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Rapid Sample Preparation and HPLC-ESITOFMS Analysis of Derivatized Amino Acids
Introduction
A wide variety of analytical methods for the analysis of amino acids have been developed over the
years, however there is still a need for faster methods as well as for more sensitive multi-analyte
methods. These needs may be fulfilled by decreasing sample preparation time, speeding up
chromatographic separations, and by choosing detectors that provide improved sensitivity over
wide mass ranges. In this work a fast and simple sample preparation method involving sample
clean up and derivatization, was combined with a fast HPLC-ESI-TOFMS separation and detection
method for the analysis of 39 amino acid standards. In this example, the fast acquisition speed and
full mass range detection of LECO’s Unique® LC-TOFMS in combination with the automated Peak
Finding and Deconvolution algorithms provided by LECO’s ChromaTOF® software were used to
detect coeluting compounds. This reduces the burden of the chromatographic system to resolve all
components, which can be difficult when analyzing very complex samples, and allows for faster
analysis times. This approach is expected to be useful for applications where screening or profiling
of large numbers of amino acids is required.
Conditions
LC Conditions (Agilent 1100 HPLC)
Column:
2.1x100 mm, 3 µm Pinnacle™ II C-18 Restek Corporation
Run Time:
15.00 minutes
Flow Rate:
0.500 mL/minute
Mobile Phase A:
10 mM ammonium formate
Mobile Phase B:
10 mM ammonium formate in methanol
Gradient Conditions:
Time
%B
0 minutes
40%
2 minutes
40%
13 minutes
80%
15 minutes
80%
5 minute re-equilibration time
Injection Volume:
5 µL
Column Temperature:
35oC
MS Conditions (Unique LC-TOFMS)
Source:
High Flow +ESI
Data Acquisition Rate:
4 spectra/second
Electrospray Voltage:
+3.50 kV
Nebulizer Pressure:
400 kPa
Desolvation Temperature:
350oC
7.00 L/minute
Desolvation Gas Flow (N2):
Interface Temperature:
105oC
Nozzle Voltage:
110V
Skimmer Voltage:
56V
Form No. 203-821-296
8/06-REV0
LECO is a registered trademark of LECO Corporation
1
Sample Preparation
Amino acid standard mixtures and internal standards were obtained from Phenomenex and
combined into a single standard mix for analysis. The standard mixtures contained the following
36 amino acids, each with a starting concentration of 200 nmol/mL
glutamine(GLN), arginine(ARG), serine(SER), citrulline(CIT), 1-methylhistidine(1-MHIS),
3-methylhistidine(3-MHIS), glycine(GLY), 4-hydroxyproline(HYP), glycine-proline
dipeptide(GPR), sarcosine(SAR), alanine(ALA), gamma-aminobutyric acid(GABA), betaaminoisobutyric acid(BAIB), alpha-aminobutyric acid(ABA), proline(PRO), methionine(MET),
ornithine(ORN), valine(VAL), aspartic acid(ASP), histidine(HIS), glutamic acid(GLU),
lysine(LYS), tryptophan(TRP), leucine(LEU), isoleucine(ILE), phenylalanine(PHE),
aminopimelic acid(APA), cystathionine(CTH), cystine(C-C), tyrosine(TYR), alpha-aminoadipic
acid(AAA), threonine(THR), asparagine(ASN), delta-hydroxylysine(HLY), prolinehydroxyproline dipeptide(PHP), thiaproline(TPR)
The internal standard mix contained the following 3 amino acids each at a starting concentration of
200 nmol/mL
Homoarginine(HARG), d3-methionine(d3-MET), homophenylalanine(HPHE)
Sample preparation consisted of a fast SPE step (ion exchange in packed pipet tips) followed by
derivatization with propyl chloroformate and subsequent clean-up by liquid-liquid extraction. The
entire procedure was accomplished in under 15 minutes total using the Phenomenex EZFast™ Kit
for amino acids. The derivatization protocol resulted in amino acid species with greater
chromatographic retention, resulting in improved separation of polar amino acids. The derivatized
amino acids may also show improved ionization in +ESI due to blocking of acidic functional groups.
Sample preparation involved the following steps.
•
100 µL of amino acid standard mix (80 to 100 µL) was spiked with 100 µL of the internal
standard mix.
•
Sample was loaded (approx 80 to 100 µL) onto a pipet tip packed with ion exchange resin.
•
Retained free amino acids were washed and then released from resin.
•
Free amino acids were then derivatized with propyl chloroformate and liquid-liquid extracted
in a biphasic aqueous/iso-octane mixture.
•
150µL of the organic phase containing the derivatized amino acids was transferred into a
vial and organic solvent was removed under a stream of nitrogen (<10 minutes).
•
Sample was re-dissolved in 7 µL of a 2:1 methanol/10mM ammonium formate solution and
stored in freezer until analysis by LC-TOFMS (no longer than 3 days).
Results
The combined amino acid standard contained a total of 36 amino acids and 3 internal standards.
Using the fast gradient described, 33 of the 39 possible compounds could be detected by +ESI
LC-TOFMS. The lack of detection of the remaining 6 amino acids was attributed to poor ionization
in the ESI source or poor compound stability. To simplify identification of all components, the
automated peak find feature in ChromaTOF was used to locate all peaks, including all coeluting
analytes. The extracted ion chromatographic (EIC) trace of all found peaks is shown in Figure 1
with the complete peak table shown in Table 1. Assignments of isomeric amino acids were made by
comparison of retention order to Phenomenex literature (application ID 14770).
Form No. 203-821-296
8/06-REV0
LECO is a registered trademark of LECO Corporation
2
TYR
CTH
C-C
APA
LEI/ILE
PHE
TRP
LYS
PRO
IS-d3MET
MET
ORN
VAL
ASP
ABA
BAIB
GLU
SAR
HYP
GLY
IS-HARG
1MHIS/3MHIS
ARG
SER
CIT
GLN
ALA
GABA
GPR/THR
IS-HPHE
HIS
Due to poor resolution of the isomeric pairs 1MHIS/3MHIS and LEU/ILE these compounds were
reported as single peaks. In addition, THR was found to exactly coelute with GPR and so could not
be automatically found. However, this compound could be detected by manual inspection of the
mass spectral data which effectively increases the total number of amino acids detected to 34
compounds. Of the remaining unidentified compounds in the sample, several major and minor
peaks were determined to be introduced during the derivatization process while the remainders are
thought to be low level sample impurities or degradants.
Figure 1. EICs for all found peaks including 34 derivatized amino acids.
Form No. 203-821-296
8/06-REV0
LECO is a registered trademark of LECO Corporation
3
Table 1. Peak List of Identified Amino Acids
Peak #
12
13
18
19
21
22
27
31
32
39
41
43
47
49
51
54
55
56
58
60
62
64
65
69
74
76
78
80
82
84
86
Name
GLN
ARG
SER
CIT
IS - HARG (NH4)
1MHIS/3MHIS
GLY
HYP
GPR (THR coelute)
SAR
ALA
GABA
BAIB
ABA
PRO
IS - d3-MET
MET
ORN
VAL
ASP
HIS
GLU
LYS
TRP
LEU/ILE
PHE
APA
IS - HPHE
CTH
C-C
TYR (NH4)
R.T.
(min:sec)
03:09.6
03:12.7
03:49.2
03:53.8
04:03.6
04:04.8
04:41.0
05:13.2
05:32.6
06:38.9
07:22.1
07:34.8
07:59.3
08:23.5
08:45.8
09:06.2
09:09.6
09:39.4
09:49.9
09:55.9
10:12.5
10:23.8
10:25.7
10:46.1
11:06.7
11:10.6
11:55.0
12:05.0
12:19.9
12:33.1
12:41.8
Unique
Mass
275.1609
303.2028
234.1349
304.1892
334.2017
298.1780
204.1288
260.1522
301.1765
218.1421
218.1450
232.1569
232.1597
232.1570
244.1597
281.1644
278.1444
347.2150
246.1741
304.1797
370.2001
318.1972
361.2365
333.1843
260.1890
294.1736
346.2199
308.1896
479.2436
497.2024
413.2314
Conclusion
By combining a fast and easy sample preparation method with a fast HPLC-TOFMS analysis, a rapid
screening method for 34 amino acid standards has been developed. To simplify compound
identification, an automated peak find algorithm was used to identify all peaks in the
chromatogram, including many highly coeluting compounds, and assign correct masses for each
peak. This approach is expected to find utility in amino acid profiling/screening applications where
rapid, automated, and easy-to-use methods are desirable.
®
Delivering the Right Results
Form No. 203-821-296
LECO Corporation • 3000 Lakeview Ave. • St. Joseph, MI 49085
Phone: 800-292-6141 • Fax: 269-982-8977 • info@leco.com • www.leco.com
ISO-9001:2000 • No. FM 24045
8/06-REV0
LECO is a registered trademark of LECO Corporation
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