Digital image colorimetry for determination of ethanol in exhaled breath condensate.

Aim: This study aimed to develop a colorimetric approach for quantifying ethanol using smartphone image analysis. Method: This research presents a straightforward smartphone-based colorimetric sensor that efficiently measures ethanol levels in exhaled breath condensate (EBC) samples. The process involved changing the acidic dichromate color in an ethanolic solution, followed by image analysis. Results: The results showed that this method was able to estimate ethanol concentrations in the range of 300-1500 and 1600-8000 µg ml-1 in EBC. Conclusion: This study was a follow-up study on the previous work published for the determination of ethanol in EBC samples and highlights the potential benefits of using digital images and smartphone applications for ethanol determination in biological samples.

Utilizing a nanocomposite aerogel grafted with Fe3O4@GO for the extraction and determination of metoprolol in exhaled breath condensate.

This article presents a solid-phase extraction method combined with a spectrofluorometric method for the extraction/pre-concentration and determination of metoprolol (MET) in exhaled breath condensate. The extraction sorbent is an agarose aerogel nanocomposite grafted with graphene oxide (GO) Fe3O4. The size and morphology of the nanosorbent were characterized via X-ray crystallography, scanning electron microscopy, Fourier-transform infrared spectrometry, and Brunauer-Emmett-Teller analysis. Factors affecting the extraction/determination of MET were optimized using the one-at-a-time method. Under optimized experimental conditions, the calibration graph was linear in the range of 0.005 to 2.0 µg mL-1 with a detection limit of 0.001 µg mL-1. The method was successfully applied for the determination of MET in biological samples taken from patients receiving MET.

Online preconcentration and chiral separation of ofloxacin in exhaled breath condensate by capillary electrophoresis.

Breath analysis is an effective method of monitoring systemic or respiratory ailments. A simple chiral capillary electrophoresis method coupled with an online field-amplified sample injection stacking method is presented for ultratrace quantification of the enantiomers of ofloxacin in exhaled breath condensate (EBC). The study is focused on the use of EBC as an easily available biological sample to monitor ofloxacin's enantiomers levels with good patient compliance. The proposed method was validated in accordance with FDA guidelines over the concentration range of 0.004-1.0 µg mL-1 of racemic ofloxacin. Inter- and intra-day precision and accuracy were within the acceptable limit (below 8.50 %). The method was specific for routine analysis of ofloxacin's enantiomers. A small volume of EBC samples from seven patients under ofloxacin therapy was analyzed using the proposed method in which the concentrations of "R" and "S" enantiomers were between 0.0026 and 0.056 µg mL-1.

A smartphone digital image colorimetric method based on nanoparticles for determination of lamotrigine.

Aim: A colorimetric approach for quantification of lamotrigine using spectrophotometric and smartphone image analysis is described in this study. Methods: For full optimization and validation procedures, UV-visible spectroscopy was used, and image analysis was carried out with the help of an app (PhotoMetrix PRO®). Then, as a multivariate calibration method, parallel factor analysis was used for data analysis. Results: The results demonstrated the capacity of these methods to estimate lamotrigine concentrations in the range of 0.1-7.0 µg.ml-1 in exhaled breath condensate, indicating the value of using digital images and smartphone applications in combination with chemometric tools. Conclusion: The image analysis can be superior for its fast and reliable lamotrigine analysis in biological samples.

A new method for investigating bioequivalence of inhaled formulations: A pilot study on salbutamol.

Purpose: An efficient, cost-effective and non-invasive test is required to overcome the challenges faced in the process of bioequivalence (BE) studies of various orally inhaled drug formulations. Two different types of pressurized meter dose inhalers (MDI-1 and MDI-2) were used in this study to test the practical applicability of a previously proposed hypothesis on the BE of inhaled salbutamol formulations. Methods: Salbutamol concentration profiles of the exhaled breath condensate (EBC) samples collected from volunteers receiving two inhaled formulations were compared employing BE criteria. In addition, the aerodynamic particle size distribution of the inhalers was determined by employing next generation impactor. Salbutamol concentrations in the samples were determined using liquid and gas chromatographic methods. Results: The MDI-1 inhaler induced slightly higher EBC concentrations of salbutamol when compared with MDI-2. The geometric MDI-2/MDI-1 mean ratios (confidence intervals) were 0.937 (0.721-1.22) for maximum concentration and 0.841 (0.592-1.20) for area under the EBC-time profile, indicating a lack of BE between the two formulations. In agreement with the in vivo data, the in vitro data indicated that the fine particle dose (FPD) of MDI-1 was slightly higher than that for the MDI-2 formulation. However, the FPD differences between the two formulations were not statistically significant. Conclusion: EBC data of the present work may be considered as a reliable source for assessment of the BE studies of orally inhaled drug formulations. However, more detailed investigations employing larger sample sizes and more formulations are required to provide more evidence for the proposed method of BE assay.

Efficient dispersive solid-phase extraction of methylprednisolone from exhaled breath of COVID-19 patients.

In the current study, bismuth ferrite nano-sorbent was synthesized and utilized as a sorbent for the dispersive solid-phase extraction of methylprednisolone from exhaled breath samples. The size and morphology of the nano-sorbent were characterized by X-ray diffraction analysis and scanning electron microscopy. Following its desorption with acetonitrile, methylprednisolone was quantified by a high-performance liquid chromatography-ultraviolet detector. Factors affecting the extraction of methylprednisolone were optimized. Under optimized experimental conditions, a linear relationship between the analytical signals and methylprednisolone concentration was obtained in the range of 0.001-0.2 µg mL-1 for exhaled breath condensate samples and 0.002-0.4 µg per filter for filter samples. A pre-concentration factor of 6.4-fold, corresponding to an extraction recovery of 96.0%, was achieved. The validated method was applied for the determination of methylprednisolone in real samples taken from the exhaled breath of COVID-19 patients under mechanical ventilation.

Propofol-induced in-situ formation of silver nanoparticles: A sensing colorimetric method.

A simple and eco-friendly colorimetric sensing method has been developed for the extremely effective detection of propofol in exhaled breath condensate (EBC). In this study, we put forward a Tollens' procedure, in which silver nanoparticles (AgNPs) were produced using propofol as a reducing agent. To verify the in-situ synthesis of AgNPs, the TEM images, and UV-Vis absorbance were recorded in the absence and presence of propofol. The solution turned from a colorless to yellow and deep yellow color due to the surface plasmon resonance absorption band of formed AgNPs. The intensity of nanoparticle absorbance was quantitatively correlated with the propofol concentration. The proposed sensor revealed good linearity over the range of 0.01-0.8 µg mL-1 at 422 nm with the detection limit of 8.8 ng mL-1 under optimum conditions. Finally, the proposed colorimetric sensor was successfully used for the determination of propofol in the EBC sample of patients receiving propofol.

A Rayleigh light scattering technique based on ß- cyclodextrin modified gold nanoparticles for phenytoin determination in exhaled breath condensate.

In the current work, a RLS technique based on ß-cyclodextrin modified gold nanoparticles was validated for phenytoin determination in the exhaled breath condensate. It relies on the complexation of ß-cyclodextrins using -OH groups with amine groups of phenytoin which results in an aggregation-induced Rayleigh light scattering intensity enhancement proportional to phenytoin addition. The method shows a linear relationship with phenytoin concentration in the range of 0.005-0.6 µg.mL-1 with a limit of detection of 0.003 µg.mL-1. The validated Rayleigh light scattering system is successfully used for phenytoin determination in the EBC of patients receiving phenytoin.

Biomarkers in exhaled breath condensate as fingerprints of asthma, chronic obstructive pulmonary disease and asthma-chronic obstructive pulmonary disease overlap: a critical review.

Asthma, chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap are the third leading cause of mortality around the world. They share some common features, which can lead to misdiagnosis. To properly manage these conditions, reliable markers for early and accurate diagnosis are needed. Over the past 20 years, many molecules have been investigated in the exhaled breath condensate to better understand inflammation pathways and mechanisms related to these disorders. Recently, more advanced techniques, such as sensitive metabolomic and proteomic profiling, have been used to obtain a more comprehensive understanding. This article reviews the use of targeted and untargeted metabolomic methodology to study asthma, COPD and asthma-COPD overlap.

Selection of Specific Aptamer against Rivaroxaban and Utilization for Label-Free Electrochemical Aptasensing Using Gold Nanoparticles: First Announcement and Application for Clinical Sample Analysis.

For the first time, a novel aptamer was designed and utilized for the selective detection of rivaroxaban (RIV) using the integration of bioinformatics with biosensing technology. The selected aptamer with the sequence 5'-TAG GGA AGA GAA GGA CAT ATG ATG ACT CAC AAC TGG ACG AAC GTA CTT ATC CCC CCC AAT CAC TAG TGA ATT-3' displayed a high binding affinity to RIV and had an efficient ability to discriminate RIV from similar molecular structures. A novel label-free electrochemical aptasensor was designed and fabricated through the conjugation of a thiolated aptamer with Au nanoparticles (Au-NPs). Then, the aptasensor was successfully applied for the quantitative determination of RIV in human plasma and exhaled breath condensate (EBC) samples with limits of detection (LODs) of 14.08 and 6.03 nM, respectively. These valuable results provide ample evidence of the green electrogeneration of AuNPs on the surface of electrodes and their interaction with loaded aptamers (based on Au-S binding) towards the sensitive and selective monitoring of RIV in human plasma and EBC samples. This bio-assay is an alternative approach for the clinical analysis of RIV and has improved specificity and affinity. As far as we know, this is the first time that an electrochemical aptasensor has been verified for the recognition of RIV and that allows for the easy, fast, and precise screening of RIV in biological samples.

Electromembrane extraction of tramadol from exhaled breath condensate and its liquid chromatographic analysis.

Tramadol has extracted from the exhaled breath condensate (EBC) samples through the supported liquid membrane consisting of 2-nitrophenyl octyl ether impregnated in the hollow fiber wall, and the lumen of the hollow fiber was filled with 20 µL of an acceptor phase. Under the optimum conditions of the electromembrane extraction, i.e. the stirring speed of 750 rpm, extraction time of 20 min, acceptor pH at 1.0, donor phase pH at 6.0, and an applied voltage of 170 V across the supported liquid membrane, a preconcentration factor of 128-fold with a extraction recovery of 64% was achieved. Acceptable linearity was obtained in the tramadol concentration range of 5-1000 ng mL-1 (R2 = 0.9999) with a limit of detection of 1.5 ng mL-1 and a limit of quantitation of 5 ng mL-1. The relative standard deviations for the intra-day and inter-day replications were obtained between 0.4% and 2.5%. The validated technique was successfully used to determine tramadol in real EBC samples.

A mixed deep eutectic solvents-based air-assisted liquid-liquid microextraction of surfactants from exhaled breath condensate samples prior to HPLC-MS/MS analysis.

A ternary solvent system-based air-assisted liquid-liquid microextraction procedure was developed for the extraction of three surfactants from exhaled breath condensate samples prior to their determination by high performance liquid chromatography-tandem mass spectrometry. In this approach, different deep eutectic solvents were synthesized based on phosphocholine chloride and fatty acids and their mixtures were used as the extraction solvents to effective extraction of the analytes. To obtain the optimum composition of the extraction solvents, a simplex centroid design approach was used. Then the effective parameters were studied by response surface methodology using central composite design. The obtained data after optimization showed that 6 times was the best extraction time for the developed procedure. When the sample solution pH was adjusted at 3.7, the method reached to higher extraction efficiency which can be related to the fact that the analytes were in the protonated forms. Increasing the sample solution temperature up to 50 °C enhanced the migration rate of the analytes into the extraction solvent and the method efficacy was increased. Also addition of sodium chloride at 2.8% (w/v) had a positive effect on the method efficiency which can be related to decreasing the analytes solubility in the sample solution. Under optimal conditions, the method showed satisfactory coefficient of determination (≥0.9979), low limit of detection (0.12-0.23 ng mL-1) and quantification (0.39-0.76 ng mL-1), acceptable repeatability in deionized water (relative standard deviation ≤ 8.2%) and in exhaled breath condensate (relative standard deviation ≤ 7.2%), and acceptable extraction recovery (75-86%) and enrichment factor (71-86). Considering these results, the developed method provided a quick and efficient way to determine surfactants in the exhaled breath condensate collected from expiratory circuit of the mechanical ventilator. It can be used in drug monitoring and clinical studies.

Development a coordination polymer based nanosensor for phenobarbital determination in exhaled breath condensate.

In this study, a fluorometric probe based on luminol-terbium coordination polymer nanoparticles (luminol-Tb CP NPs) is validated for the quantification of phenobarbital in the exhaled breath condensate collected from expiratory circuit of the mechanical ventilator (MVEBC). It relies on the coordination of phenobarbital to luminol-Tb CP NPs which casing an aggregation-induced fluorescence enhancement of probe owing to the increasing the system rigidity and the decreasing the nonradiative decay rate. As the increase in response intensity is proportional to phenobarbital amount, a method is offered for its determination in MVEBC. This method presents a linear relationship with phenobarbital concentration in the range of 0.1-10.0 mg.L-1 with a limit of detection of 0.024 mg.L-1 and the relative standard deviation of 3.6%. The proposed method is used in MVEBC of the pre-term newborn babies receiving phenobarbital under mechanical ventilator.

Exhaled breath condensate efficacy to identify mutations in patients with lung cancer: A pilot study.

Exhaled breath condensate (EBC) is used to investigate the efficacy of EBC to detect the genetic mutations in patients with lung cancer. Samples of 5 patients and 5 healthy volunteers were collected. DNA was extracted and used for amplification of hotspot regions of TP53 and KRAS genes by using PCR. We performed the mutation analysis by direct sequencing in all subjects. Detected mutations in EBC samples were compared with those of corresponding tumor tissues and there was complete agreement within the detected mutations in EBC and tumorous tissue. EBC can be used as an efficient and noninvasive source for the assessment of gene mutations in patients with lung cancer.

Breathomics: Review of Sample Collection and Analysis, Data Modeling and Clinical Applications.

Metabolomics research is rapidly gaining momentum in disease diagnosis, on top of other Omics technologies. Breathomics, as a branch of metabolomics is developing in various frontiers, for early and noninvasive monitoring of disease. This review starts with a brief introduction to metabolomics and breathomics. A number of important technical issues in exhaled breath collection and factors affecting the sampling procedures are presented. We review the recent progress in metabolomics approaches and a summary of their applications on the respiratory and non-respiratory diseases investigated by breath analysis. Recent reports on breathomics studies retrieved from Scopus and Pubmed were reviewed in this work. We conclude that analyzing breath metabolites (both volatile and nonvolatile) is valuable in disease diagnoses, and therefore believe that breathomics will turn into a promising noninvasive discipline in biomarker discovery and early disease detection in personalized medicine. The problem of wide variations in the reported metabolite concentrations from breathomics studies should be tackled by developing more accurate analytical methods and sophisticated numerical analytical alogorithms.

Simple Determination of p-Cresol in Plasma Samples Using Fluorescence Spectroscopy Technique.

The development of simple, fast, cheap and reliable analytical methods for tracing biological indicators is demanded through clinical investigations. Herein, we developed, for the first time, a cheap and specific method for the extraction and quantification of p-cresol (pC) in real plasma samples of chronic kidney disease (CKD). Plasma samples were prepared by hydrolyzing in an acidic medium to convert pCS (p-cresol sulfate) and p-Cresol glucuronide (pCG) to pC. Next, proteins of plasma samples were precipitated and then pC was extracted by acetonitrile (ACN) and saturated NaCl (as salting-out agent). Finally, fluorescence emissions were measured at λex/λem = 280/310 nm. The specificity of the method was checked by testing various possible interfering agents. The obtained results revealed a specific determination of pC. Under optimal conditions, a linear range was detected from 0.5 to 30 µg/mL of pC with a lower limit of detection (LLOQ) of 0.5 µg/mL. The reliability of the method was checked by calculating the repeatability, selectivity, and accuracy of the developed method for pC determination in plasma samples. The application of the developed method was investigated for the detection of pC in a number of CKD patients. Due to the simplicity and selectivity, the developed method could be applied for routine analysis of pC concentrations in the plasma samples of CKD patients. In addition, the developed method showed great potential for developing a point-of-care testing (POCT) device.

Development of a deep eutectic solvent-based ultrasound-assisted homogenous liquid-liquid microextraction method for simultaneous extraction of daclatasvir and sofosbuvir from urine samples.

An ultrasound-assisted homogenous liquid-liquid microextraction method using a new deep eutectic solvent was proposed for the extraction of daclatasvir and sofosbuvir from urine. The analytes were determined by high performance liquid chromatography-diode array detector. The deep eutectic solvent was prepared by mixing p-aminophenol with tetrabutyl ammonium chloride. It was used in the extraction procedure as an extraction solvent. The amine group in structure of the prepared deep eutectic solvent led to its various solubility in different pHs. In this method, urine sample was placed in a glass test tube and then mixed with sodium chloride and its temperature adjusted at 50 °C. Then, the deep eutectic solvent was dissolved in the solution by manually shaking. In the following, an ammonia solution was added to the solution and the mixture was sonicated for 4 min. After centrifugation, an aliquat of the sedimented phase was injected into the determination system. Low limits of detection (daclatasvir 1.0 and sofosbuvir 1.3 µg/L) and quantification (daclatasvir 3.3 and sofosbuvir 4.0 µg/L), high enrichment factor (daclatasvir 96 and sofosbuvir 90) and extraction recovery (daclatasvir 96 and sofosbuvir 90 %), and good percision (relative standard deviation ≤9.3 %) were obtained. The introduced method was successfully applied in the determination of daclatasvir and sofosbuvir concentrations in urine samples.

Copper nanocluster-based sensor for determination of vancomycin in exhaled breath condensate: A synchronous fluorescence spectroscopy.

A synchronous fluorescence spectroscopy (SFS) nanoprobe is developed for the determination of vancomycin in exhaled breath condensate (EBC) samples. The synthesized nanoprobe is copper nanoclusters (Cu NCs) and its SFS peak is located at 405 nm with Δλ = 80. The affinity of Cu NCs to complex formation with vancomycin results in blocking non-radiative e-/h+ recombination defect sites on the surface of NCs and consequently enhancing the SFS signal intensity. Central composite design and response surface methodology is used for the optimization of reaction conditions. Under the optimized conditions, a linear relationship is found between the SFS intensity and the concentration of vancomycin in the range of 0.1-8 µg/mL. The validated method is applied for the determination of vancomycin in EBC of newborns receiving vancomycin treatment.

Sensitive monitoring of doxorubicin in plasma of patients, MDA-MB-231 and 4T1 cell lysates using electroanalysis method.

In the present work, an innovative electrochemical sensor was fabricated based on poly toluidine blue modified glassy carbon electrode (PTB-GCE). So, PTB-GCE was used for the detection and determination of doxorubicin hydrochloride (DOX) in cell lysate, and whole human plasma samples. PTB could enhance the rate of electrochemical reaction for the electro oxidation and detection of DOX in real samples. Cyclic voltammetry (CV) technique was used for the electro polymerization of toluidine blue on the surface of GCE with the applied potential ranging from -0.6 to 0.2 V. The sensor construction steps were approved by field emission scanning electron microscopy (FE-SEM), Energy dispersive X-ray spectroscopy (EDX) and electrochemical methods. Also, CV results indicated that the DOX is oxidized via two electrons and two protons process at the optimum pH of 6.5 using PTB modified GCE. Under optimized conditions, differential pulse voltammetry (DPV) technique response exhibited linear relationship between the oxidative peak current and concentration of DOX in the range of 17 nM - 8.6 µM with low limit of quantification (LLOQ) of 17 nM for untreated and treated human plasma samples. Also, determination of DOX in MDA-MB-231 and 4T1cell lysates were performed based on its direct electrochemical oxidation on PTB-GCE. Finally, analytical validation of DOX in human bio-fluids using FDA guideline were done successfully. Results suggested that the proposed electrochemical sensor can be used to the sensitive and selective determination of DOX in biological samples. The interaction results of DOX with cancer cells indicate the developed probe can easily detect candidate drug in cancer cells with high accuracy. To the best of our knowledge this is the first report of the determination of DOX based on the direct electrochemical oxidation on PTB-GCE and determination in MDA-MB-231 and 4T1 cell lysates. It is anticipated that this research open new horizons on the design of new class of electrochemical sensors for determination drugs, and therapeutic drug monitoring (TDM) in human bio-fluids.