-based setup and included image analysis with the program ImageJ. The limit of detection of the proposed procedure was 0.15 mg g−1. The intra-day repeatability did not exceed 10.0 %. The inter-day repeatability was less than 16.5 %. The comparison of the smartphone-based procedure with high-performance liquid chromatography showed satisfactory results.

Fully Automated Multiple Standard Addition on a Centrifugal Microfluidic System.

We herein describe a novel centrifugal microfluidic system to achieve multiple standard additions, which could minimize the effects of matrix interference and consequently lead to more accurate and reliable measurements of analyte concentrations in complex samples. The system leverages laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc without the need for external pumps or pressure systems, simplifying the procedures and eliminating the need for manual intervention. The disc incorporates metering chambers with rationally designed varying sizes, which could lead to the formation of six standard addition samples very rapidly in just 2.5 min. The final solutions are designed to contain a target component at gradually increasing concentrations but have an equal final volume containing the same amount of an analyte solution, thereby equalizing the matrix effect that is supposedly caused by the unknown components in the analyte solution. By utilizing this design principle, we were able to successfully quantify a model target component, salivary thiocyanate ions, that could be used as a biomarker for exposure to tobacco smoke. Our centrifugal microfluidic system holds great promise as a powerful analytical tool to achieve fully automated diagnostic microsystems involving a standard addition process.

Development and validation of a method based on liquid chromatography-mass spectrometry for comprehensive profiling of phenolic compounds in rice

The consumption of rice grains containing phytochemicals associated with antioxidant activity can potentially reduce the risk of chronic diseases. These phytochemicals, which include phenolic acids, anthoxanthins, and anthocyanins, are present in both free and bound forms in rice grains. However, there is limited documentation on the development of a method for determining the simultaneous amount of both free and bound phenolic compounds using sensitive analytical techniques. To address this gap, a study was conducted to develop and validate a liquid chromatography-mass spectrometric detection method for quantifying eighteen phenolic compounds in rice grains, both in free and bound forms. Paracetamol was used as the internal standard to minimize the effect of matrix interferences. The method was validated by measuring its working range, linearity, accuracy, precision, limits for detection and quantification, robustness, and expanded uncertainty. The method has a wide working range of 1.0 to 30.0 mg/kg for each phenolic compound and good linearity, with regression coefficients of calibration graphs greater than 0.999. Accuracy was determined using the recoveries after fortification at three concentration levels, which were in the range of 83.1 to 101.2%. Precision was expressed as a percentage of relative standard deviation (%RSD), which was below 7.62% for all determined phenolic compounds. The detection limits (LoD) and quantification limits (LoQ) ranged between 0.53 and 0.75 mg/kg and 0.54–0.77 mg/kg, respectively. The percentages of expanded uncertainties at the coverage factor of 2 (k = 2) were below 11.7%. The method was also evaluated for its robustness by obtaining recoveries at satisfactory levels for three different kinds of cereal; wheat, maize, and millet. The validated method is an accurate and reproducible approach for the simultaneous determination of phenolic compounds present at low levels in a complex matrix such as rice. The study also found that the phenolic profiles vary among rice genotypes, which can significantly impact human health by varying the nutraceutical properties of rice grains as health-promoting aspects.

Molecularly-Imprinted SERS: A Potential Method for Bioanalysis

The most challenging step in developing bioanalytical methods is finding the best sample preparation method. The matrix interference effect of biological sample become a reason of that. Molecularly imprinted SERS become a potential analytical method to be developed to answer this challenge. In this article, we review recent progress in MIP SERS application particularly in bioanalysis. Begin with the explanation about molecular imprinting technique and component, SERS principle, the combination of MIP SERS, and follow by various application of MIP SERS for analysis. Finally, the conclusion and future perspective were also discussed.

Challenges in direct determination of Glyphosate and aminomethylphosphonic acid in water by liquid chromatography-tandem mass spectrometry at modified reverse phase columns: Effect of matrix interferences in fresh and hard water systems.

This study validated two underivatized methods (M1 and M2) according to the Eurachem guidelines to analyze the herbicide Glyphosate and its major metabolite aminomethylphosphonic acid simultaneously by liquid chromatography-tandem mass spectrometry in both fresh and hard waters. Samples were analyzed directly after filtration through 0.22 μm syringe filters in M1, while samples were acidified with acetic acid before filtration in M2. Spike recoveries were greater than 80% for Glyphosate and aminomethylphosphonic acid in both methods. The limit of quantitation was 0.5 μg/L for M1, and 0.1 μg/L for M2 by using matrix-matched calibrations. The linear regression coefficient of both methods was greater than 0.995. The expanded uncertainty was found to be less than 25% for both. Moreover, M1 has an additional mass spectral confirmation ability, and the column and the mobile phase used in M2 can be used to analyze the inert surfactant used in Glyphosate formulations, Polyethoxylated tallow amine. The accuracy of the developed methods was assured by participating in a proficiency testing program against M2 and conducting the t-test for results generated by both M1 and M2. Both methods, therefore, can be used to determine Glyphosate and aminomethylphosphonic acid content concurrently in fresh and hard waters.

Amperometric Biosensor Based on Laccase Enzyme, Gold Nanoparticles, and Glutaraldehyde for the Determination of Dopamine in Biological and Environmental Samples

The present work reports the development and application of an amperometric biosensor based on carbon paste electrode modified with laccase enzyme, glutaraldehyde, and gold nanoparticles (Lac-Glu-AuNPs/CPE) for the determination of the neurotransmitter dopamine (DA). The materials were characterized morphologically and chemically using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and cyclic voltammetry. Optimization studies were performed in order to determine the optimal amount of enzyme and pH level that can yield the best conditions of analysis. The application of the biosensor in optimal conditions using the amperometric technique yielded a linear concentration range of 8.0 × 10−7–6.2 × 10−5 mol L−1 with a limit of detection of 6.0 × 10−8 mol L−1. The proposed biosensor was successfully applied for the determination of DA in biological and environmental samples. In addition, the application of the biosensor for the conduct of electrochemical measurements showed that the sensing device has good repeatability and stability, and it does not suffer from matrix interference effects. The proposed biosensor exhibited an analytical signal of 85% after 10 days of consecutive use.

Inductively Coupled Plasma-Tandem Mass Spectrometry (ICP-MS/MS) and Its Applications

During the last about 10 years, the inductively coupled plasma tandem quadrupole mass spectrometry (ICP-MS/MS) technique with reaction cell is being utilized for the analyses of several trace elements/isotopes at very low concentration levels in a variety of matrices including geological, environmental, biological, medical, nuclear, agriculture, food, material and metallurgical sciences. Both solution nebulization, as well as direct analysis of samples by laser ablation (LA-ICP-MS/MS), are utilized for sample introduction. In fact, ICP-MS/MS technique with collision reaction cells eliminated the need for laborious chemical separations using methods such as ion-exchange or solvent extraction procedures to eliminate/minimize spectral and matrix interference effects as these interferences are removed using online chemical reactions within the instrument itself (chemical resolution). In addition, the technique is hyphenated with chromatographic techniques such as GC and HPLCfor a variety of applications. The detection limits for several elements in the periodic table lie in the pg/ml-fg/ml range, and the technique compares favourably with the expensive, complex, and superior high resolution-ICP-MS (HR-ICP-MS), both in terms of the detection limits as well as the ability to remove the most complex interferences. Thus, the ICP MS/MS made the determinations of elemental as well as isotopic concentrations very simple andeasy even in complex materials with several novel quantification approaches. This article provides an overview of these new developments during the last decade along with several applications in all areas of science and technology.

Inductively Coupled Plasma-Tandem Mass Spectrometry (ICP-MS/MS) and Its Applications

During the last about 10 years, the inductively coupled plasma tandem quadrupole mass spectrometry (ICP-MS/MS) technique with reaction cell is being utilized for the analyses of several trace elements/isotopes at very low concentration levels in a variety of matrices including geological, environmental, biological, medical, nuclear, agriculture, food, material and metallurgical sciences. Both solution nebulization, as well as direct analysis of samples by laser ablation (LA-ICP-MS/MS), are utilized for sample introduction. In fact, ICP-MS/MS technique with collision reaction cells eliminated the need for laborious chemical separations using methods such as ion-exchange or solvent extraction procedures to eliminate/minimize spectral and matrix interference effects as these interferences are removed using online chemical reactions within the instrument itself (chemical resolution). In addition, the technique is hyphenated with chromatographic techniques such as GC and HPLCfor a variety of applications. The detection limits for several elements in the periodic table lie in the pg/ml-fg/ml range, and the technique compares favourably with the expensive, complex, and superior high resolution-ICP-MS (HR-ICP-MS), both in terms of the detection limits as well as the ability to remove the most complex interferences. Thus, the ICP MS/MS made the determinations of elemental as well as isotopic concentrations very simple andeasy even in complex materials with several novel quantification approaches. This article provides an overview of these new developments during the last decade along with several applications in all areas of science and technology.

Advanced sample preparation techniques for rapid surface-enhanced Raman spectroscopy analysis of complex samples

Surface-enhanced Raman spectroscopy (SERS) is a powerful and promising analytical technique for fast, non-destructive, and sensitive analysis of trace analytes. However, the serious matrix interference effect of complex sample is a bottleneck that limited rapid SERS analysis. Therefore, it is essential to introduce proper sample preparation techniques to address the limitation for advancing SERS analysis. With adaptable sample preparation techniques, target substances could be separated and purified from complex matrices, matrix interference effect could be eliminated/reduced, and finally pure SERS spectra could be obtained. However, the process of traditional sample preparation usually requires the tedious and time-consuming operation, large solvent consumption, high labor intensity, as well as low efficiency. Given that SERS is an ultrafast analytical technique, there is a great desire to develop advanced/modern sample preparation techniques for fast and efficient analysis of complex samples. To attract much attention and raise research enthusiasm on the beginning, recent development on advanced sample preparation techniques for rapid SERS analysis of complex sample was reviewed. (1) ‘All-in-one’ strategy for simultaneous separation, enrichment, and in-situ SERS detection. (2) Integrated strategy for high-speed pretreatment and high throughput analysis. (3) Derivatization strategy switches on the SERS activity of molecules with weak responses. (4) Field-assisted strategy for preconcentration acceleration. (5) Instrument combination strategy for online processing and real-time SERS analysis. Finally, conclusions and perspectives on future development were briefly discussed.

Nanosecond Photochemical Reaction for Enhanced Identification, Quantification, and Visualization of Primary Amine-Containing Metabolites by MALDI-Mass Spectrometry.

Many metabolites, including amino acids, neurotransmitters, and pharmaceuticals, contain primary amine functional groups. The analysis of these molecules by mass spectrometry (MS) plays an important role in the study of cancers and psychogenic diseases. However, the MS-based detection and visualization of these bioactive metabolites directly from real biological systems still suffer from challenges such as low ionization efficiency and/or matrix interference effects. Here, we introduce a simple and efficient strategy, the nanosecond photochemical reaction (nsPCR)-enabled fast chemical derivatization, enabling direct MS analysis of primary amine-containing metabolites, with enhanced detection sensitivity for numerous metabolites from cell culture medium and rat brain sections. Furthermore, this nsPCR-based chemical derivatization strategy was demonstrated to be a useful visualizing tool that could provide improved spatial information for these metabolites, potentially offering alternative tools for gaining novel insights into metabolic events.

Paper-Based Simplified Visual Detection of Cry2Ab Insecticide from Transgenic Cottonseed Samples Using Integrated Quantum Dots-IgY Antibodies.

In the present study, an easy to use field-deployable methodology was developed for onsite detection of pesticidal crystal protein Cry2Ab from transgenic cotton crops to reduce seed adulteration. Anti Cry2Ab IgG and IgY antibodies were developed against recombinant Cry2Ab protein in New Zealand white rabbits and in white leg horn chickens, respectively. Carboxyl-functionalized CdTe quantum dots (QDs) were used as revealing probes, and nitrocellulose paper was used as an assay matrix. Recombinant Cry2Ab was generated in the lab and used for immunization of chicken and rabbits. After successful immunization and attaining the desired titer values (1:32 000 for IgY and 1:64 000 for IgG), eggs and hyperimmune sera were collected. Anti Cry2Ab IgY was purified as per the standardized protocols, and anti Cry2Ab IgG was purified using protein A affinity chromatography. Sensitivity of the generated antibodies was examined using indirect ELISA methods against recombinant Cr2Ab protein. Specificity evaluation was carried out against other Cry proteins including Cry2Ab, Cry4b, Cry4a, Cry1Ec, and Cry1Ac. Functionalized CdTe QDs were characterized for structure and shape as well as fluorescence properties using standard laboratory techniques. A field-deployable paper-based detection methodology was developed where IgG acted as the capturing antibody and IgY-linked CdTe QDs were used as revealing probes. The limit of detection (LOD) and quantification (LOQ) were found to be 2.91 ng/mL and 9.71 ng/mL, respectively. The effect of matrix interference was assessed on the different plant crude extracts of cottonseed materials.

A new method for the preconcentrations of U(VI) and Th(IV) by magnetized thermophilic bacteria as a novel biosorbent.

This paper proposes the use of Anoxybacillus flavithermus SO-15 immobilized on iron oxide nanoparticles (NPs) as a novel magnetized biosorbent for the preconcentrations of uranium (U) and thorium (Th). The SPE procedure was based on biosorption of U(VI) and Th(IV) on a column of iron oxide NPs loaded with dead and dried thermophilic bacterial biomass prior to U(VI) and Th(IV) measurements by ICP-OES. The biosorbent characteristicswereexplored using FT-IR, SEM, and EDX. Significant operational factors such as solution pH, volume and flow rate of the sample solution, amounts of dead bacteria and iron oxide nanoparticles, matrix interference effect, eluent type, and repeating use of the biosorbent on process yield were studied. The biosorption capacities were found as 62.7 and 56.4mgg-1 for U(VI) and Th(IV), respectively. The novel extraction process has been successfullyappliedto the tap, river, and lake water samples for preconcentrations of U(VI) and Th(IV).

Ultra-durable, multi-template molecularly imprinted polymers for ultrasensitive monitoring and multicomponent quantification of trace sulfa antibiotics.

Traditional analysis methods are susceptible to interference caused by the complexity of sample matrices, and detector surface fouling arising from nonspecific adsorption of microorganisms (in biological samples) which leads in particular to a gradual loss of sensitivity. Imprinted materials can be used to effectively reduce interference originating in the matrices. However, the poor reproducibility and multicomponent quantification of trace antibiotics represent significant challenges to the detection process. Meanwhile, the high biological risk presented by bacterial antibiotic immunity and the persistence of antibiotics in foodstuffs, especially meat, both caused by the overuse of sulfonamide antibiotics, remain urgent issues. Here, we present the first example of a method for the accurate quantification of trace sulfa antibiotics (SAs) based on multi-template imprinted polymers (MMIPs). Levels of multiple SAs have been simultaneously successfully quantified by applying MMIP extraction coupled with UPLC-MS/MS analysis. This method shows excellent linearity of detection in the range of 0.1-500 μg L-1, and ultrasensitivity with low limits of detection of 0.03 μg L-1. The maximum SA residue recovered from sample tissues by using MMIPs was 5.48 μg g-1. MMIP-coupled UPLC-MS/MS quantification of SAs is an accurate and repeatable method for the monitoring of SA accumulation in mouse tissue samples. It also provides an effective strategy for the tracking and quantification of drugs in other biological samples.

A practical solution of matrix interference effect with saline replacement in samples of chemotherapy received patients whose differential count of leukocyte cannot be measured

A practical solution of matrix interference effect with saline replacement in samples of chemotherapy received patients whose differential count of leukocyte cannot be measured

A comparison of liquid-liquid extraction and stir bar sorptive extraction for multiclass organic contaminants in wastewater by comprehensive two-dimensional gas chromatography time of flight mass spectrometry

Liquid-liquid extraction (LLE) and stir bar sorptive extraction (SBSE) are extraction methods used for the analysis of contaminants in aqueous samples. In this study, both LLE and SBSE were compared for the extraction of priority pollutants and contaminants of emerging concern (CECs) in wastewater influent and effluent samples, for analysis with comprehensive two-dimensional gas chromatography with time of flight mass spectrometry (GC × GC-TOFMS). The methods were compared for their extraction efficiency of a broad range of compounds, matrix effects, accurate and reliable quantification of targets, and sensitivity. The target analytes studied were semi-volatile organic compounds (SVOC) including polycyclic aromatic hydrocarbons, phenols, phthalate esters, anilines, ethers, aromatic nitro compounds, and nitrosamines. LLE allowed for a higher number of target analytes to be extracted with over 70% recovery and quantified more targets in the influent samples. Matrix interference effects had a negative impact on the recovery of non-polar contaminants, such as polycyclic aromatic hydrocarbons (PAHs), in the influent water samples especially with SBSE. In SBSE, 24 target analytes demonstrated significant matrix interference leading to poor analyte recovery and 13 analytes were negatively affected in the same way in LLE. Generally, polar compounds also demonstrated poor extraction with SBSE in both effluent and influent water samples. However, SBSE effluent chromatograms contained about three times as many total analytes as compared with LLE, suggesting that SBSE is more sensitive for trace contaminants in effluent samples. Based on this research, LLE is recommended for studies seeking to quantify a broad range of target analytes in complex matrices, like wastewater influent. SBSE is an appropriate method for the non-target and survey analysis of trace contaminants in less complex water samples.

An overview of therapeutic anticancer drug monitoring based on surface enhanced (resonance) Raman spectroscopy (SE(R)RS).

Therapeutic drug monitoring (TDM) is important for many therapeutic regimens and has particular relevance for anticancer drugs which often have serious effects and whose optimum dosage can vary significantly between different patients. Many of the features of surface enhanced (resonance) Raman spectroscopy (SE(R)RS) suggest it should be very suitable for TDM of anticancer drugs and some initial studies which explore the potential of SE(R)RS for TDM of anticancer drugs have been published. This review brings this work together in an attempt to draw some general observations about key aspects of the approach, including the nature of the substrate used, matrix interference effects and factors governing adsorption of the target molecules onto the enhancing surface. There is now sufficient evidence to suggest that none of these pose real difficulties in the context of TDM. However, some issues, particularly the need to carry out multiplex measurements for TDM of combination therapies, have yet to be addressed.

Quick monitoring of coloring agents in highly consumed candies using multivariate calibration

Multivariate calibration are gaining popularity in assaying food matrices. Partial least squares is a powerful multivariate calibration method that used to build a quantitative relationship between measured variables and a property of interest (i.e., concentration) of the system under study. Partial least squares PLS calibration along with UV/vis spectral data was efficient to account for indirect food matrix and direct interference effects resulted from overlapping food dyes. PLS was able to quantify tartrazine TAT, allura red AR, sunset yellow SY and brilliant black BB that added to wide selection sugar-based candies. The results indicated that 70% of samples containing single dye while 8% containing TAT-SY mix and certain samples containing TAT + SY + AR + BB. Lollypops were found to contain high levels of AR (77–120 mg/kg) and TAT (56–166 mg/kg). The maximum adulteration was 50% observed in lollypops. PLS calibration was workable to predict colorants with prediction errors of 7%. Using PLS, dyes were detected down to 0.1 mg/L with acceptable accuracy and precision. PLS showed comparable performance with liquid chromatography for dyes quantification and can substitute laborious chromatography for quick detection of coloring agents in candies.

The effect of matrix interferences on in situ boron isotope analysis by laser ablation multi-collector inductively coupled plasma mass spectrometry.

Boron isotope analysis of marine carbonates by laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) offers the potential for rapid sample throughput, and the means to examine micron-scale variations in the δ11 B signatures of fossil skeletons and shells/tests of marine organisms. Existing studies demonstrate an acceptable level of reproducibility is achievable, but also typically show a level of accuracy outside the limits required by most applications. Here we investigate matrix interference effects as a cause of inaccuracy and imprecision. Analyses were performed on a standard format Thermo Scientific Neptune Plus MC-ICP mass spectrometer coupled to a New Wave Research 193 nm ArF laser ablation system. The effects of matrix interference on δ11 B analysis were investigated through analyses of a set of reference materials with differing B/Ca ratios. Three approaches to correct for matrix-induced effects were trialled: (1) use of matrix-matched standards, (2) utilisation of the relationship between δ11 B inaccuracy and11 B/43 Ca, 11 B/40 ArCa4+ or 11 B/Cainterference from three reference materials with known δ11 B values and varying B/Ca ratios, and (3) direct characterisation of the (sloping) interference itself. Matrix interference from scattered Ca ions on 10 B can impede both the accuracy and the reproducibility of δ11 B analysis by LA-MC-ICP-MS. Based on analyses of two in-house reference materials, deep sea coral PS69/3181 and inorganic calcite UWC-1, we find approach 2, following the 11 B/Cainterference relationship, gives the best mean accuracies (within 0.4‰ of solution values) and external reproducibilities (± 0.5‰ 2 SD for PS69/3181). This approach has been applied to analyses of an annual growth cycle of a Siderastrea siderea coral and eight Cibicidoides wuellerstorfi benthic foraminifera. Both coral and foraminifera data match solution MC-ICP-MS analyses within reported uncertainties. LA-MC-ICP-MS can produce accurate and precise δ11 B data to a 0.5‰ (2σ) level on <0.3 ng B after correction for Ca interference effects.

Rapid determination of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water by direct injection-ultra performance liquid chromatography-triple quadrupole mass spectrometry

A simple method based on direct injection-ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was established for the rapid determination of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water. The water samples were filtered through a 0.22-μm filter membrane or frozen and centrifuged to remove impurities, and then, the filtrate was directly subjected to quantitative analysis without derivatization. The analytes were separated on a Metrosep A Supp 5 column (150 mm×4.0 mm, 5 μm), and gradient elution was carried out using an ammonium bicarbonate-ammonia solution as the mobile phase. The data were collected by positive electrospray ionization in the multiple reaction monitoring (MRM) mode. The results showed that the correlation coefficients (r) of the linear calibration curves were greater than 0.999 in the corresponding linear ranges (0.50-50.0 μg/L). The detection limits of the analytes were 0.05-0.09 μg/L. The recoveries of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon were in the ranges 76.3%-108%, 83.0%-107%, and 87.0%-105% at low, medium, and high spiked levels, respectively. The corresponding relative standard deviations were in the ranges 2.0%-12.3%, 2.4%-5.6%, and 2.7%-6.8%. Using this method, 34 water samples collected from Hainan Province were analyzed, among which 30 drinking water sources were found to be free from the four pesticides. Glyphosate and aminomethyl phosphonic acid were detected in three water samples near a betel nut orchard, while glufosinate and aminomethyl phosphonic acid were detected in a water sample near a banana orchard. This method is advantageous over the traditional derivatization method because of its simple operation, good reproducibility, and high accuracy; furthermore, the matrix interference effect is absent. Thus, this method is suitable for analyzing glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water samples.

Blood Plasma Separation Using a Fidget-Spinner.

In this study, we present a simple, hand-powered, and electricity-free centrifuge platform based on a commercially available "fidget-spinner." The centrifugal force provided by this inexpensive and easy-to-use toy is sufficient to separate whole blood, producing a plasma yield rate and purity of 30% and 99%, respectively, separated in as little as 4-7 min. We verified the separated plasma by performing a paper-based HIV-1 p24 capsid protein enzyme-linked immunosorbent assay, which achieved a recovery rate of up to 98%, indicating the plasma features extremely low matrix interference effects. These results demonstrate the reliability of the platform for practical use, in addition to greatly reducing the overall cost and time of analysis while retaining detection precision, making it suitable for medical applications in resource-limited regions of the world.