2.1 Optimization of extraction condition
2.1.1 Influence of extraction solvent
500 mL purified water was added into a separating funnel, and then APs and BPA working solution were added to produce the desired concentration of target compounds in the water sample (0.20 µg/L). The water sample was extracted three times using 30 mL different organic solvent with different polarities (n-hexane, toluene, dichloromethane or ethyl acetate). The effects of different extraction solvents on the recovery of target compounds are shown in Table 2. Dichloromethane displays the best extraction effect recovery rate of –target compounds, whereas the extraction efficiency of the other solvents is very low, especially for BPA (13.5 %–76.2 %). These results are consistent with standard methods (ASTM D7065 2017; JIS K 0450-10-10 2006), in which the extraction solvents are all dichloromethane. Dichloromethane was used as the extraction solvent in this method.
Table 2 Average extraction efficiency of C4-C9 APs and BPA using different extraction solvents
Extraction solvent
|
Recovery rate (%)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Dichloromethane
|
104
|
101
|
115
|
101
|
98.7
|
110
|
95.4
|
109
|
120
|
98.8
|
n-Hexane
|
83.7
|
94.1
|
108
|
97.6
|
96.5
|
107
|
87.5
|
106
|
118
|
13.5
|
Ethyl acetate
|
91.2
|
84.2
|
83.7
|
65.8
|
72.1
|
80.6
|
65.4
|
82.9
|
66.5
|
76.2
|
Toluene
|
65.3
|
88.9
|
65.0
|
78.5
|
72.0
|
83.6
|
79.4
|
88.1
|
75.4
|
55.1
|
2.1.2 Influence of extraction number
The effects of different extraction number using of dichloromethane on the recovery of target compounds were examined (Table 3). According to the results, C4-C9 APs can be extracted completely after one extraction, and BPA can be extracted mainly after two extractions. Hence, when dichloromethane is selected as the extraction solvent, the second extraction is sufficient to adhere to the necessary requirements.
Table 3 Average extraction efficiency of C4-C9 APs and BPA for different extraction number
Extraction number
|
Recovery rate (%)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Surrogate
|
1
|
97.8
|
102
|
113
|
109
|
119
|
117
|
96.9
|
117
|
103
|
76.3
|
62.6
|
2
|
1.9
|
2.0
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
1.0
|
24.9
|
27.4
|
3
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
4.6
|
5.2
|
4
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
2.1.3 Influence of extraction time
The influence of different extraction time on the recovery rate of target compounds is shown in Table 4. The duration of the extraction slightly effected extraction efficiency. As the time was prolonged, the recovery rate of the target substance increased slightly. As shown in Table 4, the results indicated that min was sufficient to fully recover the target substance from the water sample.
Table 4 Average extraction efficiency of C4-C9 APs and BPA for different extraction time
Extraction time (min)
|
Recovery rate (%)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Surrogate
|
5
|
98.9
|
101
|
109
|
101
|
118
|
112
|
95.6
|
115
|
106
|
98.7
|
91.0
|
10
|
99.7
|
104
|
113
|
109
|
119
|
117
|
96.9
|
117
|
104
|
101
|
90.0
|
15
|
104
|
109
|
118
|
117
|
122
|
127
|
98.1
|
123
|
106
|
101
|
92.3
|
2.1.4 Influence of pH value in water
The effects of acidity in water on the recovery rate of target compounds are shown in Table 5. At pH values higher than 6, the recovery of 4NP and 4nOP decreased, but that of other target compounds was not affected. Considering that acidic conditions are conducive to inhibition of bacteria in water and prevention of bacteria from consuming AP organics, pH value of water sample should be adjusted to about 2 (Martinez and Peñuela 2013).
Table 5 Average extraction efficiency of C4-C9 APs and BPA for different pH value
pH value
|
Recovery rate (%)
|
<1
|
1-2
|
2-3
|
3-4
|
4-5
|
6-7
|
4tBP
|
107
|
98.6
|
107
|
102
|
99.0
|
100
|
4nBP
|
110
|
99
|
104
|
101
|
99.4
|
96.8
|
4nPP
|
117
|
111
|
113
|
110
|
109
|
106
|
4nHexP
|
117
|
109
|
112
|
108
|
106
|
102
|
4tOP
|
122
|
106
|
115
|
110
|
109
|
110
|
4nHepP
|
125
|
115
|
120
|
114
|
113
|
105
|
4NP
|
105
|
97.3
|
105
|
101
|
97.4
|
91.3
|
4nOP
|
116
|
106
|
112
|
107
|
107
|
97.7
|
4nNP
|
121
|
110
|
121
|
115
|
120
|
108
|
BPA
|
110
|
115
|
116
|
108
|
114
|
113
|
Surrogate
|
117
|
110
|
121
|
115
|
114
|
113
|
2.1.5 Influence of extraction solvent volume
The effects of various volumes of dichloromethane on the recovery of target compounds are shown in Table 6. 20 mL of dichloromethane was determined as optimal volume to meet the extraction requirements. However, in the actual extraction process, 30 mL of extraction solvent was selected to ensure complete extraction.
Table 6 Average extraction efficiency of C4-C9 APs and BPA for different extraction solvent volume
Extraction solvent volume (mL)
|
Recovery rate (%)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Surrogate
|
20
|
95.2
|
99.7
|
111
|
97.0
|
104
|
116
|
95.2
|
96.7
|
99.0
|
98.4
|
100
|
30
|
104
|
109
|
116
|
100
|
104
|
118
|
95.2
|
96.3
|
97.6
|
101
|
103
|
40
|
107
|
111
|
118
|
101
|
103
|
117
|
98.3
|
97.9
|
97.1
|
103
|
104
|
50
|
108
|
113
|
121
|
104
|
106
|
122
|
100
|
101
|
103
|
104
|
104
|
2.1.6 Influence of salt content
Phenols are water-soluble and need salting out to improve extraction efficiency. Hence, the effects of different NaCl amount on the recovery rate of target compounds are shown in Table 7. The results showed that addition of NaCl slightly improved the recovery rate of the target compounds. This is mainly due to the recovery rate already being significantly high without addition of salt. Considering the weak salting out effect, 5 g of NaCl was added in water sample in extraction procedure.
Table 7 Average extraction efficiency of C4-C9 APs and BPA for different salt content
NaCl Dosage (g)
|
Recovery rate (%)
|
0
|
5
|
10
|
20
|
30
|
4tBP
|
97.5
|
100
|
103
|
103
|
102
|
4nBP
|
97.3
|
102
|
103
|
106
|
107
|
4nPP
|
107
|
110
|
112
|
115
|
114
|
4nHexP
|
100
|
106
|
108
|
111
|
112
|
4tOP
|
107
|
110
|
110
|
114
|
110
|
4nHepP
|
102
|
110
|
112
|
115
|
115
|
4NP
|
93.9
|
104
|
103
|
105
|
105
|
4nOP
|
95.6
|
103
|
109
|
107
|
108
|
4nNP
|
99.1
|
107
|
113
|
112
|
111
|
BPA
|
107
|
108
|
115
|
110
|
114
|
Surrogate
|
97.8
|
101
|
106
|
109
|
108
|
2.1.7 Effect of concentration degree
It is necessary to concentrate the organic extraction solution to a certain volume or redissolve the target compounds to a specific volume by solvent after being concentrated to dryness prior to analysis. In the case of C4-C9 APs, certain target compounds are liquid and volatilization occurs when the vacuum degree is appropriate. Additionally, some solid targets are powder or velvet with low density, and have drift loss with increasing vacuum degree. In our study, the extraction solution was concentrated by rotary evaporation under vacuum to either 0.5 mL concentrate or to dryness and maintained in vacuum for a period of time (from 1 min to 10 min). The influence of these methods is shown in Table 8. The recovery rate of target compounds were close to 100 %, when the organic extraction solution was concentrated to 0.5 mL. But the measured values were significantly reduced when extraction solution was concentrated to dryness and maintained in vacuum for 1 min, especially for some APs that are liquid at room temperature. Moreover, with the extension of vacuum time, the recovery rate of target compounds further decreased. Finally, in the process of concentration, the extraction solution was concentrated to 0.5 mL.
Table 8 Average extraction efficiency of C4-C9 APs and BPA for different drying process
Drying time (min)
|
Recovery rate (%)
|
Condense to 0.5 mL
|
1
|
5
|
10
|
4tBP
|
102
|
40.1
|
21.5
|
20.9
|
4nBP
|
100
|
54.0
|
22.3
|
18.8
|
4nPP
|
104
|
73.2
|
32.3
|
23.2
|
4nHexP
|
102
|
84.6
|
56.1
|
44.4
|
4tOP
|
103
|
88.1
|
60.7
|
53.7
|
4nHepP
|
104
|
90.9
|
79.4
|
74.4
|
4NP
|
100
|
92.3
|
82.5
|
74.9
|
4nOP
|
99.6
|
90.5
|
87.8
|
84.7
|
4nNP
|
104
|
95.4
|
94.1
|
92.6
|
BPA
|
102
|
93.6
|
94.6
|
92.1
|
Surrogate
|
101
|
95.1
|
96.0
|
92.9
|
2.2 Influence of derivatization time
The derivatization efficiency of C4-C9 APs and BPA for different derivatization time is shown in Table 9. The derivatization rate of the target compounds tended to be stabilized after 30 min, and the measured value was consistent with the theoretical value (100 µg/mL). Although target compounds were derivatized completely at this time, 60 min was selected as the derivatization time to ensure robustness of the method.
Table 9 The derivatization efficiency of C4-C9 APs and BPA for different derivatization time
Derivatization time (min)
|
Measured value (µg/mL)
|
5
|
30
|
60
|
90
|
120
|
4tBP
|
75.1
|
110
|
110
|
113
|
113
|
4nBP
|
84.5
|
111
|
111
|
112
|
112
|
4nPP
|
86.6
|
110
|
110
|
113
|
112
|
4nHexP
|
99.2
|
108
|
110
|
112
|
111
|
4tOP
|
101
|
112
|
115
|
118
|
113
|
4nHepP
|
102
|
108
|
109
|
111
|
110
|
4NP
|
107
|
109
|
100
|
105
|
104
|
4nOP
|
93.3
|
108
|
104
|
108
|
108
|
4nNP
|
97.7
|
106
|
105
|
107
|
106
|
BPA
|
104
|
109
|
114
|
118
|
114
|
Surrogate
|
95.7
|
109
|
110
|
114
|
110
|
2.3 Chromatogram analysis
Fig. 1 showed a chromatogram of standard solution at 100 μg/L of the target compounds in SIM mode. Under the chromatogram conditions, the peaks of each compound are independent and clear. Due to the use of derivatization treatment, there is no tailing phenomenon of phenolic compounds observed. 4NP is a mixed peak of one group of isomers (inset figure, ranging from 15.58 min to 16.15 min), and other compounds are single peaks (retention times are shown in Table 1).
2.4 Method validation and application to real samples
2.4.1 Linear correlation coefficient and limit of detection
The developed method using the above optimized conditions was validated with respect to linear correlation coefficient (R2) of standard calibration curve and limit of detection (LOD). The linear correlation coefficient (R2), LOD and limit of quantification (LOQ) of target compounds to be measured are shown in Table 10. The linear correlation coefficient (R2) values of target compounds were not less than 0.995 and standard calibration curves showed satisfactory linearity based on internal standard method.
Table 10 The linear correlation coefficient (R2), LOD and LOQ of C4-C9 APs and BPA
Compounds
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
R2
|
0.998
|
0.997
|
0.999
|
0.997
|
0.996
|
0.998
|
0.995
|
0.996
|
0.997
|
0.998
|
LOD (μg/L)
|
0.002
|
0.002
|
0.002
|
0.002
|
0.002
|
0.002
|
0.006
|
0.002
|
0.003
|
0.003
|
LOQ (μg/L)
|
0.008
|
0.008
|
0.008
|
0.008
|
0.008
|
0.008
|
0.024
|
0.008
|
0.012
|
0.012
|
LOD was confirmed according to the relevant environmental standard method (HJ 168 2020), and seven purified water samples with or without APs and BPA working solution were analyzed continuously. Due to 4NP and BPA being detected in the purified water, LOD values of 4NP and BPA were determined by analyzing seven purified water samples, whereas LOD values of the remaining target compounds were determined by analyzing seven purified water samples added with APs and BPA working solution. The concentration of remaining target compounds in these seven purified water samples is 0.010 μg/L, whereas LOQ is 4 times that of LOD (Table 10). LOD ranged from 0.002 μg/L to 0.006 μg/L, while LOQ ranged from 0.008 μg/L to 0.024 μg/L. For comparison, LOD values of related compounds in several typical literatures and standards are listed in Table 11. It can be seen that LOD values of 4tOP, 4NP and BPA are 0.001 μg/L–0.2 μg/L, 0.001 μg/L–0.9 μg/L and 0.002 μg/L–0.3 μg/L, respectively, and LOD values of 4tBP, 4nBP and 4nNP range from 0.034 μg/L to 0.077 μg/L, and LOD of this developed method is at low level.
Table 11 LOD values of target compounds in several typical literatures and standards
Literatures and standards
|
Analytes and LOD
|
Recovery (%)
|
Analysis method
|
Extraction method
|
Publication date
|
ASTM D7065
|
BPA 0.3 μg/L;4NP 0.9 μg/L;4tOP 0.2 μg/L
|
BPA 53-119; 4NP 57-110; 4tOP 56-106
|
GC/MS
|
Liquid-liquid extraction
|
2017
|
ASTM D7574
|
BPA 0.005 μg/L
|
43-120
|
HPLC/MS
|
Solid phase extraction
|
2016
|
Bolivar-Subirats et al.
|
BPA 0.002 μg/L; 4NP 0.046 μg/L; 4tOP 0.025 μg/L
|
/
|
HPLC/MS
|
Solid phase extraction
|
2021
|
Yuan et al.
|
4tBP 0.077 μg/L; 4nBP 0.034 μg/L
|
/
|
GC/MS
|
Solid phase extraction
|
2016
|
Selvaraj et al.
|
BPA 0.002 μg/L; 4NP 0.001 μg/L; 4tOP 0.001 μg/L
|
BPA 91.9-95.9; 4NP 70.5-77.7; 4tOP 64.9-78.7
|
GC/MS
|
Solid phase extraction
|
2014
|
Martinez and Peñuela
|
4nNP 0.042 μg/L
|
66.8-97.3
|
GC/MS
|
Solid phase extraction
|
2013
|
Wang et al.
|
12 of 4NP isomers 0.009-0.041 μg/L
|
69.4-129
|
GC/MS
|
Liquid-liquid extraction
|
2013
|
Shen et al.
|
BPA 0.005 μg/L;4NP 0.004 μg/L;4tOP 0.003 μg/L
|
BPA 87.8;4NP 111;4tOP 82.3; 4tOP 79.2
|
GC/MS
|
Solid phase extraction
|
2005
|
2.4.2 Recovery and precision
To further validate the developed method, precision experiment and recovery experiment were carried out. Purified water was taken as the blank matrix to carry out precision experiment, and C4-C9 APs and BPA standard working solutions was added so that the concentration of target compounds was 0.020 µg/L, 0.500 µg/L and 2.00 µg/L, respectively. These three concentration levels basically cover the concentration range of target compounds in groundwater, surface water, seawater, sewage and wastewater. The pretreatment and test were carried out according to section 1.2 and 1.3. Each concentration level was determined six times in parallel. The Average Value (AV) and RSD results are shown in Table 12. Surface water, seawater and sewage were taken as the matrix to carry out recovery experiment, and C4-C9 APs and BPA standard working solution was added so that the concentration of added target compounds was 0.5-3 times of that in the original matrix. The average recovery results are shown in Table 13. The RSD of the target compounds ranged from 0.67 % to 13.7 %, and the average recovery ranged from 68.0 % to 122 %, which indicated that the developed method showed excellent recovery and low relative standard deviation.
Table 12 Precision data
Spiked concentration (µg/L)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
0.020
|
0.025
|
9.52
|
0.020
|
4.62
|
0.025
|
3.93
|
0.023
|
2.90
|
0.024
|
2.68
|
0.500
|
0.498
|
3.20
|
0.468
|
2.15
|
0.541
|
1.88
|
0.501
|
1.92
|
0.484
|
1.96
|
2.00
|
1.77
|
2.05
|
1.85
|
2.42
|
1.93
|
2.12
|
1.97
|
2.19
|
1.93
|
1.51
|
Spiked concentration (µg/L)
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
AV (µg/L)
|
RSD (%)
|
0.020
|
0.026
|
4.69
|
0.034
|
10.1
|
0.030
|
10.2
|
0.032
|
12.3
|
0.032
|
7.08
|
0.500
|
0.516
|
1.79
|
0.533
|
2.49
|
0.528
|
2.29
|
0.574
|
3.12
|
0.431
|
2.44
|
2.0
|
2.03
|
1.74
|
1.98
|
5.32
|
2.03
|
7.20
|
2.00
|
13.7
|
1.88
|
0.67
|
Table 13 Recovery data
Water sample
|
Spiked concentration (µg/L)
|
Average recovery (%)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Seawater
|
0.05
|
106
|
96.0
|
111
|
104
|
102
|
101
|
90.7
|
112
|
99.1
|
107
|
Surface water
|
0.50
|
99.5
|
102
|
110
|
116
|
121
|
122
|
72.4
|
79.1
|
88.2
|
68.0
|
Sewage
|
2.0
|
95.9
|
102
|
104
|
107
|
104
|
111
|
97.9
|
102
|
98.6
|
91.4
|
2.4.3 Determination of real water samples
The contents of target compounds in groundwater, surface water, seawater and sewage samples are shown in Table 14. It can be seen that BPA (0.016 μg/L) was detected in groundwater, 4nBP, 4nHepP, 4NP, 4nOP and BPA (0.002 μg/L–0.077 μg/L) was detected in Bohai Seawater, 4NP and BPA (0.026 μg/L–0.032 μg/L) was detected in Yellow River water and the detection quantity of these target compounds was low. 4nPP and 4nHexP were not detected in all water samples. The measured values of 4NP (0.139 μg/L–0.965 μg/L) and BPA (0.024 μg/L–0.226 μg/L) in Guangli River water, Xiaoqing River water and two sewage samples were high. The higher pollutant content in two river water samples was attributed to the two river's function of containing pollutants discharged by chemical enterprises. However, the higher content of pollutants in two sewage samples was attributed to the large amount of detergents used in people's lives entering the sewage.
According to Directive 2013/39/EU, the annual concentration limit of 4NP is 0.3 μg/L, maximum allowable concentration is 2.0 μg/L and average annual concentration of 4OP in surface water is 0.1 μg/L (K Kern 2014). An EPA Regulation set a maximum allowable concentration of 4NP in fresh water (28 μg/L) and average annual concentration (6.6 μg/L) (EPA-822-R-05-005 2005). The contents of target compounds in groundwater, surface water and seawater in Dongying City met the strict requirements of the European Union and the United States. China's Ministry of Ecological Environment stipulated that the emission limit of BPA in wastewater is 0.1 mg/L (GB 31571 2015; GB 31572 2015) and BPA contents in two sewages also met this requirements. Considering high restrictive values of target compounds in environmental water and low LODs of this developed method, it can meet the needs of the determination of C4-C9 APs and BPA in water at home and abroad.
Table 14 Measured value of target compounds in real water sample (µg/L)
|
4tBP
|
4nBP
|
4nPP
|
4nHexP
|
4tOP
|
4nHepP
|
4NP
|
4nOP
|
4nNP
|
BPA
|
Surrogate Recovery
|
Groundwater
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
0.016
|
124 %
|
Seawater
|
n.d.
|
0.003
|
n.d
|
n.d.
|
n.d.
|
0.002
|
0.077
|
0.008
|
n.d.
|
0.011
|
104 %
|
Surface water 1
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
0.026
|
n.d.
|
n.d.
|
0.032
|
78.6 %
|
Surface water 2
|
0.031
|
0.007
|
n.d.
|
n.d.
|
0.008
|
0.002
|
0.139
|
0.002
|
0.003
|
0.068
|
92.3 %
|
Surface water 3
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
0.228
|
n.d.
|
n.d.
|
0.226
|
77.4 %
|
Sewage 1
|
0.004
|
0.008
|
n.d.
|
n.d.
|
0.019
|
0.003
|
0.965
|
0.003
|
n.d.
|
0.024
|
110 %
|
Sewage 2
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
0.344
|
n.d.
|
n.d.
|
0.156
|
88.5 %
|
Note: surface water 1 is Yellow River water, surface water 2 is Guangli River water, surface water 3 is Xiaoqing River water, sewage 1 is outlet water of sewage treatment plant in Dongying City, sewage 2 is outlet water of sewage treatment plant in Guangrao County, Dongying City.