aminoacidos

UPLC AMINO ACID ANALYSIS SOLUTION
UPLC AMINO ACID
Analysis Solution:
T he right answ er for amino acid analysis
nACQUITY UPLC
chromatographic techniques for analyzing amino acids require scientists to make significant
®
n
System
with the ACQUITY UPLC
compromises depending on your analytical priorities.
TUV Detector
With the introduction of enabling technologies such as Waters® UltraPerformance LC,®
AccQ•TagTM Ultra derivatization
and separation chemistries
including quality-control tested
columns, reagents, and eluents
n
n
ever before – without compromise – because Waters recognizes that you should
not have to make trade-offs in order to
achieve optimal analytical results.
To meet the specific and rigorous demands
of analyzing amino acids for protein
pre-configured projects,
characterization, cell culture monitoring,
and nutritional analysis of foods and feeds,
Waters has created a complete application
Connections INSIGHT
system: the UPLC® Amino Acid Analysis
Intelligent Service Delivery
Solution, part of the Waters Assured
Platform (ISDP)
Performance Solution (APS) family.
®
Application-specific
performance qualification
n
scientists can now achieve greater resolution, sensitivity, accuracy, and productivity than
EmpowerTM 2 Software
methods and report formats
n
Resolution, sensitivity, or throughput – you no longer have to choose just one. Traditional
Full system- and applicationlevel support and documentation
Assured Performanc e
Solutions
ENABLING YOU R SUCCESS WIT H P ROV EN INNOVAT IONS
Waters Assured Performance Solutions
designing instruments, chemistries, and methods for amino acids, highlighted by the development and
(APS) form a family of systems that
widespread success of our proven Pico•Tag® and AccQ•Tag chemistries. Today, Waters continues to lead
bring together industry-leading
the way in separations sciences by bringing together our most ground-breaking and popular technologies:
technologies – instrumentation,
the ACQUITY UPLC System and AccQ•Tag Ultra, the newest addition to the AccQ•Tag family of products.
UPLC resolution: Analysis of a hydrolysate standard at 10 pmol/µL
beyond, with Waters APS, you can
0.060
0.055
be assured that whenever a new
0.050
application challenge arises, Waters
0.030
0.025
0.020
The UPLC Amino Acid
Analysis Solution provides
high resolution to ensure
method robustness with good
quantitation. The short cycle
time is obtained without
compromising accurate results.
Pro
AU
0.035
Ile
Leu
Phe
0.040
Tyr
Met
Val
Deriv Peak
Cys
0.045
will have the technology and expertise
to help you conquer it.
Lys
proteomics, chemical analysis, or
Thr
Whether you work in pharmaceuticals,
scientists have come to expect from Waters.
Ala
application’s needs.
lab-to-lab, around the world. And you can always rely on the expert applications support that
Glu
in choosing the best solution for your
methodologies that assure you’ll get accurate results day-to-day, instrument-to-instrument,
Asp
these solutions remove the guesswork
results. Just as important, our system solution includes rugged and reliable performance-qualified
Arg
Gly
Designed by scientists for scientists,
enhanced chromatographic resolution to ensure accurate and precise qualitative and quantitative
Ser
application-focused performance.
acids. Derivatized amino acids are separated using the powerful ACQUITY UPLC System, which delivers
His
specifically optimized for exceptional,
The UPLC Amino Acid Analysis Solution is holistically designed and optimized for analyzing amino
NH3
tools, and services – that are
AMQ
chemistries, software, qualification
The Waters UPLC Amino Acid Analysis (AAA) Solution is the product of more than 25 years’ experience
0.015
0.010
0.005
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Minutes
Intelligently designed
The UPLC Amino Acid Analysis Solution leverages the best of Waters’ expertise in separation science,
derivatization chemistries, and information management. Your lab will reap benefits from uncompromised
chromatographic performance, accuracy, reliability, and reproducibility that cannot be found in any other
system on the market today.
ACQUIT Y UPLC System
The ACQUITY UPLC System, engineered from the ground up with robust hardware to meet the demands
of our unique sub-2 µm small-particle chemistries, is the highest performance LC system available. Its
novel design accommodates pressure-tolerant particles and fluidic modules, minimized system volume,
negligible carryover, and reduced cycle times. The system is rounded out with fast-response detectors,
integrated system software and Connections INSIGHT ISDP instrument diagnostics to ensure continuous,
consistent, and reliable operation in your lab.
AccQ•Tag Ultra Chemistry
Performance qualification
AccQ•Tag Ultra Chemistries are a comprehensive and fully-tested set of
Qualification services available for the UPLC Amino Acid Analysis
reagents, columns and eluents optimized for use with the ACQUITY UPLC
Solution meet the most stringent regulatory requirements. Installation,
System. This chemistry is based on Waters’ widely-used and understood
Operation, and Performance Qualification (IQ/OQ/PQ) protocols ensure
AccQ•Tag derivatizaton method. High resolution separations are achieved
that every element of the system is performing according to its intended
using pre-qualified AccQ•Tag Ultra UPLC columns and mobile phases.
use. An additional PQ, specific for amino acid analysis, is also available to
Derivatized amino acids are quantified to sub-picomole levels with single
verify performance and ensure accurate results.
wavelength UV detection.
Detection choices
You can analyze all the amino acids at the maximum sensitivity using
the ACQUITY UPLC TUV Detector. If you also use the system for other
applications, you can choose an FLR or PDA detector.
Derivatization with AccQ•Tag Ultra Chemistry
The AccQ•Tag Ultra reagent, or ACQ, is an N-hydroxysuccinimideactivated hetercyclic carbamate. This reagent converts both primary
and secondary amino acids in a high-throughtput batch process resulting
in exceptionally stable derivatives. The resulting derivatives are stable at
room temperature for up to one week. All components of AccQ•Tag Ultra
chemistry, including the columns, derivitatization kits, and mobile phases,
undergo strict QC testing.
H
R1
N
N
H
O
N
N
R2
NHR1R2
O
N
Amino Acid
Derivatized Amino Acid
O
O
O
N
t1/2 << 1s
AQC Reagent
+
O
HO
N
H20
t1/2 ~ 15s
O
N-Hydroxy Succinimide
O
HO
NH2
N
+
+
N
O
6-Aminoquinoline
N-Hydroxy Succinimide
CO 2
P ERFORMANCE T HAT E XCEEDS E X P EC TAT IONS
Powered by the ACQUITY UPLC System, our Assured Performance Solution for amino acid analysis delivers
uncompromised resolution, sensitivity, and throughput in a robust, reliable method.
Method robustness that minimizes variability
The UPLC Amino Acid Analysis Solution provides you with control over the major factors that commonly
cause variability in amino acid analysis. From optimized instrumentation and quality controlled chemistries
to standardized Empower Software projects and qualification tools, you will rely on a method that is
both rugged and reliable – a comprehensive and detailed approach that ensures successful analysis
of every sample.
Better resolution translates to accurate results
Sub-2 µm particle UPLC technology delivers the greatest available column efficiency and resolution.
With the system’s superior resolution of all amino acids, you will confidently achieve peak identification
and quantitation, which in turn assures the accuracy of your results. For unequivocal peak identification,
UPLC separation chemistries are fully compatible with mass spectrometry detection.
Direct cell culture sample analysis
Aliquots of cell culture media
were taken directly from
a bioreactor producing a
biopharmaceutical protein.
Samples were taken over a
period of six days and no
special sample preparation
was required before analysis
of the amino acids. The amino
acid concentration trends can
be plotted with growth as well
as response to feeding.
0.65
0.60
Ala
0.55
0.50
Day 1
Day 3
Day 6
0.45
0.35
0.05
Thr
0.10
Asp
0.15
Gly
0.20
Arg
0.25
Glu
Gln
0.30
Ser
0.00
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
M inutes
Cell Culture Media
with Feeding Schedule
1000
900
800
C o n c (m g /L )
AU
0.40
700
G ln
600
Asp
500
Ala
400
L ys
300
200
100
0
0
2
4
Day
6
8
5.00
Qualification your lab can rely on
AMQ
T hroughput greater than what’s possible
with traditional HPLC
0.060
Lys
Because we know that getting the right answer every time is imperative
Imagine achieving accurate amino acid analysis results in a fraction
for your lab, Waters has invested the time, resources, and expertise in
of the time required by conventional methods. The dramatically higher
developing comprehensive qualification services to ensure that you get
throughput possible with UPLC enables you to make more informed
the accurate and reproducible results you expect. Our application-specific
decisions faster, to perform more analyses per day, and to take on
Performance Qualification is specific to amino acid analysis. A Waters-
more projects per year.
certified engineer will qualify your system using pre-configured amino
0.055
0.050
Asp
Arg
Gly
Ser
0.025
Pro
Glu
0.030
Ala
Thr
AU
0.035
Ile
Leu
Phe
0.040
Tyr
Met
Val
Deriv Peak
Cys
0.045
acid analysis methods and protocols, a QC-tested AccQ•Tag Ultra column,
His
NH3
0.020
0.015
reagents and eluents, as well as specific qualification documentation.
0.010
0.005
Flexible methodology: Analysis of a hydrolysate standard at 10 pmol/µL and a cell culture standard at 10 pmol/µL
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Orn
0.055
0.055
Lys
AMQ
Lys
AMQ
0.060
Deriv Peak
Minutes
0.060
0.050
0.050
0.045
2.50
Ile
Leu
Phe
Trp
7.00
Cys
AABA
HyLys1
HyLys2
Pro
6.50
Asp
Glu
GABA
Tyr
2.00
Met
Val
NVa
Ser
Gln
Arg
Gly
Tau
Asn
0.015
0.015
NH3
HyPro
0.020
Ala
Thr
AU
Pro
0.030
His
Asp
0.035
0.025
His
NH3
0.020
Arg
Gly
Ser
0.025
Glu
0.030
Ala
Thr
AU
0.035
0.040
Ile
Leu
Phe
0.040
Tyr
Met
Val
Deriv Peak
Cys
0.045
0.010
0.010
0.005
0.005
0.000
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
1.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
7.50
8.00
Minutes
Lys
Deriv Peak
AMQ
Orn
Minutes
0.060
The trace on the left shows the analysis of a hydrolysate standard and the trace on the right shows a standard mixture of the larger number of amino acids found in cell culture media. The adaptation of the
method requires only a change in dilution of the Eluent A Concentrate supplied as part of the total system solution.
0.055
0.050
7.00
Cys
AABA
6.50
Asp
Glu
Pro
Met
Val
NVa
2.50
Tyr
2.00
GABA
Thr
Ser
Gln
Arg
Gly
Tau
Asn
0.015
NH3
HyPro
0.025
0.020
Ala
0.030
His
AU
0.035
HyLys1
HyLys2
0.040
Ile
Leu
Phe
Trp
0.045
0.010
0.005
0.000
1.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
7.50
8.00
GOOD SCIENCE MEANS GOOD BUSINESS
To achieve your goals, your laboratories must operate at their best. You continually balance short-term financial performance against long-term business
goals. You need to meet ever-aggressive product availability and delivery schedules, which require shorter turnaround times from product development
through production. Waters Assured Performance Solutions, including the UPLC Amino Acid Analysis Solution, simultaneously address laboratory
productivity, efficiency, and analytical quality.
Confirm quality
You may need to determine the amino acid composition of a product and
Your objective is to demonstrate process consistency. Concurrently,
compare it with the composition deduced from the gene sequence for
you have a production schedule to meet – and delays in any part of
that product or its natural counterpart. Utilizing amino acid analysis to
the process are expensive. Monitoring cell culture media for critical
provide evidence of peptide or protein composition is an important tool
nutrients allows you to optimize expression efficiencies and to make
that supports effective discovery and lead development efforts. With
timely product harvesting decisions. The UPLC Amino Acid Analysis
the UPLC Amino Acid Analysis Solution, you can characterize proteins
Solution provides the rugged, reliable performance you need to
more comprehensively and efficiently, potentially accelerating your
make the correct manufacturing decisions. You can depend on Waters
biopharmaceutical development.
instrumentation to enable you to achieve the product consistency you
0.095
Deriv Peak
0.100
AMQ
Accelerate protein characterization
0.090
0.085
0.080
0.075
0.070
0.065
0.060
AU
0.055
Glu
0.050
0.045
need for validated protocols. And when your analyses are complete,
Lys
Leu
Phe
0.040
0.035
Ile
Val
0.025
Ala
NH3
0.030
Asp
you can utilize Waters NuGenesis ® Scientific Data Management System
Tyr
Pro
His
0.005
Efficient and reliable foods and feed sample analysis: Soybean meal hydrolysate
Cys
0.010
Met
0.015
Thr
Ser
0.020
Arg
Gly
(SDMS) to automatically collect and archive results for easy future access.
0.000
-0.005
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
6.00
6.50
7.00
7.50
0.085
0.000
-0.005
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
1.50
7.50
Minutes
2.50
Val
Pro
Ala
MetSO2
3.00
3.50
4.00
4.50
5.00
5.50
7.00
7.50
Minutes
Deriv Peak
AMQ
0.100
2.00
His
0.005
-0.005
2.00
Cya
0.010
Tyr
Ile
Val
0.015
0.000
1.50
Ser
0.030
0.020
Met
Cys
His
0.005
0.035
Arg
Gly
Leu
Phe
Lys
Pro
0.010
0.040
0.025
Tyr
0.015
Ala
Ser
0.020
Thr
0.025
Arg
Gly
NH3
Asp
0.035
Asp
0.045
0.040
0.030
0.050
Leu
0.055
0.050
Phe
0.060
0.055
Ile
0.065
0.060
Lys
0.070
0.065
AU
0.075
0.070
Glu
0.080
0.075
0.045
Deriv Peak
0.090
Glu
AU
0.080
0.095
Thr
0.085
AMQ
0.100
0.090
NH3
0.095
Deriv Peak
0.100
AMQ
Minutes
Two samples of hydrolyzed soybean meal were analyzed. The trace on the left shows the untreated sample while the trace on the right shows the results of performic acid oxidation. Note the appearance of
cysteic acid and methionine sulfone in the oxidized sample.
0.095
0.090
0.085
0.080
0.075
Glu
0.070
0.065
0.050
Cya
0.005
His
0.015
0.010
Phe
Ile
Val
MetSO2
0.020
Tyr
0.025
Pro
Ser
0.030
Thr
0.035
Arg
Gly
0.040
Ala
Asp
0.045
Leu
Lys
0.055
NH3
AU
0.060
0.000
-0.005
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
Avoid costly reformulations
Turnkey application support
Amino acids are primary nutritional components in a wide variety of agri-
What do protein hydrolysates, cell culture monitoring, and nutritional
cultural feeds and food products. Are you concerned that your products are
analysis of foods and feeds have in common? The need for trouble-free
not meeting label claims for nutrient composition? With our UPLC Amino
amino acid analysis. Our UPLC Amino Acid Analysis Solution comes with
Acid Analysis Solution, you have access to Waters’ extensive experience in
Empower Software projects, methods and report template to ensure that
managing these complex analytical challenges. Our system solution will
you are up and running quickly. You do not need to be an expert in amino
enable you to get the right information, on a timely basis, so you can be
acid analysis to get accurate, precise, and reproducible results for a broad
assured that your products meet your manufacturing specifications.
range of samples.
Customized, easy-to-use data p rocessing and reporting
The UPLC Amino Acid Analysis System Solution includes a CD with
a set of processing methods and report formats that are useful for
different applications. For example, the results can be reported with a
sample chromatogram and the quantitative results can be reported in
the most useful units for the application (e.g. mg/mL or molar ratios).
Calibration reports also can be generated that summarize accuracy,
precision, and linearity for the experimental measurements.
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Waters, ACQUITY, ACQUITY UPLC, Connections INSIGHT, NuGenesis, Pico•Tag, UltraPerformance LC, and UPLC are
registered trademarks of Waters Corporation. AccQ•Tag, Empower, and The Science of What’s Possible are trademarks
of Waters Corporation. All other trademarks are the property of their respective owners.
©2006-2007 Waters Corporation. Printed in the U.S.A.
January 2008 720001837EN LB-CP