3. Results:
3.1 Test parameters
Fluorescence Detector
Signal:
Excitation: 225nm
Emission: 310nm
3.2 Linearity
3.2.1 Working linearity Range: 0.05mg/L to 2.50mg/L
3.2.2 Acceptable correlation coefficient (R2): >0.99
3.3 Instrumental Detection Limit (IDL)
3.3.1 7 repeating analysis with concentration 0.005mg/L, and after calculation (S.D x 1.645).
3.3.2 Theoretical IDL calculated by excel = 0.0005 mg/L.
3.3.3 Actual IDL after analysis by HPLC-DAD-FLD = 0.0025 mg/L achieve S/N ratio>3
3.4 Method Detection Limit (MDL)
3.4.1 Sample matrix: Yellow plastic.
3.4.2 Test Condition: 40°C for 1hr in ultrasonic bath.
3.4.3 Prepare 7 sets of sample (1.0g) and spike with 0.10mL 10mg/L Bisphenol A standard solution and follow 2.1~2.9, after calculation (S.D. x 3.14)
3.4.4 Theoretical MDLs calculated by excel are as below: (mg/L in sample solution or mg/kg in sample matrix):
Substance Average Conc. mg/L Average Recovery MDL
mg/L mg/kg
Bisphenol A 0.044 87.5% 0.019 0.374
3.4.5 Actual MDL after analysis by HPLC-DAD-FLD =0.05 mg/L (1.0mg/kg in sample matrix).
3.5 Bias Check
3.5.1 Sample matrix: Yellow plastic.
3.5.2 Test Condition: 40°C for 1hr in ultrasonic bath.
3.5.3 Prepare 7 sets of sample (1.0g) and spike with 0.50mL 100mg/L Bisphenol A standard solution and follow 2.1~2.9.
3.5.4 Recovery and RSD are calculated by excel as below:
Substance Spiked Conc. mg/L Average Recovery RSD
Bisphenol A 2.50 92.0% 3.29%
Remark: The fail limit of Bisphenol A is 50mg/kg.
3.6 Precision Check
3.6.1 Sample matrix: Yellow plastic.
3.6.2 Test Condition: 40°C for 1hr in ultrasonic bath.
3.6.3 Prepare 7 sets of sample (1.0g) and spike with 0.20mL 10mg/L Bisphenol A standard solution and follow 2.1~2.9.
For 50mg/L bisphenol A in sample, refer to data of Bias check.
3.6.4 Recovery and RSD are calculated by excel as below:
Substance Spiked Conc. mg/L Average Recovery RSD
Bisphenol A 0.10 84.9% 7.22%
Bisphenol A 2.50 92.0% 3.29%
Remark: Perform a lower concentration (spiked concentration is 0.10 mg/L) and a higher concentration (spiked concentration is 2.50mg/L)
4. Experimental:
4.1 Sample Homogeneity Check --- Sample Blank (C)
4.1.1 To the nearest mg, weigh 1 g of sample in a 200mL beaker
4.1.2 For non-polycarbonate material, add 20mL THF (5.2.4) and ultrasonic at 40℃ for 60 minutes.
4.1.3 Re-precipitated the plastic with 100mL ACN (5.2.1) with drop-wise and constant swirling.
4.1.4 Stand for 30 minutes to allow precipitate plastic residue
4.1.5 Filter the solvent mixture through filter paper (4.8) into a flat bottom flask.
4.1.6 Concentrate the filtrate to approximately 2mL with vacuum rotary evaporator (4.7) at 57oC, 300 mbar.
4.1.7 Transfer the filtrate into a 20mL volumetric flask. Rinse the flask twice with small amount of 1:1 ACN /water
4.1.8 Combine the washings and make up to the mark with 1:1 ACN / water.
4.1.9 Filter the final solution into an injection vial for HPLC-DAD-FLD analysis.
4.1.10 Repeat the steps 4.1.1 – 4.1.9 to perform 7 times of sample blank. Take an average value if any.
4.2 Spike sample (Cs)
4.2.1 To the nearest mg, weigh 1 g of sample in a 200mL beaker and spike the following amount of Bisphenol A standard solution into the beaker.
MDL BIAS PRECISION
0.10mL 10mg/L 0.50mL 100mg/L 0.20mL 10mg/L 0.50mL 100mg/L
4.2.2 Proceed the steps 4.1.2 – 4.1.10.
4.2.3 Repeat the steps 4.2.1 – 4.2.2 and prepare 7 set of spike sample.
4.2.4 Analysis with HPLC-DAD-FLD and calculate the amount of each spike sample.
4.2.5 Calculate the spike recovery in each spike sample as following equation
Spike Recovery (%) = (Cs –C)/CT x100%
Where,
C = Average concentration of the sample blank, mg/L
Cs = Concentration of spike sample, mg/L
CT = Concentration of theoretical spike amount, mg/L
4.2.6 Calculate the amount of spike sample in sample matrix, mg/kg
Bisphenol A, mg/kg = (Cs –C) x V/W
Where,
C = Average concentration of the sample blank, mg/L
Cs = Concentration of spike sample, mg/L
V = Final volume of ACN: H2O=1:1, 20mL
W = Sample weight, approximate 1.0g
4.2.7 Calculate the theoretical method detection limit (MDL) in sample matrix by the following equation
Bisphenol A, mg/kg = S.D. x t (n-1)
Where,
S.D = Standard derivation of n spike sample which calculate by 4.2.6, mg/kg
t (n-1) = The value from table of one-sided t distribution at n-1 degree of freedom and at 99% confident level
5. Conclusion:
5.1 Linearity: Working range between 0.05 mg/L to 2.50 mg/L, R2 > 0.99.
And extract 0.05 mg/L, 0.10mg/L, 0.50mg/L, and 2.50mg/L as calibration points in operation.
5.2 IDL: 0.0025 mg/L in standard solution and achieve S/N ratio>3.
5.3 MDL:
5.3.1 Spike Recovery in MDL (0.05mg/L): 87.5% average between 7 set of spike sample trials.
5.3.2 Actual MDL: After calculated by excel, 0.38mg/kg is our theoretical MDL for Bisphenol A (i.e.0.019mg/L in extract). Take a trial to spike at higher concentration in 0.05mg/L (i.e. 1.0mg/kg Bisphenol A in sample) and the result satisfies our requirement (± 50% Recovery, within 10 times of spike range).
5.4 The Reporting Limit: Since actual MDL is 0.05mg/L, the reporting limit is calculated as below:
The reporting limit, mg/kg = (Actual MDL) x V/W
Where,
V = Final volume of ACN: H2O=1:1, 20mL
W = Sample weight, approximate 1.0g
Therefore, the Reporting Limit is 1.0mg/kg.
5.5 Bias Check: Proceed 7 set of spike sample trials at each concentration. The average recovery is 92.0%. RSD is less than 10%, which satisfy our requirements (Recovery is within 80-120% and RSD<20%).
5.6 Precision Check: Proceed 2* 7 set of spike sample trials at each concentration. The average recovery is 84.9% and 92.0%, respectively. Both RSD is less than 10%, which satisfy our requirements (RSD<20%).