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Analysis of Furan and Alkylfurans in Food Samples (Part 3): Adjusting the Experimental Conditions for the Analysis of Samples with High Concentrations of Analytes and Preparing Calibration Curves

1 September 2022
  • Nathaly Reyes-Garcés, PhD
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In our previous blog post, we discussed the selection of several experimental conditions for the analysis of furan and alkylfurans in baby formula. Unfortunately, these experimental parameters are not suitable for the analysis of samples with a high concentration of target analytes, such as coffee. The high extraction capacity offered by the SPME Arrow and the high levels of certain analytes, such as furan and 2-methyl furan (typically present in hundreds of µg/kg in coffee), can easily lead to detector saturation. As a strategy to overcome this roadblock, several analysts have leaned towards the use of headspace analysis, which provides significantly lower recoveries for these target contaminants (as we already saw in our first blog post here). However, can we tweak our SPME method conditions to make it work in samples with high concentrations? The answer is YES! As we mentioned in our second blog (here), and as Colton explains in this video (here), SPME is a non-exhaustive technique where a small amount of analyte proportional to the analyte’s concentration in matrix is extracted onto/into the coating material. By adjusting the experimental conditions, we can enhance as well as decrease the amount of analyte that is extracted. As a strategy to use the SPME Arrow for coffee analysis, the following experimental parameters were adjusted: extraction time, headspace/NaCl solution volume, and split ratio.

1. Extraction Time

First, we tried decreasing the extraction time as it is the easiest way to decrease the amount of analyte extracted via SPME. Instead of exposing the SPME arrow for 10 min, an optimal time for baby formula, we chose 1 min as the extraction time for coffee samples. Opting for shorter extraction times at pre-equilibrium conditions could be a source of lower precision when manual SPME is performed; however, by employing automated systems, such as the CTC PAL autosampler, this is not a matter of concern.

2. Headspace/30% NaCl Solution Volume

The headspace volume is an important factor to take into consideration when developing a SPME method [1]. Keeping a reproducible headspace volume is critical to ensure satisfactory method reproducibility because it has an important effect on the amount of analytes extracted by the SPME device [2]. The high volatility of our target contaminants facilitates them readily partitioning into the sample’s headspace. By decreasing the volume of salt solution added and therefore increasing the headspace volume, we can dilute the concentration of the analytes in the headspace, and therefore decrease the amount of analytes extracted. For that reason, we decided to decrease the volume of salt solution added from 10 to 5 mL for the analysis of coffee samples.

3. Split Ratio

Finally, to further decrease the amount of analyte injected into the GC-MS system, the split ratio was increased from 1:10 to 1:100. This enabled us to keep the responses of analytes extracted from highly concentrated samples within the linear dynamic range of the instrument.

Once the conditions for both coffee and baby formula samples were optimized (Table 1), calibration curves were constructed by spiking 30% NaCl solution with working solutions of target analytes and internal standards. Two calibration curves were prepared, one for the analysis of low concentrations of furan and alkylfurans in baby formula, and one for the quantitation of high concentrations of target analytes in coffee [3]. Analytes were spiked by adding different volumes of working solutions to the calibration vials, as shown in Table 2. Internal standard solutions were added to each calibration vial (50 µL of the 1 µg/mL solution for the low concentration calibration curve, and 40 µL of the 25 µg/mL solution for the high concentration calibration curve). To account for variations in recoveries due to matrix differences, it was essential to use isotopically labeled analogues for almost every analyte (2-methylfuran-d6 worked for both 2- and 3-methylfuran). Calibration curves were then constructed by plotting analyte area/ISTD average area ratios (n=2) versus spiked concentration (information about working solutions preparation is presented at the end).

Table 1. Sample preparation conditions for baby formula and coffee samples


Baby formula



0.5 g

Volume of 30% NaCl solution added

10 mL

5 mL

Volume of internal standard solution added

50 µL

(1 µg/mL solution)

40 µL

(25 µg/mL solution)

Incubation time

10 min

Incubation and extraction temperature

50 ⁰C


250 rpm

HS-SPME extraction time

10 min

1 min

Desorption temperature

280 ⁰C

Desorption time

1 min

Split ratio



Table 2. Volumes of standards used to prepare calibrators.

Low concentration calibration curve

High concentration calibration curve

ng of analyte in vial

µL of working solution added (solution concentration)

ng of analyte in vial

µL of working solution added (solution concentration)

Level 1


12.5 (0.1 µg/mL)


50 (0.5 µg/mL)

Level 2


25 (0.1 µg/mL)


100 (0.5 µg/mL)

Level 3


10 (0.5 µg/mL)


10 (10 µg/mL)

Level 4


20 (0.5 µg/mL)


20 (10 µg/mL)

Level 5


40 (0.5 µg/mL)


50 (10 µg/mL)

Level 6


100 (0.5 µg/mL)


100 (10 µg/mL)

Level 7


200 (0.5 µg/mL)


200 (10 µg/mL)

Level 8


300 (0.5 µg/mL)


400 (10 µg/mL)

Level 9




800 (10 µg/mL)


Figure 1. Low range calibration curves (for baby formula analysis).


Figure 2. High range calibration curves (for coffee analysis).

As can be seen in Figures 1 and 2, we were able to obtain low and high concentration calibration curves with R2 values above 0.99 for all our target analytes. This demonstrates the suitability of the new set of conditions for samples with high concentrations of furan and alkylfurans.


By decreasing the extraction time, the volume of 30% sodium chloride solution, and increasing the split ratio to 1:100, we were able to find a suitable set of conditions for the analysis of samples with high levels of furan and alkylfurans using the SPME Arrow. We obtained low and high concentration calibration curves with R2 values above 0.99 for all our target analytes. In our next blog post, we will introduce our new application note where the results of the application of this method to real samples will be included, so please stay tuned!

Stock and Working Solutions Preparation

1 mg/mL solutions for each target analyte in methanol (MeOH) were obtained by spiking pure standards using a glass syringe. Then, 100 ppm stock solutions, one containing all target analytes and one solution with all the internal standards, were prepared in MeOH. For the calibration curve covering the low concentration range, 0.1 and 0.5 µg/mL working solutions were obtained by spiking 10 and 50 µL of the 100 ppm stock, respectively, in 10 mL of LC-MS grade water. An internal standard solution of 1 µg/mL was prepared by adding 100 µL of the 100 ppm stock solution to 9.9 mL of water. For the analysis of coffee samples, a high concentration calibration curve was run. For this purpose, aqueous working solutions at 10 and 25 µg/mL, for target analytes and internal standards, respectively, were obtained by spiking original individual methanolic solutions directly in water. A 0.5 µg/mL was prepared by diluting 500 µL of the 10 µg/mL solution in water until reaching a volume of 10 mL. The 30% NaCl solution was prepared by mixing 150 g of NaCl with 500 mL of water.


  1. Górecki, T., Pawliszyn, J., Effect of Sample Volume on Quantitative Analysis by Solid-Phase Microextraction Part 1. Theoretical Considerations. n.d.
  2. Pawliszyn, J., Handbook of Solid Phase Microextraction. Chemical Industry Press, Beijing 2009.
  3. Frank, N., Dubois, M., Huertas Pérez, J. F., Detection of Furan and five Alkylfurans, including 2-Pentylfuran, in various Food Matrices. J. Chromatogr. A 2020, 1622, 461119.

Nathaly Reyes-Garcés, PhD

Nathaly Reyes-Garcés holds a M.Sc. and Ph.D. in analytical chemistry from University of Waterloo (Canada). Her research work has been focused on the application of diverse analytical strategies to investigate complex samples of environmental and clinical interest. Nathaly has several years of hands-on experience on different sample preparation approaches, including microextraction techniques; and on gas and liquid chromatography both coupled to various detectors, and mass spectrometry analyzers. Currently, Nathaly is an LC application scientist at Restek Corporation where she works on the development of analytical workflows to support different markets, including cannabis testing.

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