How was the melamine detected?

Melamine in milk products were detected using High Performance Liquid Chromatography (HPLC) coupled with a UV detector. The protocol is as follows:

Apparatus:

1. HPLC-UV detector

2. HPLC Symmetry C18 (5 um, 3.9X 20 mm) Guard Column

3. HPLC Symmetry C18 (5 um, 4.6X 250 mm) Analytical Column

4. Solid-phase extraction (SPE) column

5. 100ml Volumetric Flask

6. 25ml Volumetric Flask

7. 50ml Centrifuge Tube

8. Vortex Shaking Machine

9. Ultrasonic bath

10. Centrifuge Machine

11. Filter Paper

Reagents:

1. Milk powder (e.g. 7 different varieties of powdered milk samples collected in this experiment)

2. Melamine (50% v/v)

3. Sodium 1-octane sulfonate (HPLC grade)

4. Methanol (HPLC grade)

5. Trichloroacetic acid

6. Citric acid monohydrate

7. Ammonia solution

8. Acetonitrile

9. DI water

HPLC system of Analysis (Summary)

Quantitative analysis:

- HPLC Waters system equipped with pump, guard column and analytical column.

- Water column heater with water temperature control system, UV-Vis Detector and waters automated gradient controller associate with computer system using Empower software

à Run and control all the calculations for the instrument.

The concentrations of the products were determined from the peak areas under the curve using the software for instrument control and data collection and processing. Solid phase extraction was performed thereafter. Before the quantitative and qualitative determination of melamine in the milk (infant milk samples), standard solutions of different standard concentrations were prepared. With those standard solutions of different melamine, calibration lines were constructed for each one of the melamine, which were later used for assessing the concentration corresponding to the different peaks in the chromatograms.

HPLC operating conditions:

v  Column used: HPLC Symmetry C18 (5 um, 3.9X 20 mm) Guard Column, HPLC Symmetry C18 (5 um, 4.6X 250 mm) Analytical Column

v  Column Temp: 50^oC

v  Flow Rate of mobile phase: 1ml/min

v  Injection Volume: 20ul

v  UV Detection: 240nm

v  Oven Temperature: 50 ^oC

Methods:

1. Preparation of standard of Melamine:

1.       100g of Melamine was dissolved with aqueous methanol in a 100ml volumetric flask. This forms  1000ug/ml melamine stock standards. Label as 'Melamine Stock Standard'.

2.       For calibration graph, stock standard was diluted appropriately to prepare working standards with concentrations of:

v  0.01ug/ml (10 000x dilution)

v  1.00ug/ml (1000x dilution)

v  2.00ug/ml (500x dilution)

v  5.00ug/ml (200x dilution)

v  10.00ug/ml (100x dilution)

v  50.00ug/ml (20x dilution)

2. Preparation of mobile phase:

1.      Buffer of 2.10 g citric acid and 2.16 g HPLC grade sodium 1-octane sulfonate were dissolved in 980 ml DI water and two ranges of pH were tested 4.5 and 3.0 the volume brought to one litter after the pH was adjusted by sodium hydroxide.

2.       920 ml of the list solution was taken and mixed with 80 ml Acetonitrile. The ratio is 92:8 (volume to volume) used for the isocratic separation mode for melamine.

3.       In the same manner, another ratio of the mobile phase buffer solution to acetonitrile (85:15) was  prepared and studied as well.

3. Sample preparations

- Food samples are typically complex matrices that are difficult to analyze due to abundance of proteins and carbohydrates

à These complex matrixes may affect the detection and enrichment of analytes

à Isolation and extraction of melamine and analogs from complex matrices is hence necessary prior to melamine determination.

- Main objectives of sample treatment (i.e. extraction, pre-concentration, and derivatization):

à To achieve lower limits of detection by removing matrix constituent

à Most sample-preparation procedures require extraction followed by one or more clean-up steps that can take from ten minutes to hours due to the complexity of the matrices.

1.       Milk powder sample blank control, free from any addition of melamine.

Label Sample ___ BLANK.

2.       Sample is spiked with 20 ul of stock standard solution and is accurately weighed into 50-ml centrifuge tubes.

3.       Add 15 ml of aqueous trichloroacetic acid and 5 ml acetonitrile into sample.

4.       Vortex sample for 1 minute.

5.       Sonicate sample in ultrasonic bath for 30 minutes.

6.       Vortex sample for 15 minutes.

7.       Centrifuge sample for 30mins @ 6000 RPM.

8.       Filter supernatants into 25 ml volumetric flasks through filter paper.

9.       Samples were brought to volume of 25ml with DI water. Label Sample ___ SPIKED.

10.       Repeat steps with all 7 milk powder samples.

4. Fortified Sample Preparation

1.       Weigh 1g milk powder sample into 50ml centrifuge tube

2.       Add 5 ml of HPLC methanol and 5 ml of DI water into the sample centrifuge tube.

3.       Vortex sample for 1 minute.

4.       Sonicate sample in ultrasonic bath for 30 minutes.

5.       Add 10 ml of 1% trichloroacetic acid to sample (1 gram in 100 ml DI water).

6.       Vortex sample for 1 minute.

7.       Sonicate sample in ultrasonic bath for 15 minutes.

8.       Centrifuge sample for 30mins @ 6000 RPM.

9.       Transfer supernatants to 25 ml volumetric flasks and fill flask to 25ml with DI water.

10.   Filter solution through a 0.45 um desk filter.

11.   Same dilution was made to bring the concentration of the sample within the range of the calibration curve.

12.   Repeat steps with all 7 milk powder samples.

5. Solid-phase extraction (SPC) for milk samples

1.       SPE column was activated prior usage by passing 3 ml of methanol and 5ml water in turn.

2.       Mixture of 5 ml sample extract and 5 ml DI water was passed through the activated SPE.

3.       Wash SPE with 3 ml methanol, then 3 ml DI water.

4.       Filter samples with 0.45 um desk filter before injection.

5.       The elution carried out with 6 ml aminated methanol solution freshly papered by mixing of 5 ml ammonia solution and 95 ml methanol.

6.       Collect eluent and dry at 50 ^oC

7.       Re-dissolve eluent in 1 ml of the mobile phase.

8.       Repeat steps with all 7 milk powder samples.

Reference:
>> Der Pharma Chemica, 2012. Identification and Determination of Melamine in Milk by High Performance 
Liquid Chromatography – UV Detector. [online] Available at: <http://derpharmachemica.com/vol4-iss2/DPC-2012-4-2-737-748.pdf> [Accessed 21 January 2013]