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ARS Home » Southeast Area » Little Rock, Arkansas » Microbiome and Metabolism Research Unit » Research » Publications at this Location » Publication #318363
Title: Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

Author
item FANG, NIANBAI - Arkansas Children'S Nutrition Research Center (ACNC)
item YU, SHANGGONG - Arkansas Children'S Nutrition Research Center (ACNC)
item RONIS, MARTIN - Arkansas Children'S Nutrition Research Center (ACNC)
item Badger, Thomas

Submitted to: Experimental Biology and Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2014
Publication Date: 4/1/2015
Citation: Fang, N., Yu, S., Ronis, M.J., Badger, T.M. 2015. Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples.Experimental Biology and Medicine. 240(4):488-497.

Interpretive Summary: High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are well accepted techniques for detecting and quantitating compounds in biological samples, such as blood, urine and tissues. However, many samples have such complex mixtures of substances that make it very difficult to identify the specific compound of interest. While studying the chemical composition of urine, researchers at Arkansas Children’s Nutrition Center in Little Rock, Arkansas have found the a process known as the matrix effect is responsible for making HPLC and LC-MS/MS measurements inaccurate. They found that because of the matrix effect, the chemical behavior of bile acids in urine interfered with reliable detection and quantification of these compounds. This is the first characterization of this matrix effect. This finding could lead to improved techniques for qualitative and quantitative analyses of blood and urine, which could greatly improve several areas, such as drug development and sports drug testing.

Technical Abstract: High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.