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John Dolan is best known as one of the worlds foremost troubleshooting authorities. Separation Science and John Dolan have collaborated to
offer this digital learning platform providing valuable advice on everyday issues, problems and challenges faced by LC practitioners.
Importantly, you will also have the opportunity to interact with John through our online questions submission system.

Peak Integration, Part 1: How It is Done

With todays data systems for HPLC, we tend to take peak measurement
for granted. Much of the time were pretty safe if we use the default
integration conditions, but this is not always the case. In this HPLC
Solutions series on integration, well look at how peaks are integrated and
what can go wrong. This instalment centres on the integration process
itself.
Click here to read>>

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HPLC Solutions Video Tutorials

Visit the new video tutorial section, where a selection

of articles are also provided in video format.
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Featured Applications

LC-MS/MS Screening of 64 New Psychoactive Substances Using Dried

Blood Spots
Reducing Cycle Time for Quantification of Human IgG s
High-Speed Quantitative Analysis of Antiepileptic Drugs Using Triple
Quadrupole LC/MS/MS

HPLC Solutions on ChromForum

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HPLC Solutions articles on Chromatography Forum.
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Peak Integration, Part 1: How It Is Done

With todays data systems for HPLC, we tend to take peak measurement for granted. Much of the time were pretty safe if we use the default integration
conditions, but this is not always the case. In this HPLC Solutions series on integration, well look at how peaks are integrated and what can go wrong.
This instalment centres on the integration process itself.
Conceptually, the measurement of a peaks height or area is pretty simple. For
peak height, just measure the vertical distance from the baseline to the highest
point. For area, start measuring the peak height when the peak starts to rise
from the baseline and stop when it returns, then add up all the individual height
measurements and call it the area.
In Figure 1 we can get an idea of what takes place. First, the system must
determine when the peak actually rises above the baseline. This is shown at the
left edge of the left-hand peak of Figure 1. One of two techniques is common.
In one, the slope of the baseline is measured over a small time segment. This
slope is compared to the slope for the next segment, and the next, and so
forth. When the slope is consistently rising (according to some pre-determined
threshold value), the peak is determined to exist, and the vertical distance from
the baseline is measured for each time segment until the peak returns to the
baseline. Alternatively, the vertical distance from the baseline is measured for each
segment. When a certain number of points in a row, for example, 5 or 10, show an
increasing trend, the peak measurement is started.
The top of the peak is determined by taking the first derivative of the peak
height for each successive segment. When this value goes to zero, it is an
indication that the sequence of measurements, or the slope, is going from
an increasing series to a decreasing one as the top of the peak is crossed. The

retention time is determined at this point, and the peak height is assigned to the
segment with the largest height. As a side note, the retention time, or highest
point in the peak, usually is ahead of the centre of mass of the peak, because
most HPLC peaks tend to tail a bit.
Pretty simple, isnt it? Well there are some complications. One is shown at the
right, where a drifting baseline is observed. Most of us operate the data system
with the autozero function turned on. This automatically sets the baseline at the
bottom of the display when an injection is made so that the chromatogram is
easier to see. However, if the baseline signal UV absorbance for an ultraviolet
detector is greater than a true zero signal, every point in the chromatogram
will have a segment height greater than zero. To correct for this, the data system
determines where the apparent baseline is and subtracts the height value for
each segment that falls between zero and the baseline (the correction trapezoid
in Figure 1). This gives a corrected signal where the baseline is zero and the peak
height and area reflect the size of the peak above the baseline.
Further corrections have to be made if the baseline is drifting, as it is seen in
the right-hand example. Determining exactly where the baseline lies and when
the peak rises from and returns to the baseline is complicated by baseline noise
and drift, as well as how badly the peak fronts or tails. It is quite amazing that the
default integration conditions actually work for most applications! Weve come

Figure 1

Figure 1: Measurement of peak height and area.

a long way from the early days of HPLC, where one way to measure the peak was to cut the peak out of the
chromatogram (recorded on a strip-chart recorder) with a pair of scissors and then weigh it on an analytical
balance. Cut-and-weigh was only one of the rather crude techniques used for quantification of peaks before
modern integrators were introduced.
In the next HPLC Solutions well look at some of the adjustments that can be made if we dont like the
default integration conditions.
To visit the HPLC Solutions video tutorials go to:
http://view6.workcast.net/?cpak=5804874214564175&pak=1939778377848693

John Dolan is best known as one of the worlds foremost HPLC troubleshooting authorities. He is also known for his
ongoing research with Lloyd Snyder, resulting in more than 100 technical publications and three books.
Contact John at [email protected]

FEATURED APPLICATIONS
LC-MS/MS Screening of 64 New Psychoactive Substances
Using Dried Blood Spots
Dried Blood Spots as an Alternative to Whole Blood
Dr. Sebastian Dresen1, Lars Ambach2, Dr. Stefan Knig2, Prof. Dr. Wolfgang Weinmann2
AB Sciex, Darmstadt, Germany and 2Institute of Forensic Medicine, University of Bern, Switzerland

Overview
A robust, fast, sensitive and specific LC-MS/MS screening
method for the determination of 64 new psychoactive substances
(NPS) in dried blood spots (DBS) has been developed and

validated using an AB SCIEX QTRAP 5500 LC/MS/MS System.

DBS provide fast and efficient sample preparation and are easy
to handle in terms of further preparation steps and shipping. Only
low sample volumes of capillary blood are required which can be
obtained through minimally invasive techniques compared to
blood sampling via venipuncture.

Introduction
In recent years there has been a significant increase of new
designer drugs alongside the classic drugs of abuse. The
number of new substances reported for the first time to the
European Monitoring Centre for Drugs and Drug Abuse
(EMCDDA) has risen from 13 substances in 2008 to 73 new
compounds in 2012 (Figure 1). Those new psychoactive
substances often consist of a broad range of substances that are
not controlled under international drug laws. Frequently, they are
chemically similar to controlled drugs to mimic the effects of
existing controlled drugs. However they are sufficiently different

Figure 2: AB SCIEX QTRAP 5500 LC/MS/MS System

that they are not covered by national drug laws.

The main groups of substances monitored by the early warning
system (EWS) operated by the EMCDDA besides synthetic
cannabinoids are the phenethylamines (with stimulant,
entactogenic or hallucinogenic effects, such as PMMA and 2C-I),
tryptamines (which have predominantly hallucinogenic effects,
such as AMT and 5-MeO-DALT), piperazines (which exhibit
predominantly stimulant effects, such as mCPP and BZP) and
cathinones [1].
Dried blood spots can be an interesting alternative to
serum/plasma or whole blood analysis for which only one or few
drops of blood are sampled on filter paper. After drying the
paper, it can be stored until preparation and analysis or can
easily be shipped. The low required blood volumes, the fast,
easy and minimally invasive sampling, as well as the stabilization
of the analytes, all form the main advantages of this technique.
Since the sample volume is very low, a highly sensitive and
selective LC-MS/MS system is required to apply this sampling
method for the detection of low concentrations of the designer
drugs [2].

Figure 1: Number of new psychoactive substances notified to the

European EWS, 20052012. Source: EMCDDA/EWS
RUO-MKT-02-1408-A

p1

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Reducing Cycle Time for

Quantication of Human IgG Using
the Agilent Bio-Monolith Protein A
HPLC Column

Alternating Column Regeneration Using an Agilent 1200

Innity Quick-Change Bio-inert 2-position/10-port Valve
and the 1290 Innity Flexible Cube

Application Note
Biotherapeutics & Biosimilars

Authors

Abstract

Sonja Schneider and Bettina Schuhn

This Application Note describes the quantication of human IgG with the

Agilent Technologies, Inc.

Agilent Bio-Monolith Protein A HPLC Column using the Agilent 1260 Innity

Waldbronn, Germany

Bio-inert Quaternary LC. Alternating column regeneration using the Agilent

1290 Innity Flexible Cube in combination with an Agilent 1200 Innity Series
Quick-Change Bio-inert 2-position/10-port valve reduced the total cycle time by
approximately 20 %. The employment of a complete bio-inert ow path ensures a
high reproducibility as metal leaching and corrosion, caused by high salt/low pH
buffers, is avoided. With this setup, good intra- and inter-column precisions were
achieved for the IgG quantication procedure.

Agilent 1290 Innity

Flexible Cube

Analytical pump

Autosampler

LC-MS/MS Screening of 64 New Psychoactive Substances Using Dried Blood Spots

Company: AB SCIEX
A robust, fast, sensitive and specific LC-MS/MS screening method for the determination of 64 new
psychoactive substances in dried blood spots (DBS) has been developed and validated using an AB SCIEX
QTRAP 5500 LC/MS/MS System. DBS provide fast and efficient sample preparation and are easy to handle in
terms of further preparation steps and shipping.
Reducing Cycle Time for Quantification of Human IgG
Company: Agilent Technologies
This application note describes the quantification of human IgG with the Agilent Bio-Monolith Protein A HPLC
Column using the Agilent 1260 Infinity Bio-inert Quaternary LC.

Click to
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AN753

Extraction of Retinol, -Carotene (Vitamin

A) and -Tocopherol (Vitamin E) from
Serum using ISOLUTE SLE+ 96-Well
Plates with APCI-LC-MS-MS Analysis
Introduction

Application Note

This application note describes the extraction of Retinol, -Carotene, and -Tocopherol from human pooled serum
using ISOLUTE SLE+ 96-well plates with LC-MS analysis.
Vitamins A and E have been shown to have antioxidant and anti-inflammatory effects that mammal
biological systems use to protect against cell
mutation and tissue damage.

The extraction of

Vitamin A and E from human serum prior to analysis

with LC-MS-MS is problematic due to matrix effects.
This method outlines a fast, high throughput sample
preparation technique that eliminates matrix
-Carotene

Retinol

interferences from serum allowing for mass spectral

-Tocopherol

detection of the target analytes at a concentration of

Figure 1. Structure of Retinol, -Carotene, a-Tocopherol

100 ng/mL or less.

Supported Liquid Extraction (ISOLUTE SLE+) plates and columns offer an efficient alternative to traditional liquidliquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced sample preparation time.

Analytes
Retinol, -Carotene, and -Tocopherol
ISOLUTE SLE+ Configuration
ISOLUTE SLE+ 400 L Fixed Well Plate, part number 820-0400-P01

ISOLUTE SLE+ Procedure

Sample pre-treatment:

Dilute human serum (200 L) 2:1 with Isopropanol (100 L).

Spike the

mixture with internal standard. Gently vortex solution.

Sample loading:

Load pre-treated samples (~300 L) onto plate followed by a short pulse of

vacuum (VacMaster-96 Sample Processing Manifold 121-9600) or positive
pressure (PRESSURE+ 96 Positive Pressure Manifold PPM-96) to initiate flow
and allow to flow under gravity for 5 minutes.

Analyte extraction:

Apply of hexane:isopropanol (v/v, 90:10) (900 L) to each well, followed by a

second application of hexane:isopropanol (v/v, 90:10) (900 L). Apply
positive pressure or pull slight vacuum as needed during collection process.

Post extraction:

Evaporate sample to dryness (SPE Dry 96 SD-9600-DHS-NA) and

reconstitute in mobile phase (300 L).
Addition of isopropanol to serum in the pre-treatment promotes protein

Sample Prep Note:

binding disruption and increased overall solubility of analytes.

Sample Throughput:

A 96 well plate can be can be fully processed in 3.2 hours. LC-MS-MS

analysis time (4 minutes/run) for 96 samples, total run time of 384
minutes (6.4 hours). Sample throughput over 24 hours will be 360 samples/
day.

www.biotage.com

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InertSustainSwiftTM C18 columns are designed to enhance your High Throughput

strategy by controlling pore size, 20 nm and surface area, 200m2/g of Evolved Surface
Silica (ESS) and carbon loading, 9.0 %. The inherent characteristics of ESS also enable
rapid re-equilibration even with gradients.

Extraction of Retinol, -Carotene (Vitamin A) and -Tocopherol (Vitamin E) from Serum using ISOLUTE
SLE+ 96-Well Plates with APCI-LC-MS-MS Analysis
Company: Biotage
The extraction of Vitamin A and E from human serum prior to analysis with LC-MS-MS is problematic due to
matrix effects. This method outlines a fast, high throughput sample preparation technique that eliminates
matrix interferences from serum allowing for mass spectral detection of the target analytes at a concentration
of 100 ng/mL or less.
InertSustainSwiftTM
Company: GL Sciences
This note explains how InertSustainSwiftTM C18 columns are designed to enhance your high throughput
strategy due to increased detection sensitivity, high plate count, very low bleed and rapid re-equilibration
ensuring accurate results are met for LC-MS and LC-MS/MS.

Click to
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LAAN-J-LM-E016

LC-MS

Liquid Chromatograph Mass Spectrometer

High-Speed Quantitative Analysis of Antiepileptic Drugs Using Triple Quadrupole

LC/MS/MS

47

This data sheet illustrates high-speed quantitative analysis of 16 anti-epileptic drugs (AEDs) using the Shimadzu UFMS
Triple Quadrupole Mass Spectrometer, LCMS-8050.
Without having any time-consuming sample pretreatment, the LCMS-8050 yielded excellent quantitative results for all 16
AEDs in plasma with a 1 uL injection volume, all in only 7 minutes.

Compound Panel
Table 1: Compounds, Ionization polarity and
MRM transition
Compounds
Levetiracetam
Zonisamide
Felbamate
Primidone

Polarity
+
+
+
+

Lamotrigine
Carbamazepine10,11-epoxide
Carbamazepine

+
+

Precursor
m/z
171.10
212.90
239.10
219.00
255.70
252.70

Product
m/z
126.15
132.00
117.10
162.15
211.05

Levetiracetam
Zonisamide

180.05

236.60

194.10

Clonazepam

316.10

270.05

Nitrazepam

281.90

236.10

Tiagabine
Diazepam
Ethomuximide

+
+
-

376.00
284.70
140.20

111.15
154.05
140.20

Phenobarbial

231.30

Topiramate

337.95

78.00

Valproic acid

143.20

143.10

Phenytoin

251.10

102.05

Felbamate
Primidone
Lamotrigine
Carbamazepine-10,11-epoxide
Carbamazepine
Clonazepam
Nitrazepam
Tiagabine
Diazepam
Ethomuximide

42.05

Phenobarbial
Topiramate
Valproic acid
Phenytoin

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

min

Figure 1: Representative chromatogram of 16 AEDs

Table 2: Analytical Conditions
Column
Mobile Phases
Gradient
Flow Rate
Column Temperature
Injection Volume
Probe Voltage
DL Temperature
Block Heater Temperature
Interface Temperature
Nebulizing Gas Flow
Drying Gas Flow
Heating Gas Flow

: Inertsil ODS-4 (50 mmL. 2.1 mmI.D., 2.0 m)

: A 10mM Ammonium acetate
: B Methanol
: 3% (0.0-0.5min) 90%(3.0-5.0min)3(5.01-7.0min)
: 0.4 mL/min
: 40
: 1 L
: 4.5 kV (ESI-positive mode) / -3.5 kV (ESI-negative mode)
: 150
: 200
: 400
: 3 L/min
: 10 L/min
: 10 L/min

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High-Speed Quantitative Analysis of Antiepileptic Drugs Using Triple Quadrupole LC/MS/MS

Company: Shimadzu
This data sheet illustrates high-speed quantitative analysis of 16 anti-epileptic drugs (AEDs) using the
Shimadzu UFMS Triple Quadrupole Mass Spectrometer, LCMS-8050. Without having any time-consuming
sample pretreatment, the LCMS-8050 yielded excellent quantitative results for all 16 AEDs in plasma with a
1 uL injection volume, all in only 7 minutes.

CONTINUE YOUR PROFESSIONAL DEVELOPMENT

HPLC Troubleshooting & Preventive Maintenance #3:
Physical Problems with HPLC Columns

Analytical Training Solutions (ATS) offers a new web-based approach to analytical technique
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John W. Dolan and Tom Jupille, LC Resources, present Session 3 of the HPLC
Troubleshooting and Preventive Maintenance: The Keys to Fixing Problems
Quickly course. This 60-minute online learning seminar covers physical problems
associated with the use of HPLC columns. This module is a genuine learning tool.
All content from this online seminar is applicable to HPLC analysis and methods
independent of vendor hardware.

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