Determining Urea Levels in Exhaled Breath Condensate with Minimal Preparation Steps and Classic LC-MS

Abstract

Exhaled breath condensate (EBC) provides a relatively easy, noninvasive method for measuring biomarkers of inflammation and oxidative stress in the airways. However, the levels of these biomarkers in EBC are influenced, not only by their levels in lung lining fluid but also by the volume of water vapor that also condenses during EBC collection. For this reason, the use of a biomarker of dilution has been recommended. Urea has been proposed and utilized as a promising dilution biomarker due to its even distribution throughout the body and relatively low volatility. Current EBC urea analytical methods either are not sensitive enough, necessitating large volumes of EBC, or are labor intensive, requiring a derivatization step or other pretreatment. We report here a straightforward and reliable LC-MS approach that we developed that does not require derivatization or large sample volume (∼36 mL). An Acclaim mixed-mode hydrophilic interaction chromatography column was selected because it can produce good peak symmetry and efficiently separate urea from other polar and nonpolar compounds. To achieve a high recovery rate, a slow and incomplete evaporation method was used followed by a solvent-phase exchange. Among EBC samples collected from 28 children, urea levels were found to be highly variable, with a relative standard deviation of 234%, suggesting high variability in dilution of the lung lining fluid component of EBC. The limit of detection was found to be 0.036 mg/mL.

Measurement of cortisol in blow samples collected from free‐swimming beluga whales (Delphinapterus leucas)

“…We examined the correlation between paired blow samples as absolute cortisol concentrations (i.e., pg/ml) or urea‐normalized cortisol concentrations (ng/mg urea). Since urea is thought to remain relatively stable and constant in an individual (Dwyer, 2004; Esther et al, 2009; Pitiranggon et al, 2014) and should be negligible in the surrounding environment, we expected to see a correlation between paired urea‐normalized cortisol concentrations, as urea should account for any seawater dilution.…”

“…Since urea is thought to remain relatively stable and constant in an individual (Dwyer, 2004;Esther et al, 2009;Pitiranggon et al, 2014) and should be negligible in the surrounding environment, we expected to see a correlation between paired urea-normalized cortisol concentrations, as urea should account for any seawater dilution.…”

“…Although several studies have used blow to examine endocrine function in cetaceans, one challenge of using this technique is the difficulty in quantifying the total volume of blow in a sample due to unknown amounts of seawater contamination and potential evaporation (Burgess et al, 2018). In human breath studies, urea is often used as a dilution marker as it is relatively stable and constant in the circulation of an individual (Dwyer, 2004; Esther et al, 2009; Pitiranggon et al, 2014). Burgess et al (2018) investigated the use of urea as a way to normalize hormone concentrations in blow collected from North Atlantic right whales ( Eubalaena glacialis ) and found that after using urea to normalize samples, hormone concentrations became more biologically relevant and varied depending on sex, age, and reproduction (i.e., a pregnant female had the highest concentration of progesterone; Burgess et al 2018).…”

The Development of Sensitive and Selective Potentiometric Biosensor for Urea Assay

“…A standard procedures with UV-Vis spectrophotometry is still been applied for urea where this analytical protocol is subject to interference from interfering species (Situmorang, et al, 2010). Another methods for urea determination have been introduces such as reflectometry (Ulianas, et al, 2011;Alqasaimeh, et al, 2007), colorimetry (Gaddes, et al, 2015;Zawada, et al, 2009;Sharma, et al, 2009), nuclear magnetic resonance (Liu, et al, 2012), LC-MS (Pitiranggon, et al, 2014), field effect transistor (Chang, et al, 2010;Lue, et al, 2011), acoustic wave (Tan, et al, 1998), and electrochemical methods (Ma, et al, 2016;Manea, et al, 2008;Ho, et al, 1999; Hilding-Ohlsson, et al, 2012; Das and Yoon, 2015). Most of the previous methods are operated with relatively expensive instrumentation.…”

Specific Metabolome Profile of Exhaled Breath Condensate in Patients with Shock and Respiratory Failure: A Pilot Study

“…EBC is suitable for metabolomics analysis and may be a good approach to studying lung reactions during shock or lung diseases, based on standardized EBC sample collection [ 18 , 19 ]. Although some metabolomics works have been published on this fluid, based on LC-MS, GC-MS [ 20 , 21 , 22 , 23 , 24 , 25 ] or NMR [ 26 , 27 ], such an approach has never been applied to explore shock pathophysiology.…”