Internal Standard Comparison
In the biphenyl separation method, all analytes performed best with
their matched ISs, though RSD and percent recovery remained in the
acceptable range for several substitutions (Table 3). For
17OHP4, the T-13C3 and
AE-13C3 ISs yielded good percent
recoveries (98.0 % and 108.9 % respectively) and RSDs (4.3 % and 1.4
% respectively), though measurements of endogenous concentrations
decreased, especially at lower concentrations. For calculating T, the
nearby 17OHP4-13C3 IS
peak performed well as an IS substitute (percent recovery = 110 % RSD =
3.7 %). For AE, the T-13C3 and
17OHP4-13C3substitutions showed good recovery (99.4 % and 107 % respectively) and
consistent endogenous AE measurements (Figure 3), but RSDs were not
considered acceptable (18.9 % and 21.0 % respectively). For
quantifying P4,
AE-13C3 was a good IS substitute, with
acceptable percent recovery (109 %), RSD (5.3 %), and
P4 measurements consistent with concentrations
determined with the matched IS. When quantified with
T-13C3 and
17OHP4-13C3,
P4 concentrations were 28.1 to 67.0 % lower than the
concentration determined with
P4-13C3 (Figure 3).
In the C18 method, the four 13C3 ISs
outperformed the two d 4 standards for
quantification of every adrenal steroid, except ALD (Figure 4). The
B-d 4 peaks had small peak areas compared to the
other analytes, which likely caused higher variability in the IS ratios
used to calculate concentrations. This is likely reflected by high RSD
values (18.5 % - 23.0 %) when using B-d 4 as an
IS for other adrenal steroids (Table 4). As a target compound, B was
best quantified with 13C3 ISs, which
were characterized by larger, more distinct peaks than
B-d 4. In comparison to an RSD of 18.8 % when
calculated with B-d 4, RSDs for B fell below 4.5
% when calculated with S-13C3,
F-13C3,
E-13C3, and
11DOC-13C3. Though ALD performed best
with ALD-d 4, this IS also had a relatively small
and broad peak, leading to more variability when used as an IS
substitute, with analyte RSDs ranging from 14.8 % to 16.5 %. Except
for quantifying 11DOC with S-13C3, the
accuracies and RSDs calculated for E, F, B, S, 11DOC, and DHEA using13C3 ISs all fell within acceptable
ranges.
For the most part, individual differences were the primary drivers in
analyte concentration. However, even when percent recovery and RSD fell
within acceptable thresholds, IS substitutions altered observed analyte
concentrations (Figure 4). In the female sample, concentrations of B
were close to the RL, 0.869 ng/g. When calculated with
B-d 4, concentrations fell below RL but exceeded
RL when calculated with S-13C3,
11DOC-13C3, and
E-13C3. In both methods, substitutions
that failed to meet acceptable RSD and/or percent recovery thresholds
altered concentrations substantially. In the C18 method, concentrations
varied from those calculated with matched ISs by as much as 63.3 %. On
average, good substitutions varied much less from matched IS analyte
concentrations (mean = 15.5 % median = 11.3 %) than poor substitutions
(mean = 31.7 % median = 30.1 %).