Zlatuse Clark (Presenter)
ARUP Laboratories
Bio: B.S. in analytical chemistry from Masaryk University, Brno, Czech Republic Ph.D. in bioanalytical chemistry from Brigham Young University, Provo, Utah Currently an R&D scientist at ARUP Laboratories, Salt Lake City, Utah
Authorship: Zlatuse D. Clark (1), Marzia Pasquali (2)
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, (2) Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112
| Short Abstract We are measuring amino acids in biological fluids using the Sciex aTRAQ labeling method and LC-MS/MS. The glycine (Gly) mass transition has a higher baseline signal than other amino acids, however, more recently, the signal intensity at the base of the Gly peak increased about 20-fold, reducing Gly peak’s signal-to-noise ratio. This change correlated with the installation of a new water purification system (Milli-Q®) supplying water for laboratory operations, including mobile phase preparation. We have evaluated water purified with different in-house systems as well as commercially available HPLC and LC-MS water. Although the baseline was significantly reduced with commercial water, it was at the expense of decreased response for all analytes. Installing filters specifically designed for LC-MS applications on our water purification system did not improve the Gly baseline. |
Long Abstract
Problem
The glycine (Gly) mass transition in our amino acids by LC-MS/MS assay has a higher baseline signal than other amino acids. Because of the aTRAQ labeling procedure, an alternate Gly transition is not available. Recently, the signal intensity at the base of the Gly peak increased about 20-fold, reducing Gly peak’s signal-to-noise ratio. This change correlated with the installation of a new water purification system (Milli-Q®) supplying water for laboratory operations, including mobile phase A preparation.
Method Information
- Urine and plasma samples treated with sulfosalicylic acid to precipitate protein, supernatant combined with Sciex aTRAQ reagent to label amino acids, and subsequently diluted with mobile phase A (MP-A)
- Shimadzu LC
- Sciex API 4000
- MP-A: 0.1% formic acid + 0.01% HFBA in water
- MP-B: 0.1% formic acid + 0.01% HFBA in methanol
- Columns:
- Sciex AAA C18 (150 x 4.6 mm, 5 um)
- Waters XSelect HSS T3 (150 x 3 mm, 3.5 um)
- 18 min gradient LC program, 0.8 mL/min and 0.65 mL/min flow rates
- Column oven 50 and 40 °C
- Injection volume 3 uL
- Quantitative SRM acquisition
Troubleshooting Steps
We investigated various sources of purified water produced in-house as well as commercially available HPLC and LC-MS water from several different vendors. We also replaced the Biopak filter on our Milli-Q® water purification system with a combination of an LC-Pak® C18 polisher and a Millipak® filter, specifically recommended for LC-MS applications.
Outcome
When the A mobile phases were prepared from in-house water sources, they all yielded similar high Gly baseline signal. Mobile phases prepared from all purchased water drastically reduced the Gly background, but also substantially suppressed the signal of all analytes and internal standards. The installation of the LC-Pak® C18 polisher and the Millipak® filter did not resolve the high baseline problem.
References & Acknowledgements:
The work presented here was supported by ARUP Institute for Clinical and Experimental Pathology®.
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