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.
Reference list
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Table 1. The m/z ion monitored and retention time for each glycol analyzed