Andrighetto Platformtechnology 2016A

PLATFORMTECHNOLOGYTOWARDSTHECHEMICAL
FINGERPRINTINGOFMETHAMPHETAMINEFROM
EPHEDRINEPATHWAYS

By
LukeMatthewAndrighetto
BachelorofForensicScience(Honours)
Submittedinfulfilmentoftherequirementsforthedegreeof
DoctorofPhilosophy(ForensicChemistry)
DeakinUniversity
June,2016

Signature Redacted by Library
Signature Redacted by Library

“Ifweknewwhatitwasweweredoing,itwouldnotbecalledresearch,
wouldit?”
AlbertEinstein
IV

TABLE OF CONTENTS

TABLEOFCONTENTS..........................................................................................................V
ACKNOWLEDGEMENTS....................................................................................................VIII
PUBLICATIONS....................................................................................................................X
ABBREVIATIONSANDACRONYMS.....................................................................................XI
ABSTRACT........................................................................................................................ XIV
CHAPTER1:......................................................................................................................... 1
INTRODUCTIONTOMETHAMPHETAMINE.........................................................................1
1.1HistoryofMethamphetamine..................................................................................2
1.2TheAustralianMethamphetamineProblem............................................................4
1.2.1IceTaskforce......................................................................................................6
1.3TheappealofMethamphetamine............................................................................7
1.4LocalSynthesis..........................................................................................................9
1.5Precursorstomethamphetamine.............................................................................9
1.5.1Pseudoephedrine/ephedrine...........................................................................10
1.5.2PhenylͲ2Ͳpropanone(P2P)...............................................................................13
1.6Detectionmethodsofmethamphetamine.............................................................14
1.6.1PoliceroadͲsidetesting....................................................................................14
1.7AnalyticalSeparationandDetectionMethodsforMethamphetamine.................15
1.8TwoͲdimensionalHPLC...........................................................................................17
1.9InͲSilicoOptimisationChromatography(computationalmodelling)......................19
CHAPTER2:....................................................................................................................... 22
SYNTHESISOFPSEUDOEPHEDRINE/EPHEDRINEFROMNITROETHANEAND
BENZALDEHYDE.................................................................................................................22
CHAPTEROVERVIEW.........................................................................................................23
2.1INTRODUCTION.......................................................................................................24
2.1.1ReductiveAminationofphenylͲ2Ͳpropanone(P2P)........................................25
2.1.2PseudoephedrineReduction...........................................................................26
2.1.3BirchReductionofpseudoephedrinetomethamphetamine.........................27
2.2EXPERIMENTAL.......................................................................................................29
Synthesisof2ͲnitroͲ1ͲphenylͲ1Ͳpropanol(mixtureofSynandAntidiastereomers)
(8).............................................................................................................................. 29
SynthesisofPhenylͲ2Ͳnitropropene(9)....................................................................31
2.3RESULTSANDDISCUSSION.....................................................................................33
2.3.1Synthesisof2ͲNitroͲ1Ͳphenylpropanol(8)......................................................33
V

2.3.1SynthesisofphenylͲ2Ͳnitropropene(9)...........................................................37
2.3.3Synthesisofnorephedrine(10)from2ͲnitroͲ1ͲphenylͲ1Ͳpropanol................41
2.3.4Synthesisofephedrine/pseudoephedrine(1)fromnorephedrine(10)..........42
2.3.4Synthesisofmethamphetamine(1)fromnorephedrine(10).........................50
2.4CONCLUSION...........................................................................................................52
CHAPTER3........................................................................................................................ 53
SYNTHESISOFPSEUDOEPHEDRINE/EPHEDRINEFROMLͲALANINE.................................53
3.1INTRODUCTION.......................................................................................................55
3.2EXPERIMENTAL.......................................................................................................57
SynthesisofMethylͲLͲalaninate(16)........................................................................58
SynthesisofMethylNͲ(tolueneͲ4Ͳsulfonyl)LͲalaninate(17)...................................58
SynthesisofMethylNͲmethylͲNͲ(tolueneͲ4Ͳsulfonyl)ͲLͲalaninate(18)...................59
SynthesisofMethyl(tertͲbutoxycarbonyl)ͲLͲalaninate(20)....................................59
Synthesisof2Ͳ(NͲ(tertͲbutoxycarbonyl)amino)propanoicacid(22)........................60
Synthesisof2Ͳ(NͲ(tertͲbutoxycarbonyl)methylamino)propanoicacid(23).............60
Synthesisof2Ͳ(methylamino)propanoicacid(NͲmethylalanine)trifluoroacetatesalt
(19)............................................................................................................................ 61
3.3RESULTSANDDISCUSSION.....................................................................................62
3.4CONCLUSION...........................................................................................................74
CHAPTER4:....................................................................................................................... 75
INͲSILICOOPTIMISATIONOF1DͲHPLCAND2DͲHPLCUSINGDRYLAB®SIMULATION
SOFTWARE........................................................................................................................ 75
4.1Introduction............................................................................................................77
4.2Experimental...........................................................................................................82
StandardsandSamplesͲExperiment1....................................................................82
HighPerformanceLiquidChromatographyͲExperiment1.....................................82
DryLab®OptimizationͲExperiment1.......................................................................83
ModelSeizureSamplesͲExperiment1....................................................................83
ChemicalsͲExperiment2.........................................................................................83
StandardsandSamplesͲExperiment2....................................................................84
ChromatographyColumnsͲExperiment2...............................................................84
2DͲHPLCͲExperiment2............................................................................................84
LCͲMSͲExperiment2...............................................................................................85
4.3ResultsandDiscussion............................................................................................86
4.3.11DͲHPLCusingDryLab®....................................................................................86
VI

4.3.22DͲHPLCusingDryLab®....................................................................................91
4.3.32DͲHPLCofarealmethamphetamineseizuresample....................................93
4.3.42DͲHPLCofasynthesisedintermediatesampletomethamphetamine.........95
4.4Conclusion...............................................................................................................99
CHAPTER5:.....................................................................................................................100
COLUMNSELECTIVITYSTUDYANDCHEMICALMAPPING..............................................100
CHAPTEROVERVIEW.......................................................................................................101
5.1Introduction..........................................................................................................102
5.2Experimental.........................................................................................................106
Chemicalsandsamples...........................................................................................106
LiquidChromatographyMassSpectrometry(LCͲMS)............................................107
LiquidChromatographyMassSpectrometry(LCͲMS)forchemicalmappingof
syntheticpathway...................................................................................................108
TwoͲdimensionalhighperformanceliquidchromatography.................................108
DataProcessing.......................................................................................................109
PrincipalComponentAnalysis(PCA).......................................................................109
5.3ResultsandDiscussion..........................................................................................110
5.3.1ColumnSelectivityStudyusingOpenMS®.....................................................110
5.3.2Chemicalmappingofimpuritiesforapathwaytomethamphetamine........122
5.3.3PreliminaryPCAStudy...................................................................................135
5.4Conclusion.............................................................................................................143
CHAPTER6:.....................................................................................................................145
CONCLUDINGREMARKSANDFUTUREWORK................................................................145
6.1ConcludingRemarks.............................................................................................146
6.2FutureWork..........................................................................................................147
REFERENCES....................................................................................................................149

VII

ACKNOWLEDGEMENTS
Iwouldliketoextendmygratitudetoanumberofpeoplefortheirhelp,supportand
advicethroughoutmyPhD.
Firstly, to my primary supervisor and friend Dr Xavier Conlan. Thank you for all your
encouragement, support, understanding and advice. Without you I would not be here
todayandwordscannotdescribehowgratefulIamforeverythingyouhavedoneforme
overthislongjourney.WehavehadalotoflaughsandgreatmemoriestogetherthatI
willforevertreasure.
TomycoͲsupervisorandfriendDrPaulStevenson.Thankyousomuchforallyourhard
work,help,supportandadvice.Itisgreatlyappreciatedandneverforgotten.Withoutyou
IwouldnothavefinishedmyPhDthisdecade.Yourbrilliantmindandprogrammingskills
havecontributedagreatdealtomyresearchandIcan’tthankyouenoughforallyour
patience.
TomycoͲsupervisorDrLukeHenderson.Thankyouforallyourhelp,supportandadvice.
WehavebeenonalongjourneytogetherfromHonourstohereandIcouldnothavegot
herewithoutyou.YoursyntheticknowledgeissecondtononeandIreallyappreciateall
yourhelpandguidance.Iwillmisstheconstantbanteringbetweenus.
TomyexternalcoͲsupervisorDrJimPearson.Thankyouforallyourhelpandadvice,itis
greatlyappreciated.Yourguidanceandinternalinformationhascontributedsignificantly
tomyresearchandIamverygrateful.
ToAlfredDeakinProfessorNeilBarnettandDrFredPfeffer.Thankyouforallyouradvice
andsupportoverthepast4years.WehavehadmanylaughstogetherandIhopefor
manymore.
TomydearfriendsNikiandNatalie.Thankyouforourdailylunchesandlaughstogether.
Ithasbeentheonlythingkeepingmesanesomedays.Iwillmissourlittlelunchdates.
ToallmyfriendsthatIhavehadtheprivilegeofworkingwithorbeside(soontobeDr
Niki Burns, Dr Zoe Smith, Dr Gregory Barbante, Dr Brendan Holland, Dr Danielle
Bassanese, Dr Jarrad Altimari, Dr Shane Hickey, Ashton Theakstone, Hannah Brozinski,
RyanRobson)thankyouformakingtheworkͲplaceanenjoyableplacetobe.
VIII

TomydearestpartnerLillian.WheredoIstart?Wordsdon’tdojusticetohowmuchyou
meantomeandIdefinitelycouldnothavefinishedthisjourneywithoutyou.Yourlove,
support,encouragementandbeliefinmeoverthepast7yearshasbeenoverwhelming
andIcannotthankyouenough.
Tomyparents.Ididit.DrLukeAndrighetto.Thankyouforallyourunconditionallove,
support,encouragementandguidanceoverthepast25years.Iliterallywouldnothave
beenhereifitweren’tforyouboth.Thankyou.
IX

PUBLICATIONS
1. L.M.Andrighetto,N.KBurns,L.C.Henderson,C.JBowen,J.R.Pearson,P.G.Stevenson
and X.A. Conlan, InͲsilico optimisation of twoͲdimensional HPLC for the
determination of Australian methamphetamine seizure samples, Forensic Science
International,266,511Ͳ516(2016).
Ͳ TheworkfromthispaperisincludedinChapter4
2. N.KBurns†,L.MAndrighetto†,S.DPurcell,N.WBarneƩ,J.Denning,P.SFrancis,X.A
ConlanandP.G.Stevenson,BlindcolumnselectionprotocolfortwoͲdimensionalhigh
performance chromatography, Talanta, 154, 85Ͳ91 (2016) DOI:
10.1016/j.talanta.2016.03.056.†(CoͲfirstauthor)
3. L.M. Andrighetto, L.C. Henderson, J.R. Pearson, P.G. Stevenson and X.A. Conlan,
Influence of base on nitroͲaldol reaction products for alternative clandestine
pathways, Australian Journal of Forensic Sciences, (2016) DOI:
10.1080/00450618.2015.1112429.
4. L.M. Andrighetto, F.M. Pfeffer, P.G. Stevenson, S.M. Hickey, G.J. Barbante, L.C
Henderson,J.RPearsonandX.A.Conlan,ThreestepsynthesisofNͲmethylalanine–
Aprecursortoephedrine,pseudoephedrine,andmethamphetamine,Journalofthe
ClandestineLaboratoryInvestigatingChemistsAssociation,24,33Ͳ37(2014).
5. L.M.Andrighetto,P.G.Stevenson,J.R.Pearson,L.C.Henderson,X.A.Conlan,DryLab®
optimised twoͲdimensional high performance liquid chromatography for
differentiation of ephedrine and pseudoephedrine based methamphetamine
samples,ForensicScienceInternational,244,302Ͳ305(2014)
X

ABBREVIATIONS AND ACRONYMS

μm Micrometre
13
CNMR CarbonNuclearMagneticResonance
1DͲHPLC OneͲDimensionalHighPerformanceLiquidChromatography
1
HNMR ProtonNuclearMagneticResonance
2DͲHPLC TwoͲDimensionalHighPerformanceLiquidChromatography
ACN Acetonitrile
amu AtomicMassUnit
ATS AmphetamineTypeSubstance
Boc TertͲbutyloxycarbonyl
Boc2O DiͲtertͲbutyldicarbonate
CDCl3 DeuteratedChloroform
d6ͲDMSO DeuteratedDimethylsulfoxide
DCM Dichloromethane
DIAD DiisopropylAzodicarboxylate
DIOSͲMSI DesorptionIonisationonSiliconͲMassSpectrometryImaging
DMSO Dimethylsulfoxide
EC Endcapped
ECL Electrochemiluminescence
EIC ExtractedIonChromatogram
EMͲSPME ElectromembraneSurroundedͲSolidPhaseMicroextraction
eq. Equivalents
ESIͲMS ElectrosprayIonisation–MassSpectrometry
Et3N Triethylamine
EtOH Ethanol
FDA FoodandDrugAdministration
g Grams
GC/FTͲIR GasChromatography/FourierTransformͲInfrared
Spectroscopy
GCͲMS GasChromatography–MassSpectrometry
h Hours
XI

HILIC HydrophilicInteractionLiquidChromatography
HIV HumanImmunodeficiencyVirus
HPLC HighPerformanceLiquidchromatography
HRMS HighResolutionMassSpectrometry
IRMS IsotopeRatioMassSpectrometry
JOEL JapanElectronOpticsLaboratory
kV Kilovolt
LCͲMS LiquidChromatography–MassSpectrometry
LCͲUV LiquidChromatographywithUltravioletͲVisibleDetection
LOD LimitofDetection
LͲPAC LͲphenylacetylcarbinol
M Molar
m/z MasstoChargeratio
MA Methamphetamine
MALDIͲMS MatrixͲassistedlaserdesorption/ionization–Mass
Spectrometry
MDMA Methylenedioxymethamphetamine
MeOH Methanol
mg Milligrams
mm Millimetre
mmol Millimole
mol Mole
Mp MeltingPoint
MS MassSpectrometry
P2P PhenylͲ2Ͳpropanone
PCA PrincipalComponentAnalysis
Pd/C PalladiumonCarbon
PDC PyruvateDecarboxylase
PE Pseudoephedrine
PFP Pentafluorophenyl
ppm PartsperMillion
psi PoundsperSquareInch
XII

r.t RoomTemperature
RP ReversePhase
SPR SurfacePlasmonResonance
TFA Trifluoroaceticacid
THC Tetrahydrocannabinol
THF Tetrahydrofuran
TIC TotalIonChromatogram
TMS Trimethylsilyl
UPLC UltraͲPerformanceLiquidChromatography
V Volts
v/v VolumeperVolume
VPFSD VictoriaPoliceForensicServicesDepartment
w/w MassperMass(WeightperWeight)

XIII

ABSTRACT

Australian clandestine drug laboratories are constantly seeking and utilising

alternative methods to produce methamphetamine. These methods are in
response to restrictions placed by government on chemicals such as
phenylͲ2Ͳpropanone(P2P)intheearly1980s,orpseudoephedrinefromthemidͲ
2000s. Law enforcement agencies have taken great interest in the nitroͲaldol
reactionoccurringbetweennitroethaneandbenzaldehydethatcanbeusedina
numberofsyntheticpathwaystomethamphetamine.Assuch,aninvestigationis
warrantedintowhatproductscanbeformedfromthisreactionandexactlyhow
thesecompoundsfitintothemanufactureofmethamphetamine.Theresulting
products, namely phenylͲ2Ͳnitropropene (from the P2P pathway) and
2ͲnitroͲ1ͲphenylͲ1Ͳpropanol (from the ephedrine pathway) are directly
dependantonwhichchemicalbaseisused;thebasemaythereforebeusedto
provideanindicationofapossiblemethamphetaminemanufacturepathwayata
clandestinelaboratory.
Restrictions on pharmaceutical drugs containing pseudoephedrine were

introducedbytheAustraliangovernmentin2006.Here,aninvestigationiscarried
outinconjunctionwithVictoriaPoliceForensicServicesDepartment(VPFSD)after
a large quantity of LͲalanine (an amino acid) was seized from a clandestine
laboratory.Asthisaminoacidcanbeboughtinlargequantitiesverycheaplyfrom
any supplement store, its potential as a precursor of a synthetic pathway to
methamphetamine was examined. This pathway focused on the formation of
pseudoephedrineviaathreestepsynthesisofNͲmethylalanine.
Asnewmethodsarebeingdevelopedforthemanufactureofmethamphetamine,
a detection system that can handle complex samples containing
methamphetamineanditsassociatedprecursorsandcuttingagentsisneededto
detectnewcompounds.InͲsilicooptimisedonedimensionalandtwoͲdimensional
high performance liquid chromatographic (2DͲHPLC) separations of a model
methamphetamine seizure sample provided an excellent match between
simulated and real separations and are described for the first time. Targeted
XIV

separation of model compounds was completed with significantly reduced

methoddevelopmenttime.ThisseparationwascompletedintheheartͲcutting
mode of 2DͲHPLC where a selected portion of a peak is transferred from one
dimension into another dimension of different retention mechanisms. C18
columns were used in both dimensions taking advantage of the selectivity
difference of methanol and acetonitrile as the mobile phases. This method
development protocol is most significant when optimising the separation of
chemicallysimilarcompounds,asiteliminateshoursoftrialͲandͲerrorinjections
commonlyrequiredtoidentifytheoptimisedexperimentalconditions.Afteronly
fourscreeninginjectionsthegradientprofileforboth2DͲHPLCdimensionswas
optimised via simulations. Importantly this process ensured the baseline
resolutionofdiastereomers(ephedrineandpseudoephedrine)in9.7minwhich
hasbeenagreatchallengeforanalysts.
Theselectionoftwoorthogonalcolumnsfor2DͲHPLCseparationofcomplexdrug
samplescanbealabourintensiveandtimeconsumingprocessasconventional
methods utilise a trialͲandͲerror approach. A novel column selectivity protocol
wasestablishedthatwascapableofidentifyingthemostappropriateorthogonal
columns,fortheseparationofinterestedcompounds.Adataprocessingpipeline,
created in the open source application OpenMS®, was developed to map the
componentsofequalmassacrossalibraryofHPLCcolumnswhilsthavinglimitsof
detection comparable to literature values across both dimensions. The
twoͲdimensional separation space was compared by measuring the fractional
surface coverage, fcoverage. A model methamphetamine sample was made and
tested with this system and identified two sets of suited column combinations
(KinetexPFP/SynergiHydroandBonusRP/PursuitXRsDiphenyl)thatpredictedan
fcoverageof0.44.OpenMS®,inconjunctionwithalgorithmsdesignedinhouse,have
allowed for a significantly quicker selection of two orthogonal columns, which
have been optimised for a 2DͲHPLC separation of methamphetamineͲtype
samples.
TheKinetexPFPandtheOnyxMonolithicC18columnswereselectedafterfurther
analysis of the stationary phases and were used in the chemical mapping of a
XV

methamphetamine pathway, using 2DͲHPLC and LCͲMS. The impurities and

byͲproducts were mapped from start to finish to create a tabular chemical
fingerprint that helped in the identification of synthetic route and precursors
used.Analternativemethodfortheidentificationofsyntheticroutewastrialled
using LCͲMS and principal component analysis on 22 real methamphetamine
seizuresamples.Thepreliminarystudywasabletodifferentiatepseudoephedrine
basedonconcentration(intensitycount),thusdemonstratingtheimportanceof
thisplatformtechnology.
This thesis presents a platform technology protocol for the comprehensive

chemicalfingerprintingofmethamphetamineseizuresamplesofinteresttothe
Victoria Police Forensic Services Department. Further key aspects of
methamphetaminesynthesishavebeendevelopedincludingtheinvestigationof
thesimpleaminoacidLͲalanineasaviablealternativeprecursor.
Highlightsofthisresearchinclude:
Ͳ The successful synthesis of novel pathways to methamphetamine from
easilyaccessiblematerials.
Ͳ The optimisation of oneͲdimensional and two dimensional HPLC
separationsusingsimulationsoftwareofforensicallyimportantsamples.
Ͳ The development of platform technology that allows for chemical
mappingofclandestinepathwaysforillicitdrugs.
Ͳ The use of principal component analysis to efficiently identify synthetic
routeinrandommethamphetamineseizuresamples.
XVI

CHAPTER1
CHAPTER 1:
INTRODUCTION TO METHAMPHETAMINE

Author’s Note: As this thesis deals with both analytical chemistry and some
general synthetic chemistry a broad introduction of methamphetamine will be
offered in Chapter 1 with a niche introduction presented at the start of each
resultschapter.

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CHAPTER1
1.1HistoryofMethamphetamine

Methamphetamineandamphetaminearehighlyaddictivepsychostimulantdrugs
that affect the neurotransmitter systems of the brain, providing the user with
increasedalertness,energyandeuphoria[1].Ephedrine(Figure1,Compound1)
was first used as a precursor for the synthesis of methamphetamine
(Figure1,Compound2)byAkiraOgata,in1919[2].Theactivealkaloidephedrine
wasdiscoveredwithintheEphedraplant(Ephedrasinica)in1885.Theuseofthe
Ephedraplanthasbeenthoroughlystudied[3]anditsusedatesbackwellover
five thousand years in Chinese medicine [4]. Upon the discovery of the active
alkaloidofephedrine,otherspeciesoftheEphedraplanthavebeenanalysedin
ordertoidentifythecompound.Itisnowknownthatonly25ofthe45speciesof
Ephedra contain ephedrine, which is found within the leaves and in low
concentrations of 1Ͳ2% [5]. Medically, ephedrine is known to offer a variety of
therapeutic benefits for nasal decongestion, appetite suppression, stimulation,
andthereliefofasthmaandbronchitis[2].
Figure1:Ephedrine1andmethamphetamine2structures.
Methamphetamine,similartoamphetaminebutmorepotent,iswatersoluble,
making it suitable for intravenous delivery into the body [6]. Because of this
methamphetaminewaswidelyusedbytheBritish,JapaneseandtheAmericans
duringWorldWarIIasanaidtokeepalliedtroopsawake.Also,Japanesesuicide
pilotsweregivenparticularlylargedosesbeforetheirmissions,enablingthemto
stayalertandflyforlonghours[2].Theendofthewarmeantthatmilitarystores
of the drug in Japan were readily available to the public on top of the already
abundant over the counter pharmacy supply [4], inducing the first
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CHAPTER1
methamphetamine epidemic in Japan. Methamphetamine abuse grew and

reacheditspeakin1954,wherethenumberofmethamphetamineabuserswas
estimatedat550,000,with55,000peoplesufferingmethamphetamineͲinduced
psychosis[7].In1940,theBurroughsWellcomeCompanyinBritain,commercially
introducedmethamphetaminetabletsbythenameofMethedrine[8],alongwith
intravenousamphetaminesofthesamenameusedforbringingpatientsoutof
anaesthesia[9].Duringthe1940’sand1950’sthismethamphetaminetabletwas
marketed as a diet aid and, asthma, attention deficit disorder, narcolepsy and
depressiontreatment[9].InAmerica,theproductwasdeemedsafebytheFood
andDrugAdministration(FDA)[9]andwithoutriskofaddiction,andassuchwas
prescribedformanydifferentailmentswithoutcautionormonitoring.Duetoits
accessibilityitwasabusedbyathletes,studentsandtruckdriversasanonͲmedical
stimulant[9].
Amphetamine inhalershave beenavailable since the early 1930s in the United
StateswiththeS.PfeifferCompanyintroducing‘Valoinhalers’.However,inthe
late 1950’s the inhalers contained 150Ͳ200 mg of methamphetamine [10],
resultingindrugabusersbreakingopentheinhalerstoingestthecontents[10].
Widespread acts of violence and crime were linked to inhaler use and abuse,
resulting in an FDA ruling in 1959 which found the inhalers available by
prescription only. The 1960s saw an increase in injectable methamphetamine
beingprescribedtotreatheroinaddictionandincreasedabuseofthistypeofthe
drug[10].Thisdecadealsomarkedtheemergenceofmethamphetaminerelated
healthandsocietalissuesinAustralia[11].The1970sbroughtaboutalegislative
changeduetotheincreaseofabuse,seeingallamphetamineproductsreceiving
ascheduleIIclassification.Thismeantthatalthoughtwotypesofamphetamine
tabletswerestillavailableuponprescription;methylphenidate(Ritalin)andthe
dietdrugphenmetrazine(Preludin)anewprescriptionhadtobefilledeverytime
thesedrugswereprescribed[12].Followingincreasingabuse,mostofthenasal
inhalerswerealsoremovedfromthemarketcompletelyby1971[13].
By the 1980s prescriptions were down 90% from those generated for
methamphetaminesinthe1960s,withthisfiguredroppingagainbyathirdaround
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CHAPTER1
1990 [14]. As restrictions were established and enforced on prescription
methamphetamine, clandestine production of the drug was on the rise, using
precursorswhichwerelegalandeasilyobtainedduringthistime[4,15]. Bythe
midͲ1980s nearly all street methamphetamine was produced in clandestine
laboratories, rather than being sourced from legally produced pharmaceuticals
(suchascoldandflumedication)[4].
1.2TheAustralianMethamphetamineProblem
Thecrystallineformofmethamphetamine,alsoknownbythestreetnames;ice,
crystalmeth,meth,glass,crystal,shard,puff,shabba,rock,yabaandtweakfirst
becameadrugofconcerninAustraliaintheearly1990sandsince2010therehas
beenanincreaseinbothsupplyanddemandforit[16].Whilethisescalationwas
observedthroughouttheworld,theincreaseinAustraliawassubstantiallymore
thanwasseeninternationally.Globallytheweightofmethamphetaminesseized
between2009and2014nearlytripled(seeFigure1.1),whilstthelatestAustralian
statisticsshowtheweightofborderseizureshasincreased60foldbetween2010
and2014.
Figure1.1:WeightofglobalseizuresofmethamphetamineandamphetamineͲ
typestimulants,intonnes[16].
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CHAPTER1
Methamphetamine is now the second most commonly used illicit drug in
Australia, second only to cannabis [17]. Ice demand and supply has also
outstripped thatof otheramphetamines,contributing79% ofallamphetamine
basedborderseizuresin2013Ͳ2014upfrom61%in2010Ͳ2011[17].Asignificant
issuewiththepolicingofmethamphetaminesupplyinAustraliaisthatitisoneof
theonlydrugswhichisbothproducedlocallyinlargequantities,andimported
fromoverseas[17].Themainprecursorforicemanufacture;pseudoephedrine,
was listed as a controlled substance under the Victorian Drugs, Poisons and
ControlledSubstancesAct1981.Thismadeithardertoobtaintheprecursorfor
manufacturingpurposesandhascontributedtoanincreaseofmethamphetamine
productbeingintroducedfromothercountries[17].
Therewereinexcessof268,000regularusersofmethamphetamineinAustralia
betweentheyears2014and2015,whichisalmosttriplethatidentifiedin2010
[18]. There were significant increases in amphetamine related ambulance
callͲouts between 2013 and 2014, including a 23% increase in metropolitan
Melbourneand27.2%increaseinregionalVictoria[18].Thisincreasehasbeen
attributed to the increase in methamphetamine use. It is now the fourth most
common drug involved in ambulance callͲouts [19]. The median purity of
methamphetamine (Figure 1.2) is also increasing significantly going from 7% in
2009Ͳ2010 to 62% in 2013Ͳ2014 [17] this may be a contributing factor to the
ambulance callouts involving methamphetamine as the drug available now is
muchmoreconcentratedthanitwaslessthantenyearsago.
Figure1.2Medianpurityofmethamphetaminebetweenyears2004Ͳ2014[17].
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CHAPTER1
1.2.1IceTaskforce
ItisforthesereasonstheAustralianGovernment,aspartofthenationaliceaction
strategycreatedthe‘NationalIceTaskforce’onthe8thApril2015.Thetaskforce
spoketoover100expertsonresearch,prevention,education,treatmentandlaw
enforcement and released a report detailing the methamphetamine related
statisticsandalistofrecommendationstohelpwiththe‘iceepidemic’.
Theexecutivesummaryoftheicetaskforcereporthighlightsmanyareaswhich
needattentiontocombattheuseoficewithinAustralia.Including;
x Theneedtosupportfamiliesandcommunitiestoenablethemtobetter
handlepeopleaffectedbyice.
x Strengtheneffortstoreducethedemandforice.
x Treatmentandsupportservicesforusers.
x Preventionoffirsttimeuse.
x CoͲordinatedandtargetedeffortstodisruptsupply.
x Betterdata,moreresearchandreportingtokeepresponsesontrack.
Inparticulartherearetworecommendationslistedbelowthathighlighttheneed
forbetteranalyticalchemistryplatformsforillicitdrugprofiling(seeFigure1.3):
Figure1.3:Extractsfromthe2016nationalicetaskforcereport[17].
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CHAPTER1
1.3TheappealofMethamphetamine
As methamphetamine is an extremely powerful stimulant, it is in very high
demanddespiteitsextremeaddictiveproperties[17].Peopleusingicearesaidto
experienceconfidence,forexample,thecaseofuseinlocalfootballwhichhas
recentlycometolight,withplayersasyoungas14takingthedrug“tofeellike
superman”whenrunningontothefootballfield[20].Othersoughtaftereffects
oficeinclude:euphoria,whichisdiminishedaftertakingthedrugregularly,much
higherdosesareneededtoachievethisfeeling[6],andenhancedsexualpleasure
[21].ThepopularityandwordͲofͲmouthamongsocietyhasledtomorepeople
wantingtotryandexperiencetheeffectsofthedrug[17].
Fromaproductionperspectivemethamphetamineisanidealdrugpresentinga
high profit margin particularly in Australia where methamphetamine sells for
$675/gAUDcomparedtoEuropeat$49/gAUD(Figure1.4)[17].Itistherefore,
feasible for dealers to carry small amounts which can be easily concealed and
transportedwhilestillmakingasufficientprofit.Unlikemanyotherillicitdrugs
methamphetaminedoesnotdependonplantbasedmaterialavailabilityandis
alsoveryflexible;itcanbeproducedonasmallscaleorlarge.Thereisnotone
single precursor or methodology for synthesis, but rather many materials and
pathwayswhichcanleadtoitsproduction.Thisflexibilityofproductionmakesit
harder for law enforcement to apprehend producers, it also means that if
restrictionsare placedon one precursor an alternative method can be used or
developed, switching to a readily available precursor, always staying one step
aheadofthelaw.Australiaisalsothemostattractiveimportdestinationdueto
relativehighwealthofthepopulationandhighdemandoftheproduct[17],the
pricepergramformethamphetamineinAustraliasubstantiallyexceedsthatof
othercountries,evenaftertakingintoaccountexternalmonetaryforces(Figure
1.4).
7|P a g e

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Figure1.4:MedianstreetpriceofagramoficeinAustraliandollars(AUD)[17].
BorderseizurefiguresinAustraliainrecenttimeshavereflectedthisincreasein
importationofmethamphetamine.Almost3tonnesofthedrughasbeenseized
attheborderbetween2010and2015(Figure1.5)[17].Theseseizureshavevaried
fromsmalltolargescales,overavarietyofsmugglingmethods,including;within
aeroplanes,shipsandmail,hiddeninfood,toys,onpeople,inmachineryandas
partsshipments[17].

Figure1.5:WeightofAustralianborderseizuresoficeandotheramphetamineͲ
typestimulantsinkilograms[17].
8|P a g e

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1.4LocalSynthesis
Inadditiontothedrugbeingimported,largeamountsofprecursorshavebeen
interceptedontheirwayintoAustralia,inanattempttocombattherestrictions
on chemical precursors and to continue drug synthesis within Australia [17].
Domestic seizures of the drug have also been rapidly increasing, which could
indicateariseinlocalsynthesis.Theweightofseizuresbetween2010and2014
has increased over five fold. Organised crime groups are at the centre of the
importation, manufacture and distribution of ice, in fact, accounting for
approximately two thirds of the groups on the ‘Australian Crime Commission’s
NationalCriminalTargetList’andthreequartersofthecrimegroupsjudgedas
highrisktothecommunity.Theorganisedcrimegroupshaveevolvedoverthe
yearsfromworkingwithintheirownnichebasetoworkingcoͲoperativelywith
othercrimegroups.Thiscollaborationisbasedonfinancialandcriminalbenefit,
ratherthanloyalty[17].
MethamphetamineproducedinAustraliaissynthesisedfromprecursorssuchas
pseudoephedrine (or less commonly ephedrine) and phenylͲ2Ͳpropanone [17].
Theseprecursorsalsohavelegitimatepharmaceuticalorpesticideuses,andas
suchwerereadilyavailableuntiltheirillicitusewasidentified[5].Ofteneitherthe
methamphetamineisimportedfromanothercountry,oraprecursormaterialis
importedforthesynthesisofthedrug[17].
1.5Precursorstomethamphetamine
The methods of methamphetamine manufacture carried out in clandestine

laboratorieshavechangedovertimeasprecursorshavebecomecontrolled[22].
As a precursor that was readily available becomes harder to obtain, other
methodstoreachthefinalproductevolvedtoensuretheproductionofthedrug
could continue [23]. As methamphetamine synthesis advances with control
changes in precursors, forensic chemists need to be able to identify new
methods/pathways in the clandestine laboratory scene [22]. Clandestine
9|P a g e

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synthesis of drugs begin with a precursor either distant (a compound which is
convertedtoanimmediateprecursor,followedbyconversiontothedrug)orfrom
immediate precursors. Immediate precursors are ideal for the manufacturer
howeverthemoretheyareused,themorechancetheyhaveofbeingrestricted
bylawenforcementauthorities[22].AnyonecanaccessstepͲbyͲstepinstructions
onthemanufactureofmethamphetaminefromarangeofprecursorsonwebsites
such as ‘Erowid’ [24]. The challenge therefore becomes obtaining these
precursors,andnotsomuchtheconversionofthemtomethamphetamine.
1.5.1Pseudoephedrine/ephedrine
In 2014, the Australian crime commission reported that from the 448 major
clandestine laboratories detected, 91% of seized methamphetamine had been
synthesisedusingpseudoephedrine[25];itissafetoassumethatitisthelargest
precursor source for methamphetamine manufacture in Australia. In Victoria,
there have been cases where pseudoephedrine was synthesised rather than
extracted from over the counter medication [personal correspondence with Dr
Jim Pearson at Victoria Police Forensic Services Department]. The selective
synthesis of the correct pseudoephedrine enantiomer for methamphetamine
becomesadifficulttaskastwostereocentres(highlightedbyredcirclesinFigure
1.6)areformedinthesynthesis.

Figure1.6:The2enantiomersand2diastereomersformedinthesynthesisof
pseudoephedrine.
10|P a g e

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One set of diastereomers form DͲmethamphetamine, a psychoactivedrug; The

other set of diastereomers (see Figure 1.7) form the significantly less active
LͲmethamphetamine,whichissimilartothepropertiesofpseudoephedrineand
islikewiseknowntobeusedforthereliefofnasaldecongestionandisinproducts
suchas‘VicksVapourInhalers’.

Figure1.7:TheformationofDͲmethamphetamineandLͲmethamphetamine
fromdifferentdiastereomers.
Clandestinesynthesisofpseudoephedrine/ephedrineisrelativelyuncommondue
tothecomplexandsometimeslengthyprocedures,resultinginracemicmixtures
which require enantiomeric purification; an extremely challenging task for
syntheticorganicchemists[26].
Theextractionprocessofpseudoephedrinefromcoldandflumedicationisquite
simplethatrequiresonlyaglassvessel,causticsoda,ethanol/methylatedspirits,
andafilter[27].ThisprocesshasbeeninvestigatedbyPigouetal.[26]anditwas
found to extract other active ingredients that in turn react in the conversion
processtomethamphetamineproducingbyproducts.Additionallyitwasfound
thattheuseofethanolormethanolasextractionsolventsleadtotheformation
of NͲethylͲephedrine and NͲmethylͲephedrine, especially when using the
hypophosphorousacidmethodtomakemethamphetamine[26].
However,inVictoriaallmedicationcontainingpseudoephedrineiscontrolledand
access to substantial amounts can become problematic. Every time a person
purchasesmedicationwithpseudoephedrine,theyarerequiredbylawtopresent
theirdriverslicenceandhavetheirdetailsrecordedonanationaldatabase.This
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alonecanbeadeterrentastheiridentityisnowonrecordforthepurchaseofthis
precursor.
Recently, an alternative fermentation method which uses cheap and easy to
sourceingredientshasbeenusedforthebiosynthesisoftheephedrineprecursor,
LͲphenylacetylcarbinol (LͲPAC) [28]. The fermentation process converts
benzaldehyde into LͲphenylacetylcarbinol (LͲPAC) which is a precursor for the
productionofLͲephedrine[28,29].Enzymesfromcertainyeaststrainsareableto
catalyse a reductive amination reaction. The same yeast (baker’s yeast Ͳ
Saccharomyces cerevisiae) is used in this process as for most alcohol
fermentations,asitisrobust.Theenzymeresponsiblefortheconversionofthe
benzaldehydeispyruvatedecarboxylase(PDC)[30].Fermentationofglucosewith
yeast results in the formation of pyruvic acid (pyruvate) which then undergoes
decarboxylation (via PDC catalysis) to form acetaldehyde, shown in Figure 1.8.
When acetaldehyde is in the presence of benzaldehyde it condenses to form
LͲPAC [26]. However, while this enzyme is able to synthesise LͲPAC it is also
associatedwiththeproductionofbenzylalcohol,anunwantedbyͲproductofthe
synthesiswhichwhencoupledwiththeoxidationofthebenzaldehydetobenzoic
acidresultsinadiminishedyieldofuseableproduct[30].
2 ADP 2 ATP
O
Glucose O PDC Catalyst
O
2 NAD 2 NADH 2 Pyruvate
H
O
H OH 2 CO2
2 Ethanol 2 Acetylaldehyde
O
OH
H Benzaldehyde
Benzyl alcohol
OH
L-PAC

Figure1.8:FermentationofprecursorLͲPAC.
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1.5.2PhenylͲ2Ͳpropanone(P2P)
PhenylͲ2Ͳpropanone (P2P) is the second most common precursor used in
clandestinemanufactureofmethamphetamine[17].Itisusuallypreparedfrom
phenylaceticacidwhichisthentreatedwithlead(II)acetateathightemperatures
orstirringamixtureofaceticanhydrideandsodiumacetate/pyridineatreflux[26,
31].TheproductionofmethamphetamineusingP2Pfrequentlyinvolvesalarge
amountofprecursor,converting10Ͳ20kilogramsofP2Ptomakeasinglekilogram
of methamphetamine [17]. The reaction between phenylacetic acid and acetic
anhydride is complex but is well described by Allen et al. [31] who proposed
mechanismillustratedinFigure1.9.

Figure1.9:Reactionbetweenphenylaceticacidandaceticanhydride[31].
PhenylͲ2Ͳpropanonecanalsobepreparedfrombenzaldehyde,whichisreacted
with nitroethane to produce ɴͲmethylͲɴͲnitrostyrene and will be discussed in
moredetailinChapter2.Thenitrostyreneproductcanthenbedirectlyreduced
toP2Pusingironandhydrochloricacid[32].
Both complex pathways (phenylacetic acid and benzaldehyde) to P2P, have
numerous side reactions resulting in characteristic impurities. Allen et al. [31]
indicated that routeͲspecific byͲproducts could be detected in crude reaction
mixtures.Specifically,withthephenylaceticacidmethodabyͲproductdetected
thathassignificantrelevanceistheproductionofdibenzylketone.Thisketoneis
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difficulttoseparatefromP2P,assuchnonͲcommercialP2Pwillprobablycontain
dibenzylketone,importantinformationforpolicetoknowwhentryingtotrace
thesourceoftheprecursor.
1.6Detectionmethodsofmethamphetamine
1.6.1PoliceroadͲsidetesting
Since1976,legislativeframeworkinVictoriahasbeeninplacetoscreendrivers
foralcohol[33].Sincethistypeoftestingcameintoeffectthenumberofdrivers
testedandfoundtobedrivingwithexcessivealcoholintheirsystemhasreduced
significantly[33].However,thedecreaseofalcoholinvolvementinroadtrauma
incidentsalsosawanincreaseintheinvolvementofothertypesofdrugssuchas
methamphetamine,amphetaminetypesubstances(ATS)andcannabis[33].
In 2000, legislation was introduced by the Victorian Government to prosecute
drivers who were impaired by other drugs, however this was based on the
recognition of impairment in drivers and enforcement was not high. In 2004
legislationwasintroducedwhichmimickedthatoftherandomalcoholscreening
for drivers using oral fluid testing at the roadside to detect the presence of
methamphetamine, methylenedioxymethamphetamine (MDMA), and cannabis
(THC)[33].Infact,outofthe4,497oralfluidsamplescollectedundertheroad
safetyactofVictoriain2015,theVictorianInstituteofForensicMedicinefound
methamphetaminein73.5%ofcases,amphetaminein71%ofcasesandcannabis
in53.9%ofcases[34].
Theroadsidescreeningisathreepartprocess,initiallyinvolvinganalcoholscreen
by the roadside then a preliminary roadside oral fluid test using an absorbent
collectorwhichtakesapproximately3to5minutes.Iftheresultsarenegativethe
driver is free to continue on, however if a target drug is detected the driver
accompaniespoliceintotheroadsidevehicletoprovideasecondoralfluidsample
whichistestedonanoralfluidscreeningdevicebyatrainedandauthorisedpolice
officer.Iftheresultispositivethedriverisinformedoftheresultsanddetailsare
collectedforchargestobelaidintheeventoflaboratoryconfirmation.Thethird
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stage in the process, where one or more of the target drugs are confirmed by
laboratoryanalysisresultsinthedriverbeingchargedwithanoffence[33].
1.7AnalyticalSeparationandDetectionMethodsfor
Methamphetamine
Methamphetaminehasreceivedagreatdealofattentionfromresearchgroups
allovertheworldastheiceepidemiccontinuestogrowandmakeheadlines[35].
Identifyingmethamphetamineindifferentmediumssuchasurine[36],saliva[34],
blood[37],plasma[38],hair[39],fingernails[40],fingerprints[41],sweat[42]
andsemen[43]havebeenareasofinterestforforensicanalysts.Becauseofthe
rangeofsampletypesandtheircomplexnature,avarietyofinstrumentationhas
beenusedfortheanalysisofsamplescontainingmethamphetamine,detailsof
whicharesummarisedinTable1.1below.
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Table1.1:Literatureworkthathasbeencarriedoutinthedetectionof
methamphetamine.
DetectionMethod Mediumtype LiteratureReference

GCͲMS Plasmaandurine [38]
GCͲMS Blood [37]
GCͲMS Hair [39]
GCͲMS Insects [44]
GCͲMS Nails [45]
HPLC Commercialtablets [46]
2DͲHPLC Streetsample [47]
LCͲMS Urine [48]
LCͲMS Fingerprint [49]
LCͲMS/MS Urine [50]
MALDIͲMS Fingerprint [41]
Chemiluminescence Urine [51]
Electrochemiluminescence(ECL) Streetsamples [52]
Electrosprayionisation(ESIͲMS) Humanurine [53]
HPLCFluorescence Humanurine [36]
FluorescencePolarisation
Purchasedstandards [54]
Immunoassay
GC/FTͲIR Purchasedstandards [55]
HeadͲspacesolidphase
PurchasedStandards [56]
microextraction/GCͲMS
Ionmobilityspectrometry Hair [57]
IsotoperatioMS Streetsamples [58]
Lasermicroscopy Hair [59]
Desorptionionisationonsiliconmass
FingerprintSweat [60]
spectrometryimaging(DIOSͲMSI)
NMR Purchasedstandards [61]
2
HNMR Syntheticpathwayproducts [62]
Capillaryelectrophoresis Urine [63]
ElectromembranesurroundedͲsolid
phasemicroextraction Urineandblood [64]
(EMͲSPME)
Surfaceplasmonresonance(SPR) Purchasedstandards [65]
FieldAssymetricIonMobility
Air [66]
Spectrometry
Colourimetrictest Mobiletelephone [67]

Interestingly,thedevelopmentofanalyticaltechnologyforforensicapplications
isoftenlimitedbytheuptakeofnewmethods,duetotheimplicationsinthecourt
room. New analytical methodologies need to be thoroughly tested and come
under strict scrutiny in cross examination of expert witnesses. Regarding
methamphetamine determination, GCͲMS is the most commonly used analysis
methodduetoitbeingoneofthefirstutilisedinthisspaceandtestedunderlaw.
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Typically GCͲMS systems are coupled with quadrapole mass spectral detectors
affordingonlylowresolutiondata,whereasLCͲMSiscommonlycoupledwithhigh
resolutiondetectors(suchastimeofflight)whichhasimprovedthedetermination
ofmethamphetamine.BespoketechniquessuchastheotherslistedinTable1.1
aretypicallyusedintheresearchdomain.Whilethesearenotcommonlyusedfor
prosecution purposes they play a strong role informing the forensic analysts
towardsreliablemethamphetaminedetection.

1.8TwoͲdimensionalHPLC
Liquidchromatographyisamethodusedbyanalyticalandsyntheticchemiststo
separatecomponentswithinamixture,basedontheretentionpropertiesofthe
mobilephase(eluent)andthestationaryphase(column)[68].Themostcommon
formofliquidchromatographyisknownas‘reversephase’whichutilisesanonͲ
polar adsorbent stationary phase with a more polar mobile phase to ensure
differentialmigration[68].Theuseofliquidchromatographywasfirstreportedin
1903byMikhailTswettwithhisworkonplantcellsandchloroplasts[68].Tswett
successfully separated plant pigments for the first time with a column, packed
with various adsorbents [68]. In 1967, Horváth and coͲworkers established the
veryfirstpressurisedchromatographysystem,knowntodayashighperformance
liquidchromatography(HPLC)[69].
TheseparationpowerofconventionalHPLCislimitedwhenitcomestoanalysing
complex samples [70]. A significant amount of effort has been dedicated to
overcoming the limits of separation and resolving power of single dimension
systems[70,71].ThishasresultedintherapiddevelopmentandevolutionoftwoͲ
dimensionalhighperformanceliquidchromatography(2DͲHPLC)
Theadvantageof2DͲHPLCoverconventionaloneͲdimensionalHPLCisitssuperior
resolvingpower,takingfulladvantageofincreasedseparationspace[70,72,73].
Traditionally, twoͲdimensional separations are a very time consuming process
[74],asnumerousparameterssuchasstationaryphaseselection,mobilephase
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selection,samplingfrequencyandanalysistimemustbeoptimisedtorealisethe
fullpotentialofthesystem[75].
TwoͲdimensionalHPLChasbeenrecentlyusedinfieldsthatrequiretheanalysis
andseparationofcomplexsamplessuchaspharmaceuticals[76],foodanalysis
[77], wine [78], metabolomics [71, 72], proteomics [79] and environmental
analysis[80].Itissignificantforatechniquetobeusedinsuchabroadrangeof
scientific fields and 2DͲHPLC has helped analysts understand and visualise the
complexityofsomesamples.
Therearetwodifferenttypesof2DͲHPLC:offlineandonlineͲbothhavingtheir
ownadvantagesanddisadvantages[81].Offlineoperatesbycollectingandstoring
fractionsastheyelutefromthefirstdimensioncolumn[81],followedbyindividual
injectionofthosefractionsintotheseconddimension.Theadvantageofthisisits
extremelylargeseparationpowerbasedonthepeakcapacitygeneratedbythe
uniquenessoftheretentionmechanisms[81],andthedownfallbeingitisalabour
intensive process (hours to weeks) with extraordinarily long analysis times,
dependingonthecomplexityoftheanalysedsample[82].
Online2DͲHPLCworksbytransferringelutingfractionsfromthefirstdimensionto
aseconddimensionwhichisrunningsimultaneouslyusingaswitchingvalveand
sampleloopsshowninFigure1.10.Thistransfercanbedonefortheentirefirst
dimension,knownas‘comprehensive’orforselectedpeaksofinterest,knownas
‘heartͲcutting’[74].Theadvantageofonlineisthefactitisfullyautomatedbythe
systemandcanbedonerapidly.However,whenusingonline2DͲHPLCthesecond
dimensionneedstobeconfiguredtoeluteacompoundbeforethenextfraction
istransferredinordertominimisethechanceofanypeakoverlapfromdifferent
fractions, placing a serious time constraint on second dimension analysis time
[74]. This emphasises the need for 2DͲHPLC systems that can operate at high
speeds,particularlyintheseconddimension[74].
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CHAPTER1
Figure1.10:Online2DͲHPLCswitchingvalve.
1.9InͲSilicoOptimisationChromatography(computationalmodelling)
Computersupported,chromatographymethoddevelopmentstartedinthemidͲ
1980s as IBM released the first PC (personal computer) [83]. TjuͲlik et al. used
computer simulations to study the abnormal peak shapes caused by
chromatographicsolvents,highlightingthatpeaksplittinganddistortioncanbe
modelledbytheeffectofthesolventonthecolumn[84].Followingthis,thefirst
simulation software (DryLab®) for HPLC was released in 1988 after significant
fundamental development by Horváth and coͲworkers into the solvophobic
interactions[85],ionogenicsubstanceinteractions[86],andionͲpairformationof
nonpolar stationary phases [87]. Snyder, Dolan and coͲworkers used Drylab®
softwareinordertointerrogateisocratic[88]andgradient[89]highperformance
liquidchromatography,highlightingtheimportanceofasimulationapproachsuch
as for significant industrial problems. This type of modelling was further
developedbyLewisetal.[90]whoincorporatedthepHofthemobilephaseinto
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thepredictionoftheseparation.Challengesinapplyingsimulationpredictionsto
largemoleculeswereidentifiedbyGhristetal.[91]whowentontoshowthat
withprecautionaryadjustmentsofthesimulatedseparationgradienttheseissues
canbeovercome.Temperaturehasbeenhighlightedasakeyparameterforthe
optimisationofsimulationsthatwasinitiallydevelopedbyHancocketal.[92]and
further reviewed by Dolan [93]. The testing of temperatures was applied to
peptides and proteins [94], highlighting that optimisation of both temperature
and gradient steepness [95] is advantageous through the application on
herbicides[96]andproteindigests[96].
Also,octanolwaterpartitionhasbeenusedasaseparationtoolforscientistsand
Giaginisetal.[97]showedtheimportanceofnͲoctanolasamobilephaseadditive
for polar compounds. The effect on nͲoctanol was shown to decrease as the
lipophilicity increased, highlighting the importance of hydrogen bonding in the
simulation mechanism for carboxylic acids. Monks et al. [98] emphasises the
robustnatureofthisprocessandincorporatesatwovariableoptimisationsystem;
oneforthecolumnandonefortheeluentbringingthemtogether.Thisconcept
is important as the ability to combine the design space and opens up the
opportunity to explore the use of this approach to a second stationary phase,
whichisrequiredfortheinͲsilicooptimisationoftwoͲdimensionalHPLC.
Simulationsoftwarehasbeenofinteresttoarangeofareasofchemistryandhas
been successfully applied to drugs and pharmaceuticals [96, 99], phenolic
pollutants [96], protein digests [96], enantiomers [100], proteinͲprotein
interactions [101], marine pigment samples [102] and fatty acid methyl esters
[96]. Additionally, there are a range of chromatography modelling software
packages available such as ‘PESOS’ made by PerkinͲElmer [103] and ‘Simplex’
madebyBerridge[104].
Thisthesiswillincorporatetwodifferentparts,asillustratedinFigure1.11.The
first part, described in chapters 2 and 3 will investigate the viability of some
contemporary clandestine methamphetamine synthetic pathways in order to
assesstheriskoftheirpotentialappearanceinfutureclandestineseizures.The
second part outlined in chapters 4 and 5 will explore the optimisation for
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advancedseparationsofcomplexseizuresamples,followedbythedevelopment
ofplatformtechnologyforthechemicalmappingandidentificationofprecursors
and synthetic route. This technology will be fully assessed in order to test its
suitabilitytogeneratemoreadvancedchemicalfingerprintinganalyticalcapacity
forseizuresampleanalysis.
Figure1.11:Generalisedoverviewofthemultidisciplinaryapproachtakenin
thisthesis.
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CHAPTER2
CHAPTER 2:
SYNTHESIS OF
PSEUDOEPHEDRINE/EPHEDRINE FROM
NITROETHANE AND BENZALDEHYDE
Author’sNote:Therearetwosyntheticchapters(2&3)includedinthisthesiswhichwere
undertakeninanalternativestyletothemoretraditionalsyntheticchemistryformat.As
thisthesisexaminesthechemicalmappingofpotentialclandestinelaboratorypathways,
isolationandpurificationofcompoundsweredeliberatelynotcarriedout,toeffectively
mapallpotentialimpuritiesfromeachstep.Thesyntheticpathwaysinvestigatedwere
performedtodetermineifitisaviablepathwaytomethamphetaminemanufacture.
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CHAPTER2
CHAPTER OVERVIEW
ThischapterprovidesabriefintroductiontotheHenryReaction(nitroaldol)and
will discuss its history, mechanism, and literature reported uses in a range of
differentfields.Itwillalsodiscussthetwomainsyntheticpathwaysusedinthe
manufacture of methamphetamine, linking the Henry reaction (using
benzaldehydeandnitroethane)tobothroutes.
A synthetic investigation will be carried out on the reaction between
benzaldehyde6andnitroethane7.Theproducts2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8
andphenylͲ2Ͳnitropropene9formedfromthisreactionwassolelydependenton
the bases used, illustrated in Figure 2.1. Coincidentally, both products are
precursors to different clandestine pathways (ephedrine and P2P) which is of
greatinteresttoVictoriaPoliceForensicServicesDepartment.
Figure2.1:Resultingclandestinepathwayswhenusingdifferentbasesinthe
reactionbetweenbenzaldehydeandnitroethane.
Likewise, a full pathway to methamphetamine was explored from the

2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8product.Chemicalmappingofeachsyntheticstep
andassociatedimpuritieswillbecarriedoutinChapter5,toidentifybyͲproducts
characteristicofthesyntheticrouteusedtogeneratetheclandestineproducts.
23|P a g e

CHAPTER2
2.1INTRODUCTION
TheHenryreaction,alsoknownasthe‘NitroͲaldol’reaction,wasfirstreportedin
1895 and is a base catalysed carbonͲcarbon bond forming reaction between
nitroalkanesandaldehydesorketones[105].Thereactioninvolvesgeneratinga
highlyreactivenitronatespeciesinsitufollowedbynucleophilicadditiontothe
carbonylgroupofanaldehydeorketone[106].TheHenryreactionisfrequently
usedduetoitshighlyfunctionalscaffoldanduniversalsyntheticcapabilitiesby
easy manipulation of both the nitro and hydroxyl functional groups to make
ɴͲnitroalcohols(conventionalproductoftheHenryreaction)[105].Nitroalcohols
can be easily converted to intermediates such as nitroalkenes (dehydration),
ɲͲnitro ketones (oxidation) and ɴͲamino alcohols (reduction) [105]. These
intermediates have found use in synthesis of pharmaceuticals (ɴͲblockers, HIV
protease inhibitor, antibiotics) [107], asymmetric synthesis and biocatalytic
applications[107,108].Asaconsequence,therehasbeenmuchefforttoimprove
the Henry reaction with research into the use of catalysts, including catalyst
controlled asymmetric reactions [106], catalyst controlled direct asymmetric
reactions [109], rare earth catalysis [110], Zinc(II) based catalysis [111], copper
based catalysis [112], cobalt based catalysis [113], organocatalysis [114], and
antiͲselectivecatalysis[115].
Benzaldehydeandnitroethanecanbeusedinanitroaldolreactiontoformeither
oftwoproducts,aɴͲnitroalcoholoraɴͲnitrostyrene(Scheme2.1).Interestingly,
eachofthesetwoproductscanbeusedinsyntheticpathwaysfortwocommon,
but alternative, precursors to the synthesis of methamphetamine, namely
phenylͲ2Ͳpropanone(P2P)andephedrine/pseudoephedrine[58].
24|P a g e

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Scheme2.1:AlternativeproductsfromtheHenryreaction.
AccordingtotheAustralianCrimeCommissionIllicitdrugdatareport2013Ͳ2014,
benzaldehyde is classed as a ‘significant precursor’ to the manufacture of
methamphetamineandapproximately10tonnesofbenzaldehydewasseizedin
Australia within the year of the report [25]. As such, the investigation of any
syntheticreactionstowardsthesynthesisofillicitdrugsinvolvingbenzaldehydeis
ofutmostimportancetoclandestinelaboratoryinvestigators.Theimplicationsof
thischemistryontheformationofmethamphetamineneedstobeconsideredas
it relates to the two major pathways, the reductive amination of P2P and the
conversionfromephedrine.
2.1.1ReductiveAminationofphenylͲ2Ͳpropanone(P2P)
PhenylͲ2Ͳpropanone (3) is an example of an immediate precursor and its
conversion to methamphetamine (2) is the more complex of the two common
methods. It initially requires the formation of an imine (4) with methylamine,
followedbyametalreduction[22],asshowninScheme2.2.
Scheme2.2:ThereductiveaminationofP2PtoMethamphetamine.
Alternatively, the Leuckart reaction can be used as shown in Scheme 2.3. This
reaction produces the NͲdialkylformamide (5) after the nucleophilic nitrogen
25|P a g e

CHAPTER2
attacksthecarbonylcarbonandsubsequentreduction,usingformicacid[15].The
intermediateproductcanthenundergoahydrolysistoformmethamphetamine.
In1981,P2P(alsoknownasphenylacetone)waslistedasacontrolledsubstance
undertheDrugsPoisonsandControlledSubstancesAct(Victoria),aswellasin
otherAustralianlegislation,inanattempttocutoffthesupplyofprecursorsto
clandestine laboratories. The act had the desired effect; with the number of
seizuresofclandestinelabsdecreasingforafewyears[22].However,anewage
in methamphetamine production arose with the use of pseudoephedrine or
ephedrineasaprecursor[22].
O
3 H
H N N H N
H NaOH
O NaOH O HCl HCl

3 5 2

Scheme2.3:TheLeuckartreactionofmethamphetaminesynthesis.
2.1.2PseudoephedrineReduction
Pseudoephedrine(andtoalesserextentephedrine)isacommoningredientin
cold and allergy medications, with many having only pseudoephedrine as the
activeingredient[116](Figure2.2).
Figure2.2:Structureofpseudoephedrine.
As P2P became more difficult to obtain in the 1980s, the development of
alternativesyntheticroutesledtoanincreaseofthesemedicationsbeingstolen,
divertedorpurchasedinbulkforuseinclandestinelaboratories.Inresponse,the
26|P a g e

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salesofproductscontainingpseudoephedrineorephedrinewerereinedinunder
strictregulationswiththeintroductionofnewlaws[22].Whilethesecompounds
arenowregulated,theyarestillavailableoverthecounteranddostillprovide
methamphetaminecookswithaprecursorforeasyconversionsonasmallscale.
There are two main processes for the conversion of pseudoephedrine to
methamphetamine; the Birch reduction (also referred to as the ‘Nazi method’)
andredͲphosphorus/hydriodicacidmethods[117].
2.1.3BirchReductionofpseudoephedrinetomethamphetamine
ThepseudoephedrinepathwayshowninScheme2.4involvespseudoephedrine
(1)beingaddedtoamixtureoflithiummetal(usuallyobtainedfrombatteries)in
anhydrousammonia.Theammoniacanbeobtainedfrompurchasedgascylinders
oralternativelygeneratedinsitufromfertilizerproducts.Whenlithiumisadded
toammoniathesolutionturnsblue;thisisshortlived,withthesolutionchanging
to grey as the reaction progresses [118]. Similar to all methods of
methamphetamine production the Birch method has both advantages and
disadvantages.Someadvantagesofthismethodarethattheresiduallithiumis
easilydecomposedbywateroralcoholandthatexcessammoniaisboiledoffin
theprocess[118].Themaindisadvantageisthatanhydrousammoniaisneeded
whichisadifficultsubstancetohandleasitiscorrosive,hazardous,andpotentially
explosive.
Scheme2.4Birchreductionofpseudoephedrinetomethamphetamine.
The second common pseudoephedrine based method used is the redͲ
phosphorus/hydriodicacidmethod,thisisanappealingmethodduetothefact
thatitisquick,canproducerelativelyhighyieldsandcanbecarriedoutina‘one
pot’process[119].Thismethodisfunctionsbyreducingthealcoholgrouptoa
27|P a g e

CHAPTER2
hydrogen [119]. There are several variations of this method, including one in
which the reactant is cold or mildly heated (<60 °C) and others which involve
heating the reaction to reflux [119]. The reaction variations involves both red
phosphorus,whichaclandestinelaboratorymayobtainfrommatchesorflares,
andhydriodicacid,eitherpurchasedorproducedinsitu[119].Anothervariation
ofthesamereactionprocessuseshypophosphorousacidandiodinetogenerate
thehydriodicacidinsitu[120].
TheimplicationsofbaseinthecommonHenryreactionwhencarriedoutusing
benzaldehyde(6)andnitroethane(7)wasinvestigated,particularlyasdescribed
inclandestineonlinedrugforumssuchasthewebsite‘Erowid’[24].Forthefirst
time a comprehensive study of the Henry reaction using alternative bases is
presentedinthischapter(Scheme2.5).

Scheme2.5:Resultingproductswhenusingdifferentbasesinthereaction
betweenbenzaldehydeandnitroethane.

Therangeofsyntheticroutesforthegenerationofmethamphetamineofferboth
asignificantchallengefortheforensicanalystswhilealsoaffordingasignificant
opportunity. The fact that there is a vast array of synthetic routes means that
analysts must have a broader understanding of the chemistry, however with a
range of routes the capacity to individualise samples is increased. Generally,
methamphetamine seizure samples in Victoria are very pure and as such it is
difficulttoidentifytherouteofmanufacture.Thisopensuptheopportunityto
use intermediate samples in order to profile the provenance of the
28|P a g e

CHAPTER2
methamphetamine.Thenatureofthistaskrequiresamultidisciplinaryapproach
andrequiresanalyticalmethodologiestoinformthesyntheticrouteinvestigation.

2.2EXPERIMENTAL

AllchemicalsweresourcedfromSigmaAldrich.All1Hspectrawererecordedona
JEOLJNMͲEX270MHz.Samplesweredissolvedindeuteratedchloroform(CDCl3)
withtheresidualsolventpeakusedasaninternalreference(CDCl3–ɷ7.26ppm).
Proton spectra are reported as follows: chemical shift ɷ (ppm), (integral,
multiplicity(s=singlet,br.s=broadsinglet,d=doublet,dd=doubletofdoublets,
t=triplet,q=quartet,m=multiplet),couplingconstantJ(Hz),assignment).

Highresolutionmassspectrometry(HRMS)wasperformedusinganAgilent6210
MSD TOF mass spectrometer with the following settings: gas temperature
(350°C),vaporizer(28°C),capillaryvoltage(3.0kV),conevoltage(40V),nitrogen
flow rate (0.5 mL minͲ1), nebuliser (15 psi). Samples were dissolved in MeOH
(0.5mgmLͲ1)priortoinjection.
Synthesisof2ͲnitroͲ1ͲphenylͲ1Ͳpropanol(mixtureofSynandAntidiastereomers)
(8)
Thesynthesisofthetitlecompoundwascarriedoutusingthreedifferentbases;
triethylamine, sodium hydroxide and sodium hydride (optimised conditions
described).The1HNMRoftheproductsfromthe3differentbaseswereidentical,
allproducinga61:39ratioofSynandAntidiasteromersandthespectraisshown
inFigure2.2.
TriethylamineBase(Et3N)
29|P a g e

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Toastirringsolutionofnitroethane(6.8mL,94.2mmol,4.0eq.)andtriethylamine
(6.6 mL, 47.1 mmol, 2.0 eq.) in dichloromethane (5 mL), benzaldehyde
(2.5mL,23.5mmol,1.0eq.)wasaddedslowlyover2minutes.Themixturewas
lefttostirfor16hourswherethesolutionthenturnedabrightyellowcolour.The
mixturewastransferredtoaseparatingfunnelwhereitwaswashedwithsodium
bisulfite(3×20mL)toremoveanyexcessbenzaldehydeandthenwashedwith
brine(3×20mL).TheorganiclayerwasdriedoverMgSO4,filteredandthesolvent
removedinvacuotoaffordthetitlecompoundasayellowoil(3.63g,85%).Syn
diastereomer;1HNMR(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),5.03(d,1H,JHH
=8.90Hz,CH),4.83Ͳ4.65(m,1H,CH),2.48(brͲs,1H,OH),1.32(d,3H, 3JHH=6.80
Hz,CH3).Antidiastereomer; 1HNMR(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),
5.40(d,1H,JHH=3.60Hz,CH),4.83Ͳ4.65(m,1H,CH),2.48(brͲs,2H,OH),1.51(d,
3H,JHH=6.80Hz,CH3).MS(ESI,m/z)calculated[C9H11NO3+Na]+204.06311found
m/z204.06493.
1
HNMRspectralpropertiesmatchedthatoftheliterature[121].
SodiumHydroxideBase(NaOH)
To a stirring solution of nitroethane (13.5 mL, 188.5 mmol, 4.0 eq.), sodium
hydroxidesolution(20mL,94.2mmol,2.0eq.)wasaddedinasingleportion.Then
benzaldehyde(5.0mL,47.1mmol,1.0eq.)wasaddedslowlyover2minutes.The
mixturewaslefttostirfor16hourswherethesolutionthenturnedabrightyellow
colour.ThemixturewastransferredtoaseparatingfunnelwhereCH2Cl2(20mL)
wasadded. Theorganiclayerwaswashedwithsodiumbisulfite(3×20mL)to
removeanyexcessbenzaldehydeandthenwashedwithbrine(3×20mL).The
organiclayerwasdriedoverMgSO4,filteredandthesolventremovedinvacuoto
affordthetitlecompoundasayellowoil(7.94g,93%).Syndiastereomer;1HNMR
(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),5.03(d,1H,JHH=8.90Hz,CH),4.83Ͳ4.65
(m,1H,CH),2.48(brͲs,1H,OH),1.32(d,3H,JHH=6.80Hz,CH3).Antidiastereomer;
1
HNMR(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),5.40(d,1H,JHH=3.60Hz,CH),
4.83Ͳ4.65(m,1H,CH),2.48(brͲs,2H,OH),1.51(d,3H,3JHH=6.80Hz,CH3).MS(ESI,
m/z)calculated[C9H11NO3+Na]+204.06311foundm/z204.06500.
30|P a g e

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1
SodiumHydrideBase(NaH)
Toastirringsolutionofnitroethane(8.08mL,113.08mmol,4.0eq.)anddryTHF
(15mL),sodiumhydride60%dispersionmineraloil(2.26g,56.54mmol,2.0eq.)
was cautiously added and left to stir until gas evolution had completed.
Benzaldehyde (2.88 mL, 28.27 mmol, 1.0 eq.) was then added under inert
conditions(N2(g))withadroppingfunnelover5minutes.Themixturewasleftto
stirfor16hourswherethesolutionwasthenquenchedwithwatercautiously(10
mL). The mixture was transferred to a separating funnel with CH2Cl2 (20 mL),
where it was washed with sodium bisulfite (3 × 20 mL) to remove any excess
benzaldehydeandthenwashedwithbrine(3×20mL).Theorganiclayerwasdried
overMgSO4,filteredandthesolventremovedinvacuo.Thecompoundwasthen
dissolvedupinhexaneandpouredthroughasilicaplugtoremovethemineraloil.
Thenethylacetatewasusedtoflushoutthetargetproductfromthesilica.The
organiclayerwasdriedoverMgSO4,filteredandthesolventremovedinvacuoto
affordthetitlecompoundasayellowoil(3.89g,76%).Syndiastereomer;1HNMR
(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),5.03(d,1H,JHH=8.90Hz,CH),4.83Ͳ4.65
(m,1H,CH),2.48(brͲs,1H,OH),1.32(d,3H,JHH=6.80Hz,CH3).Antidiastereomer;
1
HNMR(270MHz,CDCl3):ɷ7.45Ͳ7.29(m,5H,Ar),5.40(d,1H,JHH=3.60Hz,CH),
4.83Ͳ4.65(m,1H,CH),2.48(brͲs,2H,OH),1.51(d,3H,JHH=6.80Hz,CH3).MS(ESI,
m/z)calculated[C9H11NO3+Na]+204.06311foundm/z204.06493.
1
SynthesisofPhenylͲ2Ͳnitropropene(9)
Thesynthesisofthetitledcompoundwascarriedoutusingtwodifferentbases;
nͲbutylamineandammoniumacetate.The 1HNMRoftheproductsfromthe2
differentbases,wereidenticalandthespectraisshowninFigure2.3.
31|P a g e

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2.2.1nͲButylamineBase(nͲBuNH2)
Toastirringsolutionofnitroethane(2.02mL,28.27mmol,1.0eq.)andethanol(5
mL),nͲbutylamine(0.3mL,2.82mmol,0.1eq.)wasaddedinasingleportionand
lefttostirfor5minutes.Benzaldehyde(2.88mL,28.27mmol,1.0eq.)wasthen
addedslowlyover2minutes.Themixturewaslefttostiratrefluxfor8hours,
when the solution turned a bright yellow colour. The solvent was removed in
vacuo and allowed to cool. As the yellow oil cooled, yellow crystals formed to
affordthetitlecompound.(3.26g,71%).1HNMR(270MHz,CDCl3):ɷ8.10(s,1H,
CH),7.50Ͳ7.40(m,5H,Ar),2.46(s,3H,CH3).MS(ESI,m/z)calculated[C9H9NO2+H]+
164.07060foundm/z164.0636.
1
Ammoniumacetatebase(NH4OAc)
Toastirringsolutionofnitroethane(2.02mL,28.27mmol,1.0eq.),ammonium
acetate(0.22g,2.82mmol,0.1eq.)andglacialaceticacid(5mL)wasaddedina
singleportionandlefttostirfor5minutes.Benzaldehyde(2.88mL,28.27mmol,
1.0eq.)wasthenaddedslowlyover2minutes.Themixturewaslefttostiratreflux
for 4 hours, when the solution turned a bright yellow colour. The solvent was
removedinvacuoandallowedtocool.Astheyellowoilcooled,yellowcrystals
formedtoaffordthetitlecompound.(2.72g,59%). 1HNMR(270MHz,CDCl3):ɷ
8.10(s,1H,CH),7.50Ͳ7.40(m,5H,Ar),2.46(s,3H,CH3).MS(ESI,m/z)calculated
[C9H9NO2+H]+164.07060foundm/z164.0589.
1

32|P a g e

CHAPTER2
2.3RESULTSANDDISCUSSION
2.3.1Synthesisof2ͲNitroͲ1Ͳphenylpropanol(8)
In order to understand the influence of a base on the Henry reaction,
benzaldehydeandnitroethanewerereactedinthepresenceof5differentbases;
triethylamine,sodiumhydroxide,sodiumhydride,nͲbutylamine,andammonium
acetate.
Initially,thetriethylaminebase(1equivalent)wastrialledwhereitwasreacted
withoneequivalentofbenzaldehydeandoneequivalentofnitroethane(Entry1,
Table 2.1). The 1H NMR spectrum of the crude reaction product showed the
product2ͲnitroͲ1Ͳphenylpropanol8,withsomebenzaldehyde(startingmaterial)
present.Thisreactionwasthenoptimisedtominimisetheamountofunreacted
benzaldehydethatwaspresentinthecrudeextract.Theconditionstrialledare
presentedbelow(Table2.1)andthebestyieldattainedwas85%(1.0g–3.0g
scale)(Entry7,Table2.1).Foranextensivecomparisonofbases,0.2equivalents
of triethylamine was also used to ensure a range of base concentrations were
trialled(Entry9,Table2.1).
Inthecaseoflowyieldingreactions(Entries1,2&9,Table2.1)nootherproducts
were identified, just substantial amounts of recovered benzaldehyde.
Significantly, no nitrostyrene (dehydrated product) was observed in these
reactions despite the reaction of benzaldehyde and nitroethane to form
nitrostyrenesbeingmentionedthroughouttheliterature[123].

33|P a g e

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Table2.1:Triethylamineconditionsofnitroaldolreaction.
Combined Syn & Anti

OH
O Et3N NO2
+ NO2
reflux, 16 h
6 7 8

Nitroethane Triethylamine Yield

Entry Benzaldehyde(Eq.) Solvent
(Eq.) (Eq.) (%)
1 1.0 1.0 1.0 Neat 28
2 1.0 1.0 2.0 Neat 33
3 1.0 2.0 1.0 Neat 41
4 1.0 2.0 1.0 DCM 44
5 1.0 3.0 1.3 Neat 56
6 1.0 3.0 1.3 DCM 62
7 1.0 4.0 2.0 DCM 85
8 1.0 5.0 2.0 DCM 84
9 1.0 4.0 0.2 DCM 12
Analysisby1HNMRandLCͲMSconfirmedtheformationoftheproduct2ͲnitroͲ1Ͳ
phenylͲ1Ͳpropanol as diastereomers, synͲ and antiͲ. 1H NMR spectroscopy
(Figure 2.3) was used to determine a 61:39 ratio of syn/anti diastereomers for
2ͲnitroͲ1Ͳphenylpropanol 8 by integration and the chemical shifts reported by
Blayetal.[121].
34|P a g e

CHAPTER2

1E+07

1E+07
1E+07
9E+06

8E+06
7E+06
6E+06

5E+06

4E+06
3E+06
2E+06

1E+06
0
7.5 7.0 6.5 6.0 5.5 5.0 4.5

f1 (ppm)
4.0 3.5 3.0 2.5 2.0 1.5 1.0
Figure2.3:1HNMRspectrumofmixedproduct2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8,
wheregreenrepresentsSyn,bluerepresentsAntiandblackrepresentsboth.
The general mechanism for the formation of the 2ͲnitroͲ1ͲphenylͲ1Ͳpropanol

product8isshownbelowinScheme2.6,wherebytheReandSifaceattackofthe
aldehydeisresponsibleforthegenerationofthediastereomers.
Scheme2.6:Generalmechanismfor2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8.
35|P a g e

CHAPTER2
Asmorethanoneequivalentoftriethylaminewasrequiredtoobtainagoodyield,
attentionthenturnedtoastrongerbaseinordertoincreasethereactionyield.
Sodium hydroxide (an inorganic base) was used making the reaction biphasic.
Initially, the reaction was trialled with one equivalent each of benzaldehyde,
nitroethane and sodium hydroxide and as expected, the same product was
formed (2ͲnitroͲ1ͲphenylͲ1Ͳpropanol) with a large excess of benzaldehyde still
present.Thus,optimisationwascarriedoutasshowninTable2.2,wherethebest
yieldachievedwas93%(Entry4(1.0g–3.0gscale)).
Table2.2:Sodiumhydroxideconditionsofthenitroaldolreaction.

Benzaldehyde Nitroethane SodiumHydroxide Yield
Entry Solvent
(Eq.) (Eq.) (Eq.) (%)
1 1.0 1.0 1.0 H2O 32
2 1.0 1.0 2.0 H2O 46
3 1.0 2.0 1.3 H2O 78
4 1.0 4.0 2.0 H2O 93
5 1.0 4.0 0.2 H2O 14

Alargeexcess(4equivalents)ofnitroethaneand2equivalentsofthebasewas
requiredtoobtainhighyieldsfromthisreaction,indicatingthehydroxidespecies
stillmaynotbebasicenough,duetopoorsolubilityinnitroethane,todrivean
efficient reaction. Thus a stronger base, sodium hydride, was employed as
outlinedinScheme2.7below.

Scheme2.7:Nitroaldolreactionusingsodiumhydride.
36|P a g e

CHAPTER2
Benzaldehyde and nitroethane (1.0 equivalent each) were reacted with 2.0
equivalents of base (sodium hydride) under an inert atmosphere (N2) using
anhydrousTHFasthesolvent.Oncompletionofthereactionthesameproductas
describedinTable2.1andTable2.2(2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8)wasformed
inhighyield(72%).Ofnotethepresenceofsomeunreactedbenzaldehydewas
detectedinthe1HNMRspectrumandtheLCͲMSchromatogram,highlightingthat
the strength of the base had no influence on the equivalents ratio of the
benzaldehyde and nitroethane. From this it seemed, simply an excess of
nitroethaneisrequiredforefficientconversion.Inlightofthisobservation,the
reactionwasrepeatedusing4.0equivalentsofnitroethanewith1.0equivalentof
benzaldehydeand2.0equivalentsofsodiumhydridetoproduceayieldof76%.
As the nitroethane used throughout the experiments was not anhydrous, it is
possiblesomeadventitiousmoisturefromthenitroethanecouldhavequenched
someofthesodiumhydride,reducingthepotentialyield.
Accordingtotheliterature,typicallytheyieldofthenitroͲaldolisbestoptimised
byhavingasubstantialexcessofthenitroalkane(>3.0eq.)[124],whichfollows
thesametrendastheoptimisationconditions(Table2.2).
2.3.1SynthesisofphenylͲ2Ͳnitropropene(9)
Two alternative bases commonly found in clandestine literature were also
evaluated; nͲbutylamine and ammonium acetate. According to the online drug
forums [24], both of the bases are used in catalytic amounts (10 mol% and 20
mol%,respectively)toproducephenylͲ2Ͳnitropropene(9).
Initially,1.0equivalentofbenzaldehydeandnitroethanewasreacted(neat)with
0.1equivalentsofNͲbutylaminetoformphenylͲ2Ͳnitropropene9in71%yield,as
showninTable2.3.The 1HNMRspectrumshowedinFigure2.4matchedthatof
previouslyreportedliterature[122].
37|P a g e

CHAPTER2
Table2.3:NitroaldolreactionwithnͲbutylamine.
Benzaldehyde Nitroethane NͲbutylamine Yield

Entry Solvent
(Eq.) (Eq.) (Eq.) (%)
1 1.0 1.0 0.1 Neat 71
2 1.0 2.0 0.1 EtOH 70
3 1.0 2.0 2.0 EtOH nil**

**Productunidentified/complexmixtureofproducts
CH3

Figure2.4:1HNMRspectrumofphenylͲ2Ͳnitropropene9.

The same reaction was then repeated using ethanol as a solvent and 2.0
equivalentsofnitroethane,toseeiftheamountofnitroethanewouldimprove
the yield at all. This produced a yield of 70%, therefore the slight increase in
nitroethanedidnotseemtoimpacttheyieldwhenusingnͲbutylamineasthebase
38|P a g e

CHAPTER2
(Table2.3).Finally,2.0equivalentsofnͲbutylaminewastrialledasacomparison
with previous bases. Interestingly, upon analysis neither 2ͲnitroͲ1ͲphenylͲ1Ͳ
propanolnorphenylͲ2Ͳnitropropenewereobservedin1HNMRspectrum,instead
itappearedthatacompletedegradationhadoccurredinthe1HNMRspectra,with
apoormassrecovery.
The final base studied was ammonium acetate, using 0.2 equivalents with 1.0
equivalentofbothbenzaldehydeandnitroethaneinthepresenceofglacialacetic
acid (Table 2.4), producing a yield of 59% of the nitrostyrene. Likewise,
nitroethanewasincreasedto2.0equivalents,againhavingnoinfluenceproducing
ayieldof57%.Thereactionwastrialledagainusing2.0equivalentsofammonium
acetate to produce a yield of 58 %, with trace amounts of 2ͲnitroͲ1ͲphenylͲ1Ͳ
propanol9presentconfirmedvia1HNMR.
Table2.4:Nitroaldolreactionwithammoniumacetate.
Benzaldehyde Nitroethane Ammonium Yield

Entry Solvent
(Eq.) (Eq.) acetate(Eq.) (%)
1 1.0 1.0 0.2 Neat 59
2 1.0 2.0 0.2 Neat 57
3 1.0 2.0 2.0 Neat 58
The formation of phenylͲ2Ͳnitropropene 9 can occur via two alternate

mechanismsfromtheinitialnitroalcohol(8),eitheracidorbasecatalysed,both
ofwhichareoutlinedinScheme2.8.
39|P a g e

CHAPTER2

Scheme2.8:GeneralmechanismforphenylͲ2Ͳnitropropene9.

It is interesting to note that despite being vastly different bases,
phenylͲ2Ͳnitropropene 9 was only the major product when nͲbutylamine and
sodium acetate were employed, and the nitroͲaldol product was not isolated.
Though counterintuitive, when examining the nature of the other bases on an
individual basis this becomes clearer, keeping the base catalysed mechanism
outlinedinScheme2.8inmind.
Triethylamine could be too sterically hindered to facilitate deprotonation of 8,
thus preventing this dehydration pathway from proceeding despite the excess
basebeingpresentinthereactionmixture.Sodiumhydride,whileexceptionally
basic,generallyexistsforonlyashortamountoftimebeforebeingconsumedby
adventitiousmoisture,thisisespeciallythecasewhenallreagents(inthiscase
nitroethaneandbenzaldehyde)havenotbeenmeticulouslydried.Inthecaseof
sodiumhydroxidethebiphasicnatureofthisreactionisthoughttobethemajor
reason for the lack of 9 being present in the reaction mixture, as the effective
concentrationofthebaseintheorganicphasewouldbeverylowinadditionto
thecompetingdeprotonationofunreactednitroethane.
Conversely,nͲbutylamineisaprimaryaminewhichissolubleinorganicsolvents,
thustherequirementsofminimalsterichindrancearoundtheaminemoietyand
highconcentrationofthisreagentintheorganicphaseareeasilymet.Wehave
attributedtheformationof9,inthisinstance,tonͲbutylaminefacilitatingthebase
catalysed pathway outlined in Scheme 2.8. This is consistent with the use of
40|P a g e

CHAPTER2
cyclohexylaminereportedbyMcAndaandcoͲworkers[125]anditisimportantto
note that cyclohexylamine and nͲbutylamine are both small molecule primary
aminesthathaveverysimilarpKavaluesof10.64and10.59,respectively.Sodium
acetate,ontheotherhand,isonlyamildbasewithpoororganicsolubilitybutstill
capable of facilitating this reaction at high temperatures, as evinced by the
formationof9ingoodyield.Thecriticalfactorinthisreactionisthepresenceof
excessaceticacidpromotingtheprotonationof8,thishighenergyintermediate,
thenundergoesasimpledehydrationtogive9,viatheacidcatalysedpathway,
again,outlinedinScheme2.8.Importantlythisstudyusedpurestartingmaterials
and from a clandestine laboratory perspective one must also consider the
implicationsofimpurestartingmaterials.Thismayhavesomeimplicationsonthe
typesofintermediatesthatmaybepresentinrealworldclandestinesamplesand
assuchdetailedchemicalprofilingwillbeinvestigatedinChapter5.
2.3.3Synthesisofnorephedrine(10)from2ͲnitroͲ1ͲphenylͲ1Ͳpropanol
Havingidentifiedahighyieldingmethodforthesynthesisof2ͲnitroͲ1ͲphenylͲ1Ͳ
propanol,focusthenturnedtoconvertingthisproducttoephedrine.Thefirststep
wastoconvertthenitrofunctionalgroupintoaprimaryamineasshowninTable
2.5.
Table2.5:ConversionofphenylͲ2Ͳnitropropene8tothenorephedrine10.
Pd/C
Entry Solvent Time Yield
Equivalent
1 0.1 MeOH 6h Ͳ
2 0.1 MeOH 18h Ͳ
3 0.1 EtOH 6h 59%
4 0.1 EtOH 12h 86%
5 0.1 EtOH 18h 94%

41|P a g e

CHAPTER2
Acommonreductivehydrogenationmethodreportedintheliteraturewastrialled
(Entry1)asithasbeenwidelyusedonsimilarcompounds[126].2ͲNitroͲ1ͲphenylͲ
1Ͳpropanol(8)wasstirredinmethanolwith10%w/wofpalladiumonactivated
carbon, in a hydrogen environment. The 1H NMR spectrum showed this to be
unsuccessful at 6 hour and 18 hour reaction times (Entries 1 & 2). Although
methanolisthemorecommonsolventforthistypeofreactionitwasnotsuitable
with phenylͲ2Ͳnitropropene 8. Ethanol, another common literature
hydrogenationsolvent[127]wasthentrialled(entry3).1HNMRspectroscopyand
mass spectrometry matched the spectral properties reported in the literature
[128]fornorephedrine10.Usingethanol,theproductwasformedina94%yield
afterstirringfor18hours.
2.3.4Synthesisofephedrine/pseudoephedrine(1)fromnorephedrine(10)
Focusthenturnedtothesynthesisofephedrine/pseudoephedrine.Astheonly
differencebetweennorephedrine10andpseudoephedrineisthemethylgroup
ontheamine,methylationsusingmethyliodideweretrialledfirst(Table2.6).
Table2.6:Methylationofnorephedrine10.

Entry Base Solvent Time Yield

1 NaH DryTHF 8h n/a
2 NaH DryTHF 18h n/a
3 Et3N CH2Cl2 6h n/a
4 Et3N CH2Cl2 18h n/a
5 K2CO3 CHCl3 6h n/a
6 K2CO3 CHCl3 18h n/a
Initially, methylation using a strong base (Entry 1 & 2) was tried as per the
literature[129].Thisappearedtohavedeprotonatedboththeamineandhydroxyl
groups encouraging additional methylation and side products. A weaker base
42|P a g e

CHAPTER2
(triethylamine)wasthentried(Entry3&4)assuccessfulmethylationgenerates
hydriodicacid,whichwillinturnprotonatetheamine,whiletriethylamine‘mops
up’ any excess acid present. This made very little difference and the use of
triethylamine produced similar 1H NMR spectra. The next step was to try an
alternativeweakbaselikepotassiumcarbonate(Entry5&6);toavoidstripping
theprotonsfromthehydroxylgroup.Aftermassspectrometryand1HNMR,itwas
clearephedrineorpseudoephedrinewasnotformedinanyofthe6entries,which
can be expected when using methyl iodide. The second methylation
(dimethylation) can occur faster due to the ability of the monoͲmethylated
intermediatetoactasanucleophile,reactingwithmethyliodidefasterthanthe
freeprimaryamine[130].However,theformationoftwopossiblesideproducts
are suggested here, based on high resolution mass spectrometry, as shown in
Figure 2.4. Two major products were identified via LCͲMS with m/z ratios of
194.15399and180.13824,whichagreewiththeexactmassespresentedinFigure
2.5.
Figure2.5:Potentialimpuritiesandassociatedexactmassesformedinthe
methylationofnorephedrine10.
Given that methylation occurred at both the amine and hydroxyl functional
groups, a new synthetic pathway was designed to a) first protect the alcohol
group,b)hydrogenatethenitrofunctionalgrouptoanamine,c)methylatethe
amine,andfinallyd)deprotectthealcoholgroup(Scheme2.9).Thispathwaywas
designedaroundareportedmethodbyDenisetal.onaselectivenͲdemethylation
procedure[131].Therefore,ifthehydroxylfunctionalgroupwasprotectedand
43|P a g e

CHAPTER2
dimethylation occurred again, the literature method [131] could be applied to
reachthetargetproductephedrine/pseudoephedrine1.
Si Si
OH TBDMS-Cl, O MeOH, Pd/C, O
NO2 imadazole NO2 H2, 16 h NH2
42%
8 11
Si
OH O
H H
N N
1

Scheme2.9:Newpathwaytoavoidovermethylation.

Thefirststep(alcoholprotectionͲ11)successfullyproducedayieldof42%which
wasenoughtotakeontothenextstepofthereaction.However,reducingthe
nitro group to an amine proved problematic as only starting material could be
seenonthe1HNMRspectra.Thehydrogenationwasrepeatedusinganalternative
catalyst (palladium acetate) and alternative solvent (ethanol) under the same
conditions,unsuccessfully.AdifferentmethodreportedbyDesaietal.[132]was
triedasoutlinedinScheme2.10,howeverthisalsowasunsuccessful.Onlystarting
materialwasdetectedinthe1HNMRspectrum.
44|P a g e

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Scheme2.10:Reductionwithironandhydrochloricacid.

Attentionthenturnedtousingalessaggressivemethylatingagentwhichcould
potentiallylimitthenumberofpossiblealkylationsitesoncompound10.Thus,a
new synthetic pathway was designed (Scheme 2.11) for a twoͲstep, one pot
reactiontoephedrine/pseudoephedrine.
H OH
OH H
N OH
NH2 H O EtOH
30 min
10
OH OH
H
N Pd/C, H2 N
CH2
18 h
1

Scheme2.11:TwoͲsteponepotsynthesistoephedrine/pseudoephedrine.

1
Mass Spectrometry and H NMR spectroscopy confirmed that no
ephedrine/pseudoephedrine was present, however the formation of two other
sideproducts(Figure2.6)wereobserved.
Figure2.6:ImpuritiesformedfromtwoͲsteponepotreaction.
45|P a g e

CHAPTER2
As monoͲmethylation proved problematic, an alternative approach was

considered as dimethylation kept occurring. Over methylation of the primary
aminewasobservedwithbothmethyliodideandformaldehydereagents,sothe
literature was consulted for a selective demethylation method [133]. Firstly a
publishedmethod[134]wasusedtosynthesiseNͲmethylephedrine12asperthe
schemebelow(Scheme2.12).
OH HCHO/HCOOH, OH
NH2 90°C, 18 h N
90%
10 12
Scheme2.12:SynthesisofNͲmethylephedrine.
Withtheproduct(NͲmethylephedrine12)inhand,thenextstepwastotryand
selectivelyremoveonemethylgroupfromthenitrogeninCompound12asper
Denisetal.[131,133].
Scheme2.13:DemethylationofNͲmethylephedrine[131].

For this reaction, 1.1 equivalents of Diisopropyl azodicarboxylate (DIAD) were
addedtoastirringsolutionofNͲmethylephedrine12inacetoneandleftfor24
hours(Scheme2.13).The1HNMRspectrumshowedonlythepresenceofstarting
material, indicating the reaction did not work. The reaction was thenrepeated
with2.5equivalentsofDIAD,againwithnoproductobserved.
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AttentionturnedtooptimisingthepreviouslydescribedtwoͲsteponepotreaction
to ephedrine/pseudoephedrine (Scheme 2.14). As dimethylation of the amine
occurred,thereactionwasrepeated,changingthereactiontemperatureto0°C
reducingthestirringtimeto10minutesfrom30minutes,beforeaddingthePd/C.
The reasoning behind doing this reaction on ice is to slow down how fast the
formaldehyde reacts with the amine functional group, encouraging monoͲ
methylationoftheamineprovidingthedesiredproduct.

Scheme2.14:TwoStep,onepotreactiontoephedrine/pseudoephedrine.

The 1
H NMR spectrum of the product showed a mixture of
ephedrine/pseudoephedrineandNͲmethylephedrinebutduetothecomplexity,
high resolution LCͲMS analysis was carried out on the same sample for
confirmation.Asseeninthechromatogram(Figure2.7),althoughnotcleanthere
arethreemainpeaksindicatingtheformationofthreemajorproducts(A,B,C).
47|P a g e

CHAPTER2
B
C
Figure2.7:Identificationofthreemajorpeaks(A,B,C)inHighResolutionLCͲ
MSchromatogramoftwoͲstep,onepotreaction.
As this reaction is not stereoͲselective, it is expected that both ephedrine and
pseudoephedrinediastereomerswillbepresent,accountingfortwoofthepeaks
(A and B). The first coͲeluting peak had a retention time of 1.33 minutes and
associatedpseudoͲmolecularion(M+H)+massof166.12265amu;representative
ofeitherephedrineorpseudoephedrine(Figure2.8).
OH
H
N
A
Figure2.8:LCͲMSAnalysis,correspondingmassofpeakAatRt1.33min
(ephedrineorpseudoephedrine–m/z166.12265amu).
48|P a g e

CHAPTER2
ThesecondcoͲelutingpeakhadaretentiontimeof1.37minutesandassociated
massofthepseudoͲmolecularion(M+H)+m/z166.12262amu,proposedhereto
betheotherdiastereomer(Figure2.9).Thethirdandfinalpeakhadaretention
timeof1.49minutesandcorrespondingmassofthepseudoͲmolecularion(M+H)+
m/z 180.13823 amu (Figure 2.10) indicating the formation of the NͲ
methylephedrine.
OH
H
N
B

Figure2.9:LCͲMSAnalysis,correspondingmassofpeakBatRt1.37min
(ephedrine/pseudoephedrine–m/z166.12262amu).

Figure2.10:LCͲMSAnalysis,correspondingmassofpeakCatRt1.49min
(NͲmethylephedrinem/z180.13823amu).
49|P a g e

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Furtherpurificationandoptimisationwasnotnecessaryandisoutsidethisscope
ofwork,asaplausiblesyntheticroutetoephedrine/pseudoephedrinehasbeen
confirmed.Itisquiteimportantinthiscasethatpurificationisnotcarriedoutas
achemicalfingerprintcannotbeidentifiedforthispathway(furtherdiscussedin
chapter 5) without the presence of its associated impurities. In this way the
samples investigated in the platform technology developed in Chapter 5 are
representative of the types of samples that may be present in a clandestine
laboratory.
2.3.4Synthesisofmethamphetamine(1)fromnorephedrine(10)
Thenextstepwastotakethesameproductmixtureinanotherreaction(Scheme
2.15)toconfirmifanymethamphetaminecanbesynthesisedviathispathway,
makingsuretocarrythroughimpuritiesfromeachstepfortheaforementioned
chemicalmapping.
A commonly encountered reduction method (Scheme 2.15) [135] for the

conversionofpseudoephedrinetomethamphetaminewasacquired,using0.5mL
of50%w/whypophosphorousacidwasstirredonicefor15minutesbeforethe
slowadditionofIodine(0.5g).Thereactionmixtureinitiallyturnedadeepmaroon
colourbutastheiodinereacted(forminghydriodicacid)itturnedcolourless.Once
thesolutionmixtureturnedclearitwastakenofftheicebathandallowedtoreach
roomtemperature.Then0.5gofthemixtureofephedrine,pseudoephedrineand
NͲmethylephedrinewasaddedinasingleportionandsettorefluxfor1hour.The
mixture was then basified to pH 13 with 5M NaOH solution and extracted in
toluene.
Scheme2.15:Reductionofephedrine/pseudoephedrineand
NͲmethylephedrine.
50|P a g e

CHAPTER2
The 1H NMR spectrum was very complex, identification of any compound was
extremelychallengingsohighresolutionLCͲMSwasutilisedagain.Thisshowed
the formation of methamphetamine 2 (m/z 150.12755) and NͲmethylͲ
methamphetamine13(m/z164.14320)insmallamounts.However,thisreaction
formedlargeamountsofunknownbyͲproducts,whichwillbeextremelyusefulfor
thechemicalmappingofthispathway(Chapter5).Alistofidentifiedmasses(m/z)
in order of intensity (largest to smallest) is outlined here: a) 240.17432, b)
330.22116,c)256.16931,d)346.21603,e)238.15868,f)226.15874,g)302.18985,
h) 254.18988, i) 270.18491, j) 226.15874, k) 224.14308, l) 194.15372, m)
283.21646, n) 148.11197, o) 162.12749, p) 180.13795, q) 158.96384, r)
297.23206, s) 150.12755, t) 164.14320. It becomes clear that with potentially
morethan20compoundsformed,itistheimpuritiesofaseizuresamplethatmay
provide the most information about the manufacturing process of
methamphetamine.
As a full possible synthetic pathway to methamphetamine has now been
confirmed from easily accessible ingredients (benzaldehyde and nitroethane)
attentionturnedtodesigninganotherpotentialpathway.Itshouldbepointedout
thatthedifficultiesassociatedwiththeaboveprocessesmakeitunlikelythiseffort
wouldbeundertakeninaclandestinelaboratoryhoweverthesesamplesareof
interestforthedevelopmentofchemicalprofilingtechnology.
Apathwaythatremovesthehydroxylgroupbeforemethylationwouldbeidealin
order to avoid methylating both the amine and hydroxyl groups. Therefore, a
pathwaywasdesignedinwhichmethylationcanonlyoccurattheprimaryamine
ofamphetamine(Scheme2.16).

Scheme2.16:Alternativepathwaytomethamphetamine.
51|P a g e

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The first step is an exact repeat of the conditions outlined in Scheme 2.15
(reduction method of pseudoephedrine to methamphetamine) except using
norephedrine10insteadofpseudoephedrine.HighresolutionLCͲMSconfirmed
theformationofamphetamine14(m/z136.11206)astheminorproductalong
with the unknown major product mass of m/z 226.15880. The next step is to
methylate using the two step, one pot reaction which again showed a small
amountofmethamphetaminepresentwithatleast15otherimpurities.Thisis
alsoconfirmedasaplausiblepathwayforthemanufactureofmethamphetamine
2andwillbelateranalysedinordertofullyrealisethepotentialofthechemical
profilingtechnologyinChapter5.
2.4CONCLUSION
The choice of base when reacting benzaldehyde and nitroethane together is
significant to clandestine laboratory investigating chemists as it has been
demonstrated here to be a determining factor as to which precursor to
methamphetamine has been used. Synthesis using triethylamine, sodium
hydroxideorsodiumhydrideproduces2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8;aprecursor
to ephedrine/pseudoephedrine. In contrast, when using nͲbutylamine and
ammoniumacetateasthebase,phenylͲ2Ͳnitropropene9isproduced;aprecursor
toP2P.Thisinformationwillbesignificantastheidentificationofthebaseinuse
may provide additional support to the determination of the synthetic route to
methamphetamine. Likewise a full synthetic route to methamphetamine was
carriedoutandconfirmedfromthe2ͲnitroͲ1ͲphenylͲ1Ͳpropanol8product.
Theworkpresentedinthischapterhasbeenpublishedas:L.M.Andrighetto,L.C.
Henderson, J.R. Pearson, P.G. Stevenson and X.A. Conlan, Influence of base on
nitroͲaldol (Henry) reaction products for alternative clandestine pathways,
Australian Journal of Forensic Sciences, (2016) DOI:
10.1080/00450618.2015.1112429
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CHAPTER 3
SYNTHESIS OF
PSEUDOEPHEDRINE/EPHEDRINE FROM
L-ALANINE
53|P a g e

CHAPTER3
CHAPTER OVERVIEW
This chapter provides a brief literature summary of NͲmethyl amino acids and
theirprevioususesinareassuchaspeptidemanipulation.Italsohighlightsthe
increasinginterestbylawenforcementagenciesinNͲmethylalanine,duetolarge
quantitiesrecentlybeingidentifiedatclandestinelaboratories.
AsimplepathwaywasdesignedforthesynthesisofNͲmethylalaninefromalanine,
asintroducedbyPainteretal.[136]inarestrictedjournal.Painter’sstudywas
limitedtoidentificationofephedrineintraceamountsfromNͲmethylalanineand
benzaldehydeviaasimplisticGCͲMSmethod.Tofurtherthisresearch,arangeof
differentconditionsweretrialledtofullyestablishthesyntheticrouteandassess
therobustnessofthisclandestinemethod.
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3.1INTRODUCTION
TheinclusionofNͲmethylaminoacidswithinpeptideshasbeenshowntoimprove
pharmacokineticpropertiessuchasmetabolicstability,membranepermeability
and oral bioavailability [137Ͳ139]. As such, NͲmethyl amino acids have been of
greatinterestinareassuchaspeptidomimetics[140].Despitethisinterest,the
NͲmethylationofaminoacidscanbeachallengingprocessandtodate,therehave
beennumerousmethodsreportedforthisprocedureintheliterature.Methods
for the formation of NͲmethylͲamino acids include; nucleophilic substitution of
ɲͲbromoacids[141],NͲmethylationofcarbamatesandamides[142],transition
metal catalysed reductions [143], reductive aminations [139] and borohydride
reductions[144],illustratedinFigure3.1.
Figure3.1:ExamplesofsyntheticmethodsforNͲmethylaminoacids.
55|P a g e

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Alanine has been identified at several clandestine laboratories in Victoria
[personal correspondence with Dr Jim Pearson at the Victoria Police Forensic
ServicesDepartment]andisacheapaminoacid,whichisreadilyavailabletothe
public in many sports supplements or body building shops. Therefore, the
synthesis of NͲmethylalanine from alanine is a potential area of interest to
clandestineteams.Althoughtherearenumerousmethodologiesintheliterature
forthesynthesisofNͲmethylalanine,therearelimitedreportsforthesynthesisof
NͲmethylalanine from alanine itself [139]. The later editions of Uncle Fester’s
(popular ‘Meth cookbooks’) ‘Secrets of Methamphetamine Manufacture’ [145]
offeronesuchmethod usingaqueousformaldehydeandzinc[146],butasthe
authorofthetextpointsoutthiswasnotaneffectiveorpracticalprocedurefor
the preparation of NͲmethylalanine. As such, a key aim of this chapter is to
develop a simple and efficient method for the conversion of alanine to
NͲmethylalanine,aprocessthathassignificantpotentialforthemanufactureof
illicit drugs [147]. A very recent publication [148] (April 2016) in a restricted
clandestinelaboratoryjournalinvestigatedasimplemethodforthe‘clandestine
synthesisofNͲmethylalaninefrom2Ͳbromopropionicacid’,highlightingtherecent
attentionNͲmethylalanineisreceivingwithinthedrugmanufacturingscene.
Painteretal.[136]investigatedtheAkaboriͲMomotanireaction(showninFigure
3.2) on a small scale (0.2 g) with NͲmethylalanine and benzaldehyde as an
alternativemethodofsynthesisingephedrine/pseudoephedrine.
O R Et3N, reflux, OH
H
R OH 16 h N
H + N R
H R
O
R= H or alkyl

Figure3.2:AkaboriͲMomotanireaction[136].
56|P a g e

CHAPTER3
AlthoughPainteretal.[136]confirmedthepresenceephedrine/pseudoephedrine
via GCͲMS within a mixture of products, the yields, practicality, isolation or
purification of generated products from this pathway was not discussed.
Nevertheless,thepresenceofbenzaldehydeorNͲmethylalanineinaclandestine
laboratory can signify the synthesis of ephedrine/pseudoephedrine and
subsequentlymethamphetamine.
LargequantitiesofNͲmethylalaninehasbeenrecoveredinmultipleclandestine
laboratories,whichwereimportedfromothercountriesasthechemicalisvery
expensive ($894 AUD for 5 grams – Sigma Aldrich) [149]. As such, a simple
clandestinemethodforthesynthesisofNͲmethylalaninewillbeinvestigatedin
this chapter, along with the conversion of NͲmethylalanine to
ephedrine/pseudoephedrineunderalternativeconditions.
3.2EXPERIMENTAL
All 1Hand 13CNMRspectrawererecordedonaJEOLJNMͲEX270MHz.Samples

weredissolvedindeuteratedDMSO(d6ͲDMSO)ordeuteratedchloroform(CDCl3)
withtheresidualsolventpeakusedasaninternalreference(d6ͲDMSO–ɷ2.50
ppm,CDCl3–ɷ7.26ppm).Protonspectraarereportedasfollows:chemicalshift
ɷ(ppm),(integral,multiplicity(s=singlet,br.s=broadsinglet,d=doublet,dd=
doubletofdoublets,t=triplet,q=quartet,m=multiplet),couplingconstantJ
(Hz), assignment). High resolution mass spectrometry (HRMS) was performed
usinganAgilent6210MSDTOFmassspectrometerwiththefollowingsettings:
gastemperature(350°C),vaporizer(28°C),capillaryvoltage(3.0kV),conevoltage
(40V),nitrogenflowrate(0.5mLminͲ1),nebuliser(15psi).Samplesweredissolved
inMeOH(0.5mgmLͲ1)priortoinjection.

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CHAPTER3
SynthesisofMethylͲLͲalaninate(16)
Acooled(0°C)solutionofLͲalanine(2.00g,22.45mmol,1.0eq.)
inmethanol(15mL)wasstirredfor15minutesbeforethionyl
chloride(2.2mL,15.41mmol,0.7eq.)wasaddeddropwiseover
20 minutes. The mixture was refluxed for 3 hours, then brought to room
temperature and further stirred for 16 hours. The mixture was basified by
adjustingthepHto12using1MNaOH,thentransferredtoaseparatingfunnel
where the crude product was extracted 3 times with toluene (3 × 15 mL). The
organic layers were combined and dried over MgSO4, then the toluene was
removedinvacuotoaffordthetitlecompoundasanoffͲwhitesolid(2.51g,81
%).1HNMR(270MHz,CDCl3):ɷ8.76(br.s,2H,NH2),4.26(m,1H,CH),3.81(s,3H,
CH3),1.73(d,3H,JHH=7.2Hz,CH3).
1
SynthesisofMethylEͲ(tolueneͲ4Ͳsulfonyl)LͲalaninate(17)
Toacooled(0°C)solutionofmethylͲLͲalaninate(2.20
O
S O g,21.1mmol,1.0eq.)inCH2Cl2(10mL),triethylamine
N
O H (5.89mL, 42.2mmol, 2.0eq.)was addedina single
O
portion.Tosylchloride(3.62g,18.9mmol,0.9eq.)wasthenaddeddropͲwiseover
30minutesandthereactionlefttostirfor6hours.ThepHwasadjustedto11
using1MNaOHandthentransferredtoaseparatingfunnelwheretheorganic
layerwasisolatedandwashed3timeswithbrine(3×20mL).Theorganiclayer
wasthendriedoverMgSO4,filteredandthesolventremovedinvacuotoafford
thetitlecompoundasacreamsolid(3.37g,72%). 1HNMR(270MHz,CDCl3):ɷ
7.72(d,2H,JHH =7.24Hz,PhͲH),7.29(d,2H,JHH =7.23,PhͲH),3.99(m,1H,CH),
3.54(s,3H,CH3),2.42(s,3H,CH3),1.38(d,3H,JHH=7.24,CH3).
1

58|P a g e

CHAPTER3
SynthesisofMethylEͲmethylͲEͲ(tolueneͲ4Ͳsulfonyl)ͲLͲalaninate(18)
A cooled (0 °C) solution of methyl tosylͲLͲalaninate

O
S O (450 mg, 1.75 mmol, 1.0 eq.) and anhydrous THF (5
N
O mL) was stirred for 10 minutes under an inert
O
atmosphere of N2(g). Sodium hydride (126 mg, 5.25 mmol, 3.0 eq.) was added
cautiouslyandlefttostiruntilgasevolutionhadfinished.Methyliodide(1.09mL,
17.50mmol,10eq.)wasthenaddeddropͲwiseover15minutes.Themixturewas
allowed to warm to room temperature and stirred for a further 18 hours. The
mixturewasquenchedcautiouslywithwater(2.0mL)andthentransferredtoa
separatingfunnelwhereCH2Cl2(20mL)wasadded.Theorganiclayerwaswashed
with brine (3 × 20 mL) and then dried over MgSO4, filtered and the solvent
removedinvacuotoaffordthetitlecompoundasanoffͲwhitesolid(374.90mg,
79%). 1HNMR(270MHz,CDCl3):ɷ7.72(d,2H,JHH=6.9,PhͲH),7.29(d,2H,JHH=
6.9,PhͲH),3.99(m,1H,CH),3.54(s,3H,CH3),2.83(s,3H,CH3),2.42(s,3H,CH3),
1.38(d,3H,JHH=6.8,CH3).
1
SynthesisofMethyl(tertͲbutoxycarbonyl)ͲLͲalaninate(20)
O Toacooled(0°C)stirringsolutionofmethylͲLͲalaninate
O
O N (500mg,4.8mmol,1.0eq.)inCH2Cl2(5mL),Boc2O(1.05
H
O
g,4.8mmol,1.0eq.)wasdilutedinCH2Cl2(10mL)and
addeddropwiseover15minuteswithadroppingfunnel.Themixturewasleftto
stirfor16hoursatroomtemperatureandthentransferredtoaseparatingfunnel
where it was washed with brine (3 × 15 mL). The organic layer was dried over
MgSO4,filteredandthesolventremovedinvacuotoaffordthetitlecompoundas
acreamsolid(665.5mg,68%).1HNMR(270MHz,CDCl3):ɷ4.32(m,1H,CH),3.74
(s,3H,CH3),1.44(s,9H,tͲBu),1.38(d,3H,JHH=7.1,CH3).
1
59|P a g e

CHAPTER3
Synthesisof2Ͳ(EͲ(tertͲbutoxycarbonyl)amino)propanoicacid(22)
Toacooled(0°C)cloudysolutionofLͲalanine(1.0g,11.2
mmol, 1.0 eq.) in dioxane (22 mL) and water (11 mL),
Boc2O(2.45g,11.2mmol,1.0eq.)wasaddedslowlyover
5minutesunderN2(g).Themixturewaslefttostirfor3hoursatroomtemperature
untilthesolutionturnedclear,whereuponethylacetate(100mL)wasadded.The
pHwasthenslowlyadjustedto2.0with2MHCl.Themixturewastransferredtoa
separating funnel where the aqueous layer was separated and extracted with
ethylacetate(2×15mL).ThecombinedorganiclayersweredriedoverMgSO4,
filteredandthesolventremovedinvacuotoaffordthetitlecompoundasawhite
solid(1.93g,91%).Mp=79°C(80°CͲ83°C[154]).1HNMR(270MHz,d6ͲDMSO):
ɷ7.11(d,1H,3JHH=5.40Hz,NH),3.91(m,1H,CH),1.37(s,9H,tͲBu),1.21(d,3H,JHH
=5.40Hz,CH3).HRMS(ESI,m/z)calculated[C8H15NO4+Na]+212.08933foundm/z
212.08964.
1
HNMRspectralpropertiesandmeltingpointmatchedthatoftheliterature[154,
155].
Synthesisof2Ͳ(EͲ(tertͲbutoxycarbonyl)methylamino)propanoicacid(23)
Toacooled(0°C)stirringsolutionofBocͲLͲalanine(1.43
g, 7.54 mmol, 1.0 eq.) and dry THF (20 mL), sodium
hydride 60% dispersion in mineral oil (905 mg, 22.61
mmol,3.0eq.)wasaddedcautiously.Thenmethyliodide(3.9mL,60.3mmol,8.0
eq.)wasaddeddropͲwiseunderinertconditions(N2).Themixturewaslefttostir
for18hoursatroomtemperatureandwasthenquenchedcautiouslywithwater
(3mL).Themixturewastransferredtoaseparatingfunnelwherediethylether
(20mL)wasadded.Theaqueouslayerwasseparatedandcarefullyacidified(5M
HCl)topH3.0,andthenthecrudeproductwasextractedusingchloroform(2×20
mL).Thecombinedorganiclayerswerethendissolvedupinhexaneandpoured
throughasilicaplugtoremovethemineraloil.Thenethylacetatewasusedto
flushtheproductoutofthesilica.TheorganiclayerwasdriedoverMgSO4,filtered
60|P a g e

CHAPTER3
andthesolventwasremovedinvacuotoaffordthetitlecompoundasawhite
solid(1.35g,89%).Mp=90°C(89°CͲ91°C[154]).1HNMR(270MHz,d6ͲDMSO,
80°C):ɷ4.44(br.s,1H,CH),2.75(s,3H,NͲCH3),1.39(s,9H,tͲBu),1.30(d,3H,JHH
=5.2Hz,CH3).HRMS(ESI,m/z)calculated[C9H17NO4+Na]+226.10498foundm/z
226.10585.
1
HNMRspectralpropertiesandmeltingpointmatchedthatoftheliterature[154,
156].
Synthesisof2Ͳ(methylamino)propanoicacid(EͲmethylalanine)trifluoroacetatesalt
(19)
To a stirring mixture of 2Ͳ(NͲ(tertͲ

butoxycarbonyl)methylamino)propanoic acid 3 (520 mg, 2.6
mmol,1.0eq.)inCH2Cl2(20mL),20%trifluoroaceticacid(4mL)
wasaddeddropwiseover5minutes.Themixturewaslefttostirfor18hoursat
roomtemperatureandthesolventremovedinvacuo.Theproductwasdissolved
inCH2Cl2(25mL)andthesolventremovedagaininvacuoandwasrepeatedatotal
of3timestoaffordthetitlecompoundasanoffͲwhitesolid(491mg,96%).Mp=
288°C.1HNMR(270MHz,d6ͲDMSO):ɷ8.90(brs,1H,OH),3.96(q,1H,JHH=5.38
Hz,CH),3.57(br,1H,NH),2.57(s,3H,NͲCH3),1.41(d,3H,JHH=5.40Hz,CH3).13C
NMR(67.5MHz,d6ͲDMSO)ɷ13.9,55.3,99.4,171.0.19FNMR(470MHz,d6ͲDMSO)
ɷ Ͳ74.99. HRMS (ESI, m/z) calculated [C4H9NO2+Na]+ 126.05255 found m/z
126.05399.
1
HNMRand13CNMRspectralpropertiesmatchedthatoftheliterature[157].
61|P a g e

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3.3RESULTSANDDISCUSSION
Thelowsolubilityinorganicsolventsandzwitterionicnatureofaminoacidsmake
the synthetic manipulation of the carboxylate and protonated amino regions
challenging [158]. Initially, to avoid the over methylation issue presented
previouslyinChapter2(Figure2.5),theaimwastofirstprotectthecarboxylicacid
group as the methyl ester. Then perform a monoͲmethylation followed by the
deprotectionofthecarboxylicacidtoformthedesiredproductNͲmethylalanine.
A commonly used literature method [150] involving the ester formation using
thionylchlorideandmethanolwascarriedout,showninScheme3.1.Thisreaction
functionsbyfirstformingtheacidchloride,followedbymethanolundergoinga
nucleophilicattackonthecarbonylcarbon[130],whichresultsinthedeparture
ofthechlorideion(leavinggroup)andtheformationofthetargetmethylester
16.
Scheme3.1:SynthesisofmethylͲLͲalaninate16.
Thionylchloridewasaddeddropwiseover15minutestoacooledstirringsolution
0°CofLͲalanine15(1.0equivalent)inmethanol.Afterthereactionmixturewas
refluxed(3hours)itwaslefttostiratroomtemperatureforafurther14hours
andthenbasified,whichformedthedesiredproduct16in81%yield.The1HNMR
spectrummatchedthatoftheliteratureandwasquiteclean
Thenextstepwasthemethylationoftheamine(Scheme3.2),howeverinorder
topreventdimethylation,themethyliodide(1.0equivalent)wasaddeddropwise
over40minutestoacooledsolutionofmethylͲLͲalaninate16(1.0equivalent)and
62|P a g e

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triethylamine(1.1equivalents)inanhydrousTHF(Scheme3.2).Theslowaddition
of methyl iodide into a cold solution can slow down the reaction, potentially
minimisingthechanceofdiͲalkylation.
Et3N, MeI O
O
H2N N
THF, 0 °C, 12h H
O O
16
Scheme3.2:MethylationofmethylͲLͲalaninate16.
Therewerenotracesofthedesiredproductobservedinthe1HNMRspectrumof
thisreaction.Manyliteraturemethods[139]reportsuccessfulNͲmethylationof
similaraminoacids,byfirstprotectingtheprimaryaminefollowedbymethylation
andthendeprotectionofboththecarbonylandamine.
Inthiscase,theaminegroupofLͲalaninemethylester16wasprotectedusing
4Ͳtoluenesulfonylchloride(TosylChloride/TsCl),whichisagroupthathasbeen
usedintheliteraturefortheprotectionofamines[159,160](Scheme3.3).
Scheme3.3:SynthesisofmethylNͲ(tolueneͲ4Ͳsulfonyl)LͲalaninate17.
Themethylester16(1.0equivalent)wasstirredindiethyletherandtriethylamine
(2.0equivalents)onicefor20minutesbeforetosylchloride(1.0equivalent)was
added slowly and left to stir for 16 hours. The 1H NMR spectrum of the crude
productshowedthatthedesiredtosylprotectedmethylester17hadformedand
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someunreactedtosylicacidalsoremained.Therefore,thereactionwasrepeated
withalongerreactiontimeof24hoursandtheamountoftosylicacidpresent
decreased.AnaqueousworkͲupwascarriedoutwithbicarbonatetoremoveany
leftovertosylicacidorhydrochloricacidfromthecrudeproduct.
ThenextstepwastoNͲmethylatetheprotectedalanine17.Underinertconditions
(nitrogengas),methyliodide(1.0equivalents)wasslowlyaddedtoacoldstirring
solutionofanhydrousTHF,triethylamine(1.1equivalents)andmethylNͲ(tolueneͲ
4Ͳsulfonyl)LͲalaninate17(1.0equivalents).Therewerenotracesofthedesired
product18observedinthe1HNMRspectrumfromthereaction.Astriethylamine
is a weak organic base, the experiment was repeated using sodium hydride to
ensure the base would be strong enough to deprotonate the secondary amine
(Scheme3.4).
Scheme3.4:MethylationofprotectedamineofmethylNͲ(tolueneͲ4Ͳsulfonyl)LͲ
alaninate17.
Theuseofthisstrongerbasesuccessfullyformedthedesiredproductina67%
yield(0.4g–0.6gscale)asconfirmedinthe1HNMRspectrum.Thecrudeproduct
waswashed3timeswithbrineandappearedquiteclean,asillustratedinFigure
3.3.
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Figure3.3:1HNMRspectrumofmethylNͲmethylͲNͲ(tolueneͲ4Ͳsulfonyl)ͲLͲ
alaninate18.
Having successfully methylated methyl NͲ(tolueneͲ4Ͳsulfonyl) LͲalaninate 17,

attention turned to deprotecting the amine by removing the sulfonamide
protecting group from the molecule. Hydrogen bromide (HBr) has been used
frequentlywithintheliterature[161]tocleavesulfonamidegroups.Asolutionof
hydrogenbromideinaceticacidattwodifferentconcentrations(25wt.%and33
wt. %), under argon, at room temperature and a 24 hour stirring time were
trialled,showninTable3.1.
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Table3.1:Conditionsfortosyldeprotectionof18.
Entry HBrConcentration Solvent

1 25wt.%inaceticacid Neat
2 25wt.%inaceticacid DCM
3 33wt.%inaceticacid Neat
4 33wt.%inaceticacid DCM
All4experiments(Entries1–4)failedandnodesiredproduct19wasobservedin
the 1HNMRofthecrudereactionproduct.Analternativemethod[162]forthe
removalofthetosylgroupwastrialled(Table3.2).Thisreactionutilisescathodic
cleavageduetoelectrontransferandhasbeenpreviouslyusedtocleaveprimary
andsecondaryalkylphenylsulfones[162].
Table3.2:Deprotectionusingmagnesiuminabsolutemethanol.
Magnesium
Entry MagnesiumForm Time
Equivalents
1 Turnings 5.0 1h
2 Turnings 10.0 6h
3 Powder 10.0 1h
4 Powder 10.0 8h
Methyl NͲmethylͲNͲ(tolueneͲ4Ͳsulfonyl)ͲLͲalaninate 18 (1.0 equivalent) was

placedintoa10mLvolumetricflaskwithanhydrousmethanoland5.0equivalents
ofmagnesiumturnings.Theresultingsuspensionwasthensonicatedfor1hour.
After1hour,themixturewasdilutedwithdichloromethaneandpouredinto1M
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HCl,followedbytheorganicphasebeingwashedwith1Msodiumbicarbonate.
Nodeprotectedproduct19wasobservedinthe1HNMRspectrumofthereaction,
so the reaction was repeated doubling the magnesium equivalents (10.0) and
increasingthesonicationtimeto6hours,withonlystartingmaterialpresent.In
ordertoincreasetheamountofsurfaceareaallowedtoreact,magnesiumpowder
wassubstitutedforthelargerturningsandweretrialledfor1hourand8hour
sonicationtimes,howeverthesewereallunsuccessful.
Althoughtherearearangeofamineprotectinggroupswithintheliterature[159,
160],aspecificprotectinggroupthatcanbeeasilycleavedatlaterstagesofthe
synthetic pathway would be ideal. TertͲbutyloxycarbonyl (Boc) has been used
throughoutorganicchemistryfortheprotectionofamines[163,164],asremoval
isassimpleasexposingittoanacidicenvironment.Therefore,1.0equivalentof
thereagentdiͲtertͲbutyldicarbonate(Boc2O)wasdissolvedinCH2Cl2andadded
dropwisetoacooledstirringsolutionofmethylͲLͲalaninate16(1.0equivalent),
CH2Cl2andtriethylamine(1.5equivalents).Itwaslefttostirfor16hoursupon
which the 1H NMR spectrum of the crude reaction product confirmed the
successful Boc protection, and formation of target product methyl (tertͲ
butoxycarbonyl)ͲLͲalaninate20in68%yield(Scheme3.5).
Scheme3.5:BocprotectionofmethylLͲalaninate16.
Nowwiththetargetmethyl(tertͲbutoxycarbonyl)ͲLͲalaninate20inhand,thenext
step was to methylate the protected amine [165], shown in Scheme 3.6. To a
cooled stirring solution of methyl (tertͲbutoxycarbonyl)ͲLͲalaninate 20
(1.0 equivalent) and dry THF, sodium hydride (3.0 equivalents) was added
cautiously.Thenmethyliodide(1.0equivalent)wasaddeddropͲwiseunderinert
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conditions(N2).Themixturewaslefttostirfor16hoursatroomtemperatureand
wasthenquenchedwithwater(2mL)cautiously.
Scheme3.6:MethylationofBoc2OprotectedLͲalaninemethylester20.
The desired product (Compound 21) was neither observed in the 1H NMR
spectrumorLCͲMSchromatogramofthecrudereactionproduct.
Alternative approaches were then considered. As the amine is far more
nucleophilic than the hydroxyl functional group, the synthetic pathway was
repeated using Boc as the protecting group without first forming the LͲalanine
methylester(protectingthehydroxylgroup).Thatis,Boc2Owasusedtoprotect
LͲalanine,formingacarbamate[165],aprotocolthathasbeencommonlyusedto
methylatetheamineinindividualaminoacids[166].Theproceduredescribedby
Jacubecetal.[167]wascarriedoutusingdioxane:water(2:1).Importantly,only
oneequivalentofBoc2Owasusedinthisreaction,whichislessthanusual[167]
inordertolimittheamountofunreactedBoc2Ointhecrudesamplemixture;thus
obtaining a spectroscopically cleaner product. The reaction time was also
increasedslightlyfrom1hourto3hours.Usingtheseconditions,yieldsranged
from85%to91%(0.5g–1.0gscale)oftheprotectedaminecompound22,shown
inScheme3.7.

Scheme3.7:Synthesisof2Ͳ(NͲ(tertͲbutoxycarbonyl)amino)propanoicacid22.
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The second step involved the methylation of the target protected amine 22,
achieved with methyl iodide, sodium hydride and dry tetrahydrofuran, a slight
variationofknownprocedures[167,168].Thismixturewasstirredunderaninert
atmosphere(N2)inaroundbottomflask.Initiallythesamplewascooledto0°C
prior to sodium hydride and methyl iodide addition, allowed to warm to room
temperatureandfurtherstirredfor18hours.Alargeexcessofsodiumhydride
(8.0equivalents)wasused,followingpreviousmethodsregardingthemethylation
of amino acids [129]. A yield of 89% for the desired product
2Ͳ(NͲ(tertͲbutoxycarbonyl)methylamino)propanoicacid23wasachieved(Scheme
3.8).Itisunknownastowhythemethylationoftheester(compound20,Scheme
3.6)wasunsuccessfulpreviously.

Scheme3.8:Synthesisof2Ͳ(NͲ(tertͲbutoxycarbonyl)methylamino)propanoic
acid23.
Duringthecharacterisationofcompound23itwasinterestingtonotethattwo
setsofquartetswereobservedinthe1HNMRspectrumatɷ4.27ppmandɷ4.54
ppm (Figure 3.3 at 20 °C). This is attributed to the observance, on the NMR
timescale [169], of two conformers at the amide nitrogen, which suggests the
formation of a pseudoͲstereocentre at the nitrogen due to impeded pyramidal
inversion,resultinginrestrictedrotation[130].
Thisrestrictedrotationcanalsobeobservedbythesplittingassociatedwiththe
Bocprotectinggroupatɷ1.39ppm.Repeatingthe1HNMRexperimentataseries
ofincreasingtemperatures(20°C(r.t.)Æ60°C)resultedinpeakcoalescenceat
60°CforeachoftheɲͲprotonsandBocsignals,respectively.Thissuggeststhata
reasonableamountofenergywasrequiredtoallowforrapidnitrogenpyramidal
inversion or rotation and individual diastereomeric effects to no longer be
69|P a g e

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observed[170,171].PeakcoalescencefortheɲͲprotonisshownintheinsetson
thelefthandsideofthe1HNMRspectra,Figure3.4.
NͲCH3 Boc
Proton NMR - Compound 3
CH
CH3
60°C
DMSO
40°C
20°C
4.6 4.5 4.4 4.3 4.2
f1 (ppm)
H2O
4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2
f1 (ppm)

Figure3.4:1HNMRspectraofcompound23atvarioustemperatures:20qC(r.t.),
40qCand60qC.Insetshowsexpandedregionbetweentherange
ɷ4.1–4.7ppm.
The final step of the synthesis involved the deprotection of compound 23, by
simply stirring with a 20% trifluoroacetic acid in dichloromethane at room
temperature,overnight.A96%yieldofthetargetNͲmethylalanine19(isolatedas
thetrifluoroacetatesalt)wasachievedinthedeprotectionstep,showninScheme
3.9. Significantly for the clandestine drug chemist community, an immediate
precursor for the synthesis of ephedrine and pseudoephedrine has been easily
producedinanoverallyield>75%.

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Scheme3.9:Deprotectionoftertiaryaminetoformtargetcompound19.
AlthoughapathwaytoNͲmethylalanine19wascompleted,researchbyRenatoet
al. [146] also examined the reductive methylation of primary and secondary
aminesusingaqueousformaldehydeandzinc.Assuch,themethodologyreported
[146]wastrialledwithLͲalanine15asanalternativeapproach(Table3.3).
Table3.3:ReductivemethylationofprimaryamineLͲalanine.
Entry Reagents Time

1 NaOH,PhosphoricAcid 6h
2 NaOH,PhosphoricAcid 18h
3 MonosodiumPhosphate 18h
Initially 16 mL of phosphoric acid was added to 200 mL of deͲionised water,
followedbytheadditionofsodiumhydroxide(237mmol,2.1equivalents).Then
LͲalanine 15 (112 mmol, 1.0 equivalents) was added to the solution in a single
portionandfinally10mLof37%formaldehydewasaddeddropwiseandstirred
for6hoursand18hours.Unfortunately,noNͲmethylalanine19wasobservedin
the1HNMRspectraofthereportedmethodinanyofthethreeentriesfromTable
3.3.
AttentionturnedtothereactionbetweenNͲmethylalanine19andbenzaldehyde
6.TobuildontheresearchcarriedoutbyPainteretal.[136],numeroustrialsof
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thereactionwerecompletedforfuturechemicalmappingwhichwillbediscussed
inChapter5.NͲmethylalanine(Compound19)canbeusedasaprecursorforthe
synthesisofamixtureofephedrineandpseudoephedrine(Compound1aand1b).
ItcanbeproducedbythereactionofNͲmethylalaninewithbenzaldehydeusing
theAkaboriͲMomotanireactionasdescribedbyPainteretal.[136],illustratedin
Scheme3.10.

Scheme3.10:Synthesisofephedrine/pseudoephedrine.
To date, there is no sign of ephedrine/pseudoephedrine (1a and 1b) being
prepared from NͲmethylalanine 19 in this way on any significant scale. In fact,
Painter et al. [136] only identified trace amounts by GCͲMS analysis. However,
giventhepotentialforuseoftheAkaborireactiontopreparearangeofprecursor
materials by using substituted benzaldehydes, it is of great interest. If a ready
sourceofNͲmethylalanine19weretobeavailable,itwouldnotbeunexpectedto
findthisreactionbeingexploredbyillicitlaboratoriesduetoitssimplicity.
Initially,benzaldehyde6(2.0equivalents)wasaddedslowlytoastirringsolution
of toluene, triethylamine (1.1 equivalents) and NͲmethylalanine 19 (1.0
equivalent).Thesolutionwasstirredtorefluxforanhourbefore5v/v%acetic
acidwasaddedandthenlefttostirforafurther15hours.Anumberofrepeat
reactionswereperformedwithparametersalteredasperTable3.4,totestthe
robustness of this reaction. All produced ephedrine/pseudoephedrine only in
smallamounts.
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Table3.4:Ephedrine/PseudoephedrinesynthesisfrombenzaldehydeandNͲ
methylalanine.
Benzaldehyde AceticAcid LCͲMStrace

Entry Base Solvent
Equivalents v/v(%) (Ephedrine)
1 2.0 Et3N Toluene 5 Yes
2 0.5 None Toluene 5 Yes
3 2.0 Et3N Toluene 50 Yes
4 0.5 None Toluene 50 Yes
5 2.0 Et3N neat 5 Yes
6 0.5 None neat 5 Yes
7 2.0 None DMSO 0 Yes
8 0.5 None DMSO 0 Yes

Although ephedrine/pseudoephedrine (1a and 1b) were detected in all

experiments(Entries1–8),Itisimportanttonote,LCͲMSconfirmedtheformation
of at least 5 other unknown compounds (m/z 148.1036, m/z 228.1353, m/z
162.0886, m/z 254.1528, m/z 280.169) from the reaction, excluding the
ephedrine/pseudoephedrine peak (m/z 166.12263) and there were numerous
productsobservedinthe1HNMRspectrum.Furtherpurificationandoptimisation
wasnotnecessaryandoutsidethisscopeofwork,asaplausiblesyntheticroute
to pseudoephedrine has been confirmed. It is actually quite important that
purification is not carried out as a chemical fingerprint cannot be identified
(furtherdiscussedinChapter5),withoutthepresenceofitsassociatedimpurities.
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3.4CONCLUSION
The methodology presented here may be easily transferable to a clandestine
laboratoryenvironmentandpotentiallybeusedtosynthesispseudoephedrine.
TheNͲmethylalaninepathwayrequiresreadilyaccessiblereagentsandonlybasic
glasswaretobecarriedout.Althoughsomecaremustbetakenwiththeuseof
sodiumhydrideforthemethylationstepofBocprotectedLͲalanine,thiswould
not be restrictive to a clandestine operation and other bases might also prove
useful.Basedonthesynthesispresentedhereitispossibleforalaninetobecome
a molecule of interest for clandestine drug laboratories involved in the
manufacture of methamphetamine, as it has been confirmed as an alternative
routetoephedrine/pseudoephedrine.
The work presented in this chapter has been published in a restricted police
journal as: L.M. Andrighetto, F.M. Pfeffer, J.R Pearson, P.G. Stevenson, S.M.
Hickey,G.J.Barbante,X.A.Conlan,ThreestepsynthesisofNͲmethylalanine–A
precursortoephedrine,pseudoephedrine,andmethamphetamine,Journalofthe
ClandestineLaboratoryInvestigatingChemistsAssociation,24,33Ͳ37(2014).
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CHAPTER 4:
IN-SILICO OPTIMISATION OF 1D-HPLC AND

2D-HPLC USING DRYLAB® SIMULATION
SOFTWARE
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CHAPTER OVERVIEW
Thischapterincorporatesabriefliteraturesummaryoftheanalyticaltechniques
used to analyse methamphetamine samples, highlighting the need of twoͲ
dimensionalHPLC(2DͲHPLC)forcomprehensiveseparationandidentificationof
complexsamples.ThissectionexplorestheuseofsimulationsoftwareDryLab®,
anditsapplicationforinͲsilicooptimisationofamodelmethamphetaminesample
in 1DͲHPLC and 2DͲHPLC separations. An excellent match between DryLab®
predicted separations and actual separations were observed. The 2DͲHPLC
methoddevelopedwasthenappliedtoarealmethamphetamineseizuresample,
identifyingthesystemslimitofdetectioninbothfirstandseconddimensions.A
detailedexplanationoftheadvantagesanddisadvantagesof2DͲHPLCandhow
theycanimprovemodernchromatographicchemicalfingerprintingisdiscussedin
detail.
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4.1Introduction
The current limitations of methamphetamine impurity profiling have been

recentlydescribedbyStojanovskaetal.[26]whostatedthat‘futureresearchis
neededtoaddresstheknowledgegapsinregardstothemanufactureofdrugsof
currentinterest’.Globally,Australiahasoneofthehighestrecordedconsumption
ratesofmethamphetaminewithanestimated1/20residentshavingtrieditfor
recreationalpurposesand395,000reportedusersin2010[172].Gasandliquid
chromatography coupled to mass spectrometry (GCͲMS and LCͲMS), infrared
spectroscopy (IR) and capillary electrophoresis (CE) have traditionally been the
primary analytical techniques used to generate chemical information from
methamphetamineseizuresamples[173].Othertechniqueshavebeenusedto
study methamphetamine samples such as nuclear magnetic resonance
spectroscopy (NMR) [62], ion mobility spectrometry [174], isotope ratio mass
spectrometry(IRMS)[175],andhighperformanceliquidchromatography(HPLC)
[176, 177]. However, the capacity to rapidly generate comprehensive chemical
fingerprints of seized methamphetamine, and its impurities, is of upmost
importancetoAustralianlawenforcementagenciesinordertotrackandidentify
clandestinelaboratoriesandonlymultidimensionalchromatographyaffordsthis.
The relative concentrations of associated pathway impurities in the synthetic
routearesomewhatvariableandcanbedependentontheclandestinelaboratory
environment [148]. This variability, when coupled with the extensive range of
cuttingagentscommonlyusedintheproductionofmethamphetamine,formsa
chemical matrix that can be elucidated by law enforcement agencies to
individualiseseizuresamples[178].
The advent of twoͲdimensional high performance liquid chromatography

(2DͲHPLC)hasenhancedananalyst’sabilitytodevelopseparationproceduresfor
theanalysisofcomponentsincomplexforensic[47],industrial[179],biomedical
[180], and pharmaceutical [181] samples. Comprehensive twoͲdimensional gas
chromatographywithtimeofflightmassspectrometryhasbeenusedbyforensic
chemists to profile complex drug samples [182, 183]. In forensic field studies
77|P a g e

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multiͲdimensional gas chromatography has been used to monitor volatile

compoundsofinterestsuchasmoleculesfromdecayingbodies[184];however,
thisstudywaslimitedbytheinabilityoftheseparationtodealwiththeimmensely
complexsamplebackground.Despitethecomplexnatureofsamplesinforensic
science2DͲHPLChasnothadaslargeuptakeduetothetimelimitstraditionally
associated with sample analysis and the complexity of data handling [185].
However, in addition to forensic science, 2DͲHPLC has been used to elucidate
cannabinoids in hemp [73] and alkaloids in Chinese medicine [186]. A targeted
formof2DͲHPLChasbeeninvestigatedinthisthesisthatcanisolateephedrine
and pseudoephedrine from methamphetamine excipient standard mixtures,
howeverthecomprehensivechemicalinformationonaseizuresamplehasnot
beendetermined.
In order for 2DͲHPLC to be fully exploited for detailed forensic investigations
fundamental aspects of the separation process need to be optimised. This is
driven by the fact that conventional 1DͲHPLC is limited by the number of
theoretical plates (resolving power) available, the heterogeneity of the column
materialandthepotentialdisruptionofanalytesduetoviscousfingering[187].
Viscous fingering is defined as a flow instability phenomenon caused by two
differentsolventviscositiesbeingforcedtogetherthroughaporousmedium,for
example,whenaheartͲcutfromthefirstdimensionisinjectedintothesecond
dimension[188].Itisimportanttonotethatviscousfingeringislikelytobemore
problematic in 2DͲHPLC when the concentrations between dimensions is more
extremeandtheinjectionvolumesarelarge[74].Theuseoftwochromatography
columns in 2DͲHPLC dramatically increases the resolving power of complex
sampleswhendifferentseparationmechanismsareusedineachdimension[189,
190],asdemonstratedinFigure4.1.
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CHAPTER4
Figure4.1:Potentialseparationspaceof2DͲHPLCwhenusingdifferent
columnswithdifferentretentionmechanismscomparedtousingthesame
columns.
There are a range of experimental factors that influence a chromatographic

separation (solvent type, temperature, flow rate etc.). However, the most
importantvariableisthestationaryphaseandshouldbethefirstaspectaddressed
inanyoptimisationprocess[191].
Severaltechniqueshavebeenexploredtooptimisethe2DͲHPLCseparationspace
includingtheanalysisofrelativeretentionprofilesofalibraryofHPLCcolumns.
However, due to the complexity of samples requiring a multidimensional
separation this method proved to be problematic as representative standards
cannotbeeasilyfound[192];also,thesetypesofapproachesaretypicallyvery
timeconsumingduetothenumberofinjectionsandtheflowrateofthesecond
dimension[193].
TheimportanceofthisaspecthasbeenhighlightedbyStevensonandcoͲworkers
[189]whohavedevelopedafundamentalstudyhighlightingthesignificanceof
C18andC1stationaryphases,fortheseparationofPAH’sinamodelsystem.This
stresses the importance of selectivity for complex real world samples such as
those in the forensic sciences. Gilar and coͲworkers [194] developed a robust
methodforcalculatingtheorthogonalityandseparationspaceutilisationoftwo
columnsina2DͲHPLCanalysis.Thismethodwasusedtocalculatethefcoverage,also
79|P a g e

CHAPTER4
known as orthogonality (O) for the 2DͲHPLC peak capacity equation [195] by
dividing the separation space by a number of bins equal to the number of
separatedcomponentstoprovidethefractionalsurfacecoverage.Peakcapacity,
nc,isdirectlyproportionaltoorthogonality(O)asperEquation1.
௡೎ǡభ ൈ௡೎ǡమ ൈ௙೎೚ೡ೐ೝೌ೒೐

݊
Equation1: ௖ǡଶ஽ ൌ ழఉவ

where,nc,2Disthepractical2DͲHPLCpeakcapacity,
nc,iisthepeakcapacityinbothseparationdimensions,
fcoverageistheproportionalamountofseparationspaceutilisation,and
൏ ࢼ ൐istheunderͲsamplingcorrectionfactor.
Theutilisationofseparationspace,whichcanbecalculatedbyEquation2:where
єbinsisthenumberofoccupiedbinsandPmaxisthetotalnumberofbins.
σ ܾ݅݊‫ ݏ‬െ ξܲ௠௔௫

Equation2: ܱ ൌ
ͲǤ͸͵ ൈ ܲ௠௔௫
The stationary phase has the greatest impact on peak capacity, however the
solvent should not be neglected as it can act as a major driver on retention
processesforexampletheʋͲʋinteractionsthatareobservedbetweenacetonitrile
and C18 stationary phases [191]. The mobile phase associated with a
twoͲdimensional separation needs to be carefully considered to avoid solvent
mismatch which may lead to issues such as viscous fingering as identified by
Shalliker and Guiochon [196]. The selection of appropriate stationary phases,
mobile phases, and analysis time need to be considered in the optimisation of
2DͲHPLC experiments. Traditionally, 2DͲHPLC method development has taken
80|P a g e

CHAPTER4
considerable time and there exists a real need for a simplified optimisation
approach.
ThischapterintroducestheDryLab®softwarepackage[89]inordertoefficiently
optimisethegradientprofileforarapidseparationofkeycomponentsinamodel
methamphetamineseizuresample.Thiswillallowthesuperiorresolvingpowerof
2DͲHPLC to screen seizure samples and create a chemical fingerprint of
productionimpuritiesandcuttingagents.
Theinteractionbetweenthesoluteandthestationaryphaseinchromatography
is well known and has been reported by Horváth and coͲworkers [85] with
equation3:
ሾܵ‫ܮ‬ሿ
Equation3: ‫ ܭ‬ൌ
ሾܵሿሾ‫ܮ‬ሿ
ܹ݄݁‫ܭ݁ݎ‬ǡ ݅‫ݐ݊ܽݐݏ݊݋ܿ݉ݑ݅ݎܾ݈݅݅ݑݍ݄݁݁ݐݏ‬ǡ
ܵǡ ݅‫ݏ݈݁ݑ݈ܿ݁݋݉݁ݐݑ݈݋ݏ݄݁ݐݏ‬ǡ
‫ܮ‬ǡ ݅‫ݏ݈݀݊ܽ݃݅ݏݑ݋݁ܿܽ݊݋ܾݎܽܿ݋ݎ݀ݕ݄݄݁ݐݏ‬ǡ
ܵ‫ܮ‬ǡ ݅‫ݏ݊݋݅ݐܿܽݎ݁ݐܾ݊݅ܿ݅݋݄݌݋ݒ݈݋ݏ݄݁ݐݏ‬
Itiswiththismainequation,thattheDryLab®programisabletosimulate,predict
andoptimisebothoneͲdimensionalandtwoͲdimensionalHPLCseparations.
TwoͲdimensional HPLC has the capacity to separate complex mixtures via two
independentretentionmechanisms,whichallowsfortheresolutionofotherwise
coͲeluting species [197]; 2DͲHPLC has not previously been used for impurity
profilingofmethamphetaminesamples.Simulationsoftwarehasbeenusedfor
thefirsttimeonmethamphetamineseizuresamplesinordertorapidlyoptimise
an HPLC separation. This type of optimisation will ultimately lead to improved
chemicalfingerprintingofclandestinedrugseizuresamples.
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4.2Experimental
There are two major experiments in this chapter and as such will be listed
sequentially.
StandardsandSamplesͲExperiment1
All standards (methamphetamine, ephedrine, pseudoephedrine, paracetamol,

caffeine, benzyl alcohol, dimethyl sulfone, benzaldehyde, phenylͲ2Ͳpropanone
(P2P)anddiphenylacetone)wereobtainedfromSigmaͲAldrich(CastleHill,NSW,
Australia)andtheNationalMeasurementInstitute,AustralianGovernment(Port
Melbourne, Vic, Australia). High performance liquid chromatography grade
acetonitrile(ACN)andmethanolwasobtainedfromAjaxFinechem(TarenPoint,
NSW,Australia);deionizedwater(Continentalwatersystems,Vic,Australia)was
filtered through a 0.45 μm membrane filter (SigmaͲAldrich) prior to use. Stock
solutionsofallstandardswerepreparedbydissolvinginasolutionof5%aqueous
ACNataconcentrationof1mgmLͲ1andwerediluted5Ͳfoldwith5%aqueousACN
priortoinjection.
HighPerformanceLiquidChromatographyͲExperiment1
AnAgilentTechnologies(Mulgrave,VIC.,Australia)12602DͲchromatographwas
used for all separations, including: auto injector; capillary pump; binary pump;
column thermostat; 2 position 8 port switching valve; and two diode array
detectors (one in each dimension, which recorded at 254 nm). Two
chromatographycolumnswereused:thefirstdimensioncolumnconstitutedof
anAgilentPoroshell120ECͲC18(4.6×100mm,2.7μmparticlediameter);the
seconddimensionaPhenomenex(LaneCove,NSW,Australia)KinetexC18(100Å
pore diameter, 4.6 × 100 mm, 2.6 μm particle diameter). All analysis was
completedat 1 mL minͲ1 and 1 μL of all model drug solutions were injected in
triplicatetoensurereproducibility.TwoͲdimensionalHPLCwascompletedinthe
onͲlineheartͲcuttingmode,wherebyasingle20μLaliquotofthefirstdimension
(switchedat4.31min)wastransferredtotheseconddimensionviaasampleloop
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andan8port2positionswitchingvalve;valvetimingwascontrolledwithbythe
HPLCcontrolsoftware.
DryLab®OptimizationͲExperiment1
TheDryLab®usermanualrequiresscreeningdataofeachcomponentfromtwo
injectionswithdifferentgradienttimes(20and60min,respectively)tooptimise
theseparation’sgradientprofile[99].Eachstandardwasinjectedindividuallyinto
alineargradientovertheaforementionedtimeswithaninitialmobilephaseof
5%aqueousACNthatincreasedto95%aqueousACN.Retentiontime,heightsand
areasforeachinjectionwasrecordedandsubsequentlyenteredintoDryLab®.The
gradientprofileofthechromatogramgeneratedbyDryLab®wasthenmanually
adjustedinordertoproducetheoptimumrepresentativeseparationintheleast
amountoftimeandwasappliedtotheanalysisofthemodelseizuresample.
ModelSeizureSamplesͲExperiment1
Stocksolutionsofallstandardswerepreparedbydissolvinginasolutionof5%
aqueousACNataconcentrationof1mgmLͲ1andwerediluted5Ͳfoldwith5%
aqueousACN.A100μLaliquotofeachstandardwasputintoa2mLHPLCVial.
Accordingtotheoptimisedsimulationthemodelseizuremixturewasseparated
carriedoutintriplicatewith1μLinjectionsandaflowrateof1mLminͲ1witha
gradienttimeof15.57min.Theinitialconditionswere5%aqueousACNto95%
aqueous ACN over 11.57 minutes with a detection wavelength of 254 nm. The
seconddimensionwascompletedinisocraticmodewithamobilephaseof85%
aqueousmethanoluntilallpeakswereeluted;separationwasrecordedat254nm
and210nm.Bothcolumnswerethermostatedat30°Cwithinthecolumnheater.
ChemicalsͲExperiment2
MilliͲQ water was obtained inͲhouse (Continental Water Systems, Victoria,

Australia);HPLCgradeACNwaspurchasedfromSigmaͲAldrichPtyLtd(CastleHill,
NSW,Australia).
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StandardsandSamplesͲExperiment2
Paracetamol, caffeine, benzaldehyde, aspirin, creatine, procaine, NͲ

methylalanine, and diphenylacetone were obtained from SigmaͲAldrich.
Methamphetamine,pseudoephedrine,ephedrineandphenylͲ2Ͳpropanone(P2P)
were obtained from and the National Measurement Institute, Australian
Government (Port Melbourne, Vic., Australia). Stock solutions of all standards
werepreparedbydissolvinginasolutionof5%aqueousACNataconcentration
of1mgmLͲ1andwerediluted10Ͳfoldwitha5%ACNinwatersolutionandmixed
prior to injection. All methamphetamine seizure samples were provided by
VictoriaPoliceForensicServicesDepartment(Macleod,Victoria,Australia).
ChromatographyColumnsͲExperiment2
FiveHPLCcolumnsweretrialledfortheselectivitystudyofamodelseizuresample
(Table4.1)
Table4.1:TrialledHPLCcolumns.
Brand Type Size Dimensions PartNumber
Column1 Phenomenex KinetexPFP100A 2.6μm 4.6x100mm 00DͲ4477ͲE0
Column2 Phenomenex KinetexPhenylͲHexyl100A 2.6μm 4.6x100mm 00DͲ4495ͲE0
Column3 Agilent Poroshell120ECͲC18 2.7μm 4.6x100mm 695975Ͳ902
Column4 Agilent Poroshell120BonusRP 2.7μm 4.6x100mm 695968Ͳ901
Column5 Phenomenex KinetexBiphenyl 2.6μm 2.1x100mm 00DͲ4622ͲAN
2DͲHPLCͲExperiment2
Chromatographic analysis was performed with an Agilent 1260 system (Agilent
Technologies, Mulgrave, Victoria, Australia), incorporating a quaternary pump
withsolventdegasser,anautoͲsamplerandaDADmodulewhichmonitoredthe
absorbanceat254nm.Chromatographicdatawasobtainedandprocessedwith
Agilent ChemStation software. All injections were 60 μL and carried out in
triplicate.ThefirstdimensioncolumnwasaPhenomonexKinetexPFP100Å(4.6×
100 mm, 2.6 μm particle diameter) and the second dimension a Phenomonex
OnyxMonolithicC18(4.6×100mm).Thefirstdimensiongradientwascompleted
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ataflowrateof0.1mLminͲ1withaninitialmobilephaseof5%aqueousACNthat
increasedto30%aqueousACNover100minutesandthenincreasedto100%ACN
forafurther10minutes.Theseconddimensiongradientwascompletedat5mL
minͲ1 with an initial mobile phase of 5% aqueous ACN that increased to 80%
aqueousACNfor1minute.ThecomprehensivetwoͲdimensionalseparationhad
anoverallcompletiontimeof110minutes.TwoͲdimensionalHPLCwascompleted
inonͲlinecomprehensivemodewithamodulationtimeof1.5minutes,whereby
afractionvolumeof75μLwastransferredtotheseconddimensionviaasample
loopandan8port2positionswitchingvalve;valvetimingwascontrolledwithby
theHPLCcontrolsoftware.
Measurements for bins, plate heights, plate numbers and peak variances were
performed with Wolfram Mathematica 10.3 (distributed by Hearn Scientific,
SouthYarra,Victoria,Australia)usingalgorithmswritteninͲhouse.
LCͲMSͲExperiment2
Highresolutionmassspectrometry(HRMS)wasperformedusinganAgilent6210
MSDTOFmassspectrometerwiththefollowingsettings:gastemperature(350
°C),vaporizer(28°C),capillaryvoltage(3.0kV),conevoltage(40V),nitrogenflow
rate(5LminͲ1),nebuliser(15psi).
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4.3ResultsandDiscussion
4.3.11DͲHPLCusingDryLab®
DryLab® [198] is a software tool that allows scientists to optimise several
chromatographicvariablesinͲsilicotorapidlyoptimiseaHPLCseparation.Itdoes
thisbytakingtheresultsobtainedfromananalyterunattwodifferentgradient
times,tG,toobtainvaluesofk0andSforeachsolute[199]WhenpreͲelutionand
postͲelutionofthesolutescanbeignoredtheseparameterscanbeobtainedby
solvingasetofsimultaneousequations[199]:
௧బ
‫ݐ‬௚ǡଵ ൌ ሺʹǤ͵݇଴ ܾଵ ൅ ͳሻ ൅ ‫ݐ‬଴ ൅ ‫ݐ‬஽
௕ଵ
௧బ
‫ݐ‬௚ǡଶ ൌ ሺʹǤ͵݇଴ ܾଶ ൅ ͳሻ ൅ ‫ݐ‬଴ ൅ ‫ݐ‬஽
௕ଶ
where
௏బ οఃௌ
ܾ ൌ
௧ಸ ி
and tg,1 and tg,2 are the retention times for the first and second dimensions,
respectively; ȴɌ is the change in proportion of the strong mobile phase
component,Ɍ,duringthegradient;t0isthetimerequiredtoeluteanunͲretained
marker and tD is the dwell time of the system, i.e. the time required for
concentrationchangesinthemobilephasetoleavethepumpandenterthehead
of the column. Using the above equations it is relatively easy to simulate how
subtlechangestothegradientcanchangetheelutionorderandresolutionofa
HPLC separation. The software has the capacity to optimise 3 different
experimentalparameterssimultaneously,whichisveryhelpfulwhendesigninga
rapid2DͲHPLCseparation,rapidlyidentifyingcoͲelutingpeaks.DryLab®facilitates
thisprocessbygeneratingaresolutionmaptoidentifytheoptimalgradienttime,
which can then be further manipulated by adding extra gradient steps in the
gradienteditor.Targeted2DͲHPLC,knownasheartͲcutting,isaprotocolwhereby
onlyselectportionsofcoͲelutingpeaksaretransferredtotheseconddimension;
thisisimportantforrapid2DͲHPLCwhenacomprehensiveanalysisissuperfluous.
ItissignificantthatthesimulationsoftwarecouldpredictcoͲelutionandidentify
whenthisoccurssothatthetargetanalysiscouldbestreamlined.
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A model seizure sample of methamphetamine with commonly associated side

products and cutting agents was prepared for inͲsilico optimisation [47].
Specifically a sample mixture containing: methamphetamine, ephedrine,
pseudoephedrine,nicotinamide,paracetamol,caffeine,benzylalcohol,dimethyl
sulfone, benzaldehyde, P2P and diphenylacetone was prepared. Initially, each
standardwasrunindividuallyata20minutegradientanda60minutegradientto
trainthealgorithmsandthecorrespondingretentiontimesandpeakheightswere
recorded and entered into the software. DryLab® then provided the optimal
separationconditionsforbaselineseparationofallthecompounds,illustratedin
Figure4.2.Notethatnicotinamidewasnotretainedandelutedinthevoidvolume.
Note:PeakD/E(i.e.ephedrineandpseudoephedrine)coͲelutedandcouldnotbe
separatedaccordingtothesimulations.
Figure4.2:OptimisedDryLab®predictionB–Paracetamol,C–Caffeine,D/E–
Ephedrine/Pseudoephedrine,F–Methamphetamine,G–BenzylAlcohol,H–
DimethylSulfone,I–Benzaldehyde,J–PhenylͲ2Ͳpropanone,K–
Diphenylacetone.
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Totesttheaccuracyofthesimulationsoftware,amixedsolutioncontainingallof
compoundsAͲKweremadeandtheDryLab®optimalconditionsweretrialledin
anactualseparationthatisshowninFigure4.3.
Figure4.3:Separationofthemodelmethamphetaminesample.
A–Nicotinamide(B–KasforFigure4.1).
It is important to note that the optimal separation provided a relatively fast
gradienttime(i.e.tG)of11.57minutestoresolveallthepeaksofinterest,withthe
exception of the ephedrine and pseudoephedrine diastereomers (peak D/E),
whichcoͲeluted,however,theactualtimetakentoeluteall10compoundswas
only 9.7 minutes prior to the completion of the gradient. As predicted, peak
A–nicotinamide–elutedinthevoidvolume.Thisseparationwascompletedwith
an ACN mobile phase and a C18 HPLC column. The model seizure sample was
analysedintriplicatewithexperimentalparametersmatchingthoseprovidedby
thesoftware.Thedirectoverlayofthemodelmethamphetaminesampleandthe
DryLab®prediction,showsexcellentmatchingofpeaks(Figure4.4).Itisimportant
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CHAPTER4
tonotethatthelimitationsonDryLab®modellingisbasedonthetheoreticalpeak
capacityofthechromatographycolumn.
Figure4.4:OverlayofoptimisedDryLab®predictionandseparationofthe
modelmethamphetaminesample.
Importantly,thecoͲelutionoftheephedrineandpseudoephedrinewereobserved
in the real separation, as predicted by the simulation software. CoͲelution of
diastereomersisacommonprobleminchromatography,andisagreatchallenge
for the identification of the precursor involved in the synthesis of
methamphetamine. Ephedrine and pseudoephedrine were resolved from the
othercompoundsandeachotherwithasimple2DͲHPLCseparationprotocol.The
peak (D/E) at a retention time of 4.31 minutes in the first dimension was
transferred to a second dimension with a 20 μL sample loop and an 8 port, 2
position,switchingvalve.Theseconddimensionwasoperatedwithanisocratic
methanol/H2O mobile phase (85% MeOH) on a C18 column, the different
selectivitiesaffordedbythesolventspolaritywassufficienttoallowtheresolution
ofallmodelcompounds.TheseconddimensionseparationisillustratedinFigure
4.5.
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CHAPTER4
Figure4.5:Seconddimensionseparationofephedrineandpseudoephedrine
(both1.3×10Ͳ3M)fromtheoverlappingpeak(D/E).
Theephedrineandpseudoephedrinehavebeenbaselineresolvedwithretention
timesof5.05and5.38minutesrespectivelyintheseconddimension.Thisisan
importantfindingforthreereasons:
1. Thediastereomershavebeenseparated;
2. AsimpleC18:C18twodimensionalseparationwassufficientforbaseline
resolutionoftheanalytes.Itisimportanttonotethattheseparationin
thiscaseisdrivenbythedifferenceinthemobilephasesacrossthetwo
dimensions. While C18:C18 limits the ability to generate a large
separationspacethismakethesystemmuchsimplertoapply.Formore
complex samples different stationary phases would be required as
highlightedinChapter5.
3. Thetotalanalysistimefortheseparationofallmodelcompoundsinboth
dimensions, including discrimination between ephedrine and
pseudoephedrine, was completed in 10.31 minutes (4.31 min first
dimension+6.00minseconddimension).
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4.3.22DͲHPLCusingDryLab®
AstheDryLab®simulationsoftwarehasworkedextremelywellwithconventional
one dimensional HPLC, attention turned to using DryLab® as a tool for the
optimisation of a full comprehensive twoͲdimensional separation. Within this
study a full comprehensive 2D separation was developed by slowing the first
dimensionflowratedownsignificantly(0.1mLminͲ1)andtransferring4fractions
perminuteintotheseconddimension.
A model seizure sample was prepared containing known concentrations of

methamphetamine and 11 other common precursors and cutting agents:
pseudoephedrine,ephedrine,phenylͲ2Ͳpropanone(P2P),paracetamol,caffeine,
benzaldehyde,aspirin,creatine,procaine,NͲmethylalanine,anddiphenylacetone.
Afteracolumnselectivitystudywasperformed(whichwillbediscussedinchapter
5),acombinationofthePFPandECͲC18stationaryphaseswasfoundtoprovide
thegreatestsurfacespaceutilisationwithanfcoverageof0.18(seeFigure4.6).To
facilitate a rapid separation towards the goal of ‘fitͲforͲpurpose’ analysis the
monolithic[200]C18columnwastrialledinplaceoftheparticlepackedcolumn;
anfcoverageof0.18wasmaintained.Thefirstdimensionwascompletedataflow
rateof0.1mLminͲ1toallowforamodulationfrequencyof3transfersofeachfirst
dimensional peak to the second dimension. Therefore, the second dimension
mustbeperformedveryfastsothatthesecondanalysiscanbedoneinatime
equaltotheperiodoffractioncollectionminusthefractiontransfertime[70]a
speedthatcanonlybeaffordedbythemonolithiccolumn.
Figure4.6:Binsplot(PoroshellC18×KinetexPFP)oftwodimensionalHPLC
separationofthemodelmethamphetamineseizuresample.
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CHAPTER4

With this in mind, a fully comprehensive twoͲdimensional separation was
completedwithatotalanalysistimeof115minutes,theseparationisillustrated
inFigure4.7.TheretentiontimespredictedbyDryLab®areshownbythewhite
circles,whichhavebeenoverlaidontheactualtwodimensionalseparation(Figure
4.7).

Figure4.7:OverlaidDryLab®andactualtwodimensionalseparationofthe
modelmethamphetamineseizuresample.

Likewise, a more detailed 3D representation of the actual twoͲdimensional
separation,highlightingtheintensitiesofeachcompoundwascreatedandshown
inFigure4.8.
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CHAPTER4

Figure4.8:3Drepresentationofamodelmethamphetamineseizuresample
twoͲdimensionalseparation.

ThisisthefirsttimeinͲsilicosoftwarehasbeenusedtopredictatwodimensional
separation of methamphetamine samples and indeed forensically relevant
samples at large. This is of great significance for the development of 2DͲHPLC
protocols,aprocessthathistoricallytakesconsiderabletime.Thetotalmethod
developmenttimeforseparationpresentedinFigure4.8waslessthantwohours
withinͲsilicoaidedoptimisation.Thisissignificantlylessthanthetimerequired
fortypicaloptimisationofindividualsampleinjections[47].Theresultsof2DͲHPLC
separationpredictionwasimpressiveasallcomponentsofinterestwereclosely
matched.Thisisnotatrivialoutcomeduetothecomplexnatureoftheseparation
mechanisms involved allowing the user to have high confidence in the inͲsilico
prediction.
4.3.32DͲHPLCofarealmethamphetamineseizuresample
Inordertodeterminetheeffectivenessofthisprocess12realmethamphetamine
seizuresamplesprovidedbyVictoriaPoliceForensicServicesDepartmentwere
analysedusingthedevelopedmethod.Severalcomponentswereobservedinall
oftheseizuresamples;acharacteristicchromatogramispresentedinFigure4.9.
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ThetwomajorcomponentsinFigure4.9aremethamphetamine(firstdimension
retentiontime(Rt,1)of83.4min,seconddimensionretentiontime(Rt,2)of0.72
min)andpseudoephedrine(Rt,1=68.1min,Rt,2=0.66min)thatweredetermined
by comparing the retention times against known standards. With only two
detectedcomponentpeaksthissampleisparticularlyclean;thisisnotuncommon
in crystal methamphetamine samples. Trace amounts of excipients were not
observedinthisoranyoftheotherelevensamples.
Figure4.9:VictoriaPoliceForensicServicesDepartment(VPFSD)
methamphetamineseizuresample.

Ashighresolutionmassspectrometryisasignificantlymoresensitivetechnique
thanUVdetectionusingHPLC[201],LCͲMSwasusedtoanalysealltwelveseizure
samplesalsointhesearchforotherexcipients.Theassociatedmassesofall12
random seizure samples are shown in Table 4.2. As there were a few more
compounds detected using LCͲMS, it highlights the fact that there are some
detection limit issues when looking for trace amounts of excipients using a UV
detector.However,itisimportanttonotethatfor8outofthe12randomseizure
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CHAPTER4
samples, three or less compounds were detected, again highlighting the clean
natureofthesesamplesinVictoria.
Table4.2:Molecularweightsdetectedinmethamphetamine(MA)seizure
samplesincludingpseudoephedrine(PE).
SAMPLE m/z m/z m/z m/z m/z

VPFSDͲ1 MA 144.9872 168.0204 Ͳ Ͳ
VPFSDͲ2 MA 144.9872 168.0204 Ͳ Ͳ
VPFSDͲ3 MA 179.0111 335.2257 Ͳ Ͳ
VPFSDͲ4 MA 165.0756 Ͳ Ͳ Ͳ
VPFSDͲ5 MA PE 148.1124 198.0980 383.1165
VPFSDͲ6 MA PE 148.1128 198.0980 335.2248
VPFSDͲ7 MA PE 335.2255 Ͳ Ͳ
VPFSDͲ11 MA PE 133.0875 148.1118 198.0966
VPFSDͲ12 MA PE 198.0961 226.1589 383.1153

The determination of excipients is of particular importance for chemical
fingerprintingandisusefulwhenanalysingintermediatesyntheticsamplesfound
inclandestinelaboratories.
4.3.42DͲHPLCofasynthesisedintermediatesampletomethamphetamine
Toassessthelevelofchemicalcomplexitythatcanbeassessedwith2DͲHPLCan
intermediatesamplefromamethamphetaminesynthesisinthelaboratorywas
analysedandispresentedinFigure4.10.
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CHAPTER4
Figure4.10:Synthesisedintermediatesampletomethamphetamine.

Morethanfortycomponentsweredetectedbasedonpeakmaxima,determined
by examining the derivatives of the curves using algorithms described by
Stevenson et al. [202]. The components in the intermediate sample are
highlightedbywhitedotsinFigure4.10,thusemphasisingtheforensicsignificance
offingerprintingintermediateseizuresamplestobuildacomprehensivedatabase
of the chemical pathways in a clandestine laboratory. For example, if dirty
glasswarewerefoundataclandestinelaboratory,thistechnologycanbeusedto
trace back to the use of exact precursors which may be enough to determine
exactlywhatprocesseshavebeenused.

Thedistributionofthecomponentsinthemodelsystemandtherealintermediate
samplegenerallyappearinthetoprighthandquadrantofthetwodimensional
separation,duetothenonͲpolarnatureofthecomponentspresent.Whilethe
otherthreequadrantsarenotfullyexploitedhere,itisimportantforanyanalyst
tousethefullseparationprocedureincasesomeunknownpolarexcipientsare
presentandtocounteranyfuturenovelsynthesesthatmaypresentmorepolar
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CHAPTER4
components. While this will add to the overall separation time, it is of greater
importancetotheforensiccommunitytohaveacomprehensiveanalysissoone
universalmethodcanbeusedregardlessofthesyntheticroute,location,purity
andprecursors.

Anfcoverageof0.49wasfoundwhenthesurfacespaceusageoftheintermediate
separationwascompleted,farexceedingthatofthestandardmixture(fcoverageof
0.18).Whileeveryattemptwasmadetoreplicateatypicalseizuresample,the
optimisationofthemodelsampledidnotaccuratelyreflecttheseparationspace
usedbytheactualintermediatesample(Figure4.10).Thishighlightstheissues
faced when selecting columns for 2DͲHPLC and the importance of creating a
universalseparationthatcancopewithcomplexmixtures.

It is important to consider the analytical figures of merit for this type of two
dimensional separation, in order to determine if transferring peaks between
dimensionshasanyinfluenceonthedetectionlimit.Toachievethisseveralkey
excipients (paracetamol, caffeine, methamphetamine, pseudoephedrine,
procaineandaspirin)wereselectedandthelimitofdetection(LOD)inthesecond
dimension (LOD1DͲHPLC) was calculated and recorded in Table 4.3. The limit of
detectionwerecalculatedmultiplyingtheslopeofthecalibrationcurveby3.3and
dividing by the standard error [203]. The LOD of these compounds was in the
rangeof3×10Ͳ5M,whichwascomparabletoliteraturevalues[204Ͳ206].
Table4.3:2DͲHPLCLimitofDetections.
Rt Calibration LOD 1DͲHPLC LOD2DͲHPLC

EXCIPIENT R²
(min) Equation (M) (M)
Methamphetamine 2.46 0.9999 y=1×106x Ͳ 8.8 2.7×10Ͳ5 4.6×10Ͳ4
Pseudoephedrine 1.55 0.9999 y=1×106x+8.2 2.3×10Ͳ5 4.0×10Ͳ4
Aspirin 4.26 0.9998 y=918340xͲ 29.1 5.4×10Ͳ5 9.3×10Ͳ4
Caffeine 2.07 0.9985 y= 2×106xͲ 12.8 2.9×10Ͳ5 5.2×10Ͳ4
Paracetamol 0.76 0.9988 y=755343xͲ 0.61 3.4×10Ͳ5 5.9×10Ͳ4
Procaine 1.23 0.9997 y=220286x+10.6 1.2×10Ͳ4 2.0×10Ͳ3
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CHAPTER4
Theidealmodulationfrequencywhentransferringanalytesbetweendimensions
in2DͲHPLCis3–4fractionsperpeak[207].Toensurethisfrequencyismaintained
forpeaksinadjacentfractions,theamountoftransferredanalytemustatleast
matchtheseconddimension’slimitofdetection(LOD2ndͲdimension).Inthisworka
modulation frequency of 3 fractions per peak was maintained whereby a
minimum of 5.8% of the first dimension peak (fractions 1 and 3 – derived by
dividing the area of a Gaussian peak with an area of 1 into 3 evenly spaced
segments)weretransferred,withtheassumptionthatthepeakmaximaiscentred
in the 4ʍ peak width. To maintain detected 3 fractions per peak the minimum
amount of transferred material was 5.8% of the total peak area with a
concentrationatleastequaltotheLOD2ndͲdimensionafterseparation.Thus,thetotal
concentrationofcompoundinjected(i.e.theLOD2DͲHPLC)iscalculatedbyEquation
3,whichistransposedintwostepsbelow:
5.8%LOD2DͲHPLC=100%LOD2ndͲDimension(Equation3)
LOD2DͲHPLC=100%/5.8%LOD2ndͲDimension
LOD2DͲHPLC=17.2×LOD2ndͲDimension

Assuch,theLOD1DͲHPLC (singledimension)mustbemultipliedby17.2.Following
thesameproceduretheeffectwillbecomesignificantlymorepronouncedasthe
modulation frequency increases, for example if a modulation frequency of 4
fractionsperpeakwasusedtheLOD2DͲHPLCisequalto107.9×LOD2ndͲdimension.Table
4.2liststheLODs(oneandtwoͲdimensions)ofstandardsfor6commonexcipients
inthemanufactureofmethamphetamineandisintheorderof5×10Ͳ4M.

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4.4Conclusion
TheuseofinͲsilicooptimisationoftwoͲdimensionalseparationsfortheanalysis
of methamphetamine seizure samples has been shown to have a significant
impactontheexperimentaldevelopmenttime.Thegreatestchallengein2DͲHPLC
methoddevelopmenthastypicallybeenthetimerequiredtoidentifythecolumns
requiredtoprovidethegreatestseparationspaceutilisation.Thiswasovercome
herewithDrylab®optimisationsoftware.Thisimprovementinanalysistimealso
reduces the organic solvent load produced and directly impacts the laboratory
wastegenerated.ImportantlytheDrylab®optimisedinͲsilicoseparationsmatched
closely with the model and synthetic samples allowing the analyst to have
confidenceinthepredictedseparationparametersdespitethecomplexnatureof
theseparationmechanismsinvolved.A2DͲHPLCseparationwascompletedthat
isolated more than 40 individual chemical compounds from an intermediate
methamphetaminesample.Thisknowledgecanbeusedbytheforensicanalystto
createachemicalfingerprintoftheseizure,whichcantheoreticallybelinkedback
toaclandestinelaboratory.
The work presented in this chapter has been published in the following two
papers:
L.M. Andrighetto, P.G. Stevenson, J.R. Pearson, L.C. Henderson, X.A. Conlan,
DryLab®optimisedtwoͲdimensionalhighperformanceliquidchromatographyfor
differentiation of ephedrine and pseudoephedrine based methamphetamine
samples,ForensicScienceInternational,244,302Ͳ305(2014).
L.M. Andrighetto, N.K Burns, L.C. Henderson, C.J Bowen, J.R. Pearson, P.G.
StevensonandX.A.Conlan,InͲsilicooptimisationoftwoͲdimensionalHPLCforthe
determinationofAustralianmethamphetamineseizuresamples,ForensicScience
International,266,511Ͳ516(2016).
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CHAPTER5
CHAPTER 5:
COLUMN SELECTIVITY STUDY AND

CHEMICAL MAPPING
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CHAPTER5
CHAPTER OVERVIEW
This chapter brings together all the key concepts around the multidisciplinary
approach (synthetic and analytical chemistry) that has been developed in this
thesis for methamphetamine chemical fingerprinting. This chapter examines a
comprehensive column selectivity study where a novel selection protocol is
developed, using OpenMS® (open source LCͲMS data handling software). The
methodology developed in this chapter identifies the two most orthogonal
columnsinordertoprovidetheuserwiththebestseparationofcomplexsamples
ofinterest.
Thissectionwillalsoincludethechemicalmappingofafullsyntheticpathwayto
methamphetaminefromeasilyaccessibleingredientssuchasbenzaldehydeand
nitroethane. A comparative study between LCͲMS and LCͲUV Visible data was
carried out simultaneously for the mapping of the synthetic route of the
clandestine product. This enabled a comprehensive impurities table to be
generatedwhichhasbeendiscussedregardingthedirectimpacttheywillhave
towardsrealworldforensicanalysisofseizuresamples.Analternativeapproach
fortheidentificationofsyntheticrouteandprecursorswasalsotrialled,witha
preliminarystudyusingtargetedprincipalcomponentanalysis.
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5.1Introduction
AsbrieflydiscussedinChapter4,conventionalHPLCalthoughwidelyusedasa
chromatographytool,hasalimitedseparationpotentialwhenanalysingcomplex
samples.TwoͲdimensionalHPLCofferssignificantpotentialandisutilisedinorder
toseparateoutcompoundsviatwodifferentretentionmechanismsaffordedby
the two stationary phases [70]. The ability to separate complex samples is
considerably improved by using 2DͲHPLC due to the significant increase in
theoreticalpeakcapacity[70].Ideally,forthefullbenefitsofmultiͲdimensional
separations to be realised, the stationary phase properties of the selected
columnsshouldbeasdifferentaspossible[70].Theprocessofassessingthisis
often labour intensive and very time consuming, requiring a trialͲandͲerror
approachorinjectingnumerousstandardstotesteverypossiblestationaryphase
combination[192,193].
Inordertoimprovetheseparationpowerandbestutilisetheseparationspace,
the two most orthogonal columns must be selected allowing for the best
chromatography[70].Theseparationpotentialofa2DͲHPLCsystemisestablished
bythepeakcapacitywhichputsimplyisthechromatographicresolvingpower.
Thepeakcapacitydeterminestheseparationpotentialofasystem[194]andis
proportionaltothefractionalcoveragedefinedbyWatsonetal.[208]according
toEquation4:
σୠ୧୬ୱ
݂௖௢௩௘௥௔௚௘ ൌ (Equation4)
௉మ
wherefcoverageistheproportionalsurfacecoveragethatrangesfrom0to1,
єbinsisthenumberofbinsthatcontainpeaks,and
P2isthetotalnumberofavailablebins.
ThefcoverageisdirectlyrelatedtothetwoͲdimensionalpeakcapacity,highlighting
inEquation5thatthegreaterthefcoverage,thebetteruseofseparationspaceand
betteroverall2DͲHPLCseparation[194]:
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CHAPTER5
௡೎ǡభ ൈ௡೎ǡమ ൈ௙೎೚ೡ೐ೝೌ೒೐

݊௖ǡଶ஽ ൌ (Equation5)
ழఉவ
where,nc,2Disthepractical2DͲHPLCpeakcapacity,
nc,iisthepeakcapacityinbothseparationdimensions,
fcoverageistheproportionalamountofseparationspaceutilisation,and
൏ ࢼ ൐istheunderͲsamplingcorrectionfactor.
TheunderͲsamplingcorrectionfactorisdirectlyproportionaltothemodulation
frequencyperpeakfromthefirstdimensiontothesecond,asdescribedbyLiand
coͲworkers[195].Thisisachievedthroughagivennumberofbinsusedtodivide
theseparationspace,ɇbins,thatisequaltothenumberofpeaks;theareaofall
normalised bins containing peaks is then totalled giving P2 [190]. Using this
method,thefcoverageis calculatedasanumberbetween0and1,where0isno
separationand1representsfulluseoftheavailableseparationspace.
The separation space is maximised using orthogonal column stationary phases
howeveridentifyingthebesttwocolumnscanbealengthyandchallengingtask
whentheretention profilesofeachcolumnneedstobe comparedindividually
[192]. It is extremely difficult to match peaks based on area when changing
columntype,astheelutionorderandpeakprofilewillalmostcertainlychange
when optimising using the sample itself [192]. A way around this is to inject
individual standards one at a time onto each column [199] however this is
extremelytimeconsumingandinefficient.
An alternative method to monitor the peaks across each separation is by their
molecularweightwiththeaidofmassspectrometry[209].Massspectrometryis
often coupled with liquid chromatography, gas chromatography, or capillary
electrophoresistoobtainmorechemicalinformationaboutasample[210].Liquid
chromatography – mass spectrometry is superior to HPLC with UVͲabsorbance
detectionwhenanalysingcomplexsamplesasitprovidestheuserwithnotonly
retention time but also molecular weight and isotopic information, and can be
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usefulwhenusingtheselectedionmodeorextractedionmodeforconstituent
monitoring[201].Liquidchromatography–massspectrometryhasbeenusedin
this column selectivity study to monitor the same component peaks across 17
differentcolumnsandprovideasmuchinformationaboutthepeaksaspossible.
RawhighresolutionLCͲMSdataisincrediblylarge,complexandcareneedstobe
takenwhenhandlingthedatasothattheanalystcanachievethebestoutcomes.
SoftwarelikeXCMS™[211],OpenMS®[212],ProteoWizard[213],mspire[214],
MzJava [215], MzMine [216] and Pyteomics [217] have all been used for the
analysisofLCͲMSandLCͲMS/MSdata[218]andhavebeenutilisedinarangeof
fields (proteomics [217], metabolomics [219], and lipids [220]) differing in
supportedformats.
InthisinvestigationOpenMS®(version1.11.1),anopensourceprogramdesigned
to interpret and analyse mass spectrometry data, was explored to see if the
softwarecanassistinidentifyingthebestcolumncombinationforanoptimised
2DͲHPLC separation. OpenMS® was prioritised over other programs due to its
flexiblenature,largerangeofsupportedformatsanduserfriendlycharacteristics
[221].Thisprogramoffersfiltersthatcanbeappliedallowingfortheextractionof
informationtheuserdeemsimportant,suchasparticularpeaksofinterest,exact
mass measurements, mass ranges, etc. There are a many modules that can be
designedfortheanalysisofacomplexdataset,includingeliminationofnoiseand
peakpickingoperations[218].
Once the best two columns were chosen, further statistical analysis were then
trialledonmethamphetamineͲtypesamples.PrincipalComponentAnalysis(PCA)
wasalsousedtoseeifitwaspossibletopredictthesyntheticpathwayusedto
make the methamphetamine (from pseudoephedrine or P2P). Principal
ComponentAnalysisisastatisticalprocedureappliedtomultivariatedatawhich
identifiesandisolatessamplesbasedonthedegreeofvariance(ie.howsimilaror
different one sample is from another) [222]. It involves an orthogonal
transformationsothatthenewaxesliesalongthemaximumvarianceinthedata,
convertingcorrelatedintouncorrelateddata[223].PrincipalComponentAnalysis
canrevealthevariablesthatdifferentiateasamplefromanotherandcanbeused
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topredictwhatgroupsunknownsamplesaremostsimilarto.Itisimportantto
notethatthecovariancematrixmethodofPCAwasusedinthisanalysis.Assuch
the eigenvalues and eigenvectors are calculated from the covariance matrix
equation.Initially,thecovarianceiscalculatedusingequation6:
௡
‫ݒ݋ܥ‬௫௬ ൌ ෌௜ୀଵሺ‫ݔ‬௜ െ ‫ݔ‬ҧ ሻሺ‫ݕ‬௜ െ ‫ݕ‬തሻȀሺ݊ െ ͳሻ(Equation6)
ܹ݄݁‫ݒ݋ܥ݁ݎ‬௫௬ ǡ ݅‫݉݁ݐݏݕݏ݂ܽ݋݁ܿ݊ܽ݅ݎܽݒ݋݄ܿ݁ݐݏ‬ǡ
‫ݔ‬௜ ǡ ݅‫ݔ݈݁݌݉ܽݏ݂݋݁ݑ݈ܽݒ݈ܽݑ݀݅ݒ݄݅݀݊݅݁ݐݏ‬ǡ
‫ݔ‬ҧ ǡ ݅‫ݔ݈݁݌݉ܽݏ݂݋݄݊ܽ݁݉݁ݐݏ‬ǡ
݊ǡ ݅‫݁ݖ݅ݏ݈݁݌݉ܽݏ݄݁ݐݏ‬.
Thecovarianceisrepresentedinamatrixformshowninequation7:
‫ݒ݋ܥ‬௫ǡ௫ ‫ݒ݋ܥ‬௫ǡ௬
‫ ݔ݅ݎݐܽܯ݁ܿ݊݁݅ݎܽݒ݋ܥ‬ൌ ൬ ൰(Equation7)
‫ݒ݋ܥ‬௬ǡ௫ ‫ݒ݋ܥ‬௬ǡ௬
Withthecovariancematrixinhand,atranspositionisperformedasoutlinedin
equation8.Thistranspositionwhenrepresentedinthethreedimensionalspace
allows for the eigenvector to be calculated. The eigenvectors with the largest
value has the strongest correlation to the data set. When this data is plotted
betweenprinciplecomponentsthedatasetsthatarecorrelatedcanbedirectly
observed.
ሺ‫ݔ‬ଵ െ ‫ݔ‬ҧ ሻ்
‫ ݔ݅ݎݐܽܯ݁ܿ݊݁݅ݎܽݒ݋ܥ‬ൌ ܺܺ ் ൌ ሾ‫ݔ‬ଵ െ ‫ݔ‬ҧ ǡ ‫ݔ‬ଶ െ ‫ݔ‬ҧ ǥ Ǥ ‫ݔ‬௡ െ ‫ݔ‬ҧ ሿ ቎ ሺ‫ݔ‬ଶ െ ‫ݔ‬ҧ ሻ் ቏
ሺ‫ݔ‬௡ െ ‫ݔ‬ҧ ሻ்
(Equation8)
ThistypeofpredictivemodellingwasdevelopedbyHenryHotellinganditwasfirst
reportedintheliteraturein1933[224].PrincipalcomponentanalysisisawellͲ
developedtechniquethathasbeenusedinmanyanalyticalapplicationssuchas
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analysis of coffee beans [225], drugs in rats [226], heavy metals in food [227],
drugsinsmuggledpackages[228],photochemicalreactions[229],fermentedfood
productslikecow’smilkandcheese[230],profilingoftobacco[231]andoliveoils
[232].PrincipalComponentAnalysisonLCͲMSdatahassomechallengesdueto
the large data sets generated and the need for careful data alignment due to
potentialmisinformationgeneratedfromprecursorionsinMSͲMS.Typicallydata
is binned and this reduces the quality of information gained through high
resolutiondata[233].
A major difficulty in the optimisation of 2DͲHPLC operating conditions is the
selection of the most orthogonal columns. This chapter will discuss a new
approach to efficiently selecting the best column combination for 2DͲHPLC
analysisofanycomplexsamplefollowedbyashortpilotstudy,carryingoutPCA
onmethamphetaminetypesamples.Seventeencolumnswereusedtoseparatea
model methamphetamine sample using LCͲMS. OpenMS® was then used to
evaluatethedatafromeachcolumnandidentifywhichtwocolumnswhenused
together will provide the best use of separation space. Using the selected two
columns, PCA was trialled as a complementary method of swiftly identifying
routesofmanufacture.
5.2Experimental
Chemicalsandsamples
Deionised water was obtained inͲhouse (Continental Water Systems, Victoria,

Australia)andfilteredthrougha0.45μmmembranefilter(SigmaͲAldrichPtyLtd,
Castle Hill, NSW, Australia) prior to use. HPLC grade acetonitrile (ACN) was
purchased from Ajax Finechem Pty Ltd (Taren Point, NSW, Australia).
Trifluoroacetic acid was purchased from SigmaͲAldrich. All standards
(methamphetamine,ephedrine,pseudoephedrine,paracetamol,caffeine,benzyl
alcohol, dimethyl sulfone, benzaldehyde, phenylͲ2Ͳpropanone (P2P) and
diphenylacetone)wereobtainedfromSigmaͲAldrich(CastleHill,NSW,Australia)
and the National Measurement Institute, Australian Government (Port
Melbourne,Vic.,Australia).
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LiquidChromatographyMassSpectrometry(LCͲMS)
Mass spectrometry analysis was performed with an Agilent Technologies 6210
LCͲMSDTOFsystem(AgilentTechnologies,Mulgrave,Victoria,Australia).Itwas
equippedwithanAgilenttechnologies1200seriessolventdegasser,binarypump
and an Agilent technologies 1100 series ALS. Positive ion polarity was used in
conjunctionwithnitrogendryinggas(5LminͲ1)andnitrogennebulisergas(30psi).
Themodelmethamphetaminesamplewasinjected(10μL)intoalineargradient
over40minuteswithaninitialmobilephaseof5%aqueousACNthatincreasedto
100%ACN.Allanalyseswererunataflowrateof0.5mLminͲ1.Chromatographic
data was obtained and processed with Agilent ChemStation software. The 17
columnsthatcomprisedthestationaryphaselibraryforthisstudyare listedin
Table5.1.
Table5.1:Columnstrialledinstudy.
Brand Type Size Dimensions PartNumber
Column2 Agilent Poroshell120ECͲCN 2.7μm 4.6x100mm 695975Ͳ905
Column3 Phenomenex KinetexPFP100A 2.6μm 4.6 x100mm 00DͲ4477ͲE0
Column4 Phenomenex KinetexPhenylͲHexyl100A 2.6μm 4.6x100mm 00DͲ4495ͲE0
Column5 Phenomenex SynergiFusionͲ RP80A 4μm 4.6x150mm 00FͲ4424ͲE0
Column6 Phenomenex SynergiHydroͲ RP80A 4μm 4.6x150mm 00FͲ4375ͲE0
Column7 Phenomenex LunaC5100A 5μm 4.6x50mm 00BͲ4043ͲE0
Column8 Phenomenex LunaNH2100A 5μm 4.6x100mm 00DͲ4378ͲE0
Column9 Phenomenex LunaHILIC200A 5μm 4.6x150mm 00FͲ4450ͲE0
Column10 Cosmosil ʋNAP 2.5μm 4.6x100mm 08084Ͳ51
Column11 Cosmosil 5PBBͲR 5μm 4.6x150mm 05697Ͳ21
Column12 Cosmosil 5NPE 5μm 4.6x150mm 37904Ͳ01
Column14 Agilent PoroshellHPHͲC18 2.7μm 4.6x100mm 695975Ͳ702
Column15 Agilent Poroshell120,SBͲC18 2.7μm 4.6x100mm 685975Ͳ902
Column16 Agilent Poroshell120BonusRP 2.7μm 4.6x100mm 695968Ͳ901
Column17 Agilent PursuitXRs3Diphenyl 3μm 4.6x100mm A6021100X046
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LiquidChromatographyMassSpectrometry(LCͲMS)forchemicalmappingof
syntheticpathway.
MassspectralanalysiswasperformedwithaThermoFisherQͲExactivePlusUPLC
Ultimate 3000/orbitrap system (Thermo Fisher Scientific, Scoresby, Victoria,
Australia). It was equipped with a Dionex Ultimate 3000 (HPGͲ3400RS) UPLC
pump. Positive ion polarity was used in conjunction with nitrogen drying gas
(7 L minͲ1) and nitrogen nebuliser gas (30 psi). When using the Phenomenex
KinetexPFPColumn,thesampleswereinjected(10μL)intoalineargradientover
22minuteswithaninitialmobilephaseof5%aqueousACNthatincreasedto30
%ACNover20minutes,thenincreasedto100%ACNwhichwasheldforafurther
2minutes.Allanalyseswererunataflowrateof0.5mLminͲ1.Whenusingthe
Onyx C18 monolith column, the samples were injected (10 μL) into a linear
gradientover10minuteswithaninitialmobilephaseof5%aqueousACNthat
increasedto80%ACN.Massspectrometrydatawereobtainedandprocessedwith
ThermoFisher‘Xcalibur’software.
TwoͲdimensionalhighperformanceliquidchromatography
AnAgilentTechnologies(Mulgrave,Vic.,Australia)12902Dchromatographwas
used for all separations, including: an auto sampler; solvent degasser; capillary
pump; binary pump; quaternary pump; column thermostat; 2 position 8 port
switching valve; and two diode array detectors (one in each dimension, which
recorded at 254 nm). The 2DͲHPLC separations were performed using 80 μL
injectionsandacontrolledtemperatureof25°C.Thefirstdimensiongradientwas
completed at a flow rate of 0.1 mL minͲ1 with an initial mobile phase of 5%
aqueous ACN that increased to 30% aqueous ACN over 100 minutes and then
increasedto100%ACNforafurther10minutes.Theseconddimensiongradient
wascompletedat5mLminͲ1withaninitialmobilephaseof5%aqueousACNthat
increasedto80%aqueousACNfor1minute.ThecomprehensivetwoͲdimensional
separationhadanoverallcompletiontimeof110minutes.TwoͲdimensionalHPLC
was completed in onͲline comprehensive mode with a modulation time of 1.5
minutes (Kinetex PFP & Onyx Monolithic C18) and 3.0 minutes (Kinetex PFP &
SynergiHydro),wherebyafractionvolumeof75μLwastransferredtothesecond
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dimensionviaasampleloop(500μL)andan8port2positionswitchingvalve;
valvetimingwascontrolledwithbytheHPLCcontrolsoftware.
DataProcessing
ThechromatographicdataobtainedfromtheLC/MSwasanalysedwithOpenMS®
1.11.1 [212, 221]. The OpenMS® pipeline included a file converter (convert
.mzdatato.mzml),filefilter(parameterssuchasintensity,retentiontimesand
m/z values were optimised), reͲsampler (LCͲMS map is transformed into a
resampledmap),noisefilterSGolay(smoothpeaksandeliminatepeakscausedby
noise), peak picker wavelet (peaks are selected), feature finder metabo (filters
repeated masses) and data collection. All parameters within these tools were
defaultexceptinthefilefilterwherem/zvalueswerespecifiedtobe50Ͳ300and
intensityvalueswere1,000դ30,000,000.TheoutputfilesfromOpenMS®consisted
of a list of retention times for features that were common amongst the
investigatedHPLCcolumns.ThisdatawasprocessedwithWolframMathematica
10.3 (distributed by SHI UK, Milton Keynes, England) to match these retention
times and predict the relative retention behaviour using algorithms written
inͲhouse to calculate the most orthogonal columns. TwoͲdimensional contour
plotswerepreparedwithWolframMathematica10.3.
PrincipalComponentAnalysis(PCA)
PCAwascarriedouton22randommethamphetamineseizuresamplesprovided
by the Victoria Police Forensic Services Centre. This pilot study was processed
using a specialised Thermo Fisher Scientific software by the name of ‘SIEVE™’,
whichisaLCͲMSdataprocessingprogram.Thedatawasscaledandnormalised
usingtheinternalSIEVE™mechanism.Amassrangewassetasm/z166.0Ͳ166.2
inordertocarryoutatargetedPCAofpseudoephedrine.Theanalysistime(0–
15 minutes), m/z range (166.0 – 166.2), m/z tolerance (10 ppm), minimum
intensitythreshold(2.0×107),peakscans(5),signaltonoiseratio(3:1)andm/z
stepsize(10)weretheparameterschosenforthetargetedPCA.
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5.3ResultsandDiscussion
5.3.1ColumnSelectivityStudyusingOpenMS®
Whencomparingcolumncombinationsindividually,selectingtwocolumnsthat
provide the greatest separation space is a labour intensive process. As such,
monitoring the molecular masses using LCͲMS has the potential to rectify the
problemofdifferentiatingpeakelutiontimeswhencomparingall17columns.
Initially,themodelmethamphetaminesampledescribedinChapter4wasrunon
all17columns(singledimensional),withtheaidofLCͲMSasadetectionsystem.
Itisimportanttonotethatamodelmethamphetaminesamplewasusedinthe
columnselectionprocesswithOpenMS®,asdatasettrainingwasdesignedtocope
with all of the potential precursors and cutting agents related to
methamphetamine. As previously discussed in chapter 4, a real
methamphetamine seizure sample is typically very clean, only consisting of
methamphetamineacoupleofotherchemicals.Therefore,thecolumnselectivity
studywascarriedoutwithasignificantlymorecomplexmodelsampletoensure
universalapplicationforlawenforcementagencies.
Themodelmethamphetaminesamplewasinjected(5μL)ataflowrateof0.5mL
minͲ1intoalineargradientover40minuteswithaninitialmobilephaseof5%
aqueousACNthatincreasedto100%ACN.Theresultingdatafileswereuploaded
to OpenMS® which was then used to identify the fcoverage of all the column
combinations in a 2DͲHPLC separation. This allowed a large variety of columns
(272combinations)tobecomparedinasignificantlyreducedtime(20minutesof
processing)incomparisontothetraditionaltrialͲandͲerrorprocesswhichforthis
number of columns would take more than 181 hours. This is significant in an
analyticallaboratoryasitresultsintime,energy,solventandcostsavings[234].
The 9 step information pipeline that was used by OpenMS® in this work is
illustrated in Figure 5.1, which outlines the data processing pipeline utilised to
identifythemostorthogonalcolumns.
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Figure5.1:OpenMS®pipelineusedforthedataanalysis.
Thefunctionofeachsequentialstepisdescribedbelow:
x Step1–Uploadindividualcolumndatafiles(.mzdataformat).
x Step2–Convertfilefrom.mzdatato.mzmlformatforOpenMS®processing.
x Step3–Extractpeakintensities(1,000դ 30,000,000)andsetm/zrange(50Ͳ
300)ofinterest[212].
x Step 4 – Filters are applied by transposing the original LCͲMS map into a
resampledmap.
x Step5–Noisefilterisappliedtosmooththebaselineandremovepeakscaused
bynoise[16].
x Step6–PickspeaksoutofthenoiseͲfilteredandbaselineͲreduceddatathat
satisfiestheappliedfilters.
x Step7–Datareducingfilterthatremovesadditionalsmalltracesofidentical
molecularweight,leavingonlythemainsignal.
x Step8–Alltheindividualfilteredfilesarecollectedandjoinedintoasingle
datafile.
x Step9–ExportasOpenMS®datafile(.toppasformat)[212].
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The results from the pipeline were then processed using Mathematica and
algorithms designed in house by Stevenson et al. [78, 202]. Table 5.2 lists the
expectedfcoverageforeachcolumncombinationbasedonthepipelinedescribed
forOpenMS®.

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Table5.2:fcoverageofdifferentcolumncombinationscolumns1Ͳ17.
Column 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 Ͳ 0.31 0.25 0.25 0.31 0.31 0.25 0.31 0.25 0.25 0.19 0.25 0.31 0.25 0.25 0.19 0.31
2 0.31 Ͳ 0.25 0.25 0.31 0.31 0.25 0.19 0.19 0.25 0.38 0.19 0.25 0.31 0.38 0.31 0.31
3 0.25 0.25 Ͳ 0.38 0.31 0.44 0.38 0.25 Ͳ 0.31 0.31 0.25 0.31 0.31 0.19 0.19 0.38
4 0.25 0.25 0.38 Ͳ 0.25 0.38 0.19 0.19 0.25 0.31 0.25 0.31 0.31 0.25 0.25 0.31 0.25
5 0.31 0.31 0.31 0.25 Ͳ 0.25 0.25 0.25 Ͳ 0.25 0.25 0.31 0.31 0.19 0.38 0.25 0.31
6 0.31 0.31 0.44 0.38 0.25 Ͳ 0.31 0.38 Ͳ 0.38 0.25 0.25 0.25 0.19 0.31 0.38 0.25
7 0.25 0.25 0.38 0.19 0.25 0.31 Ͳ Ͳ Ͳ 0.25 0.38 0.25 0.38 0.38 0.38 0.25 Ͳ
8 0.31 0.19 0.25 0.19 0.25 0.38 Ͳ Ͳ 0.19 0.25 0.25 0.25 0.13 0.25 0.25 0.31 Ͳ
9 0.25 0.19 Ͳ 0.25 Ͳ Ͳ Ͳ 0.19 Ͳ Ͳ Ͳ Ͳ 0.25 Ͳ Ͳ Ͳ Ͳ

10 0.25 0.25 0.31 0.31 0.25 0.38 0.25 0.25 Ͳ Ͳ 0.25 0.19 0.25 0.31 0.38 0.31 Ͳ
11 0.19 0.38 0.31 0.25 0.25 0.25 0.38 0.25 Ͳ 0.25 Ͳ 0.31 0.25 0.19 0.31 0.25 0.38
12 0.25 0.19 0.25 0.31 0.31 0.25 0.25 0.25 Ͳ 0.19 0.31 Ͳ 0.25 0.28 0.13 0.38 0.25
13 0.31 0.25 0.31 0.31 0.31 0.25 0.38 0.13 0.25 0.25 0.25 0.25 Ͳ 0.25 0.25 0.38 0.31
14 0.25 0.31 0.31 0.25 0.19 0.19 0.38 0.25 Ͳ 0.31 0.19 0.28 0.25 Ͳ 0.31 0.25 0.28
15 0.25 0.38 0.19 0.25 0.38 0.31 0.38 0.25 Ͳ 0.38 0.31 0.13 0.25 0.31 Ͳ 0.38 0.31
16 0.19 0.31 0.19 0.31 0.25 0.38 0.25 0.31 Ͳ 0.31 0.25 0.38 0.38 0.25 0.38 Ͳ 0.44
17 0.31 0.31 0.38 0.25 0.31 0.25 Ͳ Ͳ Ͳ Ͳ 0.38 0.25 0.31 0.28 0.31 0.44 Ͳ
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Analgorithmbasedontheaforementionedfcoverageequations1&2,wasusedto
predictthefractionalsurfacecoveragewhencombinedinahypothetical2DͲHPLC
analysis.Fromtheseventeencolumnstestedwiththemodelmethamphetamine
sample, the two sets of column combinations that displayed the greatest
predictedfcoverage(0.44)withthebestseparationwasidentifiedas:
ͲColumn3(PhenomenexKinetexPFP)&Column6(PhenomenexSynergiHydro).
ͲColumn16(Poroshell120BonusRP)&Column17(PursuitXRs3Diphenyl).
An fcoverage of 0.44 is comparable to literature values of impressive
multiͲdimensionalseparationsinarangeofscientificfields[192,234].Burnsetal.
reported their best fcoverage as 0.43 from their 2DͲHPLC blind column selection
studyonopiates[234].Likewise,Bassaneseetal.reportedanorthogonalityand
fcoverageofapproximately0.35fromtheirworkonthetwoͲdimensionalseparation
ofextractedantioxidantsfromcoffee[192].
Columns 3 (Kinetex PFP) and 6 (Synergi Hydro) have very different chemical
propertiesandretentionmechanisms,withthegreatestpotentialtoutilisethe
most separation space available when used in combination. The Kinetex PFP
column(Figure5.2)consistsofpentafluorophenylgroupsbondedtothecoreͲshell
silicathroughCͲCbonds,withTMS(trimethylsilyl)nonͲpolarendcapping.

F F
F F
F F
F F
F F
CH3
H3C H3C CH3 CH3
CH3 H3C CH3
H3C Si
Si Si
Core-shell Silica

Figure5.2:StationaryphaseofthePhenomenexKinetexPFPcolumn.
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This stationary phase is excellent at separating aromatic compounds and
halogenatedcompoundsduetotheirʋͲʋinteractions,withtheelectronegative
fluorinegroupsofferinghighselectivityforcationiccompounds[235].Allofthe
compounds present (except NͲmethylalanine and creatine) in this model
methamphetamine sample (created in chapter 4) are aromatic compounds,
correlatingwellwiththecolumnselectionidentifiedbyOpenMS®protocol.Mirali
etal.developedafastextractionandseparationmethodforphenoliccompounds
inlentilseedsusingthesamestationaryphase[236].Thephenoliccompounds
extracted from the lentil seeds such as aminosalicylic acid (an antibiotic) and
salicin(aɴͲglucoside)areallpolararomaticcompoundswhichefficientlyseparate
usingtheKinetexPFPcolumn.TheKinetexPFPcolumnhasalsobeenusedwithin
theliteratureforsimilarcompoundsthatinallcasescontainedbothanaromatic
portionandapolarregionsuchasahydroxylmoiety.Otherresearchgroupshave
usedthesamestationaryphasewithimpurityprofilingofdrugs[237]andinthe
chemicalscreeningofpolyphenolsinbeerhops[238].
TheSynergiHydroisaC18columnwithpolarendcapping(Figure5.3)consisting
ofC18alkylchainsbondedtothesilicasurface,resultinginmethyleneselectivity
and effective separation of hydrophobic compounds through ʋͲʋ interactions
[235].
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CHAPTER5
Figure5.3:Surface(stationaryphase)ofSynergiHydrocolumn(C18withpolar
endͲcapping).

ThemainseparationpropertiesareidenticaltothatofastandardC18withaslight
variation in the secondary retention mechanism (unknown polar endcapping)
whichprovidesimprovedretentionofhydrophobiccompounds.Extremelypolar
compoundsaresometimesnotretainedandgenerallydon’tseparatewellusing
conventional C18 columns [239], whereas thepolar endcapping on the surface
improvestheretentionofthosetypesofcompounds[235].Thiscolumnhasalso
been used in the literature to separate amino acids [240], prescribed
pharmaceuticals [241] and metabolites in baby food [242], all separating
compoundsofsimilarproperties.
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Thesecondsetofcolumnswhichhadapredictedfcoverageof0.44wastheAgilent
Poroshell 120 Bonus RP and the Agilent Pursuit XRs 3 Diphenyl. The Poroshell
Bonus RP (shown in Figure 5.4) consists of stericallyͲprotected diisopropylͲC14
alkylgroupscovalentlybondedthroughanembeddedamidelinkagegrouptothe
silicaandisalsotripleendcapped[243].

Figure5.4:Surface(stationaryphase)ofPoroshellBonusRP(withtriple
endcapping).

Theembeddedamidegroupisextremelypolarthathelpstodeactivateunwanted
silanolinteractions,makingthisstationaryphaseidealfortheseparationofbasic,
neutral and highly ionisable compounds [243]. The selectivity of this column is
suitedtoeverycompoundpresentinthemodelmethamphetaminesampleand
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hasbeenusedbyresearchersforthedeterminationofprohibiteddrugsinhair
[244] and antibacterial drugs [245], which also investigate polar aromatic
compounds.
Incontrasttothepreviouscolumn,theAgilentPursuitXRs3Diphenylstationary
phase(Figure5.5)consistsoftwophenylgroupswhicharecovalentlybondedto
poroussilicaparticles[246].Thiscolumnisendcappedanddesignedtoefficiently
separatearomaticcompoundsbyutilisingstrongdipoleͲdipolehydrogenbonding
andʋͲʋinteractions[246].Ithasbeenreportedbeingusedontheseparationof
opiates[234],antibodies[247],andbitteraromaticcompoundsinwholewheat
bread[248].
CH3 H3C Si
Si
Si
CH3
Porous Silica Particle

Figure5.5:Surface(stationaryphase)ofAgilentPursuitXRs3Diphenyl(with
endcapping).
After exploring the different chemical properties of the stationary phases
(columns) with the highest predicted fcoverage, based on the compounds being
analyseditwasevidentthatthecolumnschosenbyOpenMS®canbejustifiedby
theretentionmechanismsofall4columns.InterestinglytheColumn9(LunaHILIC)
did not work well for this sample set and is generally utilised for solely polar
compounds and affords the user the ability to inject samples in strong organic
solvents[249].Thisphaseisincontrasttotheretentionmechanismshighlighted
tobeusefulhereandthisshowstheimportanceofdoingacomprehensivecolumn
selectivitystudyinordertobestunderstandfullimplicationsofstationaryphases
onasample.
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In order to visualise the predicted setting on a real sample an actual
twoͲdimensionalseparation(Figure5.6)wasperformedusingtheColumn3and
Column6combination.
Figure5.6:ComprehensivetwoͲdimensionalseparationofmodelsampleusing
thelargestfcoverageasdeterminedbyOpenMS®(whitedotsrepresentpeak
maxima–UVDetection).
From the twoͲdimensional plot, it was observed that there was a need for
improvedseparationofaclusterofpeaks(showninFigure5.6withawhitearrow),
that does not increase analysis time significantly. As the Kinetex PFP column
providedthebestfirstdimensionseparationoutofallcolumns(Figure5.7),itwas
chosen(basedonvisualinspection)astheinitialstationaryphase.
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PhenomenexȱKinetexȱPFP
50000000
45000000
40000000
35000000
30000000
25000000
20000000
15000000
10000000
5000000
0
0 5 10 15 20 25 30 35 40
RetentionȱTime
Figure5.7:LCͲMSTotalIonChromatogramofmodelmethamphetamine
sampleusingKinetexPFPcolumn.
The chromatography produced by the Kinetex PFP column is ideal for the first
dimensionasthepeaksarewellspreadoutoverthegradienttimewithseveral
baselineresolvedpeaks.ComparedtotheothercolumnstrialledfromTable1,it
is an impressive separation considering the injection volume (80 μL) which
overloadsthestationaryphase.However,thisisrequiredtoensureasufficient
concentrationcanbedetectedintheseconddimensionaftertheswitchingvalve
transferandsampledilutionwithinthemobilephase.
Attentionturnedtoselectingacolumnintheseconddimensionthatcansupport
muchfasterflowratesforimprovedanalysistime.AstheSynergiHydroisaC18
column with polar endcapping (Figure 5.3), the main separation properties are
identical to that of a standard C18 with a slight variation in the secondary
retentionmechanism(polarendcapping).
Assuch,Table5.2wasconsultedagainandthethirdbestpredictedfcoverage(0.31)
withtheKinetexPFPcolumnwastheC18columns(13and14)sothedecisionwas
madetosubstituteasolidͲcoreC18columnforaMonolithicC18columnwhich
hasthesameretentionmechanismsbutcanwithstandflowratesofupto5mL
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minͲ1 with reduced backpressure. The C18 column separates on the basis of
analytesizeandmethyleneselectivityviainteractionsbetweentheanalyteanda
chain of 18 carbons [250]. The C18 surface is widely used in modern
chromatographyandthushasmanyapplications[251].
Therefore, a comprehensive twoͲdimensional separation was carried out using

thePhenomonexKinetexPFP100Å(4.6×100mm,2.6μmparticlediameter)in
thefirstdimensionandthePhenomonexOnyxMonolithicC18(4.6×100mm)in
theseconddimension(Figure5.8).Infactwiththeuseofthemonolithcolumn,
theseconddimensionmodulationtimewasabletobereducedfrom3.0minutes
to 1.5 minutes over a 1.0 minute gradient and gave a significantly improved
separation.WhencomparedtotheprevioustwoͲdimensionalseparation(Figure
5.6),thecompoundsareconsiderablymoredistributedandmakebetteruseof
thepotentialseparationspace.Itisalsoimportanttonotethatcomprehensive
2DͲHPLCrequirestheseconddimensiontobesignificantlyshorterthanthefirst
dimension, allowing the compounds to elute in the second dimension before
another peak is transferred [70], which is satisfied using the monolithic C18
column.
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Figure5.8:TwoͲdimensionalseparationusingtheKinetexPFPandOnyxC18
Monolithiccolumns.
5.3.2Chemicalmappingofimpuritiesforapathwaytomethamphetamine
Now with an optimised twoͲdimensional separation in hand that is sculptured
around the compounds of interest (methamphetamine and its common
associatedprecursors/cuttingagents),attentionturnedtothechemicalmapping
ofanunͲpurifiedsyntheticpathwaytomethamphetamine,carriedoutpreviously
inChapter2,demonstratedinFigure5.9.
2-Nitro-1-phenylpropanol Norephedrine Ephedrine Methamphetamine

OH OH OH
H H
Benzaldehyde NO2 NH2 N N
+
Nitroethane
8 10 1 2
Figure5.9:Syntheticpathwayfrombenzaldehydeandnitroethaneto
methamphetamine.
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AsamplewastakenfromeachstepofthesyntheticpathwayfortwoͲdimensional
separation and mass spectrometry analysis. The same columns and conditions
fromthatofFigure5.8,wereusedtocarryoutatwoͲdimensionalseparationof
eachsyntheticstep.
Mass spectrometry is a significantly more sensitive detection system than UV
[201],sothesamecolumnsandscaledconditions(relativeretention)wererun
individuallyusingLCͲMSwithsubsequentcoordinatesplotted(whitedots)over
the2DͲHPLCseparationdenotingthepeaksidentifiedbymassspectrometrythat
werenotobservedintheUV.AsLCͲMSisamorewidelyusedtechniqueforthe
analysis of drugs, it was used as a comparison for the 2DͲHPLC profiling of
syntheticroutestomethamphetamine,toensurethesensitivitywassufficientto
beusedbylawenforcementagencies.Theoriginalfirstdimension(110minuteat
0.1mLminͲ1)andseconddimension(1minuteat5mLminͲ1)hadtheirruntimes
andgradientsscaledtotheoptimalflowrateof0.5mLminͲ1forLCͲMSanalysis,
resultingina22minuteanda10minuteruntime,respectively.Assuch,anymass
detected with a retention time greater than 22 minutes in the LCͲMS run was
excludedfromthe2Dplot.Thedatageneratedfromthemassspectrometrywas
significantandinordertofocusonthekeyconstituentsapeakintensitylimitof
1 × 107 counts was determined to allow for the identification of the main
components.Oncetheplatformtechnologydevelopedhereistakenupbyother
researchgroupsitwouldbeworthloweringthislimitinordertomonitorminor
constituentsandthismaybeofgreateruseoncethistechnologyisusedonlarge
setsofrealworldseizuresamples.
Thereweremassesdetectedoutsideofthescaledgradientrange(>22minutes)
which highlights the need for longer analysis times for a full comprehensive
chemical fingerprint. Again, highlighting that future method developments for
drugdetectionshouldbecarriedoutusingLCͲMSinsteadofUVasitprovidesthe
analystwithconsiderablymoreinformation.
ThisissupportedbyBarbarinetal.[201]whohaspublishedacomparisonstudy
on drug detection using LCͲUV and LCͲMS, evaluating the ability of both
techniques to detect unknown impurities. Their findings showed that although
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chemicalnoisewashigherwhenusingfullscanLCͲMS,lowlevelimpuritieswere
muchbetterdetected.Therefore,LCͲMSwascarriedoutinconjunctionwith2D
LCͲUVasadirectcomparisonforeachsyntheticstepinordertodevelopthebest
methodforthechemicalmappingofmethamphetamineimpurities.
ThefirstsequentialsampleanalysedwasbenzaldehydeandasexpectedthetwoͲ
dimensionalseparationshowninFigure5.10isquitecleanshowinganextremely
largepeakatcoordinates(firstdimensionx=95,seconddimensiony=0.95)and
asmallpeakatcoordinates(firstdimensionx=90,seconddimensiony=0.85).
Thehorizontalsignal(seconddimensiony=0.35)iscausedbythesolventfront
acrosseachdimensionandisnotsignificanttothechemicalprofiling.However,6
otherimpuritiesweredetectedusingLCͲMS,3ofwhichwereincludedinthe2DͲ
HPLCchromatogram(bottomleftofFigure5.10).Benzaldehydeelutedoutsideof
the relative scaled retention and as such was not included on the 2DͲHPLC
chromatogram. Comparatively, 7 different masses were detected using mass
spectrometry while only 2 were detected using UV detection, proving the
importanceofMSwhenitcomestochemicalprofiling.
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Figure5.10:TwoͲdimensionalHPLCseparationandtwoͲdimensionalLCͲMS
separation(whitedots)ofbenzaldehyde(m/z107.04912[M+H]+).
Rt1.78=158.96411,Rt2.47=85.05938,Rt2.98=61.02906,Rt23.09=107.04912,Rt23.74=199.16943,Rt
24.21=279.15909,Rt24.39=315.25305.
Figure5.11:ScaledfirstdimensionLCͲMSchromatogramandretentiontime
massesofbenzaldehydeusingKinetexPFPcolumnat0.5mLminͲ1.
Nitroethane was then run using both systems as well, showing that LCͲMS
detected4massesincludingnitroethane(Figure5.13),3ofwhichareillustrated
in Figure 5.12, where only one compound was detected with the UVͲvisible
detector.
Nitroethane
separation(whitedots)ofnitroethane(m/z76.03982[M+H]+).
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Rt1.70=182.98523,Rt2.45=76.03982,Rt3.13=84.08125,Rt23.73=217.17992
massesofnitroethaneusingKinetexPFPcolumnat0.5mLminͲ1.

The nitroaldol synthesis of 2ͲnitroͲ1Ͳphenylpropanol 8 is the first step that
involvesthereactionof2relativelypurecompounds,increasingtheprobabilityof
byͲproductformation.AsshowninFigure5.14,the2DͲHPLCsystemdetected4
differentcompoundsdisplayedasblueorredmarkingswhereasLCͲMSdetected
8differentcompounds(6includedfromFigure5.15).
2ͲNitroͲ1Ͳphenylpropanol8
separation(whitedots)of2ͲnitroͲ1Ͳphenylpropanol8
(m/z182.08116[M+H]+).
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Rt1.71=158.96413,Rt2.45=85.05936,Rt5.31=182.08116,Rt15.08=174.09138,Rt17.16=192.10187,
Rt19.61=248.16016,Rt23.69=174.09132,Rt24.23=164.07059
massesof2ͲnitroͲ1Ͳphenylpropanol8usingKinetexPFPcolumnat
0.5mLminͲ1.

Within the LCͲMS separation (Figure 5.15), m/z 164.07059 was found at a
retentiontimeof24.23minuteswhichisproposedtobephenylͲ2Ͳnitropropene
(Figure5.16);anintermediateproductcharacteristictothisreactionasdiscussed
inchapter2.Thisissignificantbecauseitshowsthatthereactionhasnotgoneto
completionanditnowcanbeusedasacharacteristicimpurityforthisreaction
pathway.

Figure5.16:IntermediatephenylͲ2Ͳnitropropenewithm/z164.07059.
Thenextstepanalysedwasthereductionof2ͲNitroͲ1Ͳphenylpropanol8toform
norephedrine10.Thesampleismorecomplexwithnotablymoreimpurities,with
12differentmassesdetectedusingLCͲMS,showninFigure5.18.Incontrast,UVͲ
visible detector found 4 intense peaks and 3 weak peaks; making a total of 7
compoundslocated(Figure5.17).
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Norephedrine 10
separation(whitedots)ofnorephedrine10(m/z152.10696[M+H]+).

Rt1.73=158.96409,Rt2.18=60.04504,Rt2.45=85.05933,Rt5.45=152.10696,Rt5.98=134.09642,Rt
9.95=180.13828,Rt10.23=176.10699,Rt13.55=178.12268,Rt14.35=173.10735,Rt21.82=256.13321,
Rt24.27=238.12268,Rt25.44=307.18042.
massesofnorephedrine10usingKinetexPFPcolumnat0.5mLminͲ1.

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AfteranalysisofthemassesdetectedbyLCͲMSinFigure5.18,itwasproposed
thatthem/z134.09642istheproductofthepreviousintermediate(Figure5.16)
undergoingareductionofthenitrofunctionalgrouptoaprimaryamine(Figure
5.19–StructureA).

Figure5.19:ProposedStructureA.
It is important to note that m/z 158.96409 and m/z 85.05933 impurities were
carriedthroughtothenorephedrine10samplefrom2ͲnitroͲ1Ͳphenylpropanol8.
Whenanalysingthenextstepinthepathway;themethylationofnorephedrine
10topseudoephedrine/ephedrine1,onecanseeyetanotherriseinthenumber
ofpeaksdetected.Thissuggeststhatthedeeperintothesyntheticpathwaywe
get,themoreimpuritiesandbyͲproductsareformed,makingthesamplequite
complex as shown in Figure 5.20. This is significant as this method allows the
analysttodetectwhatsyntheticpathwaywasused.
TheUVͲvisibledetectorfound9compounds,whereasLCͲMSdetected19masses
listed in Figure 5.21, in which 18 masses were overlaid on the 2DͲplot (Figure
5.20).
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Ephedrine/pseudoephedrine1
separation(whitedots)ofephedrine/pseudoephedrine1
(m/z166.12267[M+H]+).

Rt1.72=158.96130,Rt2.19=60.04502,Rt2.47=85.05933,Rt5.25=178.12270,Rt5.93=152.10701,Rt
6.21=134.09645,Rt7.61=166.12267,Rt8.48=180.13824,Rt9.96=180.13826,Rt10.35=196.13326,Rt
11.03=194.15399,Rt12.05=178.12265,Rt14.30=192.13828,Rt17.74=136.11210,Rt20.61=
206.11758,Rt21.01=284.16443,Rt21.41=256.13309,Rt21.87=256.13312,Rt24.26=238.12254.
massesofephedrine/pseudoephedrine1usingKinetexPFPcolumnat
0.5mLminͲ1.

Asthemethylationofnorephedrineisnotcontrolledbyprotectinggroups,itis
expectedthatmethylationwilloccuratdifferentsitesofthemoleculeresultingin
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CHAPTER5
StructuresB,CandDfromFigure5.22.However,StructureE(amphetamine)and
Structure F (NͲacetylmethamphetamine) appear to be common impurities
identified by other research groups in the ephedrine pathway to
methamphetamine[26,116,252Ͳ254].
Figure5.22:ProposedStructuresB–F.
It is important to note that m/z 158.96409, m/z 60.04504, m/z 85.05933, m/z
152.10696,m/z134.09642,m/z180.13828,m/z178.12268,m/z256.13321,m/z
238.12268 were all carried through to ephedrine/pseudoephedrine 1 from
norephedrine10.
The final step in a full synthetic pathway to methamphetamine looks at the
reduction of pseudoephedrine/ephedrine 1 to methamphetamine 2. Following
thesametrend,thenumberofmassesdetectedusingLCͲMShasincreasedto21
(Figure5.24),inwhich15areillustrated(whitedots)inFigure5.23alongwiththe
7compoundsbeingfoundbytheUVͲvisibledetector.
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Methamphetamine2
separation(whitedots)ofmethamphetamine2(m/z150.12771[M+H]+).

Rt2.18=60.04504,Rt5.27=178.12273,Rt5.93=194.11763,Rt6.48=134.09654,Rt7.96=166.12274,Rt
8.15=166.12271,Rt8.15=148.11215,Rt8.94=180.13840,Rt10.44=196.13332,Rt11.27=194.15410,Rt
13.45=164.14346,Rt14.61=150.12771,Rt17.75=136.11207,Rt20.61=206.11740,Rt21.01=
284.16434,Rt21.40=256.13300,Rt21.89=256.13300,Rt23.60=240.13814,Rt25.50=307.18024,Rt
26.12=282.18520,Rt26.75=284.20090.
massesofmethamphetamine2usingKinetexPFPcolumnat0.5mLminͲ1.
The literature was consulted for known impurities and byͲproducts found in
methamphetaminesamplesandthencomparedtothemassesdetectedinFigure
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5.24.StructuresG–KfromFigure5.25arewelldocumentedimpuritiesfoundin
methamphetamineseizuresamples[26,116,252Ͳ254],thatwerealsofoundin
themethamphetamine2sample(StructureG=dimethylamphetamine,Structure
H = pseudoephedrine, Structure I = n,nͲdiͲ(bͲphenylisopropyl)formamide,
StructureJ=amphetamine,StructureK=1,2ͲdimethylͲ3Ͳphenylaziridine).
Figure5.25:ProposedStructuresG–K.
There were 11 masses that were carried through to the product
methamphetamine 2 from ephedrine/pseudoephedrine 1 (m/z 60.04502, m/z
178.12270,m/z134.09645,m/z166.12267,m/z180.13824,m/z196.13326,m/z
194.15399,m/z178.12265,m/z136.11210,m/z206.11758,m/z256.13309).
A summary of LCͲMS detected masses (rounded to whole integers) in each
pathwaysteptomethamphetamineisdisplayedinTable5.3.
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Table5.3:Themappingofimpuritymassesforentirepathwayto
methamphetamine.
OH

NO2
Ph
NO2
Benzaldehyde Nitroethane 8 10 1 2

Ͳ Ͳ Ͳ m/z60 m/z60 m/z60
m/z61 Ͳ Ͳ Ͳ Ͳ Ͳ
Ͳ m/z76 Ͳ Ͳ Ͳ Ͳ
m/z85 Ͳ m/z85 m/z85 m/z85 Ͳ
Ͳ Ͳ Ͳ m/z134 m/z134 m/z134
Ͳ Ͳ Ͳ Ͳ m/z136 m/z136
Ͳ Ͳ Ͳ Ͳ Ͳ m/z148
Ͳ Ͳ Ͳ m/z152 m/z152 Ͳ
m/z158 Ͳ m/z158 m/z158 m/z158 Ͳ
Ͳ Ͳ m/z164 Ͳ Ͳ m/z164
Ͳ Ͳ Ͳ Ͳ m/z166 m/z166
Ͳ Ͳ Ͳ m/z173 Ͳ Ͳ

Ͳ Ͳ m/z174 Ͳ Ͳ Ͳ
Ͳ Ͳ Ͳ m/z176 Ͳ Ͳ
Ͳ Ͳ Ͳ m/z178 m/z178 m/z178
Ͳ Ͳ Ͳ m/z180 m/z180 m/z180
Ͳ m/z182 m/z182 Ͳ Ͳ Ͳ
Ͳ Ͳ m/z192 Ͳ m/z192 Ͳ
Ͳ Ͳ Ͳ Ͳ m/z194 m/z194
Ͳ Ͳ Ͳ Ͳ m/z196 m/z196

Ͳ Ͳ Ͳ Ͳ m/z206 m/z206
Ͳ Ͳ Ͳ m/z238 m/z238 Ͳ
Ͳ Ͳ m/z248 Ͳ Ͳ Ͳ
Ͳ Ͳ Ͳ m/z256 m/z256 m/z256
Ͳ Ͳ Ͳ Ͳ m/z284 m/z284
Ͳ Ͳ Ͳ m/z307 Ͳ m/z307
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There are 13 masses (m/z 60, m/z 134, m/z 136, m/z 164, m/z 166, m/z 178,
m/z180,m/z194,m/z196,m/z206,m/z256,m/z284,m/z307)detectedinthe
finalmethamphetamineproductthatcanalsobedetectedinprevioussteps. As
suchifanalystscanidentifyordetectanyofthesemasses,itmaybetheirpresence
isindicativethatthispathwaywasusedtomanufacturemethamphetamineand
its associated precursors can be identified. Such information may assist law
enforcement in their investigations allowing them to identify intermediate
sampleswithinaclandestinelaboratoryinordertoexpediteaprosecution.Itis
importanttonotethepresenceofephedrine/pseudoephedrine1(m/z166)was
detectedinthefinalmethamphetaminesampleandconfirmsthatthepathway
usedinthiscaseinvolvedtheuseofephedrine/pseudoephedrineasamonitored
precursor.Fromanapplicationperspectivetablessuchasthesewillbeofgreat
value to investigators and they could be generated to inform other synthetic
pathways.
5.3.3PreliminaryPCAStudy
The presence of either pseudoephedrine or P2P in a final methamphetamine
seizuresampleisanextremelyusefultooltoidentifyasyntheticroute,asalmost
allmanufactureprocessesuseoneortheother.Duetothisdistinctdifferencein
syntheticfoundation,analternatemethodofsyntheticrouteidentificationwas
attempted, that would potentially be capable of analysing hundreds of
methamphetamineseizuresamplessimultaneously.
Assuch,PCAanalysiswascarriedoutonall22seizuresamples(VPFSD1–22)
provided by Victoria Police Forensic Services Centre, as a preliminary study. A
Thermo Fisher Scientific program by the name of ‘SIEVE™’ [255] was used to
processtheLCͲMSdataproducedfromtheThermoFisherHighResolutionMass
Spectrometer (QͲExactive Plus), for simplicity of being in the same file format.
PseudoephedrinetargetedPCAwasproducedbySIEVE™inapilotstudy,setting
them/zrangeto166.0Ͳ166.2toseparatesamplespurelybasedonthepresence
ofephedrine/pseudoephedrine.
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CHAPTER5
The operation of SIEVE™ is extremely simple, user friendly and incorporates a
wizard that guides the user through a 7 step analysis process, shown in Figure
5.27.
x Step1–Opentheprogramandcreate‘NewExperiment’.

x Step2–Selecttypeofexperiment(smallmolecule)anddetectionalgorithm
(componentextraction).Thennameyourexperimentforfuturereference.

x Step3–CopyandpasteThermoFisherLCͲMSfilesforanalysis.Thenrunascan
oftherawfilestoensurecompatibility.

x Step4–Rawfilecharacterisationiscarriedout.Thatis,replicatesarenamed
andgroupedtogetherwhiletheblankrunissetasthereference.Hereiswhere
theusercanselectwhatcolourwillbelinkedtowhatsample.

x Step5–Desiredparameterslikeretentiontime(0–15minutes),m/zrange
(166.0–166.2)andm/ztolerance(10ppm)areset.

x Step 6 – This is where the component extraction algorithm parameters are
adjusted.Minimumintensitythreshold(2.0×107counts),peakscans(5),signal
tonoiseratio(3:1)andm/zstepsize(10)canallbemanipulatedforatargeted
analysis.

x Step7–Thefinalstepinvolvesclicking‘Finish’andwaitingfortheprogramto
carry out the statistical analysis and produce the results including a
normalisationofthedataandpeakalignment.
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STEP1 STEP2
STEP3 STEP4
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STEP5 STEP6
STEP7 RESULTS
138|P a g e
Figure5.27:SIEVE™7stepwizardprocess.
CHAPTER5
Analysisofallseizuresamples(1Ͳ22)wascarriedoutandprecursoramountswere
recordedinTable 5.3. Extracted ion chromatograms (EIC) for pseudoephedrine
(m/z 166.12) and P2P (m/z 135.08) were carried out on all seizure samples
individuallyandtheresultswerealsorecordedinTable5.3.Areportinglimitof
2.0×107peakintensitycountwassetinSIEVE™basedonthevisualdetectionof
thepseudoephedrinepeak.Thatis,anintensityunderthatthresholdcannotbe
seen on the LCͲMS chromatogram and was excluded and classed as trace
amounts.

Table5.3:ContentsofSeizureSamples(VPFSDͲ1–22).
Pseudoephedrine P2P
SeizureSample Methamphetamine (PeakIntensity (PeakIntensity
Counts) Counts)
VPFSDͲ1 9 3.10×104Ͳ Trace 8
VPFSDͲ5 9 3.51×108 8
VPFSDͲ6 9 5.73×108 8
VPFSDͲ11 9 6.44×108 8
VPFSDͲ13 9 8 2.51×104ͲTrace
VPFSDͲ18 9 2.55×108 8

Therewereonly4seizuresamples(VPFSD–5,6,11&18)thatsatisfiedthelimit
and are bolded in the table. For example, VPFSDͲ18 contained the smallest
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CHAPTER5
intensity count (2.55 × 108) out of the bolded seizure samples but a clear
pseudoephedrinepeak(retentiontime:1.34minutes)canbeeasilyidentifiedin
theTotalIonChromatogram(TIC)andintheExtractedIonChromatogram(EIC),
showninFigure5.28.
TotalIonChromatogram(TIC)

Pseudoephedrine
ExtractedIonChromatogram(EIC)
ofpseudoephedrine
Figure5.28:TICandEIC(pseudoephedrineͲ2.55×108)ofVPFSDͲ18seizure
sample.
By contrast, VPFSDͲ3 has the highest intensity count for pseudoephedrine
(8.32 × 106) that is below the reporting limit (2.0 × 107 counts) and as such,
pseudoephedrinecannotbeseenintheTICbutisdefinitelypresentintheEIC,as
showninFigure5.29.
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CHAPTER5
TotalIonChromatogram(TIC)

Pseudoephedrine
ExtractedIonChromatogram(EIC)
ofpseudoephedrine
Figure5.29:TICandEIC(pseudoephedrine–8.32×106)ofVPFSDͲ3seizure
sample.
This work aimed to develop the platform technology to be used as a fast,
precursorscreeningmethodforlargesamplequantities.Ifthispreliminarystudy
canseparatereportableamountsofprecursorfromtraceorabsentamounts,then
itcanbeusedasafuturetemplatetorapidlyscreenprecursors,afteradatabase
ofpathwaystandardshasbeencreated.Byalteringtheintensitythresholdand
searchingforalibraryofcharacteristicpathwayimpuritiesarobustsystemcanbe
developed to identify a manufacture route by analysing a methamphetamine
seizuresample.
Principal component analysis was carried out in triplicate using SIEVE™ (Figure
5.30) and a clear distinction of reportable amounts of pseudoephedrine was
observed;provingthisasapromisingtechniqueforfuturedevelopment.AthreeͲ
dimensional PCA model was created to help visually identify the extent of
separation. After consulting Table 5.30, it appears that the greater the peak
intensityofpseudoephedrine,thefurtherawaysamplesareplacedfromthemain
cluster.ThisissignificantbecausetheuseofephedrinetargetedPCAisclustering
samples together based on concentration and can be manipulated to target
massesofinterest.
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CHAPTER5
Figure5.30:3DPCAplotof22methamphetamineseizuresamples.
CurrentlythistypeofmultidisciplinaryapproachusingPCAandanalyticaldatais
not routinely used by Victoria Police and clearly promise is shown here. The
approachdescribedherewouldbedirectlyapplicabletocaseworkiftheVictoria
PoliceincreasedtheirtechnologicalcapacitytoincludehighresolutionLCͲMSor
tandemMSforroutineoperations.TheIcetaskforceidentifiedtheimprovements
in infrastructure required to target methamphetamine policing and this work
helps support the case. This is a targeted form of PCA and for this to be fully
realisedwithlargerealworldsamplesetsafullvalidationprocessisrequired.This
work was validated through a twoͲstep process, firstly a blind analysis of the
sampleswasperformed.Duetothefactthatthedatasetwassmallandtheunique
natureoftheseizuresamplesmeantthatgainingalargersetinashorttimeframe
was challenging and as such, secondly validation was confirmed via direct
chemical interrogation from Victoria Police sources. This was a pilot study
designedtohighlightthepotentialcapacityofPCAasatoolfordeterminationof
syntheticmanufactureroutes.Itisimportanttonotethatforfulldevelopmentfor
casework,largedatasetsandfullvalidationisrequired.
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AsdiscussedinChapter1,theAustralianIceTaskforcepublishedareportofallthe
expertfindingsin2015.Theexecutivesummaryhighlights[17]:
Ͳ IceuseinAustraliaisatextremelyhighlevelsandisincreasing.
Ͳ Themarketremainsstrongdespiteeffortsbylawenforcementagencies
todisruptthesupply.
Ͳ Ithascreatedadistinctproblemforsocietyduetoitspowerfulstimulant
andaddictionproperties.
Ͳ Methamphetamine is the only drug that is both imported and locally
manufactured in significant quantities, making the response by law
enforcementagenciesincrediblycomplex.
Ͳ Effortstosupplymustbemorecoordinatedandtargeted.
Ͳ Better data, more research and regular reporting will strengthen
Australia’sresponse.
Assuchitisevidentthatbycreatingtheplatformtechnology,itwillallowfuture
scientists, statisticians and law enforcement agencies to work collectively in
developing a more advanced multidimensional system to track the origins of
methamphetaminemanufacture.Thisissignificantasitforcesdifferentfieldsof
expertisetotacklethisiceepidemictogetherasanation.
5.4Conclusion
Current methods to identify the most orthogonal columns are time consuming
and labour intensive. The use of LCͲMS to monitor molecular masses over
differentstationaryphaseshassignificantlyimprovedthisselectionprotocol.This
allowscolumnstobeblindlyselectedforunknownsamplecomponentswithout
comparing each column pair individually. Injecting multiple standards for each
columnisstillextremelytimeconsuming,whereasallstandardscanbecombined,
requiringonlyoneinjectionforeachcolumn.Thismethodimprovesanalysistime
significantlyandsparessampleamountandsolventrequiredthusreducingthe
cost.
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CHAPTER5
Thiscolumnselectionprotocolwasusedtoacquirethemostorthogonalcolumns
formethamphetaminetypecompounds.Theywerethenappliedtoanalyseeach
stepofafullsyntheticpathwaytomethamphetamine,takingfulladvantageof
twoͲdimensionalseparationspace.AcomparisonstudybetweenLCͲUVandLCͲ
MSwascarriedoutto determinethebestmethodofmappingimpuritiesfrom
methamphetaminemanufacture.
Finally, synthetic chemistry, inͲsilico optimised 2DͲHPLC, comprehensive

selectivity studies, high resolution LCͲMS and comprehensive data analysis for
chemical mapping was developed. This significantly improving the ability to
rapidly screen precursors and associated synthetic pathways from
methamphetamineseizuresamples.
The work presented in this chapter has been published as: N.K Burns †, L.M
AndrigheƩo†,S.DPurcell,N.WBarnett,J.Denning,P.SFrancis,X.AConlanand
P.G. Stevenson, Blind column selection protocol for twoͲdimensional high
performance chromatography, Talanta, 154, 85Ͳ91 (2016) DOI:
10.1016/j.talanta.2016.03.056.(CoͲfirstauthor).
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CHAPTER6
CHAPTER 6:
CONCLUDING REMARKS AND FUTURE WORK

145|P a g e

CHAPTER6
6.1ConcludingRemarks
The work presented here fits into a broader research program within the
analyticalchemistrygroupatDeakinUniversityandthishasformedalargepart
of the progress in the area of multidimensional chromatography and chemical
mapping.Thisisthefirsttoapplyanapproachthatutilisessyntheticchemistryto
inform the chromatographic separation development processes. Significant
advancementhasbeenachievedinmethamphetamineseizuresampleprofiling
andthetechnologyplatformdevelopedisofgreatinteresttotheVictoriaPolice
Forensic Services. This work has a very high chance of having a real and direct
impactondrugprofilinginVictoriaandwillcomplementthetargetedapproach
currentlyinusetobetterinformcourtproceedingsandprojectprioritisation.This
work has also informed a parallel research program here run by one of my
publication[234]coͲauthorsNikiBurnswhoisdevelopingprotocolsforprocess
monitoringofcommercialopiatestreams.Thefundamentalapproachtakenhere
has been critical to the application of opiate monitoring which has led to the
recentdevelopmentofPCAanalysisof2DͲHPLCdata.Throughoutthisprojectcare
wastakentodevelopaplatformtechnologythatcouldbetransposedtoother
industries and it is greatly satisfying to see my approach used for process
chemistryapplications.RecentlytheleadsupervisorfromthisprojectDrXavier
Conlan has directly used my protocol to develop a research grant (Australian
Rotary Health) around mapping methamphetamine seizure samples towards
informingclinicalmentalhealthresearch.Aspartofthisproposalanagreement
was made between Deakin University and the Victoria Police Forensic Services
DepartmentenablingaccessforDeakinresearcherstomethamphetamineseizure
samples.Thishighlightshowmyworkhasalreadyleadsignificantlytoenhancing
theresearchpotentialwithinDeakinUniversity.
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CHAPTER6
6.2FutureWork
Chemistryisconstantlyevolvingandthereisalimitlessamountoffutureresearch
tobeundertakeninthechemicalmappingofillicitdrugs.Theworkpresentedin
thisthesiscanbeexpandedandafewsuggestionsareprovidedbelow.
Chapter 2 and Chapter 3 investigated viable synthetic routes to

methamphetamine from easily accessible materials; benzaldehyde, nitroethane
and LͲalanine. The reaction between benzaldehyde and nitroethane produced
differentproductssimplybyalteringthetypeofbaseused.Coincidentally,both
resulting products can be used in different pathways (P2P and Ephedrine) to
methamphetaminemanufacture.Therewere5differentbasesusedinChapter2
and a range of different bases should be trialled for a superior comprehensive
analysis.Likewisealternativeprotectinggroupsandsyntheticpathwayscouldalso
beinvestigatedtodevelopmoreefficientmethods.
Chapter 4 investigated the inͲsilico optimisation of oneͲdimensional and twoͲ

dimensionalseparationsusingasimulationsoftwarecalledDryLab®.Futurework
mayincludetriallingmorecolumnsandnewcolumnstothemarkettotestand
improvetherobustnessofthisprogram.Arangeofalternativecomplexsample
typesandsolventsshouldbeinvestigatedtoimproveonthealgorithmscreated
for accurate simulations, incorporating and multivariable analysis. Likewise, a
laboratory study should be undertaken to determine exactly how much time,
money,waste,solvent,wages,maintenancepartsandservicecontractsarebeing
saved through the use of inͲsilico optimisation, to promote reduced
environmentalwaste.
Chapter 5 investigated the chemical mapping of methamphetamine seizure

samples using, optimised 2DͲHPLC, LCͲMS and targeted principal component
analysis.Futureworktofurtherdevelopthisplatformtechnologywouldinclude
synthesisingandmappingallthedifferentknownpathwaystomethamphetamine
(>15pathways).Essentiallycreatinganenormousdatabasewhichincorporates
everystepofeverysyntheticpathway,sothatanautomatedsystemcanquickly
analyse,categoriseandinformlawenforcementagenciesastowhatrouteand
147|P a g e

CHAPTER6
precursorswereusedineachmethamphetamineseizuresample.Thepreliminary
study was carried out using high resolution mass spectrometry and typical
parameters,sothegenerationofalowresolutiondatabasewouldbeofparticular
interestforuniversalanalysisaslowresolutiondataiscommonlyusedinforensic
laboratories.AstargetedPCAwasused,nonͲtargetedPCAshouldbedeveloped
to process seizure samples to fully exploit the potential of this process. The
chemicalmappingprocesscarriedoutinChapter5shouldberepeatedwiththeLͲ
alaninepathwaytomethamphetamine,asdiscussedinChapter3. Importantly,
thedatabasewillneedtobeconstantlyupdatedlongtermasnewandalternative
syntheticpathwaysarecreatedtoovercometheuseofcontrolledsubstances.

148|P a g e

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ABBREVIATIONS AND ACRONYMS

Australian clandestine drug laboratories are constantly seeking and utilising

Restrictions on pharmaceutical drugs containing pseudoephedrine were

separation of model compounds was completed with significantly reduced

methamphetamine pathway, using 2DͲHPLC and LCͲMS. The impurities and

This thesis presents a platform technology protocol for the comprehensive

methamphetamine epidemic in Japan. Methamphetamine abuse grew and

The methods of methamphetamine manufacture carried out in clandestine

One set of diastereomers form DͲmethamphetamine, a psychoactivedrug; The

Glucose O PDC Catalyst

DetectionMethod Mediumtype LiteratureReference

Likewise, a full pathway to methamphetamine was explored from the

O NaOH O HCl HCl

Combined Syn & Anti

Nitroethane Triethylamine Yield

7.5 7.0 6.5 6.0 5.5 5.0 4.5

The general mechanism for the formation of the 2ͲnitroͲ1ͲphenylͲ1Ͳpropanol

Benzaldehyde Nitroethane NͲbutylamine Yield

Benzaldehyde Nitroethane Ammonium Yield

The formation of phenylͲ2Ͳnitropropene 9 can occur via two alternate

As monoͲmethylation proved problematic, an alternative approach was

A commonly encountered reduction method (Scheme 2.15) [135] for the

All 1Hand 13CNMRspectrawererecordedonaJEOLJNMͲEX270MHz.Samples

A cooled (0 °C) solution of methyl tosylͲLͲalaninate

To a stirring mixture of 2Ͳ(NͲ(tertͲ

Having successfully methylated methyl NͲ(tolueneͲ4Ͳsulfonyl) LͲalaninate 17,

Entry HBrConcentration Solvent

Methyl NͲmethylͲNͲ(tolueneͲ4Ͳsulfonyl)ͲLͲalaninate 18 (1.0 equivalent) was

Proton NMR - Compound 3

Proton NMR - Compound 3

Proton NMR - Compound 3

Entry Reagents Time

Benzaldehyde AceticAcid LCͲMStrace

Although ephedrine/pseudoephedrine (1a and 1b) were detected in all

IN-SILICO OPTIMISATION OF 1D-HPLC AND

The current limitations of methamphetamine impurity profiling have been

The advent of twoͲdimensional high performance liquid chromatography

multiͲdimensional gas chromatography has been used to monitor volatile

There are a range of experimental factors that influence a chromatographic

௡೎ǡభ ൈ௡೎ǡమ ൈ௙೎೚ೡ೐ೝೌ೒೐

σ ܾ݅݊‫ ݏ‬െ ξܲ௠௔௫

All standards (methamphetamine, ephedrine, pseudoephedrine, paracetamol,

MilliͲQ water was obtained inͲhouse (Continental Water Systems, Victoria,

Paracetamol, caffeine, benzaldehyde, aspirin, creatine, procaine, NͲ

Brand Type Size Dimensions PartNumber

Column1 Phenomenex KinetexPFP100A 2.6μm 4.6x100mm 00DͲ4477ͲE0

Column2 Phenomenex KinetexPhenylͲHexyl100A 2.6μm 4.6x100mm 00DͲ4495ͲE0

Column3 Agilent Poroshell120ECͲC18 2.7μm 4.6x100mm 695975Ͳ902

Column4 Agilent Poroshell120BonusRP 2.7μm 4.6x100mm 695968Ͳ901

Column5 Phenomenex KinetexBiphenyl 2.6μm 2.1x100mm 00DͲ4622ͲAN

A model seizure sample of methamphetamine with commonly associated side

A model seizure sample was prepared containing known concentrations of

SAMPLE m/z m/z m/z m/z m/z

Rt Calibration LOD 1DͲHPLC LOD2DͲHPLC

COLUMN SELECTIVITY STUDY AND

௡೎ǡభ ൈ௡೎ǡమ ൈ௙೎೚ೡ೐ೝೌ೒೐

Deionised water was obtained inͲhouse (Continental Water Systems, Victoria,

Brand Type Size Dimensions PartNumber

Column1 Agilent Poroshell120ECͲC8 2.7μm 4.6x100mm 695975Ͳ906

Column2 Agilent Poroshell120ECͲCN 2.7μm 4.6x100mm 695975Ͳ905

Column3 Phenomenex KinetexPFP100A 2.6μm 4.6 x100mm 00DͲ4477ͲE0

Column4 Phenomenex KinetexPhenylͲHexyl100A 2.6μm 4.6x100mm 00DͲ4495ͲE0

Column5 Phenomenex SynergiFusionͲ RP80A 4μm 4.6x150mm 00FͲ4424ͲE0