Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry

Abstract

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of ¹³C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For precolumn derivatization, plasma supernatants were buffered with sodium borate (pH 9.5-10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse-phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole-mass spectrometry. We hypothesized that precolumn Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective ¹³C internal standards, with limits of quantitation beyond physiological ranges. All 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly where upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all of the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. Although robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-ultraviolet and HPLC-fluorescence detector), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

Summary: We developed and validated a high-throughput method for the quantification of 19 amino acids (AA) in bovine plasma using liquid chromatography-single quadrupole-mass spectrometry (LC-MS). For a fully quantitative approach, samples or standards and internal standards consisting of 13C isotopes of each AA, are gravimetrically mixed. Plasma proteins are precipitated on acetonitrile, and AA are derivatized with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) to enhance resolution. Precolumn derivatization allows for a single strong-anion solid-phase extraction step, performed on a 96-well format aided by a positive-pressure (PP) manifold. After drying under a stream of N2, eluates are reconstituted in mobile phase and injected into the LC-MS, which is programmed with a 17.5-minute run time method divided into 4 segments. Sample preparation and analysis are accomplished in 23 minutes per sample when run in batch. Upper limits of quantitation are beyond the physiological range (except Gly) and R² ≥ 0.999 for every AA.

Figure 1

Reference chromatogram of 19 AA standards and their ¹³C isotopes analyzed using selected ion monitoring. Co-eluting peaks of a similar color correspond to the ¹³C isotopes. S2, S3, S4 = scanning segments.