application of high ph separations for the quantification of … · 2018. 6. 8. · compound name:...

©2011 Waters Corporation 1

Application of High pH Separations for the

Quantification of Small and Large Molecules

Debadeep Bhattacharya, PhD

Pharmaceutical Sciences Division

Waters Corporation

Milford, MA, USA


Bioanalysis Studies and High pH Mobile Phases with ESI Positive

Provide Information

•Regulation of metabolism

•Toxicology

•Models of disease

Experimental Tools

•Analysis & Detection

•SPE

•Chromatography

•Mass Spectrometry

Farkas, T., Peng, L., 2008 Journal of Chromatography A,

1179(2): 131-144

Mess, J., Lahaie, M., Furtado, M., Garofolo, F., 2009 Bioanalysis,

1(8): 1419-1430

Tso, J., Aga, D., 2011 Analytical Chemistry, 83(1): 269-277

Tomlinson, A., Chicz, R., 2003 Rapid Communications in Mass

Spectrometry ,17(9): 909-916

Zhou, S.; Cook, K., 2000 Journal of the American Society of

Mass Spectrometry 11: 961


Parent Drug

Increasing Ionization Efficiency ESI Positive Mode

OH Metabolite

7x increase

Parent Drug

OH Metabolite

Parent Drug 5x increase

Mather,J., Rainville, P.D., Smith, N.W., Plumb, R.S. 2010 Journal of Drug Testing and Analysis 2 (1) 11-18

pH 10 Mobile Phase

3.51 e4

pH 2.5 Mobile Phase

4.83 e3

pH 10 Mobile Phase

1.34 e5

pH 2.5 Mobile Phase

2.59 e4


pH 10 Mobile Phases Chromatographic Affect

Increasing RT of Polars Peak Shape Improvement

Acidic Acidic

Basic Basic

http://upload.wikimedia.org/wikipedia/commons/7/7b/Ranitidine_Structural_Formulae.png


Human Urine Results

Acidic Mobile Phase

Basic Mobile Phase


m/z 100.0443

Time2.00 4.00 6.00 8.00 10.00

%

0

1000.81

10.03

1.61

5.82

2.043.44

2.69

4.10

4.83

8.54

8.057.63

7.25

9.72

9.58

Samples1 2 3 4 5 6 7 8 9 10 11 12 13 14

%

0

100

Marker (4.20,100.0443) 4.873e-002

Time2.00 4.00 6.00 8.00 10.00

%

0

1002.97

0.39

1.472.31

4.01

3.30

5.18

4.57

6.90

5.919.49

8.958.697.01

8.08

9.62

9.79


m/z 962.3001

Samples1 2 3 4 5 6 7 8 9 10 11 12 13 14

%

0

100

Marker (7.44,962.3001) 9.116e-002

Time2.00 4.00 6.00 8.00 10.00

%

0

1002.97

0.39

1.472.31

4.01

3.30

5.18

4.57

6.90

5.919.49

8.958.697.01

8.08

9.62

9.79

Time2.00 4.00 6.00 8.00 10.00

%

0

1000.81

10.03

1.61

5.82

2.043.44

2.69

4.10

4.83

8.54

8.057.63

7.25

9.72

9.58


Effect of Peak Response For Alprazolam at Low and High pH


Effect of Organic Modifier on Peak Response


Bioanalytical Assay For Alprazolam With Protein Precipiated Plasma


Internal Standard and Metabolite


Salmeterol Xinoafate 0.375pg/mL

min

2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40

%

0

100

MRM of 5 channels,ES+

419.168 > 235.129

180511_spe_jm_B_V_022 Smooth(SG,2x1)

0.75 0.375

1.961e+006Salmeterol D3

3.26

39210.23

1932831

3.03

2.142.17

2.34

3.08 3.33

min

%

0

100


416.432 > 232.198


0.75 0.375

4.715e+005Salmeterol

3.27

8662.80

434484

2.42

2.12

2.94

2.502.872.77

3.223.12

3.61

3.483.44

3.943.71

Compound name: Salmeterol

Correlation coefficient: r = 0.999726, r^2 = 0.999452

Calibration curve: 5.03182 * x + 0.238402

Response type: Internal Std ( Ref 3 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

Conc

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

Re

sp

on

se

0.0

10.0

20.0

30.0

Conc

Re

sid

ua

l

-2.5

0.0

2.5

5.0

7.5

10.0


Fluticasone Propionate 0.75pg/mL

min

2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40

%

0

100


504.287 > 293.188


0.75 0.375

7.162e+0053.68

Fluticasone D3 (1)

3.06

11861.36

623557

2.42 2.772.52

2.82

3.35

min

%

0

100


501.28 > 293.185


0.75 0.375

6.864e+0042.83

2.16

2.07

2.502.432.33

2.25

2.54

2.692.57

Flut 293

3.07

1002.32

44254

2.91

2.95 3.16 3.19

3.283.40

3.49 3.61

4.334.053.94

Compound name: Flut 293

Correlation coefficient: r = 0.998297, r^2 = 0.996597

Calibration curve: 0.807755 * x + 0.317443

Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

Conc

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Re

sp

on

se

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Conc

Re

sid

ua

l

-15.0

-10.0

-5.0

0.0

5.0

10.0


Amyloid β Analysis in CSF

Data courtesy of Erin Chambers, Waters


Conclusion

Chromatographic separations and MS detection of complex samples was successfully carried out with pH 10 mobile phase coupled with ESI positive ionization.

Each set of mobile phase conditions lead to the detection of analytes not seen with the opposite pH conditions

The chromatographic performance with pH 10 mobile phases showed increased resolution, sensitivity, and selectivity


Acknowledgements

Prof. Jeremy Nicholson, Prof. Elaine Holmes, Dr. Derek Crockford, Dr. Robert Plumb, Dr Elizabeth Want (Imperial College London, UK)

Dr. Norman Smith, Prof David Cowan (Kings College London)

Prof Ian Wilson (Astra Zeneca, UK)

John Shockcor (Waters Corporation USA)

application of high ph separations for the quantification of … · 2018. 6. 8. · compound name:...

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