Ult HiUltraHigh
gh Sensitivity
Siti ity Analysis
A ly i off Testosterone
T t t

MSACL Poster # 46

1Michal Star
Star-Weinstock,
Weinstock 1Brian L.
L Williamson
Williamson, 1Subhakar Dey
Dey, 1Babu Purkayastha,
Purkayastha 2James E.
E HillHill, 2Jeanette R.
R Hill1AB SCIEX,SCIEX 500 Old Connecticut Path,
Path Framingham MA,
MA 01701
2Spot On Sciences
Sciences, 13705 Shadowglade
Shadowglade, Manor
Manor, TX 78653

ABSTRACT

Results

Free Te From Serum or Saliva

T t lTTotal
Te ffrom D
Driedi d Bl
BlooddS
Spotst (DBS)Analysis of Testosterone (Te) from both Free Te samples (FTe) and Dried Blood Spots(DBS) utilizing
derivatization
withreagentt iis ddemonstratedtili idi ti ti
ith a novell aminoxyit t d
The LLOQ after derivatization is <1 pg/mL for FTe samples and ~40 pg/mL for DBS
yg with %CV <15 at LLOQLinearityy ((R2>0.998)) is maintained over a broad dynamicrange
QQAO-derivatization and sample
p ppreparation
pworkflows are quick,q, simple,
p ,reproducible, robust and yield high recoveriesINTRODUCTION
Testosterone (Te) analysis by LC/MS/MS is becoming the analytical method of choice over immunoassays due
to its specificity and accuracy. Neutral steroid hormones undergo poor ionization efficiency in MS/MS, resulting
in insufficient sensitivityy to analyze
y samplespwhich ppossess trace concentrations. The method ppresented herein
utilizes a tag
g moietyy (quaternary(qy aminoxyy reagent)g ) which binds to the keto functionality
y of Te or any
y other ketog
y enhances its MS/MS sensitivity.
y The derivatization techniqueq enables quantitation
qsteroid and significantlyofFree Te (FTe) from samples such as equilibrium dialysates, ultra filtrates and saliva, Moreover, the derivatisation
technique enables the use of very small amounts of sample such as in Dried Blood Spots (DBS).

IS10 pg/mLATe))IS (d3 T
QAO reagentgTestosterone

QAO –TeTe

Figure2. Schematic illustration of QAO derivatization of Te.
g
The reaction is complete within 45 min at RT

Endogenous
FTe
0.94 pg/mLpg

EndogenousTeg
Measured as483.9 pg/mL

Figure8.
Concentration1-50
Fi8 Ct ti curve 150 pg/mL,/ L calibrator
libspiked
ik d into
i t ultra
lt filtrate
filt t MWCO 30 kD
kDa off ffemale
l serumL) The
Th calibrtor
lib t was d3 Te
T andd theth IS 13C
pooll (500 uL).
Te.
TeFigure
5. DBS Concentration
50Fi
5Ct ti curve off d3 Te
T 50/ L spiked
ik d intoi t 10 µL
L wholeh l bld
10000 pg/mL
blood

REAGENTS AND METHODS
A50 pg/mL
d3 Te StdFemale
Fl saliva
liendogenousdFree Te 2.121pg/mL

B
Underivatized
Te IS

20
pg/mL
Td3 TeStdFigure 3
3. MS/MS fragmentation of QAO Te.
TeB
Figure 4 illustrates the sensitivity
enhancements after derivatisation of Te(1pg on column).
Underivatized,, derivatized with Hydroxyl
yygamine and derivatized with QAO reagent
were all compared.
pThe S/N ratio of the QAO derivative issuperior due to careful selection of specificfragment ions and higher mass of thederivative that drives it farther away fromthe background noise.

Figure 1
1. Schematic description of the extraction and derivatization workflow for Free Te samples from serum
or Saliva and Dried Blood Spots
The QAO reagent (10 mg/mL), is dissolved in MeOH:5% acetic acidd3 Te is used as calibrator (Cerriliant ,99.8% pure). 13C Te used as Internal Standard (IS), Isosciences, >98% pure. Matrix: Female whole serum(Golden West Bio PS 1040)
LC Gradient Conditions: Acetonitrile / water /formic acid (0.1%) mobile phase using a C18 column (Cadenza CL). Step Gradient 1010-30%B
30%B in thefirst 0.5 min, then 30-55%B within 3 min. Shimadzu AD -30 series and QTRAP® 5500 LC/MS/MS system.

Endogenous Te
Measured as
43 pg/mL

10. Free Te analysis from female saliva
saliva.
Figure 10
1 mL saliva was extracted by the method described in Fig
Fig. 33.

Conclusions

Figure 66: 10 µL DBS
femaleblood
S fromff
A d3 TeT St
d d spiked
ik d as 50 pg/mL/ L (0.5(0 5 pg iinA.
Standard
10 µL
blood,
L bl
d on a conventionaltil filter
filt paper))

Notes:
Nt
1 Offsetting
Off tti off retentiont ti titimes were usedd tot
1.
position the peaks for visual demonstration.
2. The Hydroxyl amine (HA) Derivatization of Te
was repeated from Kushnir MM. et al. Clinical
Ch iChemistry
56:7,1138–1147
6 1138 114 (2010)

Reproducibility: At this concentrationlevel of ~1pg/mL the % CV = 14.7 (n=15)

No peak ofunderivatized Te

Structure specific fragments are selected to enhance specificity and reduce background noise.
noise The MRM
Transitions for QAOQAO- Te are 403.3
403 3->164>164.22 and 403
403.3
3->152>152.22 at CE=60eV
CE 60eVFigure 9. Ultra filtrate (30kDa MWCO)
of Female serum pool spiked with 13C
Te IS 10 pg
pg/mL. The endogenousg
FTe
was measured as 0.9 pg/mL,pg,Since the Total Te concentration was82.7 pg/mL, the % Free Te =1.14

B
B. Endogenous Te in the same DBS sample
which was calculated as 43 pg/mL
Reproducibility at the LLOQ level of ~43
43 pg/mL:%CV= 10.7
10 7 (n=22)
Figureg
4. Sensitivity
y enhancement of Testosterone uponpderivatization usingg QTRAP® 5500 LC/MS/MS.

Figure 7: Chromatogram of a DBS sample from a28 yyear old female: (A)( ) following
g derivatisation,,((B)) underivatised

The sample is extracted from a HemaSpot fan
shaped filter paper, ~8 µL whole blood in each blade
is extracted with 200 µL Hexane/10% ethyl acetate as
describe in Figure 3.

 Derivatization of Te with a novel aminoxy reagent (QAO) leads to improved LC
LC-MS/MSMS/MS properties
 This derivatisation leads to a significant
gincrease in ESI/MS/MS Te sensitivity
y which enables
analysisyof Te in Dried Blood Spotsp((<10µL)µ ) and Free Te samples.
p The LLOQ levels achieved with the QAO reagent
for FTe samples
and <50 pg
pg/mL for DBS))
g((<1pg/mLpgp
are a significant improvement over previously reported values
 QAO reagent is universal and reacts well with other compounds containing a keto or aldehydefunctionality.

TRADEMARKS/LICENSING
For Research Use Only.
y Not for use in diagnostic
gprocedures.
pp y of AB Sciex Pte. Ltd. or their respective
pThe trademarks mentioned herein are the ppropertyowners.