The worldwide newsletterfor capil lary electrophoresisVolume 6, Issue 1 • April 2002

DR. VERONICA WILEY

NSW NEWBORN SCREENING

PROGRAMME, THE CHILDREN’SHOSPITAL AT WESTMEAD,NEW SOUTH WALES, AUSTRALIA

Phenylketonuria (PKU) is arecessively inherited geneticdisorder manifested by a

compromised catabolism ofthe amino acid phenyl-alanine. The phenyl-alanine–tryosine pathwayincludes enzymatic stepsthat are particularly prone togenetic alteration. The firstenzyme in this pathway,phenylalanine 4-monooxy-genase (also called phenyl-alanine hydroxylase), catalyzesthe hydroxylation of phenyl-alanine to tyrosine. A defectin this enzyme forces catabo-lism through another path-way in which the amino acidundergoes transaminationwith α-ketoglutarate to yieldphenylpyruvate, a productthat is not metabolizedfurther. The buildup of phenylpyruvatein the blood and tissues early in lifeimpairs normal development of thebrain and causes severe mental retar-dation. If detected early, this disordercan be treated with a diet low inphenylalanine that will allow for nor-mal growth and development. It iscritical, though, that this deficiency isdetected early and treatment startedwithin the first weeks of life.

ANALYTICAL APPROACHESIn our lab, we have been develop-

ing a capillary electrophoresis-basedassay to quantitate phenylalanine incombination with tyrosine todifferentiate secondary causes ofhyperphenylalaninaemia. The follow-ing describes the methodology thatwe have developed.

NORMAL REFERENCE RANGEPhenylalanine Reference:

<200 µmol/L whole blood in infants<100 µmol/L whole blood afterfirst month of life

Tyrosine Reference:<500 µmol/L whole blood in infants<200 µmol/L whole blood afterfirst month of life

MATERIALS AND METHODSAnalysis is performed from a drop

of blood spotted onto filter paper.Amino acids are eluted from the driedblood spots with an alcohol solutionwhich also serves to precipitateprotein. This extract is then introducedinto the capillary electrophoresissystem where aromatic amino acids are

separated by micellarelectrokinetic capillarychromatography (MECC) anddetected by UV absorbance at200 nm.

EQUIPMENT USED

Hand punch (3 mm), tubetrimmer, air displacementpipette, shaker, centrifuge,ultasonicator, and theP/ACE™ 5000 System fromBeckman Coulter.

REAGENTS

Extraction Solution:70% absolute ethanol indistilled deionized water(ddH2O)

Borate Stock Buffer:233 mL of 0.1511 M sodiumtetraborate, 40 mL of

0.3875 M boric acid

EDTA—Stock Solution: 0.095 M

SDS—Stock Solution: 200 mM

H2SO4 Stock Solution: 1 M

NaOH Stock Solution: 1 M

Internal Standard:n-acetyl Anthranilic acid 2.5%

Quantitating Phenylalanine and Tyrosinefrom Dried Blood Spots

2

Phenylalanine and TyrosineStandards diluted to 100 µmol/L,300 µmol/L, 500 µmol/L,1,000 µmol/L, and 2,000 µmol/L

RUN BUFFER PREPARATION

See Table 1.

SAMPLE PREPARATION:1. Using the hand punch, punch

3 (3 mm) spots of each sample,control and standard (from filterpaper) and place into 1.5 mLmicrofuge tubes.

2. Using an air-displacement pipette(Gilson P100), add 50 µL of extrac-tion buffer to each tube.

3. Cap the tubes, place them onto theshaker, and allow samples to elutefor 1 hour.

4. Centrifuge tubes for 10 minutes at5,000 rpm.

5. In a new tube, add 30 µL internal standard + 30 µL ofeither the standard, control, or sample.

CAPILLARY ELECTROPHORESIS CONDITIONS

Detection: UV Detector—200 nm filter—10 nm bandpassCapillary: Bare fused-silica capillary, 75 µm x 37 cm

(30 cm to detector)Run pre-rinse 1: 1 min with ddH2ORun pre-rinse 2: 1 min with run buffer Applied Voltage: 12 kV for 12 minutesPost run rinse:

1.5 min with 0.1 M sulphuric acid1 min with ddH2O1.5 min with 1M NaOH

RUN STRATEGY

Samples are run in batches which include 5 sampleunknowns, a 5-point calibration curve for quantitatingphenylalanine, two positive PKU controls, and onetyrosine control. The tyrosine control gives a hightyrosine peak and a high phenylalanine peak and is keyin the confirmation process.

RESULTS AND CONCLUSIONSFigure 1 represents an 8-point calibration curve of

phenylalanine showing good linearity of detectionbetween 5 µmol/L and 2000 µmol/L which is the quantita-tive range with which we operate.

The assay shows good quantitative reproducibilitywhich is important for the development of a PKUconfirmation strategy.

Figure 2 represents an electropherogram from asample with elevated phenylalanine levels. Note the lowlevel of tyrosine relative to the phenylalanine levels—allowing us to discriminate PKU from other conditionswhich concomitantly elevate tyrosine.

Volume 6, Issue 1 • April 2002

Table 2.Assay Quantitation Specifications

Parameter Quantity C.V.

Limit of Detection < 5 µmol/L 2.7

Precision

Intraassay 100 106 9.7

500 576 3.2

2000 1956 7.3

Interassay 100 101 7.3

500 512 6.3

2000 2001 4.3

Table 1. Run Buffer Preparation

Solution Final Concentration Volume (ml)

Stock Borate Buffer 50 mM 38.76 mL

Stock SDS Solution 50 mM 25.00 mL

Stock EDTA Solution 1 mM 1.05 mL

Acetonitrile 5% (w/v) 5.00 mL

ddH20 Water Add to 100.00 mL

Phenylalanine ( mol/L) x 100

Are

a

2.5

2

1.5

1

0.5

00 2 4 6 8 10 12 14 16 18 20

Figure 1. Linearity of detection.

3

LUO GUOAN, WANG YIMING,QU JUN, WANG SHOUGANG

DEPARTMENT OF CHEMISTRY,TSINGHUA UNIVERSITY

BEIJING, CHINA

INTRODUCTION

Tooth caries are one of themost common dentalproblems in children.

This process is thought tobe a result of acidic secre-tions from bacteriaresiding in the aperturesof the teeth. Cationswithin dental plaque arethought to affect thegrowth of bacteria thatreside in caries.Therefore, it is of greatimportance to study the cations indental plaque. In this study, wepresent a new method for the rapid

analysis of cations residing in toothcaries. This is a capillary electropho-

resis- (CE) basedmethod optimized

for the analysis ofK+, Na+, Ca2+,

and Mg2+.

EXPERIMENTAL CONDITIONS

EQUIPMENT AND REAGENTS

The analysis was carried out witha P/ACE™ 5000 Series HPCE fromBeckman Coulter equipped with aUV detector and a 50 µm i.d. x 47 cmbare fused-silica capillary (Yongnianoptical fiber factory, Hebei, China)with an effective length of 40 cm.The size of the detection windowwas established as 100 x 800 µm.Data acquisition and processing wereachieved with System Gold® softwarefrom Beckman Coulter. A φ12 acido-meter from Beckman Coulter was

employed for the preparation ofthe buffer.

Standards of KCl, NaCl, MgCl2,and CaCl2 (99%, w/w) were obtainedfrom Sigma Chemical (St. Louis,

MO). Methanol (HPLC grade)was obtained from FisherScientific, Inc. (Hong Kong).

Assaying Cations from Dental Plaque Extracts

In summary, we have developed a robust assay capa-ble of monitoring whole blood phenylalanine levelsover time. The assay is able identify phenylalanine, tyro-sine, and other phenylalanine metabolites isolated fromdried blood spots and can reliably quantitate below thelevel of 120 µmol/L.

You can contactDr. Wiley by e-mail atveronicw@chw.edu.au

Editor’s Note:The P/ACE™ Series CapillaryElectrophoresis Systems have been manufactured asResearch Use Only platforms.

Figure 2. Sample highlighting elevated blood phenylalanine levels relative to tyrosine.

The dental plaque samples wereprovided by the Chinese Institute ofTraditional Chinese Medicine.

STANDARD SOLUTIONSThe standard solutions of cations

were prepared by dissolving KCl,NaCl, MgCl2 and CaCl2 in ddH2O,then the solutions were diluted to givea working concentration of 10 mM.

CE CONDITIONS

The buffer contained 10 mM ben-zylamine, 10 mM α-hydroxyaceticacid, and 40% (v/v) methanol. The pHvalue was adjusted to 4.0 with HCl.

The capillary chamber wasthermostated at 25°C using arecirculating liquid coolant and thesample was introduced at the anodeby positive pressure. The sampleintroduction parameters were 107 Pax 2 s. The separation voltage was25 kV. An indirect UV detectionmethod was employed at 214 nmwith a rise time filter of 0.1 s and anacquisition data rate of 10 Hz.

To ensure the reproducibility ofmigration time, the capillary wasrinsed daily with 0.1 M NaOH, 0.1 MHCl, water, and buffer, each for10 minutes. The capillarywas also rinsed with bufferfor 2 minutes followingtwo injection sets.

RESULTS AND

DISCUSSION

OPTIMIZING CECONDITIONS

The mobility of K+, Na+,Ca2+, and Mg2+ are all affect-ed by the concentration ofmethanol, benzylamineand α-hydroxy-acetic acidin the buffer. Since theinfluence of these com-pounds on the cationswere dissimilar, the CEconditions should be opti-mized in order to meet the

demands of the mixtureyou are assaying. In theseexperiments, we foundthat, although all of thethree components canaffect cation mobility, themethanol concentrationhad much more influenceon the selectivity ofCa2+/Mg2+ than the othertwo cations. The migrationorder of Ca2+ was prior tothat of Mg2+ when the con-centration of methanolexceeded 60% (v/v). How-ever, this migration orderreversed when theconcentration of methanolwas lower. A goodseparation of the cations of interestwas obtained by employing amethanol concentration of 40% (v/v).In this case, there was no need tomodulate the other two buffercomponents.

An electropherogram of the fourcations of interest extracted fromdental plaque is shown in Figure 1.

STANDARD CURVE

A serial dilution of standardsolutions of KCl, NaCl, MgCl2+, and

CaCl2+ were created for the con-struction of a standard curve. Theregression equations [peak–area ratio(y) vs. concentration (x) in µg/mL],their corresponding correlation coef-ficients, and the linear ranges ofthese cations are shown in Table 1.

QUANTITATION OF THE FOUR

CATIONS IN THE EXTRACT

The precision of the methodwas measured by experimentallyanalyzing the peak area from eachsample five times with the results

shown in Table 2. Theconcentrations of the fourcations in the extract areshown in Table 3.

CONCLUSIONIn this study, we

successfully established amethod for the quanti-tation of four commoncations: K+, Na+, Ca2+, andMg2+ in dental plaqueextract by CE. This will bepart of an overall strategyto assay factors impactingtooth caries in children.

4

Volume 6, Issue 1 • April 2002

Table 1. Regression Equations, Correlation Coefficients,and Linear Ranges of the Four Cations

Cation Regression Equation Correlation Linear Range

Coefficients (µg/mL)

K+ y = 0.0148x + 0.0297 0.9998 0.4–40

Na+ y = 0.0821x - 0.0257 0.9996 0.2–20

Ca2+ y = 0.0438x + 0.0035 0.9996 0.2–20

Mg2+ y = 0.1463x - 0.0023 0.9980 0.1–15

Table 2.The Precision of Four Cations (n=5)

Cation K+ Na+ Ca2+ Mg2+

Average peak area 0.017037 0.039873 0.014937 0.006717

R.S.D.% 0.178 0.515 0.113 0.113

Table 3. Concentration of the Four Cations in the Dental Plaque Extract

Cation K+ Na+ Ca2+ Mg2+

Concentration (mM) 0.030 0.0033 0.0084 0.0010

Figure 1. An electropherogram of dental plaque extract under optimumseparation conditions. CE conditions: 10 mM benzylamine, 10 mMα-hydroxyacetic acid, and 40% (v/v) methanol; pH 4.0;Voltage: 25 kV;capillary chamber temperature: 25 C; sample introduction: 107 Pa x2 seconds; UV detection @ 214 nm; acquisition frequency: 10 Hz.

5

ROLAND CHEVIGNÉ AND

FRANÇOIS DE L’ESCAILLE,ANALIS S.A. BELGIUM

The aim of a good genericanalysis strategy is to ensurethat a broad spectrum of

analytes can be resolved using as fewpermutations on a method aspossible. A case in point is the enan-tiomer methods developmentstrategy proposed by BeckmanCoulter(1), in which a single genericmethod using three forms of highlysulfated cyclodextrins (HSCDs) hasbeen shown to resolve a broad spec-trum of molecules. However, whatoptions are available to you if thisgeneric methodology fails? Thefollowing is a case-in-point exampleof a simple permutation of themethod that you may want to consid-er should no peaks appear.

Phenylglycinol is a substanceused as starting reagent for pharma-ceutical synthesis. We were recentlychallenged with the task ofdeveloping a method to resolve theenantiomers of this molecule. As wehad already demonstrated that theHSCD strategy could easily resolvethe enantiomers of 1-phenylethylalcohol, 1-phenyl-2-propanol, and1-phenylethyl amine, we felt veryconfident that this approach wouldwork well for phenylglycinol.However, to our surprise, no peakscould be detected.

Fortunately, there was a ratherelegant solution to what appearedinitially to be a problem. At pH 2.5,phenylglycinol has a positive chargeand a very high mobility, migratingvery rapidly towards the cathodic orinlet buffer vial. It was ourhypothesis that, in the above

example, the HSCD has minimal timeto interact with phenylglycinol andwas migrating off the capillary.To test this hypothesis we offerthe following suggestion: after theprimary sample injection, simplyintroduce a secondary injection ofbuffer plug using the run buffer(30 seconds at 0.1 psi). If our hypo-thesis holds true, this should allowmore time for the sample to interactwith the charged cyclodextrins.

RESULTSWith this enhancement

in place, we resolved theenantiomers of phenylglycinolwith a resolution (USP) of 1.5(see Figure 1). Got peaks? Now we do.

REFERENCE1. Methods Development Strategy for

Enantiomer Analysis Using the

P/ACE™ MDQ Chiral System. Applica-

tion Information Bulletin A-1889A,

Beckman Coulter, Inc. (2001).

Minutes

7.5 8.0 8.5 9.0 9.5 10.0

AU

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

D L

NH2

C OH2OH

H

Got Peaks?

Figure 1. Resolution of phenylglycinol enantiomers.

6

Volume 6, Issue 1 • April 2002

JOHN C. HUDSONa, ALBERT F.CHENb, AND JEFF D. CHAPMANb

aR.C.M. POLICE, TOXICOLOGY

SERVICES, REGINA,SASKATCHEWAN, CANADAbBECKMAN COULTER, INC.

INTRODUCTION

Capillary electrophoresis (CE)continues to be investigatedby many as a problem-solving

technology, resolving analytes thathave been notoriously difficult byother mechanisms of separation. Inthe last 14 years of commercial CE,this technology has found its wayinto routine analytical use for a broadspectrum of analytes – most sharingthe common traits of being chargedand polar. These assays haveincluded:• Cations, anions, and organic acids• Proteins, peptides, and

carbohydrates• Nucleic acid analysis• Enantiomer purity• Basic drug analysis, for the

purposes of purity determination,pharmacokinetic profiling, andcomprehensive drug screening.

In the field of toxicology, CE hasenabled the development of muchmore comprehensive drug screeningstrategies. This orthogonal approachprovides much greater confidence inanalyte identification. The net resultshave been fewer false negatives andvirtually an elimination of falsepositives, ultimately identifying an“unknown” more rapidly andefficiently. The strategy to which werefer is the low pH separation ofbasic drugs utilizing both an analyte’smobility and spectral signature toidentify an unknown (Hudson et al.).Figure 1 diagrammatically representsthe fundamental process by whichthis identification process is achieved.This assay exploits the high degree ofprecision and selectivity provided by

an analytes mobility coupled with aconfirmation element comprised ofmatching the UV spectra with highconfidence at a defined pH. Thisapproach has simplified the interpre-tation of results as theelectropherograms are significantlycleaner (much owing toelectrokinetic sample introduction),and has provided resolution ofcompounds that have literally stuckto the injection systems of other ana-lytical tools. This methodology alsoresolves polar metabolites from theparent drug, all within the sameanalysis—a valuable tool in toxicologyor any drug metabolism studies.

Once an unknown is tentativelyidentified, the next step becomesone of confirmation. Mass spectro-metry (MS) has been a key tool in

forensic science for the purpose ofconfirming an analyte’s identity.Important to this effort has been thecombination of MS with a front-endseparation technique, resolvinganalytes prior to MS detection andcharacterization. This approachimproves the efficiency of analyteionization and simplifies the interpre-tation of the results. In this study, wepropose the use of CE with tandemmass spectrometry as a valuable path-way towards the confirmation of ananalyte. We examine real caseextracts that have previously beenassayed using the CE–diode arraydetection (DAD) screen and evaluatethe applicability of CE–MS/MS in theconfirmation of these results.

Drug Confirmation with CE-MS/MS

Trazodone DetectedAutomatically

Mobility Search UV Library SearchDrug MobilityFentanyl 1.596Carvedilol 1.588Prenylamine 1.576Mefloquine 1.567Nialamide 1.567Procaterol 1.567

Trazodone 1.562

Encainide 1.561Flecainide 1.561Methylfentanyl 1.557Droperidol 1.554Diltiazem 1.551Nadolol 1.550

Figure 1. Drug identification study using mobility and spectral signature.

7

EXPERIMENTALThe CE-DAD screening experiments

were run by John Hudson, R.C.M.Police Toxicology Services, Regina,Saskatchewan. The methodologyused consisted of the following:

CE instrument: P/ACE™ MDQ withDAD detection (Beckman Coulter)

System software: 32 Karat™

software, version 4.0

Capillary: 75 µm x 50 cm todetector, 60.3 cm total length(bare fused-silica)

Capillary temperature: thermostat-ted, recirculating FluorInert*,25°C

Separation buffer: 100 mM sodiumphosphate buffer, pH 2.38

Sample introduction: Electro-kinetic, 10 kV for 8 seconds

Field strength: 25 kV, generatingapproximately 88 µamps current

Samples: Case extracts courtesy ofR.C.M. Police Toxicology Services

Sample preparation: Liquid/liquidextraction from whole blood, asdescribed elsewhere (Hudson, et al.)

The CE-MS/MS confirmationexperiments were by run by AlbertFu-Tai Chen, Beckman Coulter, usingthe same sample extracts as above.

Instrumentation: P/ACE MDQ withDAD Detection and MS externaldetector adapterThermo Finnigan, LCQ Advan-tage, Ion trap—MS/MS system

System software: ExcaliburSoftware, with P/ACE MDQControl Module, ver 1.0

Capillary: 75 µm x 80 cm to electro-spray inlet

Capillary temperature: Thermo-statted to electrospray inlet, recir-culating FluorInert, 25°C

Separation buffer: 100 mM sodiumphosphate buffer, pH 2.38

Sheath liquid: 0.5% acetic acid,80:20 methanol/water, infused5 µL per minute

Sample introduction: Positive pres-sure, 0.5 psi for 5 seconds; sheathgas off; spray voltage off

Electrospray: -4.5 kV

Applied voltage: 20 kV at sampleinlet, approx 43 µA currentgenerated across the capillary(net field strength: 181.25 V/cm)

RESULTS AND DISCUSSIONIn this study, we explored the use

of CE–MS/MS as a confirmationmethod for analytes identified usingthe R.C.M. Police CE-basedtoxicology screen. For this purpose,we used complex whole-blood

extracts from coded case studies andapplied a CE–MS/MS confirmationstrategy to the analysis. This strategywas treated as a confirmation—thatis, we had prior knowledge of the CEscreening results prior to CE–MS/MSanalysis. This allowed us to focus theanalysis on the molecular ions thatwe were trying to confirm.

The first whole-blood extractexamined was that from case 00-638.Figure 2 contains the UV electro-pherogram generated from the initial

Figure 2. CE-DAD screen of case extract 00-638.

Figure 3. Nefazodone metabolites.

8

Volume 6, Issue 1 • April 2002

sample screen, and highlights theidentification that the assay automati-cally assigned. In this case the assayused mobility and spectral signatureto detect the presence of nefazodonealong with two of its apparent meta-bolites, hydroxy-nefazodone andm-CPP. The internal standard,methoxamine, was used both as a

quantitative control and as areference standard for mobility deter-mination. The metabolic patternidentified here is consistent with theknown metabolic pathway fornefazodone, which is illustrated inFigure 3. To test the potential forCE–MS/MS to confirm this result, wesubjected the same sample extract to

the CE–MS/MS conditions describedabove. Figure 4 represents the resultfrom this experiment. In this case,the molecular ions (H+) wereconsistent with that of methoxamine(IS), nefazodone, OH-nephazodone,and m-CPP, and were all confirmed.While the left panel shows the datafrom monitoring the single molecularions consistent with thesecompounds, the right panelhighlights the high-resolution scan ofboth nefazodone and its polar metabo-lite, OH-nefazodone.

The second case that weexamined was that of 2001-2249.1.The CE–DAD electropherogram forthis case is presented in Figure 5. Inthis example, the “rave” drugs MDMA[3,4-methylenedioxymethamphet-amine], known on the street as Ecsta-cy, and Ketamine [2-(2-chlorophenyl)-1-(methylamino)-cyclohexanonehydrochloride], a common veterinaryanesthetic, known at these venues as“Special K,” were easily identified.This type of identification is knownto be problematic for GC–MS asMDMA and its derivatives are volatileand its metabolites are highly polar.With this methodology, analytes arerapidly resolved and identifiedwithout the need for special samplederivatization. The mobilities areidentified with a high degree ofconfidence and the UV spectra arematched with a high degree of confi-dence (as illustrated within the insetgraphs). The CE–MS and CE–MS/MSdata from this same case is shown inFigures 6 through 8.

In Figure 6, molecular ions consis-tent with methoxamine (internalstandard), MDMA and Ketamine areall identified. However, in this case,we clearly see two components witha molecular ion of 194 (H+) forMDMA, and two components with amolecular ion of 212 (H+) for theinternal standard methoxamine.Interestingly, the first molecular ion

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OH-Nefazodone

Figure 4. CE–MS of Sample 00-638.

Figure 5. CE-DAD screen of case extract 2001-2249.1.

9

of methoxamine appears to co-migratewith the second molecular ion ofMDMA. In an attempt to better under-stand this molecular ion profile, wefurther employed an MS/MS experi-ment on the second molecular ionidentified for MDMA at 194 (H+). Thisis achieved by trapping the ion andthen increasing the collision gas tofragment the parent ion into itsdaughter ions. Panel C of Figure 7represents the results of this experi-ment that clearly resolves a 194 (H+)parent ion that collapses to a 163 (H+)molecular ion with fragmentation.

This is consistent with thefragmentation pattern expected formethoxamine. We also captured thefirst molecular ion of methoxamineat 212 (H+), which appears to co-migrate with the second MDMA mol-ecular ion, and fragmented it with anincrease in collision gas (shown inpanel D). Interestingly, this parention loses H20 generating a daughterion at precisely 194( H+). Ourinterpretation of this result isunequivocal: the first resolved molec-ular ion at 194 (H+) representsMDMA while the later migrating ionis representative of the internal stan-dard methoxamine which has lostwater during its transformation to amolecular ion. What is noteworthyhere is the value of the MS/MS exper-imentation in providing us with thisunderstanding.

However, this still leaves us withthe second open question from thisdata—what is the second molecularion resolved at 212 (H+)? In this case,we simply trap the second molecularion and once again increase thecollision gas to induce fragmentationof the ion. The results of this experi-ment are highlighted in Figure 8.Panel A represents the two molecularions found at 212 (H+). Panel Bhighlights the fragmentation of thefirst ion—consistent with the loss ofwater generating a molecular ion of

194 (H+). While Panel C representsthe fragmentation pattern of thesecond molecular ion isolated at212 (H+). In this case, a uniquefragmentation pattern is generated,

creating molecular ions of 168 (H+),169 (H+), and 197 (H+), respectively.The molecular ion remainsunidentified but gives us cluestowards future experimentation.

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NL: 2.03E5

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O

NH

CH3

Cl

Ketamine

O

O

NH

CH3

H3C

MDMA

OH

CH3

NH2OC H3

H3CO

Methoxamine

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:

O

O

NH2

CH3

H3C+1

MDMA

O H

CH3

NH3OC H 3

H3CO

+1

Methoxamine

(MH) +1

MS/MS of 194

(MH) +1 - CH3

(MH) +1

(MH) +1-H2O

-H2O

MS/MS of 194

A

B

C

D

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Figure 7. CE-MS/MS of Sample 2001-2249.1.

10

Volume 6, Issue 1 • April 2002

CONCLUSIONSCE–MS/MS provided clear

confirmation of the drugs identified.using the R.CM. police comprehen-sive CE–DAD screen. Further, the useof MS/MS is of added value in inter-preting the results put forward. Thisexperiment demonstrates the feasibil-ity of CE–MS/MS as a valuable confir-mation tool in a forensic toxicologylaboratory. It is our hope this datawill inspire others to explore thisstrategy further as the potential ofthis approach appears promising.

REFERENCE1. Hudson, J. C., Golin, M., Malcolm, M.

Capillary Zone Electrophoresis in a

Comprehensive Screen for Drugs of

Forensic Interest in Whole Blood: An

Update. Can. Soc. Forens. Sci. 31 (1),

1-29 (1998).

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O H

CH3

NH3OC H3

H3CO

+1

Methoxamine

MS/MS

Methoxamine-H2O

(MH)+1-CH3

(MH)+1

(MH)+1-H2O

?

A B

C

Figure 8. CE-MS/MS of Sample 2001-2249.1.

Beckman Coulter is proud to introduce theCEQ™ 8000 Genetic Analysis System whichautomates and integrates the majority of

genetic analysis functions into one flexible,easy-to-use system. With one gel, one capillaryarray, and one software platform, users canperform denovo DNA sequencing,heterozygote detection,confirmatorysequencing, muta-tion analysis, alleleidentification, SNP(single nucleotide poly-morphism) scoring,microsatellite instability,and AFLP (amplified fragmentlength polymorphism) finger-printing.

This capillaryelectrophoresis-based systemis compact and affordable,delivering both high-resolution and high-speedDNA analysis in a singlesetup. Eight capillaries mapperfectly to a 96-well plate

format, providing the optimum flexibility to allowthe user to best manage sample workflow. The

CEQ 8000 software reduces data analysis andinterpretation time by mapping a user’s data

review strategies to an automatedprocess. With over 100 query

filters in place, users canclearly identify

criteria forqualifying

data.

The CEQ 8000 isthe latest addition to

Beckman Coulter’s suiteof genetic analysissolutions which includeautomated sample prepa-ration, centrifugation,spectrophotometry, andcapillary electrophoresistechnologies. With theCEQ 8000, users cannow focus on theirscience rather than oncomplicatedinstrumentation.

A Star Is Born

If you observe a drop in thedetection sensitivity in anassay you are developing or

routinely operating, you arefaced with the challenge ofisolating the cause of yourobservation. Does my lampneed replacing? Is less samplebeing introduced into thecapillary? Is my detectionsystem operating as designed?Is my capillary clogged? Has myassay chemistry changed?Although the list of possibilitiesmay sound daunting, it is oftenyour experience in recognizingthe signature of the change thatdrives your strategy fortroubleshooting the cause. Toassist you with this process, werecommend that you first try toisolate the issue you observe toeither your system hardware orsystem chemistry. In the case ofthe system’s detector, you canuse an OPCAL cartridge toquickly interrogate whether thedetector is operating to the systemspecification. The OPCAL cartridge isdesigned for you to use as atroubleshooting aid and can bepurchased from Beckman Coulterusing part number 144660.

PERFORMING A NOISE AND

DRIFT TESTDetector noise is one of the best

criteria to use to determine if yourdeuterium lamp needs to be replaced.With an OPCAL cartridge installed,you need only initiate a methodwhich applies a voltage of 1 kV for15 minutes while collecting data at4 Hz with 214 nm detection. In thisexperiment, there is no capillary with-in the cartridge and no buffer isrequired to bridge the circuit. Still, itis recommended you place emptycapped vials in the separationpositions for this method. The voltage

load is generated across the interfaceblock of the system and is used toaccount for detection noisecontributed by the HV power supply.By removing the “chemistry” from thesystem, you effectively isolate thenoise and drift analysis to thedetection hardware. Noise and driftcan be calculated manually throughclose inspection of the detection base-

line or you can have thesystem automaticallymeasure theseparameters using the“system suitability” func-tion of your 32 Karat™

software. If both thenoise and drift of thedetector fall within theinstrument’s specifica-tion, you can rule outthe detection hardwareas a source of the sensi-tivity change. If, howev-er, the noise level is toohigh, you may first wantto ensure the fiber opticis clean and the 214 nmfilter (if using a UVdetector) is still in goodcondition before makingany change. Of course,if the high noisepersists, you shouldchange your deuteriumlamp. Changingelectrodes may improve

performance if intermittent stepchanges are observed in the baseline.If, under these conditions, you alsoexperience excessive drift on thebaseline, this may be an indication ofsubstantial temperature variationsnear the instrument, in which caseyou will want to closely inspect theenvironment which you placed yoursystem.

11

Assessing the Health of your P/ACE MDQ Detector

P/ACE MDQ Detector Noise/Drift SpecificationsNoise* Single Wavelength UV detector < 22 µAU peak-to-peak

Diode Array Detector < 20 µAU peak-to-peak

Drift* Single Wavelength UV detector < 3000 µAU/hourDiode Array Detector < 6000 µAU/hour

* Specification met using OPCAL cartridge with a 100 x 800 µm aperturewith the following separation conditions:

Voltage = 1 kVWavelength = 214 nm, (10 nm bandwidth for DAD)Data Rate = 1 Hz, 16-25 pts/pk, normal filter setting

Volume 6, Issue 1 • April 2002

Developing innovative solutions in genetic analysis, drug discovery, and instrument systems.

*FluorInert is a registered trademark of Minnesota Mining and Manufacturing Company.All other trademarks are the property oftheir respective owners.

By now you should have received your invitation tothe Annual Beckman Coulter P/ACE User Event.This event will be held on April 16, in

conjunction with the 2002 InternationalSymposium on High Performance Capil-lary Electrophoresis and MicrocolumnTechnologies.

An important element of theannual user event is the “PuttingCE to Work Contest” where wecelebrate the successfulimplementation of CE technolo-gy. This contest is open to allP/ACE™ CEQ users andattendance at the usermeeting is not required forentry. Simply submit anexample of how you routine-ly apply CE technology inyour laboratory. As this is acontest for P/ACE users, theseparation must have beenperformed on a P/ACESeries instrument. Yourentry should fit on no more

than two pieces of A-4 or 8.5-x-11" paper. One sheetshould illustrate at least one electropherogram while thesecond sheet should describe the separation and its signif-

icance to your work. The winner will receivethe coveted “Putting CE to Work” trophy,

admiration from your peers, and, ofcourse, a nice prize to mark theoccasion.

Although the winning entry willbe selected by your peers at theP/ACE user celebration, you do notneed to be in attendance for submis-sion or consideration. However,entries must be received byBeckman Coulter, Inc., no later thanApril 12, 2002, to be considered. Toexpedite this process, we invite youto submit your entry electronicallyas an attachment to the followinge-mail address:

pacesetter@beckmancoulter.com

or you may send it via fax to:

714-773-8883

Ready Your Submission for Putting CE to Work

Beckman Coulter, Inc. • 4300 N. Harbor Boulevard, Box 3100 • Fullerton, California 92834-3100Sales: 1-800-742-2345 • Service: 1-800-551-1150 • Telex: 678413 • Fax: 1-800-643-4366 • www.beckmancoulter.com

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