RESULTS
Method validation
Specificity
The method specificity was demonstrated by injecting ultrapure water
blank samples, reference solutions of each individual glycol and a mix
solution of glycols (all at 50 µg/mL). No interference was observed at
the retention time of target analytes. Overlayed chromatograms for each
target analyte with individual solutions are presented in Figure 2.
Linearity and calibration range
The linearity of the method was determined using a 5-levels calibration
curve for each analyte. The calibration points were 10, 25, 50, 75 and
100 µg/mL and 5 replicates per calibration point were injected in the
GC-MS system in order to validate the linear range of each analyte. The
calibration curves for all target analytes were found to be linear
within the 10-100 µg/mL range and correlation coefficient (r2) for all 9
glycols were higher than 0.995. Linearity values are presented in Table
2 and the calibration curves are presented in Figure
Method sensitivity (LOQ and LOD)
The limit of quantification (10 µg/mL) was determined based on the
signal-to-noise ratio for all target analytes. The acceptance criteria
was an S/N ratio of not less than 10:1, for all analytes.
To establish the LOD concentration, serial dilutions were prepared from
Calibration solution level 1 (10 µg/mL): 5, 2, 1 and 0.5 µg/mL of each
target analyte. The LOD was defined as the lowest concentration for
which the S/N ratio was not less than 3:1.
Limits of detection of 1 and 2 µg/mL were obtained for all target
analytes. The results obtained for LOQ and LOD are presented in Table 3.
System Precision
The precision of the method was determined by GC-MS system precision, at
three levels (low-10 µg/mL, mid-50 µg/mL and high concentration-100
µg/mL). 3 replicates at each level were prepared and injected. For all
target analytes, the RSD% (based on peak area) were below 7.0% for all
three levels tested, with a combined method RSD% of 2.22 %. The
results obtained for system precision are presented in Table 4.
Robustness
According to validation guidelines, robustness is defined as a measure
of method capacity to remain unaffected by small, but deliberate
variations in method parameters and provides an indication of its
reliability during normal usage. To validate this parameter, small
variations in the instrumental method were introduced: change of carrier
gas flow rate and split ratio of the flow. A summary of the changes
introduced is presented below (Table 5).
Standard solutions of low level (10 µg/mL) and high level concentration
(100 µg/mL) of target analytes were injected in triplicate using
modified methods with small variations presented above. The RSD% for 6
injections (3 from System Precision and 3 from Robustness) was
calculated. Deviation of peak areas for all target analytes were less
than 10 %. These results proved that the method is robust. Results for
this parameter are presented in Table
Stock solution stability
A stability study was conducted based on the stock solutions prepared in
Day 1 of the validation and stored at 4°C. Calibration level 1 (10
µg/mL) and 5 (100 µg/mL) were prepared for 3 consecutive days and
analyzed in triplicate using the method developed. The solutions proved
to be stable, the concentrations were in the accepted interval of ± 20%
of target concentration. The results are presented in Figure 3 and 4.
Real sample analysis
A number of 10 different ethylene glycol-based antifreeze liquids were
purchased from Romanian market and analyzed using the validated GC-MS
method. A short description of each one of the coolants analyzed is
presented in Table 6. Each sample was prepared in triplicate and
analyzed. According to the manufacturer, for 8/10 of samples analyzed,
to reach an optimum coolant with a freezing point around 30-35°C, which
corresponds to an ethylene glycol (EG) concentration of approximately
30%, a 1:1 dilution with distilled water was necessary. Antifreeze
solution number 10 exhibited the highest EG concentration, measuring at
66.65%.
Antifreeze formulations designated as ’Ready to use liquids’ by the
manufacturer, namely, antifreeze numbers 1 and 7, contained EG
concentrations of 30.23% and 35.75%, respectively.
Notably, the presence of diethylene glycol (DEG), Triethylene glycol
(TEG), and Tetraethylene glycol (TTEG) was detected in all analyzed
samples, with concentrations ranging from 5.04% to 10.62%.
Conversely, concentrations below the limit of quantification (LOQ) were
observed in eight out of ten samples examined for DEG and in five out of
ten samples examined for propylene glycol (PG).
Isomers of dipropylene glycol and tripropylene glycol were not detected
in the analyzed samples. The results obtained for the analysis are
presented in Table 7.
CONCLUSIONS
A method that requires only a two-step dilution of anti-freeze samples
combined with direct liquid injection GC-MS was developed and validated
for the simultaneous quantification of 7 glycols (and their isomers) in
10 different types of anti-freeze liquids from Romanian market. The
results obtained in the validation procedure proved that the GC-MS
method is reliable, sensitive and precise for quantification of glycols
in real samples.
Acknowledgements
This work was supported by a grant of Ministry of Research and
Innovation, CNCS–UEFISCDI, project number 93PTE/2022 within PN III-2.1.
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Table 1. The m/z ion monitored and retention time for each glycol
analyzed