Fiber–Sample Distance, An Important Parameter To Be Considered in Headspace Solid-Phase Microextraction Applications

To define and control the parameters which impact headspace solid-phase microextraction (HS-SPME), it is important to reach the highest level of reproducibility. The present study aims to assess, for the first time, the effect of fiber–sample distance during HS-SPME in pre-equilibrium conditions. Analyses were primarily performed on mixtures of standard volatiles compounds (alkanes, alcohols, organic acids) designed in our lab and then on various food matrices (wine, chicken, cheese, tea), repeating already published experiments. Extractions were performed varying fiber penetration depths (10–60 mm) at different times (10–60 min) and temperatures of extraction (30–80 °C). The study revealed that variation of the distance between the fiber and the sample into the vial clearly impacts the results obtained during HS-SPME when conditions are such that no equilibrium is reached in HS. For example, in wine analysis, the percentage of octanoic acid at 80 °C was higher at 40 mm (7.5 ± 0.2%) than that at 20 mm (4.4 ± 0.3%). Moreover, regardless of the extraction temperature, the lower the time of extraction, the stronger the dependence on the fiber–sample distance. Indeed, at 60 °C, the obtained response factors for octadecane at 20 and 40 mm of fiber penetration were 21.8 and 44.5, respectively, after 10 min of extraction, 54.1 and 71.0 after 30 min, and 79.4 and 82.4 after 60 min of extraction. The analyses have been here corroborated by a theoretical model based on the diffusion equation. Therefore, to improve the method robustness during HS-SPME studies, we suggest specifying the fiber penetration depth or the fiber–sample distance with the other parameters of extraction.


Table of contents:
 Materials and methods:  Chemicals and reagents  HS-SPME experimental conditions  Gas chromatography-mass spectrometry (GC-MS) conditions  Statistical analyses.  Tables and Figure:  Table S1  Table S2  Table S3  Table S4  Table S5  Table S6  Table S7  Figure S1  Figure S2 S-3 either the depth of penetration of the fiber, which can be given with precision by an autosampler, or to precise the distance between the fiber tip and the top/surface of the sample (fiber-sample distance), which can be given by measuring this distance when extraction is performed manually.

Gas chromatography-mass spectrometry (GC-MS) conditions
GC-MS analyses were carried-out on an Agilent 7890B gas chromatograph system equipped with an autosampler (PAL RSI 85) and coupled to an Agilent 5977B mass selective detector (Santa Clara, California, USA). The carrier gas used was Helium, at a flow rate of 1.2 mL min -1 . After extraction, the fiber was desorbed in an injection port equipped with a liner (800 µL) at a temperature of 260°C under splitless mode. The inlet penetration depth was 40 mm, at a speed of 100 mm s -1 . The MSD transfer line was set at a temperature of 260°C. The MS source and quadrupole temperatures were 230 and 150°C, respectively. The gain factor was set at 0.05, while the resulting electron multiplier (EM) voltages were lower than 1300 V. All VOCs separation was performed using a DB -Wax column (60 m, 0.25 mm i.d., 0.25 μm film thickness) (J&W Scientific, Folsom, CA, USA) following different oven temperature program gradients.
For the analyses of alkanes, the oven was maintained at 50°C for 4 min, then raised to 325°C at a rate of 15°C min -1 , and held at 325°C for 4 min. Data were acquired in scan ion mode with a scan range of 29-400 m/z. For the analyses of the 6 VOCs, the oven temperature was kept at 35°C for 4 min, then raised to 120°C at a rate of 5°C min -1 , to 250°C at a rate of 16°C min -1 and held at 250°C for 1 min. For FFAs standards analyses, the initial oven temperature was 50°C for 3 min and increased at a rate of 5°C min -1 up to 150°C, held 1 min and then increased until 250°C at a rate of 10°C min -1 with a hold time of 7 min. MS operated in electron impact (E.I) mode and data were acquired in selected ion monitoring (SIM) mode. The selected SIM ions and time conditions for each compound are reported in Table S2.
For food samples analyses, oven temperature was maintained at 35°C for 4 min, then raised with a rate of 2.5°C min -1 to 120°C and increased at a rate of 15 °C min -1 to 250°C, held for 4 min. Data were acquired in scan, operating in E.I mode with a scan range of 29-400 m/z. Compounds identification was based on the comparison of their GC retention times and mass spectra with analytical standards and with reference mass spectra from the US National Institute of Standards and Technology (NIST, 2017). Data were analyzed by using MSD ChemStation software (Agilent, Version G1701DA D.01.00).

Statistical analysis
For food samples analyses, results were expressed in percentage and were obtained by semiquantification dividing the peak areas of the analyte of interest by the sum of the peak areas of all the identified compounds. On the other side, in standard samples analyses, results were expressed either as response factors (R.F = analyte peak area / reference compound peak area) or analyte peak areas. The results obtained from each analysis were validated by determining the S.D and the S-4 relative standard deviation (RSD % = 100 x S.D / mean). Results with RSD values ≤ 10% were considered reliable. The student t-test was used to evaluate if the differences obtained between the tested penetration distances were statistically reliable. Probability values ≤ 0.05 (p ˂ 0.05) were considered statistically significant.
S-5 Values are expressed as mean ± standard deviation (std dev). % = 100 x peak area analyte / total peak area. *: Differences were statistically significant for the VOCs considered (p ≤ 0.05).

20mm 40mm
Mean± std dev (%) Mean± std dev (%)   In a study performed on a mixture of VOCs, the RF differences was statistically significant for all 5 compounds at 250 rpm. With the increase of the stirring speed, the variations were reduced, as expected, but remained statistically significant for some analytes (Figure S2).

S-18
It is important to note that the aims of this study were neither to determine the best stirring rate for HS-SPME experiments, nor the right sample-fiber distance. Our aim was simply to demonstrate that in pre-equilibrium conditions, as for several published papers, it is important to specify the sample-fiber distance or the fiber penetration depth in order to increase the reproducibility of the researches reported in literature. In addition, few researchers have started very recently to take into consideration this parameter, giving info in their papers about the distance between the fiber tip and the sample surface or the fiber penetration depths (