In a high-sensitivity amino acid analysis (AAA)
technique that does not require sample derivatization
(AAA-Direct™, Dionex Corp., Sunnyvale, CA),
both amino acids and carbohydrates are separated
by high-performance anion-exchange chromatography
and detected by integrated pulsed amperometry
(IPAD). The highly sensitive direct detection capability
of amperometry used in the technique eliminates
the need for pre- or postcolumn derivatization.
Chemical derivatization techniques complicate
analysis, increase cost for expensive reagents, introduce
safety hazards to laboratory personnel exposed
to toxic solvents, and add a hazardous waste stream
that must be safely disposed of. These obstacles are
eliminated with the AAA technique.
Unfortunately, high concentrations of carbohydrates
in some samples may coelute with amino acids and
complicate their determination. Published techniques1–5 describe the resolution of carbohydrates
from amino acids by changing the eluent hydroxide
concentration and extending the initial isocratic
elution phase. Although this technique works well
for injected samples having carbohydrate concentrations
up to about 100 μM, decreasing hydroxide
eluent reduces detector response for the early-eluting
amino acids. When amino acid concentrations are
exceptionally low and the carbohydrate concentrations
are exceptionally high (e.g., fruit juices), it
is impossible to accurately determine amino acids
(Figure 1). These samples require the removal of carbohydrates
prior to amino acid analysis. Jandik et al.6
published an automated in-line sample preparation
procedure that eliminated carbohydrates from amino
acid samples prior to their analysis by AAA-Direct,
and Genzel et al.7 reported the successful use of the
procedure for cell culture media applications. When
carbohydrate-containing amino acid samples are
loaded on a Carbohydrate Removal Cartridge (CRC,
Dionex) under acidic conditions, the amino acids
bind to the CRC while the unbound carbohydrates
are washed to waste. After suitable carbohydrate
clearance from the cartridge, the valve switches
position and elutes amino acids from the CRC onto
the AminoPac® column (Dionex), where they are
separated by anion-exchange chromatography.
This technique was difficult to install and program, required an additional pump and two
additional high-pressure valves, and
the carbohydrate elimination feature
could not easily be turned on or off to
switch the system back to its typical
AAA-Direct setup.
Figure 1 - High carbohydrate concentrations in samples interfere with
the determination of amino acids using AAA-Direct. The figure shows that
grape juice has a high carbohydrate concentration that precludes amino
acid quantification.
This paper describes a simpler procedure
to eliminate carbohydrates prior
to AAA-Direct analysis of amino acid
using the Carbohydrate Removal
Accessory (CRA) and features of
the ICS-3000 (both from Dionex).
Using a CRA, carbohydrate-containing
amino acid samples are pumped
onto the CRC trap using the AS autosampler
sample preparation syringe
(Dionex) instead of a separate accessory
pump. The CRC replaces the
typical injection loop on the inject
valve, eliminating the need for additional
accessory valves. All controls
are automated through the software
and can be turned either on or off.
The ICS-3000 can be set up in a
dual-system format that allows two
CRA AAA-Direct applications to run
at the same time and thereby double
sample throughput. The performance
of this method is demonstrated, and
its application to challenging samples,
including cell culture media and
juices, is shown.
Experimental
Equipment and conditions
Equipment included the ICS-3000 ion chromatography system configured
for AAA-Direct, consisting of a dual
gradient pump, a detector compartment
with an electrochemical detector
outfitted with an AAA-Certified™
disposable gold working electrode, an autosampler
with sample preparation and the CRA, and
Chromeleon® software for instrument control
and data management (all from Dionex). The
AAA-Direct gradient conditions and CRA
setup are described elsewhere.8
Standards and samples
The following standards were used: arginine,
asparagine, citrulline, cysteic acid, cysteine,
fructose, fucose, galactosamine, galactose,
glucosamine, glucose, glutamine, glycine,
hydroxylysine, inositol (myo-), lactose, lysine,
maltose, mannose, methionine sulfoxide,
norleucine, ornithine, propylene glycol,
sucrose, taurine, threonine, tryptophan, tyrosine,
urocanic acid, and valine. These were
obtained from Sigma-Aldrich (St. Louis,
MO), Pfanstiehl Laboratories (Waukegan,
IL), or Fisher Scientific (Pittsburgh, PA);
hydroxyproline was from Calbiochem (San Diego,
CA); and sucralose was from McNeil Nutritionals
(New Brunswick, NJ). Standard Reference Material
2389 (amino acids in 0.1 M hydrochloric acid) was
obtained from NIST (Gaithersburg, MD). Italian
Muscat grapes were California-grown fresh produce
manually pressed, and the juice decanted. Decanted
grape juice, Bacto® Yeast Extract-Peptone-Dextrose
(YPD) broth (BD Diagnostic Systems, Sparks,
MD), McCoy’s 5A mammalian cell culture medium
(Sigma-Aldrich), and 100% carrot juice were centrifuged
at 16,000 × g for 10 min, and the supernatant
was diluted in water.
Results and discussion
CRA performance
Figure 2 - The elimination of carbohydrates and the separation of amino acids
present in YPD broth (400-fold dilution) (a) and in McCoy’s 5A medium (50-fold
dilution) (b) using 25 μL of diluted samples. AAA-Direct separations are shown
with (A) and without (B) the CRA.
The percent recovery of amino acid standards using
the AAA-Direct system with the CRA ranged from 102 to 110%, except asparagine (79%), hydroxyproline
(16%), and taurine (0%). Lower recovery of amino
acids (except taurine) did not prevent accurate quantification
of these amino acids because the slopes of the
resulting calibration curves reflected the low recovery.
The capability of the CRA to eliminate different types
of carbohydrates was investigated. Using 10 μM amino
acid standards with 1 mM glucose, fructose, sucrose,
maltose, inositol, or lactose, high amino acid recovery
was determined for each carbohydrate matrix. Slightly
elevated recovery values were observed for amino
acids coeluting with each trace carbohydrate breakthrough
peak. Accurate recovery of amino acids was
possible up to at least 56 mM glucose, except for threonine,
which coelutes with the glucose breakthrough
peak. Varying either the amino acid concentration
(1–10 μM range) or carbohydrate concentration (10
μM–56 mM) did not change amino acid recovery
from a carbohydrate matrix. The CRA eliminated
fucose, galactose, mannose, propylene glycol, and
sucralose, but not the amino sugars (galactosamine,
glucosamine), which as expected bind to the cation
exchange cartridge. Except for asparagine and
hydroxyproline, the CRA did not change amino acid
calibration curves. Retention time RSDs ranged from
0.02% to 0.40%, and the peak area and peak height
RSDs ranged from 1.2% to 4.1% and 0.7% to 9.1%,
respectively, for 15 injections of amino acid standards
(10 μM) over one day.
Application of the CRA
Figure 3 - The elimination of carbohydrates and separation of trace amino
acids present in freshly pressed Italian Muscat grape juice (100-fold dilution)
(a) and in commercial carrot juice (100-fold dilution) (b) using 25 μL of
diluted samples.
Different high-carbohydrate sample matrices were
investigated using products from the pharmaceutical,
biotechnology, and food and beverage
industries. The CRA allowed successful
amino acid determinations for all samples
investigated. Figure 2 shows the separation
of amino acids present in a fermentation
broth medium (YPD broth, 400-fold
dilution) and in a mammalian cell culture
medium (McCoy’s 5A medium, 50-fold
dilution), with and without the CRA.
Figure
3 shows the separation of amino acids present
in 100-fold dilutions of grape juice and carrot
juice, with and without the CRA. For each
of these samples, amino acid determinations at
these dilutions were impossible to perform without
the use of the CRA due to large concentrations
of glucose, sucrose, or fructose. Using the CRA,
carbohydrates were removed prior to chromatography,
thus allowing accurate amino acid determinations.
Table 1 shows the amino acid spike (5
μM) recovery from these matrices, where recovery
from YPD broth ranged from 75% (Asp) to 105%,
with an average of 91%; recovery from McCoy’s
5A medium ranged from 45% (Asp) to 112%,
with an average of 93%. The spike recovery from
grape juice ranged from 25% (Asp) to 105%, with
an average recovery of 86%. The recovery from
carrot juice ranged from 56% (Asp) to 112%, with
an average recovery of 90%.
This method was also installed on a dual ICS-3000
that allowed two AAA-Direct applications to operate
in parallel, using only one autosampler. With this
installation, throughput was nearly doubled from 18
to 35 injections per day, and the second system had
the same high performance as the first.
Conclusion
Amino acids were accurately quantified in samples
containing high concentrations of carbohydrates
using automated elimination of carbohydrates. The
method requires no sample derivatization for amino
acid detection and was successfully applied to cell
culture media and fruit and vegetable juices.
References
- Dionex Corp. Application note 150. Determination of amino acids in cell cultures and fermentation broths. Literature product no. (LPN) 1538, July 2003.
- Hong, Y.; Ding, Y.-S.; Mou, S.; Jandik, P.; Cheng, J. Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection. J. Chromatogr. A2002, 966, 89–97.
- Ding, Y.; Yu, H.; Mou, S. Direct determination of free amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection. J. Chromatogr. A2002, 982, 237–44.
- Hanko, V.P.; Rohrer, J.S. Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection. Anal. Biochem. 2003, 324, 29–38.
- Hanko, V.P.; Heckenberg, A.; Rohrer, J.S. Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection. J. Biomol. Tech.2004, 15, 315–22.
- Jandik, P.; Cheng, J.; Jensen, D.; Manz, S.; Avdalovic, N. Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples. J. Chromatogr.B2001, 758, 189–96.
- Genzel, Y; König, S.; Reichl, U. Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection. Anal. Biochem. 2004, 335, 119–25.
- Dionex Corp. Technical note 69. Carbohydrate Removal Accessory for the Determination of Amino Acids in High Carbohydrate-Containing Samples using AAA-Direct, 2006.
Mr. Hanko is Staff Chemist, and Dr. Rohrer is Director, Applications
Development, Dionex Corp., 1228 Titan Way, Sunnyvale,
CA 94086, U.S.A.; tel.: 408-737-0700; fax: 408-739-4398;
e-mail: [email protected].