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Peak Integration, Part 3:

Common Integration Errors

In the first two parts of this series (HPLC Solutions #127 and #128), we
looked at how HPLC data systems integrate chromatograms and some
of the adjustments that can be made if you dont like the way the default
settings perform. Even with proper adjustment of the settings, you may
still observe occasional or regular problems with integration. Here well
look at several common problems and how to correct them.
Click here to read>>
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Featured Applications

Veterinary Drug Analysis with Supercritical Fluid

Chromatography and Triple Quadrupole MS Analysis
Using Longer Aeris PEPTIDE Core-Shell HPLC/UHPLC
Columns for Improved Peptide Mapping
A High-Throughput SPE Method to Support the
Biomonitoring of Phthalate Metabolites in Human Urine
Using ISOLUTE ENV+ Columns Prior to LC-MS/MS

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Peak Integration, Part 3: Common Integration Errors

In the first two parts of this series (HPLC Solutions #127 and #128), we
looked at how HPLC data systems integrate chromatograms and some
of the adjustments that can be made if you dont like the way the default
settings perform. Even with proper adjustment of the settings, you may still
observe occasional or regular problems with integration. Here well look at
several common problems and how to correct them.

Figure 1 shows three examples of problems that are commonly encountered
in integrated chromatograms. In each case, the solid red line shows how the
data system selected the baseline for integration and the dashed lines are the
corrected baseline. Normally the baseline is determined by drawing a straight
line connecting the baseline before the peak to that after the peak. In example
(a) the baseline has a negative peak or dip before the peak of interest is eluted.
The data system mistakenly identified this as the low point in the baseline ahead
of the peak with an obvious error in determining the peak area. The problem was
corrected by moving the baseline to the position of the dashed red line.
The second chromatogram, (b), shows a dilemma that is often encountered.
The default integration assigned a perpendicular drop from the valley between
the two peaks, so the peak areas for the first and second peaks are divided as
shown. In this case, the proper integration is to skim the smaller peak off the tail
of the larger one. You can see that this treatment may not make much difference
in the area of the larger peak, but the original integration assigns more than twice
the appropriate area to the minor peak. This is especially critical in reporting

pharmaceutical impurities, where a batch of product may fail quality testing if

the impurity levels are reported to be higher than they actually are. In making a
decision on this, I use what I call the 10% Rule, which tells me that if the minor
peak is less than 10% of the peak height of the major one, it should be skimmed
(dashed line), whereas if it is more than 10% of the major peak, a perpendicular
drop should be used (solid line). You can see that, even though the top of the
larger peak is not visible, the larger peak is at least ten times as tall as the minor
one. Another example of the application of the 10% Rule was given in HPLC
Solutions #115.
In the last example, the data system determined that the peak in chromatogram
(c) returned to the baseline too early. This is a particularly common problem with
small peaks on a noisy or drifting baseline. In this case, the correct baseline was
drawn with the dashed line to the point where the peak actually returned to the
true baseline.
In each of these cases, manual integration was required. I regularly get
questions in the training classes I teach about this subject. In some laboratories,
workers are not allowed to manually integrate peaks because management
is afraid of negative feedback from regulatory authorities. This is being falsely
conservative. There are clear guidelines for this published in the US Code of
Federal Regulations, CFR 21 Part 11. The essential rules for manual reintegration
are (a) the person doing the integration must be identified, (b) the date and time
must be noted, (c) a copy of the original (raw) data must be preserved, and (d)
a reason for the change must be recorded. Most modern data systems include

Figure 1

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Figure 1: Three examples of problems that are commonly encountered in integrated chromatograms.

an audit function that can be turned on; this forces compliance with these
requirements.
Yes, it may be possible to have every chromatogram integrated properly, but
this is only likely when all the peaks are large and the baseline noise is minimal.
With small peaks on a noisy or drifting baseline, it is very likely that manual
integration will be required to get high quality results.

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FEATURED APPLICATIONS
Veterinary Drug Analysis with
Supercritical Fluid Chromatography
and Triple Quadrupole LC/MS

The Agilent 1260 Innity Analytical SFC Solution and

Agilent 6490 Triple Quadrupole LC/MS

Application Note
Food Testing and Agriculture

Authors

Abstract

Edgar Naegele, Joachim Thiemann,

This Application Note describes the combination of the Agilent 1260 Innity

and Thomas Glauner

Analytical SFC Solution and an Agilent 6490 Triple Quadrupole LC/MS for

Agilent Technologies, Inc.

the measurement of veterinary drugs. The connection of the SFC to the triple

Waldbronn, Germany

quadrupole MS was made through the Agilent Jet Stream Technology electrospray
ionization source. The conguration of the system is described, including a split
from the SFC to the MS and a separate make-up ow for improved ionization,
together with the method parameters. The data show limits of quantitation
(LOQs) and limits of detection (LODs) in the lower ppb range for all measured
veterinary drugs.

Veterinary Drug Analysis with Supercritical Fluid Chromatography and Triple Quadrupole MS Analysis
Company: Agilent Technologies
This application note describes the combination of the Agilent 1260 Infinity Analytical SFC Solution and an Agilent
6490 Triple Quadrupole LC/MS for the measurement of veterinary drugs.

Click to
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TN-1124

APPLICATIONS
Using Longer Aeris PEPTIDE Core-Shell HPLC/UHPLC Columns for Improved
Peptide Mapping
Michael McGinley, Deborah Jarrett, and Jeff Layne
Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

A new 3.6 m 100 HPLC/UHPLC column (Aeris PEPTIDE) has

been introduced that is specifically designed to improve separations
of peptide and peptide mapping applications. The Aeris PEPTIDE
XB-C18 column was developed to complement Aeris WIDEPORE
XB-C18 core-shell columns for protein characterization. When one
looks at peptide mapping applications, performance requirements
are significantly different versus intact protein separations, as
increased retention and selectivity are required to separate the large
number of peptides generated in peptide mapping applications.
Because increased resolution is a higher priority versus speed, a
larger particle (3.6 m) core-shell particle was developed allowing
the use of longer columns at lower backpressures. In this application
the increased resolution that longer Aeris PEPTIDE 3.6 m XB-C18
provide will be demonstrated.

backpressure amenable to using standard HPLC systems (200

bar at 1.2 mL/min). These results demonstrate the performance
advantage and utility of the Aeris PEPTIDE 3.6 m XB-C18 media
for highly complex peptide mapping mixtures where one can utilize different column lengths to optimize resolution and separation time based on the needs of a specific application.

Conclusion
Maximizing resolution between proteins and their modified
impurities is critical in obtaining useful quantitation of posttranslational modifications of biogeneric proteins. The different
applications in this technical note show the utility of Aeris
WIDEPORE columns for obtaining accurate data for intact protein
applications. The optimized geometry of the core-shell Aeris
WIDEPORE columns, as well as good selectivities of the three
separate phases offered (XB-C18, XB-C8 and C4), deliver better
resolution and recovery than existing fully porous 300 columns
for intact protein analysis. Finally, the large particle size of the Aeris
WIDEPORE column delivers a significantly lower backpressure
than sub-2 m 300 columns which allows for more flexibility in
instrument used (HPLC or UHPLC) as well as column length in
developing biogeneric protein applications.

Materials and Methods

All chemicals, standards and antibodies were obtained from Sigma

Chemical (St. Louis, Missouri). Solvents were purchased from EMD
(San Diego, California). Core-shell Aeris PEPTIDE 3.6 m XB-C18
columns (150 4.6 mm and 250 4.6 mm) were obtained from
Phenomenex (Torrance, California). Bovine serum albumin was
digested with trypsin and analyzed on an Agilent 1200 HPLC system
with autosampler, column oven, solvent degasser, and UV detector
set at 214 nm. Data was collected using ChemStation software
(Agilent, Santa Clara, California). Mobile phases used were 0.1 %
Formic acid in water (A) and 0.1 % Formic acid in acetonitrile (B)
with a gradient from 3 to 65 % B at a flow rate of 1.2 mL/min.
Gradient times were adjusted based on column length (33 to 55
minutes respectively). Column was maintained at 40 C.

Figure 1.
BSA Tryptic map separated on different length Aeris PEPTIDE 3.6 m
XB-C18 columns (150 4.6 mm top, 250 4.6 mm bottom). Note the
good separation on the shorter Aeris PEPTIDE column and the increased
resolution provided by the longer Aeris PEPTIDE (250 4.6 mm) column.
Because backpressure for the Aeris 3.6 m column is so low, one can
optimize column lengths based on their separation time and resolution
requirements.
19880

mAU

Results and Discussion

Aeris PEPTIDE 3.6 m XB-C18 core-shell particles demonstrate

similar or better performance than sub-2 m fully-porous columns
at a fraction of the backpressure, allowing the use of longer columns at backpressures compatible with existing HPLC systems.
The 3.6 m core-shell media is of particular utility for peptide map
applications where the increased resolution of longer columns is
desired (for high-speed UHPLC applications the Aeris PEPTIDE
1.7 m XB-C18 can be used instead). An example of the utility is
demonstrated in Figure 1 where 150 4.6 mm and 250 4.6 mm
Aeris PEPTIDE 3.6 m XB-C18 columns were compared for a
peptide map of BSA. The 150 4.6 mm column provides excellent separation of the peptide mixture at a low column backpressure (140 bar at 1.2 mL/min) such that a longer column could be
used to achieve additional resolution if required. When the 250
4.6 mm Aeris PEPTIDE 3.6 m XB-C18 column was used for
the separation, additional peptides were resolved while still at a

App ID 19880

20

10
7

10

11

12

min

19882

mAU

App ID 19882

30

20
12

14

16

18

20

22

min

Page 1 of 2

For additional technical notes, visit www.phenomenex.com

Click to
request copy

Application Note AN826

Biomonitoring of Phthalate Metabolites in Human Urine using ISOLUTE ENV+ Columns Prior to LC-MS/MS | Page 1

A High-Throughput SPE Method to Support the

Biomonitoring of Phthalate Metabolites in Human
Urine Using ISOLUTE ENV+ Columns Prior to
LC-MS/MS

Roy Gerona1 , Frank Kero2 Matthew Friesen1 , Victor Vandell 2 , Michael Yu2
1. UCSF Department of Laboratory Medicine, 513 Parnassus Ave, Medical Sciences Bldg S 864 San Francisco, CA, USA 94143
2. Biotage, 10430 Harris Oaks Blvd., Suite C, Charlotte, NC USA 28269

This application note describes the extraction of

nine phthalate metabolites from human urine using
ISOLUTE ENV+ solid phase extraction columns.

Introduction
Phthalates are plasticizers used in industry to adjust the
mechanical (and sometimes barrier) properties of plastics
in consumer products and packaging. Their ubiquitous
presence in our everyday lives constantly presents threats
of low level exposure through inhalation or ingestion. Thus,
large biomonitoring studies including the US National Health
and Nutrition Examination Survey (NHANES) have screened
for phthalates since 1999. Because phthalates themselves
are difficult to eliminate from sampling and processing
materials, including laboratory ware and instruments,
analysis of phthalates in human samples have focused on
their monoester metabolites. Monoethyl phthalate (MEP),
monobutyl phthalate (MBP), monobenzyl phthalate (MBzP)
and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) have
been constantly detected in human urine since the first
NHANES survey of phthalates in 1999. Phthalates exposure
has been associated with decreased anogenital distance,
lower sperm count, cryptorchidism and hypospadias among
other clinical endpoints in humans.
To facilitate the high throughput population screening
of phthalate metabolites, sample preparation methods
need to be simple, sensitive and robust to mitigate matrix
suppression and instrument down time common to many
dilute and shoot approaches to mass spectrometry. Thus, a
solid phase extraction procedure was developed for these
analytes. This application note details the optimization
strategy for nine phthalate metabolites. Proof-of-concept for
this sample preparation method was determined on a set
of real patient samples (n=5). The results were in general
agreement with previously reported concentration ranges for
these compounds. It is anticipated that this method will have
significant impact in environmental biomonitoring strategies
for these analytes.

Analytes
Monomethyl phthalate (MMP); Monoethyl phthalate (MEP);
Monobutyl phthalate (MBP); Monobenzyl phthalate (MBzP);
Monohexyl phthalate (MHxP); Mono (2-ethylhexyl) phthalate
(MEHP); Mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP);
Mono (2-ethyl-5-carboxypentyl) phthalate (MECPP);
Monoisononyl phthalate (MiNP)

MBP

MEP

MMP

MBzP

MHxP

MEHHP

MEHP

MiNP

MECPP

Figure 1. Structures of the target analytes in the phthalate metabolites panel.

1
Application Note AN826 2014 Biotage

Click to
request copy

Pharmaceutical Applications

Excellent LC-MS Separation of Penicillins and

Cephalosporins Using Ultra IBD Columns
Introduction
Antibiotics are the most widely used medications in the world.
Whether by prescription, addition to animal feed stocks, or use of
cleaning agents, everyone in the civilized world is either directly
or indirectly exposed to antibiotics in daily life. The overuse of
antibiotics, however, has allowed resistant bacteria to thrive. The
death of 12,500 people in Guatemala from an episode of Shigella
fever can be traced to a simple mutation of the bacterial strain.
Research indicated that the bacterium incorporated a single plasmid into its RNA sequence and resultantly became resistant to
four different antibiotics. This illustrates the danger of resistance
caused by adaptation. To combat resistant bacteria, new antibiotic derivatives must be created to overcome the bacterias new
defense mechanisms. Typically, HPLC columns can be used to
analyze penicillins and their structurally related cephalosporins.
However, the similarity of many derivatives may require additional interactions to effectively separate related compounds. Resteks
Ultra IBD column is better able to resolve these compounds using
polar and hydrophobic interactions.

Figure 1: Ultra IBD column separates penicillin V

from fermentation impurities.
1

Peak
1. Penicillin V

Background
Penicillins and cephalosporins represent nearly sixty percent of
antibiotics worldwide. These antibiotics possess a sulfur atom
within a five- or six-membered ring, attached to a fourmember
-lactam ring. They are produced by fermentation processes using
either selected fungi or species of Streptomyces bacteria. Derivatives are produced in two fashions:
1. Biosynthetic processThe fungus or bacteria are genetically engineered to produce a new derivative, or the starting
materials are altered to produce biosynthetic variants during
fermentation.
2. Semi-synthetic processesThe materials from a biosynthetic
process are converted to chemical derivatives. Penicillin derivatives are created from penicillin G or V, while cephalosporin
derivatives are created from cephalosporin C or cephamycin C.

Time (min)
Column
Dimensions:
Particle Size:
Pore Size:
Temp.:
Sample
Diluent:
Conc.:
Inj. Vol.:
Mobile Phase
Flow:
Detector

8
LC_0096

Ultra IBD (cat.# 9175565)

150 mm x 4.6 mm ID
5 m
100
30 C
acetonitrile:water (10:90, v/v)
1.2 mg/mL
2.5 L
10 mM ammonium formate, pH 2.5:acetonitrile (95:5, v/v)
1.2 mL/min
UV/Vis @ 270 nm

Innovative Chromatography Products

www.restek.com

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Separation of statistic MMA-MAA Copolymers using

Gradient SEC
Application Note Pharmaceutical Analysis
Author

Dr. Wolfgang Radke

contact: WRadke@pss-polymer.com

Isocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydrodynamic volume. In case of homopolymers GPC/SEC allows the fast, precise and easy
determination of the complete molar mass distribution.
Unfortunately many modern polymeric materials are copolymers and isocratic GPC/SEC does
not provide any information about the chemical composition. Here SEC-gradients or polymer
HPLC is the method of choice.

Introduction

Statistic copolymers of methyl methacrylate (MMA) and methacrylic acid (MAA) are widely used
in pharmaceutical applications. Besides the molar mass distribution the chemical composition
and the amount of comonomers in the copolymer is of importance. Separations by conventional
gradient HPLC failed, since the polar eluents required to dissolve polymers of high acid content
prevent adsorption onto the stationary phase, resulting in pronounced breakthrough peaks. The
problem can be avoided applying SEC-gradients, resulting in the desired separation according
to the amount of methacrylic acid. The system can be calibrated using reference materials of
known composition. This allows determining the average copolymer composition as well as the
compositional heterogeneity.

System Requirements
Conditions
Pump

PSS SECcurity GPC1260 binary pump

flow rate [mL/min]: 1.0
mobile phase:
Gradient: Chloroform/DMAc

Injection system

PSS SECcurity GPC1260 Autosampler

Injection interval: 32 min

Columns

Calibration
Loading

PSS PROTEEMA precolumn (8*50 mm)

PSS PROTEEMA, 3 m, 100 (8x300 mm)
Temperature: 60/C
PSS MMA-MAA copolymers of different acid content
(MAA: 9%, 25%, 31%, 42%, 48%wt)
Samples dissolved in DMAc
1 mg/mL, 100 L injection volume

Detector

PSS SECcurity ELS1000

Gas flow: 1.5 SL/min
Nebulizer temperature: 100 /C
Evaporator temperature: 200 /C.

Software

PSS WinGPC UniChrom with ChromPilot and Chemical

Heterogeneity module

PSS Polymer Standards

Service GmbH
In der Dalheimer Wiese 5
55120 Mainz | Germany

Phone +49 6131 96239-0

Fax
+49 6131 96239-11
E-Mail info@pss-polymer.com
Web www.pss-polymer.com

Polymer Standards
Service-USA, Inc.
160 Old Farm Rd, Suite A
Amherst | MA 01002 | USA

Phone +1 413 835-0265

Fax
+1 413 835-0354
E-Mail usa@pss-polymer.com
Web www.pss-polymer.com

Click to
request copy

Using Longer Aeris PEPTIDE Core-Shell HPLC/UHPLC Columns for Improved Peptide Mapping
Company: Phenomenex
A new 3.6 m 100 HPLC/UHPLC column (Aeris PEPTIDE) has been introduced that is specifically designed to improve
separations of peptide and peptide mapping applications. The Aeris PEPTIDE XB-C18 column was developed to
complement Aeris WIDEPORE XB-C18 core-shell columns for protein characterization.

A High-Throughput SPE Method to Support the Biomonitoring of Phthalate Metabolites in Human Urine Using
ISOLUTE ENV+ Columns Prior to LC-MS/MS
Company: Biotage
Phthalates are plasticizers used in industry to adjust the mechanical (and sometimes barrier) properties of plastics in
consumer products and packaging. This application note describes the extraction of nine phthalate metabolites from
human urine using ISOLUTE ENV+ solid phase extraction columns.
Excellent LC-MS Separation of Penicillins and Cephalosporins Using Ultra IBD Columns
Company: Restek
Antibiotics are the most widely used medications in the world. Whether by prescription, addition to animal feed
stocks, or use of cleaning agents, everyone in the civilized world is either directly or indirectly exposed to antibiotics
in daily life. This application note describes the LC-MS separation of penicillins and cephalosporins using Ultra IBD
columns.

Separation of Statistic MMA-MAA Copolymers using Gradient SEC

Company: PSS Polymer
Isocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydro-dynamic volume. In the case
of homopolymers GPC/SEC allows the fast, precise and easy determination of the complete molar mass distribution.
Here SEC-gradients or polymer HPLC is the method of choice.

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