Bibian van der Voorn

69 MEASUREMENT OF HUMAN MILK GC LEVELS positive ionization mode. Capillary voltage was 0.5 kV and the source temperature 120 °C. Argon was used as collision gas. Cortisol and [ 2 H 4 ]cortisol were measured using the transitions (Q1>Q3)m/z 363.2 à 121.1 and m/z 367.2 à 121.1, respectively. Cortisone and [ 2 H 8 ]cortisone were measured using the transitions (Q1>Q3)m/z 361.2 à 163.1 and m/z 369.2 à 124.1, respectively. TABLE 1. Intra- and interassay coefficients of variations (CVs) of cortisol and cortisone in human milk Intra-assay variation Mean cortisol concentration* n Intra-assay CV % Mean cortisone concentration* n Intra-assay CV % 0.56 13 13% 3.1 10 7% 3.3 38 8% 7.7 8 5% 7.0 77 4% 15.0 36 6% 23.0 48 5% 33.0 74 5% Inter-assay variation Mean cortisol concentration* n Inter-assay CV % Mean cortisone concentration* n Inter-assay CV % 6.3 18 9% 31.9 16 9% 22.0 5 4% *concentrations are reported in nmol/L Table 1 shows that the intra coefficients of variation (CV%) were 4% and 5% at a level of 7 and 23 nmol/L, respectively, for cortisol and 5% at a level of 8 and 33 nmol/L for cortisone and that the inter CV%was <9% for both cortisol and cortisone. Recovery, as judged by the recovery of spiked analytes, was 97 to 102% for cortisol and 98 to 106% for cortisone. Linearity was shown by 2-, 4- and 8-fold dilution. The difference between expected and observed values varied between 93 and 106%. The Lower Limit of Quantitation (LLOQ) with a total allowable intra-assay CV of 15% was 0.5 nmol/L for cortisol and 0.25 nmol/L for cortison, determined using diluted milk samples. In addition, the stability of cortisol and cortisone in human milk under various conditions was evaluated. We determined the stability of cortisol and cortisone in milk stored at room temperature by aliquoting one sample of fresh human milk and storing these for 0, 2, 4, 6, 8, 10, 12, 24 and 36 hours at 4°C and 20°C. After these time periods all aliquots were directly stored at −20°C. Subsequently, all samples were measured in one run. The analysis showed that both cortisol and cortisone were stable (<10% decrease) during 36h storage at 4°C and 20°C in comparison to direct storage at −20°C. Finally, we evaluated the influence of freeze-thaw cycles and saw that at least six cycles did not influence (<5% decrease) cortisol and cortisone concentrations in human milk (n = 4).