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Calibration Curve for Cyanuric acid Extraction (20-2000ng/mL) from Strata Melamine on Luna HILIC

Sample Preparation Details

Part No:8B-S049-FBJ
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 200 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Protein Precipitation:
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Protein Precipitation
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Protein Precipitation: Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Protein Precipitation
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Protein Precipitation
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Protein Precipitation
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50 uL of 4.0 ug/mL of individual stock solution of melamine and cyanuric acid and 50uL of each internal standard (at 15.0 ug/mL).
Vortex 3-5 secs.
Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step. (Spiked conc. of analyte = 200 ng, IS spiked conc.= 750 ng; pH of sample is 5.5; melamine amino-15N3, and Cyanuric-13C3 used as I.S.)
Load:
collected supernatant from centrifugation step in protein precipitation prior to SPE
Dry:2 mins at 10" of Hg
Final Prep and Analysis: Protein Precipitation:
Weigh 0.2 mg (200mg) of Baby formula (Enfamil) in a 15 ml plastic centrifuge tube, add 1 ml Lukewarm water to make a homogeneous mix, vortex and add 100uL of 0.2N HCl followed by addition of 50uL of each internal standard (at 15.0 ug/mL). Vortex 3-5 secs. Add 3 ml of ACN, vortex and centrifuge at 6000Xg for 10 mins and then collect the supernatant to carry out SPE in the subsequent step.




Inject: 15 µL on HPLC / Mass Spectrometer (MS) @ (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Cyanuric acid 200 
2 Cyanuric-13C3 750 

Application Detail (App ID: 18468)

Part No:00D-4449-B0
Dimensions:100 x 2 mm ID
Elution Type:Isocratic
Elution A:90/10 ACN/100 mM Ammonium Formate, pH 3.2
Gradient Profile:
Step No. Time (min) Pct A
1 3.5 100

Flow Rate:0.4 mL/min
Col. Temp: ambient
Detection: Mass Spectrometer (MS) @ (22°C)
Detector Info: * MS in -ve ionization till 1.7 mins then to +ve till 3.5 mins
Note:LC/MS/MS Condition:
Column: Luna 3µm HILIC, 100x2.0mm; Flow rate: 0.4ml/min
Mobile Phase: Isocratic; 90/10 ACN/100mM NH4 Formate pH 3.2; MS Detector: API 3000 LC/MS/MS with ESI (TurboIonSpray®) (www.Appliedbiosystems.com), Switch from -ve Ion Mode (for cyanuric) to Positive Ion (for Melamine);
MRM; heater gas flow 7000 cc/min; heater temperature 450 °C

Cyanuric acid QC - 200 ng
% Accuracy 100.1%
% CV 17%


Calibration Curve of Extracted Samples

conc. (ng) % Accuracy
20 104%
40 98%
200 95%
400 103%
2000 99.91%

Analyte Details

Analyte Detail not available

1
Cyanuric acid
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2
Cyanuric-13C3
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