Precision Chemistry for Particles and Surfaces
How To Guide
Last Updated: March 19, 2024
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Published: March 5, 2024
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Ensuring instruments are in proper working order is a critical aspect of making any analytical measurement. Failure to confirm system suitability can lead to uncertainty in results and incorrectly formed conclusions.
Evaluating system suitability for analytes that are challenging to handle, separate and measure requires special considerations.
This handbook highlights innovative technologies designed to increase analyte recovery, sensitivity and reproducibility by minimizing analyte-surface interactions that can lead to sample loss.
Download this handbook to discover liquid chromatography methods for:
- Separation and recovery of analytes associated with the tricarboxylic acid cycle
- Pentose phosphate pathway, glycolysis and energy metabolite separation
- Improved sensitivity, peak shape and recovery for phosphorylated and carboxylate lipids
MaxPeak Premier Columns
Small Molecule Application Notebook
CONTENTS
Introduction
Non-Specific Adsorption: Unwanted Interactions.................................................................................. 3
How Do Scientists Solve This Problem Today? ........................................................................................ 4
Precision Chemistry for Particles and Surfaces....................................................................................... 5
Fundamentals
Waters™ Premier Standards to Investigate the Inertness
of Chromatographic Systems...................................................................................................................................7
Improving Metal-Sensitive Analyte Recovery on Various LC Systems
Using MaxPeak™ Premier Columns.................................................................................................................... 8
A Comparison of MaxPeak Premier Columns with MaxPeak HPS Technology
vs PEEK-lined Column Hardware......................................................................................................................... 9
Utilization of MaxPeak HPS and Atlantis™ Premier BEH™ C18 AX Columns
to Increase Sensitivity of LC-MS Analysis...................................................................................................10
Pharmaceutical Applications
Advantages of Using MaxPeak HPS Technology for the Analysis
of Targeted Cancer Growth Inhibitor Therapies......................................................................................12
Improving Drug Metabolite Identification in Biofluids with the ACQUITY Premier
and Hybrid Surface Technology: Increased Sensitivity and Reproducibility...................13
MaxPeak High Performance Surfaces Mitigate On-column Oxidation................................14
ACQUITY™ Premier Solution Improves the UPLC-MS Analysis
of Deferoxamine - an Iron Chelating Drug...................................................................................................15
Batch-to-Batch Robustness of MaxPeak Premier Columns for the Analysis
of Dexamethasone Phosphate and Related Compounds................................................................16
Improved Chromatographic Analysis of NSAIDS Using CORTECS™
Premier Columns that Feature MaxPeak HPS Technology............................................................17
Analytical QbD Based Method Development for the Analysis
of Dexamethasone Phosphate and Related Compounds Using
Arc™ Premier MaxPeak HPS Technology......................................................................................................18
Metabolomics Applications
Utilization of High Performance Surfaces for Improved Separation
and Recovery of Analytes Associated with the TCA Cycle .......................................................... 20
Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak HPS
Technology: A Case Study on the Detection of Nucleotides ........................................................21
ACQUITY Premier LC Technology Significantly Improves Sensitivity,
Peak Shape, and Recovery for Phosphorylated and Carboxylate Lipids...........................22
Separation of Pentose Phosphate Pathway, Glycolysis, and Energy
Metabolites Using an ACQUITY Premier System with an Atlantis Premier
BEH Z-HILIC Column................................................................................................................................................... 23
Food Applications
Enhancing the LC-MS/MS Analysis of B-group Vitamins
with MaxPeak HPS Technology.......................................................................................................................... 25
Enhanced Performance of the Analysis for Veterinary Drugs
with Metal Affinity Using ACQUITY Premier and XEVO™ TQ-S Micro...................................26
The Benefits of ACQUITY Premier UPLC™ for Multi-Mycotoxin Methods..........................27
Links to Other Useful Materials....................................................................28
WHAT ARE THE CONSEQUENCES?
■ High carryover
■ On-column reactions leading to the formation of new peaks
■ Broad peaks, tailing peaks, missing peaks
■ Low area counts or reduced sensitivity with new columns
that increases over time
■ Conditioning is required with new columns
■ Poor reproducibility from injection to injection
■ Complex mobile phases required to suppress adsorption
MITIGATION OF THESE INTERACTIONS CAN
LEAD TO IMPROVEMENTS IN:
■ Sensitivity
■ Peak shape
■ Reproducibility
■ Detection
■ Analysis time
■ Carryover
What happens when you are getting this… But you need this….
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High RSD’s – 18%
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Low RSD’s – 4%
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Non-Specific Adsorption: Interactions of Analytes with Metal Surfaces
Certain analytes may interact with the surface oxide layer present on the surface of stainless steel and other metals present in the column
and HPLC system flow path.
MaxPeak Premier Columns | Application Notebook 3
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
SOLUTION HOW DOES IT WORK? WHAT IS THE CONSEQUENCE?
Passivation of surfaces with acid Removes free iron from steel surface ■ Time consuming
■ Requires strong acids
■ Not stable, needs to be repeated
Conditioning of surfaces with sample or matrix Analyte or matrix coats reactive surfaces ■ Time consuming
■ Not stable, needs to be repeated
PEEK or PEEK lined steel columns Replaces metal surface with an organic
polymer surface
■ PEEK alone is not high pressure tolerant
PEEK materials have:
— Higher dimensional variability
— Lower frit permeability
— Incompatibility with some solvents
Titanium in columns or instrument parts Titanium is less susceptible to oxidation than
stainless steel. However, the titanium oxide
surface still causes non-specific adsorption
■ Low sensitivity
■ Poor peak shape
■ Poor reproducibility
■ Possible analyte loss
Industrial coatings Covers the metals with an inert material,
e.g silica/other materials
■ MS bleed, and other unexpected problems
■ These were never designed for LC and
LC-MS applications
Additives in mobile phases Bind to metal surface to prevent
analyte adsorption
■ Ion suppression and other unknown effects
■ Continued use necessary
■ Possible solubility issues
How Do Scientists Solve This Problem Today?
MaxPeak Premier Columns | Application Notebook 4
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Precision Chemistry for Particles and Surfaces
MaxPeak Premier Columns, constructed with MaxPeak
High Performance Surfaces (HPS) Technology, were created
to address the concern of non-specific adsorption by mitigating
interactions of analytes with the metal surfaces.
These new and innovative technologies are designed to
increase analyte recovery, sensitivity, and reproducibility
by minimizing analyte-surface interactions that can lead
to sample loss.
MaxPeak Premier Columns | Application Notebook 5
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Fundamentals
Read the Full Application Note
Waters Premier Standards to Investigate the Inertness of Chromatographic Surfaces
Ensuring that instruments are in proper working order is a critical
aspect of making any analytical measurement. Failure to confirm
system suitability can lead to uncertainty in results and incorrectly
formed conclusions. Evaluating system suitability for analytes that
are challenging to handle, separate, and measure requires special
considerations. Analytes with a propensity to chelate to metals are
one such class of molecules. To better facilitate separations of these
types of molecules, Waters has designed the ACQUITY Premier
System and MaxPeak Premier Columns to have chromatographic
surfaces based on hybrid silica instead of metal or metal alloy
surfaces. When adopting MaxPeak Premier Instruments, or
attempting to make do with workarounds, analysts should consider
test approaches that would report on their system’s inertness to
metal sensitive compounds. To this end, two test standards have been
developed. Chemically, a nucleotide would be a useful test probe,
however, they are subject to hydrolysis. As an alternative, we have
made use of a non-hydrolyzable analog of adenosine diphosphate
(ADP) to improve shelf life and solution stability of the standard. This
molecule is adenosine 5’-(α,β-methylene)diphosphate (AMPcP) and
is formulated as an AMPcP-only standard, as well as an equimolar
mixture of AMPcP and adenosine.
BENEFITS
■ MaxPeak High Performance Surfaces (HPS) Technology improve
recovery and peak shape of metal sensitive analytes
■ Waters Premier Standards used as quality control reference materials
(QCRMs) with potential system suitability techniques for inert LC
instruments
■ Improved certainty in results for historically problematic metal
sensitive analytes
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AMPcP
Average peak area: 1132
AMPcP
Average peak area: 955 Adenosine
Average peak area: 1074
Adenosine
Average peak area: 1065
ACQUITY Premier
ACQUITY UPLC H-Class Bio
Overlay of five replicate injections of AMPcP and Adenosine Standard on an ACQUITY Premier System (top)
and five replicate injections on an ACQUITY UPLC H-Class PLUS Bio System (bottom).
MaxPeak Premier Columns | Application Notebook 7
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Chromatograms of the three columns tested on a Thermo Vanquish with UV detection at 260 nm. Elution Order:
1) AMPcP and 2) Adenosine. Insert is a zoomed in portion of the X-axis with overlaid chromatograms to show the
peak height and peak shape differences for AMPcP for the three columns.
Read the Full Application Note
Improving Metal-Sensitive Analyte Recovery on Various LC Systems Using MaxPeak Premier Columns
Metal adsorption can be a significant problem in LC analysis,
particularly for highly acidic probes like phosphorylated peptides,
or small molecules such as nucleotides. These probes can interact
with chromatographic metal surfaces, leading to poor peak shape,
reduced peak area, or even complete loss of recovery.1
This issue
can be mitigated by implementing MaxPeak High Performance
Surfaces (HPS) Technology, which has been applied to Waters
LC columns and LC systems. However, competitive LC systems
do not employ this technology. Here, we demonstrate the use of
MaxPeak Premier Columns on three different, non-Waters LC
systems for the analysis of a metal-sensitive analyte, adenosine
5’-(α,β-methylene) diphosphate (AMPcP). This probe has shown
strong metal-sensitivity and is more stable than compounds such
as adenosine triphosphate or adenosine diphosphate.2 Our results
show that regardless of the LC system used, a MaxPeak Premier
Column yielded higher peak areas and thus analyte recovery for
AMPcP compared to stainless steel columns. This indicates that the
advantages of the MaxPeak Premier Columns are system-agnostic
and would prove beneficial to almost all system configurations.
Chromatograms of the three columns tested on a Shimadzu Nexera-I 2040 with UV detection at 260 nm. Elution
Order: 1) AMPcP and 2) Adenosine. Insert is a zoomed in portion of the X-axis with overlaid chromatograms to show
the peak height and peak shape differences for AMPcP on the three columns.
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XSelect Premier HSS T3 Column
Zorbax SB-Aq Column
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XSelect HSS T3 Column
Zorbax SB-Aq Column
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XSelect HSS T3 Column
Zorbax SB-Aq Column
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BENEFITS
■ Increased peak area, or recovery, for AMPcP using MaxPeak Premier
Columns
■ Characterization of the extent of metal interaction using an AMPcP/
Adenosine standard on various LC systems
■ System-agnostic recovery advantages for metal-sensitive analytes
1. Jung M, Lauber M. Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak High Performance
Surface (HPS) Technology: A Case Study in the Detection of Nucleotides. Waters Application Note. 720007053EN.
2. Lauber, M.; et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters White
Paper, 720006930EN, 2020.
MaxPeak Premier Columns | Application Notebook 8
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
A Comparison of MaxPeak Premier Columns with MaxPeak HPS Technology
Versus PEEK-lined Column hardware
Read the Full Application Note
Anionic or electron-rich analytes often have poor peak shape and low
signal intensity in liquid chromatography (LC) analysis due to analyte
loss via adsorption on electron-deficient metal surfaces, such as
stainless steel.1
Alternative column hardware made entirely of PEEK
(Polyether Ether Ketone), or stainless-steel columns with a PEEK lining,
have recently been utilized for these applications to eliminate analyte
loss. However, columns that utilize PEEK materials can exhibit other
undesirable problems such as lower plate efficiency and more
column-to-column variability than traditional stainless-steel hardware.
The MaxPeak Premier Columns feature MaxPeak High Performance
Surfaces (HPS) Technology, making it possible to more easily study
metal-sensitive analytes. The MaxPeak HPS Technology provides an
effective barrier to metal-analyte interactions and any related loss of
sample due to non-specific adsorption.
BENEFITS
■ Better column efficiency
■ Better peak shape
■ Lower column backpressure
■ More consistent column-to-column reproducibility
■ Excellent analyte recovery
PEEK-lined
vendor A
MaxPeak HPS
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Chromatographic peaks: 1. Thiourea, 2. Naphthalene, 3. Acenaphthene, 4. Octanophenone
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Chromatograms for N=5 columns packed in (A) PEEK-lined Vendor A hardware, (B) PEEK-lined Vendor B
hardware, and (C) MaxPeak HPS hardware. Analyses were performed with an ACQUITY UPLC chromatograph
and 2.1 x 50 mm columns packed with C18 , 2.5 µm stationary phase. Isocratic separation conditions included
a flow rate of 0.30 mL/min, column temperature of 30 °C, 75% acetonitrile, 254 nm UV detection, and 1 µL
injection volumes.
1. Lauber, M.; et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces.
Waters White Paper, 720006930EN, 2020.
MaxPeak Premier Columns | Application Notebook 9
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Utilization of MaxPeak High Performance
Surfaces and Atlantis Premier BEH C18 AX Column
to Increase Sensitivity of LC-MS Analysis
This application brief shows the benefit of MaxPeak HPS Technology
in the mixed-mode Waters Atlantis Premier BEH C18 AX Column.
BENEFIT
■ MaxPeak High Performance Surfaces (HPS) Technology are new
and innovative technologies designed to increase analyte recovery,
reproducibility, and sensitivity by minimizing negative analyte/surface
interactions that can lead to sample losses
Peak area recovery of diazoxide (A), bicalutamide (B), salmeterol (C), and gentisic acid (D) on the
Atlantis BEH C18 AX sorbent with MaxPeak HPS Column hardware (red line) and standard column
hardware (green line).
MaxPeak Premier Columns | Application Notebook 10
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Pharmaceutical
Applications
Read the Full Application Note
Advantages of Using MaxPeak HPS Technology for the Analysis of Targeted
Cancer Growth Inhibitor Therapies
Non-specific binding of analytes to material surfaces is an inherent characteristic of conventional chromatographic columns and systems. This
phenomenon leads to reduced sensitivity and sometimes poor peak shape. The MaxPeak HPS Technology provides a solution without the need
for strong mobile phase additives, chelators, or lengthy passivation protocols. A panel of targeted cancer growth inhibitor therapies was utilized
to demonstrate the chromatographic benefits provided when using columns and systems equipped with MaxPeak HPS Technology.
BENEFITS
■ MaxPeak HPS Technology provides improved chromatographic peak performance for targeted cancer growth inhibitor therapies without the
need for strong mobile phase additives, chelators, or lengthy passivation protocols
■ The chromatographic sensitivity of cancer growth inhibitors is improved when using materials equipped with MaxPeak HPS Technology
Panobinostat
Bortezomib
Sunitinib
Imatinib
Vemurafenib
Everolimus
Idelalisib
Vismodegib
XBridge Premier BEH Shield
RP18 Column/Arc Premier System
XBridge BEH Shield
RP18 XP Column/ACQUITY Arc System
Chromatographic overlay of the panel of targeted cancer growth
inhibitor therapies analyzed using the (black) XBridge BEH Shield
RP18 XP Column/ACQUITY Arc System and the (blue) XBridge
Premier BEH Shield RP18 Column/Arc Premier System.
MaxPeak Premier Columns | Application Notebook 12
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Improving Drug Metabolite Identification in Biofluids with the ACQUITY Premier and Hybrid Surface
Technology: Increased Sensitivity and Reproducibility
Read the Full Application Note
Liquid chromatography coupled with electrospray tandem mass
spectrometry is the primary platform for drug metabolite identification
both in vivo and in vitro. Key to successful metabolite identification
is the chromatographic resolution of the drug related analytes, both
from each other and from endogenous components present in the
matrix. TRANSITion metals present in chromatography systems and
columns can act as Lewis Acids interacting with analytes containing
phospho- groups, uncharged amines, hydroxyls, and deprotonated
carboxylic acids resulting in poor chromatographic peak shape or even
severe analyte loss. The ACQUITY Premier Chromatography System
and columns employs a hybrid organic surface technology to eliminate
this type of non-specific binding. The analysis of the in vivo metabolites
of gefitinib using the ACQUITY Premier Chromatography System and
columns showed improved peak shape, increased signal response and
cleaner MS/MS spectra.
BENEFITS
■ Improved chromatographic resolution of drug metabolites
■ Increased peak response
■ Improved reproducibility
Figure 5. Improved fragment ion intensities demonstrated for gefitinib metabolite M7 (MQZP, m/z 378.1021).
Increased ion intensities relating to transitions m/z 318.0440 and 304.0284 are shown to increase for the
ACQUITY Premier HSS T3 Column.
Premier HSS T3
ACQUITY Premier ACQUITY HSS T3
MaxPeak Premier Columns | Application Notebook 13
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
MaxPeak High Performance Surfaces Mitigate On-Column Oxidation
The advent of more robust chromatographic materials has facilitated
the use of a wider range of method conditions. However, depending on
the chemical properties of the analyte, sample degradation may occur
during the chromatographic separation. This may be caused by various
factors including the use of high temperature, stability in certain mobile
phases, pH conditions, or even interactions with the stationary phase
and columns.
On-column degradation is more commonly reported for biopharmaceuticals rather than for small molecules. These larger biomolecules
can undergo conformational changes and on-column degradation with
the stationary phase and LC conditions, respectively. While reports of
on-column degradation of small molecules may be rarer, significant
cases have been observed. Instances of on-column degradation of
amino compounds have been encountered, and their degradation has
been found to be exacerbated using high pH mobile phases.
This application note demonstrates that small molecule amines can
indeed undergo on-column oxidation when separated with conventional
stainless-steel columns. However, this degradation can be mitigated
with the use of ACQUITY Premier Columns. ACQUITY Premier Columns
feature MaxPeak High Performance Surfaces, which provide a barrier
against metal-analyte interactions. This barrier, based on hybrid
organic-inorganic silica, can also help protect metal LC surfaces from
corrosion, which can further exacerbate analyte degradation. These
results demonstrate that the MaxPeak Premier Technology can reduce
UV chromatograms at 290 nm detection from the 2nd and 34th separations of clozapine, showing its Z
and E nitroso impurities, using mobile phases of ammonium hydroxide in water and acetonitrile, and an
(A) ACQUITY BEH C18 , 1.7 µm, 2.1 x 50 mm Column and an (B) ACQUITY Premier BEH C18 , 1.7 µm, 2.1 x 50 mm
Column. Separations were performed with an ACQUITY UPLC I-Class System, 0.31 mL/min flow rate, column
temperature 30 °C, gradient of 25% to 80% acetonitrile in 10.31 min, and 0.15 µg mass loads.
on-column, metal catalyzed reactions like oxidation. ACQUITY Premier
Columns should thereby be considered for use in LC work, purity
measurements, and impurity profiling or wherever analytical artifacts
might undermine the value of an assay.
BENEFITS
■ Reduction in on-column degradation products versus conventional
steel column technology
■ Improved method robustness
■ Higher fidelity data for impurity quantification
A B
Injection 2
Injection 34
Injection 2
Injection 34
MaxPeak Premier Columns | Application Notebook 14
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
ACQUITY Premier Solution Improves the UPLC-MS Analysis of Deferoxamine- an Iron Chelating Drugs
HPLC separations of metal-sensitive analytes are known to be
impacted by interactions of the analytes with the metal surfaces in
HPLC instruments and columns. This causes effects ranging from peak
broadening and tailing to loss of peak area and high injection-to-injection
variability. The ACQUITY Premier Solution mitigates these effects by
employing the novel surface technology MaxPeak High Performance
Surfaces. Here we compare UPLC separations for the iron chelating
drug deferoxamine obtained using a standard UPLC system and column
vs the ACQUITY Premier Solution. The results show that higher peak
areas and improved injection-to-injection reproducibility are achieved
using the ACQUITY Premier Solution.
BENEFITS
■ The ACQUITY Premier Solution showed higher and more consistent
peak areas for deferoxamine compared to a standard UPLC system
and column
■ No conditioning or unusual mobile phase additives were
required to achieve reproducible separations using the ACQUITY
Premier Solution
Read the Full Application Note
(A) Overlay depicting 10 replicate injections of deferoxamine mesylate obtained using the ACQUITY Premier
Solution. (B) Overlay depicting 10 replicate injections of the same analyte using a standard UPLC system and
a standard column. ACQUITY UPLC HSS T3, 100 Å, 1.8 µm, 2.1 x 50 mm Columns were used in this study. The
mass load of deferoxamine mesylate was 10 ng. Acetonitrile gradient separations were carried out with a 10
mM ammonium formate (pH 3.0) aqueous mobile phase, a flow rate of 0.5 mL/min, and a temperature of 30
°C. The deferoxamine peak was detected using an ACQUITY QDa Detector with SIR positive m/z 561.0.
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Intensity
ACQUITY Premier Solution
Avg. Peak area 4.89 E+06
RSD (n=10) = 2.1%
Intensity
Standard UPLC System
and Column
Avg. Peak area 3.01 E+06
RSD (n=10) = 16.8%
A B
MaxPeak Premier Columns | Application Notebook 15
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Batch-to-Batch Robustness of MaxPeak Premier Columns for
the Analysis of Dexamethasone Phosphate and Related Compounds
An Ultra High Performance Liquid Chromatography (UHPLC) method that uses an Arc Premier
System with MaxPeak Premier Columns was used to evaluate the batch-to-batch reproducibility
of multiple columns. Different batches of both the construction material and the packing material
of MaxPeak Premier XBridge™ BEH C18 and XSelect™ HSS T3 Columns were studied. Results
showed that MaxPeak Premier Columns were very reproducible when used for the analysis
of a mixture of metal chelating and non-metal chelating compounds. Various chromatographic
parameters including relative retention time, critical pair resolution, and peak area were all
investigated for reproducibility on the different columns. The columns showed excellent
reproducibility for all the studied chromatographic parameters. For example, the %RSD for
the peak areas for all peaks was always in the range of 0.1%–5.6% for all analytes. These findings
indicate that the batch-to-batch reproducibility of MaxPeak Premier Columns is very high and
these columns are very robust.
BENEFITS
■ Batch-to-batch reproducibility of the construction material of MaxPeak Premier XBridge
BEH C18 and XSelect HSS T3 Columns
■ Batch-to-batch reproducibility of the packing material of MaxPeak Premier XBridge
BEH C18 and XSelect HSS T3 Columns
Overlay chromatogram of 18 injections on A: three different
batches of the construction material for the MaxPeak Premier
XSelect HSS T3 Column and B: three different batches of
the construction material for the MaxPeak Premier XBridge
BEH C18 Column. Peaks according to elution order are:
hydrocortisone phosphate, betamethasone sodium phosphate,
dexamethasone sodium phosphate, dexamethasone, and
dexamethasone acetate. AU -0.010
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MaxPeak Premier Columns | Application Notebook 16
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Improved Chromatographic Analysis of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Using
CORTECS™ Premier Columns That Feature MaxPeak™ High Performance Surfaces HPS Technology
NSAIDs are common pain-relieving anti-inflammatory medications
used by millions of consumers worldwide, daily. To ensure the safety
of consumers all around the globe, methods supporting the quality
control sector of NSAIDs production are of great importance. In this
application, we develop a rapid analysis for NSAIDs featuring CORTECS
Premier Columns with MaxPeak HPS Technology. This method has
proven to be reproducible and linear in its separation and detection of
commonly used NSAIDs. Moreover, CORTECS Premier Columns that
have MaxPeak HPS Technology showed improved chromatographic
performance when compared to analysis on traditional CORTECS
Columns packed in stainless-steel column hardware.
BENEFITS
■ CORTECS Premier Columns with MaxPeak HPS Technology
improved chromatographic performance when compared to
traditional stainless-steel systems and columns
■ This method could analyze multiple NSAID compounds
in under two minutes
■ CORTECS Premier Columns with MaxPeak HPS Technology
delivers up to a 25% decrease in peak tailing and 39% increase
to height signal when compared to traditional stainless-steel
chromatographic systems
A: Chromatogram for injection five out of ten of the NSAIDs mix standard on
the ACQUITY Premier System equipped with a CORTECS Premier C18 Column.
B:Chromatogram for injection five out of ten of the NSAIDs mix standard on
the ACQUITY I-Class System equipped with a CORTECS C18 Column. Naproxen – 0.79 Fenoprofen – 1.28 Diclofenac – 1.62 Ibuprofen – 1.72 AU 0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
A ACQUITY Premier, CORTECS Premier Naproxen – 0.79 Fenoprofen – 1 2.8
Diclof neac – 1.62
Ibuprofen – 1.72
AU 0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
ACQUITYPremier, CORTECS Premier
MaxPeak Premier Columns | Application Notebook 17
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Analytical Quality By Design Based Method Development for the Analysis of Dexamethasone Phosphate
and Related Compounds Using Arc Premier MaxPeak High Performance Surfaces Technology
An Ultra High Performance Liquid Chromatography method was
developed for the analysis of a mixture of metal chelating and nonchelating compounds using the Analytical Quality by Design (AQbD)
approach. DryLab, Empower™, and Waters systems were used to
automate the method development process. The performance of the
Arc Premier System in combination with MaxPeak Premier Columns
was compared to standard stainless-steel hardware. Results showed
that MaxPeak Premier Columns on an Arc Premier System provides
great performance for the separation of metal chelating compounds
compared to stainless-steel hardware. The final method uses a MaxPeak
Premier BEH C18 Column (10 cm × 4.6 × 2.5 µm), 0.1% formic acid in
acetonitrile as an organic solvent, and 10 mM ammonium formate in
water aqueous mobile phase. The method showed excellent separations
between the peaks, ideal peak shapes, high recoveries, and good
reproducibility. For example, the USP tailing was ≤1.1 for all peaks
including the phosphorylated compounds. These findings indicate
that using the MaxPeak High Performance Surfaces (HPS) Technology
for the analysis of metal chelating compounds is greatly beneficial in
mitigating undesired interactions with metal surfaces and achieving
excellent separations.
BENEFITS
■ Show the superior advantages for using the Arc Premier System’s
MaxPeak HPS Technology for the analysis of phosphorylated
compounds
■ Show the increased efficiency of method development using
Arc Premier System in combination with Empower 3 Chromatographic
Data System (Empower CDS) and DryLab4 Software
■ Shows the seamless integration between DryLab and Empower
to fully automate the method development process
Mobile Phase B:
Methanol
Mobile Phase B:
50%Methanol:50%ACN
Mobile Phase B:
Acetonitrile
Peak tailing, poor
sensitivity, and
poor peak
recovery
Standard ACQUITY Arc and
XBridge BEH C18, 2.5 µm
Arc Premier System and
XBridge MaxPeak HPS BEH C18, 2.5 µm
Representative chromatograms from the 12 DOE runs. A: represents three experiments that were performed
on a Standard ACQUITY Arc System under different scouting conditions and B represents three experiments
that were performed on the Arc Premier System under the same conditions. Conditions in common between all
chromatograms are: Mobile phase A: 10 mM Ammonium formate in water, flow rate 0.50 mL/min, temperature
30 °C, 0.0–15 min, and 10–90% B linear gradient. Variations in conditions are detailed in the figure.
Mobile Phase B:
Methanol
Mobile Phase B:
50%Methanol:50%ACN
Mobile Phase B:
Acetonitrile
Peak tailing, poor
sensitivity, and
poor peak
recovery
Standard ACQUITY Arc and
XBridge BEH C18, 2.5 µm
Arc Premier System and
XBridge MaxPeak HPS BEH C18, 2.5 µm
MaxPeak Premier Columns | Application Notebook 18
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Metabolomic
Applications
Read the Full Application Note
Utilization of MaxPeak High Performance Surfaces for Improved Separation and Recovery of Analytes
Associated with the Tricarboxylic Acid Cycle
This application note presents a mixed-mode LC method
that is MS compatible for the analysis of TCA cycle analytes
as well as other related compounds without the use of
sample derivatization or ion-pairing reagents.
BENEFITS
■ The use of a hybrid organic-inorganic surface technology,
MaxPeak High Performance Surfaces (HPS), within the
ACQUITY Premier CSH Phenyl-Hexyl Column mitigates
analyte interactions with metal surfaces
■ A reproducible LC-MS method for the analysis of the TCA
cycle analytes as well as other related compounds without
the need for sample derivatization or ion-pairing reagents
■ Data processing by Progenesis QI using a custom database
that includes fragment and retention time match
Time
2.00 3.00
%
0
100 2.51
Time
1.00 1.50
%
0
100 1.21
Time
1.00 2.00
%
0
100 1.77
1.48
Isocitric
acid
1.4x
Citric
acid
2.1x
Malic
acid
5x
3-PG
>100x
Peak recoveries from a urine sample for isocitric acid and citric acid, malic acid, and 3-phosphoglyceric (3-PG) acid for
ACQUITY Premier CSH Phenyl Hexyl Column and a standard CQUITY CSH Phenyl Hexyl Column (filled trace). Numbers
show the improvement of peak area on the MaxPeak Premier Column.
MaxPeak Premier Columns | Application Notebook 20
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak High Performance
Surfaces (HPS) Technology: A Case Study on the Detection of Nucleotides
Read the Full Application Note
Acidic metal-sensitive analytes are challenging to robustly and sensitively
assay by LC-MS. Numerous attempts have been made to mitigate analyte
losses by modulating the chemical attraction to metal surfaces, but none
have produced a universal solution that is compatible with highly sensitive
UPLC-MS analyses. Using adenosine nucleotides as model analytes, we
have demonstrated that the Waters ACQUITY Premier LC System with
MaxPeak HPS Technology offers superior protection against metal-analyte
interactions without compromising the benefits of high-efficiency UPLC
separations with sensitive MS detection.
BENEFITS
■ This case study shows when and how chromatographic surfaces
can be of significant influence for analyte detection
■ Waters ACQUITY Premier LC System and Columns with MaxPeak
HPS Technology can dramatically improve the recovery and peak
shape of metal sensitive analytes, like nucleotides and their analogs
Time 0.20 0.40 0.60 0.80 1.00
Time 0.20 0.40 0.60 0.80 1.00
Time 0.20 0.40 0.60 0.80 1.00
% 0
100
ATP
ADP AMP
Adenosine
% 0
100
100
ATPADP
AMP
Adenosine
% 0
AMP
Adenosine
No ATP/ADP
Standard UPLC System
Standard UPLC Column
Standard UPLC System
ACQUITY Premier Column
ACQUITY Premier System
ACQUITY Premier Column
a)
b)
c)
Example chromatograms from 1 µL injections of mixture samples containing ATP, ADP, AMP,
and adenosine (20 pg/µL each).
MaxPeak Premier Columns | Application Notebook 21
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
ACQUITY Premier LC Technology Significantly Improves Sensitivity, Peak Shape, and Recovery
for Phosphorylated and Carboxylate Lipids
Phosphorylated and carboxylate lipid species are metal
sensitive and can readily adsorb to stainless steel surfaces
within the flow path of UPLC systems. This process can lead
to poor peak shape, low recovery and reduction in sensitivity.
Here we show that the ACQUITY Premier CSH C18 Column with
ACQUITY Premier System can significantly improve sensitivity,
peak shape, and recovery of phosphorylated and carboxylate
lipids compared to ACQUITY standard column and stainlesssteel ACQUITY UPLC I-Class System.
BENEFITS
The ACQUITY Premier technology provides:
■ Increased sensitivity, recovery, and reproducibility
for phosphorylated and carboxylate lipids
■ Improved peak shape and reduced tailing by minimizing
analyte-surface interaction
■ Increased lipidomics coverage by simultaneous analysis
of phosphorylated and carboxylate lipids with other
lipid classes
Negative mode base peak extracted ion chromatogram of LPA (16:0/0:0) m/z 409.2355 at a concentration of 5, 50, 100,
and 250 ng/mL. (A1) using ACQUITY standard CSH C18 Column with stainless steel surface ACQUITY System and (A2)
using ACQUITY Premier CSH C18 Column with ACQUITY Premier System.
ACQUITY standard column with
stainless steel ACQUITY system
ACQUITY Premier Column
with ACQUITY Premier System
250 ng/mL
100 ng/mL
50 ng/mL
5 ng/mL
Time
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100
Time
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100
1.00 2.00 3.00 4.00 5.00
% 0
100 A1 A2
MaxPeak Premier Columns | Application Notebook 22
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Separation of Pentose Phosphate Pathway, Glycolysis, and Energy Metabolites Using an ACQUITY
Premier System with an Atlantis BEH Z-HILIC Column
Analyses of phosphorylated metabolites are challenging because of
their interactions with metal surfaces in conventional LC systems and
columns. Here, we describe a targeted UPLC-MS/MS method for 27
pentose phosphate, glycolysis, and energy metabolites in plasma and
tissue extracts. The method leverages the benefits of MaxPeak High
Performance Surfaces Technology used in the ACQUITY Premier
System and the Atlantis Premier BEH Z-HILIC Column to mitigate
interactions of the metabolites with metal surfaces. The stability of
the column to high pH mobile phases is also key, as the best peak
sharpness, peak symmetry, and sensitivity was achieved using a pH
9 ammonium bicarbonate buffer in the mobile phase. The results
demonstrate that high efficiency, UPLC-pressure tolerant 1.7 μm
Atlantis Premier BEH Z-HILIC Columns provide excellent separations
for these challenging analytes.
Representative chromatograms of analyte standards in 50/50 ACN/water.
BENEFITS
■ A targeted UPLC-MS/MS method that provides sharp, symmetric
peaks for 27 challenging metabolites
■ The base-stable Atlantis Premier BEH Z-HILIC Column allows the use
of an optimal pH 9 buffer
■ MaxPeak High Performance Surfaces Technology in the UPLC system
and column enables excellent peak sharpness, peak symmetry, and
sensitivity
■ of phosphorylated and carboxylate lipids with other
lipid classes
ES+
ESTime
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
%
0
100
Time
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
%
0
100
1 2
3
5
6
7
8 9
10
11
12 13
14
4
16 17
18 19
20
21
22
23
2425
26
27
1 Pyruvic a2 Lactic aci3 Deoxyribo4 Dihydroxy(DHAP)
5 Ribulose phosphat6 Ribose 5-7 Glycerald8 Sedohept9 Fructose 10 2-Phosph11 3-Phosph12 Phosphoe13 Glucose 614 6-Phosph15 Fructose 16 Flavin ade17 Acetyl coe18 Dihydronidinucleoti19 Adenosin20 Nicotinam(NAD)
21 Adenosin22 Guanosin23 Adenosin24 Dihydronidinucleoti25 Nicotinamphosphat26 Guanosin27 Guanosin15
MaxPeak Premier Columns | Application Notebook 23
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Food Applications
Read the Full Application Note
Enhancing the LC-MS/MS Analysis of B-group Vitamins with MaxPeak High Performance
Surfaces Technology
Waters MaxPeak High Performance Surfaces (HPS) Technology provide an effective solution to mitigate interactions between analytes and metal
surfaces in liquid chromatography. This application note investigates the effects of the MaxPeak HPS Technology on the LC-MS/MS analysis of
B-group vitamins and demonstrates the key benefits of using the MaxPeak HPS Technology in the simultaneous analysis of B-group vitamins in
energy drinks and vitamin B complex dietary supplements. The key benefits observed include high response, improved sensitivity, less peak tailing,
better calibration linearity, and no carry-over compared to the stainless-steel surfaces in a conventional LC system setup. Greater sensitivity (3–10
times) was observed for riboflavin, thiamine, nicotinamide, flavin mononucleotide, pyridoxal 5’-phosphate, and 5-methyltetrahydrofolate using the
Waters ACQUITY Premier Solution.
BENEFITS
■ Waters ACQUITY Premier Solution improves LC-MS/MS analysis of B vitamins
■ 3 to 10 times better sensitivity were observed for six B vitamins using the MaxPeak HPS Technology than the conventional stainless-steel surfaces
■ Higher response, less peak tailing, and less carry-over are observed with Waters ACQUITY Premier Solution
Time
4.80 5.00 5.20 5.40
% 0
100 5.08
Time
2.80 3.00 3.20 3.40 3.60
% 0
100
Time
2.00 2.25 2.50 2.75 3.00 3.25 3.50
% 0
100
Time
2.00 2.25 2.50 2.75 3.00 3.25 3.50
% 0
100 2.66
Time
4.80 5.00 5.20 5.40
% 0
100 5.18
Time
2.80 3.00 3.20 3.40 3.60
% 0
100 3.05
Time
2.00 2.25 2.50 2.75 3.00 3.25 3.50
% 0
100 2.59
Time
2.60 2.80 3.00 3.20 3.40
% 0
100 2.97
FMN Thiamine PLP Pantothenic
acid
FMN Thiamine PLP Pantothenic
acid
(A)
(B)
Comparison of LC-MS chromatograms of FMN, Thiamine, PLP, and Pantothenic
acid obtained on the HPS setup (green traces) and the SOP setup (red traces).
(A) Observed during the initial injections of the same standard mix on fresh
LC systems. (B) Observed in the B vitamin analysis of the same DS sample
on LC systems that have been extensively used.
MaxPeak Premier Columns | Application Notebook 25
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Read the Full Application Note
Enhanced Performance of the Analysis for
Veterinary Drugs with Metal Affinity Using
ACQUITY™ Premier and Xevo™ TQ-S Micro
The application of MaxPeak High Performance Surfaces (HPS) reduces
interaction for metal sensitive compounds and so increases the
response of these compounds in a liquid chromatography-tandem mass
spectrometry (LC-MS/MS) analysis. Here we demonstrate the utility
of the Waters ACQUITY Premier UPLC™ System-Xevo TQ-S micro for
the analysis of a range of veterinary drugs representative of the major
classes and show increased performance of MaxPeak High Performance
Surfaces (HPS) for the known metal sensitive tetracyclines.
BENEFIT
■ Sensitivity gains for metal sensitive veterinary drugs, extending
detection limits of previous methods
(a) (b) (c)
Overlaid chromatograms showing the effect of HPS on known metal sensitive
compounds. HPS – (red) and stainless steel (green) for (a) Tetracycline
(ESI+, 445.45 > 410.00), (b) Chlortetracycline (ESI+, 479.30 > 444.18),
and (c) Oxytetracycline (ESI+, 461.26 > 426.13).
MaxPeak Premier Columns | Application Notebook 26
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
The Benefits of ACQUITY Premier UPLC for Multi-Mycotoxin Methods
Read the Full Application Note
Multi-mycotoxin methods typically present challenges, such as uneven
response for different compounds, carryover in LC systems, and matrix
effects. These factors can affect validation experiments and significantly
impact the performance of the method. To address these challenges,
Waters has developed the ACQUITY Premier System and analytical
columns, which incorporate the MaxPeak™ High Performance Surfaces
(HPS) Technology. In this work we successfully transferred a previously
developed multi-mycotoxin LC-MS/MS method on the new ACQUITY
Premier System, and we observed a significant reduction of carryover
for fumonisins by almost 80% compared to a conventional UPLC. In
addition, very good method performance was achieved, in terms of
linearity, precision, peak shape, and retention time stability.
BENEFITS
■ The ACQUITY Premier System and column effectively reduce
carryover of fumonisins compared to conventional UHPLC systems
■ On the ACQUITY Premier System, the addition of metal chelators
in the mobile phase or washing solutions was not necessary
■ Analytical throughput was improved on the ACQUITY Premier
System as the number of washing cycles can be reduced
■ Very good linearity, precision, peak shape, and retention
time reproducibility were obtained, which allowed to meet
SANTE/12089/2016 performance guidelines
Peak response and signal-to-noise ratio of aflatoxin B1 (0.75 µg/kg) and fumonisins B2 (15 µg/kg) on
the ACQUITY I-Class (left) and on the ACQUITY Premier (right). Manually-modified integrations are
characterized by yellow traces.
Standard ACQUITY I-Class ACQUITY Premier
Aflatoxin B1
Area: 63875
S/N: 38
Aflatoxin B1
Area: 77406
S/N: 45
Fumonisin B2
Area: 5363
S/N: 10
Fumonisin B2
Area: 16121
S/N: 18
Retention time (min) Retention time (min)
Retention time (min) Retention time (min)
MaxPeak Premier Columns | Application Notebook 27
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Links to Other Useful Materials
MaxPeak Premier Columns
To The Max: Eliminate Doubt with MaxPeak Premier Columns Brochure
MaxPeak Premier Columns Ordering Guide
Analytical Chemistry Article: Using Hybrid Organic-Inorganic Surface Technology
to Mitigate Analyte Interactions with Metal Surfaces in UHPLC
MaxPeak Premier Columns | Application Notebook 28
Introduction Fundamentals Pharmaceutical Metabolomics Food Applications
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
waters.com
Waters, The Science of What’s Possible, Atlantis, MaxPeak, BEH, Arc, CORTECS, XSelect, XBridge,
ACQUITY, QDa, Xevo, UPLC and Empower are trademarks of Waters Corporation. All other trademarks
are the property of their respective owners.
©2023 Waters Corporation. Produced in the U.S.A. August 2023 720007948EN GJ-PDF
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