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?

2

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

3

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

4

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.

5

NNN Urine Extracted Low QC

6

Accuracy of NNN Quantitation in 16 Lots of

Human Urine

7

Monohydroxy-3-Butenyl Mercapturic Acid

(MHBMA) In Human Urine

8

Using mixed mode SPE

Anion exchange chromatography

ESI Negative Ion mode ionization

MHBMA Extracted Samples

9

MHBMA MHBMA MHBMA

Extracted High

Standard

Extracted Urine Lot(a)

Extracted Urine Lot(b)

MHBMA heart cut on an anion exchange guard column

10

MHBMA

MHBMA Internal Standard

Arificial Urine sample

MHBMA Extracted High Standard

Concentration Artificial Urine

11

MHBMA

MHBMA

Arificial Urine Sample

Urine Sample

Accuracy of Matrix Effect for MHBMA in Human Urine

12

Sub-pg/mL Method Example

13

Derivatized 3-OH-BaP

14

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 )

15

3-OH BaP Extracted Blank (in artificial urine )

16

3-OH BaP Extracted Human Urine Sample

Approximately 150 fg/mL

17

Accuracy of Matrix Effect Data in Nine Lots Of

Human Urine

18

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

19

3-HPMA RP chromatography using 2 stable

label internal standards

20

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

21

3-HPMA RP-UHPLC Chromatography Using the

Stable labeled Internal standard

22

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

23

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

24

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

25

Acknowledgments

Alan Dzerk

Christine Kafonek

Kirk Newland

Patrick Miller

Veniamin Lapko

Paul Brown

26