Analysis of Choline and its Metabolites on the Finnigan LTQ Gargi Choudhary Diane Cho Thermo Electron, San Jose, CA Abstracted from a poster presented at Montreux 3 LC/MS Symposium, with co-authors: Brad Hart, Marie Caudill 1 1 Human Nutrition and Food Sciences Dept of Cal Poly Pomona
Goal: What we set out to demonstrate The applicability of the Finnigan LTQ to a quantitative method previously established on the LCQ Advantage The sensitivity and dynamic range of choline and its metabolites on the Finnigan LTQ The precision and quantitative characterization of the LTQ The limits of detection (LD) and quantitation (LQ) for this method on the LTQ 2
Background: What s the interest in cholines and its metabolites? Choline Part of several major phospholipids that are critical for normal cell membrane structure and lipoprotein function (1). A precursor of betaine, which is used by kidney to maintain water balance (2), and by liver as a source of methyl groups for methionine formation (3). Also used to produce acetylcholine, an important neurotransmitter (4). If deprived of choline, cells die of apoptosis (5-7)and humans develop liver dysfunctions (8). While the essentiality of cholines in several animals has long been recognized, a dietary recommendation for humans was only recently recommended by the US National Academy of Sciences (9). 3
Choline Compounds Analyte Structure MH+ Normalized Collision energy Product ions Betaine H N 118 58 and 59 Choline H N 14 Acetylcholine 146 32 87 N Generated with Mass Frontier software 4
Choline Compounds (2) Analyte Structure MH+ Normalized Collision energy Product ions Phosphochloline H P H N 184 32 86 Glycerophospho choline H H P H N 258 27 14 CDP choline H 2 N N N H H P H P N 489 27 1 Generated with Mass Frontier software 5
LC Conditions Surveyor MS Pump and Surveyor Autosampler Mobile Phase A: Acetonitrile/water/ethanol/1M ammonium acetate/glacial acetic acid (/127/68/3/2 v/v) B: Acetonitrile/water/ethanol/1M ammonium acetate/glacial acetic acid (//85/27/18 v/v) Column x 2.1 mm id packed with 5 µm Silica particles Injection volume: 1 µl Gradient: At % B for.5 min and µl/min; to % B in 5. min at µl/min; to % B in 2 min at 3 µl/min at % B for 3 min at µl/min to % A in 1 min at µl/min at % A for 6 min at µl/min 6
Conditions Electrospray, positive ionization Capillary temperature 31 C Spray voltage 4.5 kv Sheath gas Aux gas 1 7
LC/MS/MS Analysis of 3 Cholines RT:. - 6. SM: 7G 2.57 Acetylcholine Base Peak m/z= 86.-87. F: ITMS + c ESI Full ms2 146.@32. [.-1.] MS Genesis new_sample_16 2.34 2. Betaine Choline. 1. 2. 3. 4. 5. 6. Time (min) Base Peak m/z=57.-58.+58.- 59. F: ITMS + c ESI Full ms2 118.1@. [.-122.] MS Genesis new_sample_16 Base Peak m/z= 59.-. F: ITMS + c ESI Full ms2 14.1@42. [.-18.] MS new_sample_16 87.2 59.1.8 Relative Abundance.12 128.9146.9 7 9 11 13 1 58.6 86.1.7 11.5 1 75.11 86.7 55 65 75 85 95 15 8
LC/MS/MS of 3 Cholines RT:. - 4. SM: 7G Phosphocholine 2.47 Base Peak m/z= 85.-86. F: ITMS + c ESI Full ms2 184.@32. [.- 187.] MS new_sample_14 Glycerophosphocholine 2.48 Base Peak m/z= 13.-14. F: ITMS + c ESI Full ms2 258.@27. [ 7.- 261.] MS new_sample_14 Relative Abundance CDP choline 1.97 Base Peak m/z=.-1.+417.- 418. F: ITMS + c ESI Full ms2 489.@27.[ 1.-493.] MS new_sample_14 1. 2. 3. 4. Time (min).9 86.12 14.5 418.26 489.46 196.94.9 112.6 124.21 9 1 2 3 4 m/z 1 1 1 1 1 m/z m/z
Calibration Curve for Acetylcholine 44 Y =.41943*X R^2 =.9996 W: Equal Area Ratio 36 32 28 24 16 12 8 4 Acetylcholine N Dynamic Range fmol 1 nmol (> five orders of magnitude) 3 7 9 Amount (pgs on-column) 1
Same-Day Assay Precision for Acetylcholine Amount on column % CV fmol 11.6 fmol 1 pmol 1 pmol pmol 1 nmol 1 nmol 7.56 5.2 1.32 1.85 2.3 1.82 * For three injections 11
Calibration Curve for Glycerophosphocholine 2.8 Y =.252578*X R^2 =.999 W: Equal Area Ratio 2.4 2. 1.6 1.2.8 H Glycerphosphocholine H P H N Dynamic Range fmol-1 nmol (five orders of magnitude).4. 3 7 9 Amount (pgs on-column) 12
Same-Day Assay Precision for Glycerophosphocholine Amount on column % CV fmol 3.95 fmol 5.63 5 pmol 2.14 pmol 3.88 pmol 3.9 1 nmol 2.74 5 nmol 1.24 1 nmol 1.12 * For three injections 13
LD for Acetylcholine and Glycerphosphocholine S/N: 9 Acetylcholine 25 fmol on-column Base Peak m/z= 86.-87. F: ITMS + c ESI Full ms2 146.@32. [.-149.] Relative Abundance S/N: 13 Glycerophosphocholine fmol on-column Base Peak m/z= 13.-14. F: ITMS + c ESI Full ms2 258.@27. [7.-261.] 1. 2. 3. 4. 5. Time (min) 6. 14
LD* and LQ* Analyte Acetylcholine Glycerophosphocholine LD (on-column) 25 fmol fmol LQ (on-column) fmol fmol * In water 15
Choline and Betaine from Pooled Liver Extract S:N Comparison Finnigan LTQ Finnigan LCQ Advantage RT: 4. - 12. X 5.5 RT: 4. - 12. 9 S/N: 263 Base Peak F: ITMS + c ESI Full ms2 14.1@. [.-18.] MS 9 S/N:47 Base Peak F: + c Full ms2 14.1@35. [.-11.] MS Relative Abundance 7 3 9 Relative Abundance 7 3 Choline 1 1 9 S/N: 71 X 1 Base Peak F: ITMS + c ESI Full ms2 118.1@38. [.-122.] MS 9 S/N: 7 Betaine Base Peak F: + c ESI Full ms2 118.1@38. [.-1.] MS 7 7 3 3 1 1 4 5 6 7 8 1 11 12 Time (min) 4 5 6 7 8 9 1 11 12 Time (min) 16
Choline and Betaine from Pooled Liver Extract # Scans Across the LC Peak Finnigan LTQ Finnigan LCQ Advantage Relative Abundance 9 7 3 1 RT: 4. - 12. 9.9 7.53.7 Base Peak F: ITMS 59.98 + c ESI Full ms2 14.1@. [ 9.-18.] MS AqStd_nmol_2 Relative Abundance 7 3 1 Choline Base Peak F: + c Full ms2 14.1@35. [.-11.] MS aqstd_nmol_2 9 7.34 58.99 Base Peak F: ITMS + c ESI Full ms2 118.1@38. [.-122.] MS AqStd_nmol_2 9 6.68 58.13 Betaine Base Peak F: + c ESI Full ms2 118.1@38. [.-1.] MS aqstd_nmol_2 7 7 99.92 9.6. 9.67 99.82 Quan on this?? 3 3 1 1 7 8 9 1 Time (min) 4 5 6 7 8 9 1 11 12 Time (min) 17
Conclusions Full scan MS/MS analysis with the Finnigan LTQ linear ion trap mass spectrometer provides the selectivity and sensitivity for the analysis of choline compounds Accurate quantitation of choline compounds with a linear dynamic range over five orders of magnitude is achieved on the Finnigan LTQ. The Finnigan LTQ provides excellent within-day assay precision for the analysis of choline compounds. The CV for glycerophosphocholine is less than 6% for the entire linear dynamic range. The CV for acetylcholine is less than 8% except near LQ where the CV is 11.6%. The limit of quantitation is determined from the calibration curves as fmol for acetylcholine and fmol for glycerophosphocholine. The LD for acetylcholine is determined to be 25 fmol (on-column) with a signal to noise ratio of 9:1, whereas that for glycerophosphocholine is determined to be fmol (on-column) with a signal to noise of 13:1 18
Acknowledgements Marie Caudill, Cal Poly Pomona University Brad Hart Jonathan McNally Jae Schwartz Lester Taylor Rohan Thakur 19
References 1. Zeisel, S.H. Nutrition, 16, 669-671. 2. Burg, M. Am. J. Physiol. 1995, 268, F983-F996 3. Dudman, N; Guo, X; Gordon E,; Dawson, P; Wilcken D. J. Nutr. 1996, 126,1295S-13S 4. Haubrich, D. R; Wang, P. F.; Herman, R. L.; Clody, D.E., Life Sci. 1975, 17, 739-747 5. Holmes-McNary, M. Q,; Loy, R.; Mar, M. H.; Albright, C. D.; Zeisel, S. H. Dev. Brain Res. 1997, 11, 9-16. 6. Yen, C. L.; Mar, M. H.; Zeisel, S. H. FASEB J. 1999, 13, 135-142 7. Albright, C. D.; Salganik, R. I.; Kaufmann, W.K.; Vrablic, A. S.; Zeisel, S. H. J. Nutr. Biochem. 1998, 9, 476-481
References 8. Zeisel, S. H.; DaCosta, K. A.; Franklin, P.D.; Alexander, E. A.; Lamont, J. T.; Sheard, N. F.; Beiser, A. FASEB J. 1991, 5, 93-98 9. Institute of Medicine: National Academy of Sciences U.S.A. Dietry reference intakes of folate thiamin riboflavin, niacin, vitamin B12, panthothenic acid, biotin, and choline; National Academy press: Washington, DC 1998 1. Koc, H.; Mar, M. H.; Ranasinghe, A.; Swenberg, J. A.; Zeisel, S. H.; Anal. Chem. 2, 74, 4734-47. 21