the science required for successful bioanalytical …...n’-nitrosonornicotine (nnn) chemical...
TRANSCRIPT
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The Science Required For Successful
Bioanalytical Methods
Ridha Nachi
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Scope Of Presentation
Using pH to manipulate selectivity
Enhancing sensitivity
Are all stable-labeled internal standards
equivalent?
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N’-nitrosonornicotine (NNN) Chemical Structures
Target LLOQ 0.75 pg/mL in human urine
Combining similar column chemistries under
different pH conditions to manipulate selectivity
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N
NN
O
N
NN
ONNNC9H11N3O
177.23 Da
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NNN Extracted Urine Sample
Chromatography on an anion exchange column separated the two forms of NNN
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NNN
NNN
NNN
NNN
NNN
NNN
High Standard
Urine sample Lot (a)
Urine sample Lot (b)
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Two-dimensional chromatography of extracted urine samples on a
Polar RP pre-column.
Compared acetic acid and formic acid for the heart-cut.
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NNN Urine Extracted Low QC
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Accuracy of NNN Quantitation in 16 Lots of
Human Urine
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Monohydroxy-3-Butenyl Mercapturic Acid
(MHBMA) In Human Urine
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Using mixed mode SPE
Anion exchange chromatography
ESI Negative Ion mode ionization
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MHBMA Extracted Samples
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MHBMA MHBMA MHBMA
Extracted High
Standard
Extracted Urine Lot(a)
Extracted Urine Lot(b)
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MHBMA heart cut on an anion exchange guard column
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MHBMA
MHBMA Internal Standard
Arificial Urine sample
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MHBMA Extracted High Standard
Concentration Artificial Urine
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MHBMA
MHBMA
Arificial Urine Sample
Urine Sample
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Accuracy of Matrix Effect for MHBMA in Human Urine
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Sub-pg/mL Method Example
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Derivatized 3-OH-BaP
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Derivatization added a new functional group which
is easily ionized
Improved fragmentation- MS/MS efficiency in
positive ion mode
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3-OH BaP LLOQ S/N= 23 (in artificial urine )
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3-OH BaP Extracted Blank (in artificial urine )
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3-OH BaP Extracted Human Urine Sample
Approximately 150 fg/mL
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Accuracy of Matrix Effect Data in Nine Lots Of
Human Urine
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Summary for 3-OH-BaP Method
An ultra-sensitive method for 3-OH-Bap with
LLOQ of 50 fg/mL was developed and validated
Robustness was demonstrated by a batch
passing rate over 98%
ISR was successfully performed with this
method
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3-HPMA RP chromatography using 2 stable
label internal standards
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3-HPMA IS Label 1
3-HPMA
3-HPMA IS label 2
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Matrix Effect Test in Human Urine Comparing the
Performance of Two Different Labeled Internal
Standards
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3-HPMA RP-UHPLC Chromatography Using the
Stable labeled Internal standard
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LLOQ in Artificial
Urine
Low QC in Human
Urine
High QC in
Human Urine
3-HPMA IS
3-HPMA IS
3-HPMA IS
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Structures of 3-HPMA and Internal Standards
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d6-3HPMA failed to accurately quantify 3-HPMA
matrix effect test in human urine when [15N13C3]3-
HPMA reliably tracked the analyte
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3-HPMA Method Results
A LC-MS/MS method for the simultaneous
analysis of CEMA, 3-HPMA and HBMA with
improved selectivity has been developed and
validated
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Conclusion
Using pH to manipulate selectivity provided
better data selectivity for the assay of NNN
Derivatization provided an excellent solution to
enhancing sensitivity to reach sub pg/mL levels
C13 and N15 labeled internal standards which
coelute with the analytes provide the most
robust bioanalytical methods
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Acknowledgments
Alan Dzerk
Christine Kafonek
Kirk Newland
Patrick Miller
Veniamin Lapko
Paul Brown
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