\r\n\t\u00a0\r\n<\/p>\r\n\r\n
\r\n\tThe time during which the stability is tested, is important, but it is usually not specified in the guidelines and\u00a0only brief recommendations are given. The reason being that depending on the particular situation, the suitable duration of the stability study can be very different<\/strong> and should be decided by the analyst<\/strong>\u00a0based on the situation at hand.\u00a0\u00a0Usually when the\u00a0stability of the analyzed sample solutions or standard solutions is evaluated, the injections of the sample solutions and standard solutions are carried out overnight \u00a0(i.e. the duration of the study is 10-20 h).\u00a0Testing of not yet processed samples should be carried out covering the actual duration of the sample storage during the proposed research study. Analyte concentration should be evaluated over a specific storage period and in conditions that would improve the stability over time, should be identified.\r\n<\/p>\r\n\r\n \r\n\tThe experimental design\u00a0<\/strong>of the stability\u00a0testing should take into\u00a0<\/strong>account. The most important parameters\u00a0\u2013 storage time\u00a0<\/strong>and\u00a0 temperature. <\/strong>Stability<\/strong>\u00a0results should be used as the indicator to adjust one or the other in order to improve the\u00a0<\/strong>stability<\/strong>\u00a0of the analyte or internal standard.<\/strong>\r\n<\/p>\r\n\r\n \r\n\tStability should be studied at least at two concentration levels \u2013 low and high concentration level and in a matrix\u00a0matching the \u201creal-life\u201d matrix.\r\n<\/p>\r\n\r\n \r\n\tFor doing this,\u00a0the native blank biological matrix should be used and the analyte should be spiked into the matrix at these concentration levels.\r\n<\/p>\r\n\r\n \r\n\tBench-top stability or short-term stability at room temperature or sample processing temperature will indicate the analyte stability under sample preparation conditions. Freeze-thaw stability is usually evaluated during three thawing cycles to predict the\u00a0influence of\u00a0possible delays and glitches on the sample handling.\r\n<\/p>\r\n\r\n \r\n\tStability should be evaluated at different time points and samples should be analyzed in six [ref 2] or miminum of three [ref 7, ref 8] replicates. However, EU regulation 2021\/808 [ref 5] finds evaluation from at least five replicates sufficient.\r\n<\/p>\r\n\r\n \r\n\tThe analyte or internal standard stability in the test or reference solutions, ST<\/em>%, expresses the part of the analyte or internal standard in a sample that does not decompose before the actual LC-MS analysis of the sample.\u00a0In the absence of decomposition,\u00a0ST<\/em>% = 100%.\r\n<\/p>\r\n\r\n \r\n\t\u00a0\r\n<\/p>\r\n\r\n\r\n\t