IMPROVING VOLATILE COMPOUNDS SAMPLING FOR ADVANCING VOLATILOME RESEARCH

VOLATILOMICS IS A BRANCH OF METABOLOMICS, FOCUSED ON THE STUDY OF THE VOLATILOME, THE COMPLETE SET OF VOLATILE ORGANIC COMPOUNDS (VOCS) PRODUCED BY LIVING ORGANISMS. AMONG OTHERS, A MAJOR CHALLENGE IN VOCS ANALYSIS ARE THEIR LARGE NUMBER, TRACE-LEVEL CONCENTRATIONS AND THE DIFFICULTY IN RETAINING VERY VOLATILE ORGANIC COMPOUNDS (VVOCS).THIS THESIS EXPLORES INNOVATIVE APPROACHES FOR SOLVING THESE CHALLENGING ASPECTS. TWO PRE-CONCENTRATION TECHNIQUES AND AN ORIGINAL SAMPLING METHODOLOGY WERE INVESTIGATED, NAMELY SOLID PHASE MICROEXTRACTION (SPME) AND THERMAL DESORPTION (TD), BOTH COUPLED TO GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS), APPLIED TO TWO DIFFERENT MATRICES, CELL CULTURES AND EXHALED BREATH. THE FIRST STUDY EMPLOYED SPME TO PROFILE THE VOLATILOME OF HUMAN MELANOMA CELLS VOLATILOME, TO CHARACTERISE THEIR UNALTERED METABOLISM AND ASSESS ALTERATIONS INDUCED BY A KNOWN STRESS INDUCER, THAPSIGARGIN (TG). EXTERNAL PARAMETER ORTHOGONALIZATION (EPO) WAS APPLIED TO REMOVE BATCH EFFECT, WHILE ANOVA-SIMULTANEOUS COMPONENT ANALYSIS (ASCA) QUANTIFIED THE CONTRIBUTION OF BOTH BATCH AND TG-TREATMENT. CLASSIFICATION ANALYSIS, USING PARTIAL LEAST SQUARES-DISCRIMINANT ANALYSIS (PLS-DA), EVALUATED CLASS SEPARATION AND VARIABLE CONTRIBUTIONS. THE MODEL, VALIDATED THROUGH REPEATED DOUBLE CROSS-VALIDATION AND PERMUTATION TESTING, ACHIEVED AN OVERALL ACCURACY ABOVE 98%. VARIABLE IMPORTANCE IN PROJECTION (VIP) SCORES IDENTIFIED 19 KEY FEATURES, WHOSE TRENDS WERE THEN EXAMINED USING A UNIVARIATE APPROACH. THE SECOND STUDY FOCUSED ON DEVELOPING A TD TUBE USING AN INNOVATIVE CLASS OF MATERIALS, ACTIVE CARBON FIBERS (ACFS), CHARACTERISED BY A VERY HIGH SPECIFIC SURFACE AREA AND SHARP PORE SIZE DISTRIBUTION. SEVERAL ACF-BASED TUBES WERE DEVELOPED USING FIVE DIFFERENT TYPES OF FIBERS (IN BOTH FABRIC AND NON-FABRIC FORMATS) AND COMPARED WITH CONVENTIONAL CARBON-BASED MULTI-BED TUBES (MBTS). PRELIMINARY TESTS USING STANDARD GAS MIXTURES WERE CONDUCTED UNDER THREE DIFFERENT EXPERIMENTAL SETUPS, COVERING BOTH DRY AND 100% RELATIVE HUMIDITY CONDITIONS. THE IMPACTS OF THE DEVELOPED WATER-REMOVAL STRATEGY AND STORAGE CONDITIONS ON ANALYTE RECOVERIES WERE ALSO EVALUATED. BASED ON THESE RESULTS, THE BEST-PERFORMING ACF MATERIAL WAS SELECTED FOR APPLICATION TO A REAL EXHALED BREATH SAMPLE FROM ONE HEALTHY VOLUNTEER, SHOWING SUPERIOR TRAPPING EFFICIENCY FOR VVOCS COMPARED WITH TRADITIONAL MBTS. TWO LARGER-SCALE APPLICATIONS WERE THEN CARRIED OUT, USING ESTABLISHED BREATH ANALYSIS PROTOCOLS: THE LACTULOSE BREATH TEST AND THE PEPPERMINT BREATH TEST. THE FIRST APPLICATION INVOLVED BREATH SAMPLING FROM 30 VOLUNTEERS, AFTER OVERNIGHT FASTING AND FOLLOWING LACTULOSE INGESTION. HYDROGEN AND METHANE WERE MEASURED ACCORDING TO THE STANDARD DIAGNOSTIC PROTOCOL TO DISTINGUISH POSITIVE FROM NEGATIVE SUBJECTS, AND THE VOC PROFILES (COLLECTED USING THE SELECTED ACF-BASED TUBE) WERE ANALYSED FOR TWO TIME POINTS (BEFORE AND AFTER LACTULOSE INTAKE). THE SECOND APPLICATION EXAMINED PEPPERMINT OIL-RELATED VOCS WASHOUT IN THREE VOLUNTEERS. HERE, ACF-BASED TUBES WERE COMPARED TO POLYMER-BASED TENAX® TUBES. ASCA CONFIRMED THE SIGNIFICANCE OF BOTH SAMPLING TIME AND ADSORBENT TYPE, WHILE UNIVARIATE ANALYSIS ALLOWED US TO INVESTIGATE THE TREND OF EACH COMPOUND. OVERALL, ACF AND TENAX® EXHIBITED HIGHLY COMPARABLE PERFORMANCE. THE THIRD PROOF-OF-CONCEPT STUDY EXAMINED A “PAIRED SAMPLING” APPROACH THAT COMPARED AMBIENT AIR WITH A HELIUM–OXYGEN MIXTURE (HELIOX) DURING EXHALED-BREATH COLLECTION. THE LOW DENSITY OF HELIUM WAS LEVERAGED TO IMPROVE THE CAPTURE OF VOCS ORIGINATING FROM ALVEOLAR GAS EXCHANGE. WHEN SAMPLES COLLECTED DURING NITROGEN/OXYGEN SYNTHETIC-AIR INHALATION WERE COMPARED, ONLY COMPOUNDS KNOWN TO BE ASSOCIATED WITH BLOOD–ALVEOLAR EXCHANGE SHOWED SIGNIFICANT INCREASES IN RELATIVE ABUNDANCE IN THE HELIOX SAMPLES, WHEREAS COMPOUNDS ORIGINATING FROM EXTERNAL SOURCES SHOWED EITHER NO SIGNIFICANT CHANGE OR A DECREASE.