Furthermore, maintaining the milk at pH 8.5C9.0 reduced degradation of residues in vials placed in autosampler up to 24 h, and ensured repeatability of injections in the LC instrument. 99 to 104% and precision between 1 and 10%, RSD for both compounds. The limit of quantitation (LOQ) was 0.0020 mg kg?1 and other tested parameters (linearity, sensitivity, specificity, matrix effect, robustness, etc.) satisfied acceptance criteria. AP1903 Stability assessment using spiked samples revealed the compounds were stable in natural and pasteurised milk for 4 weeks at C80 C storage temperature. Maintaining samples at pH 8.5C9.0 further improved stability. Analysis of 516 milk samples from the field study found that NBPT and NBPTo concentrations were below the LOQ of 0.0020 mg kg?1, thus suggesting very low risk of residues occurring in the milk. The method developed is quick, robust, and sensitive. The method is deemed fit-for-purpose for the simultaneous determination KIAA1704 of NBPT and NBPTo in milk. and NBPTo 151.90 92.90 = 30), %68C11231C117RSD (%)1316 Open in a separate window 2.3.2. Matrix Effect The impact AP1903 of matrix effects was assessed by fortifying 30 different negative raw milk samples at 0.0020 mg kg?1 post-extraction and comparing the response of each sample against solvent standard at the same level. The results showed there was both enhancement and suppression effects for both NBPT (ME range = 68C112%) and NBPTo (ME range = 31C117%). This is because the individual raw milk samples came from different sources and in different batches. The phenomenon of matrix components suppressing or enhancing the signal responses of target analyte ions in a bioanalysis conducted with LC-MS technique is known to occur frequently [18,22]. A key recommendation to compensate for this matrix effect is by adopting a matrix-matched calibration. In this study, a matrix-matched calibration was applied, which minimised the effects within the acceptable range of 80C120%. 2.3.3. Trueness and Precision In order to demonstrate the trueness and precision of the method under within laboratory repeatability (RSDr) and reproducibility (RSDwR), mean recoveries and relative standard deviations were calculated for all the spike levels tested in the ten analytical runs (Table 1). The results obtained showed that trueness for the high (0.0020 mg kg?1) and low (0.0250 mg kg?1) levels tested were satisfactory and met the acceptability criteria (80C120%), with NBPT values ranging from 87C118% (low level) and 92C118% (high level), while NBPTo ranged from 80C118% (low level) and 91C118 (high level). Furthermore, the method precision also showed overall satisfactory results of 8% (NBPT) and 10% (NBPTo) for both RSDr and RSDwR. Having met the precision acceptability criteria of 20%, the method developed proved to be accurate and precise for the quantitation of NBPT and NBPTo residues. The criteria for determining the robustness of this method was also satisfied by the values obtained in the average recoveries and RSDwR, which were also within acceptable limits. Adding to the robustness of the method, a new column from a different batch, introduced during the validation runs, caused a shift in the retention times of both NBPTo (1.10 to 1 1.19 min) and NBPT (2.10 to 2.19 min) in milk matrix. However, the difference in the retention time shifts were AP1903 0.1, which is the allowable limit recommended in SANTE/12682/2019 guidelines. Furthermore, different batches of ultrafiltration tubes and mobile phase solvents, supplied by different vendors were used during analysis. The changes in brands and batches of consumables had a negligible effect on the results obtained. This further validated the robustness of the method. 2.3.4. Limit of Quantitation The limit of quantitation (reporting limit) was determined as the lowest spike level meeting the method performance criteria of trueness and precision within laboratory repeatability and reproducibility. The value corresponded to 0.0020 mg kg?1, which is the second calibration level in the 8-point calibration employed in this study. Trueness and precision was monitored in all the ten validation runs. The LOQ obtained in this method is much lower than that obtained in a previous study  (0.050 mg kg?1). The difference might be because both studies used LC-MS/MS instruments from different manufacturers with different method parameter settings, etc., which most likely resulted in varying levels of detection capacity. 2.4. Application of Method 2.4.1. Stability Assessment of Standards in Solvent and in Milk Matrix Understanding the stability of the compounds under different conditions (storage temperature, solvent types, sample matrix, pH and storage time) is essential in ensuring accuracy of experimental results . Stability studies also enable identification of optimum storage conditions for experimental samples to ensure target analytes for quantitation are not degraded before and during.