*3.2.2 2DLC analysis*

The 2DLC system consisted of two LC-20AB binary gradient pumps (Shimadzu Technologies), one six-port two-position switching valve (VICI Valco Instruments, Houston, TX, USA), a SIL-20A autosampler, two DGU-20A3 degassers, a CTO-20A column oven, and two SPD-M20A diode array detectors.

 A scheme of the 2DLC system is shown in **Figure 6**. For the first dimension (1D), a Venusil XBP-C4 analytical column (4.6 mm × 100 mm, 5 μm) coupled with a C4 guard column was used. One aspect is for separation of proteins and additives, and the other is for five proteins analysis. The target fractions (polar substances) from 1D were enriched by a trapping column (ODS C18, 4.6 mm × 50 mm, 5 μm) and switched into the 2D through a six-port valve. A Hypersil ODS-2 C18 column (4.6 × 150 mm, 5 μm) was used to completely separate the seven food additives in 2D. The mobile phase consisted of ACN/water/TFA (10/90/0.1, v/v/v) (solvent A) and ACN/water/TFA (90/10/0.1, v/v/v) (solvent B) for 1D and ammonium acetate (25 mM, pH 6.6) (solvent A) and ACN/water (50/50, v/v, pH 7.2) with 25 mM ammonium acetate (solvent B) for 2D. The column temperature was 40°C. The detection wavelengths for 1D was 214 nm, for 2D were 254 nm and 278 nm. The eluted program is shown in **Figure 6**. The injection volume was 5 μL, and each sample or standard was injected in triplicate.

**Figure 6.** 

*Schematic representation (a–c) and gradient, flow rates, and switching times (d and e) of the stop-flow heartcutting 2DLC system.* 

### **3.3 Optimization of chromatographic conditions**

In order to accurately quantify the five proteins and seven additives in milk powder samples using the 2DLC method, some important parameters were optimized, including the stationary phase, mobile phase, and switching time.

 According to the literature, a shorter switching time in the 2DLC system means better shape of the peaks in the 2D chromatogram [15]. Because the milk powder matrix is so complex, the ideal 1D column would be able to separate the proteins and additives into two groups. The additives with higher polarity were concentrated within a short period of time and eluted rapidly, while the proteins were separated completely after the elution of additives by adjusting the mobile phase. In order to achieve this goal, two columns were tested: a Venusil XBP-C4 column (4.6 mm × 100 mm, 5 μm) and a Venusil XBP-C8 column (4.6 mm × 100 mm, 5 μm). These two types of columns could separate the seven additives and five proteins as two groups. The seven additives were concentrated at 2.0–5.0 min on the C4 column and 2.0–6.0 min on the C8 column. Therefore, the Venusil XBP-C4 column was chosen as the 1D column because of the shorter switching time. **Figure 7A** shows the chromatogram of the seven additives and five major proteins. The seven additives were focused at the first minutes, and the five major proteins could be separated later. For 2D separation, Hypersil ODS-2 C18 column showed better separation performance for the seven additives. A trapping column was used as the interface between 1D and 2D, which should result in better enrichment of the targets [16]. For online 2DLC, the choice of the mobile phase is very important. Because of protein separation, ACN was chosen as the organic mobile phase. 0.1% v/v TFA was added to all mobile phases to improve the protein separation effect.

The mobile phases for 1D were A1, ACN/water (10/90 v/v, 0.1% TFA), and B1, ACN/water (90/10 v/v, 0.1% TFA). Solvent B1 was set at 25% from 0 to 5 min in order to elute the additives quickly. Due to the little polarity difference of proteins, a gentle gradient of 0.14% B min<sup>−</sup>1 was used to achieve good separation of the five proteins, which was consistent with the literature [17–19]. As shown in **Figure 7**, the proteins were eluted in the following order: αs2-CN, αs1-CN, α-Lac, β-CN, β-LgB, and β-LgA. It should be noted that there were no standards for αs1-CN and αs2-CN

*Analysis of Additives in Milk Powders with SPE-HPLC or 2D-HPLC Method DOI: http://dx.doi.org/10.5772/intechopen.86445* 

### **Figure 7.**

*The 2DLC chromatogram of the 12 mixed standards substances. The 1D chromatogram of seven additives (2.0– 5.0 min) and five proteins on the 1D C4 column (4.6 mm × 100 mm, 5 μm) (up) and the 2D chromatogram of the seven additives on the C18 analytical column (down). (1) Maltol, (2) saccharin sodium, (3) benzoic acid, (4) sorbic acid, (5) ethyl maltol, (6) ethyl vanillin, and (7) vanillin.* 

 proteins, only for their mixture [10]. The chromatographic profiles showed no carryover effects of these proteins. A shoulder for the αs1-CN standard can be seen due to the presence of its two variants (αs1-CN and αs2-CN), which are very difficult to separate completely. From the different findings from previous reports, the monomorphic α-Lac was eluted firstly than β-CN [17–19]. The three shoulders of β-CN corresponded to its variants. As previously reported, γ-CN is the proteolytic product of β-CN, so they could be eluted together [18]. For β-Lg, variant B eluted before variant A, which is consistent with the literature [18]. During the process of quantitative analysis, αs1-CN and αs2-CN were quantified together, as for the three variants of β-CN.

 Acetic ammonia is often used as the modifier in liquid chromatography separation. To obtain better separation of the seven additives in 2D, a series of acetic ammonia concentrations (15, 20, 25, 30 mM) were tested. When 25 mM acetic ammonia was added, the baseline was much more stable, and the peak shape was greatly improved. Therefore, the 2D mobile phase were as follows: A2, 25 mM acetic ammonia, and B2 ACN/water (50/50 v/v) with 25 mM acetic ammonia. The gradient program is shown in **Figure 6**. The initial mobile phase of 1D was optimized and set at 25% B1. If lower than 25% B1, elution of the seven additives would be taken too long in the 1D column, which could lead to sample loss in the trapping column before switching; if higher than 25% B1, maltol and saccharin sodium could be separated incompletely in 2D because of ACN in the trapping column.

The switching time is a key parameter in this method. Three switching time (2.0–4.5, 2.0–5.0, and 2.0–5.5 min) were tested. When the switching time was between 2.0 and 4.5 min, maltol and saccharin sodium were separated incompletely, and the sorbic acid peak was less sharp than that for 2.0–5.0 min; when between 2.0 and 5.5 min, some analytes were lost in the trapping column. Therefore, 2.0–5.0 min was chosen as the final switching time for the experiment. **Figure 7** shows the chromatogram of the 12 mixed standard substances using the optimized 2DLC method. In **Figure 7A**, the seven additives were eluted between 2.0 and 5.0 min due to their higher polarity, and the proteins were separated on the 1D column (8.0–30.0 min); **Figure 7B** shows the 2D chromatogram of the seven additives that were switched from the 1D column at 2.0–5.0 min. The whole analysis process was less than 30 min, which provide a highly efficient analysis method.

## **3.4 Comparison of analysis parameters for 1DLC and 2DLC**

The matrix effect, linearity, LOD, intra- and interday precision, and accuracy were validated under the optimized conditions for 1DLC and 2DLC.

The method validation parameters of 1DLC and 2DLC were shown in **Table 4**. The correlation coefficient values (*R*<sup>2</sup> ) for both methods are higher than 0.9988 for all the additives. And *R*<sup>2</sup> of ethyl maltol in 1DLC is lower than that in 2DLC.

Considering the complexity of milk powder, the possibility of a matrix effect was investigated by comparing the slope ratio of the calibration curves for the seven additives obtained in the presence and absence of blank milk powder [20]. For example, the slope ratio is closer to 1.0, which means a lower matrix effect in the method. The results in **Table 4** show that 2DLC (slope ratio: 0.94–1.09) had a lower matrix effect than 1DLC (slope ratio: 0.84–1.21). The sample matrix effect for the determination of the seven additives for both 1DLC and 2DLC can be seen in **Figure 8**. The milk powder sample matrix chromatogram of 2DLC (b′) is much clean and flat than that in 1DLC (a′), and there has no interference peak for the analytes. Although the matrix effect of 2DLC is low, we still chose the matrix-matched standard curve for the sample analysis [19]. The LOD values of the 2DLC method were higher than that of the 1DLC method, as the peak width obtained with the new method is broader than that with the conventional method. Those are the advantages and disadvantages of these two methods.

 **Table 5** shows the precision and recovery results of 1DLC and 2DLC. The intraday and interday data showed that the precision of the two methods is satisfactory. However, the recovery of the 2DLC method (89.6–103.5%) was much better than that for the 1DLC method (65.5–99.2%), which is mainly benefit from the "one-step" sample preparation method. Analytes may be lost during the processes of traditional sample pretreatment (such as solid-phase extraction, liquid–liquid extraction, and precipitation). In this method, the whole analysis time was less than 1 h. So, 2DLC is much more efficient than 1DLC. Overall considering the environmental protection and time saving, the automation offered by 2DLC possesses more advantages.

### **3.5 Commercial sample analysis**

Four different commercial milk and milk powder samples purchased from local supermarkets were analyzed using the developed 2DLC method. The chromatograms are shown in **Figure 9**. **Figure 9A** and **B** were infant formula milk powder (IFMP), **Figure 9C** was skimmed milk powder, and **Figure 9D** was fresh bovine milk. Benzoic acid and ethyl vanillin were detected only in the IFMP 1 sample. α-CN, β-CN, and α-Lac were detected in the four milk products. β-LgB and β-LgA were detected in the IFMP 2 and SMP samples.


### *Analysis of Additives in Milk Powders with SPE-HPLC or 2D-HPLC Method DOI: http://dx.doi.org/10.5772/intechopen.86445*


**Table 4.** 

*Method validation parameters of 1DLC and 2DLC (n = 3).* 

**116** 

*Analysis of Additives in Milk Powders with SPE-HPLC or 2D-HPLC Method DOI: http://dx.doi.org/10.5772/intechopen.86445* 

### **Figure 8.**

*1DLC (A) and 2DLC (B) chromatograms for testing sample matrix effect. (a′ and b′) Sample matrix without standard substances. (a and b) Sample matrix with standard substances. Chromatographic peaks: (1) benzoic acid, (2) sorbic acid, (3) saccharin sodium, (4) maltol, (5) ethyl maltol, (6) vanillin, and (7) ethyl vanillin.* 



### **Table 5.**

*Accuracy of the two methods (n = 6).* 

**Table 6** showed the contents of the five major proteins and the seven additives. The contents of α-CN and β-CN were much higher than that of α-Lac, β-LgB, and β-LgA in all the milk products. The contents of α-Lac, β-LgB, and β-LgA were lower in the infant formula milk powder than that in the skimmed milk powder. The results are consistent with those from the literature [17], which is probably due to the denaturation of the thermosensitive whey proteins [21] or intentional removal of β-LgB to prevent allergic reactions [22].

In order to evaluate the accuracy of protein determination using the proposed method in this work, four brands of commercial milk products were analyzed using both the 2DLC and Kjeldahl methods. **Table 7** shows the total major protein contents in the various milk matrices determined by these methods, 2DLC, the Kjeldahl method, and TPC, as given by the manufacturers. The RSD of the three groups were less than 3%, which means that the milk protein contents were similar for our method and the Kjeldahl method as well as that given by the manufacturers.

*Analysis of Additives in Milk Powders with SPE-HPLC or 2D-HPLC Method DOI: http://dx.doi.org/10.5772/intechopen.86445* 

### **Figure 9.**

*Chromatograms of four brands of commercial milk and milk powders. (A) Infant formula milk powder 1, (B) infant formula milk powder 2, (C) skimmed milk powder, and (D) bovine milk. (1) α-Casein (α-CN), (2) α-lactalbumin (α-Lac), (3) β-casein (β-CN), (4) β-lactoglobulin B (β-LgB), and (5) β-lactoglobulin A (β-LgA).* 


*a IFPM, infant formula powder milk; SMP, skimmed milk powder; BM, bovine milk.* 

*b ND, not detected.* 

*c The values of the concentration are means ± SD (n = 3).* 

### **Table 6.**

*Contents of food additives determined in milk powder samples by 2DLC (n = 3).* 


*a TMPC, the total major protein concentrations.* 

*b TPC, the total protein concentration.* 

*c Powder milks in g/100 g and liquid milks in g/100 ml.*

*d RSD among the data determined by the two methods and indicated by manufacturers.* 

*e The values of the concentration are means ± SD (n = 3).* 

### **Table 7.**

*Comparison between the total major protein concentrations (TMPC) in the various milks determined with 2DLC method and the total protein concentration (TPC) determined with Kjeldahl method and TPC given by the manufacturers.* 
