title: Optimisation and validation of a HS-SPME-GC-IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: Application in a pilot study to discriminate individuals with smoking habits.
abstract: A new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: isabel_135@hotmail.com.
authors.forename: Isabel
authors.initials: I
authors.lastname: Calejo
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; Portuguese Catholic University, CBQF-Center for Biotechnology and Fine Chemistry, School of Biotechnology, Rua Arquiteto Lobão Vital, 4202-401 Porto, Portugal.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Moreira
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Ana Margarida
authors.initials: AM
authors.lastname: Araújo
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; FP-ENAS, CEBIMED, Fundação Ensino e Cultura Fernando Pessoa, Universidade Fernando Pessoa, Porto, Portugal.
authors.forename: Márcia
authors.initials: M
authors.lastname: Carvalho
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Maria de Lourdes
authors.initials: Mde L
authors.lastname: Bastos
authors.affiliation: UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: pguedes@ff.up.pt.
authors.forename: Paula Guedes
authors.initials: PG
authors.lastname: de Pinho
mesh_terms: D000328:Adult; D000447:Aldehydes / Q000652:urine; D015415:Biomarkers / Q000652:urine; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000592:standards*; D006801:Humans; D006898:Hydroxylamines / Q000652:urine; D007659:Ketones / Q000652:urine; D008297:Male; D008875:Middle Aged; D010865:Pilot Projects; D012907:Smoking / Q000652:urine*; D052617:Solid Phase Microextraction / Q000592:standards*; D055549:Volatile Organic Compounds / Q000652:urine*; D055815:Young Adult; D005466:Fluorocarbons
chemical_list: D000447:Aldehydes; D015415:Biomarkers; D006898:Hydroxylamines; D007659:Ketones; D055549:Volatile Organic Compounds; C008664:nonanal; C547093:2-methylbutanal; C010277:Florox Reagent; D005466:Fluorocarbons; C038029:4-heptanone; C017439:isobutyraldehyde; C027576:4-hydroxy-2-nonenal
keywords: Carbonyl compounds; Central Composite Design (CCD); HS-SPME–GC–IT/MS method; Smoking habits; Urinary biomarkers