Materials and methods
Chemicals
The subject of the experiment was zinc dialkyldithiophosphate (ZnDDP), used as an anti-wear agent for automobiles, and various types of compounds exist depending on the type of the alkyl group. Among various ZnDDP compounds, this experiment was conducted on Zinc O, O-dibutyl dithiophosphate manufactured by BOC Science. As an internal standard (IS), pentachlorophenol (98%, Sigma-Aldrich, St. Louis, USA), was used. The solvents used for extraction were HPLC grade, and methanol, dichloromethane, and hexane (n-hexane), used as extraction solvents in solid-phase extraction and liquid–liquid extraction, were obtained from JT Baker (Phillipsburg, NJ, USA). The solvents included: tetrahydrofuran (JT Baker, Phillipsburg, NJ, USA), toluene (JT Baker, Phillipsburg, NJ, USA), acetonitrile (Burdick & Jackson, MI, USA), acetone (Burdick & Jackson, MI, USA), ethyl ether (Burdick & Jackson, MI, USA), ethyl acetate (Sigma-Aldrich, St. Louis, USA), and dimethyl sulfoxide (Duksan Pure Chemical Co., Ansan, South Korea). HPLC grade ones were purchased, and they were used without further purification. The engine oils and transmission oils, used in the experiments, were the products used in our vehicle, including two engine oils (gasoline 0W-20 and diesel 0W-30) and two transmission oils (manual DCTF TGO & automatic SP4MI).
Instrument and parameters
In this study, gas chromatography-mass spectrometry (GC–MS) was used to analyze the anti-wear agent in automotive engine oil and to calculate its content. Gas chromatography was performed, using Chromatec-Crystal 9000 (C9000) from JSC SDO Chromatec (Russia), and the column was HP-Ultra 2 capillary column (25 m × 0.11 mm × 0.2 film thickness) from J & W Scientific (Folsom, CA, USA). AS-2M (3D) auto-sampler manufactured by JSC SDO Chromatec (Russia) was used as a sample injector. A total of 2 µl samples were injected and analyzed in splitless mode (Added: SIM mode). The inlet temperature was set at 250℃, and the flow rate of helium carrier gas was 1 ml/min. The solvent delay time was 3.0 min, and a one-ramp temperature gradient was used. The initial temperature was maintained for 3 min at 50 ℃; the heating rate was 8 ℃/min to 300 ℃, and the holding time was 10 min. A Chromatec-Crystal mass detector manufactured by JSC SDO Chromatec (Russia) was used as a mass spectrometer. As for the ionization method, electron impact ionization (EI) was used at 70 eV. The MSD ion source temperature was maintained at 200 ℃ in full-scan mode (25–1000 m/z) for qualitative and quantitative analysis.
Sample preparation methods
Standard sample manufacturing
A standard sample was prepared by diluting Zinc O, O-dibutyl dithiophosphate, used as an anti-wear agent in 10 ml of solvent to 500 µg/ml (extraction solvent: hexane and methanol).
Liquid–liquid extraction (LLE)
LLE, which extracts analytes from liquid samples, first extracted the 500 µg/ml standard sample dissolved in hexane, using a separation funnel using various solvents (DMSO, acetonitrile, and methanol). The recovery rate was measured.
Solid-phase extraction (SPE)
A 24-port SPE Vacuum Manifold from Honeywell Burdick & Jackson was used for SPE (Standard sample extraction). 10 ml of hexane was flowed at a rate of 5 ml/min to condition a SPE cartridge. Zinc O, O-dibutyl dithiophosphate, used as an anti-wear agent in the activated cartridge, was diluted in 10 ml of hexane to 500 µg/ml and poured into the cartridge by flowing that at a rate of 5 ml/min. As an extraction solvent, 10 ml of methanol was extracted at a rate of 5 ml/min.
Validation test
Recovery
To confirm the recovery, ZnDDP standard samples were dissolved in methanol and added at the concentrations of 50 µg/ml. Pentachlorophenol 10 µg/ml was added to sample. After the extraction (pretreatment) process was added, they were analyzed five times. The control group was extracted by adding the internal standard material at the same concentrations and then pretreated by adding the ZnDDP standard samples. Relative recovery was measured, using the area ratio of the ZnDDP standard sample and the internal standard of the comparison group and the control group.
Linearity
To prepare the calibration curve, the ZnDDP standard samples were dissolved in methanol to make 8-point standard samples from 0.2 to 500 µg/ml at 10 µg/ml of pentachlorophenol to each sample. The samples were prepared by addition. The analysis was repeated three times with GC–MS, and the concentration of the calibration curve was selected to 8 points (0.2, 1, 5, 10, 50, 100, 200, and 500 µg/ml) for ZnDDP based on calibration limits. Linearity was evaluated by the correlation coefficient, which is the R2 value of the calibration curve.
Limit of detection and quantification
In order to measure the limit of determination (LOD) and limit of quantification (LOQ), concentrations of the ZnDDP standard samples in methanol were added at 0.2, 1, 5, 10, 50, 100, 200, and 500 µg/ml. The analysis was repeated three times per each concentration. Concentrations with a signal to noise (S/N) of 3.3 were selected as detection limits, and concentrations with that of 10 were selected as limit of quantification.
Precision and accuracy
In order to evaluate the precision, 500 µg/ml ZnDDP standard sample was added repeatedly five times and expressed as a relative standard deviation (% RSD, CV). Accuracy was selected based on the calibration limits: low-quality control (LQC) concentration at 1 µg/ml; medium-quality control (MQC) concentration at 50 µg/ml; and high-quality control (HQC) concentration at 100 µg/ml. Percent bias (%) was:
$$\frac{{\left( {{\text{Average measurement concentration}} - {\text{known concentration}}} \right)}}{{{\text{known concentration}}}} \times 100$$
Analytical procedures (Actual sample extraction)
10 ml of hexane was flowed at a rate of 5 ml/min to condition the SPE cartridge. The activated cartridge was injected with 500 µl of gasoline and diesel engine oils, and transmission oils, DCTF and SP4MI. 10 ml of hexane was flowed at a rate of 5 ml/min to remove the base oil, which is an analyte. 10 ml of methanol, an extraction solvent, was extracted at a rate of 5 ml/min. The analytical procedure is shown in Fig. 1.