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©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
©2011 Waters Corporation 2
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
©2011 Waters Corporation 3
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
©2011 Waters Corporation 4
pH 10 Mobile Phases Chromatographic Affect
Increasing RT of Polars Peak Shape Improvement
Acidic Acidic
Basic Basic
©2011 Waters Corporation 5
Human Urine Results
Acidic Mobile Phase
Basic Mobile Phase
©2011 Waters Corporation 6
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
©2011 Waters Corporation 7
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
©2011 Waters Corporation 8
Effect of Peak Response For Alprazolam at Low and High pH
©2011 Waters Corporation 9
Effect of Organic Modifier on Peak Response
©2011 Waters Corporation 10
Bioanalytical Assay For Alprazolam With Protein Precipiated Plasma
©2011 Waters Corporation 11
Internal Standard and Metabolite
©2011 Waters Corporation 12
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
MRM of 5 channels,ES+
416.432 > 232.198
180511_spe_jm_B_V_022 Smooth(SG,2x1)
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
©2011 Waters Corporation 13
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
MRM of 5 channels,ES+
504.287 > 293.188
180511_spe_jm_B_V_022 Smooth(SG,2x1)
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
MRM of 5 channels,ES+
501.28 > 293.185
180511_spe_jm_B_V_022 Smooth(SG,2x1)
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
©2011 Waters Corporation 14
Amyloid β Analysis in CSF
Data courtesy of Erin Chambers, Waters
©2011 Waters Corporation 15
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
©2011 Waters Corporation 16
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)