Stronger ESI signal for deuterated substances
There are a couple reasons this could be happening.
The most interesting possibility: isotope effects during ion fragmentation
You are performing LC-MS/MS, i.e., your mass spectrometer is chemically reacting the estradiol and estradiol-$d_5$ with gas molecules in the collision cell of the instrument.  This process, known as CID or collisionally induced dissociation, will break chemical bonds in the parent molecule and form fragment ions.
On triple-quad and qTOF instruments, the CID is of the &quot;beam&quot; or non-resonant type.  This means a fairly large amount of energy is imparted to parent ions.  In general, this means that a wide variety of fragment ions are formed.  This is true of estradiol; publicly available fragmentation spectra show many fragment ions for this compound (see several GNPS spectra for example).
Deuteration is likely to change the relative propensity of the different fragmentation pathways.  For example, these reactions could represent the fragmentation of a parent ion $\ce{P}$ into two different fragments $\ce{F1}$ and $\ce{F2}$.
$$\ce{P -&gt; F1}$$
$$\ce{P -&gt; F2}$$
Isotopically normal $\ce{P}$ might give an intensity ratio of say 1 to 1 for $\ce{F1}$ and $\ce{F2}$.  But due to deuterium isotope effects, the pathway to $\ce{F1}$ may be much less favored in deuterium-labeled $\ce{P}$.  This could lead to an enhanced signal for $\ce{F2}$ as a much higher fraction of the $\ce{P}$ ion remains available to proceed down that fragmentation pathway.
However, a three-fold increase is pretty large.  I was able to find good measurements of various deuterated tocopherols, and judging from Table 1 in that paper, the response ratio for $d_0$ (unlabeled) tocopherol vs. $d_9$ is 11.8/0.967/10, or 1.22, not 3.  For measurement of trimethylamine N-oxide, authors note &quot;the intensity ratios of the two product ions are different between TMAO and $d_9$-TMAO due to the difference in bond energy among C and H and C and D (deuterium)&quot; but did not quantify this difference.  Eyeballing Figure 1, the intensity ratio of the light and heavy fragment is about 2.5:1 for $d_0$ but 1.4:1 for $d_9$.  None of those values are close to 3-fold.  But maybe estradiol and its site of deuteration and its fragmentation pathway differ and that a value of 3-fold is possible.
Errors in sample preparation
Maybe the concentration of one of your samples is off.  How many times have you independtly prepared the standards and repeated the measurement?
Ways to tell
If you can see a signal with a single quadrupole (i.e. do MS1 instead of MS/MS), do you see the same three-fold difference?  If so, error in sample prep is likely to blame.  If not, then isotope effects during ion fragmentation are to blame.