**2.2 Comparison of sample preparation methods**

 For method A (liquid-liquid extraction), the extraction time of 10, 15, 20, 25, 30, 35, 40, 45, and 50 min was investigated. The results are illustrated in **Figure 1**, indicating 40 min was the best choice. The different extraction temperatures from 25 to 40°C were tested, and the results showed that there were no obvious differences in recoveries of each preservative. For method B (precipitation based on sodium tungstate) and method C (precipitation based on potassium ferrocyanide), the results showed that there were no significant differences between these two methods. The recoveries of benzoic acid, sorbic acid, and saccharin sodium were more than 80%. However, the recovery of lysozyme (<30%) indicated that both of the precipitate-based methods were not suitable for enzyme [14] and it might be coprecipitated with the proteins in milk powder. For method D-F (SPE), three kinds of cartridges (CNWBOND LC-C18 SPE, Poly-Sery HLB SPE, and Poly-Sery MAX SPE) and their elution solvents applied were investigated. Porous silica particles surface bonded with C18 was the most commonly used sorbents. Poly-Sery HLB is the kind of polymeric sorbents that was reported as being superior to the silica-based C18. The major difference between the HLB and MAX sorbents is the presence of the anion-exchange groups that provide high selectivity for acidic compounds. So, the three kinds of cartridges were tested to evaluate their applicability. Four elution solvents with different polarity were tested: 70% acetonitrile, 80% acetonitrile, 90% acetonitrile, and 100% acetonitrile. The recoveries of each food additive showed that Poly-Sery HLB with 80% acetonitrile provided the best results.

**Figure 2** shows the recovery results of each food additive obtained by the six different abovementioned methods, respectively. Each sample was analyzed in triplicate. Both method A (liquid-liquid extraction) and method D (Poly-Sery HLB SPE)

### **Figure 1.**

*Effects of extraction time on the six food additives recovery obtained with method A in the two milk samples (n = 3). (A) Whole milk powder and (B) skimmed milk powder (SMP). The six food additives: benzoic acid, sorbic acid, natamycin, lysozyme, aspartame, and saccharin sodium.* 

have good recoveries (>80%) for all six analytes, but results of our study showed that the average RSD% for method A of the six analytes were all between 3.7 and 5.4%, which is more than that of HLB SPE method (2.3–3.8%). Considering the environmental and economic costs, method D (Poly-Sery HLB SPE) was employed in our further study.

### **2.3 SPE (Poly-Sery HLB)-HPLC-DAD method validation**

 The proposed SPE (Poly-Sery HLB)-HPLC-DAD method was validated in terms of linearity, limit of detection, limits of quantity (LOQ ), within- and betweenday precision, and accuracy. **Figure 3** shows the chromatograms of the mixed standard solutions. We can see that the six preservatives and sweeteners were

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

baseline separated and had a good resolution (R ≥ 2.6) under the chromatographic condition.

The linearity of the method was evaluated under 210 nm with the mixed standard solutions, pooled whole milk powder matrices,and pooled skimmed milk powder matrices. The LODs and LOQs in each case were estimated based on S/N = 3 and 10, respectively. The results including the regression equations, the linear ranges, and regression coefficients are summarized in **Table 1**.

 The precision and accuracy were tested in two milk powder matrices, that is, the whole milk powder and the skimmed milk powder. **Figures 4** and **5** showed the

### **Figure 2.**

*Recovery results of the six different sample preparation methods. The left group in A–F represents the whole milk powder matrix, and the right group in A–F represents the skimmed milk powder matrix. The six food additives: benzoic acid, sorbic acid, natamycin, lysozyme, aspartame, and saccharin sodium. (A) Method A (liquid-liquid extraction). (B) Method B (precipitation based on sodium tungstate). (C) Method C (precipitation based on potassium ferrocyanide). (D–F) Method D-F (solid phase extraction): (D) Poly-Sery HLB SPE, (E) CNWBOND LC-C18 SPE, and (F) Poly-Sery MAX SPE.* 

### **Figure 3.**

*Chromatogram of mixed standard solutions. (1) Benzoic acid, (2) sorbic acid, (3) saccharin sodium, (4) natamycin, (5) aspartame, and (6) lysozyme.* 

representative chromatograms. All chromatographic peaks were separated completely and had a good resolution (R ≥ 1.5). Recovery studies of benzoic acid, sorbic acid, natamycin, lysozyme, saccharin sodium, and aspartame were evaluated by analysis of blank pooled samples spiked with 10 μg g<sup>−</sup><sup>1</sup> (lower level), 50 μg g<sup>−</sup><sup>1</sup> (middle level), and 100 μg g<sup>−</sup><sup>1</sup> (upper level) of each analyte. And the data were calculated based on the matrix-matched regression curves and summarized in **Table 2**. The intraday precision was studied at lower, middle, and upper concentration levels (n = 5). The interday precision was analyzed with spiked samples at 50 μg g<sup>−</sup>1 for six-day determinations (**Table 3**).


### **Table 1.**

*Comparison of the cost-effectiveness of six methods.* 

### **Figure 4.**

*Chromatograms of the blank whole milk powder sample and spiked whole milk powder sample. (A) The spiked whole milk powder sample: (1) benzoic acid, (2) sorbic acid, (3) saccharin sodium, (4) natamycin, (5) aspartame, and (6) lysozyme (50 μg g<sup>−</sup><sup>1</sup> of each food additives). (B) The blank whole milk powder sample.* 

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

### **Figure 5.**

*Chromatograms of the blank skimmed milk powder sample and spiked skimmed milk powder sample. (A) The spiked skimmed milk powder sample: (1) benzoic acid, (2) sorbic acid, (3) saccharin sodium, (4) natamycin, (5) aspartame, and (6) lysozyme (50 μg g<sup>−</sup><sup>1</sup> of each food additives). (B) The blank skimmed milk powder sample.* 


*(A) Aqueous, (B) pooled whole milk powder, and (C) pooled skimmed milk powder.* 

*b y is the average peak area of each analyte (n = 3), and x is the mass concentration of the analyte in μg L<sup>−</sup><sup>1</sup>*

*.* 

### **Table 2.**

*Linearity and LOD of the developed method (n = 3).* 


**Table 3.** 

*Precision and accuracy of the assay for whole milk powder and skimmed milk powder analysis (n = 3).* 
