2.4. Method validation
Analyses were performed over a period of 2 weeks. Each batch consisted of replicate analyses of blanks (limit of detection), standard solutions (sensitivity and linear range) and both spiked and unspiked samples (recovery and precision).
2.4.1. Linearity and range
Linearity was addressed by preparing eight standard solutions of sodium nitrite and sodium nitrate ranging between 0.0125–10.0 and 0.025–15.0 mg/L. A linear regression analysis of analyte concentration versus peak response was performed.
2.4.2. Detection limits
The detection limits were calculated as the concentration corresponding to three times the background noise of the blank.
2.4.3. Quantification limits
The quantification limits were calculated as the concentration corresponding to ten times the background noise of the blank.
2.4.4. Recoveries
The experimental recovery was obtained from difference between two measurements (sample and spiked samples), according to the following relationship:
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The recovery ranges for nitrite and nitrate estimated immediately after sample preparation.
2.4.5. Precision
A total of three analyses were performed for one ham sample to evaluate the relative standard deviation (RSD%) of the method.
2.4.6. Reference methods
As no certified reference materials were available and in order to evaluate the accuracy of the results obtained by HPLC, the reference methods of the International Standards Organization ISO 3091–1975 and ISO 2919–1976 [24] and [25] were used for the determination of nitrite and nitrate levels, respectively in meat products.