Protocol for the preparation of calibration curves for volatile analysis (SPME-GC-MS and SPME-GC-FID)

*In order to minimize competition phenomena between volatiles compounds it was decided to divide the stock solution in two mixtures (SM A and SM B).

Note: For calculation of the concentrations, use the exact concentration of SM A and SM B.

Preparation of Standard Mixtures (SM) at 10000 mg/kg

For the low concentration mixture (SM A):

1.

Put an empty vial of 20 mL in the analytical balance and tare it.

2.

Weigh 5.000 ± 0.001 g of refined olive oil in the vial.

3.

Put approx. 0.100 ± 0.001 g for each standard, exactly weighed, for the mixture A (low concentration, 10 compounds) in the vial.

4.

Add refined olive oil up to reach approx. 10.000 ± 0.001 g, exactly weighed.

5.

Close the vial (cap + septum).

6.

Shake for 30 seconds on the agitator (e.g. vortex).

Note: Do not forget to write down the weights for concentration calculation.

For the high concentration mixture (SM B):

1.

Put an empty vial of 20 mL in the analytical balance and tare it.

2.

Weigh 5.000 ± 0.001 g of refined olive oil in the vial.

3.

Put approx. 0.100 ± 0.001 g for each standard, exactly weighed, for the mixture B (high concentration, 8 compounds) in the vial.

4.

Add refined olive oil up to reach approx. 10.000 ± 0.001 g, exactly weighed.

5.

Close the vial (cap + septum).

6.

Shake for 30 seconds on the agitator (e.g. vortex).

Note: Do not forget to write down the weights for the concentration calculation.

Preparation of dilutions from the two SM (A and B)

Note: the following procedure has to be applied in the same way, both from SM A and SM B

SM1 (200 mg/kg):

1.

Place a vial (labelled as SM1) of 20 mL on the analytical balance and tare it.

2.

Weigh 5.000 ± 0.001 g of refined olive oil.

3.

Put approx. 0.200 ± 0.001 g of SM, exactly weighed, in the vial.

4.

Add refined olive oil up to reach approx. 10.000 ± 0.001 g, exactly weighed.

5.

Close the vial (cap + septum).

6.

Shake for 30 seconds on the agitator (e.g. vortex).

Note: Do not forget to write down the weights for the concentration calculation.

SM2 (20 mg/kg):

1.

Place a vial (labelled as SM2) of 20 mL on the analytical balance and tare it.

2.

Weigh 5.000 ± 0.001 g of refined olive oil.

3.

Put approx. 1.000 ± 0.001 g of SM1, exactly weighed, in the vial.

4.

Add refined olive oil up to reach approx. 10.000 ± 0.001 g, exactly weighed.

5.

Close the vial (cap + septum). Shake for 30 seconds on the agitator (e.g. vortex).

Note: Do not forget to write down the weights for the concentration calculation.

SM3 (2 mg/kg):

1.

Place a vial (labelled as SM3) of 20 mL on the analytical balance and tare it.

2.

Weigh 5.000 ± 0.001 g of refined olive oil.

3.

Put approx. 1.000 ± 0.001 g of SM2, exactly weighed, in the vial.

4.

Add refined olive oil up to reach approx. 10.000 ± 0.001 g, exactly weighed.

5.

Close the vial (cap + septum).

6.

Shake for 30 seconds on the agitator (e.g. vortex).

Note: Do not forget to write down the weights for the concentration calculation.

Preparation of dilutions for building the calibration curves

For the low concentration mixture (SM A), it is necessary to prepare the following 12 dilutions starting from SM1, SM2 or SM3 (see the procedure and Table 2): 0.05, 0.10, 0.15, 0.20, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 5.00, and 10.00 mg/kg. For the high concentration mixture (SM B), it is necessary to prepare 12 dilutions starting from SM1, SM2 or SM3 (see the procedure and Table 2): 0.20, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 5.00, 10.00, 15.00, 20.00, and 25.00 mg/kg.

1.

Place each vial (20 mL) on the analytical balance and tare it.

2.

Weigh 1.000 ± 0.001 g of refined olive oil.

3.

Add 0.100 ± 0.001 g of IS dilution¹.

4.

Add the appropriate weight (Column 5 of Table 2) of SMx (Column 1 of Table 2) in the vial. The approximate obtained concentration is indicated in the column 6 of Table 2, although the exact concentration needs to be calculated.

5.

Add refined olive oil up to reach approx. 2.000 ± 0.001 g, exactly weighed (Column 3 of Table 2 shows the amount of refined olive oil approximately to be added).

6.

Close the vial quickly with cap and septum.

7.

Shake the vials gently and softly (never spread the oil through the vial walls or the septum).

Note: Do not forget to write down the weights for the concentration calculation; ¹, Preparation of IS dilution explained in the section “Standard solution and sample preparation (SPME-GC-MS and SPME-GC-FID)”, IS in refined olive oil at a concentration of about 50 mg/kg. Fig. 1 shows the chromatograms of SM A and SM B, added with IS and both SM at 10.00 mg/kg, analysed by SPME-GC-MS.

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Fig. 1. Chromatograms of SM A and SM B, added with IS, at 10 mg/kg (SPME-GC-MS). *Compounds deriving from the SPME Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) fiber.

Sequence of the GCanalysis for building the calibration curves

In the sequence of the chromatographic analyses, set the higher concentrations at the end of the sequence, and analyse the blank samples (empty vials) and blank refined oil (oil without compounds added). The following example is for the low concentration mixture (SM A):

1.

Blank (empty vial).

2.

Blank of the matrix (refined olive oil: 2.0 g).

3.

Blank of the matrix + IS (refined olive oil: 1.9 g + IS: 0.1 g).

4.

Blank (empty vial).

5.

0.05 mg/kg vial.

6.

0.10 mg/kg vial.

7.

0.15 mg/kg vial.

8.

0.20 mg/kg vial.

9.

Blank (empty vial).

10.

0.25 mg/kg vial.

11.

0.50 mg/kg vial.

12.

1.00 mg/kg vial.

13.

1.50 mg/kg vial.

14.

Blank (empty vial).

15.

2.00 mg/kg vial.

16.

2.50 mg/kg vial.

17.

5.00 mg/kg vial.

18.

10.00 mg/kg vial.

19.

Blank (empty vial).

Standard solution and sample preparation (SPME-GC-MS and SPME-GC-FID)

Caution: work at controlled room temperature (T = 20-25°C) due to the volatility of the standards. Samples must be at room temperature before sample preparation.

○

Preparation of the stock standard solution

Weigh 15.000 ± 0.001 g of refined olive oil in a vial, then add 0.100 ± 0.001 g, exactly weighed, of 4-methyl-2-pentanol and add refined olive oil up to reach 20.000 ± 0.001 g, exactly weighed (approximate concentration of 5000 mg/kg). Take note of exact weights (± 0.001 g) for calculation of concentration. A rapid preparation of the stock standard solution is advisable to avoid evaporation of internal standard.

○

Preparation of standard solution

Weigh 5.000 ± 0.001 g of refined olive oil in a vial, then add 0.100 ± 0.001 g of the stock standard solution of 4-methyl-2-pentanol, exactly weighed, and add refined olive oil up to reach 10.000 ± 0.001 g, exactly weighed (approximate concentration of 50 mg/kg). Take note of exact weights (± 0.001 g) for calculation of concentration. A rapid preparation is advisable to avoid evaporation of internal standard.

○

Sample preparation

Weight exactly 1.900± 0.001 g of virgin olive oil (sample) in a 20 mL glass vial and add 0.100 ± 0.001 g of 4-methyl-2-pentanol standard solution (5.1.2) as internal standard (IS approximate concentration = 2.5 mg/kg). Take note of exact weights (± 0.001 g) for calculation of concentration. Hermetically close the vial with polytetrafluoroethylene septum (PTFE). Shake the vial manually, but very gently and softly (never spread the oil through the vial walls or the septum). Leave for 10 min at 40°C under agitation (250 rpm in continuous is suggested) to allow for the equilibration of the volatiles in the headspace. After the equilibration time, the septum covering each vial is pierced with a SPME needle and the fiber exposed to the headspace for 40 min.

•

Gas chromatography analysis (choose one of two options):

•

With FID: The volatiles adsorbed by the fiber are thermally desorbed in the hot injection port of a GC for 5 min at 250°C with the purge valve off (splitless mode) and deposited onto a capillary column of a GC with a FID at 260ºC. The carrier gas is helium (or hydrogen if the equipment and installation are adapted and allow for safety procedure), at a flow rate of 1.5 mL/min (this is a suggestion, the flow rate must be adjusted to optimize the separation of peaks depending on the use of helium or hydrogen as carrier gas). The oven temperature is held at 40°C for 10 min and then programmed to increase by 3°C/min to a final temperature of 200°C. A cleaning step can be added (20°C/min to 250°C and hold the temperature for 5 min).

•

With MS: The volatiles adsorbed by the fiber are thermally desorbed in the hot injection port of a GC for 5 min at 250°C with the purge valve off (splitless mode) and deposit onto a capillary column of a GC with a mass spectrometry (MS) detector. The transfer line temperature is set at 260 ºC. The temperature of the ion source is set according to the technical specifications of the instrument. The carrier gas is helium (or hydrogen if the equipment and installation are adapted and allow for safety procedure), at a flow rate of 1.5 mL/min (this is a suggestion, the flow rate must be adjusted to optimize the separation of peaks depending on the use of helium or hydrogen as carrier gas). The oven temperature is held at 40°C for 10 min and then programmed to increase by 3°C/min to a final temperature of 200°C. A cleaning step can be added (20°C/min to 250°C and hold the temperature for 5 min).

•

Peak identification

To identify with greater certainty each extracted compound, the LRI are determined. The mixture of n-alkanes is injected in the GC; the retention times of the alkanes are used in the following equation, obtaining the LRI of each analyte extracted.LRI=100×z+100×((RTanalyte−RTz)(RTz+1−RTz))z is the number of carbon atoms of the alkane that elutes before the molecule; the RT_analyte, the RT_z and the RT_z+1 are the retention times of the analyte of interest, of the alkane that elutes before it and the one that elutes after it.

•

Quantitative analysis

The quantification of selected volatile compounds is carried out by calibration based on the internal standard (IS) and the external calibration curve. The IS is also added in the sample because it is necessary for evaluating the goodness of the fiber sampling and the whole analytical procedure. Thus, an abrupt change in the peak area assigned to IS, or in its usual RT, would mean an issue that needs to be checked.

External calibration curve (A_Analyte/A_IS vs. C_Analyte):

A_Analyte/A_IS = m·C_Analyte, (Y = m·x)

C_Analyte = (A_Analyte/A_IS)/m

Where:

C_Analyte is the concentration of the analyte.

A_Analyte is the area corresponding to the analyte.

A_IS is the area corresponding to the IS; m, is the slope of the calibration curve (built for the selected analyte).

Note: Analysis should be carried out in triplicate. The means will be calculated from the areas of those triplicates.

Note: An abrupt reduction of the IS area can be observed in the case of lampante olive oils with a high median level of main defect.

Data and results about the validation of the method is reported in Aparicio Ruiz et al., 2022 [1] and Casadei et al., 2021 [2] (see References).