Page 74 - Application Notebook - Solution for Food Safety

LAAN-A-LC-E290

Application High Performance Liquid Chromatography

News Analysis of Melamine and Its Related Substances in

Fertilizers
No.L514

Calcium cyanamide is effective as a fertilizer, pesticide, mAU
soil amendment, and for many other uses, and it is 3 ˙ Peaks
essential compound for producing high quality 10.0 1) Cyanuric acid
vegetables. Recently, high levels of melamine were 2) Ammelide
3) Melamine
discovered as a byproduct in some calcium cyanamide 7.5 4) Ammeline
hydrate products, pelletized by adding water to calcium
cyanamide. Due to the risk of agricultural products 5.0 1 2 4
absorbing the melamine from the soil, it has been
identified as a potential public health risk. For example, 2.5
if both melamine and its related substance cyanuric acid
are ingested at the same time, they can form crystals 0.0
that can impede kidney function. 1)
As a result, the Food Safety and Consumer Affairs 0.0 2.5 5.0 7.5 10.0 12.5 15.0
min
Bureau in the Ministry of Agriculture, Forestry and
Fisheries in Japan issued a notice specifying a 0.4 % Fig. 2 Chromatogram of Standard Mixture (1 mg/L each)
provisional maximum allowable concentration of
melamine in calcium cyanamide. 2) Area (×100,000) Area (×100,000)
This article describes an example of pretreating and 1.75 Cyanuric acid 2.50 Ammelide
analyzing melamine and its related substances, namely R = 0.99999 2.25 R = 0.99999
2
2
ammeline, ammelide, and cyanuric acid, in fertilizer, in 1.50 2.00
accordance with the testing methods supervised by the 1.25
Food and Agricultural Materials Inspection Center 1.75
(FAMIC) in Japan for fertilizers and other substances 1.00 1.50
(2016, 8.1.c) 3), 4), 5) 1.25
0.75
1.00
Q Analysis of Standard Solution 0.50 0.75
The structure of melamine and its related substances is 0.25 0.50
shown in Fig. 1. The analytical conditions are indicated 0.25
in Table 1. The chromatogram of the standard mixture 0.00 0.00
4.0
5.0
4.0
5.0
solution of melamine and its related substances (1 mg/L 0.0 1.0 2.0 3.0 Conc. (mg/L) 0.0 1.0 2.0 3.0 Conc. (mg/L)
each) is shown in Fig. 2. For more details regarding the Area (×100,000) Area (×100,000)
procedures used to prepare the standard solution and 3.5
mobile phase, refer to the applicable test methods. 6.0 Melamine Ammeline
2
2
Calibration curves for melamine and its related 5.0 R = 0.99999 3.0 R = 0.99999
substances are shown in Fig. 3. Calibration curves were 2.5
prepared for a concentration range of 0.05 to 5 mg/L. 4.0
The results indicated good linearity, with a contribution 2.0
2
rate (R ) over 0.9999. 3.0
1.5
2.0
Substance R1 R2 R3 1.0
Cyanuric OH OH OH 1.0 0.5
acid
Ammelide OH OH NH2 0.0 0.0 1.0 2.0 3.0 4.0 5.0 0.0 0.0 1.0 2.0 3.0 4.0 5.0
Conc. (mg/L) Conc. (mg/L)
Ammeline OH NH2 NH2
Melamine NH2 NH2 NH2 Fig. 3 Linearity (0.05 to 5 mg/L)
Fig. 1 Chemical Structure of Melamine and Q Repeatability
Its Related Substances The relative standard deviation (%RSD) results for
peak area after analyzing the standard solution
Table 1 Analytical Conditions
(0.1 mg/L) six consecutive times were very good, with
System : Prominence 0.41 % for cyanuric acid, 0.42 % for ammelide,
Column : TOSOH, TSKgel Amide-80 0.52 % for melamine, and 0.56 % for ammeline.
(250 mm L. × 4.6 mm I.D., 5 μm)
Guard Column : TOSOH, TSKgel guardgel Amide-80 When pretreated as indicated in Fig. 4, the 0.1 mg/L
(15 mm L. × 3.2 mm I.D.) concentration of the standard solution is equivalent to
Mobile Phase : (Sodium) phosphate buffer pH 6.7±0.2 / a 0.02 % concentration of melamine and other
Acetonitrile = 1/4 (v/v) related substances in fertilizer.
Flowrate : 1.0 mL/min
Column Temp. : 40 C
Injection Vol. : 10 μL
Detection : UV-VIS detector (SPD-20A) at 214 nm