24 KUBÁŇ, ĎURČ, LAČNÁ, GREGUŠ, FORET, ET AL. The use of MII-pH is, however, rather unpleasant and up to one third of subjects report discomfort. In this paper, the replacement of the above-mentioned MII-pH method with a significantly less invasive technique for analyzing EBC was attempted. EBC was collected using a specially designed miniature sampler [4, 9, 10] that was previously developed in our laboratory. EBC samples were collected from several healthy volunteers and a group of patients suffering from symptoms attributed to gastroesophageal reflux disease. Capillary electrophoresis was used to ana- lyze the ionic content of EBC samples and differences in selected ion concentrations were identified between the groups. 2. Experimental 2.1 Instrumentation An in-house-built CE instrument was used for all elec- trophoretic separations at ± 15 kV using a high voltage power supply unit (Spellman CZE2000R Start Spellman, Pulborough, UK). Two Pt wires (with an outer diameter (OD) of 0.5 mm, 3 cm in length, Advent Research Ma- terials Ltd., Eynsham, UK) were inserted into the elec- trolyte vials to serve as electrodes. Fused-silica (FS) cap- illaries (with an inner diameter (ID) of 50 µm, OD of 375 µm, 39 cm in total length, Polymicro Technologies, Phoenix, AZ, USA) were used for the separation. All CE experiments were performed at ambient temperature. A custom-made contactless conductometric detector (CCD) (Version 5.06, ADMET s.r.o., Prague, Czech Republic) operating at a frequency of 1.8432 MHz and a voltage of 50 Vp−p was used for the detection of the separated ana- lytes. Data were collected using an Orca 2800 24-bit A/D converter and ECOMAC software ver. 0.254 (ECOM spol s.r.o., Prague, Czech Republic). In all experiments, hy- drodynamic injection was applied to the samples, con- sisting of the elevation of the sample vial to a height of 10 cm for 20 s. The sampler for the collection of EBC has been de- veloped in our group previously [9]. Briefly, it was con- structed from a 2 ml syringe (B. Braun Melsungen AG, Melsungen, Germany) cooled by a 5 cm-long hollow alu- minum cylinder with an OD of 2.5 cm and an ID identical to the OD of the syringe (see Fig. 1). The cylinder was kept in dry ice at -80 °C prior to the collection of EBC and an insulation sleeve was used during its collection. A straw with an OD of 6 mm, and a wall thickness of 0.2 mm (purchased in a local store) was used to exhale the air through the sampler. The end of the syringe was enclosed with a parafilm septum to avoid loss of EBC. 2.2 Chemicals All chemicals were of reagent grade (Sigma-Aldrich, Steinheim, Germany) and deionized (DI) water (Purite, Figure 1: The photograph and scheme of the EBC sampler used in this work. Neptune, Watrex, Prague, Czech Republic) was used for the preparation of stock solutions and dilutions. Back- ground electrolytes (BGEs) for CE measurements were prepared daily by diluting 100 mM of stock solutions of 2-(N-morpholino)ethanesulfonic acid (MES), L-histidine (L-His) and 18-Crown-6 to the required concentrations. Cetyltrimethylammonium bromide (CTAB) was prepared as a 10 mM stock solution in 5 % acetonitrile. The separation electrolyte that was used for separa- tion and quantitation of inorganic anions, cations and or- ganic acids consisted of 20 mM MES, 20 mM L-His, 2 mM 18-Crown-6 and 30 µM CTAB. 2.3 Capillary-conditioning procedure Prior to its first use, the separation capillary was precon- ditioned by flushing it with 0.1 M NaOH for 30 mins., then with DI water for 30 mins. and finally with a solu- tion of BGEs for 10 mins. Between two successive injec- tions, the capillary was flushed with the BGE solution for 1 min. At the end of a working day, the capillaries were washed with DI water for 15 mins., followed by the ap- plication of a vacuum for 5 mins. before being stored dry overnight. 2.4 Sample-collection procedure The EBC was collected in the morning, the subjects did not eat or drink beforehand. The EBC was collected using the previously described EBC-sampler. The subject was asked to exhale deeply 3-5 times into the sampler, but depending on the lung capacity of each, the number of exhalations was modified to collect approximately 100 µl of their EBC. After the sampling of EBC was completed, the condensate from the walls of the syringe was forced to the tip of the syringe by the plunger and transferred to the sample vial for CE analysis. Part of the EBC sample was also used in another experiment published elsewhere, in which the pH was measured using a microelectrode. Hungarian Journal of Industry and Chemistry CE ANALYSIS OF EBC IN GERD DIAGNOSIS 25 Figure 2: Separation of a model mixture consisting of 4 in- organic anions and 4 organic acids. BGE: 20 mM MES/L- His, 2 mM 18-Crown-6, 30 µM CTAB. HV: positive +15 kV, C4D detection. Ion concentrations in the parentheses in µM: acetate (200); chloride, nitrate, lactate, propionate, butyrate (25); nitrite, sulfate (10). 2.5 Selection of patients and healthy individ- uals In this initial screening, a group of healthy individuals and a group of patients diagnosed with asthma and pul- monary fibrosis were selected. The healthy individuals had no history of GERD or heartburn and were free from other related symptoms. The healthy individuals selected were from approximately the same age group as the pa- tients, i.e., the average age of healthy subjects was 48 and that of patients was 56. The patients suffered from asthma and pulmonary fibrosis and were from the Department of Pulmonary Diseases and Tuberculosis at the Faculty Hos- pital Brno. 3. Results and Analysis 3.1 Electrolyte selection To separate the ionic content of the EBC, the BGEs composed of MES and L-His was chosen as this elec- trolyte allows for the analysis of anions, organic acids, and cations with good degrees of sensitivity. MES and L- His fulfill the criteria for suitable coions and counterions in CE separation using contactless conductivity detection (C4D). The conductance of the respective BGE compo- nents is low, thus analytes including small organic acids (acetate, lactate, butyrate) will be detected as positive peaks. The addition of 18-Crown-6 to the separation elec- trolyte improves the resolution of K+ and NH + 4 cations Figure 3: Separation of a model mixture of 5 inorganic cations. BGE: 20 mM MES/L-His, 2 mM 18-Crown-6, 30 µM CTAB. HV: negative -15 kV, C4D detection. Ion con- centrations in the parentheses in µM: ammonium (1000); calcium (25); potassium, sodium and magnesium (10). and does not influence the selectivity of other measured cations to a great extent, nor does it influence the sepa- ration of anions and organic acids. CTAB is added to the separation electrolyte to decrease the electroosmotic flow (EOF). It should be noted here that although this separation electrolyte allows for the simultaneous separation of an- ions and cations using dual-opposite end injection [11], in this work anions and cations were determined separately by switching the high-voltage polarity. The separation of a model mixture consisting of 8 anions in less than 2.5 mins. is shown in Fig. 2. The ion concentrations were selected to be similar to those found in EBC samples. The separation of cations using the same conditions but using reverse-polarity volt- age is shown in Fig. 3. The ammonium cation is usually present at a concentration 100 times greater than other cations in the EBC, its concentration was thus also in- creased to 1000 µM to reflect the expected concentration. Note that even with this significant excess the separation of NH + 4 from K + is sufficient and the BGE composition can be used for real sample analysis. 3.2 Analysis of anions in EBC samples From each subject, the EBC sample was collected as de- scribed previously. About 50 µl of the sample was trans- ferred into an Eppendorf tube and hydrodynamically in- jected into the CE system. The EBC samples were ana- lyzed first for anions and organic acids using positive HV polarity and then for cations using negative HV polarity, 46(1) pp. 23-27 (2018) 26 KUBÁŇ, ĎURČ, LAČNÁ, GREGUŠ, FORET, ET AL. Figure 4: Separation of anions in EBC samples of healthy persons as well as patients with acid reflux or weakly acid reflux. BGE: 20 mM MES/L-His, 2 mM 18-Crown-6, 30 µM CTAB. HV: positive +15 kV, C4D detection. with the HV electrode placed in the detection vial. The concentrations of all 13 ions in the samples were eval- uated using calibration curves measured with standard solutions. The parameters such as the calibration curve, regression coefficient (R2) and concentration range are shown in Table 1. In this initial screening the analysis of several samples from the healthy group and patients that were diagnosed as having the acid reflux or weakly acid reflux was attempted and the ions that would be signifi- cantly different in these groups identified. A series of electropherograms of anions and organic acids in a healthy individual, patient with acid reflux and patient with weakly acid reflux is shown in Fig. 4. One can clearly see that the concentrations of several anions, most notably chloride, nitrate and organic acids, are sig- nificantly higher in the group of patients than in that of the healthy volunteers. Some small inorganic anions such as nitrite and nitrate are often found in the EBC of patients with respiratory diseases as markers of nitrosative stress. On the other hand, the presence of an increased concen- tration of chloride and organic acids may be a significant step towards the diagnosis of GERD. 3.3 Analysis of cations in EBC samples The analysis of cations in the same samples can be seen in Fig. 5. Although higher concentrations of the ammo- nium cation were found in patients compared to healthy individuals, the content was not significantly different from that of healthy individuals, and this applied for other cations (K+, Na+, Ca2+, Mg2+) as well. Figure 5: Separation of cations in EBC samples of healthy persons as well as those with acid reflux and weakly acid reflux. BGE: 20 mM MES/L-His, 2 mM 18-Crown-6, 30 µM CTAB. HV: negative -15 kV, C4D detection. 4. Discussion A limited number of subjects were tested and the determi- nation of whether a simple EBC sample could be used as a surrogate for more invasive, expensive and tedious diag- nostic methods attempted. The analysis of anions showed some promising results with regard to the small inorganic anions (chloride and nitrate) collected and the concentra- tion of organic acids that were elevated in the patients Table 1: Calibration equations of all separated anions, cations and organic acids. BGE: 20 mM MES/L-His, 2 mM 18-Crown-6, 30 µM CTAB. HV: positive +15 kV or negative -15 kV, C4D detection. Ion Calibration equation R 2 range (µM) Cl − y = 0.0278x + 0.0258 0.9957 0.6-25 NO − 2 y = 0.0270x + 0.0023 0.9994 0.6-25 NO − 3 y = 0.0315x + 0.0166 0.9987 0.6-25 SO 2− 4 y = 0.0656x + 0.0143 0.9980 0.25-10 acetate y = 0.0156x + 0.0200 0.9995 1-200 lactate y = 0.0207x + 0.0097 0.9996 0.6-25 propionate y = 0.0189x + 0.0024 0.9983 0.6-25 butyrate y = 0.0188x − 0.0001 0.9990 0.6-25 NH + 4 y = 0.0211x − 0.1437 0.9993 25-1000 K + y = 0.0347x + 0.0013 0.9994 0.25-10 Ca 2+ y = 0.0528x + 0.0059 0.9995 0.6-25 Na + y = 0.0356x + 0.0079 0.9996 0.25-10 Mg 2+ y = 0.0618x − 0.0054 0.9995 0.25-10 Hungarian Journal of Industry and Chemistry CE ANALYSIS OF EBC IN GERD DIAGNOSIS 27 suffering from GERD symptoms, compared to healthy volunteers. Unfortunately, with regard to the analysis of cations, the differences were not significant and cations were deemed unsuitable. 5. Conclusion Capillary electrophoretic analysis of EBC was used for the first time in an attempt to distinguish the groups of patients with GERD from healthy individuals. CE with C4D was used for the analysis of small inorganic anions, cations and organic acids present in the EBC samples. Although it has been shown that selected samples had elevated concentrations of chloride, nitrate, butyrate and propionate, the number of subjects was too small to draw definite conclusions concerning the discriminatory power of these ions. It also seems that although there is a dif- ference between groups of healthy individuals and suf- ferers of acid reflux/weakly acid reflux, the results with regard to acid reflux and weakly acid reflux are very sim- ilar and these two groups cannot be distinguished based on the current data. A larger scale clinical study, in which the EBC of patients and healthy individuals is scrutinized with regard to the ion content, pH and concentration of other biomarkers (for instance pepsin in saliva) to obtain statistically significant results for evaluation is currently being undertaken. Nevertheless, the initial data is some- what promising for EBC as a useful non-invasive alterna- tive for other methods used in GERD diagnostics. Symbols BGE background electrolyte CE capillary electrophoresis C4D contactless conductivity detection CTAB cetyltrimethylammonium bromide DI deionized EBC exhaled breath condensate GERD gastroesophageal reflux disease L-His L-histidine ID inner diameter MES 2-(N-morpholino)ethanesulfonic acid OD outer diameter Acknowledgement This research was supported by grant no. 17-31945A from the Ministry of Health of the Czech Republic. 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A, 2002 964, 227–241 DOI: 10.1016/S0021-9673(02)00656-8 46(1) pp. 23-27 (2018) Introduction Experimental Instrumentation Chemicals Capillary-conditioning procedure Sample-collection procedure Selection of patients and healthy individuals Results and Analysis Electrolyte selection Analysis of anions in EBC samples Analysis of cations in EBC samples Discussion Conclusion