**7. Experimental part**

#### **7.1. Analytical method for multi-marker screening**

The following analytical methods combined with various pre-treatment methods are currently referenced in the literature for the determination of biomarkers present in EBC: HPLC-MS, GC-MS and EIA (ELISA). Based on validation parameters (e.g., accuracy, precision, limit of quantification (LOQ), limit of detection (LOD), linearity, selectivity, etc.) the methods described above can be compared.

LC-MS method in a highly selective and accurate SRM mode affords both quantitative and qualitative information about the monitored biomarkers and today seems to be method of the first choice. Liquid chromatography can be used in UHPLC, which is characterized by the fact that the separation of substances occurs at higher flow rate of the mobile phase (1 mL/min) on LC columns with smaller average particle size of the stationary phase (diameter of particles <2 μm) and by shortening the time of LC-MS analysis. When using the so-called "stableisotope-dilution assay," the accuracy and precision of the LC-MS method can be increased by suitable deuterated internal standards. However, the main disadvantage of the LC-MS analysis is the inclusion of the pre-treatment step (SPE, immunoaffinity extraction, etc.), when the EBC sample is recommended to exclude a contact to room temperature, ideally temperature above 0°C. This problem can be prevented by using the 2D technology for liquid chromatography. In the first dimension, an on-line SPE is carried out and the subsequent dimension uses the UHPLC. For detection of selected biomarkers, 2D UHPLC-MS method was developed and because of the sensitivity of biomarkers mentioned above, it is highly recommended.

Analysis of substances were realized on the LC-MS system consisting of quaternary pump and mass spectrometer operating on the principle of triple quadrupole equipped with electrospray ionization (ESI). To implement multimarker screening, it was necessary to carry out two types of analyses. The first one were determined substances containing amino group in its structure. The second one serves to determine substances with aldehyde and carboxylic groups. These two analyses were necessary because of the different conditions of derivatization reactions (acid vs. alkaline environment) and the resulting liquid chromatography at different conditions (different composition of the mobile phase used on different chromatographic columns).

#### *7.1.1. Determination of the amino compounds*

in case of patients suffering from bronchial asthma. The results of another conducted study

The majority of pulmonary diseases is also characterized by alternations in the protein profile of the patients. Many of these changes are measurable in EBC and can be used for monitoring of pathological process occurring (mainly) in the respiratory tract. The changes in the structure and concentration levels of various proteins have recently become a popular and reliable tool for monitoring of the process and molecular alterations in lungs and airways. Based on the proteomic analysis of EBC, 44 unique proteins [45, 46] have been detected so far. Many of these proteins might become steady biomarkers of inflammation or oxidative stress, when scanning of the differences between the proteome profiles of healthy control subjects and subjects with various pulmonary diseases may represent a significant shift toward detecting

Serotonin (5-hydroxytryptamin (5-HT)) is a neurotransmitter that is predominantly located in central nervous system and gastrointestinal tract (GIT). In GIT, 5-HT regulates bowel movements. In CNS, it is responsible for the regulation of mood, sleep, muscle contraction, and some cognitive functions (involving memory and learning abilities). It is also present in thrombocytes, where it is involved in the regulation of homeostasis and coagulation [47].

5-HT plays a significant role in many pathological and neuropsychiatric diseases [47, 48]. The serotonergic substances are also important in pharmacology. The genes that code various components of 5-HT system are the subject of the study as they could be factors of depression, schizophrenia, obsessive–compulsive disorder, aggression, alcoholism, migraine, and autism [49].

The following analytical methods combined with various pre-treatment methods are currently referenced in the literature for the determination of biomarkers present in EBC: HPLC-MS, GC-MS and EIA (ELISA). Based on validation parameters (e.g., accuracy, precision, limit of quantification (LOQ), limit of detection (LOD), linearity, selectivity, etc.) the

LC-MS method in a highly selective and accurate SRM mode affords both quantitative and qualitative information about the monitored biomarkers and today seems to be method of the first choice. Liquid chromatography can be used in UHPLC, which is characterized by the fact that the separation of substances occurs at higher flow rate of the mobile phase (1 mL/min) on LC columns with smaller average particle size of the stationary phase (diameter of particles

showed that significantly increased levels of GSSG occur in EBC of alcoholics [44].

**6.5. Other molecules determined in EBC**

154 Biomarker - Indicator of Abnormal Physiological Process

new prognostic and/or diagnostic biomarkers.

**7.1. Analytical method for multi-marker screening**

methods described above can be compared.

*6.5.1. Proteins and metabolites*

*6.5.2. Serotonin*

**7. Experimental part**

For the derivatization of compounds containing an amino group in its structure (*o*-tyrosine (*o*-Tyr), 3-nitrotyrosine (3-NO2 -Tyr), 3-chlorotyrosine (3-Cl-Tyr), hydroxyguanosin (8-OHG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were used as derivatization reagent 3-aminopyridyl-N-hydroxysukcinimidyl carbamate (= APDS). To 500 μl of the EBC sample-containing deuterium labeled analyte analogues was added to 450 μl of borate buffer (pH 8.5) and 50 μl of APDS derivatization agent (concentration of 1 mg/ml of acetonitrile). Derivatization reactions were carried out for 10 min at 4°C. Thus prepared sample was subjected to LC-ESI-MS/MS analysis on chromatographic column XTerra MS (C18 50 × 1 mm × 3.5 μm) (Waters, Republic of Ireland). The substances were subjected to analysis where isocratic elution method with a mobile phase composed of acetonitrile: water (60:40—v/v) (water = 0.1% formic acid) was used. The column was tempered to 25°C. Mobile phase flow rate was 150 μl/min. The volume of the analyzed samples was 10 μl. Mass spectrometer parameters were optimized to the following values: capillary voltage −2500 V, the inlet capillary temperature 300°C, the temperature of the evaporator HESI 300°C, sheath gas (nitrogen) pressure 45 psi, auxiliary gas (nitrogen) 10 ArbU. Measurement parameters were optimized for use in neutral loss mode in the interval 250–500 Da (Q1) → 130– 380 Da (Q3) with CID energy 15 eV in the negative electrospray ionization (ESI-).

#### *7.1.2. Determination of aldehydes and carboxylic acids*

Derivatization of aldehydes (n-aliphatic aldehydes (C6–C12), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal (4-HHE) and substances with a carboxyl group in its structure 8-isoprostane (8-iso-PGF2α), cys-LTs, LTB<sup>4</sup> was carried out using derivatization with Girard's reagent T (GirT) in the presence of N-(3-dimethylaminopropyl)- N′-ethylkarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide. To the sample containing 100 μl of EBC with deuterium-labeled internal standards were added 10 μl of each derivatization reagent GirT (c = 100 μl/ml), reagent EDC together with 10 μl of sulfo-Nhydroxysuccinimide, 10 μl of 1% hydrochloric acid and 270 μl of propan-2-ol. Derivatization proceeded for 30 minutes and in such way prepared sample was immediately analyzed by LC-ESI-MS/MS. Chromatographic column used was a Thermo Hypercarb (100 × 21 mm × 5 μm) with pre-column Hypercarb (Thermo Electron Corporation, USA). For separation of substances, was used the isocratic elution method with a mobile phase composed of methanol: water (40:60—v/v) (pH adjusted with ammonium hydroxide to 9). Flow rate of mobile phase was 150 μl/min. Chromatographic column temperature was 30°C and the sample volume was 10 μl. Mass spectrometer parameters were optimized to the following values: capillary voltage 3000 V, capillary inlet temperature 300°C; HESI evaporator temperature 300°C, sheath gas (nitrogen) pressure 45 psi and auxiliary gas (nitrogen) 10 ArbU. Measurement parameters were optimized for use in neutral loss mode in the interval 150–750 Da (Q1) → 91–691 Da (Q3) with CID energy − 16.5 eV in the positive electrospray ionization (ESI+).

**a.** Severe refractory asthma.

**c.** Moderate persistent asthma.

control group were involved.

**e.** Non-asthmatics—healthy control subjects.

**8.2. Monitoring of efficacy of the used pharmacotherapy**

The developed method was used in a parallel study. The study was conducted to prove whether the method could be applied for monitoring of efficacy of the used pharmacotherapy. In this case, *per oral* and inhaled glucocorticoid treatments have been compared. Results of the study are present in **Figure 9**. In the clinical study of 35 patients with *per oral* glucocorticoid therapy, 35 patients with inhaled glucocorticoid therapy and 32 people from the healthy

Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate

http://dx.doi.org/10.5772/intechopen.74402

157

From the results, it is obvious that the PCA analysis divided the subjects into three groups. The first group contained only healthy control subjects; however, the two remaining have

**Figure 8.** Statistically evaluated clinical results: levels of cys-LTs and LXs in different asthma phenotypes.

**b.** Severe asthma.

**d.** Mild asthma.
