• Application Note

Compilation of Amino Acids, Drugs, Metabolites and Other Compounds in Masstrak Amino Acid Analysis Solution

Compilation of Amino Acids, Drugs, Metabolites and Other Compounds in Masstrak Amino Acid Analysis Solution

  • Paula Hong
  • Kendon S. Graham
  • Alexandre Paccou
  • Thomas E. Wheat
  • Diane M. Diehl
  • Waters Corporation

For research use only. Not for use in diagnostic procedures.

Abstract

The MassTrak Amino Acid Analysis Solution provides a reproducible, robust method with easy identification of the common physiological amino acids. Common compounds detected with the MassTrak AAA Solution include a wide number of pharmaceuticals and metabolite side products found in biological fluids. This compilation provides a guide to more than 100 such compounds, beyond the standard amino acids, that may appear in the analysis of biological fluids with the MassTrak AAA Solution.

Introduction

Physiological amino acid analysis is commonly performed to monitor and study a wide variety of metabolic processes. A wide variety of drugs, foods, and metabolic intermediates that may be present in biological fluids can appear as peaks in amino acid analysis, therefore, it is important to be able to identify unknown compounds.1,2,3 The reproducibility and robustness of the MassTrak Amino Acid Analysis Solution make this method well suited to such a study as well.4

Experimental

Compound sample preparation

A library of compounds was assembled. Each compound was derivatized individually and spiked into the MassTrak AAA Solution Standard prior to chromatographic analysis. The elution position of each tested compound could be related to known amino acids.

  1. Each compound was prepared at 500 μM in 0.1 N HCl.
  2. Each compound was derivatized according to the standard as described in the MassTrak AAA Solution User’s Guide at a concentration of 250 μM.
  3. Each compound was also derivatized in conjunction with the MassTrak AAA Standard: 70 μL of borate buffer, 5 μL of MassTrak AAA Standard (250 μM) and 5 μL of compound (500 μM) were combined prior to derivatization.

Sample preparation utilized the MassTrak AAA derivatization kit and Total Recovery vials. The amino acids were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (US Patent 5,296,599 and European Patent EP 0533 200 B1). The protocol followed that described in the MassTrak AAA Solution User’s Guide.

LC conditions

LC System:

Waters ACQUITY UPLC System with TUV

Column:

MassTrak AAA Column 2.1 x 150 mm, 1.7 μm

Column Temp:

43 ˚C

Flow Rate:

400 μL/min.

Mobile Phase A:

MassTrak AAA Eluent A Concentrate, diluted 1:10

Mobile Phase B:

MassTrak AAA Eluent B

Weak Needle Wash:

5/95 Acetonitrile/Water

Strong Needle Wash:

95/5 Acetonitrile/Water

Gradient:

MassTrak AAA Standard Gradient (as provided in kit)

Detection:

UV @ 260 nm

Injection Volume:

1 μL

Injection Mode:

Partial Loop with Needle Overfill (PLNO)

Results and Discussion

A wide variety of antibiotics, pharmaceutical compounds and metabolite by-products are found in biological fluids. The retention times of a number these compounds have been cataloged for a number of amino acid analysis methods.1,2,3 A similar study was conducted for the MassTrak AAA Solution.

The compounds listed in Tables, 1, 2 and 3 were analyzed using the MassTrak AAA Solution standard. Retention times were verified for the individual standards, as well as combined with the MassTrak AAA standard. The latter analysis accurately documented coelutions, which include partial resolution. The following tables are compilations of the interference compounds in alphabetical order ( Table 1), and elution order (Table 2). Unreactive compounds are listed in both Table 1 and Table 3.

Table 1. Compounds in alphabetical order

Table 2. Compounds in order of retention time (RT)

Several compounds produce atypical results upon reaction with the derivatization reagent. These compounds may form isomers or undergo derivatization of an alcohol. Some isomers yield two peaks in the chromatogram (ex. pipecolinic acid). For these compounds, both peaks have been documented and are labeled as Peak 1 and Peak 2. Other compounds, such as argininosuccinic acid, react upon heating to form anhydrides. These secondary products have also been reported.

A number of compounds are directly detectable due to their chemical structure within these chromatographic conditions. These compounds, for example xanthine and caffeine, are not derivatized but appear in the chromatogram.

Conclusion

The MassTrak Amino Acid Analysis Solution provides a reproducible, robust method with easy identification of the common physiological amino acids. These characteristics allow for routine preliminary identification of common interferences and other compounds by relative retention time. This same approach or set of experiments is required for ion exchange methods using protocols, columns and instruments that differ from the historical database. Given the characteristics of ion exchange, this process can be time consuming and variable.

Common compounds detected with the MassTrak AAA Solution include a wide number of pharmaceuticals and metabolite side products found in biological fluids. This compilation provides a guide to more than 100 such compounds, beyond the standard amino acids, that may appear in the analysis of biological fluids with the MassTrak AAA Solution. The listed relative retention times also provide a reliable starting point for identification of such compounds in authentic samples.

References

  1. S.A. Cohen, M. Meys, T.L. Tarvin, Pico Tag Method: A Manual for Advanced Techniques for Amino Acid Analysis, 1989, Millipore Corp., p 80-90.
  2. S. Ian, S. Martin, H. Mick, “Drug Interference in Amino Acid Analysis,” SSIEM 2007, National Metabolic Biochemistry Network, Training Documents.
  3. S.J. Rehfeld, H. Loken, W.D. Korte, Letters to the Editor: “Interference by Antibiotics in Amino Acid Analysis,” Clin. Chem., 20 (1974), 11, 1477.
  4. P. Hong, T. E. Wheat, D. M. Diehl, Analysis of Physiological Amino Acids with the MassTrak Amino Acid Analysis Solution, 2009, Waters Corp., Application Note, 720002903EN.

720003142, July 2009

Back To Top Back To Top