2 advances in microsampling for in vivofiles.alfresco.mjh.group/.../thecolumn_june052015nasm.pdf ·...

Mastering MicrosamplingAdvances in microsampling for

pharmacokinetic studies

2 Advances in Microsampling for In Vivo Pharmacokinetic Studies Stuart Kushon1, Daniel Kassel2, Hong Xin3, and Nurith Amitai3, 1Neoteryx LLC, 2SciAnalytical Strategies, 3Explora BioLabs Volumetric absorptive microsampling (VAMS) is gaining traction because it delivers the benef ts of dried blood spots (DBS) and overcomes its limitations while generating comparable PK data to conventional sampling methods. This article explains more.

Cover Story

Features

21 Recent Developments in Pharmaceutical Analysis (RDPA 2015) A look to the upcoming symposium on the Recent Developments in Pharmaceutical Analysis (RDPA 2015), which will be held 28 June to 1 July 2015 in Perugia, Italy.

15 Using GPC/SEC for Excipient Characterization Stephen Ball, Malvern Instruments This article looks at how gel permeation/size-exclusion chromatography (GPC/SEC) can be applied to measure characteristics such as molecular weight (MW), MW distribution and structure, and degree of branching for polymeric excipients.

Regulars8 News

Analysis of ink samples using microdestructive CE, detecting parabens in plastic teething toys, and identifying fracking contamination of drinking water using GC×GC–TOF-MS are featured this week.

11 Tips & Tricks GPC/SEC Answering Common Questions About GPC/SEC Columns

Daniela Held and Wolfgang Radke, PSS Polymer Standards Service GmbH A selection of commonly asked questions about GPC/SEC from users.

22 CHROMacademy

Find out what’s new on the professional learning site for chromatographers.

23 Training Courses and Events

25 Staff

5 June 2015 Volume 11 Issue 10

ES625487_LCTC060515_001.pgs 06.01.2015 20:23 ADV blackyellowmagentacyan

Advances in Microsampling for In Vivo Pharmacokinetic Studies

Pharmacokinetic (PK) studies are performed throughout the drug discovery process. In general, 250 µL of whole blood is retrieved at each time point, processed to plasma, and stored at -80 °C prior to bioanalysis. Microsampling with dried blood spots (DBS) is an attractive alternative to the conventional whole blood plasma workup. DBS reduces the need to collect large volumes of blood and therefore the number of animals required. However, DBS technology has not been fully embraced as a result of its well-documented hematocrit bias and the labour-intensive sample manipulation required. A new approach — volumetric absorptive microsampling (VAMS) — is gaining traction because it delivers the benef ts of DBS and overcomes its limitations while generating comparable PK data to conventional sampling methods.

Stuart Kushon1, Daniel Kassel2, Hong Xin3, and Nurith Amitai3, 1Neoteryx LLC, Torrance, California, USA, 2SciAnalytical Strategies, La Jolla,

California, USA, 3Explora BioLabs, San Diego, California, USA.

Pharmacokinetic (PK) studies are routinely

performed throughout the drug discovery

process, from screening during the lead

generation phase to comprehensive PK

studies in candidate selection. During hit and

lead generation, compounds are synthesized

at the milligram level for intravenous (IV)

and oral (PO) dosing in a rat or mouse (for

example 1 mg/kg for IV and 5 mg/kg for

PO). Blood samples are typically taken over

a def ned time course and three animals are

used to obtain an average drug exposure

at each time point. A typical study design

for discovery PK is shown in Table 1. In

general, for rodent PK studies, 250 µL of

whole blood is retrieved at each time point,

processed to plasma (yielding approximately

100 µL), and stored at -80 °C prior to

bioanalysis. Liquid chromatography coupled

to tandem mass spectrometry (LC–MS–MS)

bioanalysis is performed to assess the

pharmacokinetic properties of the molecules

and their potential as lead candidates.

In general, compounds exhibiting >20%

oral bioavailability, moderate to low

clearance, and moderate to long terminal

half-life are prioritized for further prof ling

in pharmacodynamics (PD) and eff cacy

studies. With a plethora of high-sensitivity

LC–MS–MS systems available today, blood

volume is no longer a critical determinant

of bioanalytical success, especially for

discovery PK studies. Despite these analytical

advances, the “one mouse, one time point”

study design described above remains

common practice.

The Promise of Dried Blood Spotting

Microsampling and, more specif cally,

the dried blood spot (DBS) technique Ph

oto

Cre

dit

: R

alf

Hie

mis

ch/G

ett

y I

ma

ge

s

2

Kushon et al.2 News8 Tips & Tricks: GPC/SEC11 Ball1588 11RDPA 2015 Event Preview21 CHROMacademy22 Training and Events23 Staff252222 2323


The Column www.chromatographyonline.com

Standard curve for acetaminophen (mouse plasma)(a)

5.3

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

1.0

0.5

0.0

200 400 600 800 1000 1200 1400 1600 1800

Analyte Conc./IS Conc. (ng/mL)

An

aly

te A

rea

/ I

S A

rea

Standard curve for acetaminophen (mouse whole blood)(b)

An

aly

te A

rea

/ I

S A

rea

2000 2200 24000

500 1000 1500 2000 2500 3000 3500 4000 4500

Analyte Conc./IS Conc. (ng/mL)

50000

Figure 1: Standard curves for acetaminophen: Standards met the criteria of the analytical assay, all measured to within +/- 30% of their target values. R values for plasma and whole blood were 0.9975 and 0.9986, respectively. Standard curves were generated at the front-end and back-end of the study samples.

Kushon et al.

3


You‘ll fi nd the ideal GPC/SEC solution at PSS under:

Phone + 1 413 853 0265

www.pss-polymer.com

[email protected]

PSS are world leaders in macromolecular characteri-

zation and have the expertise to help you with your

analysis requirements. We offer a range of products

and services from instruction courses and training thru

contract analysis, consulting, method development

and qualifi cation services all the way up to supplying

turnkey GPC/SEC and LC/2D systems.

All this comes with the personal and direct support

of our dedicated team of innovative and pioneering

specialists. Is there any better way of achieving your

analysis goals?

Driving GPC/SEC forward

Thinking Forward.

GPC/SECTheory or practice?If there is one thing we can do, it’s both.

ES625700_LCTC060515_V3.pgs 06.01.2015 21:37 ADV blackyellowmagentacyan


have gained considerable attention as an

alternative to the conventional whole-blood

plasma workup used for PK analysis. There

are several compelling reasons for the

interest in DBS. First, with improvements

in LC–MS–MS technology, the blood

volume requirements for bioanalysis are

signif cantly smaller. It is somewhat ironic

that in the last 10 years, the sensitivity

of triple quadrupole mass spectrometers

(the “gold standard” for PK bioanalysis)

has increased 100× to 1000× relative to

earlier generation instruments while blood

sampling protocols have stayed the same

for many in vivo DMPK and toxicology

groups. Second, DBS, relative to plasma,

offers a simplif ed and cost-effective sample

collection, storage, and shipping process.

Third, and perhaps most importantly, DBS

offers the very real opportunity to reduce

the number of animals required for PK,

toxicokinetic (TK), PD, and eff cacy studies.

For example, many groups still perform the

conventional “one mouse, one time point”

PK study design. As shown in Table 1, for a

standard mouse PK study design, a total of

27 mice are required for each dosing group.

Microsampling blood collection enables the

use of far fewer animals because multiple

blood samples can be collected from the

same animal. The use of fewer animals also

eliminates inter-animal variability and is

therefore likely to produce more consistent

data. For PD and eff cacy studies, PK data

generally comes from satellite PK groups.

This is because the traditional larger

volume blood collections preclude the

ability to acquire this information from the

main study animals. The fact remains that

large volumes of whole blood continue to

be used out of habit rather than out of

necessity and this has translated into the

use and sacrif ce of more animals than

necessary.

With the clear benef ts of DBS and

microsampling, the legitimate question to

ask is why haven’t more groups in drug

discovery fully embraced the technology?

There are several reasons for this. One of

the major reasons is the well-documented

hematocrit bias of DBS cards. The blood

hematocrit (HCT), or the volume percentage

of the whole blood that is comprised of red

blood cells, inf uences the viscosity of blood,

and therefore dramatically inf uences the

extent to which a given volume of blood

spreads out onto a DBS card. High-HCT

blood will be viscous and tend to not

spread out across the card, while low-HCT

blood will be more f uid and will spread

out farther. Therefore, a given volume of

blood will generate different diameter spots

on the DBS card depending on its HCT.

In a typical DBS workf ow a small circular

Kushon et al.

4


Calibration-based measuring techniques require assumptions—which aren’t always correct. Tat’s

why every major pharmaceutical and biotechnology company, as well as most federal regulatory

agencies are switching from relative methods to Wyatt Technology’s absolute measurements.

Our DAWN® multi-angle light scattering (MALS) instruments allow you to determine abso-

lute molecular weights and sizes without relying on so-called standards and tedious column

calibration. Wyatt’s instruments measure all of the quantities required for determining ab-

solute molar masses directly. So visit www.wyatt.com and request our free 32-page Ultimate

Guide to Light Scattering. Our booklet is bound to lift your spirits.

“I can’t believe I published the wrong molecular weight.”“It wasn’t your fault, Max. It was your assumption’s fault.”

DAWN HELEOS II. The most advanced multi-angle light scattering instrument for macromolecular characterization.

Optilab T-rEX. The refractometer with the greatest sensitivity and range.

ViscoStar II. The viscometer with unparalleled signal-to-noise, stable baselines and a 21st-century interface.

Eclipse. The ultimate system for the separation of macromolecules and nanoparticles in solution.

DynaPro Plate Reader II. Automated dynamic light scattering for 96 or 384 or 1536 well plate samples—now with an onboard camera!

© 2008 Leo Cullum from cartoonbank.com. All Rights Reserved. DAWN, Optilab, DynaPro and the Wyatt Technology logo are registered trademarks, and ViscoStar and Eclipse are trademarks of Wyatt Technology Corporation.



punch is removed from the overall spot for

analysis. The effective volume contained in

this subpunch is not uniform from sample to

sample because it is a function of the overall

spot size (which is controlled by the HCT of

the blood sample).

To reduce this HCT bias, whole-spot

collections can be made from a DBS

card. This approach provides a uniform

volume; however, it is diffi cult to automate

because of the varied spot sizes and the

fact that liquid handlers commonly found

in laboratories are not amenable to the

card format. Another reason for the slow

adoption of DBS is that procedures and

techniques for preparing animals for

microsampling must be developed

and routinized through training. In

Microsampling Group 2

Microsampling Group 1Plasma Group 1

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Co

nce

ntr

ati

on

(n

g/m

L)

Time (h)

0 1 20.5 1.5 2.5

Figure 2: Overlay of concentration versus time profi le for microsampling group 1 (cardiac puncture, whole blood wicked onto device tips) and 2 (saphenous vein whole blood wicked onto device tips) and plasma group 1 (cardiac puncture, whole blood processed to plasma). The 5 min (0.08 h) microsampling group 2 time point shows slightly higher concentration than plasma and whole blood for study 1 animals.

Kushon et al.

5


LCGC Web VideosTUNE IN TODAY!

ChromTube is LCGC’s platform

for informative videos from

premier suppliers in the

separation science field. Watch

videos in your areas of interest:

Mass Spec, Sample Prep, GC,

and HPLC/UHPLC .

LCGC TV’s editorial videos bring

you insights from leading

analytical scientists, updates on

new research and methods as

well as best practices and

practical advice you can apply

in your lab.

WATCH NOW!LCGC TV: www.chromatographyonline.com/lcgctv

ChromTube: www.chromatographyonline.com/chromtube



addition, sample manipulation, including

card punching, sample extraction, and

handling prior to bioanalysis is more

labour-intensive when using DBS cards

compared to the simple plasma crash

approach that is widely adopted and

effectively used by discovery groups to

generate PK in discovery.

Recently, a novel volumetric absorptive

microsampling (VAMS) approach has been

developed to simplify the blood collection

process and offer other advantages. An

inert, porous, hydrophilic material is used

to absorb a set volume of blood, which

rapidly “wicks” up the volume independent

of hematocrit. Extensive trials have shown

that over a wide range of HCT values (20%

to 70%) there is no HCT bias associated

with the 10 µL volume capture.1,2 Another

important differentiating feature of this

approach to dried matrix microsampling is

that it simplif es the post-sample collection

handling and sample extraction protocol.

Simple sample handling and extraction is

critical in the drug discovery environment

because time is of the essence when

screening compounds. In the following case

study we show how microsampling using

the VAMS approach can be applied to

simplify blood collection.

Case Study: Volumetric Absorptive

Microsampling (VAMS)

In the past, because of the HCT bias

issue, there were major concerns that a

dried matrix sampling technique could

not produce data leading to the same

decisions as those resulting from a standard

plasma PK data set. To investigate if the

volumetric absorptive microsampling (VAMS

approach could deliver comparable data,

PK prof les were generated and compared

for acetaminophen following intravenous

dosing in mice using a conventional design

(standard plasma processing, n = 21

animals) versus a dried whole-blood design

(VAMS microsampling, n = 3 animals for

entire time course).

Table 1: Standard PK study design.

Group No. Route Time Points No. of Rats No. of Mice

1 IVpre-dose, 0.08 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h

N = 3 N = 27

2 POpre-dose, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h, 24 h

N = 3 N = 27

Volume of blood drawn at each time-point = 250 µL, processed to plasma, stored at -80 °C prior to bioanalysis.

Method: Acetaminophen was formulated

in saline to a concentration of 5 mg/mL and

a 2 mg/kg dose was administered into the

tail vein of the mouse. In group 1, three

animals were sacrif ced at each time point.

A total of 21 mice were required for the

entire time course (predose, 0.08 h, 0.25 h,

0.5 h, 1 h, 2 h, and 4 h). Time points past

4 h were not necessary because of the

short half-life of acetaminophen in mice.

Group 2 animals received acetaminophen

through the same route of administration.

However, just three mice were used

in this study, with each animal bled at

every time point across the entire time

course. For group 1, at the specif c time

point, blood was harvested by cardiac

puncture. A Mitra microsampling device

(Neoteryx LLC) using VAMS technology

was dipped into the collected blood and

set aside to dry. The remaining blood

was processed to plasma and stored at

-80 °C until analysis. LC–MS–MS analysis

was performed on a Symbiosis HPLC

(Spark Holland) coupled to an API4000

QTRAP triple quadrupole ion trap mass

spectrometer (Sciex). Chromatographic

separations were performed using a 2 mm

× 50 mm, 5-μm Kinetex C18 column

(Phenomenex). D4-acetaminophen was

used as the internal standard. Mobile phase

A was water containing 0.1% formic acid.

Mobile phase B was acetonitrile containing

0.1% formic acid. The gradient was 1% B

to 70% B in 2.5 min following an initial

hold at 1% B for 0.5 min. For group 2,

blood was retrieved at the specif c time

point by saphenous vein sampling. Plasma

and dried whole-blood standard curves

were generated for acetaminophen over

the concentration range of 5–5000 ng/

mL, as shown in Figure 1. The r-value for

the mouse plasma and mouse whole blood

was 0.9975 and 0.9986, respectively. These

standard curves were then used to quantify

the plasma and whole blood exposures,

respectively, for acetaminophen following

2-mg/kg intravenous dosing.

Shown in Table 2 are the PK parameters

for group 1 plasma (conventional) and

Table 2: Standard PK study design.

Group No.AUC (0-t)(ng*h/mL)

CLs(mL/min/kg)

T½(h)

Vd(mL)

Plasma Group 1 481 70 0.62 1270

Microsampling Group 1 432 77 0.2 983

Microsampling Group 2 739 45 0.4 835

Kushon et al.

6




t = 0.08 h was 1830 ng/mL, and at

t = 0.25 h was 1000 ng/mL. In the case

of group 1 animals, the concentration

of acetaminophen at the t = 0.08 h

time point was 1320 ng/mL. The 0.5 h,

1 h, and 2 h time points showed similar

whole-blood concentrations between the

two groups. The differences are likely not

attributable to the bioanalytical assay;

rather, they may more likely be explained

by the source of whole-blood retrieval

(saphenous vein versus cardiac puncture)

and/or the fact that the group 2 study

was conducted on a different day with a

different (and fresh) preparation of test

article. The PK parameters for group 2

are shown in Table 2. Consistent with the

higher exposure at the early time points,

the AUC was higher relative to group

1 and the clearance lower. Importantly,

the conclusions from this group 2 are

consistent with group 1 — that is, the

compound exhibits moderate to high

clearance, high volume of distribution,

short half-life and moderate exposure after

intravenous dosing of acetaminophen at

2 mg/kg.

Conclusion

The data presented in this article suggest

that volumetric absorptive microsampling

technology is a very useful collection

dried whole blood (VAMS). The area

under the curve (AUC), systemic clearance

(CLs), and volume of distribution (Vd)

were comparable. Only the terminal

(elimination) half-life was significantly

different (T½ = 0.6 h for plasma versus

0.2 h for dried whole blood). Importantly,

in the context of drug discovery, the

interpretation of data would be the same

— that is, the compound exhibits high

clearance, high volume of distribution,

and short half-life in the mouse. Given the

concordance between the mouse plasma

and mouse whole-blood pharmacokinetics

incorporating a conventional “one mouse,

one time point” paradigm, a group 2

study was performed. For group 2, the

pharmacokinetics of acetaminophen using

only three mice was evaluated by doing

serial bleeding via saphenous vein sampling

directly onto the microsampling devices.

Shown in Figure 2 is the VAMS

microsampling whole blood versus time

profile for acetaminophen incorporating

an n = 3 study design as compared

to group 1. The mouse group 2 dried

whole-blood concentration versus

time profile was similar to the group 1

result, the primary difference being the

measured concentration at the 0.08 h and

0.25 h time points. The acetaminophen

dried whole-blood concentration at

technique for pharmacokinetic studies

involving mice, greatly reducing the

animal number requirement and opening

the door to the possibility of combining

PK and PD and efficacy assessments in

the same study animals. This advance in

microsampling could quite possibly change

the “one mouse, one time point” paradigm

commonly found in early discovery

pharmaceutical development. Ultimately,

pharmaceutical companies will need to

decide whether the potential differences

in quantitative bioanalytical data are

truly meaningful and might present any

legitimate risk to the drug discovery and

development process as well as if those

differences outweigh the benefits that

dried matrix microsampling provide.

References

1. Neil Spooner, Philip Denniff, Luc Michielsen, et al.,

Bioanalysis 7(6), 653–659 (2015).

2. Philip Dennif and Neil Spooner, Anal. Chem.

86(16), 8489–8495 (2014).

Stuart Kushon currently holds the position

of senior research scientist at Neoteryx

LLC, a company dedicated to developing

novel microsampling solutions for the

pharmaceutical and clinical markets.

Kushon is a physical organic chemist with

over 10 years of experience developing

products for the direct detection and

analysis of targets including: viral, bacterial,

and protein pathogens as well as small

molecules that serve as diagnostic markers

for disease states.

Daniel Kassel founded SciAnalytical

Strategies in 2013, a bioanalytical,

biomarker discovery, and consulting

firm that serves the pharmaceutical,

biotechnology, and clinical industries.

Kassel also maintains a half-time

appointment as director of research and

development for InSource Diagnostics,

a research-driven clinical diagnostics

organization.

Hong Xin is a biotechnology entrepreneur

specializing in preclinical research with

extensive experience in contract research

and operations, with academic knowledge

across multiple disciplines including cancer

biology, cell biology, molecular biology

and pharmacology. Xin currently works at

Explora BioLabs as COO.

Nurith Amitai is a biomedical scientist

with 10 years of predoctoral and

postdoctoral training, who previously held

the position of Scientist I at Explora BioLabs

in San Diego, California, USA.

E-mail: [email protected]: www.neoteryx.com

Kushon et al.

7



Ph

oto

Cre

dit

: R

UT

H J

EN

KIN

SO

N/S

PL

/Ge

tty Im

ag

es

Microdestructive Capillary Electrophoresis Analysis of

Ink Samples

A novel microdestructive capillary electrophoresis (CE) method

for the analysis of blue pen ink strokes has been published in the

Journal of Chromatography A.1 The analysis of pen inks can be

challenging because ink compositions are usually under patent,

the samples are small, and inks can degrade over time.

CE has previously given promising results when applied to the

analysis of questionable documents, according to coauthor Matías

Calcerrada. He told The Column: “We believe that CE could be an

eff cient analytical technique for casework involving questioned

documents, due to advantages such as its minimal sample

consumption and ability to detect and quantify different analytes

found in ink formulations.”

The team developed a microdestructive sample treatment

method using a scalpel to scratch 0.3 mg of an ink stroke from

paper, prior to analysis using CE with a DAD detector. Samples

of ink from 34 blue pens from different technologies (ballpoint,

rollerball, marker) with different composition (gel, water-based,

oil-based) were analyzed to discriminate between different

technologies and inks. Calcerrada said: “We believe that the

results published demonstrate that the method could be applied

in forensic casework after applying other non-destructive

methodologies, which are always recommended before destroying

the sample.” — B.D.

Reference

1. Matías Calcerrada, Journal of Chromatography A 1400, 140–148 (2015).

Parabens Detected in Plastic Teething ToysParabens are commonly used in cosmetics and personal care products to protect against microbial growth, but a new study

published in the Journal of Applied Toxicology suggests that manufacturers may also be using parabens in plastic teething

products designed for infants.1 Scientists applied an effect-directed approach (to determine endocrine disrupting ability) to

analyze teething products prior to chemical analysis using gas chromatography coupled to mass spectrometry (GC–MS). Of

10 products were tested, two were found to contain endocrine disrupting chemicals (EDCs).

EDCs are natural or synthetic compounds that disrupt the normal functioning of the human endocrine system, often

imitating natural hormones, and are recognized as a contributing factor to a range of diseases. In light of this, the use of

phthalates in toys is restricted in the European Union to reduce childhood exposure. However, there is a huge variety of

possible EDCs used in plastics, meaning that exposure is likely to be underestimated because analysis focuses on common

culprits such as Bisphenol A and phthalates. Corresponding author Martin Wagner from Goethe University Frankfurt am

Main, Germany, told The Column: “Our research is about the EDCs we do not know yet: Instead of analyzing well known

compounds, we use in vitro bioassays to screen all types of samples for hormonal activity and try to identify the causative

chemicals using non-target chemical analysis afterwards (effect-directed analysis).” He added: “In that sense, our research

demonstrates that there are far more EDCs out there (which we currently overlook) and that they come

from unexpected sources (who would have suspected a plastic to contain parabens).”

Samples were taken from 10 plastic and one natural rubber teething soothers purchased

in Germany in 2012. Methanol extracts and water eluates from the samples were screened

using a Yeast Estrogen Screen (YES) and a Yeast Antiandrogen Screen (YAAS). One sample

was shown to possess both estrogenic and antiandrogenic activity, and another was

found to be antiandrogenic. GC–MS analysis showed the presence of ethyl-, methyl-, and

propylparaben in one product. Wagner told The Column: “In [the] case of one product we

found three different parabens. These are well-known EDCs, which are normally used

as preservatives in cosmetics.” He added: “In [the] case of the second product, we

demonstrated that this teether leached six different EDCs, all of which remain so

far unidentif ed. In a broader sense, our study demonstrates that babies will be

exposed to EDCs when chewing on plastic teethers. Because they are especially

susceptible, we should aim at minimizing their exposure to EDCs.” — B.D.

Reference1. E. Berger et al., Journal of Applied Toxicology DOI 10.1002/jat.3159 (2015).

8




Identifying Fracking Contamination of Drinking Water Using GC×GC–TOF-MSFracking activity in the northeast of the

USA has rapidly increased over recent years,

with over 8000 Marcellus wells drilled in the

Marcellus Formation in Pennsylvania, USA,

alone.1 Fracking is attracting more media

attention because it opens up more natural

resources but has also been associated with

environmental contamination. Scientists

in Pennsylvania investigating an isolated

contamination incident have published

data from their study, demonstrating

the application of comprehensive 2D gas

chromatography coupled with time-of-f ight

mass spectrometry (GC×GC–TOF-MS) to

investigate fracking contamination in a

drinking water source.1

The study published in the journal PNAS

reports that in 2011 the Pennsylvania

Department of Environmental Protection

(PADEP) documented the contamination of

an aquifer used as a source of drinking water

with natural gas from Marcellus Shale gas

wells. In the year prior, three households

discovered white foam in their drinking water

wells drawn from an aquifer, and subsequent

analyses performed by regulators detected

natural gas contamination, thought to be

attributable to the drilling of new gas well

pads. The regulators were not, however,

able to determine the cause of the white

foaming. In 2012, the company responsible

for the gas well leak acquired the three

households affected by the drinking water

contamination.

Co-author Frank Dorman from Pennsylvania

State University told The Column: “We did

the work because we were initially interested

in the potential for source identif cation and

apportionment in the event of an accidental

discharge of the f uids used in the drilling

industry. Our opinion was that this needed

to be done in a ‘true discovery’ approach,

because there is really little to no disclosure

as to what exact chemicals are used at the

potential sites. This was especially true when

we started this work ~5 years ago.”

The team performed GC×GC–TOF-MS

analyses on water samples collected from

one of the originally contaminated wells, two

of the replacement wells that were drilled

as a replacement for the homeowners, a

natural spring, and water wells near the

contaminated area. GC×GC–TOF-MS analyses

detected an “unresolved complex mixture”

(UCM) of over 1000 different hydrocarbons

in the contaminated samples that was

similar to the UCM of hydrocarbons prof led

in f owback/production water provided by

other gas fracking companies in the area.

The commercially available shale gas-drilling

additive, 2–BE, was also detected at low levels

in the contaminated water samples.

Dorman told The Column that the UCM

“signature” detected in the f owback/

production and well samples could be used as

a diagnostic to detect the impact of shale-gas

operations on surface or groundwater. He

said: “We are continuing to work on being

able to quantitatively describe the hydrocarbon

signatures using GC×GC–HRTOF-MS

through the development of mass-defect

plots (Kendrick diagrams), and then

hopefully developing the ability to determine

goodness-of-f t of the hydrocarbon signature

of reference samples to f eld samples.”

In an FAQ document published by the

authors, they state that it is not possible to

“unambiguously” determine the source of the

2-BE or UCM in the drinking water, and that

the gas company responsible for the leak has

since made changes to gas well construction

practices.2

In terms of future work, Dorman explains

that the team are exploring the possibility of

bioremediation to remove organic content prior

to recycling of water at the well sites. — B.D.

References1. G.T. Llewellyn et al., PNAS 112(20), 6325–6330

(2015).

2. FAQ Document: http://www.

appalachiaconsulting.com/home/whats_new/

pnasarticlefaqs

Ph

oto

Cre

dit

: D

wig

ht

Na

dig

/Ge

tty I

ma

ge

s

News

9




News In Brief

Like us Join us Follow Us

Sciex and New Objective Partner UpSciex (Massachusetts, USA) has announced a

partnership with New Objective (Massachusetts,

USA) that will enable Sciex to offer high

performance nanospray ionization technology

with their LC–MS systems. Gary Valaskovic, Ph.D.,

President and co-founder of New Objective, said:

“Partnering with Sciex is a great opportunity

for us to forge new relationships through their

market leading position in quantitative analysis.”

www.sciex.com

One-Step QuEChERS Sample PreparationThe sample preparation method Quick, Easy,

Cheap, Effective, Rugged, and Safe (QuEChERS)

offers high recovery, reproducibility, and lower

costs. A new study published in the Journal of

Chromatography A proposes a new strategy to

combine the two steps involved — extraction

and purif cation — into one magnetic solid-phase

extraction step.

DOI:10.1016/j.chroma.2015.04.021

Thermo Fisher Scientif c Expands in Middle EastThermo Fisher Scientif c (California, USA) has

opened a new Customer Experience Center

(CEC) at the life sciences hub DuBiotech in

Dubai. The 7000-square-foot facility will expand

the company’s ability to offer demonstrations

and training in the region, as well as enable

collaborations with universities in the region.

news.thermof sher.com

LCGC TV HighlightsPrinciples of In-Tube Extraction for

Headspace Sampling of Beer

Torsten C. Schmidt of the University Duisburg-Essen in Germany, recently developed an in-tube extraction (ITEX) method for headspace sampling of beer prior to gas chromatography–mass

spectrometry (GC–MS). In this short video, he explains the principles of the technique and its advantages over other methods. Watch Here>>

Luigi Mondello on the Fundamentals of 2D LCComprehensive liquid chromatography (2D LC) has the potential to increase peak capacity resolution when separating complex mixtures, especially in food analysis. Luigi Mondello from the University

of Messina, Italy, describes the fundamental principles of 2D LC, and explains the advantages over 1D LC.

Watch Here>>

Peaks of the WeekCurrent Trends in Mass Spectrometry Supplement: An Accurate-Mass Database for Screening

Pesticide Residues in Fruits and Vegetables by Gas Chromatography–Time-of-Flight Mass

Spectrometry — The main objective of this study was to evaluate the capabilities of gas

chromatography (GC) with time-of-f ight mass spectrometry (MS) for screening pesticides in fruits and

vegetables using a purpose-built accurate-mass database. Read Here>>

The LCGC Blog: Troubleshooting Retention Time Issues in Reversed Phase HPLC — While some

of the relationships between parameters in gradient HPLC are complex, others are reasonably

straightforward. Here, Tony Taylor explains, through an example, how adjusting the f ow rate can

improve resolution between critical peak pairs. Read Here>>

LC Troubleshooting: Calibration Problems — LCGC Europe columnist John W. Dolan discusses a case

study to show how to troubleshoot calibration problems in the laboratory. Data is presented from a

routine liquid chromatography method in a clinical laboratory. Read Here>>

Ph

oto

Cre

dit

: Stu

art

De

e/G

ett

y I

ma

ge

s.

News

10



Tips & Tricks GPC/SEC: Answering Common Questions About GPC/SEC ColumnsDaniela Held and Wolfgang Radke, PSS Polymer Standards Service GmbH, Mainz, Germany.

The hardest part of any gel permeation chromatography/size-exclusion chromatography (GPC/SEC) separation is selecting the right

columns and developing a robust method. Here, we present a selection of commonly asked questions from users together with our

answers based on our experiences.

Gel permeation chromatography/

size-exclusion chromatography (GPC/SEC)

is performed to determine the complete

molar mass distribution. It can be applied

over a wide range of molar masses for

different types of natural and synthetic

macromolecules soluble in mobile phases

of very different polarities. GPC/SEC is

often used in quality control (QC), but

developing a robust and high-resolution

method that delivers precise results, which

are reproducible in the long-term, is a

challenging task. It is therefore of no surprise

that many users need expert advice when

making the choice of the optimum column

(set) from the large selection available. At a

recent event we were asked a lot of good

questions that we want to share here.

Q. When avoiding high backpressure

and shear is it suff cient to run the

GPC/SEC at a lower f ow-rate (so long

as you have enough time) or is larger

particle size the better choice?

A: Macromolecules can be very sensitive,

so forcing high molar mass or stiff polymer

chains through a liquid chromatography (LC)

system at a very high pressure can result in

chain degradation and generate results only

for the fragments. The overall pressure in

a system depends mainly on the f ow-rate,

mobile-phase viscosity, temperature, inner

diameter, number of columns applied, and

particle size of the column stationary phase.

Small particles should be avoided when

analyzing high molecular weights.1 It is

recommended to use larger particle sizes

when running very high molar mass samples

because this will reduce shear. Also note

that in this case column frits with larger

porosity are used and this further reduces

shear stress. For really high molar masses a

combination of both large particle sizes and

low f ow-rate is ideal, if time permits. If you

are using highly viscous solvents, running at

higher temperatures (to reduce mobile phase

viscosity) is also recommended.

For lower molar mass samples, where

high resolution is required (for example to

separate oligomers) the application of larger

particles to overcome back pressure issues

is not recommended. Larger particles result

in lower plate counts, thereby reducing

resolution. Nevertheless most small molar

mass samples will also prof t from higher

temperatures and lower f ow rates for highly Ph

ot o

Cre

dit

: p

orc

ore

x/G

ett

y Im

ag

es

11




viscous solvents because both approaches

increase resolution as a result of better mass

transport.

Q. Could you please comment on the

pros and cons of mixed bed column vs.

individual pore size columns?

A: This is a tough question because

philosophy plays a part here and there are

many aspects to consider.

Let us start with the often-mentioned

expectation of a “linear calibration

curve”. Many people seem to feel more

comfortable with linear relations, even

though the requirements for simple

mathematics has diminished with the

widespread use of computers in the

laboratory that can handle more complex

algorithms.

Unfortunately, the relation between the

logarithm of the molar mass and the elution

volume is not linear. GPC/SEC calibration

curves are typically sigmoidal in shape,

where the logarithm of the molar mass is

plotted versus the elution volume. Most of

the time polynomial functions of 3rd (cubic),

5th, or 7th order are used to f t the data.

This is a good approach as long as the slope

of the calibration curve is also reviewed to

avoid overf tting.2 The approach also allows

the full use of the complete separation

range of the column. Please note that linear

columns are also non-linear at the high and

low molar mass end, therefore a different

f tting approach is required if samples elute

in that region.

Linear or mixed-bed columns are the result

of intense work by column manufacturers.

The production involves either a special

synthesis route or, much more often, the

careful blending of individual pore sizes. The

main advantage of linear columns is that

they can separate over a wide molar mass

range with a constant resolution, and are

ideal for routine QC or as screening columns

if users have to deal with very different

molar masses. You can easily increase the

resolution by adding other linear columns

of the same type. However, it is very

diff cult to alter the molar mass separation

range when higher or lower molar masses

need to be separated. The risk of porosity

mismatch is extremely high, for example

when combining linear columns with

individual columns ideally suited for oligomer

separations.3

The main advantage of individual pore-size

columns is that they provide a highly

eff cient separation but in a limited molar

mass range. Individual pore size columns

are therefore often combined in column

banks. Columns can be added and removed

to alter the molecular weight to tailor it to

the application and the time requirement.

Tips & Tricks: GPC/SEC

12




When following this approach an additional

advantage is that the column with the

largest porosity can be used f rst to separate

the high molar mass (and therefore high

viscous) chains to avoid viscous f ngering.

The best column type is therefore very

dependent on the requirements of the

laboratory.

Q. Do you expect polymeric columns

to be stable and tolerant to switching

eluents? Or do you like to have a

column set devoted to a particular

solvent and then switch columns

when switching solvents? What if

you repeatedly change between pure

tetrahydrofuran (THF) and THF with

small amounts of additives, such as

acids or amines?

A: In general, with solvents that are of a

similar polarity to the packing material —

such as THF, chloroform, dichloromethane, or

toluene for styrene-divinylbenzene columns —

exchanging the solvents should not harm the

columns. However, it is advisable to exchange

solvents slowly at reduced f ow-rates of

0.3–0.5 mL/min. The solvent leaving the

column should go directly to the waste and

the detectors should be disconnected. There

is no reason in principle not to exchange

solvents; however, time might be an issue

because completely re-establishing swelling

equilibrium after going from one solvent to

another often takes longer than reaching a

stable RI-baseline.

For solvents that differ substantially in

solvent polarity from the column material,

we recommend using different columns

because of the different swelling of the gel.

We even recommend ordering such columns

in the solvent of use. Please note that in many

of these cases a different stationary phase

polarity might be the better choice to avoid

interactions.4

For the exchange between pure solvent and

solvent with additives (amines, acids, salts)

we do not see any problems with switching

solvents back and forth.

In any case, columns should be stored in

pure solvents without additives (no salts,

amines, acids). The exception is columns for

aqueous applications where a small amount of

methanol or NaN3 (0.05 g/L) should be added

to avoid growth of algae.

Q. Does changing solvent composition

by addition of salts or other co-solvents

require recalibration of the detectors?

Should the standards be run with the

same modif er as you use for your

sample?

A: Calibration is always an issue in GPC/SEC.

There are two types of calibrations that can

be applied:


13


Conference Office: Symporg SA - Rue Rousseau 301201 Geneva / SwitzerlandTel. +41 22 839 84 [email protected]

Venue: International Conference Center in Geneva (CICG) - www.cicg.ch

Conference Chair: Prof. Gérard Hopfgartner, University of Geneva

www.hplc2015-geneva.org

HPLC 2015Geneva, Switzerland

High Performance

Liquid Phase Separations

& Related Techniques

21 - 25 June 2015

The program will be built around three main themes.A) Core Separation Technology, understanding the fundamental aspects to drive innovationB) Multidimensional separations, mass spectrometry, pushing the limits in separation, detection, identification and data processingC) High Impact Sample Preparation, Separation and Detection, on the edge of current and future applications



E-mail: [email protected]: www.pss-polymer.com

1. Nearly all users perform a column

calibration where they measure the

elution volumes of calibration standards

with different molar masses and plot

the logarithm of the molar mass (or size)

against the elution volume to construct

a calibration curve (see also question

above).

2. In some cases, users need to also

calibrate their detectors in a high

performance liquid chromatography

(HPLC) type of detector calibration.

This is often required when

doing multi-detection GPC/SEC

(triple-detection, light-scattering, or

viscometry) or when they want to

determine concentrations or perform

copolymer analysis. In these cases

different concentrations are measured to

determine the detector responses.

In both cases we always recommend to

apply the same conditions for calibration

as for operation. So the standards should

be run with the same modif ers or with

the same co-solvents, and standards and

samples should be prepared from the solvent

bottle that also supplies the pump.

Q. Do you f nd some salts more/less

corrosive to the instrument than others?

For example, dimethylformamide (DMF)

with LiBr is hard on the instrument, but

gives good results.

A: Halides are usually more corrosive than

other salts, and chlorides are more aggressive

than bromides. Unfortunately, LiCl and LiBr

have better solubilities than other lithium

salts in commonly applied organic solvents;

in addition, because lithium is more superior

in breaking down aggregates than other

counter ions, there are not too many options.

In aqueous solvents, neutral salts like

nitrates or sulphates can be applied instead

of NaCl to reduce the danger of corrosion.

The main reason for salt addition is to shield

electrostatic interaction and reduce the

breakdown of aggregates.

An important factor when using salt

solutions is to ensure that the system always

runs with fresh solutions and that it is not

left to stand for a long time. Always apply a

low f ow-rate to avoid salt crystallizing out

and heavy corrosion. If the system is not

in use for a long period of time it should

be transferred into pure solvent before

switching it off.

Summary

As with other analytical techniques, small

details can have a big impact in GPC/SEC.

Selecting the best column option and

performing proper method development

is def nitely time-consuming, but can save

much more time afterwards. A stable,

robust method is much cheaper in the long

run than choosing the wrong settings in

the beginning. It is therefore important to

always ask questions and so we encourage

all readers to share their questions with us

because there are a lot more answers that

can help users make educated choices.

References

1. D. Held, The Column 10(10), 12–15 (2014).

2. D. Held, The Column 4(6), 18–21 (2008).

3. T. Hofe, The Column 4(4), 20–23 (2008).

4. T. Hofe and G. Reinhold, The Column 12, 30–33

(2007).

Daniela Held studied polymer chemistry

in Mainz, Germany, and works in the

PSS solutions department. She is also

responsible for PSS webinars, education

programmes, and customer training.

Wolfgang Radke studied polymer

chemistry in Mainz, Germany, and

Amherst, Massachusetts, USA, and is

head of the PSS application development

department. He is also responsible for the

PSS customer-training programmes, and for

customized trainings.


14



Using GPC/SEC for Excipient Characterization

The properties of polymeric excipients can directly affect the clinical eff cacy, safety, and quality of a f nished pharmaceutical product and are routinely identif ed as critical quality attributes (CQAs). This article looks at how gel permeation/size-exclusion chromatography (GPC/SEC) can be applied to measure characteristics such as molecular weight (MW), MW distribution and structure, and degree of branching. Case study data for the measurement of poly-lactic acid (PLA) and poly-lactic glycolic acid (PLGA) highlights the detailed information that can be accessed.

Stephen Ball, Malvern Instruments, Malvern, UK.

Polymeric excipients are an important

addition to the sophisticated tableting

blends of today. Ingredients such as

poly-lactic acid (PLA), poly-lactic glycolic acid

(PLGA), and hydroxyl methyl cellulose and

its derivatives enable formulators to achieve

closely controlled drug release prof les

using a growing range of manufacturing

techniques that includes spray drying,

hot-melt extrusion, and lipid-based drug

delivery. The properties of these polymers

can directly affect the clinical eff cacy, safety,

and quality of the f nished pharmaceutical

product and are therefore often identif ed

as critical quality attributes (CQAs). CQAs

for polymer excipients typically include

molecular weight (MW), MW distribution,

and structural characteristics such as degree

of branching.

Gel permeation/size-exclusion

chromatography (GPC/SEC) is a powerful

technique for the characterization of

polymers and other macromolecules. In this

article we examine its application in the

analysis of polymeric excipients. Case study

data for the measurement of PLA/PLGA

highlights the detailed information that can

be accessed.

The Vital Role of Excipients

The workf ows associated with the

development of oral solid dosage forms,

whether innovator or generic, are

increasingly well established and are rooted

in a Quality by Design (QbD) approach.1

These workf ows emphasize the need for

detailed characterization of the excipient, as

well as the active ingredient, both alone and

within the blend. The resulting information

supports the development of a detailed

understanding of how the drug product

will behave and of a specif cation for each

component that will ensure successful drug

delivery and the necessary quality control.

Traditionally excipients have been used

simply as “bulking agents” — in this case

the impact of excipient properties on the

safety, eff cacy, and quality characteristics of

the drug product may be relatively limited.

However, in modern formulations polymeric

excipients often play a far more active role

controlling the drug delivery prof le and

other aspects of drug product performance.

Polymers are now routinely used to:2

• Formulate coatings that control the

rate of dissolution of the tablet in the

stomach.

• Develop spray-dried solid dispersions for

the delivery of sparingly soluble drugs.

• Improve blend ý ow properties.

• Tailor the taste and texture of tablet for

improved patient acceptability.

• Improve the stability of tablets

containing moisture-sensitive active

ingredients.

Precisely differentiating between grades

of excipient, choosing an optimal candidate,

and ensuring the consistency of supply

is critical to the manufacturing process;

however, it can be complicated by two

factors. Firstly, a number of pharmaceutical

excipients are derived from natural polymers,

such as naturally occurring celluloses, which

can limit the manufacturer’s ability to control

polymer properties. There is a choice of

different grades and sources but only a

limited ability to precisely tailor features

such as branching and MW distribution. The

second factor stems from the way in which

the pharmaceutical industry is structured. An

active ingredient often undergoes substantial

development in-house, including the

rigorous investigation of all CQAs, resulting Ph

oto

Cre

dit

: sa

mxm

eg

/Ge

tty I

ma

ge

s

15




in a complete understanding of how the

drug substance behaves and, ideally, a

detailed def nition of the structure-function

relationships that def ne its performance.

In contrast an excipient tends to be bought

in, sometimes from multiple suppliers,

making it diff cult to secure supplies that

enable a rigorous investigation of the

effect of excipient properties. For example,

assessing the impact of branching relies

on sourcing excipients with different

degrees of branching. Furthermore, once an

excipient has been identif ed it can be quite

challenging to scope and control the degree

of variability associated with the supply.

Therefore, although a polymer excipient

may be a more straightforward chemical

entity than the active ingredient, and indeed

have a less direct impact on drug product

performance, there are unique challenges

associated with its characterization.

An Introduction to GPC/SEC

GPC/SEC is a two-step analytical technique

where samples are f rst separated into

fractions on the basis of hydrodynamic size

(by passing through a packed chromatography

column), which are subsequently characterized

using one or more detectors. Measuring

the amount of sample in each sized fraction

50

1

0

10 20

106

0.01

0.1

1

105

10-3

10-4

3.0039E-5

104

1000

100

10

0.2227

1

0

30

0

1

100

0

-3.4617 -0.5150

5.7551

-1.0494 -110.4509

40

30

20

Vis

com

ete

r -

DP (

mV

)

Refr

act

ive in

dex

(mV

)

Mo

lecu

lar

weig

ht

(Da)

Intr

insi

c vi

sco

sity

(d

L/g

)

Low

an

gle

lig

ht

scatt

eri

ng

(m

V)

Rig

ht

an

gle

lig

ht

scatt

eri

ng

(m

V)

10

Figure 1: A chromatogram and derived data for a PLA sample dissolved in THF demonstrating the excellent data quality achieved.

Ball

16


detailed certificates of analysis • acknowledged by regulatory authorities w

orld

wid

e • p

ha

rma

ce

utic

al im

purities • custom synthesised impurities • pharmaceutical primary standards

Pharmaceutical impurities and primary reference standards 2015

LGC Quality - ISO Guide 34 • GMP/GLP • ISO 9001 • ISO/IEC 17025 • ISO/IEC 17043

REQUEST O

CATALOGU

PHARMACOPOEI

REFERENCE STANDA


© LGC Limited, 2015. All rights reserved. LGC Standards is part of the LGC Group. 4365/LB/0515

www.lgcstandards.com • Science for a safer world

Is quality control giving you a headache?

• Primary standards produced

under ISO Guide 34

• Impurities linked to the European

Pharmacopoeia (Pharm. Eur.)

wherever possible

• Acknowledged by regulatory bodies

• Detailed CoA with additional

documentation on request

• $VVLVWDQFH�LQ�¿QGLQJ�WKH�ULJKW�

material to suit your analyses

LGC has 20+ years experience in

manufacturing and supplying reference

materials for the pharmaceutical sector.

Available online at www.lgcstandards.com

detailed certificates of analysis • acknowledged by regulatory authorities world

wid

e • p

ha

rma

ce

utic

al im

puritie

s • custom synthesised impurities • pharmaceutical primary standards

Pharmaceutical impurities

and primary reference

standards 2015


REQUEST OUR

CATALOGUE

PHARMACOPOEIAL

REFERENCE STANDARDS

From Aspirin to Xylazine: LGC has over 3,500 reference materials for APIs

and their impurities including the top generic painkillers, plus a selected

range of primary standards.

@LGCStdsPharma



enables determination of the size distribution,

and more importantly MW and MW

distribution data.

Traditional GPC/SEC systems use a

single-concentration detector, typically

a refractive index (RI) detector. With this

set-up, column calibration with appropriate

standards provides the correlation needed

to estimate a relative (to the standard)

MW distribution. These MW data are

only accurate if the relationship between

molecular size and weight is the same for

the sample as it is for the standard. This is a

crucial limitation especially when gathering

precise data for the detailed comparison of

relatively similar excipients, and when the

calibration standards are sub-optimal for the

polymers of interest.

The use of multiple detectors can directly

address this limitation — for example, a

light-scattering detector in combination

with a concentration detector enables

the direct measurement of absolute MW

with minimal, non-specif c calibration.

Further complementary additions include

a viscometer to enable the measurement

of structural features such as branching or

conformation.

GPC/SEC systems with sensitive

multi-detector characterization

capabilities are consequently useful for

QbD applications. The following case

study demonstrates the application of

multi-detection GPC/SEC for the analysis

of PLGA and PLA, polymers that are used

routinely in pharmaceutical formulation.

Case Study: Analyzing PLA/PLGA

Samples

Produced by polymerizing lactic acid or

lactic and glycolic acid respectively, PLA

and PLGA are used to formulate controlled

drug release systems and to manufacture

medical components such as absorbable

surgical thread and implants.3 Derived from

renewable and natural resources, they are

commonly classif ed as “green polymers”

because of their biodegradability and

biocompatibility. The properties of PLGA

can be controlled by varying the ratio of

lactic to glycolic acid used in its production

to manufacture polymers with different

MW, MW distribution, and structure.

Multi-detection GPC/SEC can be applied

to measure and sensitively compare the

properties of the resulting materials.

Method: Samples of PLA and of PLGA

were analyzed using an OMNISEC GPC/

SEC system with triple detector array (RI,

light-scattering, and viscometer detectors) (all

Malvern Instruments). Tetrahydrofuran (THF)

was used as the solvent for the samples

and as the mobile phase. The analyses were

performed at 30 oC using samples prepared

Ball

17


LIVE WEBCAST: Wednesday, July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST

Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series

Series Part 4 Event Overview:Safeguarding the environment and the global food supply requires continuous monitoring of more and more compounds. Pesticide residues analysts are chal-lenged to detect, identify, and quantify hundreds of diferent pesticides with a fast turnaround time (often within 24 hours of receipt). Data processing and analysis can become a bottleneck. Method development, data analysis and pro-cessing can be simplifed with the use of comprehensive compound databases, pre-determined methodologies, and intuitive software. This webinar will provide pesticides residue analysts with valuable information on software method development and data processing for the analysis of pesti-cide residues in food for both LC–MS and GC–MS. Technical experts will review the latest in software advances to help with data interpretation and reporting. They will guide you through industry-relevant trends and new techniques that allow you to see more, do more, and be more productive. Example workfows will be shown, along with data analysis using the latest in compound databases and spectral libraries.

Who Should Attend:

n Researchers and analysts in pesticide analysis

n Food scientists interested in learning the latest technologies for sample preparation of food matrices

n Anyone struggling with sample preparation challenges for pesticide residue analysis in food

Series Moderator

Richard Fussell, Ph.D.Global Vertical Marketing Manager, Food and Beverage, Chromatography & Mass Spectrometry Division, Thermo Fisher Scientifc

Presenter:

Charles YangSenior Marketing Specialist, Environmental and Food Safety, Thermo Fisher Scientifc

LCGC Moderator:

Laura Bush: Editorial Director, LCGC

Key Learning Objectives:

n Learn how to quickly set up acquisition methods with TraceFinder software

n Proceed to rapidly analyze data with integrated software workfows

n Learn about the latest in comprehensive compound databases and libraries for triple-quadrupole and HRAM LC and GC mass spectrometry

For questions, contact Kristen Moore at [email protected]

Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series

Sponsored by

Presented by

Part 1: Sample Prep Tips and Tricks Using QuEChERS and Accelerated Solvent ExtractionON-DEMAND WEBCAST, originally aired March 24, 2015

Part 2: Workflow Guide for the use of LC-MSON-DEMAND WEBCAST, originally aired April 29, 2015

Part 3: Maximizing Analysis Efficiency through New GC-MS ApproachesON-DEMAND WEBCAST, originally aired June 17, 2015

Part 4: Latest Developments & Future Directions in Data Processing & Analysis Software for LC-MS/MS & GC-MS/MSWed., July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST

Register for the Pesticide

Residue Analysis Webinar Series

Register for the Pesticide

Residue Analysis Webinar Series

Latest Developments and Future Directions in Data Processing & Analysis Software for LC–MS-MS and GC–MS-MS



and measured at concentrations in the range

1–5 mg/mL.

THF is a common solvent for many GPC/

SEC applications that readily dissolves both

PLA and PLGA; however, the RI sensitivity (dn/

dc) for this sample/solvent combination is low

(approximately 0.045 mL/g to 0.051 mL/g).

This means that any change in polymer

concentration induces a relatively small change

in RI, requiring a very sensitive RI detector to

measure concentration changes under these

conditions. Likewise, dn/dc values also impact

the response of light-scattering detectors.

A compromise was therefore forced on

the analyst by this dn/dc issue. They would

either have to analyze the samples at a high

concentration, which overloads the SEC

columns and distorts the MW distribution

obtained, or have to switch to an alternative

solvent, normally acetone, which is far less

suitable for the proper dissolution of the

sample. However, the results gathered here

using a multi-detector GPC/SEC approach

demonstrate that it is suff ciently sensitive to

enable the use of the preferred THF solvent.

Results: Figure 1 shows typical

chromatograms for a PLA sample, with

traces from each detector in the array, and

derived values of MW and intrinsic viscosity

(IV) plotted as a function of retention time.

Excellent data quality is seen across all the

measurements made with each detector, as

3.162

Molecular Weight (Da)

PLGA 5050 low mid MW

PLGA 5050 low mid MW

PLGA 5050 high mid MW

PLGA 5050 high mid MW

PLGA 6535 mid MW

PLGA 6535 mid MW

PLGA 7525 high MW

PLGA 7525 high MW

Intr

insi

c vis

cosi

ty (

dL/

g)

0.3162

1.0569

0.1

104 3.164 105 3.165 106149.863

Figure 2: M-H plots for the four PLGA samples with different lactide:glycolide compositions show that changes in composition are associated with differences in the structural characteristics of the polymer.

Ball

18




evidenced by the clean stable baseline and the

smooth well-defi ned signal peaks. Similar data

quality is also observed in the measurement of

the PLGA samples (data not shown).

Using the MW and IV data generated by

these measurements it is possible to construct

a Mark—Houwink (M-H) plot, a logarithmic

plot of IV against MW, to investigate the

structural differences between samples. IV is

inversely proportional to the molecular density

in solution so both the gradient and intercept

of a M-H plot reveal information about the

structural characteristics of a polymer. Three

different types of PLGA sample were measured

to investigate the impact of composition on

structure. These included copolymers with

lactide:glycolide compositions of 50:50;

65:35; and 75:25, respectively. For the 50:50

copolymer, two samples were analyzed that

had different overall MW, a low-to-mid MW

sample, and a mid-to-high MW sample. This

gave four samples in total and for each one

duplicate injections were made on the GPC/

SEC system to check repeatability.

The results indicate that increasing the

amount of lactide in the polymer decreases

its coil density in solution. Samples with

a 75:25 lactide:glycolide ratio exhibit the

highest IV at any given MW while those

that have a 50:50 composition have a

much lower IV, at an equivalent MW. This

means that any change in the copolymer

composition will change the molecular

structure of the polymer. More practically,

the data provide insight as to why these

polymer samples will behave differently,

thereby supporting the manipulation of

polymer properties to control drug delivery

performance. It is also worth noting that

this easy differentiation of copolymer

content by the M-H plot is independent

of the molecular weight, as shown by the

consistent, overlapping plots of the two

50:50 samples with different molecular

weights. In other words, the exact excipient

MW distribution and structure (composition)

can be determined, or compared to a

reference, in a single analysis.

Table 1: Data from multiple injections of the same PLGA sample.

Parameter Average Value Unit RSD (%)

Mn 26,102 g/mol 2.32

Mw 44,722 g/mol 0.37

Mz 67,009 g/mol 1.22

(η)w 0.36 dL/g 1.37

Rh.w 6.07 nm 0.50

Ball

19


Subscribe to LCGC North AmericaÕs e-Newsletters

Unique electronic newsletters covering the most up-to-date industry topics delivered on a weekly basis

e-Separation Solutions

e-Separation Solutions is LCGCÕs weekly e-newsletter covering all of the hottest chromatography topics,

techniques, and applications. Each e-newsletter is dedicated to a different area of separation science.

e-Application Note Alert

LCGCÕs e-Application Note Alert is a monthly e-newsletter showcasing useful applications by technique.

Webcasts | Events | Chromatographyonline.com | Application Notes | The Peak | Subscribe | Contact Us

Follow us on:

Technology Forum

Bioanalysis: LC–MS-MS, Sample Prep, and Dried Blood Spot Analysis Bioanalysis uses a variety of separation techniques to analyze samples ranging from plasma and urine to dried blood spots. Participants in this Technology Forum are Ling Bei, Patrik Appelblad, and Dave Lentz of EMD Millipore; Nadine Boudreau of PharmaNet Canada; Diab Elmashni, Jeff Zonderman, and Simon Szwandt of Thermo Fisher Scientific; and Debadeep Bhattacharya of Waters Corporation. More...

Name your budget or application—the Agilent 1200 Infinity Series has you covered. From our affordable 1220 Infinity LC starting at just $15,000 to our cutting-edge 1290 Infinity LC, we have a solution that’s right for you. Plus, our most popular 1200 Infinity Series LC configurations are now available with a 3-5 year up-and-running guarantee. Read more.

Featured News

Health Sciences Unit LaunchedLGC has launched a new business unit, Health Sciences, which combines the group's sport, food, consumer safety and pharmaceutical testing activities within a single entity. More...

KNAUER is now offering its PLATINblue UHPLC systems with the MSQ Plus mass detector in a special package deal. PLATINblue systems and the MSQ Plus are an ideal combination for high-throughput applications. For a limited-time only, the UHPLC-MS package is being offered at a very special price. Don’t miss out!

Performance Materials Supplier AcquiredAmerican-based Avantor Performance Materials will acquire Polish Performance Materials Supplier (POCH S.A.). More...

Your brilliance. Our know-how. Collaborative Life Science. It all joins forces at EMD Millipore. Now your organization can leverage the combined synergies of two leading Life Science companies – for deep insight and know-how along every step of the biotherapeutic value chain. Find out how at www.emdmillipore.com

Featured Vidcast

Using LC–MS with Online Sample Preparation to Survey Metabolites Formed In VitrolAn Interview with Samuel Yang, University of Texas at Arlington. More...

Featured Products

EXP® Products for UHPLC

The EXP® Product Family offers hand-tight fittings, filters,

traps and guards – rated to 20,000+ psi. This advanced

GC HPLC Sample Prep GPC Hyphenated Miscellaneous

Subscribe Go to Application Note Library

Featured Application Note:Screening and Quantification of Multiple Drugs in Urine Using Automated Online Sample Preparation

and Tandem Mass Spectrometry Barbora Brazdova and Marta Kozak, Thermo Fisher Scientific

Learn about a 9-min, sensitive (LOQ 1—50 ng/mL) method to quantitate 30 immunosuppressant

drugs using TurboFlow technology and LC–MS-MS.

Evaluation of the Ultra Inert Liner Deactivation for Active Compounds Analysis by GCLimian Zhao, David Mao, and Allen Vickers, Agilent Technologies

Endrin and DDT breakdown and active semivolatiles tests were used for the Ultra Inert liner deactivation performance evaluation. The results indicate that the Ultra Inert deactivated liners provide superior inertness for analysis of active compounds.

Food Analysis of PAHs Using GCxGC-TOFMS and QuEChERSLECO CorporationThe combination of QuEChERS extraction and GCxGC-TOFMS is a fast and accurate method for

detecting and identifying PAH contaminants in complicated foodstuff matrices such as liquid infant formula and blended blueberries.

Screening and Identification with High Confidence Based on High Resolution and Accurate Mass LC–MS-MSAndre Schreiber and David Cox, AB SciexThis note describes a workflow and tools to identify targeted and nontargeted pesticides in fruits and vegetables. High resolution, accurate mass LC–MS-MS data is mined using advanced

software tools to identify components based on retention times, accurate mass, isotopic pattern, and MS-MS library searching.

Highly Sensitive UV Analysis with the Agilent 1290 Infinity LC System for Fast and Reliable Cleaning Validation – Part 1Edgar Naegele and Katja Kornetzky, Agilent TechnologiesThis application note demonstrates high sensitivity measurement of pharmaceutical compounds

with the Agilent 1290 Infinity LC. It also demonstrates a performance comparison of different flow cells with the Agilent 1290 Infinity LC diode array detector (DAD) for highly sensitive UV measurement including calibration, validation, and determination of LOD and LOQ.

Pesticides in Fatty MatricesDon Shelly, UCT"Fat is where it's at" when it comes to finding most pesticides. Extracting the pesticides and not

the lipids can be a challenge! This months featured application is easy, quick, effective, rugged, and inexpensive.

Which GPC Column First? Bruce Kempf, Tosoh BioscienceMost manufacturers recommend the installation of SEC columns in order of decreasing pore size when running columns in series. Scientists at Tosoh Bioscience tested the validity of this recommendation.

Ultra-Fast Analytical Method for the Sample Cleanup and LC–MS-MS Analysis of

Chloramphenicol in Shrimp and Other Marine Food ProductsPhilip J. Koerner, Matthew Trass, Liming Peng, and Jeff Layne, PhenomenexA method for the analysis of chloramphenicol in shrimp has been developed with a limit of quantitation (LOQ) of 0.001 ng/g in shrimp (0.001 ppb) based on the calibration standards. This is 300 times lower than the current U. S. Food and Drug Administration (USFDA) method. The method described uses Strata-X solid phase extraction (SPE) cartridges for sample cleanup and

concentration, followed by ultra-fast LC–MS-MS analysis (<5 min) using a Kinetex core-shell column.

Topics and categories include: HPLC • GC • Sample Prep • LC-MS and GC-MS • Emerging techniques

www.chromatographyonline.com/enews



can also allow formulators to gather the

information needed to fully scope excipient

performance, and to swiftly specify an

optimal excipient for any given drug product.

References

1. “Analytical techniques with a place in the oral

solid dosage formulation toolkit’ Whitepaper

available for download at: http://www.malvern.

com/en/support/resource-center/Whitepapers/

WP141223AnalTechOralToolkit.aspx

2. A. Siew, Pharmaceutical Technology Europe 39(1),

(2015).

3. M. Chaubal, Drug Delivery Technology 2(5), (2002).

Stephen Ball is Product Marketing Manager,

Nanoparticle and Molecular Characterization,

at Malvern Instruments. He holds a degree

in computer aided chemistry from the

University of Surrey, UK, which included

a year in industry working as a research

chemist for the Dow Chemical Company

in Horgen, Switzerland. Before joining

Malvern Instruments, he worked for Polymer

Laboratories as an applications chemist,

before taking on a marketing position as

a product manager for light scattering

instrumentation at Agilent Technologies.

In an extension of the study, multiple

injections of the same PLGA sample were

performed to directly assess reproducibility.

Table 1 shows data from 10 repeat injections,

each of 100 µL, for a PLGA sample

containing 50% lactic acid, measured at a

concentration of 2.132 mg/mL. A relative

standard deviation of 0.53% for the 10

adjacent samples demonstrates the excellent

repeatability obtained. Such repeatability with

automation substantially lightens workload

associated with the rigorous and extensive

experimentation needed to adopt QbD.

Conclusion

The performance of many sophisticated

pharmaceutical products relies on the use

of polymeric excipients. The MW, MW

distribution, and structural characteristics

of such excipients def ne their behaviour

and are therefore identif ed routinely as

CQAs for the product. GPC/SEC is an

established technique for the measurement

of these properties and as a result has an

important role to play in pharmaceutical

formulation. The technique when applied

with multiple detection systems can provide

high sensitivity, which enables the precise

differentiation of excipient grades and

greater f exibility in solvent choice, and

highly automated repeatable measurement,

for eff cient, high productivity analysis. It

E-mail: [email protected] Website: www.malvern.com

Ball

20



Recent Developments in Pharmaceutical Analysis (RDPA 2015)

E-mail: [email protected]

Website: rdpa2015.chimfarm.unipg.it

Ph

oto

Cre

dit

: R

aq

ue

l Lo

na

s/G

ett

y I

ma

ge

s

A symposium on the Recent

Developments in Pharmaceutical

Analysis (RDPA 2015) will be held at

the University of Perugia, Perugia,

Italy, from 28 June to the 1 July 2015.

The Scientific and Organizing

Committees invite you to RDPA 2015,

which will be held in Perugia, Italy,

a beautiful city with an outstanding

architectural heritage.

The programme will include plenary

and keynote lectures, as well as oral and

poster presentations on a wide range

of topics including: advanced methods

and instrumentation; hyphenated

techniques; fundamentals (theories,

retention models, chemometrics); (bio)

pharmaceutical analysis; food analysis,

nutraceuticals, functional food, natural

products; proteomics, glycomics,

metabolomics; biomarker discovery;

and sample preparation, validation,

quality by design, and data processing.

Participation of young researchers,

both from industry and university,

will be facilitated by low registration

fees.

Great opportunities to meet colleagues

in informal discussions will be made

easy by an attractive location, taking

advantage of a city that offers a

multitude of cultural, historical, and

artistic attractions, all at walking

distance from the symposium venue.

A look to the upcoming symposium on the Recent Developments in Pharmaceutical Analysis

(RDPA 2015), which will be held 28 June to 1 July 2015 in Perugia, Italy.

21



powered by

SPEMSGCHPLC IR

HPLC Troubleshooter

Step 1

Step 2

Step 3

Try our Interactive HPLC TroubleshooterGet answers fast, reduce downtime and improve ef ciency

Still got a problem?Ask our experts.

If you have a specif c enquiry, or just need more information, one of our technical experts

will contact you within 24 hours and will work with you until your problem has been resolved.

“Ask the Expert” is available only to Premier Members.

To f nd out more about CHROMacademy Premier membership contact:

Glen Murry on +1 732 - 346 - 3056 | e-mail: [email protected]

To discuss CHROMacademy Group or Corporate memberships contact:

Allen Basis on +1 732 - 346 - 3013 | e-mail: [email protected]

We developed the CHROMacademy Troubleshooters with busy

chromatographers in mind. In 3 simple steps we can help you

overcome your instrument, separation, and quantitation issues.

1. Select your chromatographic symptoms.

2. Select your instrument symptoms.

3. The troubleshooter returns a list of possible causes.

Each cause has a concise summary of the problem and

recommended solutions. These solutions are supported by

over 1000 references, feature articles, and CHROMacademy

content written by our experts.

The CHROMacademy troubleshooters are an on-line interactive

version of the much-loved troubleshooting poster you see on lab

walls all over the world.

Our troubleshooters are available to Lite and Premier members and

are used globally as a resource for analytical scientists who wish to

more accurately diagnose issues with equipment and separations.

See a list ofprobablecauses and therecommendedsolutions.

Select yourinstrumentsymptoms.

Select yourchromatographicsymptoms.

22



The Column www.chromatographyonline.com Training & Events

23


Training CoursesGCThe Technique of GC in 3 Parts, Fundamentals, Troubleshooting, and Method Development 10 June 2015

Radisson Blu Hotel,

Manchester Airport, UK

Website: www.hichrom.co.uk

GC–MS Interpretation12 August 2015

Hilton Glasgow Grosvenor,

Glasgow, UK

Website: http://www.crawfordscientific.

com/training-online-calendar.asp

Hands-on GC–MS Theory and Methods 26 August 2015

The Open University,

Milton Keynes, UK

Website: http://anthias.co.uk/training-

courses/handson-GC-MS-theory-methods

Gas Chromatography: Fundamentals, Troubleshooting, and Method Development8–11 September 2015

Axion Analytical Laboratories,

Chicago, Illinois, USA

Website: http://proed.acs.org/course-

catalog/courses/gas-chromatography-

fundamentals-troubleshooting-and-

method-development/

HPLC/LC–MSLC–MS for the Chromatographer28 July 2015

Thermo Scientific, Runcorn, UK

Website: http://www.crawfordscientific.

com/training-online-calendar.asp

How to Develop Validated HPLC Methods: Rational Design with Practical Statistics and Troubleshooting14–15 October 2015

MicroTek, Edison,

New Jersey, USA

Website: http://proed.acs.org/course-catalog/

courses/how-to-develop-validated-hplc-

methods-rational-design-with-practical-

statistics-and-troubleshooting/

The Theory of HPLCOn-line training from CHROMacademy

Website: http://www.chromacademy.com/

hplc-training.html

Fundamental LC–MSOn-line training from

CHROMacademy


mass-spec-training.html

HPLC TroubleshooterOn-line training from

CHROMacademy


hplc_troubleshooting.html

METHOD VALIDATIONValidation and Transfer of Methods for Pharmaceutical Analysis30 September–2 October 2015

Hilton Garden Inn, London Heathrow

Airport (formerly Jurys Inn Heathrow),

London, UK

Website: http://www.

mournetrainingservices.co.uk/course_list.

html#vampa

SAMPLE PREPARATIONSolid-Phase ExtractionOn-line training from

CHROMacademy


sample-prep-training.html

Hands-on Sample Preparation5–8 October 2015

The Open University,

Milton Keynes, UK

Website: http://anthias.co.uk/training-courses/

hands-on-sample-preparation

MISCELLANEOUSLight Scattering and Viscometry Hands-On Training29–30 June 2015Mainz, Germany

Website: www.pss-polymer.com

Light Scattering Training24–25 September 2015Santa Barbara, California,

USA

Website: http://www.wyatt.com/

training/training/light-scattering-

training.html

Please send your event and training course information to Kate Mosford [email protected]


The Column www.chromatographyonline.com Training & Events

24


21–25 June 2015 42nd International Symposium on High Performance Liquid Phase Separations

and Related Techniques (HPLC 2015)

International Conference Centre, Geneva, Switzerland

Tel: +41 22 839 84 84


Website: www.hplc2015-Geneva.org

23–28 August 201535th International Symposium on Halogenated Persistent Organic Pollutants

(Dioxin 2015)

Hotel Maksoud Plaza, Sao Paulo, Brazil

Tel: +55 11 3056 6000


Website: www.dioxin2015.org

3–6 November 2015Recent Advances in Food Analysis (RAFA)

Clarion Congress Hotel, Prague, Czech Republic

Tel: +386 1 4760 265

E-mail: [email protected].

Website: www.rafa2015.eu

4–6 November 201531st Montreux Symposium on LC–MS and MS–MS

Aldershof Convention & Exhibition Centre, Berlin-Aldershof, Germany


Website: www.lcms-montreux2015.de

Event News

* Five years free access to CHROMacademy only available to customers affliated with an academic or research

institution, conditions apply.

** A valid university e-mail address is required.

Five years FREE access for all university students & staff at www.chromacademy.com/agilent

BUILDING BETTER SCIENCE

facebook.com/Agilent.Tech

facebook.com/CHROMacademy

Agilent Products are for Research Use Only.

Not for use in diagnostic procedures.

Information, descriptions and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc. 2013

Published in USA, 5991-1198ENUC, 2013

ENJOY THE BENEFITS OF

CHROMACADEMYCOMPLIMENTS OF AGILENT TECHNOLOGIES

Stay connected with Agilent and

CHROMacademy via Twitter,

Facebook, LinkedIn and YouTube.

Join the conversation today!

twitter.com/AgilentChem

(@AgilentChem) or

twitter.com/AgilentLife

(@AgilentLife)

twitter.com/CHROMacademy

(@CHROMacademy)

linkedin.com/company/Agilent-

Technologies

YouTube.com/AgilentChem or

YouTube.com/AgilentLife

CHROMacademy is an intuitive, comprehensive e-learning and trouble-shooting

platform with more than 3,000 pages of content for HPLC, GC, sample preparation,

and hyphenated techniques. No other online resource offers separation scientists

more live streaming events, a knowledge base, practical solutions, and new

technologies in one easy to navigate website.

• Monthly webcasts increase your knowledge from the comfort and convenience

of your desk

• Multi-media and video tutorials enhance your skills at your own pace

• Interactive experiments, lab simulations, and tools improve your productivity

• Applications, news, and feature articles from world leaders in analytical science

• Troubleshooting modules based on real-world laboratory experience, help you

solve common problems

• Access to over 5,000 articles and application notes from LCGC

• ...and more.

In addition, CHROMacademy has an interactive discussion forum where visitors

can get technical and application assistance from other users or provide advice to

members who are looking for help.

All of this can be yours for free*, thanks to Agilent Technologies. Simply log on to

www.chromacademy.com/agilent, complete** and submit the form, and receive

your complimentary five year membership worth US $1,475.


Mission StatementThe Column (ISSN 2050-280X) is the analytical chemist’s companion within the dynamic world of chromatography. Interactive and accessible, it provides a broad understanding of technical applications and products while engaging, stimulating and challenging the global community with thought-provoking commentary that connects its members to each other and the industries they serve.Whilst every effort is made to ensure the accuracy of the information supplied, UBM Life Sciences accepts no responsibility for the opinions and statements expressed.Custom Reprints: Contact Brian Kolb at Wright’s Media, 2407 Timberloch Place, The Woodlands, TX 77380. Telephone: 877-652-5295 ext. 121. Email: [email protected].

©2015 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced in any material form (including photocopying or storing it in any medium by electronic means and whether or not transiently or incidentally to some other use of this publication) without the written permission of the copyright owner except in accordance with the provisions of the Copyright, Designs & Patents Act (UK) 1988 or under the terms of a licence issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP, UK.Applications for the copyright owner’s permission to reproduce any part of this publication should be forwarded in writing to Permissions Dept, Honeycomb West, Chester Business Park, Wrexham Road, Chester, CH4 9QH. Warning: The doing of an unauthorized act in relation to a copyright work may result in both a civil claim for damages and criminal prosecution.

Contact InformationGroup PublisherMichael J. [email protected]

Sales Manager Gareth [email protected]

Sales Executive Liz [email protected]

Sales Operations ExecutiveSarah [email protected]

Editor-in-ChiefAlasdair [email protected]

Managing EditorKate [email protected]

Associate EditorBethany [email protected]

UBM Life SciencesHoneycomb West,Chester Business Park,Wrexham Road,Chester, CH4 9QH, UKTel: +44 (0) 1244 629 300Fax: +44 (0) 1244 678 00

Group PublisherMichael J. [email protected]

Associate PublisherEdward [email protected]

East Coast Sales ManagerStephanie [email protected]

Account ExecutiveLizzy [email protected]

Editorial Director,Analytical SciencesLaura [email protected]

Group Technical Editor Stephen A. [email protected]

Managing EditorMegan L’[email protected]

Administation and Sales OfficesWoodbridge Corporate Plaza,485 Route 1 South,Building F, First floor, Iselin,NJ 08830, USATel: +1 732 596 0276Fax: +1 732 225 0211

Corporate Office, 641 Lexington Ave., 8th Floor, New York, NY 10022-4503, USA

UBM Life Sciences:

Chief Executive Officer Joe Loggia

Executive Vice-President, Life Sciences

Tom Ehardt

Executive Vice-President Georgiann DeCenzo

Executive Vice-President Chris DeMoulin

Executive Vice-President, Business Systems

Rebecca Evangelou

Executive Vice-President, Human Resources

Julie Molleston

Executive Vice-President, Strategy & Business

Development Mike Alic

Sr Vice-President Tracy Harris

Vice-President, General Manager Pharm/

Science Group Dave Esola

Vice-President, Legal Michael Bernstein

Vice-President, Media Operations

Francis Heid

Vice-President, Treasurer & Controller

Adele Hartwick

UBM Americas:

Chief Executive Officer Sally Shankland

Chief Operating Officer Brian Field

Chief Financial Officer Margaret Kohler

UBM plc:

Chief Executive Officer Tim Cobbold

Group Operations Director Andrew Crow

Chief Financial Officer Robert Gray

Chairman Dame Helen Alexander

Eu

rop

eN

ort

h A

meri

ca

25



2 advances in microsampling for in vivofiles.alfresco.mjh.group/.../thecolumn_june052015nasm.pdf ·...

Documents