ABSTRACT

A capillary zone electrophoresis method was developed for determination of melamine in food samples, such as quail egg and milk products. The CE procedure was performed on fused silica capillary (41 cm × 75 μm I.D.) at 17 kV using pH3.1 60mmol/L phosphate buffer as run buffer and detecting at 200 nm. The proposed method showed good linearity (0.5 - 10.0 μmol/L) and low LOD (0.5 μmol/L) with good reproducibility (RSD% was 2.4 and 3.2 for migration time and peak area respectively), which made it suitable for quantity control of the related food product.

Keywords:Capillary Zone Electrophoresis; Melamine; Food Products

1. Introduction

Melamine (MEL), a nitrogen-rich (66% by mass) chemical intermediate, which is often used to produce plastics and melamine-formaldehyde resins, had been found in foods as an illegal addictive to increase the apparent protein content in related foods. MEL is less harmful, however, it could form insoluble harmful crystals via reacting with its metabolite (cyanuric acid) and later one could cause damage to the organs (such as kidney), and even lead to death in some cases [1] -[3] . Therefore, it is critical to establish a simple, rapid, potent method for detecting MEL especially in low concentration.

Varieties of assay methods, such as liquid chromatography [4] [5] , gas chromatography [6] -[8] , capillary electrophoresis [9] -[13] , etc. coupled with kinds of detectors, such as ultra-violet detector [10] [11] , diode array detector [12] , electrical chemical detector [13] and mass spectrum [4] -[8] etc., had been selected and applied to determine MEL in milk and related products [4] [7] -[9] [13] , infant formula [10] [11] , pet food [5] [12] to name a few. Among these methods, sample treatment steps seemed to be one of the crucial factors to maintain better separation. However, some of them were complicated and time-consuming. For example, in HPLC methods, additional extraction steps, such as liquid-liquid [4] , liquid-solid extraction [5] at cation-exchange [14] or hydrophilic modes [6] , were normally needed for clearance of interferents from complicated samples. Moreover, in some cases (like GC mode [6] -[8] ), special sample treatments, such as derivatization, were demanded to obtain better separation. All these additional sample treatment steps limited their applications to some extent.

In our works, CE, which has several advantages of high performance, high resolution, rapid, smaller sample volumes and solvent consumption comparing with traditional analytical techniques, was used for determination of MEL in real sample (taking quail egg and pure milk as examples). The sample treatment procedure as well as factors that influence separation (pH and concentration of the buffer, separation voltage etc.) were carefully optimized. Under optimization condition, base-line separation of MEL can be easily obtained within 15-min. The proposed method showed a good linearity (0.5 - 10.0 mmol/L, R² = 0.9907) and reproducibility (RSD% for migration time and peak area was both less than 3.2%), lower limits of detection (LOD = 0.5 μmol/L) which makes it suitable for assay of MEL in related foods.

2. Materials and Methods

2.1. Reagents

MEL was purchased from the Sigma-Aldrich (USA, sigma-Aldrich, 99.0%). Phosphoric acid, sodium phosphatedibasic, sodium dihydrogen phosphate and sodium hydroxide (NaOH) were analytical reagents (Tianjin kermel). Acetone, acetonitrile, and methanol, obtained from Hongyan company (Tianjin, chromatographic grade). All reagents and solutions were used directly. Water used throughout was purified by OLST M6 (Ao Lian Co, Xian, China) with a conductivity of 18.2 Ωcm.

2.2. Apparatus

A K1030 Capillary electrophoresis (CE) system (KaiAo, CHINA), equipped with an ultraviolet (UV) detector (LC-900, KaiAo, CHINA), was used for CE experiments. Fused silica capillary (75 μm I.D. and 365 μm O.D.,) was obtained from Yongnian optical fiber company (Hebei, China) with a total length of 50 cm and the effective length of 41 cm from the inlet. Blender was from Shangliang electronic company (MEI JIA BAO, A-288, CHINA), A PB-10 pH meter (Sartorius, Germany) was employed for pH measurements. Centrifugation was performed on a 5810R (Eppendorf, Germany).

2.3. Sample Preparation

Milk samples were treated with different organic solvent or TCA at certain ratio (see section 3.3.1). And then samples were blended and sonicated again for 10 min at 20˚C. Finally, samples were centrifuged at 12,000 rpm for 10 min at 4˚C. The supernate was filtered by 0.45 μm syringe filter before injection; Quail eggs (obtained from a local market) were peeled, blended and sonicated for 10 min at 20˚C, then were treated as milk samples. For recovery test, MEL was added to the real sample prior the sample treatment.

2.4. Electrophoresis Procedure

A pH3.1 60.0 mmol/L phosphate buffer was served as ruining buffer. Sample was injected into capillary hydrodynamically at 50 mBar for 5 seconds and detected at 200 nm, 20˚. For the first usage, the capillary was activated with 1.0 mol/L NaOH and purified water for 10 min respectively. To maintain good reproducibility, an additional rinse step (specific pressure) using 0.1 mol/L NaOH (5min) followed with purified water (5 min) and run buffer (5 min) was recommended.

3. Results and Discussion

3.1. Optimization of Sample Treatment

3.1.1. Selection of Deprotein Reagent

As the real samples contain large amount of interferents, such as nucleic acid, protein, fat, etc., which would ruin the separation and detection, in this section, kinds of organic solvents, including methanol, acetonitrile, and acetone (the percent of organic solvent was set to 70%) as well as trichloroacetic acid (TCA), were tested for their abilities to reduce the possible interference. Our data showed that all these four sample treatment protocols could perform satisfied cleaning results. However, the recoveries (present via peak heights) obtained from these treatment methods differed (Figure 1, the shown data throughout this paper was obtained based on average of at least three parallel samples). It could be seen that, acetone treated sample showed the highest peak height among all the selected sample treatment methods and was regarded as the best one for further usage.

3.1.2. Ratio between Acetone and Real Sample

In this section, the ratios between acetone and real sample were investigated in a certain range (from 30% to 70%, percent of acetone in total volume). The electropherograms were shown in Figure 2. It could be seen that when the percent of acetone was less than 50%, the MEL could not be separated from interferents (arrow pointed in Figure 1); on the contrary, when it was larger than 50%, interferents were well separated from MEL. For the selection of acetone percent, a 60% was considered as the best one which maintained better Rs (3.57) and highest theoretical plate number (N) value (123,000) and less dilution to samples.

3.2. Selection of Separation Conditions

3.2.1. pH Value of Run Buffer

The pH value of run buffer was one of the important parameters that affect the separation and determination efficiency, as it influenced electro-osmotic flow (EOF) and the status of charged electrical species. Yan et al investigated the influence of pH in the range of 2.4 to 4.0, however, it's not adequate, as MEL contains three amino groups and the pKa values is around 5.0 - 5.5. In our works, a wider pH value ranging from 2.4 to 6.0 was carefully studied (Figure 3). It could be seen that, in pH range of 2.7 - 3.8, MEL (# in Figure 3) could be well separated from the interferent (* in Figure 3). It also could be seen that the migration times were similar to each other which may due to the slightly increase of EOF and decreased of positive charges of MEL when the pH increased. Furthermore, a change of peak order between MEL and the interferent was firstly observed in our works when the pH values went upper than 4.0 (at pH3.8, the peak of interferent appeared at the right side of MEL peak, but at pH4.5, it moved to the left side). For selection of best pH, the resolution (Rs, between MEL and interferent) combined with N values were evaluated. Our data (not shown) indicted that the Rs values went up as the pH values increased from 2.4 to 3.1, and went down when the pH increased continuously. Finally, a buffer pH of 3.1 was selected for its better N value (~115,000 ± 11,400, n = 3) and largest Rs value (3.71).

3.2.2. Selection of Buffer Concentration

The viscosity of the buffer and the thickness of the electric double layer were strongly related to buffer concentration, as a result, buffer concentration could affect EOF and hence the separation result. Normally, for selection of buffer concentration, the lowest concentration which provided a good separation was preferred, as the high concentration buffer may cause extra Joule heat generation, which in turn, caused peak broaden and decreasing in separation efficiency. In this section, a concentration range of 10 mmol/L to 100 mmol/L phosphate buffer at pH3.1 was investigated. Our works indicated that when the buffer concentration was higher than 10 mmol/L better separation could be easily obtained and when the buffer concentration was higher than 80 mmol/L, the current was too high (>200 μA) to maintain good separation (data not shown). The influences of buffer concentrations on migration times and N values were shown in Figure 4. It could be seen that the migration times increased while the buffer concentration became lager, (resulted in decreased EOF); And that the N values got its maximum value when the concentration set to 60 mmol/L (higher concentration led to higher Joule heat generation and poor efficiency), based on this, a buffer concentration of 60 mmol/L was selected for further studies.

3.2.3. Selection of Separation Voltage

In theory, higher voltage reduces analysis time, however, it may lead to significant losses of resolution and peak efficiencies if excessive Joule heat generated within the capillary. Based on this, the choice of separation voltage should be optimized with considering total analytical times, separation efficiency and other factors, like buffer

concentration etc, so that an acceptable level of current could be controlled. In this section, several voltages were investigated and the results were shown in Figure 5. As was shown, N values dropped while the voltages increased, especially when the voltages became larger that 17 kV (the current reached 180 μA). It’s also could be seen that the migration times dropped with increasing separation voltages and the total analytical times shortened, however, it’s at the expense of decreased N values and Rs (data not shown). A voltage of 17 kV was finally selected as its maintained shorter analysis time and satisfied N values (~155,400).

4. Method Validation

The linearity between peak area and the concentrations was obtained from the real samples adding different amount of MEL at a final concentration of 0.5 - 10 μmol/L of MEL. For measuring the reproducibility of the