Table 3. Inter- and Intra-batch Reproducibility of Full Process Replicates for LC-MS Quantification of Estrogen Metabolites.

On each of 3 days, three replicate samples of charcoal stripped human urine (CSHU) were spiked with 250, 1000 and 6400 pg/mL estrogen metabolites. Variability in LC-MS measurements are reported as CV (%). CV values should not exceed 20%; if it does then the source of the variation should be identified [30].

Metabolite	Inter-Batch CV (%)			Day 1 Intra-Batch CV (%)			Day 2 Intra-Batch CV (%)			Day 3 Intra-Batch CV (%)
	250 pg/mL	1000 pg/mL	6400 pg/mL	250 pg/mL	1000 pg/mL	6400 pg/mL	250 pg/mL	1000 pg/mL	6400 pg/mL	250 pg/mL	1000 pg/mL	6400 pg/mL
E3	13	10.1	12.5	1.4	1.2	3.5	5.8	7.2	3	4.2	2.2	7.6
E1	9.5	7.5	9.7	0.4	3.1	3.9	4.2	4.6	4.1	7.6	3	7.3
2-OHE1	7.1	5.5	6.9	3.1	4.6	3.3	3.6	7.7	4.9	4.9	0.9	7.8
16-ketoE2	5.3	5	7.3	0.7	1	4.8	6.6	6.5	3.4	5.7	6.2	6.2
4-OHE1	4.4	4.5	7.1	3.1	3	3.9	3.5	6.4	5	5.6	3.2	8
16α-OHE1	11	8.1	10.6	1.3	1.7	4.3	4.9	5.3	4.7	5.3	2.3	7
2-MeOE1	15.9	5.7	6.8	16.1	8.5	5.2	4.6	6.3	3.1	5.3	1.6	7.1
4-MeOE2	5.6	3.5	6.8	1.2	2.3	4.7	4.3	4.7	2.2	9.3	2.1	7.7
E2	13.6	10.5	10.9	1.7	1.7	4.5	6.4	6.8	3.6	4.6	2.5	8.4
16-epiE3	5.9	16.1	15.4	2.3	6	3.9	4.6	11.5	7.8	4.4	4.7	7.9
17-epiE3	16.1	20.1	15.6	4.8	6.4	6.3	6.8	12	8.5	14.9	20.1	4.5
4-MeOE1	12.7	11.5	11.4	0.7	1.9	4.5	4.3	4.4	2.8	5.1	2.7	5.8
2-OHE2	11.9	10.7	9.9	2.4	2.1	3.7	6.2	7	3	7.7	6.8	8.8
3-MeOE1	10.5	8.6	9.3	0.9	6.1	3.8	5	6	2.9	4	5.1	7.3
2-MeOE2	9.8	7.4	8.4	1	2.8	3.7	5.7	5.2	3.3	5.7	1.2	5.6

^*No standard was available for 17-epiE3, used D₂-16-epiE3.