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METHODS FOR DETERMINATION OF MELAMIN IN MILK

CÁC PHƯƠNG PHÁP XÁC ĐỊNH MELAMIN TRONG SỮA

PHAM BA LICH K57T- Advanced Chemistry Program

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INTRODUCTION

Melamin (2,4,6-triamino-1,3,5-triazine ) MEL C3H6N3

Applications: Manufacture of melamine-formaldehyde resins for surface coatings, laminates, and adhesives and in theproduction of flame retardants (Chang 1994)

Problems: increases the [N] → a false increase in [protein]. When meeting strong acids or alkali, melamine yields ammeline, ammelide and cyanuric acid by hydrolysis.

Melamine could form insoluble complexes with cyanuric acid, depending on urine pH, which could lead to crystallization and subsequent tissue injury , such as urolithiasis and bladder cancer, renal stone caused by melamine in the milk powder

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Methods

GC/MS, (HPLC)/MS, capillary zone electrophoresis/MS (CE/MS or UV), potentiometry (Liang et al. 2009) [ shortages: complicated preconcentration, time-consuming steps and high-cost instruments ]

Electrochemical methods: simplicity, low-cost, accurateness,sensitivity and high stability.

Phương pháp cực phổ xung vi phân (Differential PulsePolarography - DPP)

Solid Phase Microextraction at Bismuthyl Chloride Modified Graphite Epoxy Composite Electrode

Electrochemiluminescence of [Ru(bpy)3]2+ at bare and single-wall carbon nanotube modified glassy carbon electrodes

Oligonucleotides film modified gold electrodes

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Differential Pulse Polarography – DPP - PREPARATION electrochemical analyzer was used for DPP and CV

measurements. A three electrode system was used, consisting of a platinum counter electrode, an Ag/AgCl (3 M NaCl) reference electrode and a DME as a working electrode.

Stock solution in EtOH–water (50:200, v/v) to 0.01 M. Britton–Robinson (B–R) buffer, pH 11.2 ( 2.3 mL of glacial CH3COOH, 2.7 mL H3PO4 (85%) and

2.47 g of boric acid to 1.0 l; pH was adjusted by 2.0 M NaOH.)

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Analytical Procedure supporting electrolyte : HClO4, HCl, acetate,

phosphate, ammonia or B–R buffer, de-oxygenated with highpurity nitrogen (99.999) for about 5 min.

scanning potential : 0.0 V → −1,400 to −2,200 mV (vs. Ag/AgCl) depending on the pH of the solution.

Polarographic responses of 1×10-5 to 1×10-3 M melamine at the DME

=> Optimal condition: Linear range: 1.0 to 66.4uM ( R = 0.999) Melamine peak potential : -50mV, scan rate of 5

mVs-1, a pulse amplitude of 50 mV.

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Effect of pH and Buffer Solution acidic solution : No peak of melamine was

observed PO4

3- and NH3 buffers: polarographic currents were very weak. The B–R buffer was chosen for its wide pH range applicability.

peak current of melamine increased in the pH range 10.5–11.2, decreased strongly at pH values greater than 11.2 and there was no peak at pH 13.

=> optimum conditions: pH 11.2 B–R buffers, at a reduction potential of

−50 mV, 2 s drop time, 50 mV pulse amplitude.

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Interference Studies electro-active ions [ Cu(II), Fe(III), Co(II), Ni(II), Pb(II), Zn(II),

Cd(II), Mn(II), Se(IV)] => optimum conditions for polarographic determination10

μM melamine, Cu (−182 mV), Fe (−1,440 mV), Cd (−660 mV) and Zn (−1,351 mV) appeared at a more negative potential than melamine (−50 mV).

Thus, the polarographic peaks of these ions did not overlap the melamine peak, and therefore they had no significant effect on the polarogram of melamine.

Al(III), K(I), Ba(II), Na(I), Ca(II), Mg(II), NO3-, SO4

2-, Cl− and I− are polarographically inactive species and therefore had no serious effect on the determination of melamine.

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No interferences were observed in a blank extracted from the milk and milk powder.

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Conclusion

Linear range: 1.0 to 66.4uM ( R = 0.999); LOD : 0.3uM ; LOQ : 1.0uM

Linear dynamic range: : For milk 1-58uM ; Milk powder samples 10-57uM

Melamine peak potential : -50mV, scan rate of 5 mVs-1, a pulse amplitude of 50 mV.

Advantages: the possibility determining the trace quantities of melamine directly from natural samples (milk and milk powder) without any previous treatment, such as extraction, clean-up and derivatization or pre-concentration, which are tedious, time-consuming and can lead to pollution

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Oligonucleotides film modified gold electrodes Mica – coated by MEL brings (+) charge Oligonucleotides (d(T)20) (a 6-mercaptohexyl linker at the 5’ end, stock

solution (20uM) brings (-) charge Buffer: 0.01 M phosphate (pH 7) whereas pKa(MEL) = 8. electrochemical probe of [Fe(CN)6]3-/4- - cyclic voltammetry – electrical

sensing morphologic differences between melamine and melamine interacted with

oligonucleotides were observed by AFM ( atomic force microscopy)

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Preparing dT20/Au and MEL-mica dT20 modified gold electrode:

(1) Cleaned by 70% H2SO4 conc, 30% H2O2 (10min).

(II) Rinsed ultrasonically EtOH+ water. (III) Cycled 0.2 -1.5 V in 0.1 M H2SO4 at 100 mV/s until

reproducible voltammograms were obtained. (IV) Self-assembled monolayer: 15uL d(T)20 solution onto the

gold electrode’s surface (16 h immobilization period), soaked in sterilized water 2 h, dried with N2.

Cleaned mica substrates : dried by N2 + a drop of MEL-PO4

3- (1,5h)→ Cleaned by Distilled H2O→ dried by sterile atm => MEL-modified mica substrates.

MEL-modified mica substrates + dT20 -PO43-/Au (pH = 7, 1-4h) => AFM

analysis

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Effect of IT and C on d(T)20 immobilization

Incuabation time (IT): the d(T)20 molecules formed much more clusters to form a higher surface coverage with the increasing incubation time. ( 1h: 8.2nm ; 2h: 32.8nm).

Concentration(C): different [d(T)20] (5.4pM and 11pM) at RT => more clusters formed on the melamine-modified mica surface with the increasing [d(T)20] since the assembly becomes more convenient at higher [d(T)20] .

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Optimal conditions Amount of dT20 : [d(T)20] ( 1-40uM) tends to increase

semicircle diameter but plateau at 20uM. => apply 20uM

Accumulation potential and time: + AP: [Fe(CN)6]3-/4- -PBS buffer- (pH7.0) (1.6uM MEL)

AP = 0.2 to 1.0 V, t =30 s =>The peak current increased with increasing accumulation potential and reached the highest at 0.8 V. +T applied: 0-300s where accumulation potential 0.8V=> peak current increase slowly, but 30s is rapid detection.

pH: pKa(MEL) = 8; pH >8 => more saturated or (-)MEL ; pH < 8: more (+)MEL. => pH = 7 is chosen.

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RECAP Due to complementary triamino triazine unit, melamine can

be immobilized onto the oligonucleotides modified electrode via electrostatic interaction + H-bonding.

=> Increase in the peak currents of ferricyanide, which could be used for electrochemical sensing of melamine.

The redox peak currents of ferricyanide were linear with the concentration of melamine in the range from 3.9 × 10-8 to 3.3 × 10-6 M (R =0.990). The detection limit was 9.6 × 10-9 M.

The proposed electrochemical biosensor is rapid, convenient and low-cost for effective sensing of melamine. Particularly, determination of melamine in milk products with 95% recovery.

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THANKS YOU FOR YOUR ATTENTION

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SPIKED MILK AND MILK POWDER Milk: 1.0 mL of milk + 1.0 mL 1×10-2 M melamine in 8.0 mL

acetonitrile Homogenizing(20 min)→ centrifuge(20 min 9,000 rpm (6,758.32×gunits) to remove the milk protein residues.

0.1 mL of melamine-free aliquots→ polarographic cell (9.9 mL of B–R buffer at pH 11.2. Differential pulse polarogram was recorded,

milk sample that contains 1×10-3 M melamine was added two times. We added 0.1 mL of spiked melamine sample to the polarographic cell containing 9.9 mL electrolyte and 0.1 mL of melamine-free aliquots.

In the polarography cell, [melamine] in milk sample is 1.0×10-5 M. Milk powder sample: 0.5g - 1.0 m 1×10-2 M melamine + 9.0 mL of

acetonitrile. Vortexing for 1 min + centrifuged for 20 min, 9,000 rpm (6,758.32×g units. Take 0.1-mL aliquots then added to the cell containing 9.9 mL of B–R buffer solution at pH 11.2. Differential pulse polarograms were recorded and melamine determination in melamine-spiked milk powder was performed from the peak current at about −65 mV using melamine standard additions.