[ application note ]

[ application note ]
I NT RODUCTI ON
The analysis of amino acids is important in a wide range of applica-
tion areas including foods, nutraceuticals, pharmaceuticals, and
various biological applications. The current methodology for amino
acid analysis is liquid chromatography (LC) with pre- or post-column
derivatization for the purposes of improving sensitivity and/or
increasing retention of the analytes of interest. While being accu-
rate, these methods are often time-consuming and labor intensive.
The direct analysis of underivatized amino acids is very attractive,
as the elimination of derivatization brings the advantages of sim-
plicity, flexibility, as well as the desired sensitivity and separation
speed. In addition, the removal of the derivatization step reduces
the possibility of altering the sample through contamination or
degradation.
This application note describes a new UPLC

/MS/MS methodology
for direct amino acid analysis. The use of UltraPerformance LC

technology combined with MRM mass spectrometric detection allows
for direct amino acid analysis with high selectivity and sensitivity.
To achieve adequate retention for underivatized amino acids with
reverse-phase, UPLC columns were used with an ion pairing agent.
EXPERI MENTAL
UPLC conditions
LC system: Waters

ACQUITY UPLC

system
Column: ACQUITY UPLC BEH C
18
,
2.1 x 50 mm, 1.7 m, 45 C
Flow rate: 0.8 mL/min (no split)
Mobile phase: A: 0.1% Pentadecafluorooctanoic
Acid (PDFOA),
99.5%:0.5% water/acetonitrile with
0.1% formic acid
B: 0.1% PDFOA,
10%:90% water/acetonitrile with
0.1% formic acid
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
The ACQUITY UPLC system with the Quattro Premier XE.
DI RECT UP LC / MS/ MS ANALYSI S OF AMI NO ACI DS
Peter Alden, Kate Yu, Rob Plumb
Waters Corporation, Milford, MA, USA
[ application note ]
MS conditions
MS system: Waters Quattro Premier XE
mass spectrometer
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
Interscan delay: 5 ms
Interchannel delay: 10 ms
Acquisition mode: MRM
Dwell volume: Func1 Func2 Func3 Func4
20 ms 40 ms 50 ms 30 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 acidic, basic, and neutral amino acids. The standard
mixture was diluted from 1:100 to 1:500,000 in 0.1% formic acid
to generate the calibration curves.
MS/MS methods
The Quattro Premier XEs MRM transitions and optimization condi-
tions were obtained by infusing the individual amino acid standard
solutions. For the majority of the amino acids analyzed, the stron-
gest transition was the molecular ion [M+H]
+
minus 46, due to the
loss of formic acid from the parent amino acid.
RESULTS AND DI SCUSSI ON
The MS method was comprised of four ESI
+
functions covering the
full run time. This allowed for the maximum dwell time for each
analyte, thus giving maximum sensitivity for each amino acid. The
resulting chromatograms contained sufficient data points (>20)
across the peaks for accurate and reproducible quantitation. Figure
2 shows the MRMs obtained for the standard amino acids from all
four ESI
+
functions.
The best overall amino acid separation was obtained using PDFOA
as the ion pairing reagent. Even acidic and polar amino acids
(typically only weakly retained) exhibited sufficient retention for
separation and quantitation.
The optimized amino acid method utilized a 2.1 x 50 mm, 1.7 m
ACQUITY UPLC BEH C
18
column for high resolution, short run times,
and high throughput. The analysis required a 4.5-minute gradient
with an 8-minute total run time; longer gradients could have been
employed to further enhance the resolution.
Retention Time (minutes)
0 4.5 3.8 2.5 1.0
Aspartic Acid
Serine
Glutamic Acid
Glycine
Threonine
Cysteine
Alanine
Histidine
Arginine
Lysine
Phenylalanine
Leucine
Isoleucine
Valine
Methionine
Proline
Tyrosine
Time
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.34
0.40
0.46
0.49
0.56
0.56
0.83
Time
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.25
1.31
1.66
1.97
Time
2.5 3.8
%
2.5 3.8
%
3.06
3.23
3.16
Time
3.8 4.5
%
3.8 4.5
%
3.8 4.5
%
4.08
4.11
4.12
156.1 > 109.9
175.2 > 70.0
146.9 > 83.9
132.1 > 85.8
117.8 > 71.9
166.1 > 119.9
150.1 > 103.9
115.8 > 69.9
182.2 > 136.0
90.0 > 43.9
121.8 > 75.8
120.0 > 73.9
76.0 > 30.0
148.1 > 83.8
106.0 > 59.9
134.1 > 73.9
MS Func 1
Time
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0 4.5 3.8 2.5 1.0
Time
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.34 0.34
0.40 0.40
0.46 0.46
0.49 0.49
0.56 0.56
0.56 0.56
0.83
Time
0.83
Time
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.0 2.5
%
1.25 1.25
1.31 1.31
1.66 1.66
1.97
Time
1.97
Time
2.5 3.8
%
2.5 3.8
%
2.5 3.8
%
2.5 3.8
%
3.06
3.23
3.06
3.23
3.16
Time
3.8 4.5
%
3.16
Time
3.8 4.5
%
3.8 4.5
%
3.8 4.5
%
3.8 4.5
%
3.8 4.5
%
4.08 4.08
4.11 4.11
4.12
156.1 > 109.9
175.2 > 70.0
146.9 > 83.9
132.1 > 85.8
117.8 > 71.9
166.1 > 119.9
150.1 > 103.9
115.8 > 69.9
182.2 > 136.0
90.0 > 43.9
121.8 > 75.8
120.0 > 73.9
76.0 > 30.0
148.1 > 83.8
106.0 > 59.9
134.1 > 73.9
4.12
156.1 > 109.9
175.2 > 70.0
146.9 > 83.9
132.1 > 85.8
117.8 > 71.9
166.1 > 119.9
150.1 > 103.9
115.8 > 69.9
182.2 > 136.0
90.0 > 43.9
121.8 > 75.8
120.0 > 73.9
76.0 > 30.0
148.1 > 83.8
106.0 > 59.9
134.1 > 73.9
Time
0.0 1.0
%
Time
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
0.0 1.0
%
MS Func 2 MS Func 3 MS Func 4
Figure 2. MRMs for all of the amino acids in the standard
mixture. Optimum sensitivity was obtained through the use
of several timed functions to maximize dwell time.
[ application note ]
Quantification
Figure 3 highlights four typical calibration curves (data points
in triplicate) for amino acids obtained with this method. The
responses show good linearity with detection limits ranging from
approximately 3 to 80 pg on-column. The quantification results for
the protein hydrolysate amino acid standards are summarized in
Table 1. Sensitive and linear results were obtained for nearly all
of the amino acids evaluated. Although all amino acids may be
detected using these conditions, the method is most applicable to
acidic, uncharged polar and non-polar amino acids.
Figure 3. Four example calibration curves for amino acids
demonstrate the linearity and wide linear range of the
UPLC/MS/MS method.
Example calibration curves
1/500K to 1/100 dilution, 3 injections per conc.
pmol/mL
0 5000 10000 15000 20000 25000
R
e
s
p
o
n
s
e
25000
50000
75000
100000
125000
150000
175000
200000
0 5000 10000 15000 20000 25000 0
25000
50000
75000
100000
125000
150000
175000
200000
0
1/20,000 to 1/100
Alanine
pmol/mL
0 5000 10000 15000 20000 25000
R
e
s
p
o
n
s
e
0
200000
400000
600000
800000
1000000
1200000
0 5000 10000 15000 20000 25000 0
0
200000
400000
600000
800000
1000000
1200000
0
1/50,000 to 1/100
Phenylalanine
pmol/mL
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
R
e
s
p
o
n
s
e
0
10000
20000
30000
40000
50000
60000
70000
80000
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0
0
10000
20000
30000
40000
50000
60000
70000
80000
0
1/100,000 to 1/500
Tyrosine
pmol/mL
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
R
e
s
p
o
n
s
e
0
100000
200000
300000
400000
500000
600000
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
0
100000
200000
300000
400000
500000
600000
1/500,000 to 1/500
Proline
Table 1. The quantification
results for the 17 amino acids
contained in the Pierce Standard
H mix: LODs of 3 to 80 pg of
amino acids on-column were
obtained.
Quantification Results
Standard Mol. Wt. T
R
(Min) LOD ng/mL LOD (pg) R
2
Alanine 89.09 0.83 11.3 56.5 0.990
Arginine 174.2 4.12 0.871 4.35 0.887
Aspartic Acid 133.1 0.33 0.665 3.32 0.979
Cysteine 121.16 0.57 15.1 75.5 0.937
Glutamic Acid 147.13 0.45 0.735 3.67 0.947
Glycine 75.07 0.49 1.48 7.40 0.986
Histidine 155.16 4.08 0.775 3.87 0.905
Isoleucine 131.17 3.06 16.4 82 0.984
Leucine 131.17 3.23 16.4 82 0.991
Lysine 146.19 4.11 0.73 3.65 0.837
Methionine 149.21 1.66 0.746 3.73 0.992
Phenylalanine 165.19 3.16 8.26 41.3 0.990
Proline 115.13 1.31 0.575 2.87 0.995
Serine 105.09 0.40 0.525 2.62 0.971
Threonine 119.12 0.56 0.595 2.97 0.980
Tyrosine 181.19 1.25 4.53 22.6 0.993
Valine 117.15 1.97 2.92 14.6 0.995
[ application note ]
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
CONCLUSI ON
A simple and rapid direct amino acid analysis method was devel-
oped that is applicable to acidic, uncharged polar, and non-polar
amino acids. High sensitivity and selectivity was achieved without
the need for pre-/post-column derivatization or split flow prior to
the MS. Typical limits of detection observed ranged from 3 to 80 pg
on-column for amino acids that work well with this method. Non-
polar amino acids tended to exhibit slightly higher LODs than other
amino acids analyzed.
Additionally, excellent linearity over a wide concentration range
was demonstrated for most of the amino acids evaluated. A sepa-
rate method
1
was developed to obtain optimum results with basic
amino acids.
References
1. Alden P, Yu K, Plumb R. Waters Application Note 720002003EN, Mar. 2007.
Waters, UPLC, UltraPerformance LC and ACQUITY UPLC
are registered trademarks of Waters Corporation. Quattro
Premier and The Science of Whats Possible are trademarks of
Waters Corporation. All other trademarks are the property of
their respective owners.
2007 Waters Corporation. Produced in the U.S.A.
March 2007. 720002002EN. LB-PDF