Rapid Separation and Detection of Amino Acids by HPLC-HPIMS · and detection of amino acids is essential for a variety of applications in the medical, pharmaceutical, and food industries.3-5.

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Rapid Separation and Detection of Amino Acids by HPLC-HPIMSTM

In addition to being the structural units that make up proteins, amino acids play important roles in gene expression, cell signaling, and immunity; also serving as metabolic intermediates in the synthesis of neurotransmitters and other biomolecules.1,2 Due to their biological significance, efficient separation and detection of amino acids is essential for a variety of applications in the medical, pharmaceutical, and food industries.3-5

Most High-Performance Liquid Chromatography (HPLC) methods for amino acid analysis (including commercial amino acid analyzers) require long run times and involve some form of derivatization to enhance separation or detection.1-6 Like many compounds, underivatized amino acids lack a strong chromophore, fluorophore, or electroactive moiety for photometric, fluorometric, or amperometric detection, respectively.1,3,4,7 In addition to being time consuming and inconvenient for the analyst, issues with derivative instability, side reactions, and reagent interferences can result in the presence of extra peaks in the chromatogram that can complicate interpretation and reduce analytical confidence.1,3-6 For these reasons, direct detection is preferred whenever possible.

Figure 1: Excellims’ IA 3100 HPLC-HPIMSTM displayed with a Shimadzu Prominence UFLC system.

Excellims’ award-winning IA3100 (Fig. 1) provides a robust and efficient alternative for amino acid analysis that brings the rapid orthogonal separation and detection capabilities of High-Performance Ion Mobility Spectrometry (HPIMSTM) to any existing HPLC system. This additional dimension of separation allows the analyst to condense long chromatographic methods by performing a partial separation with reverse phase (RP) chromatography and completing the separation in the gas-phase using ion mobility spectrometry. Using this technology, it is now possible to obtain complete separation of all 20 common amino acids in less than 15 minutes – twice as fast as state-of-the-art amino acid analyzers.9,10

With HPLC-HPIMSTM, as the sample elutes from the LC column, it flows through an adjustable flow splitter that diverts a small portion of the eluent (2 to 8 μL min-1) to the electrospray ionization (ESI) source, which converts the sample to gas-phase ions. The ionized analytes then enter the drift tube of the HPIMSTM and separate according to gas-phase mobility by the action of two opposing factors: 1) A strong electric field pulls the ions toward the detector with a force proportional to the charge on the ion; 2) The ions are slowed down to different extents by collisions with molecules of drift gas as they travel down the drift tube. The probability of these collisions is proportional to the cross-sectional area of the molecule reflecting its structural shape. Thus, to a first approximation, the mobility separation is dependent on the charge and cross-sectional area (i.e., shape) of the ion. A Faraday plate detector at the end of the drift tube records the time at which each ion hits the detector and converts the current generated into an intensity value that is displayed on the IMS spectrum. The drift time of each ion can then be compared with standard or user-defined database values to determine the identity of the compound.

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To demonstrate the utility of combining HPLC and HPIMSTM for orthogonal separation and detection, consider the separation that each individual method provides by itself (Fig. 2, 3).

Figure 2: RP-HPLC separation of 20 common amino acids (100 µM each) using a Thermo Aquasil C18 column (100 x 4.6 mm, 5.0 µm). Method: H2O (A), ACN (B); Step Gradient: Hold 0% B for 3.5 min, 5% B for 2.5 min, 18% for 4 min, 35% for 4 min, 65% for 4 min, linear gradient to 100% B over 2 min and hold for 3 min; Injection: 5.0 µL).

Table 1: Amino Acid Separation and Detection Methods Method Separation Detection

IEC + Standard column + Efficient + Reproducible

- Requires derivatization for UV or fluorometric detection - Requires desalting of eluent prior to ESI-HPIMSTM, ELSD, or CLND12

IPC

+ Standard RP column5,6,7 - Long re-equilibration2,6,7 - Poor reproducibility and intersystem transferability6 - Ion-pairing agents toxic and expensive!

- Poor sensitivity direct UV detection5 - Ion-pairing agents suppress ionization, which decreases sensitivity and complicates quantitation12

Specialty Column

+ Efficient + Reproducible - Expensive - Single-purpose

- Poor sensitivity direct UV detection5

- Often designed to be used with inorganic buffers that are incompatible with ESI-HPIMSTM, ELSD, and CLND

RP - Insufficient retention of polar amino acids2,7,10

- Eluent compatible with ESI-HPIMSTM, ELSD, and CLND

Complete separation of 20 amino acids by HPLC

takes approximately 30 minutes and can be accomplished using ion exchange chromatography (IEC), ion-pairing chromatography (IPC) on a RP-e C18

or porous graphitic column, or other methods that employ specialty amino acid columns.7,10,11 Traditional reverse phase chromatography is ineffective as it provides insufficient retention of the most polar amino acids.2,7,10 In the chromatogram in Figure 2, the first ten amino acids elute in under two minutes and seven of the ten co-elute as a single LC peak. (See Table 1 for a comparison of advantages and disadvantages of the separation and detection options for each type of chromatography).

Although this separation is not trivial, it is generally recognized that the challenge of amino acid analysis lies not in their separation, but in their detection.1,3 The detector that is most commonly used for amino acid analysis is the electrochemical detector; however, this method of detection requires an experienced operator. In addition, the number of amino acids that can be detected at carbon-based electrodes is limited and the electrodes are prone to fouling.1,3 UV and refractive index detectors are also commonly used, but they respond to amino acids with poor sensitivity and changes in solvent composition elicit a strong response which further clouds the weak analyte signal.1,3,4,7 These strong solvent responses can be seen in Figure 2 at 8, 12, 16, and 21 minutes. For this reason, these detectors are generally considered to have poor compatibility with step-gradient elution and are incompatible with all other gradient elution profiles.3,5,7 In contrast, direct detection by ESI-HPIMSTM is highly sensitive, reproducible, and has the added benefit of providing a rapid orthogonal separation based on size-to-charge ratio (Fig. 3). While many of the amino acids can be rapidly separated and detected using HPIMS alone, some amino acids exhibit ion mobility coincidences, necessitating a complementary method to resolve them. Thus, the addition of the gas-phase separation capabilities of HPIMSTM to HPLC allows for greater flexibility in the chromatographic separation as compounds that co-elute in the HPLC can be resolved by the HPIMSTM detector. One capability that distinguishes ion mobility from other orthogonal separation techniques is its ability to rapidly separate isomers and other molecules with slight structural differences that do not separate by HPLC. For example, the



HPIMSTM separates leucine and isoleucine as shown below (Fig. 4) with just a few second acquisition.

Figure 3: HPIMSTM separation of 20 common amino acids on Excellims’ GA2100 stand-alone ion mobility spectrometer equipped with a DirectsprayTM ionization source. Sample: 100 µM each in 50:50 MeOH/H2O, Flow Rate: 1.0 µL min-1; Source: 3.0 kV; Drift Tube: 8.0 kV; Desolvation/Drift Region Temp: 180 oC; Drift Gas: 2.0 L min-1, air; Exhaust Flow: 1.8 L min-1.

Therefore, this isomer capability enables

complete two-dimensional separation of amino acids using a standard reverse phase column without the need for ion-pairing agents. Ion-pairing agents are generally added to increase retention of polar compounds that are poorly retained in reverse phase columns, thus enabling their separation.2,4,5 Long-chain perfluorinated carboxylic acids are often used as ion-pairing agents as they are volatile enough to be used with ELSD, CLND, and instruments that rely on electrospray ionization for sample introduction, such as MS and HPIMSTM.2,4 However, ion-pairing chromatography has a number of major drawbacks with respect to efficiency, reproducibility, and detection sensitivity.2,6,7 Depending on the chain length of the ion pairing agent used, it can take up to 3 hours for the ion-pairing agent to re-equilibrate with the stationary phase after each run, which severely limits throughput.2,7 Furthermore, variation in the extent of re-equilibration results in poorly reproducible retention times, complicating data analysis. These ion-pairing agents have also been shown to reduce ionization efficiency by up to 80%, which negatively impacts detection sensitivity.12 However, if ESI-HPIMSTM is used for detection, these polar amino acids can be separated by gas-phase ion mobility, eliminating the need for ion-pairing agents

and the problems that come with using them. This simplification of the chromatographic separation conditions results in a more robust and efficient amino acid separation. In addition, the mobility data from the HPIMSTM provides an additional metric for compound identification that enhances detection confidence.

Since HPLC separations are carried out on a timescale of minutes, whereas IMS separations are completed in milliseconds, dozens of IMS spectra can

Figure 4: HPIMSTM separation of leucine and isoleucine with Excellims GA 2100 stand-alone ion mobility spectrometer equipped with a DirectsprayTM electrospray ionization source. Sample: 100 µM each in 80:20 MeOH/H2O, Flow Rate: 1.0 µL min-1; Source: 2.4 kV; Drift Tube: 10.0 kV; Desolvation/Drift Region Temp: 180 oC; Drift Gas: 1.4 L min-1, air; Exhaust Flow: 1.2 L min. -1

Figure 5: A three-dimensional HPLC-HPIMSTM spectrum showing the separation of 20 amino acids. HPLC retention time is on the y-axis, ion drift time is on the x-axis, and signal intensity is on the z-axis. The HPLC separation conditions are identical to those listed in Figure 2. The UV absorbance detector trace at 190 nm (blue) and 210 nm (red) is displayed on the left. The HPIMSTM separation was conducted on Excellims’ IA3100 equipped with an adjustable flow splitter and an InfusionTM electrospray ionization source. Sample Flow: 4.0 µL min-1, Sheath Flow: 2.0 µL min-1 ACN; Source: 2.4 kV; Drift Tube: 10.0 kV; Desolvation/Drift Region Temp: 180oC; Drift Gas: 2.0 L min-1, air; Exhaust Flow: 1.2 L min-1.



be acquired across each chromatographic peak. These IMS “slices” can be stacked in-parallel to create a three-dimensional contour plot (Fig. 5).

Each peak in the contour plot has a unique drift time and retention time that can be used to identify each amino acid in the spectrum. This identification is readily accomplished using the user-defined compound library feature in Excellims VisION Control software. To populate this library, run standards of each amino acid and enter a compound identifier with the calibrated drift time. The software will automatically assign the peaks in the spectrum based on this library data and will even perform quantification if calibration data is supplied.

Most of the samples for which amino acid analysis is of interest are present in complex biological or other matrices. Thus, the practical utility of HPLC-HPIMSTM as a method for amino acid analysis is dependent on its ability to handle samples in complex matrices, such as fermentation and cell culture media. The efficiency of these bioprocesses is currently limited by the availability of on-line monitoring data that can guide decision making in real time. Lysogeny broth (often referred to as Luria Broth or simply LB broth) is a common nutrient mixture used in E. coli cell culture work. Although a variety of formulations exist, a typical recipe contains 10 g L-1 NaCl (~170 mM), 10 g L-1 tryptone, and 5 g L-1 yeast extract. Tryptone is produced by the tryptic digestion of casein protein and serves as an amino acid fuel source for growing bacteria. The digestion products are predominantly free amino acids and some small peptides. Yeast extract is produced by the autolysis of yeast and provides an assortment of free amino acids, peptides, carbohydrates, vitamins, and other organic compounds necessary for growth. The depletion of these nutrients and accumulation of waste products are essential parameters influencing the success of these processes and must be monitored and managed to optimize process

efficiency. As Figure 6 shows, the integrity of the HPLC-HPIMSTM method for amino acid analysis is preserved despite the complex biological matrix of the cell culture medium. A variety of small molecules and tryptic peptides from the yeast extract and the tryptone can be seen in the inset, but they do not

appear to overlap with the amino acid signals. The Excellims IA3100 is a promising alternative to traditional HPLC methods for amino acid analysis that involve derivatization. Not only does it deliver results in half the time of industry-leading amino acid analyzers, the orthogonal separation provided by HPIMSTM enhances detection confidence by providing an additional metric for compound identification. This orthogonal separation also enables greater matrix compatibility without tedious and time-consuming adjustments to HPLC methods. Authors: William J. Warren, Anthony J. Midey, Adam M. Graichen, and Ching Wu*, Excellims Corp., Acton, MA

Intensity Scaling: 5.8x

Figure 6: A three-dimensional HPLC-HPIMSTM spectrum of Lysogeny broth obtained on an Excellims IA3100 interfaced with a Shimadzu Prominence UFLC. The instrument parameters are the same as those used in Figure 5. No modifications to the HPLC or HPIMSTM separation conditions were necessary to accommodate for the matrix.



References:

1. Agrafiotou, P.; Sotiropoulos, S.; Pappa-Louisi, A. Direct RP-HPLC determination of underivatized amino acids with online dual UV absorbance, fluorescence, and multiple electrochemical detection. J. Sep. Sci. 2009, 32, 949–954

2. Chaimbault, P.; Petritis, K.; Elfakir, C.; Dreux, M. Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids. J. Chromatogr. A 2000, 870, 245-254

3. Petritis, K.; Elfakir, C.; Dreux, M. A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids. J. Chromatogr. A 2002, 961, 9-21

4. Petritis, K.; Chaimbault, P.; Elfakir, C.; Dreux, M. Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry. J. Chromatogr. A 2000, 896, 253-263

5. Alarcon-Flores, M.I.; Romero-Gonzales, R.; Frenich, A.G.; Martinez Vidal, J.L.; Reyes, C. Rapid determination of underivatized amino acids in fertilizers by ultra high performance liquid chromnatography coupled to tandem mass spectrometry. Anal. Methods 2010, 2, 1745-1751

6. Yokoyama, Y.; Amaki, T.; Horikoshi, S.; Sato, H. Optimum Combination of Reversed-Phase Column Type and Mobile-Phase Composition for Gradient Elution Ion-Pair Chromatography of Amino Acids. Anal. Sci. 1997, 13, 963-967

7. Petritis, K.N.; Chaimbault, P.; Elfakir, C.; Dreux, M. Ion-pair reversed phase liquid chromatography for determination of polar underivatized amino acids using perfluorinated carboxylic acids as ion pairing agent. J. Chromatogr. A 1999, 833, 147-155

8. Hitachi High-Tech Amino Acid Analyzer L-8900. http://www.hitachi-hitec.cohttp://www.hitachi-hitec.com/global/science/lc/l8900.htmlm/global/science/lc/l8900.html

9. membraPure ARACUS – Fully Automatic Amino Acid Analyzer.

http://www.membrapure.com/chromatography/amino-acid-analyzer/

10. Chaimbault, P.; Petritis, K.; Elfakir, C.; Dreux, M. Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospraty mass spectrometry. J. Chromatogr. A 1999, 855, 191-202

11. Yan, D.; Li, Guo.; Xiao, X.; Dong, X.; Li, Z. Direct determination of fourteen underivatized amino acids from Whitmania pigra by using liquid chromatohgraphy-evaporative light scattering detection. J. Chromatogr. A 2007, 1138, 301-304

12. Gustavsson, S.A.; Samskog, J.; Markides, K.; Langstrom, B. Studies of signal suppression in liquid chromatography-electrospray ionization mass spectrometry using volatile ion-pairing reagents. J. Chromatogr. A 2001, 937, 41-47


Rapid Separation and Detection of Amino Acids by HPLC-HPIMS · and detection of amino acids is essential for a variety of applications in the medical, pharmaceutical, and food industries.3-5.

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