M1067. Comparison of two Amino Acid Analysis Methods: Pre-column Derivatization/RP-UPLC vs. Post-column Derivatization/Cation-Exchange HPLCShiqian Zhu, Paushika Shah, Lei Zhang, André Johnson and James BourdageBiotechnology Services, Covance Laboratories Inc., Greenfield, Indiana

Amino Acid %Recovery of Two Methods

0.0

20.0

40.0

60.0

80.0

100.0

120.0

%Re

cove

ry

Post-column/HPLC 100.8 99.7 91.4 98.6 97.4 102.8 96.5 98.5 110.2 99.5 101.2 100.8 102.3 99.1 101.3 101.0

Pre-column/UPLC 104.2 93.7 86.2 102.3 97.6 101.4 95.5 99.7 108.2 94.7 96.2 89.4 88.7 108.3 90.8 92.1

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg

Figure 3. %Recovery of 16 amino acids.

% Recovery of 16 amino acids in BSA hydrolysates determined by both methods (n=3) is provided in Figure 3. The % Recovery of all amino acids was 91.4% - 110.2% for post-column/HPLC and 86.2% - 108.2% for pre-column/UPLC, both within 85% - 115%.

Figure 1. Pre-column derivatization vs. post-column derivatization.

IntroductionAmino acid analysis (AAA) refers to the methodology used to determine the amino acid composition or content of proteins or peptides.

Amino acids are the basic constituents of proteins. For that reason, qualitative and quantitative analysis of the amino acid composition of hydrolyzed samples of pure proteins or peptides is used to identify the material and to directly measure concentration. In addition, amino acids are also intermediates in many metabolic pathways; the amino acids are, therefore, measured as elements of physiological and nutritional studies.

The procedure of AAA includes Hydrolysis, Separation, Detection and Quantification. A protein or peptide sample is hydrolyzed to its individual amino acid constituents before amino acid analysis. The free amino acids are then separated by HPLC and detected by an appropriate detector. Pre-column and post-column derivatization methods have long been used to improve the sensitivity and selectivity (Figure 1).

In the past decade, UPLC (ultra-high performance liquid chromatography), a variant of HPLC, has become more and more popular in analytical laboratories. Using sub-2 micron particles, high linear velocity mobile phases, and instrumentation that operates at high pressures, UPLC can dramatically increase the resolution, sensitivity, and speed of analysis compared to traditional HPLC.

The purpose of this study is to compare two Amino Acid Analysis methods, 1) reverse-phase UPLC coupled with pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization and 2) cation-exchange HPLC coupled with post-column ninhydrin derivatization, which has been considered as a reference method due to its robust nature.

Figure 2. Example chromatograms.

Table 1. Methods and Materials

Method Pre-column/UPLC Post-column/HPLC

InstrumentWaters® ACQUITY UPLC™ System

Hitachi L-8800 amino acid analyzer

ColumnWaters AccQ∙Tag Ultra C18 1.7µm 2.1 x 100mm

Hitachi AAA special analysis column 4.6 x 60mm

Detection Wavelength 260nm 440nm (570nm for Pro)

Derivatization reagent6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)

Ninhydrin

Run time 9.5min 84.8min

Injection Volume 1 µL 20 µL

StandardWaters Amino Acid Standard

Thermo Scientific amino acid standard

Internal Standard NorValine NorLeucine

Sample Bovine Serum Albumin (BSA) hydrolysate

ResultsThe example chromatograms of amino acid standard (200nmole/mL) from both methods are shown in Figure 2. The resolution and sensitivity of pre-column/UPLC method is significantly better than that of post-column/HPLC method.

Methods and MaterialsMethods and Materials are summarized in Table 1.

Table 2. Linearity and Instrument Repeatability Results

Linearity (R2)Instrument Repeatability (%RSD)

Retention Time Peak Area

Amino Acid Pre-UPLCa Post-HPLCb Pre-UPLCa Post-HPLCb Pre-UPLCa Post-HPLCb

His 0.9998 1.0000 1.0% 0.0% 0.4% 0.7%

Ser 1.0000 1.0000 0.4% 0.1% 0.3% 0.5%

Arg 0.9998 1.0000 0.4% 0.0% 0.3% 0.7%

Gly 0.9999 1.0000 0.3% 0.1% 0.3% 0.6%

Asp 0.9995 1.0000 0.3% 0.2% 0.3% 0.5%

Glu 0.9996 1.0000 0.3% 0.1% 0.3% 0.5%

Thr 1.0000 1.0000 0.4% 0.1% 0.4% 0.5%

Ala 0.9998 1.0000 0.6% 0.2% 0.3% 0.6%

Pro 0.9999 0.9998 0.4% 0.1% 0.3% 0.8%

Cys 0.9998 0.9999 0.2% 0.0% 0.4% 0.7%

Lys 0.9986 1.0000 0.2% 0.0% 0.7% 0.7%

Tyr 0.9996 1.0000 0.2% 0.0% 0.3% 0.6%

Met 0.9999 1.0000 0.2% 0.0% 0.3% 0.6%

Val 0.9999 1.0000 0.2% 0.0% 0.3% 0.6%

Ile 1.0000 1.0000 0.2% 0.0% 0.3% 0.6%

Leu 1.0000 1.0000 0.2% 0.0% 0.3% 0.6%

Phe 0.9996 1.0000 0.2% 0.0% 0.4% 0.6%

a Pre-column derivatization/UPLC method.b Post-column derivatization/HPLC method.

ConclusionsThe linearity, repeatability and recovery of both methods met the requirement for intended use to determine protein content and amino acid composition.

Compared to each other, both methods showed their advantages and disadvantages.

For post-column/HPLC method, the derivatization reaction is automated (on-line) and not affected by the sample matrix, which offers excellent quantitative performance and reproducibility. However the option of derivatization reagent is limited and the sensitively is relatively low. In addition, the total run time of each sample using post-column/HPLC is as long as 84.8 min; and the method requires a dedicated instrument.

Pre-column/UPLC method provides significantly better resolution and sensitivity with much shorter run time (9.5 min). The consumption of derivatization reagent can be very small, allowing using more expensive reagent. Therefore there are more options of derivatization reagent for pre-column/UPLC method, as long as the unreacted derivatization reagent can be separated from amino acids on the column. However, the derivatization step of pre-column/UPLC method is labor-intensive and derivatization reaction efficiency may be affected by sample matrix.

In summary, after the reaction system is optimized, post-column/HPLC method is a good choice for routine quantitative analysis; pre-column/UPLC method can be used for screening derivation reagents and studying special amino acids, and for the sample with limited amount.

The instrument repeatability was evaluated by %RSD of retention time and peak area of each amino acid in 5 consecutive injections of the standard. The linearity of standard curve for each amino acid was evaluated in the range of 20nmol/mL to 200nmol/mL. The RSD% and determination of coefficient (“r2”) results are listed in Table 2. The determination of coefficient of each amino acid standard curve is > 0.99; the RSD% of retention time and peak area of each amino acid peak is ≤ 1.0% for both methods.

Presented at the2012 AAPS National Biotechnology ConferenceSan Diego, California21-23 May 2012

M1067. Comparison of two Amino Acid Analysis Methods: Pre-column Derivatization/RP-UPLC vs. Post-column Derivatization/ Cation-Exchange HPLCShiqian Zhu, Paushika Shah, Lei Zhang, André Johnson and James BourdageBiotechnology Services, Covance Laboratories Inc., Greenfield, Indiana

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