the science required for successful bioanalytical …...n’-nitrosonornicotine (nnn) chemical...

The Science Required For Successful

Bioanalytical Methods

Ridha Nachi

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

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)

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

MHBMA Extracted Samples

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MHBMA MHBMA MHBMA

Extracted High

Standard

Extracted Urine Lot(a)

Extracted Urine Lot(b)

MHBMA heart cut on an anion exchange guard column

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MHBMA

MHBMA Internal Standard

Arificial Urine sample

MHBMA Extracted High Standard

Concentration Artificial Urine

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MHBMA

MHBMA

Arificial Urine Sample

Urine Sample

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

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

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

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

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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