The presence and role of the intermediary CO reservoir in heterogeneous electroreduction of CO2.

SignificanceThe electroconversion of CO2 to value-added products is a promising path to sustainable fuels and chemicals. However, the microenvironment that is created during CO2 electroreduction near the surface of heterogeneous Cu electrocatalysts remains unknown. Its understanding can lead to the development of ways to improve activity and selectivity toward multicarbon products. This work introduces a method called on-stream substitution of reactant isotope that provides quantitative information of the CO intermediate species present on Cu surfaces during electrolysis. An intermediary CO reservoir that contains more CO molecules than typically expected in a surface adsorbed configuration was identified. Its size was shown to be a factor closely associated with the formation of multicarbon products.

Fabrication and Performance of Bubble-Containing Multicompartmental Particles: Novel Self-Orienting Carriers.

In this work, a class of bubble-containing multicompartmental particles with self-orienting capability is developed, where a single bubble is enclosed at the top of the super-segmented architecture. Such bubbles, driven by potential energy minimization, cause the particles to have a bubble-upward preferred orientation in liquid, enabling efficient decoding of their high-density signals in an interference-resistant manner. The particle preparation involves bubble encapsulation via the impact of a multicompartmental droplet on the liquid surface and overall stabilization via rational crosslinking. The conditions for obtaining these particles are systematically investigated. Methodological compatibility with materials is demonstrated by different hydrogel particles. Finally, by encapsulating cargoes of interest, these particles have found broad applications in actuators, multiplexed detection, barcodes, and multicellular systems.

Analyzing fuzzy boundary value problems: a study on the influence of mitochondria and ER fluxes on calcium ions in neuron cells.

Cytosolic-free calcium ions play an important role in various physical and physiological processes. A vital component of neural signaling is the free calcium ion concentration often known as the second messenger. There are many parameters that effect the cytosolic free calcium concentration like buffer, voltage-gated ion channels, Endoplasmic reticulum, Mitochondria, etc. Mitochondria are small organelles located within the nervous system that are involved in processes within cells such as calcium homeostasis management, energy generation, response to stress, and cell demise pathways. In this work, a mathematical model with fuzzy boundary values has been developed to study the effect of Mitochondria and ER fluxes on free Calcium ions. The intended findings are displayed utilizing the physiological understanding that amyloid beta plaques and tangles of neurofibrillary fibers have been identified as the two main causes of AD. The key conclusion of the work is the investigation of [Formula: see text] for healthy cells and cells affected by Alzheimer's disease, which may aid in the study of such processes for computational scientists and medical practitioners. Also, it has been shown that when a unique solution is found for a specific precise problem, it also successfully deals with any underlying ambiguity within the problem by utilizing a technique based on the principles of linear transformation. Furthermore, the comparison between the analytical approach and the generalized hukuhara derivative approach is shown here, which illustrates the benefits of the analytical approach. The simulation is carried out in MATLAB.

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products.

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

An Algebraic Blueprint for Predicting Turnover Numbers and Endpoints in Photocatalysis.

Photocatalysis is a contemporary research field given that the world's fossil energy resources including coal, mineral oil and natural gas are finite. The vast variety of photocatalytic systems demands for standardized protocols facilitating an objective comparison. While there are commonly accepted performance indicators such as the turnover number (TON) that are usually reported, to date there is no unified concept for the determination of TONs and the endpoint of the reaction during continuous measurements. Herein, we propose an algebraic approach using defined parameters and boundary conditions based on partial-least squares regression for generically calculating and predicting the turnover number and the endpoint of a photocatalytic experiment. Furthermore, the impact of the analysis period was evaluated with respect to the fidelity of the obtained TON, and the influence of the data point density along critical segments of the obtained fitting function is demonstrated.

An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling.

The development of liquid chromatography UV and mass spectrometry (LC-UV-MS) assays in pharmaceutical analysis is pivotal to improve quality control by providing critical information about drug purity, stability, and presence and identity of byproducts and impurities. Analytical method development of these assays is time-consuming, which often causes it to become a bottle neck in drug development and poses a challenge for process chemists to quickly improve the chemistry. In this study, a systematic and efficient workflow was designed to develop purity assay and purification methods for a wide range of compounds including peptides, proteins, and small molecules with MS-compatible mobile phases (MP) by using automated LC screening instrumentation and in silico modeling tools. Initial LC MPs and chromatography column screening experiments enabled quick identification of conditions which provided the best resolution in the vicinity of the target compounds, which is further optimized using computer-assisted modeling (LC Simulator from ACD/Labs). The experimental retention times were in good agreement with the predicted retention times from LC Simulator (ΔtR < 7%). This workflow presents a practical workflow to significantly expedite the time needed to develop optimized LC-UV-MS methods, allowing for a facile, automatic method optimization and reducing the amount of manual work involved in developing new methods during drug development.

Assessment of legacy and emerging PFAS in the Oder River: Occurrence, distribution, and sources.

The purpose of the study was to evaluate the occurrence and distribution of emerging contaminants, poly- and perfluoroalkyl substances (PFAS), in the Polish Oder River, aiming to uncover new insights into their environmental impact. The research aimed to identify potential sources of PFAS, assess water quality levels, and verify compliance with European Union environmental quality standards. The concentrations of 25 PFAS (20 legacy and 5 emerging) in 20 samples from intakes upstream and downstream of urban areas were analyzed using novel, developed in these studies, environmental analytical procedures involving solid phase extraction and liquid chromatography-tandem mass spectrometry. The presence of 14 PFAS was confirmed, and the concentration of Σ14PFAS ranged from 7.6 to 68.0 ng/L. The main components were short-chain analogs. PFBA was the most abundant, accounting for about one-third of all PFAS detected. An exception was observed in the waters of the Gliwice Canal, where ADONA represented half of the detected Σ14PFAS. Alternative PFOS replacements were found in all samples. In 11 of 20 water samples, environmental quality standards for PFOS exceeded the limit of 0.65 ng/L. In 5 of 9 cases, the ability of urban areas to increase PFAS levels in the river was determined. 9.5%-54.4% share of alternative PFAS in relation to the sum of the targeted PFAS showing their increasing use as substitutes for phased-out PFOS. Hierarchical cluster analysis was used to identify potential sources of PFAS. Analysis revealed that PFAS in the Oder River most likely originated from domestic and agricultural wastewater, as well as chemical industry discharges. However, the occurrence of PFAS in the Oder River is low and comparable to other recent European studies. These findings provide valuable insights for environmental management to mitigate the risks associated with PFAS pollution in Polish rivers. Moreover, the developed analytical procedure provides a valuable tool that can be successfully applied by other researchers to monitor PFAS in rivers around the world.

Critical review on airborne microplastics: An indoor air contaminant of emerging concern.

Airborne Microplastics (MPs), an emerging environmental issue, have gained recent attention due to their newfound presence in indoor environments. Utilizing the Web of Science database for literature collection, the paper presents a comprehensive review of airborne MPs including emission sources, assessment methods, exposure risks, and mitigation strategies. This review delves into the diverse sources and mechanisms influencing indoor airborne MP pollution, underscoring the complex interplay between human activities, ventilation systems, and the characteristics of indoor environments. Major sources include the abrasion of synthetic textiles and the deterioration of flooring materials, with factors like carpeting, airflow, and ventilation significantly impacting MP levels. Human activities, such as increased movement in indoor spaces and the intensive use of plastic-based personal protective equipment (PPE) post-pandemic, notably elevate indoor MP concentrations. The potential health impacts of airborne MPs are increasingly concerning, with evidence suggesting their role in respiratory, immune, and nervous system diseases. Despite this, there is a scarcity of information on MPs in diverse indoor environments and the inhalation risks associated with the frequent use of PPE. This review also stresses the importance of developing effective strategies to reduce MP emissions, such as employing HEPA-filtered vacuums, minimizing the use of synthetic textiles, and enhancing indoor ventilation. Several future research directions were proposed, including detailed temporal analyses of indoor MP levels, interactions of MP with other atmospheric pollutants, the transport dynamics of inhalable MPs (≤10 µm), and comprehensive human exposure risk assessments.

The carcinogenic PAHs in breads, amount, analytical method and mitigation strategy, a systematic review study.

Bread is one of the most consumed foods all over the world. Several contaminants are identified in bread. Polycyclic aromatic hydrocarbons (PAHs) is one of these contaminants. This systematic study evaluates the amount of four carcinogenic PAHs (PAH4) in various types of breads. To conduct this study, a comprehensive search was carried out using keywords of polycyclic aromatic hydrocarbons, PAHs, PAH4, and bread, with no time limitations. 17 articles were selected and fully evaluated. The observed range of PAH4 concentrations in bread varied from non-detected (ND) to 20.66 µg/kg. In the sample preparation process for analysis, an ultrasonic bath was predominantly utilized. Most chromatographic methods are able to measure PAHs in food, but the GC-MS method has been used more. To mitigate PAH levels in bread, it is suggested to incorporate antioxidants during the bread-making process. Furthermore, the type of bread, the type of fuel used to bake the bread, the temperature and the cooking time were some of the factors affecting the amount of PAH. Restricting these factors could significantly reduce PAH content. Regarding the risk assessment conducted in the manuscript, it was determined that industrial breads are usually considered safe. However, some traditional breads may pose risks in terms of their potential PAH content.

Differential role of potential stressors, underlying degradation mechanism, characterization of degradants using LC-MS/MS, and establishment of a stability-indicating analytical method for duvelisib.

Duvelisib (DUV) was first approved globally in 2018. An extensive literature search revealed that the differential role of a potential degradation medium in altering the shelf-life of DUV due to its exposure during storage has not been identified till date. Moreover, its degradation impurities and degradation mechanism are not known. In addition, no analytical method has been reported for the quantification of DUV in the presence of its degradation impurities. Therefore, the aim of this study was to identify the impact of different potential degradation media on the stability of DUV, establish the degradation mechanism, and identify its major degradation impurities. The aim was also to establish a stability-indicating analytical method for the quantification of DUV in the presence of its degradation impurities. This study is the first to report the structure of degradation impurities and the step-by-step degradation mechanism of DUV. This information will be useful for the scientific community and manufacturers in optimizing the formulation parameters and/or storage conditions. The validated method can be employed for analysis of stability study and routine quality control samples of newer DUV formulations in pharmaceutical industries. The identified impurities may serve as impurity standards for specifying their limits in the drug after required qualification studies.

LC-MS/MS determination of N-acetyl-l-cysteine in chicken plasma.

N-acetyl-l-cysteine (NAC) shows beneficial effects in cases of aflatoxicosis and heat stress in poultry but little is known about its pharmacokinetics in chickens. Therefore, the study aimed to develop and validate a sensitive LC-MS/MS analytical method for quantitative analysis of NAC in chicken plasma. A split calibration curve approach was used for determination of NAC in chicken plasma. Standard curves for low (0.05-2.5 µg/ml) and high (2.5-100 µg/ml) ranges of concentrations were prepared. The standard curves for low (r2 = 0.9987) and high (r2 = 0.9899) concentrations were linear within the tested range. The limits of detection (LOD) and of quantification (LOQ) for the standard at low concentrations were 0.093 and 0.28 µg/ml, respectively. The accuracy was from 97.35 to 101.33%. The values of LOD and LOQ for the standard at high concentrations were 0.76 and 2.30 µg/ml, respectively. The accuracy was between 99.77 and 112.14%. The intra- and inter-day precisions for all concentrations from both standards did not exceed 8.57% and 10.69%, respectively. The recovery for all concentrations was between 92.45 and 105.52%. The validated method for determination of NAC in chicken plasma can be applied in future pharmacokinetic studies in chickens without dilution of samples and their repeated analysis.

A liquid chromatography-tandem mass spectrometry method development for the quantification of favipiravir drug and its related impurities in rat plasma and its application to pharmacokinetic studies.

Favipiravir is an antiviral drug used for the treatment of virus-based diseases such as influenza. In this context, the development of a reliable liquid chromatography-tandem mass spectrometry method for the quantification of the drug and its impurities is necessary, particularly following the COVID-19 pandemic. Chromatographic separation was achieved on an inertial ODS column using gradient elution with a buffer containing triethylamine in high-performance liquid chromatography water and adjusting its pH with formic acid. The mixture of buffer and acetonitrile was used as a mobile phase with a flow rate of 1 ml/min at ambient temperature. The separation of favipiravir and its related impurities from remdesivir as an internal standard was achieved. The results indicated that all the variables, like precision, accuracy, linearity, matrix effect and stability, were successfully achieved within the limits of US Food and Drug Administration guidelines. This study could provide a new protocol for the development of new analytical methods for the detection of favipiravir and its impurities.

Delineation of prototypical degradation mechanism, characterization of unknown degradation impurities by liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry and stability-indicating analytical method of selumetinib.

Selumetinib (SELU) was recently approved by the US Food and Drug Administration (US FDA) in 2020. However, the degradation impurities of SELU have not been characterized or identified to date. The mechanism for impurity formation and the degradation behavior have not been previously studied. This study aims to elucidate the prototypical degradation mechanism of SELU. Furthermore, the degradation impurities have been identified using LC-quadrupole-time-of-flight tandem mass spectrometry and are reported in this article for the first time. In addition, a stability-indicating analytical method (SIAM) has been developed for this drug. Forced degradation studies revealed the degradation of SELU under various stress conditions, including hydrolytic stress (acid and base), oxidative stress, and photolytic stress (ultraviolet and visible). Three degradation impurities were identified. This article presents the first validated SIAM, capable of accurately quantifying SELU in the presence of its degradation impurities. Furthermore, we have proposed the degradation pathway for SELU and its degradation impurities, a first in the field. The developed SIAM can find applications in process development and quality assurance of SELU in both research laboratories and pharmaceutical industries. Moreover, the identified degradation impurities may serve as impurity standards for quality control testing in pharmaceutical industries.

Facile Access to Solifenacin Impurity K: One-Step Synthesis and an HPLC-MS Method for Its Determination.

Solifenacin (SFC) is a potent muscarinic antagonist that effectively reduces bladder muscle contraction, thereby alleviating symptoms such as frequency of micturition and urgency. Oxidation of SFC leads to the formation of impurities like Impurity K. Effective analysis and control of this impurity is crucial for ensuring compliance with regulatory standards and safeguarding patient health. To address these challenges, we propose a novel one-step synthesis of Impurity K from SFC. Impurity K was synthesized using cerium(IV) ammonium nitrate (CAN) in water/acetonitrile as the solvent. Additionally, we describe a new HPLC-MS method for the detection of Impurity K in solifenacin succinate tablets.

Recent Advances in the Determination of Major and Trace Elements in Plants Using Inductively Coupled Plasma Optical Emission Spectrometry.

Interest in measuring major and trace elements in plants has increased in recent years because of growing concerns about the elements' contribution to daily intakes or the health risks posed by ingesting vegetables contaminated by potentially toxic elements. The recent advances in using inductively coupled plasma atomic emission spectrometry (ICP-OES) to measure major and trace elements in plant samples are reviewed in the present work. The sample preparation before instrumental determination and the main advantages and limitations of ICP-OES are described. New trends in element extraction in liquid solutions using fewer toxic solvents and microextractions are observed in recently published literature. Even though ICP-OES is a well-established and routine technique, recent innovations to increase its performance have been found. Validated methods are needed to ensure the obtaining of reliable results. Much research has focused on assessing principal figures of merit, such as limits of detection, quantification, selectivity, working ranges, precision in terms of repeatability and reproducibility, and accuracy through spiked samples or certified reference materials analysis. According to the published literature, the ICP-OES technique, 50 years after the release of the first commercially available equipment, remains a powerful and highly recommended tool for element determination on a wide range of concentrations.

Effect of Hemodynamic Monitoring Systems on Short-Term Outcomes after Living Donor Liver Transplantation.

Background and Objectives: To evaluate the effects of the pulse index continuous cardiac output and MostCare Pressure Recording Analytical Method hemodynamic monitoring systems on short-term graft and patient outcomes during living donor liver transplantation in adult patients. Materials and Methods: Overall, 163 adult patients who underwent living donor liver transplantation between January 2018 and March 2022 and met the study inclusion criteria were divided into two groups based on the hemodynamic monitoring systems used during surgery: the MostCare Pressure Recording Analytical Method group (n = 73) and the pulse index continuous cardiac output group (n = 90). The groups were compared with respect to preoperative clinicodemographic features (age, sex, body mass index, graft-to-recipient weight ratio, and Model for End-stage Liver Disease score), intraoperative clinical characteristics, and postoperative biochemical parameters (aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, prothrombin time, international normalized ratio, and platelet count). Results: There were no significant between-group differences with respect to recipient age, sex, body mass index, graft-to-recipient weight ratio, Child, Model for End-stage Liver Disease score, ejection fraction, systolic pulmonary artery pressure, surgery time, anhepatic phase, cold ischemia time, warm ischemia time, erythrocyte suspension use, human albumin use, crystalloid use, urine output, hospital stay, and intensive care unit stay. However, there was a significant difference in fresh frozen plasma use (p < 0.001) and platelet use (p = 0.037). Conclusions: The clinical and biochemical outcomes are not significantly different between pulse index continuous cardiac output and MostCare Pressure Recording Analytical Method as hemodynamic monitoring systems in living donor liver transplantation. However, the MostCare Pressure Recording Analytical Method is more economical and minimally invasive.

Validated green and white RP-HPLC method for the estimation of Zolmitriptan in marketed dosage form.

OBJECTIVE: To develop and validate a rapid, accurate, economical, effective and greenery RP-HPLC method for the determination of Zolmitriptan in tablet dosage form. MATERIAL AND METHOD: RP-HPLC method was developed using Luna (C18) (4.6×250mm, 5µm) column and Sodium phosphate buffer (pH 4.7): Methanol [75: 25, v/v] was used as mobile phase at a flow rate of 1.0mL/min. The detection was carried out at 227nm. Further, eco-friendliness, productivity and performance of the optimized analytical method were assessed by green and white tools. RESULTS: The retention time of Zolmitriptan was found to be 3.25min with acceptable chromatographic parameters. The optimized RP-HPLC method was more eco-friendly, efficient, throughput and practicable than the reported methods as confirmed by AES, AGREE, GAPI and RGB tools. Further, the proposed analytical method showed all the validation parameters within the acceptance limit of ICH Q2 R1 guidelines. The linear regression analysis indicated a good linear response in the 10 to 120µg/mL concentration range with R2 of 0.99998. The percentage content and percentage assay of Zolmitriptan in Zomig-5mg tablet was found to be 103.36±0.356% and 97.86±0.693%. CONCLUSION: The developed and validated method has several advantages compared to the reported HPLC methods and is useful in the systematic analysis of Zolmitriptan in its dosage form.

Self-Powered Electrochemiluminescence for Imaging the Corrosion of Protective Coating of Metal and Quantitative Analysis.

In almost all electrochemical systems for electrochemiluminescence (ECL) analysis, electrodes are connected with an external power source, either directly or via wireless energy transfer circuit. That is inconvenient and makes some applications impossible. Herein, we both use galvanized iron with two different metals as both power source and electrodes to achieve a self-powered ECL and exploit ECL for the imaging of the corrosion of protective coating of widely used metal (e.g. galvanized iron) for the first time. The self-powered ECL enables the visualization of the deterioration of galvanic coating on iron using a smartphone and the detection of ascorbic acid with a linear range of 0.5-100 µM and a limit of detection of 0.31 µM. The devices based on self-powered approach do not require external power supply, thus effectively reducing their volume and cost. The self-powered ECL holds great promise for metal corrosion imaging and analytical applications.

Magnitude of Pulse Pressure Variation is Associated with Qp:Qs Imbalance during Pediatric Cardiac Surgery: A Two-Center Retrospective Study.

Background: Pulse pressure variation (PPV) is based on heart-lung interaction and its association with the imbalance between pulmonary and systemic blood flow (Qp:Qs) has been understudied. We hypothesized that (1) baseline PPV (after induction of anesthesia) is different in a mixed congenital heart disease population with different Qp:Qs, (2) baseline PPV is different between a pooled group with high Qp:Qs and one with low Qp:Qs, and (3) a systemic-pulmonary shunt procedure results in reduced PPV compared to baseline. Methods: We retrospectively reviewed the medical charts of children who presented to the operating room for cardiac surgery between 2010 and 2018. General patient characteristics, PPV, and other hemodynamic parameters following the induction of general anesthesia were retrieved. Patients were grouped according to the type of congenital heart disease, and whether the Qp:Qs ratio was higher or lower than 1. We also identified patients who received a systemic-pulmonary shunt in order to evaluate changes in PPV. Results: A total of 1253 patients were included in the study. Baseline PPV differed significantly according to the type of congenital heart disease, with atrial septal defect showing the lowest PPV (9.5 ± 5.6%) and tricuspid valve malformation the highest (21.8 ± 14.1%). The high Qp:Qs group (n = 932) had significantly lower PPV compared to the low Qp:Qs group (n = 321; 11.8 ± 5.7% vs. 14.9 ± 7.9%, respectively; p < 0.001). PPV decreased significantly following systemic-pulmonary shunt. Conclusions: PPV was associated with Qp:Qs imbalance in children undergoing general anesthesia for cardiac surgery. A lower PPV was associated with increased Qp:Qs. Clinicians should take this into account when using PPV to evaluate volume status and when conducting clinical trials in a mixed population of patients with congenital heart disease.