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

LC Conditions

Column

Luna 3 µm HILIC 200 Å, LC Column 100 x 2 mm, Ea

Brand

Luna

Part No

00D-4449-B0

Phase Name

HILIC

Elution Type

Isocratic

Mobile Phase

A: 90/10 ACN/100 mM Ammonium Formate, pH 3.2

Gradient Profile

Step No.

Time(min)

%A

%B

1

3.5

100

0

Flow Rate

0.40 mL/min

Temperature

22°C

Detection

Mass Spectrometer (MS) @ (22°C)

Detection Info

Sample Notes

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%

Sample Preparation Details

Pretreatment Note

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.

Condition 1

3 mL Methanol at 1.0 mL/min

Condition 2

3 mL Water at 1.0 mL/min

Load

Load 3.50 mL Sample diluted in Acetonitrile:Water

Wash 1

1 mL Acetonitrile:Water (50/50); 2 aliquots of 500 uL at 1.0 mL/min

Wash 2

0.5 mL Methanol:Water (50/50); 1 aliquot of 500 uL at 1.0 mL/min

Wash 3

2 at 1.0

Dry

2 mins at 10 of Hg

Elution 1

0.5 mL Methanol at 1.0 mL/min

Elution 2

1.0 mL 5% Ammonium Hydroxide in Methanol (aliquots of 0.5 mL) at 1 mL/min

Evaporation

Evaporate to dryness

Reconstitute

1 mL 90/10 : ACN/100mM ammonium formate, pH 3.2