Simultaneous Analysis of Melamine and Cyanuric acid on Luna 3u 100x2.0 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

0

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 +ve Ion at 1.7 mins (for Melamine);MRM; heater gas flow 7000 cc/min; heater temperature 450 ° C

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

Water

Load

Load 1.00 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 at 1.0 mL/min

Wash 3

2 at 1.0

Dry

Dry for 2 minutes under 10 of Hg

Elution 1

0.5 mL Methanol at 1.0 mL/min

Elution 2

1.0 mL 5% Ammonium Hydroxide in Methanol (2 aliquots of 500 uL) at 1.0 mL/min

Evaporation

Evaporate to dryness

Reconstitute

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