Sep 02, 2025
Standardized SPME-GC-MS Protocol for the Detection of Volatile and Semi-Volatile Compounds in Human Serum
- Lilia C. Soler-Jiménez1,
- Juan Vázquez-Martínez2,
- Víctor . Vidal-Martínez1
- 1Departamento de Recursos del Mar, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV–IPN) Unidad Mérida, Carretera antigua a Progreso Km. 6, Cordemex, Mérida, Yucatán C.P. 97310, México.;
- 2Departamento de Ingeniería Química, TecNM/ITS Irapuato, Silao-Irapuato km 12.5 El Copal, 36821 Irapuato, Guanajuato.
Protocol Citation: Lilia C. Soler-Jiménez, Juan Vázquez-Martínez, Víctor . Vidal-Martínez 2025. Standardized SPME-GC-MS Protocol for the Detection of Volatile and Semi-Volatile Compounds in Human Serum. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg319mel25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2025
Last Modified: September 02, 2025
Protocol Integer ID: 224592
Keywords: Chagas disease, VOCs, SPME, GC-MS, serum, biomarker discovery, mass spectrometry, studies of disease biomarker, disease biomarker, disease biomarker discovery, gas chromatography, human blood serum, human serum, biological sample, standardized method for the extraction, standardized spme, ms protocol for the detection, using solid phase microextraction, solid phase microextraction
Abstract
This protocol describes the standardized method for the extraction and analysis of volatile and semi-volatile compounds in human blood serum using Solid Phase Microextraction (SPME) with direct fiber immersion, followed by Gas Chromatography-Mass Spectrometry (GC-MS). The method is optimized for use with a DVB/CAR/PDMS fiber and a GC-MS system equipped with an HP-5 column. It enables reproducible, non-targeted metabolic profiling for applications in disease biomarker discovery and environmental exposure assessment. The method is optimized for biological samples and can be applied to studies of disease biomarkers, such as Chagas.
Guidelines
Ethical compliance: The serum samples analyzed in this study were collected and handled under approval of the institutional ethics committee, following established ethical guidelines and with informed consent from all participants. Users intending to apply this protocol must ensure that their own sample collection complies with ethical standards, including review and approval by an Institutional Review Board (IRB) or equivalent ethics committee, and with international guidelines such as the Declaration of Helsinki.
Materials
Materials
- Human serum samples (≥500 μL each)
- 10 mL headspace vials with PTFE/silicone septa
Reagents
- Methanol (HPLC grade)
- Helium gas (99.999% purity)
- Calibration standards (optional)
Equipment
- SPME fiber: DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) (80 μm / 23 ga, Supelco or equivalent)
- GC-MS system (e.g., Agilent 7860 GC with 5977B MSD)
- GC column: HP-5MS, 30 m × 0.25 mm i.d., 0.25 μm film thickness
- Vortex mixer
- Thermostatic water bath or agitator block (40–50 °C)
- Centrifuge
- Micropipettes and tips
Equipment Setup
- Injection mode: splitless
- Injection temperature: 270 °C
- Temperature program:
o 50 °C (2 min hold)
o Ramp 8 °C/min to 100 °C
o Ramp 15 °C/min to 280 °C (4 min hold)
o Ramp 20 °C/min to 300 °C (10 min hold)
- Carrier gas: helium, 1 mL/min
- MS mode: Full scan (m/z 50–650)
Troubleshooting
Safety warnings
SAFETY INFORMATION
- Treat all human serum samples as potentially infectious material; follow institutional biosafety level 2 procedures.
- Methanol is toxic and flammable; use in a fume hood and wear PPE (lab coat, gloves, safety glasses).
- Dispose of all biological and chemical waste following institutional and national regulations.
Ethics statement
This study did not involve the use of animals. Human serum samples were collected and handled under the approval of the institutional ethics committee, in compliance with national and international ethical guidelines. All participants provided informed consent prior to sample collection.
Before start
- Ensure that all serum samples were collected with proper ethical approval (IRB or equivalent) and informed consent from participants.
- Serum samples should be stored at –80 °C until analysis. Thaw samples on ice immediately before processing to avoid degradation of volatile compounds.
- Verify that the SPME fiber (DVB/CAR/PDMS, 80/23 mm) is properly conditioned according to the manufacturer’s instructions before use.
- Confirm that the GC-MS system is calibrated, the column is installed correctly, and high-purity helium gas is available as the carrier.
- Perform all sample handling in accordance with biosafety guidelines for human-derived material.
Procedure
Thaw serum samples on ice.
In a 1.5 mL tube, mix:
o 100 µL of blood serum
o 200 µL of HPLC-grade methanol
Vortex for 30 seconds.
Let stand at room temperature for 15 minutes to allow protein precipitation.
Centrifuge at 12,500 rpm for 10 minutes.
Transfer the clear supernatant to a 0.5 mL Eppendorf tube.
Store the supernatant at 4°C if not analyzed immediately (up to 24 hours).
Precondition the DVB/CAR/PDMS fiber as per manufacturer instructions.
Introduce the DVB/CAR/PDMS SPME fiber directly into the serum supernatant.
Allow extraction to proceed for 30 minutes at room temperature.
Immediately transfer the fiber to the GC inlet for thermal desorption.
Optional: For volatile-only profiling, perform headspace extraction at 40 °C.
Desorb the fiber in the GC injector at 270 °C for 5 min in splitless mode.
Run GC-MS with the specified temperature program.
Acquire mass spectra in full scan mode (m/z 50–650).
Perform peak deconvolution.
Identify compounds using the NIST library (Salek et al 2013).
Normalize peak areas and calculate retention indices (Sun and Stremple 2003).
Compare VOC profiles between Chagas-positive and negative groups.
Troubleshooting
Critical Notes
Perform all extractions in triplicate to assess reproducibility.
Regularly clean the injector port and replace the septum to avoid carryover.
Ensure fibers are properly conditioned before use.
Store samples and extracts cold to avoid degradation.
Protocol references
Sun, G. & Stremple, P. Retention index characterization of flavor, fragrance and many other compounds on DB-1 and DB-XLB. J&W Scientific, 91 Blue Ravine Road, Folsom (2003).
Salek, R.M. et al. The role of reporting standards for metabolite annotation and identification in metabolomic studies. GigaSci 2, 13 (2013). https://doi.org/10.1186/2047-217X-2-13