Download Direct UPLC/MS/MS Analysis of Basic Amino Acids

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D i r e c t U P L C / M S / M S A n alys i s o f Bas i c A m i n o A c i ds
Peter Alden, Kate Yu, Rob Plumb
Waters Corporation, Milford, MA, USA
INT RO DUC T ION
The analysis of amino acids is important for a wide range of application areas including foods, nutraceuticals, pharmaceuticals,
and various biological applications. The traditional methodology
for amino acid analysis has been liquid chromatography (LC) with
pre- or post-column derivatization for the purposes of improving
sensitivity and increasing retention of the analytes of interest.
More recently, direct UPLC®/MS/MS methods utilizing ion pairing
reagents have been described which offer a simple alternative to
derivatization approaches1. Although this direct method works
well for a wide range of amino acids, chemical interferences were
observed from blank injections when analyzing basic amino acids
at the low pH (~3) normally used.
The ACQUITY UPLC system with the Quattro Premier XE.
This application note describes a modified direct UPLC/MS/MS
method that is applicable for the direct analysis of basic and wellretained non-polar amino acids. Figure 1 shows the amino acids
that were used to demonstrate this analysis method.
E X P E RIM ENTA L
UPLC conditions
N
O
N
N
N
N
O
O
N
Arginine (basic)
O
N
Histidine (basic)
N
O
O
Phenylalanine
(non-polar)
N
O
O
Lysine (basic)
N
Figure 1. Arginine, phenylalanine, histidine, and lysine amino
acids were used in the analysis.
LC system:
Waters ® ACQUITY UPLC® system
Column:
ACQUITY UPLC BEH C18,
2.1 x 50 mm, 1.7 µm, 45 °C
Flow rate:
0.8 mL/min (no split)
Mobile phase:
A: 0.1% pentadecafluorooctanoic acid,
99.5%:0.5% water/acetonitrile with 0.1% formic acid, pH 5.8
(with NH4OH)
B: 0.1% pentadecafluorooctanoic acid,
10%:90% water/acetonitrile with 0.1% formic acid, pH 5.8
(with NH4OH)
[ application note ]
Gradient:
Time (min) %A %B
Curve
0.0 99.9
0.1
6
0.5 98
2
6
2.0 80 20
6
4.0 60 40 6
4.5
0.1
99.9 6
8.0
99.9
0.1
1
R E SU LT S AND DISCUSSION
This direct amino acid method, which is run at a pH of 5.8, results
in shorter retention times than those obtained at lower pHs. Some
acidic and very polar amino acids may not be retained under these
conditions. For this reason, this method is only applicable for the
analysis of basic and some non-polar amino acids.
Figure 2 shows the MRMs for lysine, histidine, phenylalanine, and
MS conditions
arginine obtained from the injection of the listed mass-on-column
MS system:
Waters Quattro Premier™ XE
of amino acid standards (in black). Also shown are the MRMs from
mass spectrometer
a blank injection of 0.1% formic acid (in blue).
Ionization mode:
Electrospray positive
Capillary voltage: 0.5 kV
Source temp.: 130 °C
Desolvation temp.:
400 °C
Desolvation gas:
1000 L/hr
Cone gas flow:
50 L/hr
The limits of detection determined for these four amino acids are
The use of the MRM mode results in excellent sensitivity and specificity for the amino acids while maintaining a short analysis time
and high throughput. If desired, the gradient time can be shortened
to further reduce analysis times.
Interscan delay:
5 ms
well below the levels typically required for the most common amino
Interchannel delay:
10 ms
acid applications.
Data acquisition mode:
MRM
Func1
Func2
Dwell time:
60 ms
100 ms
Sample preparation
The amino acid separation was developed using the Pierce Amino
Acid Standard H Mix (protein hydrolysate) as a guide to optimize
resolution of the amino acids of interest. The standard mixture was
diluted from 1:500 to 1:500,000 in 0.1% formic acid to generate
calibration curves. Although the Pierce Standard H contains 17
amino acids, only lysine, histidine, phenylalanine, and arginine
were analyzed in this example. The MRM transitions and MS optimization conditions were obtained by infusing individual amino acid
standard solutions into the mass spectrometer.
[ application note ]
Quantification
Figure 3 shows the four calibration curves (data points in triplicate)
for the amino acids analyzed with this method. The responses for
the amino acids show good linearity with correlation coefficients of
approximately 0.995 for all four amino acids. The sensitivity of the
method for these compounds is summarized in Table 1. Detection
limits of 0.39 pg to 2.17 pg on-column were obtained for injections
of standards.
Figure 3. Four calibration curves for the amino acids demonstrating
the linearity and wide linear range of the method.
Amino Acid
Figure 2. The MRMs for lysine, histidine, phenylalanine, and
arginine standards are shown (black). The blue traces were
obtained from the corresponding blank injections.
LOD
LOD
µM
pg on-column
Phenylalanine
0.0005 µM
0.41 pg
Arginine
0.0025 µM
2.17 pg
Histidine
0.0005 µM
0.39 pg
Lysine
0.0010 µM
0.73 pg
Table 1. The detection limits for the four amino acids evaluated.
[ application note ]
CONC LUSION
A simple and rapid UPLC/MS/MS method for the direct analysis
of basic amino acids was developed. Chemical interferences from
blank injections, commonly observed with lower pH methods, were
eliminated. High sensitivity and selectivity was achieved without
the need for pre- or post-column derivatization and without the
need to split flow prior to the mass spectrometer. Limits of detection observed for the amino acids evaluated ranged from 0.39 pg
to 2.17 pg on-column. In addition, excellent linearity over a wide
concentration range was observed for the amino acids analyzed.
References
1. Alden P, Yu K, Plumb R, Waters Application Note, No. 720002002EN,
Mar. 2007.
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©2007 Waters Corporation. Produced in the U.S.A.
March 2007. 720002003EN. LB-PDF
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