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THE APPLICATION NOTEBOOK Ð SEPTEMBER 2015 3

ES667470_lcgcAN0915_003.pgs 09.01.2015 23:33 ADV blackyellowmagentacyan

THE APPLICATION

NOTEBOOK

Medical/Biological

7 Versatility of the Multi-Mode Scherzo SS-C18 in

the Retention of Polar Neurotransmitters

Robert Puryear, Piotr Macech, Eric Wendt, and Itaru Yazawa, Imtakt USA

8 The Application of QuEChERS in the Extraction

of Anabolic Steroids in Whole Blood

Xiaoyan Wang, UCT, LLC

Chiral

9 Detection of Chiral Drugs Using HPLC with CD Detection

JASCO

10 Characteristics and Application of a

Chiral Column, Chiral CD-Ph

Xiaomi Xin and Taketoshi Kanda,

Frontier Science Business Division, Shiseido Co. Ltd.

Environmental

12 Pressurized Liquid Extraction of Soil Samples for

the Analysis of Diesel Range Organics by GC–FID

FMS

13 Hexavalent Chromium Determination Hamilton

PRP-X100 Anion Exchange HPLC Column

Derek Jensen and Mark Carrier, Hamilton Company

14 An Automated Extraction Solution

for Imidazolinone Herbicides

Tyler Trent, Teledyne Tekmar

4 THE APPLICATION NOTEBOOK – SEPTEMBER 2015

TABLE OF CONTENTS


Food and Beverage

15 Aflatoxin B1, B2, G1, G2 Food Safety Testing Prior to

Automated SmartPrep® Extractor Solid-Phase Extraction

Toni Hofhine*, Elizabeth Krantz†, Cheri A. Barta†, Pamela Doolittle†,

Robert Buco‡, Richard Koeritz‡, Zachary Lilla‡, Jennifer Claus§, Kenneth

Espenschied§, and Michael Ye§, *HorizonTechnology, †University of

Wisconsin, ‡Shimadzu Scientific Instruments, and §Sigma-Aldrich

16 Analysis of L-Theanine in Tea by HPLC

with Post-Column Derivatization

Maria Ofitserova, PhD, and Sareeta Nerkar, PhD, Pickering Laboratories

17 A New HILIC Column for Saccharide Analysis

Melissa Turcotte* and Naoya Nakajima†,

*Showa Denko America, Inc., and †Showa Denko K.K.

Pharmaceutical/Drug Discovery

18 Authentication of Traditional Chinese

Prescriptions Using Comprehensive 2D-LC

Sonja Krieger, Agilent Technologies Inc.

19 Ex vivo Stabilization of Small Molecule

Compounds and Peptides in EDTA Plasma for

LC–MS-MS Analysis Using Frozen Aliquotting

Glenn Smith and Joseph Fraone, CryoXtract

20 Accurate Pain Management Analysis in Under

5 Min on Raptor™ Biphenyl Superficially

Porous Particle LC Columns

Sharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation

22 Fast Analysis of IgG Charge Heterogeneity

by Ion Exchange Chromatography

Tosoh Bioscience LLC

24 Separation of Macrocyclic Lactones

Diamond Analytics

25 Faster Analysis and Higher Efficiency with

Thermally Stable HPLC Columns

Dwight Stoll and Peter W. Carr, ZirChrom Separations, Inc.

THE APPLICATION NOTEBOOK – SEPTEMBER 2015 5

TABLE OF CONTENTS


Polymer

26 Light Scattering for the Masses®:

Quality Control of Epoxy Resins

Wyatt Technology Corporation

General

27 Nanometer Internal Diameter Fused Silica Capillary Tubing

Joe Macomber and Corey Hoshiwara, Molex

Articles

28 A Life with the Boys

Mary Ellen P. McNally, PhD

32 Why and How to Avoid Ionic Contamination

in Water Used for LC–MS Analyses

Anastasia Khvataeva-Domanov and Stéphane Mabic

37 Using Compact Mass Spectrometry for Detection

and Quantification of Cannabis-Related Compounds

Daniel Eikel, Simon J. Prosser, and Jack D. Henion

Departments

43 Call for Application Notes

Cover Photography: Getty Images


TABLE OF CONTENTS



MEDICAL/BIOLOGICAL

Polar neurotransmitters are diff cult to retain using ODS

columns. In this work we show two different methods to retain

these polar analytes on the multi-mode Scherzo SS-C18 column.

The versatility in retention characteristics with simple changes

in mobile phase is highlighted.

Found in the highly aqueous extracellular space of the synaptic terminals

of neurons, neurotransmitters are themselves polar and suited for this

environment. This presents a signif cant challenge for chromatographic

separation methods because polar analytes are diff cult to retain using

conventional hydrophobic ODS stationary phases. Use of the multi-

mode column Scherzo SS-C18, however, improves retention by the

addition of high density strong anion (SAX) and strong cation exchange

(SCX). The presence of three different types of stationary phase provides

a wide range of retention mechanisms and mobile phase options. Here

we show two different methods and their impact on retention of some

common neurotransmitters.

Imtakt USA1104 NW Overton St., Portland, OR, 97209

tel. (888) 456-HPLC, (215) 665-8902, fax (501) 646-3497

Website: www.imtaktusa.com

Versatility of the Multi-Mode Scherzo SS-C18 in the Retention of Polar NeurotransmittersRobert Puryear, Piotr Macech, Eric Wendt, and Itaru Yazawa, Imtakt USA

Figure 1: Scherzo SS-C18 100 mm × 3.0 mm.

Figure 2: Scherzo SS-C18 150 mm × 3.0 mm. See Figure 1 for analyte list.

Results and Discussion

Chromatograms of neurotransmitters separated using Scherzo SS-C18

under two different sets of conditions are shown in Figure 1 and Figure 2.

Both methods shown are a combination of reversed phase and ion

exchange, where organic solvent and high salt solutions are used as

eluents. The key differences in these methods are that Figure 1 utilizes

higher salt concentration, while Figure 2 utilizes stronger organic sol-

vent. In Figure 1, by using a higher salt concentration in the B solution,

analytes with a signif cant electrostatic interaction, are less retained. The

exceptions are Glu, L-DOPA, and GABA, which show greater retention.

These compounds are more strongly retained under weaker organic sol-

vent (acetonitrile vs. methanol), as in Figure 2, pointing to their sensitivity

to hydrophobic retention mechanisms. The elution order changes for L-

glutamic acid, GABA, 5-HIAA, acetylcholine chloride, L-tryptophan, and

serotonin from Figure 1 to Figure 2 clearly indicate that while the electro-

static interactions are present, hydrophobic interactions dominate.

Conclusion

The results presented here show successful retention and separation

of small, polar, and biologically important molecules. The f exibility of

the mixed mode column used in this study allows precise tailoring of

separation conditions for analytes and/or instrumentation. Comparison

of these two methods show that minor adjustments in the organic or the

ion exchange component

of the eluent can result in

dramatic changes in elu-

tion order when using a

multi-mode column like

Scherzo SS-C18.

Experimental Conditions

Noted in Figures 1 and 2.



MEDICAL/BIOLOGICAL

This application utilizes the original, non-buffered QuEChERS (acro-

nym for quick, easy, cheap, effective, rugged, and safe) technique

to quantify anabolic steroids in human whole blood. Previous ex-

traction techniques typically involve a protein precipitation step fol-

lowed by a liquid-liquid extraction (LLE) or solid phase extraction

(SPE). Target analytes are extracted from human whole blood by

combining 1 mL of sample with acetonitrile (MECN), magnesium

sulfate (MgSO4), and sodium chloride (NaCl) to enhance phase

separation and partioning of the anabolic steroids into the organic

layer. Following adequate shaking and centrifugation, further dis-

persive SPE is conducted on the sample to absorb residual water

and remove matrix co-extractives. This ultimately results in a clean,

f nal extract for LC–MS-MS analysis.

QuEChERS Procedure

1) Add 2 mL of MECN to 15-mL centrifuge tube containing 400

MgSO4 and 100 mg NaCl (ECQUUS1015CT).

2) Add internal standards (IS), and appropriate amounts of steroids

spiking solution to fortif ed samples.

3) Add 1 mL of negative whole blood into the 15-mL tubes.

4) Cap and shake for 1 min at 1000 strokes per min using a Spex

2010 Geno-Grinder.

5) Centrifuge at 3000 g for 5 min.

dSPE Cleanup

6) Transfer 1 mL of the supernatant to a 2-mL dSPE tube contain-

ing 150 mg MgSO4 and 50 mg of PSA (CUMPS2CT).

7) Shake 1 min at 1000 strokes/min using the Spex 2010 Geno-

Grinder.

8) Centrifuge at 3000 g for 5 min.

9) Transfer 0.4 mL of the cleaned extract into a 2-mL auto-sampler

vial; add 0.4 mL of reagent water, and vortex for 30 s.

10) The samples are ready for LC–MS-MS analysis.

The Application of QuEChERS in the Extraction of Anabolic Steroids in Whole Blood Xiaoyan Wang, UCT, LLC

UCT, LLC 2731 Bartram Road, Bristol, PA 19007

tel: (800) 385-3153; Email: [email protected]

Website: www.unitedchem.com

Extraction/Analytical Materials

ECQUUS1015CTEnviro-Clean® 15 mL centrifuge tube with 400

mg MgSO4 and 100 mg NaCl

CUMPS2CTEnviro-Clean® 2 mL dSPE tube with 150 mg

MgSO4 and 50 mg PSA

SLC-18100ID21-3UMSelectra® C18 HPLC column, 100 × 2.1 mm,

3 μm

Instrumental

LC–MS-MS: Agilent 1200 Binary Pump SL with AB Sciex API 4000

QTrap MS/MS

Column: UCT Selectra® C18 LC column, 100 × 2.1 mm, 3 μm

Guard Column: UCT Selectra® C18 guard column, 10 × 2.1 mm, 3 μm

Injection Vol.: 10 μL

Mobile Phase A: 0.1% formic acid in water

Mobile Phase B: 0.1% formic acid in methanol

Column Flow rate: 0.30 mL/min

Results

Conclusion

Excellent recoveries ranging from 81.4% to 101.6% were obtained

using UCT’s QuEChERS extraction kit (ECQUUS1015CT) and

dispersive clean-up products (CUMPS2CT) in conjunction with

the Selectra® C18 HPLC column (SLC-18100ID21-3UM) for the

extraction and analysis of anabolic steroids in human whole blood.

Table I: Recovery and RSD% of spiked serum

Compound

Spiked at

10 ng/mL

Spiked at

50 ng/mL

Spiked at

200 ng/mL

Recovery

%

RSD%

(n=6)

Recovery

%

RSD%

(n=6)

Recovery

%

RSD%

(n=6)

17-Hydroxypro-

gesterone89.6 6.6 99.2 5.7 99.3 3.2

Androstanedione 93.5 9.2 95.7 3.3 94.3 1.5

Boldenone 91.2 8.2 101.6 2.9 99.4 1.4

Methandienone 94.7 6.5 97.2 3.3 96.1 3.0

Methenolone 98.2 4.5 96.0 4.7 95.3 3.9

Norethandrolone 94.0 6.7 98.5 5.1 99.8 4.0

Nandrolone 96.4 9.6 92.3 1.1 89.8 1.6

Progesterone 101.6 5.0 95.5 1.3 94.8 4.0

Stanozolol 85.1 5.9 92.1 3.4 91.3 2.2

Testosterone 92.4 6.3 95.0 3.4 95.1 2.4

Trenbolone 81.4 9.0 93.2 6.9 95.0 3.0

Epitestosterone 89.8 5.4 97.6 4.4 99.3 2.8



CHIRAL

The need for chiral detection in the development of pharmaceuticals is

increasing due to regulatory changes and the demand for single-isomer

pharmaceutical drugs. These shifts require a means for chiral detec-

tion and compound separation because there is little control over which

chiral form of a chemical compound is formed during the drug’s pro-

duction. At the molecular level chiral partners have identical physical

properties and therefore are very diff cult and expensive to separate.

Both Prilosec (omeprazole) and Nexium (esomeprazole) are

proton pump inhibitors used to treat ulcers, heartburn, acid

ref ux, and Zollinger-Ellison syndrome. Nexium’s active ingredient,

esomeprazole, is the S-enantiomer of omeprazole, the active

ingredient in Prilosec. Nexium was one of the f rst pharmaceuticals

to be repatented and marketed as a pure enantiomer of a formerly

mixed enantiomer drug. Nexium has a superior clinical eff cacy

due to its higher and more consistent bioavailability. We aimed to

test the enantiomeric purity of both drugs using an HPLC system

with circular dichroism (CD), f uorescence (FP), and optical rotation

(OR) detectors.


Prescription Nexium and Prilosec were examined by grinding the

tablets and dissolving them in ethanol, and sonicating for 20 min.

These solutions were brought to a f nal concentration of 1 μg/μL with

an injection size of 2 μL. Separations were accomplished using a

ChiralPak AD column (4.6 × 250 mm) with a 100% EtOH mobile

phase and a f ow rate of 0.9 mL/min. A JASCO HPLC system with

CD, OR and FP detectors was used to perform the analysis.


The Nexium and Prilosec chromatograms are shown in Figures

1 and 2. As expected, the Prilosec chromatograms showed the

inclusion of both enantiomers while in the Nexium results only the

esomeprazole enantiomer was detected.

The results are clearest in the CD chromatograms. While the

FP detector was as sensitive as the CD detector with respect to

concentration, it was not able to clearly identify which enantiomer

was which. On the other hand, while the OR detector was capable

of discriminating the enantiomers, it was far less sensitive than the

CD detector with respect to concentration.

Conclusion

An HPLC system outf tted with a CD detector is able to effectively

determine the enantiomeric purity of certain chiral drugs more

effectively than similar systems with either FP or OR detectors.

JASCO currently offers the world’s only CD detector for HPLC.

Detection of Chiral Drugs Using HPLC with CD Detection JASCO

JASCO, Inc.28600 Mary’s Court, Easton, MD 21601

tel. (800) 333-5272, fax (410) 822-7526

Website: www.jascoinc.com/applications

Figure 2: Nexium chromatogramsTop: CD detector / Middle: FP detector / Bottom: OR detector

Figure 1: Prilosec chromatogramsTop: CD detector / Middle: FP detector / Bottom: OR detector.



CHIRAL

“Chiral CD-Ph” is silica-based chiral packing material modif ed

by phenyl-carbamated β-cyclodextrin (CD) as the enantio-

mer selector. Chiral CD-Ph is useful for the chiral separation

of the racemic substance with benzene ring in its structure.

It has superior capacity of chiral recognition derived from

balanced hydrophobicity and polarity from the phenyl car-

bamate and the inclusion function from the central cavity

in the structure of cyclodextrin. In addition, Chiral CD-Ph is

applicable in both of the normal-phase and reversed-phase

mode; hence enable to provide separation conditions best

matching the solubility of different samples. Chiral CD-Ph

column is easy-to-use with excellent durability.

In recent years, in the course of pharmaceuticals purif ed, as the

need to chiral purif cation decreased since the improving of the

asymmetric synthetic techniques, the desire to make medicine ef-

fective yet with a smaller number of doses and promised safety has

raised. In response, complicated drug design has become more nec-

essary. Against this background, chiral separation has become an

essential technique for the development of high value-added agents.

Shiseido succeeded in the development of packing material Chi-

ral CD-Ph from a precisely classif ed high-purity spherical silica with

phenyl-carbamated β-cyclodextrin chemically bonded as a chiral

selector. The Chiral CD-Ph column is designated as one of USP

parking L45. In this report, the basic characteristic of Chiral CD-Ph

is reported with the applications in the separation of enantiomers

and the durability evaluation.

Experiment and Results

1. Synthesis of Chiral CD-Ph

The packing material Chiral CD-Ph was synthesized by the following

steps.

• First, synthesizie a saline coupling agent of carbamated

β-cyclodextrin.

• Bond to the precisely classiå ed high-purity spherical silica.

• Make the carbamated β- cyclodextrin phenylated.

The synthesized chiral β-cyclodextrin is shown in Figure 1. The

phenyl-carbamated β-cyclodextrin (CD) is bonded to base silica

as the chiral selector via a spacer. The balance between the hy-

drophobicity and polarity of phenylcarbamate assures sample

retention, and the central cavity of β-cyclodextrin presents excel-

lent chiral recognition with inclusion.

2. Applications in chiral separations

1) Chiral separation under-phase normal-phase mode.

Neutral compound hexobarbital was separated under C2H

5OH

/ n-hexane = 40 / 60. The chromatogram is shown in Figure. 2.

Good separation and peak shape are obtained.

2) Application in reverse-phase separation

The neutral compound of carvone was separated under mo-

bile phase of CH3OH / H

2O = 70 / 30, whereas, the basic com-

Characteristics and Application of a Chiral Column, Chiral CD-PhXiaomi Xin and Taketoshi Kanda, Frontier Science Business Division, Shiseido Co. Ltd.

Figure 1: Structure of Chiral CD-Ph.

Figure 2: Chiral separation of Hexobarbital.

Figure 3: Chiral separation of Carvone.



CHIRAL

Shiseido Co., Ltd.

4F Shiodome Bl., 1-6-2, Higashi-shinbashi, Minato-ku, Tokyo, Japan

tel. +81-3-6253-1412, email: [email protected]

Website: http://hplc.shiseido.co.jp/e/

pounds of norfenefrine and atropine were separated under 0.5

mol/L NaClO4 / CH

3CN = 70 / 30, 50 mmol/L KH

2PO4 / CH

3OH

= 20 / 80 respectively. The compounds were certainly separat-

ed under water/acetonitrile system, or water/methanol system.

With all compounds, good separation with good peak shape are

reached (Figures 3, 4, 5). In addition, Miura and colleagues (1)

reported that Chiral CD-Ph was applied in the separation of a

second-generation antihistamine fexofenadine. The chiral sepa-

ration of fexofenadine is carried out by using a plasma sample

of 400 μL after treated by solid-phase extraction, which suggest

that Chiral CD-Ph is also useful in clinical samples.

3) Durability

In the durability test under reversed phase mode, propranolol

was used as a sample to perform 1-month continuous running

under the mobile phase of 50 mmol/L KH2PO

4 / CH

3CN = 70/30,

the column pressure and peak shapes were evaluated as indi-

cators of durability. There was no change observed in column

Figure 4: Chiral separation of Atropine.Figure 6: Durability test of Chiral CD-Ph in reversed-phase condition.

Figure 5: Chiral separation of Norfenefrine.

pressures and peak shapes between the test start and the end

after 1-month of the test, which suggests that Chiral CD-Ph is a

durable column (Figure 6).

Conclusion

Chiral CD-Ph is packing material for chiral separation created from

a precisely classif ed high-purity spherical silica with phenyl-carba-

mated β-cyclodextrin chemically bonded as a chiral selector. It is

capable of applying under normal phase as well as reversed-phase

mode, and excels at the chiral separation of basic and neutral com-

pounds. Chiral CD-Ph column is recommended as an easy solution

for chiral separation with the excellent selectivity as well as durability.

Reference

(1) M. Miura, T. Uno, T. Tateishi, and T. Suzuki, J. Pharm. Biomed. Ana. 43,

741–745 (2007).



ENVIRONMENTAL

Pressurized Liquid Extraction of Soil Samples for the Analysis of Diesel Range Organics by GC–FID FMS

Diesel range organics or DRO is the cumulative analysis of extractable

alkanes between the ranges C10 through C28 as measured on a f ame

ionization detector. Within this range, specif c fuel oil identif cations are

possible through pattern or “f nger print” matching. Not regulated un-

der RCRA, various individual US state methods exist, many expanding

analysis to the oil range organics (ORO) or C25–C35.

The following application note focuses on the DRO constituents

which due to their increased volatility, can pose greater analyti-

cal diff culties than the OROs. The approach of pressurize liquid

extraction paired with a gentle evaporation at low temperatures was

chosen for optimal recoveries.

Equipment

• FMS, Inc. PLE system

• FMS, Inc. SuperVap® Concentrator

• FMS, Inc. concentrator tubes

• Agilent 7890 GC with FID detector

Consumables

• Fisher Optima* Methylene Chloride

• Agilent Hydromatrix®

• Restek Ottawa Sand

• Aliphatic HC standard (1000 µg/mL)

• O-Terphenyl Standard (2000 µg/mL)

• Diesel Reference Material

Sample Prep

1. 9 × 10 g samples were weighed out of both Ottawa sand

and soil matrices

2. All samples spiked with O-terphenyl surrogate solution.

3. 5 replicates of each matrix were spiked with Aliphatic HC solution.

4. 3 replicates of each matrix were spiked with diesel reference material.

5. Samples mixed with Hydromatrix® and transferred to 40 mL

extraction cells.

PLE Procedure

Cells loaded onto PLE extraction system and DRO program initiated.

Solvent: Methylene Chloride

Extraction Temp.: 150 °C

Extraction Time 15 min

Flush volume: 2 × cell volume

N2 Purge: 1 min

FMS, Inc.580 Pleasant Street, Watertown, MA 02472

tel. (617) 393-2396, fax (617) 393-0194

Website: www.fms-inc.com

Figure 1: FMS Inc. PLE w/SuperVap®.

Mean Recoveries

Analyte Sand Soil

Total DRO HC 82.2% 88.4%

OTP 88% 92%

Diesel Ref. 82.3% 87.9%

Results run in triplicate by both GC/Ion Trap and GC/FPD

SuperVap

1. Preheat temp: 10 min at 35 °C

2. Evap. mode w/sensor temp: 35 °C

3. Nitrogen Pressure: 5 psi

4. Samples reduced to 1 mL

5. Samples reduced to 1 mL å nal volume

Results

Conclusions

Analysis of aliphatic hydrocarbon spikes showed good DRO range re-

coveries for both sand and soil matrices, with all individual alkanes in

the C10–C28 range recovering within 70–130%. Blank analysis not

only yielded good recovery for OTP, but no detectable DRO range was

measured. Lastly the analysis of a diesel reference material resulted in

calculated concentrations very close to the known reference amount

(within 20% for both matrices).



ENVIRONMENTAL

Chromium (Cr) is a metal with an interesting relationship to

the environment. Whereas trivalent chromium (Cr(III)) is an

essential nutrient, hexavalent chromium (Cr(VI)) is a poison

to humans and aquatic life and poses serious environmental

and ecological threats. Recent studies of various ground and

drinking water sources have detected toxic levels of Cr(VI).

This dangerous trend has gained the attention of national

and worldwide health organizations, such as the United

States Environmental Protection Agency (EPA) and the US

Food and Drug Administration (FDA), who seek to under-

stand how widespread the problem is. The California De-

partment of Public Health included Cr(VI) as an unregulated

chemical requiring monitoring in 2001. Based on recent data,

3107 of 6565 public wells in Los Angeles, San Bernardino,

and Fresno counties had Cr(VI) concentrations above 1 µg/L.

A Public Health Goal of 0.02 µg/L was published in July 2011.

Analysis of chromium species is made challenging due to the na-

ture of the element and diverse sample matrices. Because chro-

mium exists in two oxidation states, it is important to differentiate

between the nutrient, Cr(III), and the poison, Cr(VI) in samples. An

HPLC–ICP-MS method using the Hamilton PRP-X100 has been

developed in order to determine relative abundance of Cr(III) and

Cr(VI) in diverse sample matrices.

Trivalent chromium (Cr(III)) is stabilized as a chelation complex by

incubating the sample at 70 °C in 0.2 mM EDTA. The Cr(III)-EDTA

complex is suitable for binding to an anion exchange resin. Resolu-

tion of the two species then becomes a straightforward isocratic

separation. Although UV and conductivity are suitable for detecting

chromium species, inductively coupled plasma-mass spectrometry

(ICP-MS) is the method of choice for trace analysis.

Hexavalent Chromium DeterminationHamilton PRP-X100 Anion Exchange HPLC ColumnDerek Jensen and Mark Carrier, Hamilton Company

Hamilton Company4970 Energy Way, Reno, NV 89502

tel. (775) 858-3000, (800) 648-5950

Website: www.hamiltoncompany.com

1

2

2 4 6Time (min)

1) Cr(VI) and 2) Cr(III) EDTA separation on Hamilton PRP-X100

Experimental ConditionsColumn: Hamilton PRP-X100, 5µm,4.6 x 250 mmPart Number: 79181Flow Rate: 1.0 mL/minMobile phase: 2mM (NH

4)

2CO

3 for 0–3 min

40mM (NH4)

2 CO

3 for 3–14 min

2 mM (NH4)

2 CO

3 for 13–17 min

Injection volume: 50 µL, 100µg/Lof each standard

Detection: ICP-MS

8 10



ENVIRONMENTAL

Imidazolinone herbicides effectively control a wide-range of weed

species. This group of herbicides is comprised of f ve main chemical

compounds: imazamox, imazapic, imazapyr, imazaquin, and ima-

zethapyr (1). Each compound has an identical imidazolinone ring

structure with an attached carboxylic acid group. They only differ by

the functional group attached to the Imidazolinone ring structure.

This project will evaluate the performance and versatility of the

AutoMate-Q40 for the extraction of Imidazolinone herbicides. Liquid

chromatography coupled to a triple-quadrupole mass spectrometer

(LC–MS-MS) was employed for the detection of these herbicides

in agricultural commodities. Quantiå cation was based on matrix-

matched calibration curves with the use of internal standard to en-

sure method accuracy. By using the AutoMate-Q40 to streamline

this extraction, it provides us with appropriate analytical results fall-

ing in the established method guidelines (recovery range of 70–

120% and an RSD <20%) for the target compounds.

Sample Preparation and Results

Figure 1 shows the sample preparation and extraction steps that

are needed to extract imidazolinone herbicides from the carrot body

and the carrot leafy stem.

Automating the imidazolinone herbicide extraction enables fast,

easy, reliable, and highly reproducible extractions. The AutoMate-

Q40 offers labor saving, and improves the repeatability and consis-

tency between the extracted samples.

A precision and accuracy study was performed using the AutoMate-

Q40. The system was able to fortify the carrots and the leafy stems,

samples at 12.5 ng/g and 25.0 ng/g. This is accomplished through the

systems automated standard addition feature. All control samples used

for this study showed no signiå cant residue of Imidazolinone herbicides.

Table I shows that when using the AutoMate-Q40 to extract imid-

azolinone herbicide residues from carrots recoveries ranging from

86.5% to 105.0%. Table I also shows that the results have excellent

precision ranging from 2.4% to 10.4%.

Conclusion

Automation of this imidazolinone herbicide extraction method pro-

duced reliable results for the spiked samples. Automating this ex-

traction shows the versatility of the AutoMate-Q40 and how it can

be adapted to other extractions. The AutoMate-Q40 led to improved

repeatability, a reduction in the likelihood of human error and the

potential for signif cant labor savings.

Precision and accuracy were assessed for the carrots analyzed.

Results for the automated procedure were well within the criteria set

forth in this study: Average recoveries for the range of commodities

were between 86.5% and 105.0% with good precision (ca. 6% RSD).

References

(1) R. Krieger, F.G. Hess, J.E. Harris, K. Pendino, and K. Ponnock, “Handbook

of Pesticide Toxicology,” Imidazolinones 1, 1641–1642, (2001).

(2) C. Lamberth, Bioactive Heterocyclic Compound Classes: Agrochemicals, 47–49

(2012).

(3) U. Schirmer, Modern Crop Protection Compounds: Herbicides 1, 88–91, (2012).

Acknowledgments

A special thanks to Rick Jordan and Paciå c Agricultural Laboratory for their time and

support. Without their help and knowledge, this project wouldn’t have been possible.

Teledyne Tekmar4736 Socialville Foster Rd., Mason, OH 45040

tel. (513) 229-7000

Website: www.teledynetekmar.com

An Automated Extraction

Solution for Imidazolinone

Herbicides Tyler Trent, Teledyne Tekmar

Imidazolinone Herbicide Extraction

Weigh 4 grams homogenized samples into 50.0 mL extraction tubePlace extraction tube in the AutoMate-Q40

Add an amount of DI water to each sample to equal a total moisture of8.0 mL*

Add 2.0 mL of 0.5M buffered extraction solventAdd 10.0 mL of MeOH

Shake the sample for 2.0 minutesCentrifuge sample for 2.0 minutes

Transfer 5.0 mL to a 15.0 mL dSPE cleanup tube#

Shake the dSPE cleanup tube for 1.0 minutesCentrifuge dSPE cleanup tube for 1.0 minutes

Take an aliquot dilute 10X for analysis by LC-MS/MS

*e.g for a 4.0 g sample with 80% moisture, 4.0 * 0.8 = 3.2 g H2O.Therefore, the amount of water added to the sample will be 8.0 mL - 3.2 mL = 4.8 mL of water to sample.

#dSPE cleanup will be chosen base on sample type, either1.) 900 mg of MgSO4, 150 mg PSA and 45 mg of GCB

2.) 900 mg of MgSO4 and 150 mg PSA

Figure 1: Imidazolinone herbicide extraction.

Table I: Imidazolinone herbicides carrot results

Compound

Body of carrot* Leafy stem of carrot†

12.5 ng/g spike 25.0 ng/g spike 12.5 ng/g spike 25.0 ng/g spike

%

Recovery

%

RSD

%

Recovery

%

RSD

%

Recovery

%

RSD

%

Recovery

%

RSD

Imazapic 105.0 4.8 88.7 7.0 98.6 10.4 93.0 2.4

Imazapyr 86.5 4.8 87.1 9.0 100.0 9.1 89.7 3.9

Imazaquin 105.2 5.9 100.5 2.7 101.0 8.0 89.7 7.6

Imazethapyr 94.7 8.6 89.2 9.2 97.5 6.2 92.7 4.5

Imazamox 101.4 7.2 99.6 7.4 98.8 5.7 92.8 2.7

*Cleanup contained MgSO4 and PSA; †cleanup contained MgSO4, PSA, and GCB



FOOD & BEVERAGE

Mycotoxin testing awareness has increased as countries involved in

world trade of raw agriculture and processed consumer products rely

on a safe global food supply. Several mycotoxins are naturally produced

and monitored; however, af atoxins, produced mainly by the Aspergillus

species, are considered to be the most important mycotoxin group in

the world’s food supply to monitor. The frequency of occurrence and

negative health impacts to both animals and humans has spiked

interest in the af atoxins B1, B2, G1, and G2 levels present within

foods commonly exported to determine if these levels meet the current

acceptable regulatory limits specif ed by each country.

Preparation of samples for af atoxins B1, B2, G1, and G2

testing generally requires liquid-liquid extraction (LLE) as a sample

preparation step and solid phase extraction (SPE) as a sample cleanup

step to f rst remove as much as possible, any sample matrix effects,

such as color, protein, fat, etc. Proper cleanup of samples allows

af atoxins B1, B2, G1, and G2 to be easily detected and quantif ed

by HPLC analysis. An alternative methodology to the more common

immunoaff nity cartridge for sample cleanup, is to bind the interfering

compounds and immediately elute the af atoxins from a non-gel,

standard solid phase extraction (SPE) cartridge. The effectiveness

of the Supel™ Tox Af aZea SPE Cartridges in combination with the

SmartPrep Extractor System from Horizon Technology eff ciently

prepared a variety of foodstuffs samples representing a quadrant

of moisture and viscosity levels prior to analysis using HPLC with

f uorescence detection. Sample matrices chosen to represent the

quandrant were almond milk, fresh corn, peanut paste, and curry

powder. Data presented concludes that af atoxins B1, B2, G1, and G2

are effectively recovered using the novel and simple sample matrix

preparation roadmap to meet both United States and European

Union limits. Table I includes the results for spikes of af atoxin B1 and

a mixed spike to assess performance using the US and EU criteria.

Almond milk and curry powder were spiked at (8 µg/kg B1, 20 µg/

kg total US; 4 µg/kg B1, 10 µg/kg EU).

Fresh corn and peanut paste were spiked at (8 µg/kg B1, 20 µg/kg

total US; 1.6 µg/kg B1, 4 µg/kg EU).

Reference

(1) “A Unique and Simple Matrix Preparation Roadmap for Aflatoxin B1, B2, G1,

G2 Food Safety Testing Prior to Automated SmartPrep® Extractor Solid Phase

Extraction,” Application Note AN0981410_01, www.horizontechinc.com, (2014).

Horizon Technology, Inc.16 Northwestern Drive, Salem, NH 03079

tel. (603) 893-3663

Website: www.horizontechinc.com

Af atoxin B1, B2, G1, G2 Food Safety Testing Prior to Automated SmartPrep® Extractor Solid-Phase ExtractionToni Hofhine*, Elizabeth Krantz†, Cheri A. Barta†, Pamela Doolittle†, Robert Buco‡, Richard Koeritz‡, Zachary Lilla‡, Jennifer Claus§, Kenneth Espenschied§, and Michael Ye§,

*Horizon Technology, †University of Wisconsin, ‡Shimadzu Scientif c Instruments, and §Sigma-Aldrich

Table I: Calculated recoveries and %RSD for US and EU performance criteria of aflatoxins in sample matrices

Spiked Recovery

Results

Automated SmartPrep Extractor SPE Results (n=3)

US Advisory Limits % Recovery and %RSD Values (No

Acceptance Ranges Stated)

European Advisory Limits % Recovery and % RSD Values (70 –

110% Recovery & 20% RSD Acceptance)

Aflatoxin B1 RSD Total Aflatoxins RSD Aflatoxin B1 RSD Total Aflatoxins RSD

Almond milk 93.7 5.10 93.1 4.90 97.0 7.94 97.0 7.96

Fresh corn 86.5 4.78 88.0 4.87 81.0 3.83 85.3 1.46

Peanut paste 85.6 4.35 86.2 5.05 81.0 9.14 73.2 5.97

Curry powder 117 1.91 97.1 2.61 110 6.44 101 5.83



FOOD & BEVERAGE

Theanine is a neurologically active amino acid found in tea plants. L-

Theanine is a dominant amino acid in green tea and is responsible

for its unique pleasant taste as well as known relaxation effect. The-

anine also has been proven to reduce physical and mental stress,

and improve cognition and mood.

Cation-exchange chromatography using post-column Ninhydrin

reagent Trione and UV detection has shown unmatched reproduc-

ibility and selectivity in the analysis of free amino acids in complex

matrices. The Pinnacle PCX post-column derivatization system al-

lows shortening run times by utilizing column temperature gradients.

We introduce a simple and robust method for analysis of L-The-

anine in addition to the other free amino acids in tea leaves.

Method

Sample Preparation

Homogenize 2 g of dry tea leaves with 25 mL of Li220 for 5 min.

Centrifuge and f lter through 0.45 µm nylon f lter.

Analytical Conditions

Column: High-eff ciency

Lithium cation-exchange

column, 4.6 × 75 mm, Cata-

log number 0354675

Flow rate: 0.55 mL/min

Mobile phase: See method

Analysis of L-Theanine in Tea by HPLC with Post-Column DerivatizationMaria Of tserova, PhD, and Sareeta Nerkar, PhD, Pickering Laboratories

Pickering Laboratories, Inc.1280 Space Park Way, Mountain View, CA 94043

tel. (800) 654-3330, (650) 694-6700

Website: www.pickeringlabs.com

-0. 0 10.0 20.0 30.0 40.0 50.0 60.0

As

pa

rtic

Ac

id

Th

reo

nin

eS

eri

ne As

pa

rag

ine

Glu

tam

ic A

cid

Glu

tam

ine

Th

ea

nin

e

Va

lin

e

Pro

lin

eG

lyc

ine

Ala

nin

e

Cy

sti

ne

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leu

cin

eL

eu

cin

eT

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sin

e

Ph

en

yla

lan

ine

Arg

inin

e

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e

His

tid

ine

Am

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nia

a-A

min

o-n

-bu

tyri

c A

cid

70.0

Try

pto

ph

an

Eth

an

ola

min

e

y-A

min

ob

uty

ric

Ac

id

Figure 2: Chromatogram of white tea sample.

Table I: HPLC gradient

Time

(min)

1700–1125

(%)Li365 (%) Li375 (%)

RG003

(%)

0 100 0 0 0

10 100 0 0 0

19 40 60 0 0

32 0 100 0 0

43 0 100 0 0

43.1 0 0 100 0

57 0 0 100 0

57.1 0 0 70 30

72 0 0 70 30

Post-Column Conditions

Post-column System: Pinnacle PCX

Reactor Volume: 0.5 mL

Reactor Temperature: 130 °C

Reagent: Trione Ninhydrin reagent

Flow Rate: 0.3 mL/min

Detection: UV-vis 570 nm for primary amino acids,

440 nm for secondary amino acids

Run time: 72 min

Equilibration time: 12 min

Table I: Column oven program

Time (min) Temperature (°C)

0 34

6 34

17 65

25 70

70 70

71 34

0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

As

pa

rtic

Ac

id

Th

reo

nin

eS

eri

ne

As

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ine

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ic A

cid

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ine

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e

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e

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ine

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nin

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e

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ine

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tid

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Am

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a-A

min

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-bu

tyri

c A

cid

Figure 1: Chromatogram of green tea sample.



FOOD & BEVERAGE

Due to the highly polar nature of saccharides, the analysis of sugars is

typically achieved using hydrophilic interaction chromatography (HILIC).

Using a high polarity packing material, separation is based on the

hydrophilic interactions of the sugars on the stationary phase surface

typically using an eluent with a majority of polar organic solvent.

HILIC columns with amino functional groups are often used to pre-

vent the separation of anomers; however, one drawback of the amino

functional group on the packing material is the low recovery rate of

reducing sugars, such as glucose and mannose. Reducing sugars

are able to adhere to the packing material through the formation of a

Schiff base, and must be hydrolyzed with acid for removal.

Shodex introduces the VG-50 4D column packed with a durable

polymer based packing material modif ed with chemically stable tertiary

amino functional groups. Shodex VG-50 4D is suitable for saccharide

analysis, has demonstrated the separation of reducing sugars, and has

been compared to two competitor silica-base amino columns.


The analysis of four saccharides is accomplished with Shodex VG-

50 4D (4.6 mm I.D. × 150 mm, 5 μm), a polymer-based amino

A New HILIC Column for Saccharide AnalysisMelissa Turcotte* and Naoya Nakajima†,

*Showa Denko America, Inc., and †Showa Denko K.K.

Shodex™/Showa Denko America, Inc.420 Lexington Avenue, Suite 2335A, New York, NY, 10170

tel. (212) 370-0033 x109

Website: www.shodex.net

Figure 2: Comparison of Shodex VG-50 4D and silica-based amino columns demonstrating recovery ratio of mannose. Recovery ratio = (peak area mannose)/(peak area sucrose).

Figure 1: The analysis of sugars using VG-50 4D and two silica-based amino columns. Column: Shodex VG-50 4D (top) and silica-based ami-no columns (middle and bottom); column temperature: 40 °C; injec-tion volume: 5 µL; eluent: CH3CN/H2O = 80/20; f ow rate: 0.4 mL/min for VG-50 4D and 1.0 mL/min for silica columns. Detector: Shodex RI. Sample: 1. fructose, 2. mannose, 3. glucose, and 4. sucrose.

TM

HILIC column. The separation is compared to the same analysis

using two silica-based amino columns under the same conditions.

Column temperature was 40 ºC and f ow rate was 0.4 mL/min for

the VG-50 4D analysis and 1.0 mL/min for the silica-based amino

analyses. Eluent conditions are 80% acetonitrile in water. Injection

volume of 5 μL of 5 mg/mL of each sugar was used for each experi-

ment. The HPLC system was coupled with RI detector.

Results

The saccharides, fructose, mannose, glucose, and sucrose, were

analyzed successfully by HPLC and RI detection with VG-50 4D and

two silica-based amino columns (Figure 1). A comparison of the re-

covery ratio of mannose, a reducing sugar, in relation to sucrose, a

nonreducing sugar, is demonstrated in Figure 2. Shodex VG-50 4D

demonstrates baseline separation of the four sugars with an elution

volume of 20 min and allows up to 90% recovery rate of mannose.

Silica-based amino column 1 shows baseline separation of the four

compounds. However, the recovery ratio of reducing sugars is 40%.

Silica-based amino column 2 cannot separate the four sugars, and

the recovery ratio is less than 20%.

Conclusions

Shodex VG-50 4D, a polymer-based hydrophilic interaction (HILIC)

chromatography column suitable for saccharide analysis, has

demonstrated the recovery ratio of reducing sugars compared with

silica-based amino columns.



PHARMA/DRUG DISCOVERY

This application note shows the comprehensive 2D-LC analysis

of the traditional Chinese prescription Si-Wu-Tang and the in-

dividual herbs contained in Si-Wu-Tang. The possibility for au-

thentication of traditional Chinese prescriptions is investigated.

The authentication of traditional Chinese prescriptions is a challeng-

ing task because of the highly complex nature and the natural vari-

ability of the herbs used in Chinese herbal medicine (CHM). Gen-

erally, chromatographic f ngerprinting is regarded as an effective

method for authentication (1–4). Si-Wu-Tang is composed of the

four herbs Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix

Paeoniae Alba, and Radix Rehmanniae Preparata. For each herb,

characteristic components are detected in Si-Wu-Tang as a means

of authentication. Additionally, changes following the omission and

replacement of one herb from Si-Wu-Tang are examined.


Comprehensive 2D-LC analysis was achieved with the Agilent 1290

Inf nity 2D-LC Solution. In the f rst dimension an Agilent ZORBAX

RRHT SB-Aq column (2.1 × 100 mm, 1.8 µm) was used with a

gradient of water and methanol, each with 0.1% formic acid, at a

f ow rate of 0.05 mL/min. The second dimension separation used an

Agilent Poroshell 120 Bonus-RP column (3.0 × 50 mm, 2.7 µm) with

shifted gradients of water and acetonitrile, each with 0.1% formic

acid, at a f ow rate of 2.5 mL/min. Modulation was realized using the

Agilent 2-position/4-port-duo valve, equipped with two 40 µL loops. A

modulation time of 30 s was employed. Detection was performed at

254 nm as well as using Q-TOF mass spectrometry in positive and

negative ionization mode. Samples were prepared as decoctions, in

the same manner as they are prepared for pharmaceutical use.

Results

To enable authentication of Si-Wu-Tang, a separate comprehensive

2D-LC analysis of each herb contained in Si-Wu-Tang was per-

formed. Detection of accurate masses by Q-TOF mass spectrometry

in connection with literature data enabled the tentative identif cation

of characteristic components of each herb. The peaks that were

tentatively identif ed and further high abundant peaks were select-

ed to construct a template for each herb. Each template was then

matched to the peaks detected in Si-Wu-Tang in terms of f rst and

second dimension retention times as well as agreement of the base

peak in the respective mass spectra. Figure 1 shows the analysis of

Si-Wu-Tang with the peaks matched from the templates of the indi-

vidual herbs. Several peaks detected in Si-Wu-Tang can be attrib-

uted to more than one individual herb, for example, senkyunolide A

from Radix Angelicae Sinensis and Rhizoma Chuanxiong.

Authentication of Traditional Chinese Prescriptions Using Comprehensive 2D-LCSonja Krieger, Agilent Technologies Inc.

Agilent Technologies Inc.5301 Stevens Creek Blvd., Santa Clara, CA 95051

Website: www.agilent.com

Figure 1: Comprehensive 2D-LC analysis of a decoction from Si-Wu-Tang with MS detection in positive (a) and negative (b) ionization mode. Peaks matched from the templates of the individual herbs are marked: Radix Angelicae Sinensis (yellow), Rhizoma Chuanxiong (black), Radix Paeoniae Alba (white), and Radix Rehmanniae Preparata (red).

Generally, 75% or more of the template peaks could be matched

to peaks detected in Si-Wu-Tang. This shows the possibility to detect

characteristic components of an individual herb in a traditional Chi-

nese prescription. Additionally, adulteration through omission and

replacement of one herb from Si-Wu-Tang could be detected by

the matching of a considerably reduced number of template peaks.

Conclusions

Comprehensive 2D-LC is ideally suited for the analysis of complex

samples such as the traditional Chinese prescription Si-Wu-Tang.

The analysis of Si-Wu-Tang and its individual herbs provides a

means of authentication. Further, it is illustrated that one or a few

marker compounds for each herb are not suff cient for authentica-

tion when those compounds are not uniquely contained in one herb.

References

(1) P.S. Xie, and A.Y. Leung, Journal of Chromatography A 1216, 1933–1940 (2009).

(2) X.M. Liang et al., Journal of Chromatography A 1216, 2033–2044 (2009).

(3) D.Z. Yang et al., Journal of Chromatographic Science 51, 716–725 (2013).

(4) Y.Z. Liang et al., Journal of Chromatography B 812, 53–70 (2004).

8

Gallic acid

2–1

3

4

4 9

6–2–2

10–6

8

5 Albiflorin/Paeoniflorin

1

9

11

1Senkyunolide ASenkyunolide A

Z-LigustilideZ-Ligustilide

12

11 7

1023

Gallic acid

5

3

4 Echinacoside

1 14 7

23

15

8

16 7

2–114

12

Catechin

10 9

15

19

Ferulic acid

Ferulic acid

61

14 12 13 18

4 6

Albiflorin/

Paeoniflorin

(a)

(b)




A novel methodology for stabilizing small molecule and pep-

tide drugs was investigated by GSK and CryoXtract Instruments.

The application of automated frozen aliquotting using the CXT

750 Frozen Sample Aliquotter to the analytical preparation was

observed to improve the stability of labile compounds in both

human and rat EDTA plasma.

The ex vivo lability of some drug compounds and molecules in bio-

analytical assays can add complexity and uncertainty to results and

can pose signif cant challenges in drug development efforts. This

can be especially true for compounds that are subjected to long-

term cryogenic storage and that may need to be tested multiple

times (e.g. samples collected for clinical trials). Frozen aliquotting

technology may offer a simple solution for stabilizing target com-

pounds in frozen biological specimens, helping to streamline bio-

analytical assay development and execution. The CXT 750 Frozen

Sample Aliquotter, an automated instrument capable of generating

quantitative aliquots of frozen plasma without thawing the sample,

was evaluated at GlakoSmithKline for integration into biaonalytical

workf ows and the stabilization of labile compounds in EDTA plasma.


A test mixture of labile small molecule compounds and peptides was

spiked into human and rat plasma at a single concentration and fro-

zen in 2 mL cryogenic vials. The samples were then subjected to four

rounds of freeze-thaw aliquotting (liquid aliquotting of a thawed sample)

and frozen aliquotting on CryoXtract’s CXT 750 Frozen Sample Aliquot-

ter. In both cases, aliquots of 100 µL volumes were generated per sam-

ple and time-point. Both freeze-thaw aliquots and frozen aliquots were

precipitated and then analyzed by liquid chromatography tandem mass

spectrometry (LC–MS-MS). Compound recovery for each aliquotting

cycle was based on the peak area counts generated by the LC–MS-MS.

Results

In general, compounds that exhibited an extreme decrease in re-

covery when subjected to freeze-thaw aliquotting exhibited signif -

cantly better recovery when processed on the CXT 750. Figure 1

conveys the analytical recovery of caffeic acid, a small molecule

drug compound, in human and rat EDTA plasma when subjected

to multiple freeze-thaw cycles or frozen aliquotting. In both spe-

cies, caffeic acid is nearly undetectable by LC–MS-MS after three

freeze-thaw cycles. However, approximately 75% of the compound

is detected when processed by frozen aliquotting on the CXT 750.

Additional data for several more compounds tested can be found at

http://www.cryoxtract.com/pdf/Cryoxtract_ISBER2015_small_mol-

ecule_GSK_Poster.pdf.

Conclusions

The capability of the CXT 750 to generate frozen aliquots of a speci-

f ed volume (precision data available in the full application note)

and improve the stability of labile compounds for bioanalysis shows

great promise for streamlining development and execution of ana-

lytical protocols in the regulated drug analysis arena.

CryoXtract Instruments, LLC 5 Constitution Way, Woburn, MA 01801

tel. (339) 298-5615

Website: www.cryoxtract.com

Ex vivo Stabilization of Small Molecule Compounds and Peptides in EDTA Plasma for LC–MS-MS Analysis Using Frozen Aliquotting Glenn Smith and Joseph Fraone, CryoXtract

Figure 1: Peak area counts show a marked decline across time points zero through four in freeze-thaw aliquots. In comparison, frozen aliquots of human and rat plasma samples produced on the CXT 750 showed sig-nif cant improvements in stability. Caffeic acid was not recoverable by LC–MS-MS after two to three freeze-thaw cycles, whereas 75% of compound remained recoverable after four frozen aliquotting rounds.




Accurate Pain Management Analysis in Under 5 Min on Raptor™ Biphenyl Superf cially Porous Particle LC ColumnsSharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation

Pain management LC analyses can be diff cult to optimize

due to the limited selectivity of C18 and phenyl-hexyl phas-

es. In contrast, the selectivity of Raptor™ Biphenyl superf -

cially porous particle (SPP) LC columns provides complete

resolution of isobaric pain medications with a total cycle

time of 5 min.

Accurate, reliable analysis of pain medications is a key component

in monitoring appropriate medical use and preventing drug diver-

sion and abuse. As the demand for fast, multicomponent methods

grows, LC–MS-MS methods are increasingly desired for pain man-

agement and therapeutic drug monitoring due to the low detection

limits that can be achieved with this highly sensitive and selective

technique. However, despite the selectivity offered by mass spec-

trometry, hydrophilic matrix components can still interfere with ear-

ly-eluting drug compounds resulting in ion suppression. In addition,

isobaric pairs must be chromatographically separated for positive

identif cation. The need for highly selective and accurate methods

makes LC column selection critical.

While C18 and phenyl-hexyl phases are frequently used for bioan-

alytical LC–MS-MS applications, Restek’s Biphenyl phase offers bet-

ter aromatic retention and selectivity for pharmaceutical and drug-

like compounds, giving it a signif cant advantage over other phases

for the analysis of pain management medications or other drugs

of abuse. The Biphenyl phase, originally developed a decade ago

by Restek, has recently been combined with Raptor™ SPP (“core-

shell”) silica particles to allow for faster separations without the need

for expensive UHPLC instrumentation. Here, we demonstrate the

fast, selective separation of commonly tested pain drugs that can be

achieved using the new Raptor™ SPP Biphenyl LC column.


A standard containing multiple pain management drugs was pre-

pared in blank human urine and diluted with mobile phase as fol-

lows, urine:mobile phase A:mobile phase B (17:76:7). The f nal

concentration for all analytes was 10 ng/mL except for lorazepam,

which was 100 ng/mL. Samples were then analyzed by LC–MS-

MS using an AB SCIEX API 4000™ MS-MS in ESI+ mode. Chro-

matographic conditions, retention times, and mass transitions are

presented here and in Tables I and II:

Column: Raptor™ Biphenyl, 50 mm × 3.0 mm i.d. × 2.7 µm

Sample: Fortif ed urine

Inj. vol.: 10 μL

Inj. temp.: 30 °C

Mobile phase A: Water + 0.1% formic acid

Mobile phase B: Methanol + 0.1% formic acid

Results

As shown in Figure 1, 18 commonly tested pain management drugs

were analyzed with the last compound eluting in less than 3.5 min,

giving a total cycle time of 5 min on Restek’s Raptor™ SPP Biphe-

nyl LC column. Analyte retention times are presented in Table II.

Important isobaric pairs (morphine/hydromorphone and codeine/

hydrocodone) were completely resolved and eluted as symmetrical

peaks, allowing accurate identif cation and integration. In addition,

early-eluting compounds such as morphine, oxymorphone, and

Figure 1: Baseline resolution of isobaric pain management drugs in sub-5-min runs on the Raptor™ Biphenyl column.

Table I: Mobile phase gradient

Time (min) Flow (mL/min) %A %B

0.00 0.6 90 10

1.50 0.6 55 45

2.50 0.6 0 100

3.70 0.6 0 100

3.71 0.6 90 10

5.00 0.6 90 10




hydromorphone are separated from hydrophilic matrix interferenc-

es, resulting in decreased ion-suppression and increased sensitivity.

Similar analyses on C18 and phenyl-hexyl columns often exhibit

poor peak shape and resolution (for example, peak tailing between

closely eluting isobars), which makes identif cation and accurate

quantif cation more diff cult.

Conclusions

Complete separation of critical pain management drug analytes

from hydrophilic matrix components and isobaric interferences was

achieved using the new Raptor™ SPP Biphenyl LC column in less

than 5 min. The fast, complete separations produced in this method

allow accurate quantif cation of pain management drugs and sup-

port increased sample throughput and improved lab productivity.

To learn more, visit www.restek.com/raptor

Restek Corporation110 Benner Circle, Bellefonte, PA 16823

tel. (800) 356-1688, fax (814) 353-1309

Website: www.restek.com/raptor

Table II: Analyte retention times and transitions

Peaks tR (min) Precursor Ion Product Ion 1 Product Ion 2

Morphine* 1.34 286.2 152.3 165.3

Oxymorphone 1.40 302.1 227.3 198.2

Hydromorphone* 1.52 286.1 185.3 128.2

Amphetamine 1.62 136.0 91.3 119.2

Methamphetamine 1.84 150.0 91.2 119.3

Codeine* 1.91 300.2 165.4 153.2

Oxycodone 2.02 316.1 241.3 256.4

Hydrocodone* 2.06 300.1 199.3 128.3

Norbuprenorphine 2.59 414.1 83.4 101.0

Meprobamate 2.61 219.0 158.4 97.2

Fentanyl 2.70 337.2 188.4 105.2

Buprenorphine 2.70 468.3 396.4 414.5

Flurazepam 2.73 388.2 315.2 288.3

Sufentanil 2.77 387.2 238.5 111.3

Methadone 2.86 310.2 265.3 105.3

Carisoprodol 2.87 261.2 176.3 158.1

Lorazepam 3.03 321.0 275.4 303.1

Diazepam 3.31 285.1 193.2 153.9

*An extracted ion chromatogram (XIC) of these isobars is presented in the inset of Figure 1.




Fast Analysis of IgG Charge Heterogeneity by Ion Exchange Chromatography Tosoh Bioscience LLC

Therapeutic antibodies are enjoying high growth rates in the phar-

maceutical market. A majority of the top bestselling global drug

brands are monoclonal antibodies (mAbs). The main product char-

acteristics to be analyzed in process monitoring and quality control

of these antibodies are aggregation, fragmentation, glycosylation,

and charge heterogeneity. Charge isoforms of proteins result from

deamidation of asparagine or glutamine residues or from incom-

plete removal of the C-terminal lysine residue. Besides isoelectric

focusing, ion exchange chromatography is the method of choice to

analyze charge heterogeneity of proteins.

TSKgel® STAT columns are packed with a non-porous polymer

based stationary phase. Proprietary surface modif cation technology

ensures a high density of charged groups. Compared to traditional

porous ion exchange phases, non-porous particles yield outstanding

resolution of charged species at shorter analysis time. TSKgel STAT

columns are available with a weak cation exchanger containing a

weak acid (carboxymethyl) and as a strong cation exchanger contain-

ing a bonded sulfonic acid group (sulfopropyl). Best performance is

achieved when the columns are used with low dead volume chroma-

tography systems, such as modern UHPLC systems. This application

note presents the fast analysis of mAb charge heterogeneity by cation

exchange chromatography using TSKgel STAT columns.

Figure 2: Analysis of mAb B on weak and strong ion exchange columns at pH 7.

Figure 3: Dependence of cation exchange separation on mobile phase pH.

Figure 1: Analysis of mAb A on weak and strong ion exchange columns at pH 7.





Columns: TSKgel SP-STAT, 7 μm, 4.6 mm ID × 10 cm

TSKgel CM-STAT, 7 μm, 4.6 mm ID × 10 cm

Mobile phase: A: 10 mmol/L sodium phosphate buffer, pH 7.0

(Figures 1, 2, 3)

10 mmol/L sodium phosphate buffer, pH 6.0

(Figure 3)

10 mmol/L sodium acetate buffer, pH 5.0

(Figure 3)

B: 100 mmol/L phosphate, pH 7.0 + 500 mmol/L

NaCl (Figures 1, 2, 3)

100 mmol/L phosphate, pH 6.0 + 500 mmol/L

NaCl (Figure 3)

100 mmol/L acetate, pH 5.0 + 500 mmol/L NaCl

(Figure 3)

Gradient: 0–100% B in 30 min

Flow rate: 1 mL/min

Detection: UV @ 280 nm

Injection vol.: 10 μL

Samples: mAb A (2 g/L), mAb B (2 g/L)

Results

The charge isoforms of two monoclonal antibodies were separated on a

TSKgel CM-STAT weak cation exchange column and a TSKgel SP-STAT

strong cation exchange column. Figure 1 shows the analysis of mAb A

on both columns at pH 7. For this IgG, the weak cation exchange col-

umn delivers a better separation of the basic variant from the main peak.

As shown in Figure 2, this is not the case for mAb B, where the

strong cation exchange column shows a better separation. Retention

and resolution of the charged isoforms are dependent on the buffer pH,

as can be seen in Figure 3 for the analysis of mAb B on TSKgel SP-STAT.

Conclusions

Weak and strong cation exchange columns provide different selectivi-

ties for the analysis of charge heterogeneity of proteins. In order to

reach the best separation of acidic and basic isoforms from the main

peak, both types should be evaluated at various pH values of the mo-

bile phase during method development. TSKgel STAT series columns

provide a high resolution of isoforms in a short analysis time and are

ideally suited for the QC of biotherapeutics by UHPLC or HPLC.

Tosoh Bioscience and TSKgel are registered trademarks of Tosoh

Corporation.

Tosoh Bioscience LLC3604 Horizon Drive, Suite 100, King of Prussia, PA 19406

tel. (484) 805-1219, fax (610) 272-3028

Website: www.tosohbioscience.com




Avermectins are a series of 16-membered macrocyclic lactone derivatives that are used extensively in animal and crop protection. They occur in nature and can be produced as fermentation by-products by the micro-organism, streptomyces avermitilis. Examples of well-known avermectins and derived products include ivermectin, eprinomectin, selamectin, doramectin, and abamectin. All the avermectins have high anthelmintic and insecticidal properties even at low dose levels and residues of these veterinary drug components reach the environment through manufacturing and animal waste and may potentially affect terrestrial and aquatic life forms.

Several crop protection companies have strong interest in the synthesis, production, and analysis of these compounds. Therefore, there exists a need to develop a fast and eff cient analytical method capable of determining avermectin residues on animals, in food, and in the environment. The HPLC method presented here is fast and eff cient and can be used for residual analysis as well as for quality control of avermectins in drug formulations. The peaks produced are sharp resulting in high sensitivity.

The mixture can be baseline separated using gradient elution with an MS-compatible mobile phase containing acetonitrile, methanol, and water. What is even more interesting is that the four avermectin products considered and up to 20 separate degradation products could be identif ed.

HPLC Conditions

Column name: FLARE C18 MMColumn dimensions: 4.6 × 150 mm (15698-14-2, 3.6 μm, 120 Å) HPLC system: Agilent 1200Inj. vol. and conc.: 5.0 μL, ~0.2 mg/mL of each major analyte in MeOHDetection: UV at 244 nmFlow rate: 1.0 mL/minSolvents: A: 50 mL ACN, 100 mL MeOH, 850 mL H2O; B: 600 mL ACN, 200 mL MeOH, 200 mL H2OGradient: 0.00 min, 10% B; 10.00 min, 90% B; 10.01 min, 10% B; 18.00 min, 10% BTemperature: 35 °C

The FLARE C18 MM column is manufactured with 3.6 µm diamond core-shell particles that have 120 Å pores. The particle geometry and tight particle size distribution contribute to high packing den-sity and column eff ciency with reduced plate height, h, of ~2. The columns come in various lengths and diameters and are compatible

Diamond Analytics11260 S 1600 W., Orem, UT

tel. (801) 235-9001, fax (801) 235-9141

Website: www.diamond-analytics.com

Separation of Macrocyclic Lactones Diamond Analytics

Figure 1: Structures of selected macrocyclic lactones.

Figure 2: Separation of macrocyclic lactones using FLARE C18 MM column.

Figure 3: Exploded view showing peaks between 8 and 12 min.

with 100% aqueous to 100% organic solvents. They are pH and temperature stable and available to ship worldwide.




Faster Analysis and Higher Eff ciency with Thermally Stable HPLC ColumnsDwight Stoll and Peter W. Carr, ZirChrom Separations, Inc.

In his article, published in 2000, David V. McCalley found large

increases in the eff ciency for basic compounds at elevated

temperature (1). McCalley suggested both that basic compounds

should be analyzed at high temperature and that columns should

be developed that are stable at high temperature.

In addition to improved eff ciency, high temperature operation

allows for dramatic improvements in analysis speed. Raising the

temperature decreases mobile phase viscosity, allowing for increased

eluent f ow rate (and faster analysis) without excessive back pressure.

Experimental

A mixture of antihistamines was separated at room temperature using

a ZirChrom®-PBD column. The separation conditions were as follows:

Column: 4.6 mm × 100 mm ZirChrom-PBD

Mobile Phase: 29/71 ACN / 50mM Tetramethylammonium

hydroxide, pH 12.2

Injection Vol.: 0.5 µL

Pressure Drop: 195 bar

Detection: UV at 254 nm

The initial separation is shown in Figure 1a. Then, the temperature

was increased to 50 ºC, and the eluent f ow rate also increased

to maintain the same system back pressure. The separation (not

shown) was more than twice as fast, with the resolution of the two

closely eluting compounds maintained.

Finally, the temperature was increased to 80 ºC, again increasing

the eluent f ow rate to maintain system back pressure. Now the

separation is f ve times faster, maintaining the same resolution of

the closely eluting peaks (see Figure 1b).

Note that even temperature-sensitive compounds can benef t from

modest increases in temperature, making faster analysis possible.

ZirChrom’s technical support group has extensive experience in this

area, and would be happy to help you with your particular application.

ZirChrom columns combine the high eff ciency usually associated

with silica columns with complete chemical and thermal stability.

References

(1) D.V. McCalley, J. Chrom. A 902, 311–321 (2000).

Figure 1: Separation of antihistamines at room temperature (a) and at 80 °C (b). 1 = Doxylamine, 2 = Methapyrilene, 3 = Chlorpheniramine, 4 = Triprolidine, 5 = Meclizine.

ZirChrom Separations, Inc.617 Pierce Street, Anoka, MN 55303

tel. 1 (866) STABLE-1

Website: www.zirchrom.com



POLYMER

Epoxy resins are an important group of synthetic resins. They f nd

applications in a wide variety of industries, and are used for the

production of coatings, adhesives, laminates, casting materials, etc.

Traditionally, simple analytical characteristics (for example,

content of epoxy groups, chlorine, α-glycol groups, viscosity

or softening point) are used to identify epoxy resins. But these

quantities are often unable to distinguish among particular samples

and to find relations between the epoxy structure and its properties.

For vir tually all synthetic resins, there are no suitable

size-exclusion chromatography (SEC) calibration standards

available. The preparation of standards by fractional precipitation is

laborious and frequently leads to unsatisfactory results. Polystyrene

calibration standards are used universally, but are far from the true

values of the resins they purport to mimic, so the absolute analysis

can only be completed by DAWN or miniDAWN multi-angle light

scattering (MALS) coupled to SEC.

In this application note, the chromatograms recorded from the

SEC analysis of an epoxy resin sample are depicted in Figure 1.

The light scattering signal proves conclusively that the miniDAWN

MALS detector can be used to detect even very low molar masses

of the epoxy. In fact, the Mn value determined by SEC–MALS agrees

with those determined by vapour pressure osmometry (VPO), which

measures absolute Mn directly.

Figure 2 contrasts differential molar mass distribution curves of

two epoxy resin samples with identical epoxide equivalent weights.

It demonstrates that the capabilities brought to SEC by MALS is the

most direct way to correctly determine the molar mass of synthetic

resins. The MALS detection reveals differences that are not seen

by traditional analytical methods or by using SEC with column

calibration and RI detection alone.

Light Scattering for the Masses®:Quality Control of Epoxy Resins Wyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, CA 93117

Tel:+1 (805) 681 9009 fax: +1 (805) 681 0123

Website: www.wyatt.com

Figure 1: Light scattering (top) and RI (bottom) chromatograms from SEC of bisphenol A based epoxy resin. Mn (SEC–MALS) = 1520 g/mol, Mn (VPO) = 1470 g/mol.

Figure 2: Differential molar mass distributions of two epoxy resin samples with equal epoxide equivalent weights of 1670.



GENERAL

Synthetic fused silica capillary tubing continues to be a vital

material in the separation sciences. In this application note,

we present and discuss recent data on cutting edge, nano-

meter internal diameter capillary tubing.

Scientists employ fused silica capillary tubing routinely in a wide

range of analytical applications, encompassing GC, CE, capillary

LC, and CEC. It has proven as an ideal substrate for interfacing

in hyphenated techniques. As the separation sciences and related

analytical technologies evolve, a general trend toward smaller bore

capillary tubing is clearly evident.

Polymicro TechnologiesTM capillary tubing leads the f eld in meeting

this market demand by offering 25 standard products with internal di-

ameters (i.d.) of less than 50 µm, with six of those having i.d. tolerances

of ± 1 µm. The next logical innovation is the sub-1-µm regime. For

many years it was felt that nanometer i.d. capillary, or “nano-capillary,”

could not be produced as the open bore of the capillary would close

shut during the manufacturing process. After innovative developments,

nano-capillary has been drawn several times in small quantities.

In this application note we discuss details of a 1 km-long experi-

mental run of polyimide coated, fused silica-based nano-capillary.

Experimental

Nano-capillary tubing used in this study was produced on a standard

Polymicro Technologies drawing tower using proprietary, optimized

draw parameters. The fused silica employed was the same glass type

used for similar small i.d. capillary. The objective was to produce tub-

ing of <1 µm, but specif c nanometer level targeting was not possible

due to the delay between production sampling and SEM verif cation.

Individual samples were measured using a Hitachi S4500, using a

NIST traceable standard from Geller MicroAnalytical Laboratory.

During the draw, individual batches of capillary were collected

in pre-determined lengths of 100 m. Each batch was further seg-

mented and end measurement samples taken every 25 m. The i.d.

of each sample was then determined by SEM yielding 5 data points

every 100 linear meters.

Results

A chart of i.d. data collected from the 10 batches produced is shown

in Figure 1. The i.d. target of <1 µm was met on all data points. The

i.d. range within each 100 m long batch varied from a low of 40 nm

to a high of 314 nm, with six of the batches having ranges of <125

nm. Table I shows a summary of the average i.d. by spool and the cor-

responding standard deviation. The i.d. initially trended downward from

~900 nm to nominally 200 nm and then slowly trended upward into

the 800 nm range. A review of all collected data suggests that a higher

frequency of measurement linearly is needed (i.e. every 10 m), which

may allow for tolerances approaching ± 100 nm.

Conclusion

It is evident that through state of the art process control and me-

trology, providing nano-capillary tubing is possible. Finished spool

length and tolerances need to be def ned. The data presented in

this note represents one of several studies currently under way.

Molex and Polymicro Technologies™ are trademarks or registered trademarks of Molex, LLC.

Nanometer Internal Diameter Fused Silica Capillary TubingJoe Macomber and Corey Hoshiwara, Molex

Molex 18019 N. 25th Ave., Phoenix, AZ 85023

tel. (602) 375-4100

Website: www.molex.com/capillarytubing

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 25 50 75 100

Inn

er

Dia

mete

r (µ

m)

Spool Position (m)

ID Variance Over Fixed Lengths of 100 meters

Figure 1: i.d. data for 100 m long batches from a 1 km draw of nano-capillary.

Table I: Average i.d., range, and standard deviation for 10 batches of nano-capillary

Spool 1 2 3 4 5 6 7 8 9 10

Average 0.893 0.532 0.239 0.228 0.231 0.485 0.572 0.541 0.711 0.766

Range 0.040 0.314 0.085 0.061 0.124 0.292 0.214 0.082 0.258 0.100

Std. Dev. 0.020 0.123 0.036 0.025 0.056 0.118 0.083 0.033 0.106 0.038



Mary Ellen P. McNally, PhD

A Life with the BoysA memoir of Phyllis BrownÑa pioneering chromatographer, a devoted mentor, and a champion for women in science

There are a lot of misconceptions about women in science, said Phyllis Brown in a 2014 interview (1). At the time, she had just published a book about

caring for her husband while he suffered from Alzheim-er’s disease, and she was planning to write another book, about her career as a scientist. “I think my next book will be called A Life with the Boys,” she said.

Sadly, Brown passed away before that sec-ond book became a reality. This renowned chromatographer died on Wednesday, July 8, 2015, at the age of 91, in Providence, Rhode Island.

In ref lect ing on Brown and her career, I think about her in two ways. First, I think about her as an out-standing chromatog-

rapher, who championed high performance liquid chromatography (HPLC) and showed how it could be used for the analysis of com-plex real-world samples in fields like biochemistry and medicine—at a time when many were only testing it with simple, clean samples. Equally, I remember her as pioneer who saw the particular challenges faced by women in sci-ence and who supported and championed other women in the field.

A Pioneer in HPLC, Applying It to Messy, Real-World SamplesBrown, an excellent scientist and a prolific scientific au-thor who had more than 200 scientific peer-reviewed articles to her credit, was the author of f ive books in chromatography and capillary electrophoresis (CE), and was a longtime coeditor, with Professor Eli Grushka, of 30 volumes of the much-loved Advances in Chroma-tography book series. She received a consistent f low of awards for her work. These recognitions include a Ful-bright Fellowship, the Tswett Medal in Chromatography, the Dal Nogare Award in Chromatography (she is one of only three women to have won this award since it began almost 50 years ago), the Governor’s Medal for Contributions to Science and Technology in the State of Rhode Island, the Scholarly Achievement Award for

Excellence in Research at the University of Rhode Island, a Brown University Citation for Outstanding Research in Chemistry, the Csaba Horváth Medal for Separation Science, and the Eastern Analytical Symposium’s Award for Achievements in Separation Science. She was also a member of LCGC’s Editorial Advisory Board for 27 years, from 1983 to 2010.

Brown received her BS in chemistry from George Wash-ington University. But it was not until her four children had reached ages 9–14 that she went back to get her PhD in chemistry from Brown University, where she later became an assistant research professor. In 1973, she joined the fac-ulty of the University of Rhode Island and was promoted to full professor in 1980, retiring in 2001.

Brown was a pioneer in the application of HPLC and CE to biochemistry and medical research. In the infancy of HPLC in the early 1970s and CE in the early 1980s, she saw the tremendous potential of these techniques for biotech-nology and had the foresight to predict that they would open up new horizons in medical research.

Specifically, Brown made significant contributions to separation science in three main areas. One was the devel-opment and optimization of HPLC and CE for biotech-nology, biochemistry, and pharmaceutical research and for use in clinical chemistry and medicine. The second was the use of these assays for nucleic acid constituents in pharmacology and in investigations of disease processes. The third was in studies of the mechanisms of reversed-phase LC retention of purines, pyrimidines, and their nucleotides.

Indeed, an important part of Brown’s pioneering work was her application of HPLC to messy, real-world samples in biochemistry, starting with the first paper she published on the topic in 1970 (2). In a 2012 interview in LCGC (3), Brown talked about her willingness to apply the method to difficult analytical problems at a time when the fact that she was a woman—and an older woman at that, hav-ing gone back to get her PhD after raising her children—already made her subject to doubt.

It is a wonder I ever survived as a chromatographer.

Almost everyone in the field was an analytical chemist,

an engineer, or a physical chemist, and of course they

were all male. Along I came, an organic chemist with an

interest in biochemistry and pharmacology, and an older

woman at that. In addition, they were interested either

in the theory of chromatography or in the instrumen-

tation and I was interested in applying their toy to real

Phyllis Brown (1924–2015)



problems. Their chromatograms,

mainly of standards, were neat and

clean with nice sharp peaks. My

chromatograms of real samples

were broad and sloppy looking

with peaks of strange sounding

compounds. The “old boys” didn’t

know what to make of me.

Joy Miksic, who was Phyllis Brown’s first graduate student at URI in 1978, remembers her mentor’s confidence in the value of HPLC during the early days of the technique, in the late 1960s, when it was still very challenging to use. “Those were days of ubiquitous leaks, drifting baselines, 2-hour anal-ysis times, and irreproducible col-umns,” recalls Miksic, now the prin-cipal of Bioanalytical Solutions, LLC.

“In spite of those early issues, Phyllis had absolute confidence in the future of HPLC. She offered her help to any-one who had an idea to improve the technique.”

The authority Brown had acquired in the field also impressed Miksic.

“When Phyllis walked into a room or a vendor booth, people gathered,” recalls Miksic. “Her enthusiasm and positive energy could be felt loud and clear as she praised the technique and

declared it a breakthrough for analy-sis of biological samples.”

Bob Stevenson, editor emeritus of American Laboratory and a Califor-nia Separation Science Society Fel-low, also valued Brown’s efforts to develop and champion the technique.

“‘Mother’ Phyllis was such a great leader in the early days of HPLC,” he said. “I often reflect on the early lead-ers, especially those who explored and taught the basics. Yes, we have lost a great one.”

A Champion of Women, and All Young ScientistsBrown didn’t just fight to advance HPLC. She learned, early on, the dis-advantages she faced as a woman in science, as she realized she was being excluded from the community of the

“old boys,” as she explained in that 2012 interview (3):

For the most part, they ignored

me. In fact, for several years they

didn’t tell me when the HPLC

meetings were held. They were

mainly by invitation and I didn’t

get invited. It took me a while to

learn that most of the plans for the

coming year were made in the bar

in the evenings. By plans, I mean

who was going to be invited to

speak at next year’s meeting, who

they were going to support for

awards, editorial boards, and so

on. I then learned to go to the bar,

sip my drink, and try to be part of

“the boys.”

Once she learned what one had to do, she did it. And then she made it a mission to teach those lessons to younger scientists, particularly women, and to support their careers. In particular, she encouraged young women in chromatography to take advantage of the recognition that came from presenting and publishing their work and being visible at scien-tific meetings.

“Phyllis Brown was an extremely knowledgeable analytical chem-ist and chromatographer, and she generously advised and inspired many younger scientists,” said Peter Schoenmakers, a professor at the Uni-versity of Amsterdam. “She was a role model for women in chromatography, after struggling many years for full acceptance in a male-dominated field. Phyllis Brown was very willing to share these experiences with younger women as well.”

“Phyllis was a champion of women in science long before the current STEM movement picked up steam,”

Brown graduating with her PhD from Brown University in 1968, with Bob Hope and Irving Fain.

Brown celebrating with fellow University of Rhode Island professor Sze Yang after receiving the Connecticut Separation Sci-ence Council award in 2004.



agreed Patrick D. McDonald, a cor-porate fellow emeritus at Waters Cor-poration, who was an undergraduate classmate with Phyllis at Brown Uni-versity.

And she encouraged all scientists, female or male. “Dr. Brown person-ally guided and inspired all of us, and everyone she met with love and enthusiasm, inspiring the confidence that we could succeed at anything if we tried hard enough,” remembers Rick Hartwick, who completed both his masters and PhD under Brown at the University of Rhode Island and later cofounded PharmAssist Ana-lytical Laboratory.

Michael Swartz of Boston Analyti-cal sought out Brown to be his PhD advisor, because he knew she had mentored other nontraditional stu-dents like him who returned to school having a full-time job or a family, or both. “In your lifetime you will meet only a handful of people that truly make a difference in your life and for me, Dr. Brown is certainly one of those people,” he said. “Her influ-ence in my life and career has, and will continue to have, an everlasting effect.”

John V. Amari, now at the pharma-ceutical company Shire, is grateful

for the opportunities Brown provided him as a graduate student to learn outside the classroom, through men-toring undergraduates and collabo-rating with another department. He also remembers her wise words. “She frequently said ‘you never know what you’ll be doing,’” he recalls. “How true that statement was and still is.”

Karyn Usher, an associate profes-sor at Metropolitan State University in St. Paul, Minnesota, also remem-bers Brown’s sage advice. The first was to never let anyone tell her that she could not achieve her goals. “She also told me that it was important to always look for opportunities and to be open to deviate from my master plan, as long as it felt right,” Usher said. “To this day, I follow this advice and have found it to be quite useful.”

Brown’s support for women, how-ever, went far beyond mentoring.

“Sometimes she acted quietly behind the scenes, and we may not have been aware that she was responsible for our invitations to international symposia or to contribute to special journal issues or books,” noted Vicki McGuf-fin, professor emerita at Michigan State University. “But at other times she was quite vocal, especially about the need to nominate women for

awards. Because of her efforts, women are represented among the recipients of many national and international awards in separation science.”

And some, like Christina Robb, acknowledged that it took time for them to realize the importance of what Brown did for women in science. Robb was Brown’s last PhD student and is now an Assistant Agricultural Scientist II for the Connecticut Agri-cultural Experiment Station.

“I came from an undergraduate experience where there were lots of women in the physical sciences,” said Robb. “At the time I came to work for Phyllis, in terms of the women in science issue, I didn’t quite connect. However, I realized over the years, I was exactly what she was fighting for.”

Ron Majors, retired from Agilent, remembers the “Women in Chroma-tography” luncheon Brown organized at the HPLC ’86 conference in San Francisco that he chaired.

“The restaurant was filled to capac-ity with nearly every woman at the symposium in attendance and, to my surprise, many noted male chro-matographers were there as well—and the food wasn’t free!” he said.

“Although there were no formal pre-sentations, the presence of Phyllis and

Brown at the Eastern Analytical Symposium in 2006, when she won the Eastern Analytical Symposium’s Award for Achievements in Separation Science.

Brown hosting her annual graduation brunch for all the graduating students and their families of the chemistry de-partment in 2001 at the Larchwood Inn, in Wakef eld, Rhode Island.



so many women gave a not-so-subtle message about increasing the pres-ence and acceptance of more women in chromatography to an increasingly less skeptical primarily male audi-ence.”

A Short, But Significant WalkApryl l Sta lcup, a professor at the University of Cincinnati and the di-rector of the Irish Separation Science Cluster and Professor of Chemical Sciences at Dublin City University in Ireland, did not attend that luncheon at the 1986 HPLC conference, but she felt its impact—and especially of what Brown did after the luncheon. When it was time to return to the plenary session, Brown arranged to have al l the women walk into the room at the same time.

The first time I heard of Phyllis

Brown was when I was a graduate

student working at NIST. I was

the only woman present, having

lunch with several very prominent

chromatographers. They were all

talking about Phyllis organizing

all the women chromatographers

at a recent conference to enter

the room at the same time, and

they were wondering what it all

meant. I thought this was very

interesting because before enter-

ing graduate school, I had worked

in an environmental lab where the

guys with my same level of train-

ing got to attend conferences but

the company didn’t want to pay for

an extra hotel room for me. After I

heard this story about what Phyl-

lis Brown did, for the first time, I

didn’t feel so alone. I felt that it was

ok to be on this path.

I was among the female chroma-tographers who entered that room together. That was an empowering and pivotal moment. Like Apryll, after that event, I didn’t feel alone anymore. It was just a short walk, and the destination was simply a seat in the room. There were no speeches. But the message was felt: We are here. We matter. We are connected. Phyllis was our gatherer, and her gathering inspired and pro-voked us.

Brown’s efforts to support women in science, making our presence felt by the men and connecting us to each other, occurred not just in group events like the gesture of entering the room together, but through regular, consistent, and

personal support. She weaved the thread of connection consistently, calling to ask about a paper she saw that we published, and concluding the conversation by talking about efforts going on for women scien-tists. In her discussions, science was first. But the discussion of how we could encourage young women in the field and focus more energies toward enabling women to be more active in the scientific community was never left out. The science was Phyllis’s passion; giving women a voice in science was her goal.

So, thank you, Phyllis Brown, for your great science, the incredible strides you made in taking chroma-tography to the practical realm of the imperfect sample, and for publish-ing and continuing to publish your experiments, your learnings, and your theories. But also, thank you for bellying up to the bar and sipping those drinks, for the march into the room, for the countless subsequent women’s lunches you organized at meetings. You made a difference. You connected, you asked, and you pushed and cajoled so that gender would not make a difference in sci-entific and technological advance-ment. And you expected us to do the same. We have, we are, we will.

References

(1) ht t p : //s dppub l i sh i n g so lu t ion s . c om /

author-corner-phyllis-brown-author-of-

help-me-im-sl ipping-ta lk s-about-f ind-

ing-solace-in-writing-and-her-hope-that-

her-book-will-help-caregivers/ (2014).

(2) P.R. Brown, J. Chrom. 52, 257 (1970).

(3) K.M. Usher, LCGC North Am. 30(11),

982–985 (2012). http://www.chroma-

tographyonline.com/phyllis-brown-story-

serend ipit y-per se vera nce -v i s ion-a nd-

chromatography.

Mary Ellen P. McNally, PhD, is a

Technical Fellow with DuPont Crop

Protection at the Stine Haskell

Research Center in Newark, Delaware.

She is an editorial advisory board

member of LCGC North America.

Direct correspondence to:

[email protected] ◾

Brown in her off ce in the early 2000s.



Why and How to Avoid Ionic Contamination in Water Used for LC–MS AnalysesIonic contaminants in the water used in ultrahigh-pressure liquid chromatography (UHPLC) analyses with mass spectrometry (MS) detection lead to adduct formation and reduced analytical signals because of ion suppression. In MS, the preferred ion type is the protonated molecular ion, especially in peptide analysis, since the partially mobile proton charge enables more meaningful fragmentation analysis, as compared to a sodiated peptide ion. Moreover, the occurrence of protonated analyte signals demon-strates that solvents and reagents, as well as the MS instrument used in analyses, were clean and did not contribute any contaminating cationic components to the analytical process. In the experiments presented here, it was observed that the signal intensi-ties of the protonated species decreased as the sodium ion concentration in the water increased. This was accompanied by an increase in the intensity of sodiated adducts.

Anastasia Khvataeva-Domanov and Stéphane Mabic

Water plays an essential role in liquid chroma-tography–mass spectrometry (LC–MS), where it is used extensively in the workf low. Con-

taminants in the water can affect the quality of data and instrument performance; therefore, it is recommended and prudent to use only the highest purity solvents. Or-ganic contamination of the water used in high perfor-mance liquid chromatography (HPLC) is an important issue and has been addressed accordingly (1), but the ionic purity of the water should also be considered, es-pecially when MS is used as a detection technique (2). Ionic contaminants lead to adduct formation and reduced analytical signals because of ion suppression.

Electrospray ionization (ESI) remains the most popu-lar MS technique. In positive-ion analyses, it is ideal to have only protonated peaks of the parent ion or its frag-ments in the mass spectrum. The presence of metal ad-duct peaks, such as sodium adducts (M+Na), makes data analysis more challenging and complicated. Metal ions may come from several possible sources (3) such as sol-vent reservoirs, gloves, the analyst, and the solvents used in preparing the mobile phase. Therefore, using ultrapure water free of metal ions will contribute to the success of any LC–MS analysis.

ExperimentalExperiments to Evaluate the Effect of

Ionic Contamination in Water on MS Data

Two compounds, bradykinin fragment 1-7 and Glu1-fi-brinopeptide B, were used to investigate the effect of ionic contamination on LC–MS analyses. Different samples of peptides were prepared and infused directly to a mass spec-trometer. The experimental details for peptide analyses are described in Table I.

Experiments to Identify the

Levels of Ions in Ultrapure Water

To evaluate the level of sodium in ultrapure water, water sam-ples from an EMD Millipore Milli-Q Advantage A10 water purification system were analyzed using an Agilent 7700s inductively coupled plasma–mass spectrometry (ICP-MS) instrument (4). The calibration standards used in experi-ments were a mixture of Agilent and Spex CertiPrep, and containers were all perfluoralkoxy (PFA) polymer precleaned with ultrapure water.

Water Purification Systems

All ultrapure water samples (resistivity of 18.2 MΩ·cm and total oxidizable carbon [TOC] below 5 ppb) from EMD Millipore water purification systems were analyzed immediately after water collection.

Results and DiscussionEffect of Ionic Contamination in Water on MS Data

The quality of LC–MS data is influenced by many factors, such as instrumentation, experimental parameters, sample prepara-tion, the quality of reagents used, and the quality of solvents (5). Water is used extensively in reversed-phase LC–MS work-flows; therefore, its purity plays a critical role in instrument performance and the quality of data generated.

The purity of water for LC–MS work is mainly assessed through organic contamination, which can be expressed by the level of TOC (6), or by certificates of analysis reporting results of suitability tests.

The second approach to evaluate water quality for LC–MS analyses is to assess its level of ionic purity. Thus, for bottled water, certificates of analysis usually detail maxi-mum concentrations for certain ions because when stored in a glass bottle, the water will leach out contaminants

ES667449_lcgcAN0915_032.pgs 09.01.2015 23:32 ADV blackmagentacyan


from the container even though the water may have been of very high pu-rity immediately after production. For example, standard glass bottles leach out alkali, contaminating ultrapure water and leading to a higher count of adducts. Since the quality of bottles can vary strongly from one to another, the nature of ions selected to report in the certificate of analysis depends on the quality of the bottle used to store the water. Table III compares the spec-ifications for some metal ions in ultra-high-pressure liquid chromatography (UHPLC)–MS-grade, LC–MS-grade bottled waters from three vendors, and in fresh ultrapure water.

Because ionic impurities present in water increase its conductivity, the con-ductivity parameter, or alternatively its inverse, resistivity, can be used to char-acterize the ionic purity of water.

Thus, at 25 °C, conductivity of 0.055 µS/cm, or resistivity of 18.2 MΩ·cm, implies that the water is ul-trapure, whereas at 1 ppb of Na+ in ul-trapure water, resistivity decreases to 17.6 MΩ·cm, and at 5 ppm, dramati-cally drops down to 0.093 MΩ·cm; these values can be calculated based on the concentration of sodium, its charge, and mobility (7). Therefore, when fresh ultrapure water of 18.2 MΩ·cm resistivity measured via an in-line monitor was used to dissolve a

peptide sample (bradykinin fragment 1-7), and infused directly to a mass spectrometer, the resulting spectrum was clean (Figure 1a). The parent and fragment peaks represented only pro-tonated species. However, when the

water used was contaminated with sodium ions, the spectrum was more complex with the presence of sodium adduct peaks (Figure 1b).

The presence of metal ions can sup-press the signal of the protonated ion

Figure 1: Mass spectra of bradykinin (fragments 1-7). Direct infusion, ESI+, using (a) 96% fresh ultrapure water, 4% acetonitrile and (b) 96% 1 ppm

Na+, 4% acetonitrile.

[M-115]+

[M-115]+

m/z 538 ???

m/z 703 ???

[M+H]+

[M+H]+

[M-202]+

[M-202]+ [M-115+Na]+

[M+Na]+

4 105

(a) (b)

3.5 105

2.5 105

1.5 105

3 105

2 105

1 105

0.5 104

m/z (amu)

Inte

nsi

ty (

cps)

0

500 550 600 650 700 750 800 850 900

4 105

3.5 105

2.5 105

1.5 105

3 105

2 105

1 105

0.5 104

m/z (amu)

Inte

nsi

ty (

cps)

0

500 550 600 650 700 750 800 850 900

Table I: Direct infusion experiments

First set of experiments Second set of experiments

Sample Bradykinin (fragment 1-7)Monoisotopic Mass 756.40 Da

[M+H] ion (m/z 757)[M-115+Na] ion (m/z 664)[M-202] ion (m/z 554)

Glu1-f brinopeptide BMonoisotopicMass 1569.65 Da

[M+H] ion (m/z 1570)[M+2H] ion (m/z 785)[M+Na+H] ion (m/z 796)[M+2Na] ion (m/z 807)[M+3Na-H] ion (m/z 818)

Instrument Applied Biosystems API 2000 Waters Synapt HDMS

Ionization mode ESI+ ESI+

Flow 10 μL/min 10 µL/min

Solvents (a) 96:4 fresh ultrapure water–acetonitrile

(b) 96:4 1 ppm Na+ in ultra-pure water–acetonitrile

(a) 50:50 fresh ultrapure water–acetonitrile

(b) 50:50 1 ppb Na+ in ultrapure water–acetonitrile

(c) 50:50 100 ppb Na+ in ultrapure water–acetonitrile

(d) 50:50 1000 ppb Na+ in ultrapure water–acetonitrile

Sample concentration

1 μM 500 pM



Figure 2: (a) Mass spectra of protonated molecular ion [M+2H] of Glu1-fibrinopeptide B sample, (b) and (c) extracted ion chromatograms of

[M+Na+H] ion as examples of the effect of ionic concentration on MS detection.

100

%

0760 765 770 775 780 785 790

785.820786.822

787.835796.826

m/z

(a)

776.819 813.384 816.782 818.784

795 795 800 805 810 815 820 825 830 835 840 845 850 855 860

100

%

0785 786 787 788 789 790

785.820786.315

786.822787.339

m/z

(b)

787.835

1 ppb of sodium in the water–acetonitrile mixture TOF MS ES+2.20e5100

%

0785 786 787 788 789 790

785.820786.326

786.832787.349

m/z

787.845


%

0785 786 787 788 789 790

785.830 786.326786.832

787.339

m/z

787.835


100

%

0785 786 787 788 789 790

785.830 786.326786.832

787.339

m/z

787.835


%

0796 797 798 799 800 801

796.826 797.315

797.325

786.825

797.835

797.825

m/z

796 797 798 799 800 801m/z

796 797 798 799 800 801m/z

796 797 798 799 800 801m/z

(c)

798.314


%

0

796.826

796.826

796.316

798.335 799.605 800.616


%

0

797.325797.825 787.335 798.813 799.605 800.606


%

0

796.826797.325

797.825798.335 798.824 799.303

799.605 800.606

100

%

0760 765 770 775 780 785 790

785.820786.832

796.826

m/z

776.829799.605

859.809

795 795 800 805 810 815 820 825 830 835 840 845 850 855 860

100

%

0760 765 770 775 780 785 790

785.830786.832

787.835

m/z

807.815 813.615 827.642804.812796.826

859.798

795 795 800 805 810 815 820 825 830 835 840 845 850 855 860

TOF MS ES+1.57e5

100

%

0760 765 770 775 780 785 790

785.830

771.571

786.832

787.835

m/z

807.815818.805 827.631799.605

796.826859.603

795 795 800 805 810 815 820 825 830 835 840 845 850 855 860

TOF MS ES+1.37e5


1000 ppb of sodium in the water–acetonitrile mixture



TOF MS ES+1.86e5




peak of interest. The effect of the so-dium ion on the signal intensity of Glu1-fibrinopeptide B was analyzed by varying the ion concentration in the water–acetonitrile mixture that was used to dissolve the peptide. It was observed that signal intensity of the [M+2H] molecular ion decreased with the increase of sodium ion con-centration in the water. In parallel, it was observed that signal intensities of the sodium adducts increased with the increase in Na+ concentration (Figures 2a, 2b, and 2c).

Further signal intensities of Glu1-fibrinopeptide B such as the [M+2H], [M+Na+H], [M+2Na], and [M+3Na-H] ions were recorded for each analyzed sample and presented as a function of Na+ concentration in water (Figure 3).

Specifically, the presence of 1 ppb of Na+ decreased the [M+2H] signal intensity by 5%. But the decrease in signal was 20% with 100 ppb Na+, and 30% when it was 1000 ppb. Altogether the presence of sodium results in more complex spectra, leading to difficulties in data characterization, analyte quan-tification, and subsequently more time spent on data analysis. Therefore, using water in LC–MS analyses that is free of ions is of high importance. In addition, there are a few key considerations to keep in mind when choosing the source of ultrapure water, and also when han-dling it to minimize contamination.

Five Tips to Avoid Ionic

Contamination in LC–MS Practice

1. Choose the Best Source of Ultrapure Water Common choices are freshly produced ultrapure water from a laboratory

water purification system, and bottled water, such as UHPLC–MS grade or LC–MS grade. Fresh ultrapure water produced using an optimal combina-tion of purification technologies is usually of very high purity, with ionic levels below 1 ppb at 18.2 MΩ·cm of resistivity, which can be monitored online at the moment of water collec-tion. In the case of bottled water, spec-ifications of ionic purity for laboratory water are usually provided and can be easily consulted to assess the potential risk of ionic contamination on LC–MS analyses.

Because ultrapure water is a very aggressive solvent, it tends to absorb

ions and organic compounds from the container. Thus, maximum concentra-tions of ions in final ultrapure product water depend significantly on the level of extractables released by the contain-ers used to collect freshly produced ultrapure water, or to store it, in the case of bottled water. Table IV shows the concentration of Na+ in freshly pro-duced ultrapure water and ultrapure water after storage in a glass bottle for one day.

As shown in Table IV, the Na+ con-centration in freshly produced ul-trapure water was 0.020 ppb. After stor-age in a glass bottle, the concentration increased to 0.212 ppb.

Figure 3: Signal intensities of 500 pmol Glu1-fibrinopeptide B in 50:50 (v/v) acetonitrile–water.

The mixture was spiked with different amounts of sodium ions and injected directly into the mass

spectrometer.

200,000

150,000

100,000

50,000

818 (M+3Na-H)807 (M+2Na)796 (M+Na+H)785 (M+2H)

0 ppb 1 ppb 100 ppb 1000 ppb

0

Table II: Freshly produced ultrapure

water sources

Experiment Water Purif cation System Used

Bradykinin

direct infusion

and ICP-MS

Elix + Milli-Q Advan-

tage A10 (Q-Gard

and Quantum TEX

cartridge, Millipak

f nal f lter)

Glu1-f bri-

nopeptide B

direct infusion

Milli-Q Integral

(Quantum TEX car-

tridge)

Table III: Specifications for some metal ions in UHPLC–MS-grade or LC–

MS-grade water and fresh ultrapure water produced by a laboratory water

purification system specifically for LC–MS practice*

Maximum Concentration (ppb)

Metal Ion

UHPLC–MS Grade Wa-ter Brand X

LC–MS Grade Water

Brand Y

LC–MS Grade Water

Brand Z

Fresh Ultrapure Water for LC–MS Analyses

Aluminum 20 500 10 1

Calcium 50 100 100 1

Iron 30 100 5 1

Potassium 50 100 10 1

Magnesium 20 100 20 1

Sodium 50 100 200 1

*Specif cations of different sources of laboratory water dedicated for MS analyses were collected from off cial websites of manufacturers but their names are not provided to avoid direct comparison.



2. Choose Glassware of the Highest Quality That Leaches the Minimum Amount of ContaminationIt is a lso recommendable to have dedicated glassware for LC–MS prac-tice, and glassware should be cleaned thoroughly before use.

3. Keep Your Work Area CleanMoreover, since laboratory air is characterized by a lot of contamina-tion and as ultrapure water absorbs contamination from the air includ-ing volatile molecules (8), the work-ing area should be kept clean (9). Appropriate covers and caps for mo-bile-phase reservoirs and glassware can help to avoid contact between ultrapure water and the laboratory air, as well. It is also a good idea to wear gloves, and choose ones that are powder-free and have the few-est metal or ion extractables (for ex-ample, polyethylene) because simply touching glassware with a bare hand can transfer enough salt to cause a significant appearance of metal ad-duct ions.

4. Select Reagents and Organic Solvents of the Highest PurityHere, to evaluate the purity level nec-essary for sensitive MS applications, a certificate of analysis is a good source of information. Also, it is recom-mended to obtain details from the sup-plier beforehand concerning the level of leachables and extractables from solvent and reagent packaging.

5. Routine MaintenanceWhen using a laboratory water puri-fication system, make sure that it un-dergoes routine maintenance to ensure the highest water quality.

Ion Removal in Laboratory

Water Purification Systems

The laboratory water purif ication systems used in this study combine the technologies shown in Table V to efficiently remove different types of contaminants from tap water feed. The removal of ions is carried out by using reverse osmosis, electrodeioni-zation, and ion exchange resins.

To ensure the efficiency of the pu-rification process and make sure that the ultrapure water contains the low-est possible ionic content, resistivity monitoring can be used. Ultrapure water of 18.2 MΩ ·cm resist iv it y refers to ultrapure water free of a significant level of ions for LC–MS analyses.

ConclusionThe presence of ions in the water used for LC–MS analyses inf luences the quality of data by forming metal ad-ducts and suppressing analyte signals. To avoid ionic contamination, it is recommended to use fresh ultrapure water when preparing mobile phases. It is also important to apply best labo-ratory practices.

AcknowledgmentsThe authors would like to acknowledge Jens Baumgaertner of Merck KGaA in Darmstadt, Germany, and Maricar Tarun-Dube of EMD Millipore Corpo-ration in Danvers, Massachusetts, for their help with this article.

References(1) M. Tarun, C. Monferran, C. Devaux, and

S. Mabic, LCGC The Peak, 7–14 (2009).

(2) B.O. Keller, J. Sui, A.B. Young, and R.M.

Whittal, Anal. Chim. Acta 6(27), 71–81

(2008).

(3) M. Oehme, U. Berger, S. Brombacher, F.

Kuhn, and S. Kolliker, TrAC, Trend Anal.

Chem. 21, 322–331 (2002).

(4) A. Khvataeva-Domanov, G. Woods, and

S. Mabic, “Choosing Optimal High Purity

Water Source in Accordance to ICP-MS

Application Needs and Laboratory Envi-

ronment,” presented at the Winter Confer-

ence on Plasma Spectrochemistry, Amelia

Island, Florida, 2014.

(5) M.J. Capdeville and H. Budzinsk, TrAC,

Trend Anal. Chem. 30, 586–606 (2011).

(6) C. Regnault, I. Kano, D. Darbouret, and

S. Mabic, J. Chromatogr. A. 1030, 289–295

(2004).

(7) E. Riché, A. Carrié, N. Andin, and S.

Mabic, High-Purity Water and pH Am. Lab.

(Boston) 16, 22–23 (2006)

(8) N. Nioradze, R. Chen, N. Kurapati, A.

Khvataeva-Domanov, S. Mabic, and S.

Amemiya, Anal. Chem. 89, 4836–4843

(2015).

(9) I. Rodushkin, E. Engstrom, and D.C. Bax-

ter, Anal. Bioanal. Chem. 396, 365–377

(2010).

Anastasia Khvataeva-

Domanov and Stéphane

Mabic are with Millipore S.A.S. in

Saint-Quentin-en-Yvelines, France.

Direct correspondence to:


Table IV: Concentration of sodium

ions in fresh ultrapure water and in

the same ultrapure water that was

stored in a glass bottle for one day

Sample Na+ (ppb)

Freshly produced ultra-pure water

0.020

Ultrapure water in a glass bottle after one day

0.212

Table V: Water purification technologies used in the production of ultrapure

water from tap water

Pretreatment (f lter, activated carbon)

• Filtration• Removal of oxidizing agents

Reverse osmosis • Removal of the bulk of contaminants (95–99%): organics, ions, microorganisms, particulates

Electrodeionization • Further removal of ions, and charged organic species

UV (254 nm) • Prevents bacterial contamination

Virgin ion-exchange resins • Removal of trace amounts of ions

Synthetic activated carbon • Removal of trace levels of organics

UV (185, 254 nm) • Photo-oxidation to further remove organics

Point-of-use end f lters • Prevents particle and bacterial contamination• Allows removal of trace levels of organics



Using Compact Mass Spectrometry for Detection and Quantification of Cannabis-Related CompoundsThe transition of cannabis from an illegal drug to a drug for medical and even rec-reational use raises challenging questions for regulatory agencies and analyti-cal chemists alike. Here, we show a selection of analytical techniques based on compact mass spectrometry (MS) in combination with three different sample inlets (atmospheric solids analysis probe, thin-layer chromatography [TLC], and classical liquid chromatography [LC]) for the detection and quantification of can-nabinoids and pesticides in cannabis-related material and contraband.

Daniel Eikel, Simon J. Prosser, and Jack D. Henion

In the United States, cannabis is now on its way to become a normal crop and consequently many of the current ly discussed chal lenges that relate to

cannabis and its products have long been solved for other comparable crops. What makes cannabis unique though, and significantly more complicated, is its cur-rently fragmented legal status as well as its various forms of consumption and aims. Is it a medicine or a recreational drug? Is it a tobacco product? Is it food? Does it have parallels to alcohol? Such questions should ultimately be answered and will dictate how it will be regulated in the marketplace and what agency should be responsible for its monitoring.

One of the issues with cannabis that is often over-looked is the changing cannabinoid make up over the last decades. Cannabis produces upward of 400 com-pounds (approximately 80 compounds unique to this plant) that interact to varying degrees on the natural cannabinoid receptors in the human brain as well as the nervous and immune system (the endocannabinoid system [1]). Unfortunately, cannabis strains with ex-tremely high psychotropic tetrahydrocannabinol (THC) levels have been bred in the past for illegal recreational drug use (some reports show cannabis strains with THC content as high as 30Ð40% [2]). But those strains have little benefit for medicinal use because their potent psy-chotropic effect is undesirable for patients and because other cannabinoids, terpenes, and secondary plant me-tabolites such as cannabidiol (CBD) are down-regulated at the same time and these are likely much more impor-tant for medicinal effects (3,4).

Another emerging problem is the contamination of the cannabis product with pesticides. Because of its sta-tus as illegal drug on the federal level there is no United States Environmenta l Protection Agency (US EPA)

guidance as to what pesticide can be used during the growth of the plant or what residue level would be ac-ceptable (and monitored by the Food and Drug Admin-istration [FDA] or the U.S. Department of Agriculture [USDA]). Consequently, it is i l legal to use any pesti-cides on cannabis plants and it should be an organically grown product (5). However, a test analysis of a very small number of cannabis samples from dispensaries in Los Angeles, California, in 2009 showed that two out of three samples had significant levels of the insecticide bifenthrinÑone sample exceeded the legal limit for an edible crop by a factor of 1600 (6). It is noteworthy that the major consumption of cannabis is through smok-ingÑnot ingestionÑand it has been shown that pesti-cides can pass into the mainstream smoke at rates up to 69%, depending on smoking style (filter or filterless, waterpipe and so on) (7). In other words, two of the three samples obtained in the above small-scale study would be considered a consumer health risk. This is even more concerning in light of patients with com-promised immune systems taking medical cannabis to improve their health status.

Finally, it is important to note that despite changes in some states, cannabis is still an illegal drug in a ma-jority of states and on the federal level. The challenges described above can of course be monitored with ana-lytical chemistry workf lows that satisfy the different requirements for• simple and legally defensible cannabis detection in

contraband material;• detection of pesticides and their quantification;

• and characterization of the major cannabis compo-

nents to provide adequately labeled products that in-formed consumers can choose from where it is legal to do so.



Here, we discuss three analytical workf lows based around compact mass spectrometry (MS) with dif-ferent sample inlet systems ranging from the more recent atmospheric solids analysis probe (8), a thin-layer chromatography (TLC) extraction device, and a more classica l high performance liquid chromatography (HPLC) inlet to demonstrate cost effective and easy-to-use solutions for law enforcement, producers, and dispensaries of cannabis.

ExperimentalAtmospheric Solids Analysis Probe

and Compact MS Analysis of Contra-

band Material and Surfaces of Interest

The glass capi l lary of the atmos-pheric solids analysis probe (ASAP, Advion, Inc.) was either placed into direct contact with the contraband material or rubbed over the index finger or shirt sleeve of a volunteer that had been in contact with the materia l . The capi l lary was then placed i nto t he sou rce housi ng holder with commencement of MS acquisition. The sample inlet (Fig-ure 1) uses hot nitrogen gas to rap-idly vaporize the analytes present

on t he t ip of t he g lass capi l lar y and transports the vapor stream to a corona discharge needle where molecu les are ionized by atmos-

pheric pressure chemical ionization

(APCI). Ions are transferred into the MS analyzer through the inlet capil-

lary of the mass spectrometer. With this technique the separat ion of analytes happens exclusively during the evaporation stage and by mass-to-charge ratio in the gas phase.

TLC and Compact MS

Analysis of Cannabinoids

Cannabinoid standards and a make-up sample of a leaf extract fortified with cannabinoids were separated on TLC silica gel 60 F254 (Merck) with a developing solvent of 80:20 pet roleu m et her (60 –80 bp)–d i-oxane (Sigma Aldrich). The TLC-compact MS analysis used a Plate Express (Advion, Inc.) extraction device with a 200-µL/min solvent f low of methanol–0.1 vol% formic

Figure 1: Schematic of the ASAP ion source inlet as deployed on a single-quadrupole compact

mass spectrometer (expression, Advion Inc.).

Figure 2: Selection of cannabinoids investigated in this study.

Sample

Corona discharge needle

MS inlet

Hot N2

Probe with melting point capillary inserts

H3C

H3C

H3C

H3C

H3C

H3C

H2C

H2C

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

-∆9-THC (THC)

MW = 314.22

(C21

H30

O2)

-∆9-THC acid (THC-acid)

MW = 358.21 (C22

H30

O4)

Cannabidiol (CBD)

MW = 314.22

(C21

H30

O2)

Cannabidiolic acid (CBD-acid)

MW = 358.21

(C22

H30

O4)

Cannabinol (CBN)

MW = 310.19

(C21

H26

O2)

H

H

H

H

HH

H

H

O

O

OH

OH OH

OH

O

O

HO

HO

HO

HO

HO

O

Figure 3: Shirt sleeve (left) and index finger (right) sampled with the atmospheric solids analysis

probe after exposure to cannabis material from handling confirmed contraband material.



acid and an extraction head with an area of approximately 1 mm ×

2 mm.

HPLC and Compact MS Analysis of

Cannabinoid and Pesticide Standards

Sa mples were a na lyzed w it h an Agilent model 1220 HPLC gradient system and a 100 mm × 2.1 mm Su-pelco Titan column (Sigma-Aldrich) at a column temperature of 50 °C, a f low rate of 250 µL/min and a 5-min gradient from 35% to 90% B. Sol-vent A consisted of water–0.1 vol% formic acid, and solvent B consisted of acetonitrile–0.1 vol% formic acid. For the analysis of pesticides, the gradient was adjusted to start at 10% B.

A mass range of m/z 100 to m/z

1000 was scanned using both polar-ity switching and in-source collision-induced dissociation (CID). Selected ion monitoring (SIM) scanning in the negative ion mode MS was used at m/z 313.2 (THC and CBD) and m/z 309.2 (CBN) for the quantita-tive analysis approach for cannabi-noids and SIM scanning in positive ion mode was used at m/z 202.1 for carbaryl, m/z 222.1 for carbofuran, m/z 300.0 for captan, and m/z 331.1 for malathion (all compounds from Sigma-Aldrich). All MS methods used the expression CMS mass spec-trometer (Advion Inc.).


T he combi nat ion of t he at mos-pheric solids analysis probe and a compact mass spectrometer (Figure 1) provides a rapid analysis system that can detect the presence of can-nabis in contraband material and ot her sur faces of interest . Even short exposure of fingers and shirt s leeves to t he contraband mate-rial during its handling provided sufficient residue of cannabinoids (Fi g u re 2) to m a k e suc c e s s f u l swabs. Tests from the index finger and sleeve of a shirt (Figure 3) re-sulted in MS data in both negative and posit ive ful l-scan mode that showed the expected m/z signals for THC, CBD, CBN, and the acids of CBD and THC (the acids from

Figure 4: Atmospheric solids analysis probe and compact MS data obtained from a skin and cloth

swab. Top left shows the control for both negative and positive ion mode compact MS as well

as the skin sample exposed to cannabis contraband material (top right). Having both ionization

modes available unequivocally distinguishes the control from sample from exposed skin. Bottom

row shows exposed shirt sleeve sample positive for cannabinoids at the same m/z signals of 311.2

and 315.2 for CBN and CBD/THC for positive ion mode as well as m/z 309.2, 313.2 and 357.2 for

CBN, CBD/THC, and CBD-acid, THC-acid, respectively, in negative ion mode compact MS.

98Control Exposed skin

Exposed skin

Exposed cloth

Exposed cloth

Control

85

73

77

61

46

31

15

0

61

49

37

24

260 280 300 320 340 360 380 400

257.2

275.3

315.3

315.4

329.3

309.2

313.2

311.2

327.1

281.2

279.1

285.1267.2269.4

255.3

315.3

311.2

316.4

329.3

327.2357.4

313.2

311.2

309.2

314.3279.1

372.3

373.2

311.2

313.4355.2

281.2

255.2

253.2

267.2269.2

279.2

283.2

295.2313.3

311.2

371.3

12

89

1.5E9

1E9

500E6

500E6

500E6

%

% %

71

54

36

18

0

89

%

71

54

36

18

0

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

m/z

260 280 300 320 340 360 380 400m/z

260 280

280 300 320 340 360

300 320 340 360 380 400m/z

m/z

280 300 320 340 360m/z

260 280 300 320 340 360 380 400m/z

Figure 5: Schematic of the extraction device used to sample directly from developed TLC plates

and analyze spots of interest in a mass spectrometer (left, Plate Express, Advion, Inc.). On the

right, a TLC plate is shown after separation of a cannabinoid standard mixture, marking off three

analyte spots for CBN, THC, and CBD under UV light (Rf 0.41, 0.44, and 0.47, respectively) and

extraction of one selected area representing THC.

Compact mass spectrometer

Six-portvalve

Isocratic pump1–200 µL/min80:20 Acetonitrile–0.1% formic acid

TLC plate

Sealing face

Rf

0.47

0.44

0.41Elution head



skin were not detectable because of ion suppression, presumably from fat t y acids—Fig ure 4). The con-traband material itself resulted in very strong mass spectrometry sig-nals at the expected m/z (data not shown). The method is therefore sufficiently sensitive and selective for rapid screening of cannabinoids present in either raw material, sus-pect contraband, or other foren-sic samples. It is noteworthy that a compact MS system can also be outfitted for transportability by ap-plying a mobile attenuation module, which would make the compact MS system comply with MIL-STD-810 (rev G) and allow for mobile analy-sis systems f it for f ield work. The atmospheric solids analysis probe and compact MS work was executed in a United States Drug Enforce-ment Agency (DEA)-approved lab-oratory with a real life contraband sample.

Further analysis using a simple form of chromatographic separa-tion by thin-layer chromatography (TLC) showed that cannabinoids can be separated from each other and the matrix components (Figure

Figure 7: (a) Typical HPLC–compact MS chromatogram for the cannabinoid analytes THC, CBD, and CBN in negative ion mode single reaction

monitoring (SRM) with the upper trace being the extracted ion chromatogram (XIC) of m/z 313.2 and the lower trace XIC of m/z 309.2. Good

linearity calibration functions can be obtained for all three compounds ([b] THC, [c] CBD, and [d] CBN) covering a range from 2.5 to 250 ng on

column—sufficient to analyze plant material with as little as 0.1% w/w content.

2E6

2.0E+07

1.5E+07

1.0E+07

5.0E+06

0.0E+000 50 100 150 200 250 300

1.5E6

1E6

0.00 1.67 3.33 5.00

Time (min)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Peak a

rea (

cts)

6.67 8.33 10.00

0.00

0 50 100 150 200 250 300

1.67 3.33 5.00

Time (min)

Amount on column (ng)

0 50 100 150 200 250 300


6.67 8.33 10.00

500E3

CBD, Rt = 6.7 min

(a) (b)

Peak a

rea (

cts)


(c) (d)

THC, Rt = 7.9 min

Tetrahydrocannabinol THC

Cannabinol CBNCannabidiol CBD

CBD, Rt = 7.5 min

y = 68,280.68x + 629,386.31

R2 = 0.97

y = 1,410.685.97x + 19,611.383.59

R2 = 0.97y = 184,676.74x + 75,870.46

R2 = 0.98

20E6

6.0E+07

5.0E+07

4.0E+07

3.0E+07

2.0E+07

1.0E+07

0.0E+00

Peak a

rea (

cts)

4.0E+08

3.5E+08

3.0E+08

2.5E+08

2.0E+08

1.5E+08

1.0E+08

0.0E+00

5.0E+07

15E6

10E6

5E6

0E0

Figure 6: Top trace shows the CMS full scan data for the extracted THC TLC spot indicated in

Figure 5 with the characteristic (M-H)- m/z at 313.2. Bottom trace shows the in-source CID full-

scan MS data obtained from the same TLC spot. Fragmentation information can greatly add to

the identification and characterization of natural products in the TLC and compact MS workflow.

Inte

nsi

ty (

%)

Inte

nsi

ty (

%)

95

87

72

58

43

29

14

0100 200 300 400

313.2

316.9

245.1203.1

187.0

181.0

191.1

358.2

313.2

191.1

314.2

179.1

113.1

248.9

81

68

54

41

27

14

0100 200 300 400

m/z

m/z

TLC–MS

In-source CID-MS



5). Using an on-l ine TLC extrac-tion device, spots of interest can be transferred to the mass spectrom-eter for in depth interrogation, for

example, Figure 6 shows the analy-sis of the corresponding THC spot at Rf 0 .4 4 in negat ive ion mode MS with addit iona l informat ion

by in-source fragmentation. How-ever, THC and CBD (as well as their acids) are isobaric compounds and cannot be dist inguished by mass

Figure 8: Typical HPLC–compact MS selected ion monitoring (SIM) chromatogram of analytical standards of four example pesticides

(m/z 202.1 for carbaryl eluted at a 6.56 min retention time, m/z 222.1 for carbofuran at 6.48 min, m/z 300.0 for captan at 5.94 min,

and m/z 331.1 for malathion at 7.27 min). The calibration function for all four pesticides selected show good linearity in the 1–500 ng

on column range (data not shown) and the given example represents a level of 10 ppm in cannabis sample plant leaf material. Such

sensitivity would be sufficient for detection and quantification of pesticides cannabis if a maximum residue tolerance levels were

established.

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

Inte

nsi

ty (

c/s)

1.5E6

1E6

0.00 3.33 6.67 10.00 13.33

HN

HN

O

O

N SCl

ClCl

H

H

OO

O O

O

Carbarylm/z 202.1 at 6.56 min

Carbofuranm/z 222.1 at 6.48 min

Captanm/z 300.0 at 5.94 min

Malathionm/z 331.0 at 7.27 min

500E3

Time (min)

40E6

20E6

0.00 3.33 6.67 10.00 13.33

Time (min)

1E6

50E3

0.00 3.33 6.67 10.00 13.33

Time (min)

O O

OO

O

O

P

S

S

5E6

0.00 3.33 6.67 10.00 13.33

Time (min)



alone. In positive ion mode ioniza-

t ion, even their fragmentation is identical (data not shown, compare to reference 10) and only negative ion mode fragmentation offers some d is t i nc t ion si nce CBD requ i res higher collisional energy to result in the same fragment ions. The ad-vantages of a TLC and compact MS natural product analysis l ie in its low cost and simple integration into standard laboratory workf lows with a quick and unequivocal compound identification; however, quantifica-tion is not usually done by TLC and compact MS.

For a more det a i led c a n nabi-noid make-up analysis, a chroma-tographic separation was therefore used combining classica l HPLC coupled with compact MS analysis. With this technique, we were able to obtain a separation of all inves-tigated analytes either in the time or mass-to-charge domain within a 5-min gradient HPLC run. Result-ing calibration functions showed a good linear correlation (Figure 7) with an assay sensitivity sufficient to identify cannabinoid components with as little as 0.1% (w/w). With the same system setup, but a sl ight ly different gradient start point, a se-lection of four example pesticides can also be detected at the 10 ppm level (Figure 8), which is entirely suff icient to screen for i l legal pes-ticide use.

Because Advion does not have a DEA licensed facility and our home state of New York has not legalized

cannabis, the TLC–compact MS and HPLC–compact MS work was car-ried out with analytical standards as well as maple tree leaf samples only. Typical alternatives to meas-ure the cannabinoid make-up could use gas chromatography (GC), usu-ally in combination with MS detec-tion and electron impact ionization

(11–13). However, these methods tend to require more extensive sam-ple preparation and derivatization

compared to LC–MS.

ConclusionCompact mass spectrometry inter-faced with suitable sample introduc-tion techniques can be part of an efficient workf low for screening of cannabinoids in contraband materi-als. This approach is also amenable to the quantification of cannabinoid composition and the detection and quantif ication of pesticides al low-ing compliance with current regu-latory guidelines. The approach is cost effective, easy to perform, and can be transported to the field. We suggest this approach can be use-ful for both law enforcement and forensic applications as well as for growers and dispensers of cannabis where it is legal. In particular, the product character izat ion of can-

nabis seems an inevitable necessity given the vast dif ferences in can-nabinoid ma ke-up bet ween ca n-nabis strains and its signif icantly dif ferent aim (and desirable com-position) between recreational and medical use. Pesticide detection is also a requirement to help charac-terize the origin of the cannabis

material in question and should be part of the product characterization

to improve consumer health protec-tion, particularly when considering cannabis for medical use.

AcknowledgmentsWe would like to thank Dr. George Mailin and the Morris State College Equine Drug Testing and Research program for the opportunity to an-alyse authentic cannabis forensics samples in its DEA licensed facility. We also thank LuAnn Yaerik from Sigma-Supelco for the generous gift of a Titan HPLC column as well as Changtong Hao, Jamey Jones, and Amy Boardman-Lummus from Ad-vion Inc., for their assistance during this project.

References(1) A.A. Izzo, F. Borrel li, R. Capasso, V.

DiMarzo, and R. Mechoulam, Trends

Pharmacol. Sci. 30(10), 515–527 (2009).

(2) A. LaFrate, “Marijuana Testing Yields

Fascinating Results,” presented at the

249th National Meeting & Exposition of

the American Chemical Society (ACS),

Denver, Colorado, 2015, http://bit.ly/

MarijuanaTestingACS.

(3) E .B. Russo, Br. J. Pharmacol . 163,

1344–1464 (2011).

(4) Understanding Medical Cannabis 2nd edition

(Steep Hill Halent, Elemental Wellness, San Jose,

California, 2014) https://drive.google.com/file/

d/0B3DPJKkBZdbYdjh3ZDJXY1U4OWM/

edit?usp=sharing/&pli=1.

(5) Thomson, “Medica l Marijuana Cul-

tivation and Policy Gaps,” California

Research Bureau 2012.

(6) N. Skeet, “City Attorney Explains Medi-

cal Marijuana Issue on NBC,” http://lac-

it yorgat t y.blogspot.com/2009/10/cit y-

attorneyexplains-medical.html, 2009.

(7) N. Sullivan, S. Elzinga, and J.C. Raber,

Journal of Toxicology (2013) http://dx.doi.

org/10.1155/2013/378168.

(8) C.N. McEwen, R.G. McKay, and B.S.

Larsen, Anal . Chem. 77, 7826 –7831

(2005).

(9) United Nations Off ice on Drugs and

Crime, “Recommended Methods for

the Identif ication and Analysis of Can-

nabis and Cannabis Products” (2009).

Ava i lable at : ht tps://w w w.unodc.org/

documents/scient i f ic /ST-NA R-40-Eb-

ook.pdf.

(10) S. Broekner, S. Herre, and F. Pragst,

Forensic Sci . Int. 10(218[1–3]), 68–81

(2012).

(11) J. Omar, M. Olivares, J.M. Amigo, and

N. Etxebarria, Talanta 121, 273–280

(2014).

(12) J. Omar, M. Olivares, M. Alzaga, and

N. Etxebarria, J. Sep. Sci. 36(8), 1397–

1404 (2013).

(13) B.K. Logan, L.E. Reinhold, A. Xu, and

F.X. Diamond, J. Forensic Sci . 57(5),

1168–1180 (2012).

Daniel Eikel is the product

manager with Advion Inc., in Ithaca,

New York. Simon J. Prosser is

the vice president of marketing with

Advion Inc. Jack D. Henion is

the chief scientific officer with Advion

Inc. Direct correspondence to:



THE APPLICATION NOTEBOOK

Call for Application Notes

LCGC is planning to publish the next issue of

T e Application Notebook special supplement in

December. T e publication will include vendor

application notes that describe techniques and

applications of all forms of chromatography and

capillary electrophoresis that are of immediate in-

terest to users in industry, academia, and govern-

ment. If your company is interested in participat-

ing in these special supplements, contact:

Michael J. Tessalone, Group Publisher,

(732) 346-3016

Edward Fantuzzi, Associate Publisher,

(732) 346-3015

Stephanie Shaf er, East Coast Sales Manager,

(774) 249-1890

Lizzy T omas, Account Executive,

(574) 276-2941

Application Note Preparation

It is important that each company’s mate-

rial f t within the allotted space. T e editors

cannot be responsible for substantial editing

or handling of application notes that deviate

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Each application note page should be no more

than 500 words in length and should follow

the following format.

Format

• Title: short, specif c, and clear

• Abstract: brief, one- or two-

sentence abstract

• Introduction

• Experimental Conditions

• Results

• Conclusions

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• Two graphic elements: one is the company

logo; the other may be a sample chromato-

gram, f gure, or table

• T e company’s full mailing address,

telephone number, fax number,

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All text will be published in accordance with

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matical accuracy, although the content will not

be edited. Text should be sent in electronic for-

mat, preferably using Microsoft Word.

Figures

Refer to photographs, line drawings, and

graphs in the text using arabic numerals in

consecutive order (Figure 1, etc.). Company

logos, line drawings, graphs, and charts must

be professionally rendered and submitted as

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of 300 dpi. Lines of chromatograms must be

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tion. Provide peak labels and identif cation.

Provide f gure captions as part of the text,

each identif ed by its proper number and title.

If you wish to submit a f gure or chromato-

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Each table should be typed as part of the main

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T e deadline for submitting application notes for the December issue of T e Application Notebook is:

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Table I: Factor levels used in the designs

Factor Nominal value Lower level (−1) Upper level (+1)

Gradient profile 1 0 2

Column temperature (°C) 40 38 42

Buffer concentration 40 36 44

Mobile-phase buffer pH 5 4.8 5.2

Detection wavelength (nm) 446 441 451

Triethylamine (%) 0.23 0.21 0.25

Dimethylformamide 10 9.5 10.5

Figure 1: Chromatograms obtained using the conditions under which the ion sup-pression problem was originally discov-ered. The ion suppression trace is shown on the bottom. Column: 75 mm × 4.6 mm ODS-3; mobile-phase A: 0.05% heptaf uo-robutyric acid in water; mobile-phase B: 0.05% heptaf uorobutyric acid in aceto-nitrile; gradient: 5–30% B in 4 min. Peaks: 1 = metabolite, 2 = internal standard, 3 = parent drug.



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