FAAS experiments were performed in a flame atomic absorption spectrometer (AA240FS; Varian, Mulgrave, Australia) fitted with a deuterium lamp for background correction. Ca and Na were determined in absorption and emission mode, respectively. The experimental conditions used (wavelength, lamp current for Ca and acetylene and air flow rates) were those recommended by the manufacturer.

All solutions were made with deionized water (Milli-Q Plus, Millipore, Barueri, São Paulo, Brazil). Calcium (0.25 - 4 mg·L^–1) and sodium (0.1 - 0.6 mg·L^–1) aqueous standard solutions were prepared after successive dilutions of the metals standard stock solutions of 1000 mg·L^–1 (Merck, Darmstadt, Germany).

Milk samples were furnished by a dairy company located at São Carlos (São Paulo State, Brazil). The samples were previously analyzed by several parameters and the results were in agreement with Brazilian’s rules [21] which are compatible with international’s rules. The samples were adulterated with whey, tap-water, synthetic milk, synthetic urine, hydrogen peroxide, urine and commercial NaOH (caustic soda) in different percentage of adulteration (5%, 15%, 25%, 35% and 50% (v/v)), except for the caustic soda. In this case, the fresh milk was turned sour (at ambient temperature) and NaOH (10 mol·L^–1) was added to establish the original pH. Synthetic milk and synthetic urine were prepared according to literature [15,22]. Whey solutions were made according to the manufacturer’s recommendations. Commercial NaOH (10 mol·L^–1) and tap-water were used to simulate the adulteration procedure. For adulteration with hydrogen peroxide, the p.a. reagent was applied.

In order to develop a fast and direct method to detect milk adulteration, determination of Ca and Na were performed without acid digestion process. The samples were just diluted in deionized water until the Ca and Na concentrations were agreeing with the linear equipment range. The method was validated by the comparison to the standard additions data.

Milk is a complex matrix that contains compounds may be interfering in the analytical results. Several procedures of sample preparation have already used to reduce these interfering, and most of them involve a step of digestion to eliminate the organic matrix [23,24]. In this study, a simple dilution of the milk in water was tested and results obtained were compared with the standard additions. Standard addition can be applied to the most analytical techniques in instead of a calibration curve to solve the matrix effect problems.

Figure 1 shows the results obtained with the aqueous standards calibration and the standard addition methods for Ca. The aqueous standards calibration curves (black circles) were prepared by appropriate dilution of Ca standard stock solution in water (0.25, 0.5, 1, 2 and 4 mg·L^–1) and the standard addition (black squares) by addition 0.0, 0.2, 0.8 and 1.6 mg·L^–1 of Ca in milk samples. The parallel behavior of the regression lines and the similar angular coefficient (0.123 and 0.118), indicating that the method developed can be applied to quantify Ca and Na in milk.

Table 1 shows Ca and Na concentrations in pure and milk adulterated with water, whey, synthetic urine, synthetic milk, hydrogen peroxide and caustic soda. The concentrations of Ca and Na in pure milk were 1131 ± 29 mg·L^–1 and 794 ± 23 mg·L^–1, respectively. Ca concentrations varied from 1342 ± 97 (for milk adulterated with whey at 5%) to 531 ± 32 mg·L^–1 (for milk adulterated with water at 50%) and Na from 1659 ± 86 (for milk adulterated

with urine at 5%) to 592 ± 20 mg·L^–1 (for milk adulterated with synthetic milk at 50%). The largest decrease in the Na and Ca concentrations were observed in samples adulterated with synthetic milk and tap-water. These effects can be associated to increase in the milk volume due the addition of these liquids adulterants. On the other hand, the largest increase in the Na and Ca concentrations was observed for Na in the milk adulterated with caustic soda. Na concentrations varied from 794 ± 23 (for pure milk) to 2219 ± 67 mg·L^–1.

Significant differences in the Ca and Na concentrations were also observed in the samples adulterated with whey. In this case, the addition of whey results in a nonlinear variation of Ca and Na concentration. According to the manufacturer, whey is composed by 1086 and 796 mg·g^–1 of Na and Ca, respectively. Therefore, the adding of whey provides not only the dilution of samples but also an increase in the Ca and Na concentrations. Same behavior (nonlinear variation) was observed in the samples adulterated with hydrogen peroxide and synthetic urine. Figure 2 shows the variation of Ca and Na concentrations in milk adulterated with synthetic urine. Results obtained showed that the Na concentration increased significantly after the first addition of synthetic urine (from 794 ± 23 to 1659 ± 89 mg·L^–1) and, after that, the concentration decreased until 884 ± 23 mg·L^–1 with an adulteration of 50%. For Ca, the behavior was the opposite. Ca concentration decreased to 925 ± 39 mg·L^–1 after adulteration of 5% synthetic urine and increased to 959 ± 100 mg·L^–1 with adulteration of 25%.

A paired t-test was used for comparing the concentrations of Ca and Na obtained in pure and adulterated milk. Results showed that the Ca concentrations in pure milk was statistical different to the samples adulterated with water and synthetic milk (calculated t values higher than 2.776) (Table 1). Na concentrations in pure and adulterated milk were statistical different just in the samples adulterated with NaOH and synthetic urine. Adulteration

by addition of whey and hydrogen peroxide were the most difficult to identify by variation of Na and Ca concentrations. According to the results, significant difference in the Na and Ca were obtained just for some selected levels analyzed.