Ult Hi hS iti it A l i fT t t Ultra High Sensitivity Analysis of Testosterone Ultra High Sensitivity Analysis of Testosterone MSACL Poster # 46 MSACL Poster # 46 1 Michal Star Weinstock 1 Brian L Williamson 1 Subhakar Dey 1 Babu Purkayastha 2 James E Hill 2 Jeanette R Hill 1 Michal Star-Weinstock, 1 Brian L. Williamson, 1 Subhakar Dey, 1 Babu Purkayastha, 2 James E. Hill, 2 Jeanette R. Hill 1 AB SCIEX 500 Old Connecticut Path Framingham MA 01701 1 AB SCIEX, 500 Old Connecticut Path, Framingham MA, 01701 2 Spot On Sciences 13705 Shadowglade Manor TX 78653 2 Spot On Sciences, 13705 Shadowglade, Manor, TX 78653 ABSTRACT TtlT f D i d Bl dS t (DBS) Results Free Te From Serum or Saliva ABSTRACT Total Te from Dried Blood Spots (DBS) Results Free Te From Serum or Saliva Analysis of Testosterone (Te) from both Free Te samples (FTe) and Dried Blood Spots Analysis of Testosterone (Te) from both Free Te samples (FTe) and Dried Blood Spots (DBS) tili i d i ti ti ith l i ti d t td (DBS) utilizing derivatization with a novel aminoxy reagent is demonstrated A IS The LLOQ after derivatization is <1 pg/mL for FTe samples and ~40 pg/mL for DBS A 10 pg/mL Linearity (R 2 >0.998) is maintained over a broad dynamic range with %CV <15 at LLOQ IS (d T) QAO-derivatization and sample preparation workflows are quick, simple, IS (d 3 Te) reproducible, robust and yield high recoveries QAO reagent reproducible, robust and yield high recoveries QAO Te Testosterone INTRODUCTION QAO –Te Endogenous INTRODUCTION Figure 2. Schematic illustration of QAO derivatization of Te. Endogenous Te Endogenous FTe Testosterone (Te) analysis by LC/MS/MS is becoming the analytical method of choice over immunoassays due The reaction is complete within 45 min at RT Measured as FTe 0.94 pg/mL 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 483.9 pg/mL to its specificity and accuracy. Neutral steroid hormones undergo poor ionization efficiency in MS/MS, resulting in insufficient sensitivity to analyze samples which possess trace concentrations. The method presented herein utilizes a tag moiety (quaternary aminoxy reagent) which binds to the keto functionality of Te or any other keto- steroid and significantly enhances its MS/MS sensitivity. The derivatization technique enables quantitation of Fi 8C t ti 1 50 / L lib Free Te (FTe) from samples such as equilibrium dialysates, ultra filtrates and saliva, Moreover, the derivatisation Fi 5 DBS C t ti fd T 50 Figure 8. Concentration curve 1-50 pg/mL, calibrator ik di t lt filt t MWCO 30 kD ff l technique enables the use of very small amounts of sample such as in Dried Blood Spots (DBS). Figure 5. DBS Concentration curve of d 3 Te 50- 10000 / L ik di t 10 L hl bl d spiked into ultra filtrate MWCO 30 kDa of female serum l (500 L) Th lib t d T d th IS 13 C 10000 pg/mL spiked into 10 μL whole blood pool (500 uL). The calibrtor was d 3 Te and the IS 13 C Te REAGENTS AND METHODS Te. REAGENTS AND METHODS F l li Figure 9. Ultra filtrate (30kDa MWCO) 50 pg/mL A Female saliva d Figure 9. Ultra filtrate (30kDa MWCO) of Female serum pool spiked with 13 C 50 pg/mL d 3 Te Std 20 endogenous Free Te 21 B of Female serum pool spiked with C Te IS 10 pg/mL. The endogenous FTe d 3 Te Std Underivatized pg/mL d T Free Te 2.1 pg/mL was measured as 0.9 pg/mL, Underivatized Te IS d 3 Te Std pg/mL Since the Total Te concentration was Std 82.7 pg/mL, the % Free Te =1.14 Figure 3 MS/MS fragmentation of QAO Te Figure 3. MS/MS fragmentation of QAO Te. Reproducibility : At this concentration Structure specific fragments are selected to enhance specificity and reduce background noise The MRM No peak of level of ~1pg/mL the % CV = 14.7 (n=15) Structure specific fragments are selected to enhance specificity and reduce background noise. The MRM Transitions for QAO- Te are 403 3->164 2 and 403 3->152 2 at CE=60eV No peak of underivatized Te Transitions for QAO Te are 403.3 >164.2 and 403.3 >152.2 at CE 60eV underivatized Te B Endogenous Te B Endogenous Te Measured as Figure 4 illustrates the sensitivity Measured as 43 pg/mL Figure 10 Free Te analysis from female saliva Figure 4 illustrates the sensitivity enhancements after derivatisation of Te 43 pg/mL Figure 10. Free Te analysis from female saliva. 1 mL saliva was extracted by the method described in Fig 3 enhancements after derivatisation of Te (1pg on column). 1 mL saliva was extracted by the method described in Fig. 3. Underivatized, derivatized with Hydroxyl amine and derivatized with QAO reagent Conclusions were all compared. The S/N ratio of the QAO derivative is Figure 7: Chromatogram of a DBS sample from a Derivatization of Te with a novel aminoxy reagent (QAO) leads to improved LC-MS/MS properties superior due to careful selection of specific Figure 7: Chromatogram of a DBS sample from a 28 year old female: (A) following derivatisation, Derivatization of Te with a novel aminoxy reagent (QAO) leads to improved LC MS/MS properties This derivatisation leads to a significant increase in ESI/MS/MS Te sensitivity which enables fragment ions and higher mass of the (B) underivatised 6 10 Sf f analysis of Te in Dried Blood Spots (<10μL) and Free Te samples. derivative that drives it farther away from Figure 6: 10 μL DBS from female blood A d T St d d ik d 50 / L (0 5 i The LLOQ levels achieved with the QAO reagent (<1pg/mL for FTe samples and <50 pg/mL for DBS) the background noise. The sample is extracted from a HemaSpot fan A. d 3 Te Standard spiked as 50 pg/mL (0.5 pg in 10 L bl d ti l filt ) are a significant improvement over previously reported values Nt shaped filter paper, ~8 μL whole blood in each blade 10 μL blood, on a conventional filter paper) QAO reagent is universal and reacts well with other compounds containing a keto or aldehyde Notes: 1 Off tti f t ti ti dt is extracted with 200 μL Hexane/10% ethyl acetate as B Endogenous Te in the same DBS sample functionality. Figure 1 Schematic description of the extraction and derivatization workflow for Free Te samples from serum 1. Offsetting of retention times were used to describe in Figure 3. B. Endogenous Te in the same DBS sample which was calculated as 43 pg/mL Figure 1. Schematic description of the extraction and derivatization workflow for Free Te samples from serum or Saliva and Dried Blood Spots position the peaks for visual demonstration. which was calculated as 43 pg/mL or Saliva and Dried Blood Spots Reproducibility at the LLOQ level of ~43 pg/mL: TRADEMARKS/LICENSING The QAO reagent (10 mg/mL), is dissolved in MeOH:5% acetic acid 2. The Hydroxyl amine (HA) Derivatization of Te Reproducibility at the LLOQ level of 43 pg/mL: %CV= 10 7 (n=22) TRADEMARKS/LICENSING d 3 Te is used as calibrator (Cerriliant ,99.8% pure). 13 C Te used as Internal Standard (IS), Isosciences, >98% pure. Matrix: Female whole serum (Golden West Bio PS 1040) was repeated from Kushnir MM. et al. Clinical Ch i 6 1138 114 (2010) %CV= 10.7 (n=22) For Research Use Only. Not for use in diagnostic procedures. (Golden West Bio PS 1040) LC Gradient Conditions: Acetonitrile / water /formic acid (0.1%) mobile phase using a C18 column (Cadenza CL). Step Gradient 10-30%B in the Chemistry 56:7,1138–1147 (2010) The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. Figure 4. Sensitivity enhancement of Testosterone upon LC Gradient Conditions: Acetonitrile / water /formic acid (0.1%) mobile phase using a C18 column (Cadenza CL). Step Gradient 10 30%B in the first 0.5 min, then 30-55%B within 3 min. Shimadzu AD -30 series and QTRAP® 5500 LC/MS/MS system. derivatization using QTRAP® 5500 LC/MS/MS.
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Ult Hi h S iti it A l i f T t tUltra High Sensitivity Analysis of TestosteroneUltra High Sensitivity Analysis of Testosterone MSACL Poster # 46g y y MSACL Poster # 461Michal Star Weinstock 1Brian L Williamson 1Subhakar Dey 1Babu Purkayastha 2James E Hill 2Jeanette R Hill1Michal Star-Weinstock, 1Brian L. Williamson, 1Subhakar Dey, 1Babu Purkayastha, 2James E. Hill, 2Jeanette R. Hill1AB SCIEX 500 Old Connecticut Path Framingham MA 017011AB SCIEX, 500 Old Connecticut Path, Framingham MA, 017012Spot On Sciences 13705 Shadowglade Manor TX 786532Spot On Sciences, 13705 Shadowglade, Manor, TX 78653
ABSTRACT T t l T f D i d Bl d S t (DBS)Results Free Te From Serum or SalivaABSTRACT Total Te from Dried Blood Spots (DBS)Results Free Te From Serum or Saliva
Analysis of Testosterone (Te) from both Free Te samples (FTe) and Dried Blood SpotsAnalysis of Testosterone (Te) from both Free Te samples (FTe) and Dried Blood Spots (DBS) tili i d i ti ti ith l i t i d t t d(DBS) utilizing derivatization with a novel aminoxy reagent is demonstrated A IS The LLOQ after derivatization is <1 pg/mL for FTe samples and ~40 pg/mL for DBS A
10 pg/mLLinearity (R2>0.998) is maintained over a broad dynamic range with %CV <15 at LLOQ
IS (d T )y ( ) y g
QAO-derivatization and sample preparation workflows are quick, simple, IS (d3 Te)Q p p p q , p ,reproducible, robust and yield high recoveries QAO reagentreproducible, robust and yield high recoveries
QAO TeTestosterone
g
INTRODUCTIONQAO –Te
EndogenousINTRODUCTION Figure 2. Schematic illustration of QAO derivatization of Te. Endogenous TeEndogenous FTe
Testosterone (Te) analysis by LC/MS/MS is becoming the analytical method of choice over immunoassays dueg
The reaction is complete within 45 min at RTg
Measured as FTe0.94 pg/mLTestosterone (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 483.9 pg/mLpg
to its specificity and accuracy. Neutral steroid hormones undergo poor ionization efficiency in MS/MS, resulting in insufficient sensitivity to analyze samples which possess trace concentrations. The method presented herein y y p p putilizes a tag moiety (quaternary aminoxy reagent) which binds to the keto functionality of Te or any other keto-g y (q y y g ) y ysteroid and significantly enhances its MS/MS sensitivity. The derivatization technique enables quantitation of
Fi 8 C t ti 1 50 / L libg y y q q
Free Te (FTe) from samples such as equilibrium dialysates, ultra filtrates and saliva, Moreover, the derivatisation Fi 5 DBS C t ti f d T 50
Figure 8. Concentration curve 1-50 pg/mL, calibrator ik d i t lt filt t MWCO 30 kD f f ltechnique enables the use of very small amounts of sample such as in Dried Blood Spots (DBS). Figure 5. DBS Concentration curve of d3 Te 50-
10000 / L ik d i t 10 L h l bl dspiked into ultra filtrate MWCO 30 kDa of female serum
l (500 L) Th lib t d T d th IS 13C10000 pg/mL spiked into 10 µL whole blood pool (500 uL). The calibrtor was d3 Te and the IS 13C TeREAGENTS AND METHODS Te.REAGENTS AND METHODS
F l li Figure 9. Ultra filtrate (30kDa MWCO)50 pg/mLA Female saliva
d
Figure 9. Ultra filtrate (30kDa MWCO) of Female serum pool spiked with 13C50 pg/mL
d3 Te Std 20 endogenous Free Te 2 1B
of Female serum pool spiked with C Te IS 10 pg/mL. The endogenous FTe d3 Te Std
Underivatized pg/mL d T
Free Te 2.1 pg/mL
pg gwas measured as 0.9 pg/mL, Underivatized
Te IS d3 Te Std
pg/mL pg ,Since the Total Te concentration was
Std 82.7 pg/mL, the % Free Te =1.14
Figure 3 MS/MS fragmentation of QAO TeFigure 3. MS/MS fragmentation of QAO Te. Reproducibility: At this concentration
Structure specific fragments are selected to enhance specificity and reduce background noise The MRM No peak of level of ~1pg/mL the % CV = 14.7 (n=15)Structure specific fragments are selected to enhance specificity and reduce background noise. The MRM Transitions for QAO- Te are 403 3->164 2 and 403 3->152 2 at CE=60eV
No peak of underivatized TeTransitions for QAO Te are 403.3 >164.2 and 403.3 >152.2 at CE 60eV underivatized Te
BEndogenous Te
BEndogenous TeMeasured as
Figure 4 illustrates the sensitivityMeasured as 43 pg/mL Figure 10 Free Te analysis from female salivaFigure 4 illustrates the sensitivity
enhancements after derivatisation of Te43 pg/mL Figure 10. Free Te analysis from female saliva.
1 mL saliva was extracted by the method described in Fig 3enhancements after derivatisation of Te (1pg on column).
1 mL saliva was extracted by the method described in Fig. 3. (1pg on column). Underivatized, derivatized with Hydroxyl , y yamine and derivatized with QAO reagent Conclusionsgwere all compared.pThe S/N ratio of the QAO derivative is Figure 7: Chromatogram of a DBS sample from a Derivatization of Te with a novel aminoxy reagent (QAO) leads to improved LC-MS/MS propertiessuperior due to careful selection of specific
Figure 7: Chromatogram of a DBS sample from a 28 year old female: (A) following derivatisation,
Derivatization of Te with a novel aminoxy reagent (QAO) leads to improved LC MS/MS propertiesThis derivatisation leads to a significant increase in ESI/MS/MS Te sensitivity which enables
fragment ions and higher mass of the y ( ) g ,
(B) underivatised6 10 S f f
g yanalysis of Te in Dried Blood Spots (<10µL) and Free Te samples.
derivative that drives it farther away from ( )
Figure 6: 10 µL DBS from female blood A d T St d d ik d 50 / L (0 5 i
y p ( µ ) pThe LLOQ levels achieved with the QAO reagent (<1pg/mL for FTe samples and <50 pg/mL for DBS)
the background noise. The sample is extracted from a HemaSpot fan A. d3 Te Standard spiked as 50 pg/mL (0.5 pg in 10 L bl d ti l filt )
g ( pg p pg )are a significant improvement over previously reported values
N tshaped filter paper, ~8 µL whole blood in each blade 10 µL blood, on a conventional filter paper) QAO reagent is universal and reacts well with other compounds containing a keto or aldehyde
Notes: 1 Off tti f t ti ti d t
is extracted with 200 µL Hexane/10% ethyl acetate as B Endogenous Te in the same DBS sample
functionality.
Figure 1 Schematic description of the extraction and derivatization workflow for Free Te samples from serum1. Offsetting of retention times were used to describe in Figure 3.B. Endogenous Te in the same DBS sample
which was calculated as 43 pg/mLFigure 1. Schematic description of the extraction and derivatization workflow for Free Te samples from serum or Saliva and Dried Blood Spots
position the peaks for visual demonstration. which was calculated as 43 pg/mLor Saliva and Dried Blood Spots
Reproducibility at the LLOQ level of ~43 pg/mL: TRADEMARKS/LICENSINGThe QAO reagent (10 mg/mL), is dissolved in MeOH:5% acetic acid 2. The Hydroxyl amine (HA) Derivatization of Te Reproducibility at the LLOQ level of 43 pg/mL:
%CV= 10 7 (n=22)TRADEMARKS/LICENSING
d3 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)
was repeated from Kushnir MM. et al. Clinical Ch i 6 1138 114 (2010)
%CV= 10.7 (n=22)For Research Use Only. Not for use in diagnostic procedures.(Golden West Bio PS 1040)
LC Gradient Conditions: Acetonitrile / water /formic acid (0.1%) mobile phase using a C18 column (Cadenza CL). Step Gradient 10-30%B in theChemistry 56:7,1138–1147 (2010)
y g pThe trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.Figure 4. Sensitivity enhancement of Testosterone upon LC Gradient Conditions: Acetonitrile / water /formic acid (0.1%) mobile phase using a C18 column (Cadenza CL). Step Gradient 10 30%B in the
first 0.5 min, then 30-55%B within 3 min. Shimadzu AD -30 series and QTRAP® 5500 LC/MS/MS system.p p y pg y p
derivatization using QTRAP® 5500 LC/MS/MS. g
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