Rethinking Solid-Phase Microextraction (SPME) for Bioanalysis: Workflow Optimisation in Automated Vacuum-Assisted Cooling SPME for Fatty Acid Monitoring in Faecal Waters
7 affiliations
- 1Unit of Microbiomics and Research Unit of Microbiome, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy
- 2Department of Life Sciences, Health and Health Professions, Link Campus University, 00165 Rome, Italy
- 3Department of Experimental and Clinical Medicine, University of Florence, 50121 Florence, Italy
- 4Division of Occupational Medicine, Careggi University Hospital, 50134 Florence, Italy
- 5Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy
- 6Research Unit of Microbiome, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy
- 7Research Unit of Genetics of Complex Phenotypes, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy
Abstract
Short-chain fatty acids (SCFAs) are key biomarkers of gut microbiota and intestinal health, yet their analysis is challenging due to high volatility, hydrophilicity, and the complexity of the faecal matrix. This study introduces a novel, green, and fully automated workflow for monitoring SCFAs in faecal water samples. It combines microwave-assisted derivatisation, vacuum-assisted cooling multiple solid-phase microextraction (Vac-HS-cooling-MSPME), and fast gas chromatography–mass spectrometry (GC-MS). On-sample derivatisation with 2,3,4,5,6-pentafluorobenzyl bromide was optimised via design of experiments, reducing reaction time from 3 h to just 2 min at 100 °C while preserving high yields and selectivity. Integration of Vac-HS-cooling-MSPME with a fast GC column (5 m × 0.1 mm ID × 0.4 µm) increased sample throughput from two to approximately six samples per hour, with total chromatographic times of 5.24 min. MSPME specifically improved sensitivity for low-response analytes such as pentafluorobenzyl formate. The method demonstrated excellent linearity (R2 > 0.998), low limits of detection (0.002–0.09 mg/L), good intra- and inter-day precision, accuracies exceeding 85%, and consistent recovery for each analyte (86–90%). The AGREE greenness score improved from 0.65 to 0.68, confirming alignment with green analytical chemistry principles. This system offers a high-throughput, sensitive, and eco-friendly tool for SCFA quantification, confirmed by the application on real paediatric faecal water samples.
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BibTeX
@article{putignani2026rethinking,
title = {{Rethinking Solid-Phase Microextraction (SPME) for Bioanalysis: Workflow Optimisation in Automated Vacuum-Assisted Cooling SPME for Fatty Acid Monitoring in Faecal Waters}},
author = {Lorenza Putignani and Stefano Dugheri and Niccolò Fanfani and Giovanni Cappelli and Ilaria Rapi and Michele Santillo and Giorgio Marrubini and Chiara Marangelo and Pamela Vernocchi and Federica Del Chierico and Maria Rita Braghini and Giulia Andolina and Anna Alisi and Antonio Baldassarre and Nicola Mucci},
journal = {Molecules},
year = {2026},
month = oct,
doi = {10.3390/molecules31203716},
url = {https://www.mdpi.com/1420-3049/31/20/3716},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Rethinking Solid-Phase Microextraction (SPME) for Bioanalysis: Workflow Optimisation in Automated Vacuum-Assisted Cooling SPME for Fatty Acid Monitoring in Faecal Waters
AU - Lorenza Putignani
AU - Stefano Dugheri
AU - Niccolò Fanfani
AU - Giovanni Cappelli
AU - Ilaria Rapi
AU - Michele Santillo
AU - Giorgio Marrubini
AU - Chiara Marangelo
AU - Pamela Vernocchi
AU - Federica Del Chierico
AU - Maria Rita Braghini
AU - Giulia Andolina
AU - Anna Alisi
AU - Antonio Baldassarre
AU - Nicola Mucci
PY - 2026
DA - 2026/10/01
JO - Molecules
DO - 10.3390/molecules31203716
UR - https://www.mdpi.com/1420-3049/31/20/3716
AB - Short-chain fatty acids (SCFAs) are key biomarkers of gut microbiota and intestinal health, yet their analysis is challenging due to high volatility, hydrophilicity, and the complexity of the faecal matrix. This study introduces a novel, green, and fully automated workflow for monitoring SCFAs in faecal water samples. It combines microwave-assisted derivatisation, vacuum-assisted cooling multiple solid-phase microextraction (Vac-HS-cooling-MSPME), and fast gas chromatography–mass spectrometry (GC-MS). On-sample derivatisation with 2,3,4,5,6-pentafluorobenzyl bromide was optimised via design of experiments, reducing reaction time from 3 h to just 2 min at 100 °C while preserving high yields and selectivity. Integration of Vac-HS-cooling-MSPME with a fast GC column (5 m × 0.1 mm ID × 0.4 µm) increased sample throughput from two to approximately six samples per hour, with total chromatographic times of 5.24 min. MSPME specifically improved sensitivity for low-response analytes such as pentafluorobenzyl formate. The method demonstrated excellent linearity (R2 > 0.998), low limits of detection (0.002–0.09 mg/L), good intra- and inter-day precision, accuracies exceeding 85%, and consistent recovery for each analyte (86–90%). The AGREE greenness score improved from 0.65 to 0.68, confirming alignment with green analytical chemistry principles. This system offers a high-throughput, sensitive, and eco-friendly tool for SCFA quantification, confirmed by the application on real paediatric faecal water samples.
SN - 1420-3049
ER -