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.

A colorimetric nanoprobe based on dynamic aggregation of SDS-capped silver nanoparticles for tobramycin determination in exhaled breath condensate.

A colorimetric nanoprobe was developed for the quantification of tobramycin in exhaled breath condensate (EBC). The nanoprobe consists of silver nanoparticles (Ag NPs) modified with sodium dodecyl sulfate (SDS), which is applied in the presence of sodium metaborate. Characterization of the synthesized SDS-capped Ag NPs by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX) showed that the nanoparticles were well synthesized with nearly uniform size and an average diameter of < 30 nm. Interaction of sodium metaborate with the SDS-capped Ag NPs and tobramycin results in aggregation of the nanoparticles and consequently decreases the absorbance intensity, leading to the production of a new absorbance peak and a color change from yellow to purple. The absorbance intensity was recorded at λmax = 400 nm and 522 nm and λ522/λ400 was used as the analytical signal. The experimental parameters were investigated and optimized using a multivariate optimization method (central composite design). The current nanoprobe gives a linear response for tobramycin from 1.0 to 50.0 ng mL-1 with a detection limit of 0.5 ng mL-1. The intra- and inter-day relative standard deviations for five replicated analyses of 10.0 ng mL-1 tobramycin are 2.8% and 4.2%, respectively. Graphical abstractSchematic representation of SDS-capped silver nanoparticles's response to tobramycin in the presence of sodium metaborate.

In-situ microscale spectrophotometric determination of phenytoin by using branched gold nanoparticles.

A rapid method for the sensitive detection of phenytoin (PHT) by branched gold nanoparticles (B-AuNPs) is described. These nanoparticles were synthesized by adding methanol as the reducing agent and poly(ethylene glycol) as the stabilizer at 70 °C. The B-AuNPs are red in color with an absorption maximum at 540 nm when prepared in situ. However, the color becomes increasingly weaker when PHT is present in increasing concentrations. This method can determine PHT over the 67-670 ng·mL-1 concentration range, with detection limit of 21 ng·mL-1. The relative standard deviation for five replicate measurements at 68 and 530 ng·mL-1 of PHT was 3.2% and 1.2%, respectively. The method was applied to the determination of PHT in plasma samples of epileptic patients, and the results were in agreement with those obtained by a standard official method. Graphical abstract Branched gold nanoparticles (AuNPs) prepared in situ have a red color with an absorption maximum at 540 nm. The color becomes increasingly weaker with decreasing the intensity of the characteristic SPR band when PHT is present in increasing concentration. The current assay is capable of determining PHT over the 67-670 ng·mL-1 concentration range with a limit of detection of 21 ng·mL-1.

A single-shot diagnostic platform based on copper nanoclusters coated with cetyl trimethylammonium bromide for determination of carbamazepine in exhaled breath condensate.

A fluorescent nanoprobe is designed for the determination of carbamazepine (CBZ) in exhaled breath condensate (EBC) of patients receiving CBZ. The probe consists of copper nanoclusters (Cu NCs) coated with cetyl trimethylammonium bromide. The interaction of probe with CBZ results in blocking non-radiative e-/h+ recombination defect sites on the surface of Cu NCs and consequently enhancing the blue-green fluorescence of Cu NCs (excitation/emission wavelengths: 290/480 nm). The experimental conditions were optimized using a response surface methodology (central composite design). Under the optimized conditions, the calibration plot is linear in the 0.2 to 20 µg mL-1 CBZ concentration range and the detection limit is as low as 0.08 µg mL-1. The intra-day and inter-day relative standard deviations for six replicated measurements of 10 µg mL-1 CBZ are 3.9% and 4.8%, respectively. The method was applied for the determination of CBZ level in EBC of patients receiving CBZ. The accuracy of the method was confirmed by HPLC-UV analysis as a reference method. Graphical abstract Graphical abstract contains poor quality and small text inside the artwork. Please do not re-use the file that we have rejected or attempt to increase its resolution and re-save. It is originally poor, therefore, increasing the resolution will not solve the quality problem. We suggest that you provide us the original format. We prefer replacement figures containing vector/editable objects rather than embedded images. Preferred file formats are eps, ai, tiff and pdf. "Figures 1 contains poor quality of text inside the artwork. Please do not re-use the file that we have rejected or attempt to increase its resolution and re-save. It is originally poor, therefore, increasing the resolution will not solve the quality problem. We suggest that you provide us the original format. We prefer replacement figures containing vector/editable objects rather than embedded images. Preferred file formats are eps, ai, tiff and pdf.A new PDF format of Graphical Abstract was provided in attachment section. Schematic presentation of cetyl trimethylammonium bromide coated copper nanocluster's response to carbamazepine.

Non-volatile compounds in exhaled breath condensate: review of methodological aspects.

In contrast to bronchial and nasal lavages, the analysis of exhaled breath condensate (EBC) is a promising, simple, non-invasive, repeatable, and diagnostic method for studying the composition of airway lining fluid with the potential to assess lung inflammation, exacerbations, and disease severity, and to monitor the effectiveness of treatment regimens. Recent investigations have revealed the potential applications of EBC analysis in systemic diseases. In this review, we highlight the analytical studies conducted on non-volatile compounds/biomarkers in EBC. In contrast to other related articles, this review is classified on the basis of analytical techniques and includes almost all the applied methods and their methodological limitations for quantification of non-volatile compounds in EBC samples, providing a guideline for further researches. The studies were identified by searching the SCOPUS database with the keywords "biomarkers," "non-volatile compounds," "determination method," and "EBC."

Extraction and Analysis of Methadone in Exhaled Breath Condensate Using a Validated LC-UV Method.

PURPOSE: A combined microextraction and separation method is presented for the determination of methadone in exhaled breath condensate (EBC) which is a promising non-invasive biological component for monitoring drug concentrations. METHODS: In this work, dispersive liquid-liquid microextraction (DLLME) and ultrasonic liquid-liquid microextraction (ULLME) procedure coupled with a validated liquid chromatography method were used for analysis of methadone in EBC collected using an in-house cold trap setup. The method has been validated according to the FDA guidelines using EBC-spiked samples and tested on a number of EBC samples collected from patients. RESULTS: The best DLLME conditions involved the use of a disperser solvent of methanol (1 mL), extraction solvent of chloroform (200 mL), EBC sample pH of 10.0 and centrifugation at 6000 rpm for 5 minutes. The conditions for ULLME were 150 mL of chloroform and the samples were sonicated for 4 minutes. The method was validated over the concentration range of 0.5-10 mg/L-1 in EBC. Inter- and intra-day precision and accuracy were less than 5 % where the acceptable levels are less than 20%. Furthermore, the validated method was successfully applied for the determination of methadone in patients' EBC samples. CONCLUSIONS: The outcomes indicate that the developed LC-UV combined with DLLME and/or ULLME extraction methods can be employed for the extraction and separation of methadone in EBC samples.

Quantification of morphine in exhaled breath condensate using a double network polymeric hybrid hydrogel functionalized with AuNPs.

BACKGROUND: Morphine serves as a foundation for creating other opioid derivatives, such as hydro/oxymorphine and heroin, which possess enhanced pain-relieving properties but are also prone to addiction and abuse. In cases of morphine overdose, it not only affects multiple immune functions but can also cause severe health complications. Given these concerns and the widespread use of morphine, it is crucial to develop efficient, uncomplicated, and precise methods for accurately detecting morphine in various biological and pharmaceutical samples. RESULTS: In this investigation, a novel gold nanoparticle (AuNPs)-based double network hydrogel (DNH) nanoprobe has been fabricated for sensitive quantification of morphine in exhaled breath condensate samples. For that, gelatin/agarose DNH was fabricated through a one-step heating-cooling method in the presence of AuNPs, providing not only chemical stability but also prevent the AuNPs aggregation during synthesis process. In this method, the absorbance intensity of the nanoprobe gradually decreased with increasing morphine concentration due to the interaction morphine with AuNPs surface plasmon. The aggregation of AuNPs by addition of morphine was verified by UV-Vis spectrophotometry. The sensor displayed high sensitivity with detection limit of 0.006 µg.mL-1 in the linear range from 0.01 to 1.0 µg.mL-1. A reliable performance was attained for the spectrophotometric method for determination of morphine in the real samples.

Design and development of metal-organic framework-based nanocomposite hydrogels for quantification of deferiprone in exhaled breath condensate.

In this study, a novel fluorescence nanoprobe based on Materials of Institute Lavoisier (MIL-101) metal-organic frameworks embedding into the agarose hydrogel is fabricated using a hydrothermal technique. It uses for sensitive quantification of deferiprone in exhaled breath condensate (EBC) samples. The morphology and characterization of MIL-101/agarose nanocomposite hydrogel is studied by transmission electron microscopy, dynamic light scattering instrument, powder X-ray diffraction analysis, and Fourier transform infrared spectroscopy. The probe shows a reasonable fluorescence intensity quenching in the presence of deferiprone due to the interactions between iron centers in MIL-101 (Fe) and deferiprone, which likely form non-fluorescent complexes. The proposed nanoprobe demonstrates a linear calibration curve from 0.005 to 1.5 µg mL- 1 with a detection limit of 0.003 µg mL- 1. The intra- and inter-day precision of the reported method are 0.3% and 0.4% (n = 5, deferiprone concentration = 1.0 µg mL- 1), respectively. This method demonstrates high sensitivity and specificity towards deferiprone in the EBC samples and also presents a sensing platform with simplicity, convenience, fast implementation, and cost-effective in medical monitoring.

Development of a nanocomposite hydrogel catalyzed H2O2/TMB system for determination of chlordiazepoxide in exhaled breath condensate.

In this study, an enzyme mimic catalyzed H2O2-tetramethylbenzidine system based on UiO-66/Au NPs-PVA nanocomposite hydrogel was employed as an optical probe for chlordiazepoxide sensing. An excellent detection limit of 0.0032 µg mL-1 with a linear range of 0.005-2.0 µg mL-1 was obtained for chlordiazepoxide in exhaled breath condensate samples under optimal conditions. The validated system showed good repeatability, simplicity, and stability toward chlordiazepoxide sensing in the exhaled breath condensate of patients receiving this drug.

Utilizing nitrogen, sulfur, phosphorus, and chlorine co-doped carbon dots as a fluorescent probe for determination of vancomycin in exhaled breath condensate.

Vancomycin is employed to treat infections caused by gram-positive bacteria. Ensuring precise vancomycin dosages is essential to avoid the emergence of bacterial resistance. In the current study, a fluorescent nanoprobe was designed for vancomycin determination in exhaled breath condensate samples. The nanoprobe was based on carbon dots (CDs) doped with nitrogen, sulfur, phosphorus, and chlorine (NSPCl-doped CDs). Vancomycin significantly reduced the fluorescence of NSPCl-doped CDs and presented a quenching process in the analytical response of the probe within a concentration range of 0.01-2.0 µg mL-1 due to forming a non-fluorescent complex. The nanoprobe's intra-day and inter-day relative standard deviations were 1.4 % and 3.2 %, respectively. This nanoprobe was successfully used to determine vancomycin in the patients receiving this drug collected from the expiratory circuit of the mechanical ventilator.

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.

A cross-sectional study on metoprolol concentrations in various biological samples and their inter-correlations.

BACKGROUND: Concentrations of metoprolol in exhaled breath condensate (EBC) have not been investigated. Herein, we aim to determine the metoprolol levels in EBC, plasma, and urine samples. METHODS: Biological samples were collected from 39 patients receiving metoprolol. Metoprolol was determined using liquid chromatography mass spectrometery. The obtained metoprolol levels in biological fluids were investigated for possible inter-correlations. RESULTS: Acceptable linearity was obtained with coefficient of determinations equal to 0.9998, 0.9941, and 0.9963 for EBC, plasma, and urine samples, respectively. The calibration curves were linear in the ranges of 0.6-500, 0.4-500, and 0.7-10,000 µg·L- 1 regarding EBC, plasma, and urine samples, respectively. The detection and quantification limits were (0.18, 0.12, and 0.21 µg·L- 1) and (0.60, 0.40, and 0.70 µg·L- 1) for EBC, plasma, and urine samples, respectively. The relative standard deviations for the intra- and inter-day replications were obtained between 5.2 and 6.1 and 3.3-4.6%, respectively. The obtained mean metoprolol levels in EBC, plasma, and urine samples of 39 patients were 5.35, 70.76, and 1943.1 µg·L- 1. There were correlations between daily dose and plasma and urinary concentrations of metoprolol in the investigated samples, whereas no significant correlation was observed for daily dose and EBC levels. The correlation among plasma-urine levels was significant, however, the non-significant correlation was obtained between plasma and EBC concentrations. CONCLUSION: Metoprolol levels varied widely due to the metabolic pattern of the Azeri population, different dosages received by the patients, formulation effects, age, sex, and interactions with the co-administered drugs. A poor correlation of EBC-plasma concentrations and a significant correlation of plasma-urine concentrations were observed. Further investigations are required to provide the updated services to personalized medicine departments.

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.

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.

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.

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.

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.

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.