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ANALYTICALMETHODSFOR THERAPEUTICDRUG MONITORINGAND TOXICOLOGY

Copyright C 2011byQ.AlanXuandTimothyL.Madden.Allrightsreserved.

PublishedbyJohnWiley&Sons,Inc.,Hoboken,NewJersey.

PublishedsimultaneouslyinCanada.

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Includesbibliographicalreferencesandindex. ISBN978-0-470-45561-6(cloth)

1.Drugs–Analysis.2.Drugs–Toxicitytesting.I.Madden,TimothyL.II.Title. [DNLM:1.DrugMonitoring–methods.2.Toxicology–methods.WB330X85a2010] RS189.X82010

615 .19–dc222010019511

PrintedintheUnitedStatesofAmerica. 10987654321

ToHongqin,Michael,andAndyfortheirloveandsupport.

—QAX

ToMary,Andy,Colleen,andAmy,whohelpmekeepmysanity.

—TLM

Prefacexiii

MONOGRAPHS

Abacavir3

Abecarnil6

AcamprosateCalcium6

AcebutololHydrochloride8

Aceclofenac10

Acemetacin11

Acenocoumarol13

Acetaminophen13

Acetazolamide15

Aconitine16

Acrivastine16

Acyclovir17

Albendazole18

Albuterol19

AlcuroniumChloride20

AlfentanilHydrochloride20

AlfuzosinHydrochloride21

Allobarbital22

Allopurinol22

Alprazolam23

Alprenolol24

AmbroxolHydrochloride25

Amikacin25

AmilorideHydrochloride27

AmiodaroneHydrochloride27

Amisulpride29

AmitriptylineHydrochloride30

AmlodipineBesylate34

Amobarbital35

Amoxapine35

Amoxicillin36

Amphetamine36

AmphotericinB38

Ampicillin39

Amprenavir40

Amsacrine43

ApomorphineHydrochloride44

Aripiprazole44

ArotinololHydrochloride46

Artemisinin47

Artesunate47

Aspirin48

AtazanavirSulfate49

Atenolol50

Azathioprine54

Azithromycin54

Baclofen56

Barbital57

BenactyzineHydrochloride57

Bendroflumethiazide58

Benzthiazide59

BenzylpenicillinPotassium60

BetaxololHydrochloride60

Biapenem61

BisoprololFumarate62

Bromazepam63

Bromisoval63

Bromperidol64

BrompheniramineMaleate65

BuflomedilHydrochloride65

Bumetanide66

Buparvaquone67

BupivacaineHydrochloride67

Buprenorphine68

BupropionHydrochloride69

BuspironeHydrochloride69

Busulfan70

Caffeine73

CandesartanCilexetil74

Canrenone74

Capecitabine75

CapreomycinSulfate75

Carbamazepine76

Carbidopa80

CarbinoxamineMaleate81

Carboplatin81

Carbromal81

CarteololHydrochloride82

Carvedilol82

CaspofunginAcetate83

Cathine84

Cefaclor84

Cefadroxil85

Cefalexin86

CefazolinSodium87

Cefdinir88

CefditorenPivoxil89

CefepimeHydrochloride89

Cefixime92

CefotaximeSodium92

CefozopranHydrochloride93

Cefpiramide93

CefpiromeSulfate94

CefpodoximeProxetil95

Cefprozil95

Ceftazidime96

Ceftibuten97

CeftiofurHydrochloride98

CeftizoximeSodium99

CeftriaxoneSodium100

Cefuroxime101

Celecoxib102

CeliprololHydrochloride103

CephalexinHydrochloride103

CetirizineHydrochloride104

Chloramphenicol105

Chlordiazepoxide105

ChlorhexidineAcetate106

Chlorothiazide106

ChlorphenamineMaleate107

Chlorpromazine108

Chlorpropamide109

Chlorthalidone110

Cilnidipine111

Cimetidine111

Ciprofloxacin112

Cisapride115

Cisplatin115

CitalopramHydrobromide116

Clarithromycin118

ClavulanatePotassium119

ClemastineFumarate120

ClenbuterolHydrochloride122

ClinafloxacinHydrochloride123

Clobazam123

Clofazimine124

ClomipramineHydrochloride125

Clonazepam127

Clonidine129

Clopamide129

Cloperastine130

Clotiapine131

Cloxacillin131

Clozapine132

Cocaine134

Codeine134

Cotinine135

Cyclopenthiazide135

Cyclophosphamide136

Cyclosporine137

CyproheptadineHydrochloride138

Cytarabine139

DabigatranEtexilate141

Dapsone141

Daptomycin142

Darunavir142

DebrisoquineSulfate142

Decitabine143

DelavirdineMesylate145

Derxazoxane145

DesipramineHydrochloride146

Desloratadine148

Dexamethasone149

DexfenfluramineHydrochloride149

DextromethorphanHydrobromide150

DextromoramideTartrate151

DiamorphineHydrochloride152

Diazepam153

DibenzepinHydrochloride155

DiclofenacSodium156

Diclofenamide157

DicloxacillinSodium158

Didanosine159

Digoxin161

DiltiazemHydrochloride162

Dimethylformamide163

Dimethylsulfoxide164

DiphemanilMetilsulfate164

DiphenhydramineHydrochloride165

DiphenylpyralineHydrochloride166

Diprophylline167

Dipyridamole167

Dipyrone168

Docetaxel169

DonepezilHydrochloride169

DopamineHydrochloride170

Doripenem171

DosulepinHydrochloride172

DoxapramHydrochloride173

DoxazosinMesylate173

DoxepinHydrochloride174

Doxifluridine176

Doxofylline177

DoxorubicinHydrochloride178

Doxycycline179

DoxylamineSuccinate179

Droloxifene180

Droperidol180

Drospirenone181

EcabetSodium183

Efavirenz183

EmedastineDifumarate186

Emtricitabine187

EnalaprilMaleate188

Enoxacin189

Enoxolone(GlycyrrheticAcid)190

EphedrineHydrochloride191

Epinephrine192

EprosartanMesylate193

ErlotinibHydrochloride194

ErtapenemSodium195

EscitalopramOxalate197

EsmololHydrochloride197

Estazolam198

EthacrynicAcid199

EthambutolHydrochloride200

Ethionamide200

Ethosuximide202

Ethotoin203

Etodolac204

Etoposide205

Everolimus206

Famotidine208

FaropenemSodium208

Felbamate209

Felodipine210

Fenoterol211

Fenretinide212

FentanylCitrate212

FexofenadineHydrochloride214

Flavopiridol215

FlecainideAcetate216

Fleroxacin217

Floxacillin218

Fluconazole218

Flucytosine220

FlunarizineHydrochloride221

Flunitrazepam221

Fluorouracil222

FluoxetineHydrochloride223

FlupentixolDecanoate226

FluphenazineHydrochloride227

FlurbiprofenSodium228

FluvoxamineMaleate229

FormicAcid230

Furosemide231

Gabapentin233

Ganciclovir234

GarenoxacinMesylate234

Gatifloxacin235

GemcitabineHydrochloride236

Gemfibrozil237

Glibenclamide237

Glimepiride238

Gluconolactone239

Griseofulvin239

Haloperidol241

Heptabarbital243

HomochlorcyclizineHydrochloride243

Hydrochlorothiazide244

Hydrocortisone245

Ibafloxacin247

Ibudilast247

Ibuprofen248

IfenprodilTartrate248

Ifosfamide249

ImatinibMesylate250

Imidafenacin251

Imipenem252

Imipramine253

Indapamide256

IndinavirSulfate257 Indomethacin260 Iomeprol262 IprindoleHydrochloride262 Irbesartan263 Isepamicin263 Isoniazid264 Itraconazole265

KanamycinA266

KetamineHydrochloride266

Ketanserin267

KetobemidoneHydrochloride267

Ketoconazole268

Ketoprofen269

KetorolacTromethamine270

KetotifenFumarate271

LabetalolHydrochloride273 Lacidipine274 Lamivudine275 Lamotrigine278 Lansoprazole280 LapatinibDitosylate281 LercanidipineHydrochloride281 Levetiracetam282 Levocetirizine283 Levodopa283 Levofloxacin285 LevomepromazineHydrochloride286 Levonorgestrel288 Lidocaine288 LomefloxacinHydrochloride290 Lopinavir292 Loratadine295 Lorazepam295 LosartanPotassium297 Loxapine297 Lysergide298

ManidipineHydrochloride299 Maprotiline299 Mefruside301

MelitracenHydrochloride301 Meloxicam302

MelperoneHydrochloride303 MepindololSulfate304 Mercaptopurine304 Meropenem305 Mesalazine306 Mesuximide307

MetforminHydrochoride308

MethadoneHydrochloride309

MethamphetamineHydrochloride311

Methcathinone312

Methotrexate313

Methyclothiazide313 3,4-Methylenedioxyamphetamine314 3,4-Methylenedioxymethamphetamine315

MethylephedrineHydrochloride317

MetoclopramideHydrochloride317

Metolazone318

Modafinil330

Pentoxifylline380 PerazineDimalonate380 Perifosine381 Perphenazine381

PethidineHydrochloride382

Phenazone383

PhencyclidineHydrochloride383

Phenethylamine384

Pheneturide384

PhenobarbitalSodium385

PenicillinV386

Phenprocoumon386

Phenylethanolamine387

Phenytoin388

Pimozide389

Pindolol390

Pipamperone391

PiperacillinSodium391

PipethanateEthobromide392

Pirarubicin393

Piroxicam394

Posaconazole394

Prednisolone395

Pregabalin397

Primidone398

Probenecid399

ProcainamideHydrochloride399

ProguanilHydrochloride400

PromethazineHydrochloride401

Propofol401

PropranololHydrochloride402

Protionamide404

ProtriptylineHydrochloride404

Pseudoephedrine405

Pyrazinamide407

Pyrimethamine407

QuetiapineFumarate409

Quinethazone410

QuinineSulfate411

RaltegravirPotassium412

RanitidineHydrochloride413

ReboxetineMesylate413

Resveratrol413

Ribavirin414

Rifampin414

Rifapentine415

Riluzole416

Risperidone416

Ritonavir418

RizatriptanBenzoate421

Rofecoxib421

Rogletimide422

RopivacaineHydrochloride422

SalicylicAcid424

Saquinavir425

SecbutabarbitalSodium427

SecobarbitalSodium428

Serotonin428

SertralineHydrochloride429

SildenafilCitrate430

Sirolimus431

SodiumValproate432

SotalolHydrochloride433

Sparfloxacin435

Spironolactone436

Stavudine436

SufentanilCitrate439

Sulfadiazine440

Sulfadoxine440

Sulfamerazine441

Sulfamethizole441

Sulfamethoxazole442

Sulfaquanidine443

Sulpiride444

SultoprideHydrochloride444

SumatriptanSuccinate445

TacrineHydrochloride446

Tacrolimus446

Tadalafil447

TamoxifenCitrate447

TegaserodMaleate448

Telithromycin448

Telmisartan449

Temazepam449

Teniposide450

Tenofovir451

TerazosinHydrochloride452

TerbutalineSulfate452

TertatololHydrochloride453

Testosterone453

TetracaineHydrochloride454

Theobromine454

Theophylline455

Thioguanine455

Thioridazine456

Thiotepa456

TiaprideHydrochloride457

TimololMaleate458

Tipranavir458

Tobramycin459

Tolbutamide459

Toloxatone460

Topiramate461

ToremifeneCitrate461

Torsemide462

Tosufloxacin462

TramadolHydrochloride463

Triamterene463

Triazolam464

Trimethoprim465

Trimipramine466

TriprolidineHydrochloride468

Tryptophan468

TyramineHydrochloride470

ValacyclovirHydrochloride471

ValganciclovirHydrochloride471

ValproicAcid472

VancomycinHydrochloride472

VenlafaxineHydrochloride473

VerapamilHydrochloride475

Vigabatrin475

ViloxazineHydrochloride476

VincristineSulfate477

VinorelbineTartrate477

Voriconazole478

Vorinostat478

WarfarinSodium480

Zalcitabine481

Zidovudine481

Zolmitriptan484

Zonisamide485

Zopiclone485

Zotepine486

ZuclopenthixolHydrochloride486

Index489

PREFACE

AnalyticalMethodsforTherapeuticDrugMonitoringand Toxicology isacompilationofsummarizedanalyticalmethodsthatwerepreviouslypublishedintheliteratureand weredesignedprimarilytoservetheneedsofpharmacologists,toxicologists,andallotheralliedhealthprofessionalsinvolvedinthedevelopment,use,ormonitoringof pharmaceuticals.Thematerialispresentedasstructured monographson511differentdrugentitiesdetailing964 differentanalyticalmethods.

Therapeuticdrugmonitoringplaysacriticalrolein today’sindividualizedmedicine.Itconsistsofanassessmentoftheclinicalindicationforperformingtherapeutic drugmonitoring(TDM),chemicalanalysisofthebiologicalsamples,andinterpretationoftheseresultsforpossibletherapeuticintervention.Amongthese,determinationofdrugconcentrationinaparticularhumanbiologicalmatrixisoneofthemostcriticalcomponentsoftherapeuticdrugmonitoring.Manyanalyticalprocedureshave beendevelopedfortherapeuticdrugmonitoring.Asanalyticaltechniquesandinstrumentationhaveadvanced, thisareaofTDMhasprobablygrownmorerapidlythan anyother.These“growth”techniqueshaveincludedvarioustypesofimmunoassay,gaschromatography(GC), high-performanceliquidchromatography(HPLC),electrophoresis,andliquidchromatography–massspectrometry(LC-MS).

Briefly,immunoassaymeasuresadrugmoleculeasan antigenusingaparticularantibody,withdetectioncarried outbyultravioletlightabsorption,radioactivity,orfluorescencepolarization.Immunoassayrequireslittleorno samplepreparationandisrapidandeasytouse,buta lackofspecificitycanbeproblematicwiththistechnique. Electrophoresishashighefficiencyinliquid-phaseseparationandtheabilitytoseparatecompoundsfromsmall inorganicionstolargebiomolecules.

Gaschromatographyisausefulanalyticaltoolintherapeuticdrugmonitoring.Inthistechniquedrugsare rapidlyseparatedonaGCcolumnheatedbetween200◦ C and350◦ C.Detectorsaretheflameionizationdetector (FID),theelectron-capturedetector(ECD),thenitrogen–phosphorusdetector(NPD),andthemassspectrometer (MS).Amongthem,FIDandECDdetectorsarethemost commonlyusedfordruganalysis.Thelengthofcapillary columnsallowsforcolumnstensofmeterslong,which

provideenormouschromatographicresolvingpower,but thehightemperaturesinvolvednecessitatetheanalysisof thermallystabledrugsonly.

High-performanceliquidchromatography(HPLC)is themajoranalyticaltoolusedintherapeuticdrugmonitoring.Itisreliable,robust,anddependable.Ultraviolet–visible,fluorescence,refractiveindex,electrochemical,and photodiode-arraydetectorsarethemostfrequentlyused detectors.Becauseofalargeselectionofcolumnsandmobilephases,HPLCmethodsarebothversatileandsensitive.Furthermore,thecombinationofHPLCwithmass spectrometry,whichishighlysensitiveandselective,is becomingthemethodofchoicefortherapeuticdrugmonitoringandtoxicologystudies.

Thisbookisuniquebecauseitcontainsanalytical methodsthatincludenotonlyHPLCmethodsbutalso GC,immunoassay,electrophoresis,andnewerultra-highperformanceliquidchromatography–ultraviolet(UPLCUV),andUPLC–tandemmassspectrometry(MS/MS) methods.Oursummariesareindetailedtextformat,providingthereaderwithathoroughdescriptionofmethod validation.ItisintendedforusebyknowledgeableanalystsskilledinanalyticaltechniquessuchasHPLC, UPLC,GC,LC-MS/MS,GC-MS/MS,immunoassay,and electrophoresis.

EachmonographincludesmajorChemistry,Method(s), andReference(s)sections,whichincludethefollowing items:

Chemistry

1.USadoptedname

2.Drugcategory

3.Chemicalname

4.Othernames

5.Molecularformula

6.Molecularweight

7.ChemicalAbstractsService(CAS)number

8.Appearance

9.Solubilities

10.p Ka value(s)

Method(s)

11.Individual(s)whodevelopedorpublishedthe method

12.Drugthatwasanalyzed

13.Descriptionofanalyticalsystemcomponents

14.Descriptionofmajoroperatingparameters

15.Standardsolutionpreparation

16.Samplepreparation(dilution,liquid–liquid,or solid-phaseextraction,derivatization,etc.)

17.Dynamicconcentrationrangeofthecalibration curve

18.Correlationcoefficientandlinearity

19.Coefficientofvariationoftheassay

20.Intradayandinterdaycoefficientsofvariationin samples

21.Limitofdetectionandlimitofquantification

22.Interference

Reference(s)

23.References

TheinformationintheChemistrysectionwasobtained fromstandardreferenceworks[1–3].IntheMethodSection,wehaveincludedalloftheinformationascompletely aspossiblefromtheoriginalpublishedarticles.Unfortunately,notallofthepublishedanalyticalmethodshaveincludedalloftheinformationalitemsthatwethinkareappropriatefortherapeuticdrugmonitoringandtoxicology. Usersofthisbookmustalsobeawarethatthepresentation

ofananalyticalmethodintheliteraturedoesnotexempt themfromverifyingtheadequacyofthemethodintheir ownhands.Theanalyststillmustdeterminefundamental performanceparametersofthemethod,includingselectivity,sensitivity,precision,reproducibility,accuracy,limitof detection,andlimitofquantification.However,theseare morelikelytobeeasilyachievedwhenthemethodhas beendemonstratedtoworkinanotherlaboratory.

Theauthorsofthisworkacknowledgethatthereare undoubtedlynumerousanalyticalmethodsthathavebeen developedfortherapeuticdrugmonitoringandtoxicology study;however,spacedoesnotallowacompletecompilationofallthesemethods.Wehaveincludedmonographsof drugsthatwethinkaremostcommonlyused.

TheauthorswouldliketothanktheirscientificcolleaguesDr.MaryJohansen,Dr.KirkCulotta,Edward Felix,JihaiPang,JayThapar,CaseySchultz,Mitsumasa SakamotoattheUniversityofTexasM.D.AndersonCancerCenter,andMr.JonathanRoseatWiley-Blackwellfor theirencouragementandsupportinthiswork.

REFERENCES

1.O’NeilMJ,ed., TheMerckIndex,14thed.,WhitehouseStation, NJ:Merck&Co.,Inc.,2006.

2. AHFS2010DrugInformation,Bethesda,MD:AmericanSocietyofHealth-SystemPharmacists,2010.

3.SweetmanS,ed., Martindale:TheCompleteDrugReference, 36thed.,London:PharmaceuticalPress,2009.

March,2010

Q.ALAN XUAND TIMOTHY L.MADDEN

MONOGRAPHS

ABACAVIR

CHEMISTRY

Abacavirisanantiretroviral.Itschemicalnameis {(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H -purin-9-yl] cyclopent-2-enyl}methanol.OthernamesincludeAbakavirandZiagen.ItsmolecularformulaisC14 H18 N6 O, withamolecularweightof286.3andaCASnumberof 136470-78-5.

METHODS

Assay1 Dogan-Topaletal.[1]reportedanHPLCmethod forthesimultaneousdeterminationofabacavir,efavirenz, andvalganciclovirinhumanserum.AnAgilentTechnologies1100seriesliquidchromatographwasequippedwith amodelG1315Bdiode-arraydetector(DAD)andmodel G1329ALSautosampler.Thestationaryphasewasa WatersSpherisorbcolumn(250 × 4.6mm,5 mparticle size).Themobilephaseconsistedofacetonitrile,methanol, andmonobasicpotassiumphosphatebuffer(pH5.0) (40:20:40,vol/vol/vol)andwasisocraticallydelivered at1.0mL/min.Theruntimewas15min.Theinjection volumewas20 L.

Stocksolutionsofabacavir,efavirenz,valganciclovir, andfluvastatin(internalstandard)at1.0mg/mLeach werepreparedinmethanol–water(50:50,vol/vol).Workingsolutionswerepreparedbydilutingstocksolutions withmethanol–waterandwerestoredat 20◦ C.Standardswerepreparedbyspikingdrug-freeserumwith workingsolutions.Astandardorserumsample(1.0mL) wasmixedwith1.0mLofacetonitrile,vortexedfor5min, andcentrifugedat5000 g for10min.Thesupernatant wascollectedandassayed.Retentiontimesforabacavir, efavirenz,valganciclovir,andfluvastatinwere4.1,11.6, 3.4,and5.5min,respectively.

Acalibrationcurveforabacavirwasconstructedinthe concentrationrangeof50–30,000ng/mL.Thecorrelation coefficientwas0.999.Thecoefficientofvariationoftheassaywas0.3%.Recoveryofthedrugfromserumwasbetter than98.8%.Limitofdetectionandlimitofquantification were3.80and12.68ng/mL,respectively.Nointerference wasfoundfromendogenoussubstances.

Assay2 Verweij-vanWissenetal.[2]developedanHPLCUVmethodforthesimultaneousdeterminationofabacavir,didanosine,lamivudine,stavudine,andzidovudine inpatientplasmasamples.TheliquidchromatographconsistedofThermoElectronmodelP4000solventdelivery pump,modelAS3000autosampler,modelUV2000programmablewavelengthUVdetector,andaChromJetintegrator.ThestationaryphasewasaWatersSymmetryShieldRP18column(150 × 4.6mm,3.5 mparticle size)coupledwithaSymmetryShieldRP18guardcolumn (20 × 3.9mm,3.5 mparticlesize).Thecolumntemperaturewassetat30◦ C.SolventAwasamixtureof

20mMpotassiumacetatebuffer(pH4.60)andacetonitrile (95:5,vol/vol),andsolventBconsistedof20mMpotassiumacetatebuffer(pH4.60)andacetonitrile(76:24, vol/vol).Themobilephasewasdeliveredat100%Afrom 0to10min,decreasedlinearlyto0%from10to24min, returnedto100%Afrom24to26min,andreequilibrated at100%Aforanadditional9min.Theflowratewas 1.0mL/min.UVdetectionwasperformedat260nm.

Astocksolutionofthesedrugs(0.5mg/mLeach)was preparedinmethanol/water(1:9).Standardswerepreparedbyfortifyingblankhumanplasmawiththestock solution.Allsolutionswerestoredat 20◦ C.Waters OasisMAXsolid-phaseextraction(SPE)columnswere conditionedwith0.5mLofmethanolfollowedby0.25mL ofwater.Aplasmasample(0.5mL)andanaliquot(0.5mL) ofwaterwereloadedontoapreconditionedSPEcolumn, drawnthroughundervacuum,washedwith0.15mLof water2times,driedundervacuumfor5min,elutedby 0.25mLofmethanol/water(80:20,vol/vol)twice,evaporatedtodrynessat40◦ Cunderastreamofnitrogen,reconstitutedin0.2mLofacetonitrile/water(5:95,vol/vol), vortexedfor20s,centrifugedfor5min,andassayed.The injectionvolumewas25 L.Undertheseconditions,retentiontimesforlamivudine,didanosine,stavudine,zidovudine,andabacavirwereabout4.3,5.3,6.2,18.7,and 21.3min,respectively.

Calibrationcurvesforabacavirwerelinearovertheconcentrationrangeof0.015–5mg/L.Averagerecoveryfor abacavirwas101.0%.Theaccuracyrangedfrom97%to 100%.Intradayandinterdaycoefficientsofvariationwere lessthan1.9%and2.3%,respectively.Thelimitofquantificationwas0.015mg/L.

Thisassaywasfreeofinterferencefromacetaminophen,acyclovir,amphotericinB,amoxycillin,amprenavir, atovaquone,caffeine,calciumfolinate,carbamazepine, clarithromycin,clindamycin,clofazimine,dapsone,domperidone,efavirenz,erythromycin,ethambutol,famotidine,fluconazole,ganciclovir,indinavir,isoniazid,itraconazole,ketoconazole,lidocaine,lopinavir,methadone, nelfinavir,nevirapine,ofloxacin,oxazepam,pentamidine, phenobarbital,phenytoin,pyrazinamide,pyrimethamine, rifabutin,rifampicine,ritonavir,saquinavir,sulfamethoxazole,sulfametrol,tenofovir,trimethoprim,valproicacid, zalcitabine,anditsmetabolites.Over1500patientsampleswereanalyzedusingthisassayinpharmacokinetic studies.

Assay3 Aymardetal.[3]developedanHPLCmethodfor simultaneousdeterminationof12antiretroviraldrugsin humanplasma.Fortheanalysisofamprenavir,indinavir, nelfinavir,ritonavir,saquinavir,andefavirenz,aThermoQuestliquidchromatographicsystemequippedwithan isocraticP1000pump,AS3000autosmpler,PC1000integrator,UV1000variable-wavelengthUVdetector,anda Waterscolumnheaterwasused.Thestationaryphasewas aWatersSymmetryC18 column(250 × 4.6mm,5 mparticlessize)protectedbyaWatersGuard-Pak Bondapak C18 precolumn.Thecolumntemperaturewasmaintained

at37◦ C.Themobilephasewascomposedof0.04Mdibasic sodiumphosphatebufferwith4%(vol/vol)0.25Moctanesulfonicacidandacetonitrile(50:50,vol/vol)andwas deliveredat1.3mL/min.UVdetectionwascarriedoutat 261nmbetween0and9min,at241nmbetween9and 20min,andat254nmbetween20and32min.Theinjectionvolumewas100 L.

Forthedeterminationofabacavir,didanosine,lamivudine,stavudine,zidovudine,delavirdine,andnevirapine, aliquidchromatographicsystemconsistingofthreeBeckmanmodel114Mpumps,aWatersmodelWISP717Plus autosampler,amodel481variable-wavelengthUVdetector,aShimadzuRF551fluorescencemonitor,twoLea SwitchI&Tswitchvalves,aCilCluzeauCroco-Cilcolumnheater,andaBeckmanSystemGold2integratorwas used.ThestationaryphasewasaWatersSymmetryShield C18 column(250 × 4.6mm,5 mparticlesize)protected byanUpChurchfilter(2 m).Thecolumntemperature wasmaintainedat30◦ C.Themobilephaseconsistedof monobasicpotassiumphosphatebufferwith1%(vol/vol) 0.25Moctanesulfonicacidandacetonitrile.Mobilephase 1contained5%(vol/vol)acetonitrileandwasdeliveredat 1mL/minfrom0to12min,mobilephase2contained20% (vol/vol)acetonitrileandwasdeliveredat1mL/minfrom 12to35min,andmobilephase3contained70%(vol/vol) acetonitrileandwasdeliveredat1.2mL/minfrom35to 40min.UVdetectionwasperformedat260nm.Thefluorescencedetectorfordelavirdinewassetatanexcitation wavelengthof305nmandemissionwavelengthof425nm, respectively.Theinjectionvolumewas150 L.

Stocksolutionsofabacavir,didanosine,lamivudine,and stavudineat1mg/mLwerepreparedinwaterandstoredat 4◦ C.Stocksolutionsofzidovudine,delavirdine,efavirenz, amprenavir,indinavir,nelfinavir,ritonavir,andsaquinavir at1mg/mLandneviapineat5mg/mLwerepreparedin methanolandstoredat 20◦ C.Workingsolutionsofdidanosine,stavudine,andzidovudineat100 g/mLwere preparedbydilutingstocksolutionswithwater.Working solutionsofsaquinavirandefavirenzat500 g/mLwere preparedbydilutingstocksolutionswithmethanol.All workingsolutionswerestoredat4◦ C.Standardswerepreparedbyspikingdrug-freehumanplasmawithworking solutions.Aplasmasampleorstandard(1mL)wasloaded ontoaJ.T.BakerC18 extractioncolumnthatwaspreconditionedwith3mLofmethanolfollowedwith3mL ofdistilledwater,drawnthroughthecolumnunderpressure,washedwith2mLofdistilledwater,driedunder vacuumfor1min,andelutedwith2.6mLofmethanol. Theeluatewasdividedintotwoaliquotsof1and1.6mL. Thesetwosolutionswereevaporatedtodrynessat40◦ C underagentlestreamofnitrogen.Oneresiduewasreconstitutedwith200 Lofwaterfortheanalysisofabacavir, didanosine,lamivudine,stavudine,zidovudine,andnevirapine.Theotherresiduewasreconstitutedwith150 L ofmobilephasefortheanalysisofamprenavir,indinavir, nelfinavir,ritonavir,saquinavir,andefavirenz.Retentiontimesforindinavir,amprenavir,ritonavir,efavirenz, saquinavir,nelfinavir,lamivudine,didanosine,stavudine, zidovudine,abacavir,andnevirapinewere4.8,5.6,12.9, 15.2,16.8,29.2,8.5,9.6,11.1,17.4,20.9,and27.9min, respectively.

Acalibrationcurveforabacavirwasconstructedin theconcentrationrangeof20–2,000ng/mL.Thecorrelationcoefficientwasgreaterthan0.998.Within-day andbetween-daycoefficientsofvariationwerelessthan 9.5%and9.9%,respectively.Thelimitofquantificationwas20ng/mL.Therewasnointerferencewiththe analysisofamprenavirfromthefollowingcoadministereddrugsexceptsulpiride:acebutolol,acetaminophen, acetylcysteine,acetylsalicylicacid,acyclovir,albendazole, alimemazine,alizapride,amikacin,amiodarone,amphotericinB,ampicillin,bepridil,buprenorphine,butobarbital,caffeine,calciumfolinate,captopril,carbamazepine, carbutamide,chloroquine,ciprofloxacin,clindamycin,clofazimine,clofibrate,clonazepam,clonidine,cloxacillin, clozapine,cocaine,codeine,cortisol,cyamemazine,dantrolene,dexamethasone,dextropropoxyphene,diazepam,diclofenac,digoxin,dihydroergotamine,diltiazem,doxycycline,ethambutol,flecainide,fluconazole,flunitrazepam, fluoxetine,fluvoxamine,foscarvir,furosemide,ganciclovir,gentamicin,glibenclamide,granisetron,halofantrine,haloperidol,imipramine,indomethacin,interferonalfa,isoniazid,itraconazole,josamycin,ketoconazole, levomepromazine,lidocaine,loperamide,loratadine,losartan,mefloquine,meprobamate,methadone,methylprednisolone,metoclopramide,metronidazole,mianserin,moclobemide,morphine,nifedipine,niflumicacid,nitrofurantoin,omeprazole,paroxetine,pentamidine,phenobarbital, phenytoin,piracetam,prazosin,prednisolone,prednisone, primidone,propranolol,quinidine,quinine,ranitidine,ribavirin,rifabutine,rifampicin,roxithromycin,salicylicacid, simvastatin,sulfadiazine,sulfamethoxazole,thalidomide, theophylline,trimethoprim,valproicacid,venlafaxine,vigabatrin,viloxazine,zolpidem,andzopiclone.

Morethan500plasmasampleswereassayedoneach columnwithoutsignificantlossofresolution.

Assay4 Sauxetal.[4]reportedthesimultaneousdeterminationofabacavir,didanosine,emtricitabine,lamivudine, stavudine,tenofovir,andzidovudineinhumanplasma byhigh-performanceliquidchromatographywithtandemmassspectrometry.AThermoFinniganAccelaliquid chromatographwascoupledwithaThermoFinniganTSQ QuantumDiscoveryMaxtriplequadrupolemassspectrometerthroughaheated-electrosprayionization(HESI) interface.ThestationaryphasewasaWatersAtlantisT3 column(100 × 2.1mm,3 mparticlesize).Thecolumn temperaturewasmaintainedat40◦ Candautosampler temperature,at10◦ C.SolventAwas0.05%formicacidin water,andsolventBwas0.05%formicacidinmethanol. Themobilephasewasdeliveredat0.250mL/minat5% Bfrom0to3min,linearlyincreasedfrom5%to40%B from3to8min,andkeptat5%Bfrom8to14min.The injectionvolumewas10 L.

Themassspectrometerwasoperatedinthepositive mode:sourcetemperature50◦ C,capillarytemperature 270◦ C,capillaryvoltage5kV,nebulizinggas(nitrogen) 35psi(lb/in.2 ),auxiliarygas(nitrogen)30units,andcollisiongas(argon)1.5mTorr.Abacavirwasmonitoredin theselected-reactionmonitoring(SRM)mode: m/z 287.1 → 190.0atcollisionenergy20Vandtubelens120V. 6- -Hydroxytheophyline(internalstandard)wasobserved

inSRMmode: m/z 225.1 → 181.3atcollisionenergy18V andtubelens110V.

Stocksolutionsofabacavirandtenofovirat1000 g/mL eachwerepreparedinwateranddidanosine,emtricitabine,lamivudine,stavudine,andzidovudineat1000 g/mLinmethanol.Workingsolutionsofzidovudine, stavudine,andabacavir(40 g/mLeach),lamivudine,didanosine,andemtricitabine(20 g/mLeach),andtenofovir(10 g/mL)werepreparedbydilutingstocksolutions withwater.Astocksolutionof6- -hydroxytheophyline(internalstandard)at1000 g/mLwaspreparedinwater anddilutedto2 g/mLwithwater.Analiquot(100 L) ofdrug-freehumanplasmawasspikedwith50 Lofa workingsolution,whereasaplasmasample(100 L)with 50 Lofwater,mixedwith50 Loftheinternalstandard, protein-precipitatedwith500 Lofacetonitrile,vortexed for30s,allowedtostandatroomtemperaturefor15min, andcentrifugedat2200 g for10minatambienttemperature.Thesupernatantwascollected,evaporatedtodrynessat35◦ Cunderastreamofnitrogen,reconstitutedin 500 Lofwater,andcentrifugedat2200 g for10min. Thesupernatantwasassayed.Retentiontimesforabacavirandtheinternalstandardwere8.36and8.21min, respectively.

Acalibrationcurveforabacavirwasconstructedinthe concentrationrangeof0.020–4.000 g/mL.Thecorrelationcoefficientwas0.995.Within-dayandbetween-daycoefficientsofvaiationwerelessthan4.62%and10.00%, respectively.Therecoveryrangedfrom83.8%to90.6%.

Theobservedionsuppressiondidnotinfluencequantitationoftheanalytes.Nointerferencewasfoundfrom endogenoussubstancesorotherdrugssuchastipranavir, darunavir,fosamprenavir,ritonavir,lopinavir,saquinavir, atazanavir,indinavir,efavirenz,nevirapine,etravirine, enfivirtide,raltegravir,voriconazole,posaconazole,caspofungin,amphotericinB,fluconazole,ceftazidime,ceftriaxone,cefotaxime,ciprofloxacin,perfloxacin,rifampicin, rifabutin,andisoniazid.Morethan1000sampleswere analyzedfortherapeuticdrugmonitoringusingthis method.

Assay5 Notarietal.[5]developedanHPLC-UVassayfor thesimultaneousanalysisof16anti-HIVdrugs—abacavir, amprenavir,atazanavir,didanosine,efavirenz,emtricitabine,indinavir,lamivudine,lopinavir,nelfinavir,nevirapine,ritonavir,saquinavir,stavudine,zalcitabine,and zidovudine—inhumanplasma.AWatersliquidchromatographconsistingofaWatersmodel600pump,model717 Plusautosampler,andmodel2487UV–visibledetectorwas used.ThestationaryphasewasaWatersSymmetryC18 column(250 × 4.6mm,5 mparticlesize)protectedbya WatersSentryguardcolumn(20 × 3.9mm)ofthesame packingmaterial.Themobilephaseconsistedof0.01M monobasicpotassiumphosphatebuffer,andacetonitrile andwasdeliveredat1.0mL/mininagradientmode.The percentageofthephosphatebufferwasdecreasedfrom 94%to40%in10min,maintainedat40%phosphatebuffer for10min,furtherdecreasedto0%in5min,andreturned to94%phosphatebufferin10min.Thetotalruntimewas 40min.UVdetectionswereperformedat240and260nm. Theinjectionvolumewas20 L.

Stocksolutionsof16drugsat1.0mg/mLeachwereindividuallypreparedinmethanol.Workingsolutionswere preparedbydilutingstocksolutionsinmethanolandwere storedat4◦ C.Standardswerepreparedbyspikingdrugfreehumanplasmawithworkingsolutions.Analiquotof 600 Lofplasmaorstandardwasmixedwith100 Lof methanol,vortexedfor1min,andcentrifugedat13,000 rpm(rev/min)for6min.Thesupernatantwasdiluted with1mLofwater;loadedontoaWatersOasisHLB cartridge(30mg/1mL),whichwaspreconditionedwith 1mLofmethanolfollowedby1mLofwater;washedwith 1mLof5%methanolinwater;elutedwith550 Lof 0.01monobasicpotassiumphosphatebuffer,followedby 2mLofabsolutemethanol;evaporatedtodrynessat36◦ C underasteamofnitrogen;reconstitutedwith100 Lof methanol;andassayed.Undertheseconditions,retention times(inminutes)were4.1(lamivudine),6.2(zalcitabine), 7.8(emtricitabine),8.6(didanosine),9.7(stavudine),15.1 (abacavir),16.2(zidovudine),16.6(nevirapine),18.1(indinavir),19.2(saquinavir),19.9(amprenavir),21.1(nelfinavir),23.1(ritonavir),24.5(lopinavir),28.4(efavirenz), and32.0(atazanavir).

Calibrationcurvesforabacavirwereconstructedover therangefrom0.025to10 g/mL.Correlationcoefficients were0.9959.Therecoveryinplasmawas93.6%.Theaccuracyexpressedastherelativepercentageerrorwas 8.8%.Intradayandinterdaycoefficientsofvariationwere 8.9%and9.9%,respectively.Thelimitofquantification was0.025 g/mL.Thisassaywasusedroutinelyatthe researchers’institutefortherapeuticdrugmonitoringin HIV-infectedpatients.

Assay6 Rezketal.[6]reportedthesimultaneousdeterminationofzalcitabine,lamivudine,didanosine,stavudine, zidovudien,abacavir,andnevirapineinhumanplasmaby HPLCwithUVdetection.AnAgilentseries1100liquid chromatographconsistingofabinarypump,adegasser, anautosampler,andaUVphotodiode-arraydetectorwas used.ThestationaryphasewasaWatersPolaritydC18 column(150 × 3.9mm,5 mparticlesize)withaguard column(20 × 3.9mm,5 mparticlesize)ofthesamepackingmaterial.Thecolumntemperaturewasmaintained at40◦ C.MobilephaseAwas10mMammoniumacetate bufferadjustedtopH6.5withdilutedaceticacid.MobilephaseBwasamixtureof200mLofmobilephaseA, 500mLofacetonitrile,and300mLofmethanol.Themobilephasewasdeliveredat1.1mL/mininagradientmode. ThemobilephaseBwasdeliveredat4%for15min,linearlyincreasedto64%overnext15min,heldat64% for3min,andpumpedat4%foranother7min.UVdetectionwasperformedat269nmfrom0to11min,at 250nmfrom11to14min,at271nmfrom14to24min, andat230nmfrom24to33min.Theinjectionvolume was80 L.

Stocksolutionsofzalcitabine,didanosine,andstavudineat1.0mg/mLeachwereseparatelypreparedinwater.Stocksolutionsoflamivudine,abacavir,zidovudine, andnevirapineat1.0mg/mLwereindividuallyprepared inamixtureofmethanolandwater(60:40).Working solutionswerepreparedbydilutingstocksolutionswith water.Standardswerepreparedbyspikinghumanplasma

withworkingsolutions.Astocksolutionofhexobarbitalat 1.0mg/mLwaspreparedinacetonitrile.Aworkingsolutionofhexobarbitalat2.0 g/mLwaspreparedbydiluting stocksolutionwith25mMammoniumacetatebuffer(pH 7.0)andwasusedasaninternalstandard.Analiquotof 1.0mLofplasmasampleorstandardwasspikedwithinternalstandard;loadedontoaVarianBondElutC18 solidphaseextractioncartridge(100mg/1mL),whichwaspreconditionedwith1mLofmethanolfollowedwith1mL of100mMammoniumacetatebuffer(pH7.0);passed throughthecartridge;washedwith1mLof100mMammoniumacetatebuffer(pH7.0);driedundervacuumfor 1min,elutedwith800 Lofmethanol;evaporatedtodrynessat40◦ Cunderastreamofnitrogen;reconstitutedwith 100 Lofmobilephase;vortexedfor30s;andcentrifuged at18,000 g for3min.Thesupernatantwasseparatedand assayed.Undertheseconditions,retentiontimesforzalcitabine,lamivudine,didanosine,stavudine,zidovudine, abacavir,nevirapine,andhexobarbitalwere5.9,8.6,13.6, 15.7,23.8,25.1,27.3,and30.6min,respectively.

Calibrationcurvesforabacavirwereconstructedover therangefrom10to10,000ng/mL.Correlationcoefficientsweregreaterthan0.998.Theaccuracywas101%. Intraassayandinterassaycoefficientsofvariationwere lessthan7%and7.6%,respectively.Thelimitofquantificationwas10ng/mL.Therewerenointerferences fromeitherendogenouscompoundsinplasmaordrugs suchasindinavir,amprenavir,saquinavir,nelfinavir,ritonavir,lopinavir,delavirdine,efavirenz,tenofovir,and atorvastatin.

REFERENCES

1.Dogan-TopalB,OzkanSA,UsluB,Simultaneousdeterminationofabacavir,efavirenzandvalganciclovirinhumanserum samplesbyisocraticHPLC-DADdetection, Chromatographia 66:S25–S30(2007).

2.Verweij-vanWissenCPWGM,AarnoutseRE,BurgerDM,SimultaneousdeterminationoftheHIVnucleosideanaloguereversetranscriptaseinhibitorslamivudine,didanosine,stavudine,zidovudineandabacavirinhumanplasmabyreversed phasehighperformanceliquidchromatography, JChromatogr B 816:121–129(2005).

3.AymardG,LegrandM,TrichereauN,etal.,Determination oftwelveantiretroviralagentsinhumanplasmasampleusingreversed-phasehigh-performanceliquidchromatography, JChromatogrB 744:227–240(2000).

4.SauxTL,ChhunS,ReyE,etal.,Quantificationofseven nucleoside/nucleotidereversetranscriptaseinhibitorsinhumanplasmabyhigh-performanceliquidchromatographywith tandemmass-spectrometry, JChromatogrB 865:81–90 (2008).

5.NotariS,BocediA,IppolitoG,etal.,Simultaneousdeterminationof16anti-HIVdrugsinhumanplasmabyhighperformanceliquidchromatography, JChromatogrB 831: 258–266(2006).

6.RezkNL,TidwellRR,KashubaADM,SimultaneousdeterminationofsixHIVnucleosideanaloguereversetranscriptase inhibitorsandnevirapinebyliquidchromatographywithultravioletabsorbancedetection, JChromatogrB 791:137–147 (2003).

ABECARNIL

CHEMISTRY

Abecarnilisa -carbolinecompoundandisstudiedasananxiolyticandanticonvulsant.Itschemical nameisisopropyl6-(benzyloxy)-4-(methoxymethyl)-9Hpyrido(3,4-b)indole-3-carboxylate.Othernamesinclude AbecarniloandZK112119.Itsmolecularformulais C24 H24 N2 O4 ,withamolecularweightof404.5andaCAS numberof111841-85-1.

METHOD

Assay1 Krauseetal.[1,2]describedthedetermination ofabecarnilbyHPLCwithfluorescencedetection.Aliquid chromatographicsystemwascomposedofaWatersmodel 6000Apump,model710BWISPautosampler,andaKratos modelMS970fluorescencedetector.Thestationaryphase wasaSpherisorbODSIIcolumn(125 × 4.6mm,5 m particlesize).Themobilephaseconsistedofmethanoland 0.01Mammoniumcarbonatebuffer(70:30,vol/vol)and wasisocraticallydeliveredat1.5mL/min.Fluorescence detectorwassetatanexcitationwavelengthof295nmand anemissionwavelengthof418nm.Theinjectionvolume was200 L.

5-Benzyloxyabecarnilwasusedasaninternalstandard. Plasmaorurine(0.5mL)wasmixedwith2.5mLofdiethylether,shakenfor15min,centrifugedatroomtemperatureat1500 g for10min,andfrozen.Theorganic phase(toplayer)wascollected,evaporatedtodrynessunderastreamofnitrogen,reconstitutedwith250 Lofthe mobilephase,andassayed.

Alinearcalibrationcurveforabecarnilwasconstructed intheconcentrationrangeof0.1–200ng/injection.The recoveryofthedrugfromplasmawasabout90%.The between-day(interday)coefficientofvariationwasless than11%.Thelimitofdetectionwas0.2–0.5ng/mL.No interferencewasfoundfromendogenoussubstances.

AsimilarmethodwasusedbyKararaetal.[3].

REFERENCES

1.KrauseW,SchuttB,DukaT,Pharmacokineticsandacute tolerationofthe -carbolinederivativeabercarnilinman, Arzneim-Forsch 40:529–532(1990).

2.KrauseW,MengelH,NordholmL,Determinationof -carbolinederivativesinbiologicalsamplesbyhighperformanceliquidchromatographywithfluorescencedetection, JPharmSci 78:622–626(1989).

3.KararaAH,FryeRF,HayesPE,etal.,Pharmacokineticsof abecarnilinpatientswithrenalinsufficiency, ClinPharmacol Ther 59:520–528(1996).

ACAMPROSATECALCIUM

CHEMISTRY

Acamprosatecalciumisusedintreatmentofalcohol dependence.Itschemicalnameiscalcium3-acetamido1-propanesulfate.OthernamesincludeAcamprosatum

Calcicum,Campral,andZulex.Itsmolecularformula isC10 H20 CaN2 O8 S2 ,withamolecularweightof400.5 andaCASnumberof77337-73-6.Acamprosatecalcium isawhitepowder.Acamprosatecalciumisfreelysolubleinwaterandpracticallyinsolubleinethanoland dichloromethane.

METHODS

Assay1 Luoetal.[1]developedaLC-MS/MSmethod forthedeterminationofacamprosatecalciuminhuman plasma.AShimazdu10AVPliquidchromatographincludedmodelLC10ADVPpump,modelSIL-HTcautosampler,andmodelCTO10ASVPcolumnoven.ThestationaryphasewasaPhenomenexGeminiC18 column(50 × 3.0mm,3 mparticlesize).Thecolumntemperature wasmaintainedat50◦ C.Themobilephaseconsisted of10mMammoniumacetateandmethanol(95:5), adjustedtopH7.4withammonia.Theflowratewas 0.2mL/min.

AnAPI3000tandemmassspectrometercoupledtothe liquidchromatographwasoperatedinanegativeelectrosprayionizationmode.Acamprosatecalciumwasmonitoredinmultiplereactionmonitoringmode: m/z 180 → 80.Theoperatingparameterswereasfollows:nebulizer gas13,curtaingas10,coreenergy 55V,capillaryenergy 3.6kV,collisionenergy 32V,andsourcetemperature 450◦ C.

Acamprosatecalciumstocksolution200mg/Lwaspreparedinwaterandstoredat4◦ C.Workingsolutions werepreparedbydilutingthestocksolutionwithwater. Standardswerepreparedbyspikingtheblankhuman plasmawithworkingsolutions.Patientplasmaorstandards(150 L)weremixedwith500 Lofacetonitrileand centrifugedat16◦ Cat10,000rpmfor8min.Supernatants (500 L)werecollected,evaporatedtodrynessat50◦ Cina waterbathunderastreamofair,reconstitutedin100 Lof themobilephase,mixedwith1.0mLofdichloromethane, andcentrifugedat16◦ Cat2000rpmfor3min.Supernatantswerecollectedandassayed.Theinjectionvolume was15 L.Undertheseconditions,theretentiontimeof acamprosatecalciumwas2.4min.

Acalibrationcurveforacamprosatecalciumwasconstructedintherangefrom2to2048 g/L.Thecorrelationcoefficientwas0.9999.Within-dayandbetween-day coefficientsofvariationwerelessthan4.0%and11.6%, respectively.Recoveriesofacaprosatecalciuminplasma rangedfrom83.6%to94.4%.Thelimitofdetectionwas 2.0 g/L.

Assay2 Giraultetal.[2]describedthedetermination ofacamprosatecalcium(calciumacetylhomotaurinate)in humanplasmaandurinebygaschromatography–mass spectrometry(GC-MS).AHewlett-Packard5985Bgas chromatograph–massspectrometerwasutilized.Theinjectionportwasmaintainedat320◦ C.Theoventemperaturewasprogrammedfrom240◦ Cto310◦ Cat10◦ C/min andheldat310◦ Cforadditional2min.Heliumwasused asthecarriergas.ThestationaryphasewasaChrompack fused-silicacapillarycolumn(25m × 0.35mm)wall-coated

withanOV1701liquidphase.Thefilmthicknessandinnersidediameterofthecapillarycolumnwere0.2 mand 0.25mm,respectively.

Themassspectrometerwassetinthenegative-ion chemicalionizationmodeandoperatedatanelectronenergyof100eV,anemissioncurrentof300 A,andanionsourcetemperatureof150◦ C.4-Acetylaminobutanesulfonicacidwasusedasaninternalstandard.Thedrugand internalstandardweremonitoredinasingle-ionmonitoring(SIM)mode: m/z = 424and317forthepentafluorobenzoyldi-n-butylamidederivativeofacamprosatecalciumand m/z = 438and311forthederivativeofinternal standard.

Stocksolutionsofacamprosatecalciumat1 g/mLand internalstandardat10 g/mLwereindividuallyprepared inwaterandstoredinthedarkat4◦ C.Standardsinplasma werepreparedbyspiking1mLoftheblankhumanplasma with50 Lofinternalstandardanddesiredamoutsof thestocksolutionofthedrug.Standardsinurinewere preparedbyfortifying50 Lofblankhumanurinewith 50 Lofinternalstandardandvariousamountsofthe drugstocksolution.Thedrugwasextractedfromplasma andurineandderivatizedasdescribedbelow.Aplasma sampleorstandard(1mL)wasmixedwith1mLofacetonitrile,vortexedfor20s,andcentrifugedat1600 g for 15min.Thesupernatantwascollected,extractedwith 5mLofmethylenechloridefor10min,andcentrifugedfor 15min.Theaqueouslayerwasremovedandhydrolyzed with0.2mLof10Mhydrochloricacidat100◦ Cfor2h. Aurinesampleorstandard(50 L)washydrolyzeddirectly.ThehydrolyzedsolutionwasappliedtoaJ.T.Baker C18 disposablesolid-phaseextractioncartridgethatwas preconditionedwith1mLofmethanolfollowedby1mL ofdistilledwater.Theeluatewascollectedandalkalized with5Msodiumhydroxidesolution.Thissolutionwas mixedwith20 Lofpentafluorobenzoylchloride,adjusted topH3with2Mhydrochloricacid,washedtwicewith 3mLdiethylether,andcentrifuged.Theaqueouslayer wascollected,mixedwith0.1mLof10%tetrabutylammoniumhydrogensulfatesolutionand5mLofmethylene chlorideatroomtemperaturefor10min,andcentrifuged for10min.Theorganiclayerwascollected,evaporated todrynessat45◦ Cunderagentlestreamofnitrogen,redisolvedin50 Lofthionylchloride,incubatedat80◦ C for30min,mixedwith0.2mLof2Mdi-n-butylaminein acetonitrile,acidifiedwith1mLof20%phosphoricacid, extractedwith4mLofpentane,andcentrifuged.Theorganiclayerwascollected,evaporatedtodrynessundera streamofnitrogen,reconstitutedin0.3mLofethylacetate,andassayed.Theinjectionvolumewas1 L.Undertheseconditions,retentiontimesofacamprosatecalciumandtheinternalstandardwere5.5and6.2min, respectively.

Acalibrationcurveforacamprosatecalciumwas constructeddailyintheconcentrationrangeof3.12–800ng/mL.Thecorrelationcoefficientwasgreaterthan 0.9999.Thecoefficientofvariationoftheassaywasless than7.9%.Therewasnointerferencewiththeanalysisof thedrugfromendogenouscompounds.Thismethodwas appliedtothepharmacokineticstudyofacamprosatecalciumin24healthyvolunteers.

8ACEBUTOLOLHYDROCHLORIDE

REFERENCES

1.LuoM,QinY-P,YuQ,etal.,Determinationofacamprosate calciuminhumanplasmabyRP-HPLC-MS/MS, ChineseJNew Drugs 16:163–166(2007).

2.GiraultJ,GobinP,FourtillanJB,Determinationofcalcium acetylhomotaurinateinhumanplasmaandurinebycombinedgaschromatography-negative-ionchemicalionization massspectrometry, JChromatogr 530:295–305(1990).

ACEBUTOLOLHYDROCHLORIDE

CHEMISTRY

Acebutololisacardioselective -blocker.Itschemicalname is(±)-3 -acetyl-4 -(2-hydroxy-3-isopropylaminopropoxy)butyranilidehydrochloride.OthernamesincludeAbutol, Prent,andSectral.ItsmolecularformulaisC18 H28 N2 O4 · HCl,withamolecularweightof372.9andaCASnumber of34381-68-5.Acebutololhydrochlorideisawhiteor almostwhitecrystallinepowder.Acebutololhydrochloride hassolubilitiesof200mg/mLinwaterand70mg/mLin alcohol.Itisveryslightlysolubleinacetoneanddichloromethaneandpracticallyinsolubleinether.Acebutololhas anapparentpK a of9.4inwater.

METHODS

Assay1 Umezawaetal.[1]describedthesimultaneousdeterminationoffourß-blockers,acebutolol,labetalol, metoprolol,andpropranololinhumanplasma,usingLCMS/MS.AnAgilent1100seriessystemconsistingofa modelG1315Adiode-arraydetector,modelG1313Aautosampler,andmodelG1322Avacuummembranedegasserwasutilized.ThestationaryphasewasaShodex MSpakGF3104Bcolumn(50 × 4.6mm,6 mparticle size)protectedbya2- mSUMIPAXPG-ODSinlinefilter. SolventAwas10mMammoniumacetateaqueoussolution andsolventB,acetonitrile.Themobilephasewasdelivered at0.55mL/mininagradientmodeat100%Afrom0to 3min,decreasedto0%Ain1min,andkeptat0%Afrom 4to9.5min.Thecolumnwasreequilibratedat100%Afor 5.5min.

AnAppliedBiosystems/MDSSCIEXAPI2000triple quadrupolemassspectrometerequippedwithaTurboIonSprayionsourcewasoperatedinthepositivemode:TurboIonSpraytemperature490◦ C,ionsourcevoltage3kV, ringvoltage390V,nebulizergas(high-purityair)20psi, heatergas(high-purityair)80psi,curtaingas(high-purity nitrogen)40psi,orificevoltage41Vforacebutolol,25V forlabetalol,40Vformetoprolol,51Vforpropranolol,and 40Vforpindolol,collisiongas(nitrogen)4,collisionenergy 29eVforacebutolol, 22eVforlabetalol, 25eVfor metoprolol, 25eVforpropranolol,and 25eVforpindolol.Quantificationwasperformedinselective-reaction monitoring(SRM)modeusingiontransitionsat m/z 337 → 116foracebutolol, m/z 329 → 311forlabetalol, m/z 268 → 116formetoprolol, m/z 260 → 116forpropranolol, and m/z 249 → 116forpindolol,respectively.

Stocksolutionsofacebutolol,labetalol,metoprolol,propranolol,andpindolol(asinternalstandard)at1mg/mL wereseparatelypreparedinmethanol.Workingsolutions werepreparedbydilutingstocksolutionswith10mM ammoniumacetateinwater.Calibratorswereprepared byspikingdrug-freeplasmawithworkingsolution.An aliquotof1mLofaplasmasampleorcalibratorwasmixed with3mLof13.3mMammoniumacetateaqueoussolution andcentrifugedat9000rpmfor10min.Thesupernatant wasfilteredthrougha0.2- mWhatmanGD/Xsyringefilter(13mm)andassayed.Theinjectionvolumewas100 L. Undertheseconditions,retentiontimesoflabetalol,metoprolol,acebutolol,propranolol,andpindololwere6.6,6.9, 7.2,7.8,and7.9min,respectively.

Calibrationcurvesforacebutololwereconstructedin therangefrom10to1000ng/mL.Correlationcoefficients weregreaterthan0.9992.Theeffectofionsuppression ontheanalysisofacebutololwaslessthan29.8%.The recoveryofacebutololfromplasmarangedfrom74.4to 89.9%.Theaccuracyrangedfrom94.0%to111%.Intradayandinterdaycoefficientsofvariationwerelessthan 2.9%and5.6%,respectively.Thelimitofdetectionwas 1ng/mL.

Assay2 Vienoetal.[2]developedanLC-MS/MSmethod forthedetectionofacebutolol,atenolol,metoprolol,sotalol, carbamazepine,ciprofloxacin,ofloxacin,andnorfloxacinin drinkingwater,surfacewater,andsewagetreatmentplant water.AnAgilent1100seriessystemconsistingofabinary pump,vacuumdegasser,autosampler,andathermostated columnovenwasused.ThestationaryphasewasanAgilentZorbaxXDBC18 column(50 × 2.1mm,5 mparticle size)protectedbyanAgilentnarrowboreguardcolumn (12.5 × 2.1mm,5 mparticlesize).Thecolumntemperaturewasmaintainedat30◦ C.Themobilephaseconsisted ofacetonitrileand1%aceticacidinwaterandwasdeliveredat250 L/mininagradientmodefrom3%to28% acetonitrilein12minandto53%acetonitrileinanother 5min,keptat53%acetonitrilefor1min,andthenreturnedtotheinitialconditionin1min.Thecolumnwas equilibratedat3%acetonitrilefor8min.

AMicromassQuattroMicrotriplequadrupolemass spectrometerequippedwithanelectrosprayionizationinterfacewasoperatedinpositivemode:desolvationgas640 L/h,nebulizinggas30L/h,collisiongas(argon)2.8 × 10 3 mbar,sourcetemperature120◦ C,anddesolvationtemperature325◦ C.Conevoltage(V)andcollisionenergy(eV) were28and20foracebutolol,30and23foratenololol, 25and15formetoprolol,30and23forsotalol,25and 15foralprenolol(internalstandard),29and18forcarbamazepine,35and21fordihydro-carbamazepine(internalstandard),30and17forciprofloxacin,28and16for norfloxacin,29and18forofloxacin,and28and18for enrofloxacin(internalstandard).Quantificationwasperformedinmultiple-reactionmonitoring(MRM)modeusing iontransitionsat m/z 336.8 → 116.0foracebutolol, m/z 267.0 → 144.9foratenolol, m/z 267.9 → 190.9formetoprolol, m/z 254.8 → 132.9forsotalol, m/z 249.9 → 172.9 foralprenolol, m/z 237.0 → 193.9forcarbamazepine, m/z 239.0 → 193.9fordihydrocarbamazepine, m/z 331.9 → 287.9forciprofloxacin, m/z 319.8 → 275.9fornorfloxacin,

m/z 361.8 → 317.9forofloxacin,and m/z 359.9 → 315.9 forenrofloxacin,respectively.

Stocksolutionsofdrugswerepreparedinmethanol, exceptthatantibioticswerepreparedinamixtureof methanoland0.01Mhydrochloricacid(1:1,vol/vol)and storedat 18◦ C.Workingsolutionswereprepareddailyby dilutingstocksolutionswiththesamesolvents.Standards werepreparedbyspikingnoncontaminatedgroundwater withworkingsolutionsandinternalstandards.Asample (100,250,500,and1000mLforsewageinfluent,sewage effluent,surfacewater,andgroundwater,respectively)was adjustedtopH10.0with2Msodiumhydroxidesolution, spikedwith500ngoftheinternalstandards,andfiltered througha0.45 mSchleicher&SchuellGF6filterthat waspreviouslywashedwith n-hexane,acetone,methanol, andwater.ItwasthenloadedontoaWatersOasisHLB solid-phaseextractioncartridge(3mL,60mg)bymeans ofpolytetrafluoroethylene(PTFE)(Teflon)tubesatflow ratesof2,5,10,and20mL/min(sewageinfluentwater, sewageeffluentwater,surfacewater,andgroundwater,respectively),whichwaspreconditionedsequentiallywith 2mLof n-hexane,2mLofacetone,10mLofmethanol, and10mLofnoncontaminatedgroundwater(pHadjusted to10.0);pulledthroughthecartridge;washedwith2mLof 5%methanolin2%aqueousammoniumhydroxide;dried withastreamofnitrogenfor30min;elutedwith4 × 1mLofmethanol;evaporatedtoneardrynessundera streamofnitrogen;reconstitutedwith20 Lofmethanol and480 Lof1%aceticacid;andassayed.Underthese conditions,retentiontimesofsotalol,atenolol,norfloxacin, ofloxacin,ciprofloxacin,enrofloxacin,acebutolol,metoprolol,alprenolol,carbamazepine,anddihydrocarbamazepine were3.3,4.4,9.8,9.8,10.2,10.9,11.1,11.2,15.4,17.6,and 17.8min,respectively.

Calibrationcurvesforacebutololwereconstructedin therangefrom0.82to6000 g/L.Correlationcoefficients weregreaterthan0.996.Averagerecoveriesofacebutolol fromgroundwaterandtapwater,surfacewater,sewageeffluentwater,andsewageinfluentwaterwere93%,105%, 78%,and64%,respectively.Limitsofquantificationin drinking(potable)water,surfacewater,sewageeffluent water,andsewageinfluentwaterwere0.4,0.8,2.1,and 6.4ng/L,respectively.

ThisLC-MS/MSmethodwassuccessfullyappliedfor thedeterminationofacebutololinsewageinfluentwater, sewageeffluentwater,andtheirrecipientrivers.

Assay3 Delamoyeetal.[3]developedanHPLCmethod forsimultaneousdeterminationof13ß-blockersand onemetabolite:atenolol,sotalol,diacetolol,carteolol, nadolol,pindolol,acebutolol,metoprolol,celiprolol,oxprenolol,labetalol,propranolol,tertatolol,andbetaxolol. ASpectraliquidchromatographicsystemconsistingofa modelP1000XRquaternarygradientpump,modelAS3000 autoinjectorwitha100- Lloop,andmodel6000LP photodiode-arraydetectorwasused.Thestationaryphase wasaThermoHypersilHypurityC18 column(250 × 4.6mm,5 mparticlesize)protectedbyaC18 precolumn (4 × 4.4mm,5 mparticlesize).Thecolumntemperature wasmaintainedat35◦ C.UVdetectionwasperformedat 220nm.Theinjectionvolumewas80 L.

Stocksolutionsofthesecompoundsat1.0g/Leachwere preparedinmethanol.Workingsolutionswerepreparedby dilutingstocksolutionswithmethanol.Astocksolutionof medroxalolat50.0mg/Linmethlanolwasusedasaninternalstandard.Thesestockandworkingsolutionswere storedat 20◦ C.Analiquotof1mLofplasma,standard, orcontrolwasspikedwith20 Loftheinternalstandard,mixedwith500 Lof1Msodiumcarbonate(pH 9.7),extractedwith7mLofchloroform–pentanol–diethyl ether(6:2:1,vol/vol/vol),shakenfor15min,andcentrifugedat3000 g for5min.Theorganicphasewascollected,mixedwith250 Lof0.05Mphosphoricacid(pH 2.1),shakenfor10min,andcentrifugedat3000 g for5min. Theaqueousphasewascollectedandassayed.Underthese conditions,retentiontimesforatenolol,sotalol,diacetolol, carteolol,nadolol,pindolol,acebutolol,metoprolol,celiprolol,oxprenolol,medroxalol,labetalol,propranolol,tertatolol,andbetaxololwere5.1,5.6,7.9,9.2,9.9,10.5,14.5, 15.4,18.8,20.4,21.2,21.8,24.6,25.1,and25.9min, respectively.

Calibrationcurvesforacebutololwereconstructedover therangefrom25to1000ng/mL.Themeancorrelation coefficientwas0.999.Themeanaccuracywas100.1%at 100ng/mL.Themeanrecoveryrangedfrom90to113%. Intradayandinterdaycoefficientsofvariationwere6.27% and6.60%,respectively.Limitsofdetectionandquantificationwere6and25ng/mL,respectively.

Assay4 Leeetal.[4]developedanLC-MS/MSmethod forthesimultaneousdeterminationof12ß-blockersand ß2 -agonistsinsewagesamples.AWaters2695separation modulewasused.ThestationaryphasewasanAgilent ZorbaxSBC8 column(150 × 2.1mm,3.5 mparticlesize) protectedbyaSBC8 guardcolumn(12.5 × 2.1mm,5 m particlesize).Thecolumntemperaturewasmaintained at35◦ C.MobilephaseAwasamixtureofwater,acetonitrile,andformicacid(94.5:5.0:0.5,vol/vol/vol),andmobilephaseBwasamixtureofacetonitrileandformicacid (99.5:0.5,vol/vol).Themobilephasewasdeliveredina gradientmodefrom100%Ato75%Ain13min,heldat 75%Afor13min,andthenpumpedat100%Aforanother14min.Theflowratewas0.2mL/min.Theinjection volumewas10 L.Thetotalruntimeofaninjectionwas 40min.

AMicromassQuattroUltimatriplequadrupolemass spectrometerequippedwithanelectrosprayionization sourcewasoperatedinthepositive-ionmode.Themajor parametersweresetasfollows:nebulizergas(nitrogen) 50L/h,desolvationgas(nitrogen)500L/h,sourcetemperature120◦ C,desolvationtemperature350◦ C,capillary voltage3.45kV,coneenergy60V,andcollisionenergy 17kV.Acebutololwasanalyzedinthemultiple-reaction monitoring(MRM)modeationtransitions m/z 337 → 116forquantitationand m/z 337 → 260forconfirmation.

Individualstocksolutionsofacebutolol,alprenolol, atenolol,bisoprolol,clenbuterol,fenoterol,labetalol,metoprolol,nadolol,pindolol,propranolol,sotalol,terbutaline, andtimololat1000 g/mLwerepreparedinacetonitrile ormethanol.Astocksolutionofsalbutamolwasprepared at500 g/mL.Thesestocksolutionswerestoredat 20◦ C. Workingsolutionswerepreparedbymixinganddiluting

thesestocksolutionswithmobilephaseB.Analiquotof 250mLofsewagesamplewasfilteredthrougha1.2- m GF/Cfilter(90mmi.d.)withalayerofCelite;acidified topH3with1Mhydrochloricacid;loadedontoaWaters OasisMCXcartridge(6mL,150mg,30 m)ataflowrate of10–15mL/min,whichwaspreconditionedwith6mLof methanolfollowedby10mLofwateratpH3;driedfor 10minundervacuum;washedwith100mLofwaterat pH3followedby6mLofmethanol;elutedwith8mLofa mixtureofdichloromethane,2-propanol,andammonium hydroxide(78:20:2,vol/vol/vol);evaporatedtodryness at40◦ Cunderastreamofnitrogen;reconstitutedin1.0mL ofmobilephaseB;filteredthrougha0.45- mnylonsyringe filter;andassayed.Undertheseconditions,retentiontimes foracebutolol,alprenolol,atenolol,bisoprolol,labetalol, metoprolol,nadolol,pindolol,propranolol,sotalol,timolol, clenbuterol,fenoterol,salbutamol,andterbutalinewere 17.36,24.32,8.96,21.77,22.11,18.02,13.60,14.15,23.87, 7.85,17.02,17.91,11.50,6.97,and6.53min,respectively. Calibrationcurvesforacebutololwereconstructedover therangefrom50to500pg/ L.Themeanrecoveryofthe drugfromwaterrangedfrom88%to95%.Thelimitof detectionwas9ng/L.

REFERENCES

1.UmezawaH,LeeX-P,ArimaY,etal.,Simultaneousdeterminationofß-blockersinhumanplasmausingliquid chromatography-tandemmassspectrometry, BiomedChromatogr 22:702–711(2008).

2.VienoNM,TuhkanenT,KronbergL,Analysisofneutral andbasicpharmaceuticalsinsewagetreatmentplantsand inrecipientriversusingsolidphaseextractionandliquid chromatography-tandemmassspectrometrydetection, JChromatogrA 1134:101–111(2006).

3.DelamoyeM,DuverneuilC,ParaireF,etal.,Simultaneousdeterminationofthirteenß-blockersandonemetabolitebygradienthigh-performanceliquidchromatographywith photodiode-arrayUVdetection, ForensicSciIntnatl 141:23–31 (2004).

4.LeeH-B,SrafinK,PeartTE,Determinationofß-blockersand ß2 -agonistsinsewagebysolid-phaseextractionandliquid chromatography–tandemmassspectrometry, JChromatogrA 1148:158–167(2007).

ACECLOFENAC

CHEMISTRY

AceclofenacisaNSAID(nonsteroidalanti-inflammatory drug).Itschemicalnameis[o-(2,6-dichloroanilino) phenyl]acetateglycolicacidester.Othernamesinclude Aceclofar,Aceclofenaco,Aceclofenacum,Beofenac,and Preservex.ItsmolecularformulaisC16 H13 Cl2 NO4 ,witha molecularweightof354.2andaCASnumberof89796-996.Aceclofenacoccursasawhiteoralmostwhitecrystalline powder.Aceclofenacispracticallyinsolubleinwater.Itis solubleinalcoholandfreelysolubleinacetone.Aceclofenac shouldbestoredinairtightcontainersandprotectedfrom light.

METHODS

Assay1 Jinetal.[1]reportedanHPLCmethodforthedeterminationofaceclofenacinhumanplasma.AShimadzu LC10AsystemequippedwithamodelSPD10AUVdetectorwasused.ThestationaryphasewasaShimadzuODS column(150 × 4.6mm,5 mparticlesize).Themobile phaseconsistedofmethanoland0.1Mammoniumacetate aqueoussolution(pH6.0)(7:3,vol/vol)andwasisocraticallydeliveredat1.0mL/min.UVdetectionwasperformed at275nmand0.005AUFS(absorbanceunitsfullscale). Theinjectionvolumewas20 L.

Analiquotof0.5mLofaplasmasamplewasspiked with75 Lof1Mhydrochloricacid,vortexed,mixedwith 3.5mLofether,shakenfor3min,andcentrifugedat3000 rpmfor10min.Analiquotof3mLofthesupernatantwas collected,evaporatedtodrynessat37◦ Cunderastream ofnitrogen,reconstitutedwith0.2mLofmobilephase, andassayed.Undertheseconditions,theretentiontimeof aceclofenacwasabout6.9–8.4min.

Acalibrationcurveforaceclofenacwasconstructedin theconcentrationrangefrom0.05to40.0mg/L.Thecorrelationcoefficientwas0.9999.Theaveragerecoveryof aceclofenacfromplasmawas82.5%.Intradayandinterdaycoefficientsofvariationwerelessthan7.1%and9.3%, respectively.Thelimitofquantificationwas0.05mg/L. Theanalysisofaceclofenacwasnotaffectedbyendogenouscompoundsinplasma.

Assay2 Leeetal.[2]describedthesimultaneousdeterminationofaceclofenacanddiclofenacinhumanplasma bynarrowboreHPLCusingacolumn-switchingtechnique.AShiseidoNanospaceSI-1seriesliquidchromatographconsistingoftwomodel2001pumps,model2002 UV–visibledetector,model2003autosampler,model2004 columnoven,model2012high-pressureswitchingvalve, andmodel2009degassingunitwasutilized.ThestatinaryphasewasaPhenomenexLuna2phenylhexylnarrowborecolumn(100 × 2mm,3 mparticlesize).The columntemperaturewasmaintainedat30◦ C.Themobile phaseconsistedofacetonitrileand0.02Mpotassiumphosphatebuffer(pH7)(33:67,vol/vol)andwasdeliveredat 0.2mL/min.UVdetectionwasperformedat278nm.

Stocksolutionsofaceclofenacanddiclofenacat1mg/mL werepreparedinmethanol.Standardswerepreparedby spikingdrug-freehumanplasmawithstocksolutions.An aliquotof100 Lofaplasmasamplewasfilteredthrough a0.2- mmembranefilterandintroducedontoaCapcell PakMFPh1precolumn(20 × 4mm)toremoveproteins usingamixtureofacetonitrileand0.1Mpotassiumphosphatebuffer(pH7)(14:86,vol/vol)at0.5mL/minfrom 0to6.0min.ThevalvewasswitchedtoaCapcellPakC18 UG120column(35 × 2mm)toconcentratedrugsfrom6.0 to8.8min.From8.8to17min,thevalvewasswitched totheanalyticalcolumntoseparatedrugsusingthemobilephaseat0.2mL/min.Undertheseconditions,retentiontimesofdiclofenacandaceclofenacwereabout13and 14.3min,respectively(estimatedfromthepublishedchromatogram).

Acalibrationcurveforaceclofenacwasconstructed intherangefrom50to10,000ng/mL.Thecorrelation

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Plainly Ark declined to believe that his lifelong friend was dead, and it seemed useless to impress him with the undoubted fact. He complained that the policeman would not allow him to enter the Vicarage, and that no one would take any notice of his protestations that Leigh was not dead. Rupert, although in a hurry to return to his unfinished breakfast, stayed to persuade Titus to take a more reasonable view of the situation.

"Dr. Tollart says that Mr. Leigh has passed away. Besides the knock on the head he had heart disease, and either the one or the other was enough to kill him."

"Dr. Tollart," grunted Ark stolidly, "he be better wi beer than wi curing folk. I nivir heard tell as Muster Leigh had heart-badness. He be aloive, I tell ee, Squoire."

"Well, Titus, have your own way. But it will be your duty within a couple of days if not less, seeing that the weather is hot, to put our late vicar in his family vault."

"Oh, I'll put him there, Squoire; but he bain't dead fur all that. Hor! Hor! Hor!"

With another shrug Rupert passed on, and returned to The Big House to find Dorinda. She greeted him hastily and appeared to be very dismayed at the dreadful news of the vicar's murder. "Who could have hurt him, Rupert?" she asked, again and again. "He had no enemies. He would not have harmed a fly."

"I'm sure I can't tell you, dear. Kensit seems to think that it was a burglar did the trick."

"But there was nothing in the Vicarage to rob," protested Dorinda.

"Just what I say. However, some burglar from London might have believed that Leigh was a miser and had treasure."

"Has any stranger from London been seen about the village?"

"No. Kensit can't make head nor tail of it," Rupert shook his head and thought for a moment. "Unless some very startling evidence turns up, Dorinda, I don't believe that the truth will ever become known. What does your father say, dear?"

"Nothing. You know father did not care much for Mr. Leigh. He told me that he was sorry, but that Leigh was a fool, or he would have locked up his house regularly every night."

"Your father hasn't much sympathy, Dorinda."

"He never has. You know how badly he thinks of everyone. What is to be done about the murder, Rupert?"

"The Inspector from Tarhaven is coming to-day, and he will arrange for an inquest this afternoon or to-morrow. Upon what evidence is obtainable will depend the next step. I expect the body"--Dorinda quivered and turned pale--"will be buried almost immediately."

"Why. Don't they keep bodies a week?"

"Sometimes. But in this case, Tollart says that the sooner poor Leigh is buried the better. The corpse"--Rupert hesitated--"won't keep."

"Oh, don't"--Dorinda made a wry face--"poor Mr. Leigh. He was such a good man, Rupert. Who inherits his books, which are all he has left."

"I think there's a distant cousin of sorts, a ship captain. He won't benefit much by Leigh's death. I wonder if the old man made a will."

"Oh, yes. He told me a year ago that he had, but did not mention to whom he had left his library. You are the executor."

"Am I, indeed? That is news to me, as Leigh never asked my permission. However"--Hendle was thinking of the probability of his ancestor's will being among the papers and books--"it is just as well under the circumstances."

"What do you mean by that?"

Hendle tugged at his moustache and replied in an embarrassed fashion, "Oh, nothing, only I can look after things better than a stranger, you know. By the way, Dorinda, I forgot to tell you that Carrington is coming down by the midday train."

"Coming again so soon," said Dorinda, remembering her father's warnings against the barrister, "and why?"

"Only about some business I went up to town about yesterday," answered Rupert confusedly. "Will you walk with me to the station to meet him?"

"No," said the girl promptly. "I don't want to meet Mr. Carrington again. I don't like him overmuch."

"Ah, you've been listening to your father, dear. Mallien likes no one."

"I saw Mr. Carrington myself, Rupert, and I didn't like him. I don't require my father to judge for me."

"What a spitfire you are!" laughed Hendle, putting his arm round her waist.

"Because I want you all to myself, and I think Mr. Carrington is not a good friend for you."

"Jealous."

"Sensible. There, Rupert, don't worry me." She slipped out of his arms, much to his surprise, and he showed his feelings so visibly

that she colored. "I am rather out of sorts this morning," she said hurriedly. "Father has been rather trying."

"Never mind, dear; in a month you will be with me forever."

"I hope so," sighed Dorinda, "but somehow this death of the vicar suggests to me the possibility that something will occur to prevent our marriage."

"Oh, nonsense!" Rupert stared. "What could prevent our marriage?"

"It's only a feeling," persisted Dorinda, "and I dare say it is a foolish, silly feeling; but it's here for all that," and she laid her hand on her heart.

Rupert took as much pains to argue away this fancy as he had done to argue away the fancy of Titus Ark. But Dorinda was quite as stubborn in her belief that evil fortune was coming to prevent the marriage, as the sexton was that Leigh was alive. Finally, because Rupert laughed at her, she parted from him rather irritated at the corner, where he branched off to the station road. She would not even look back when her lover went away, and Rupert walked on to meet Carrington with the reflection that women were kittle cattle, as the Scotch say. As a rule, Dorinda was amiable and calm, so it seemed strange that she should be so easily annoyed this morning. But there was a reasonable excuse after all, as Hendle concluded, since the girl, always having been markedly friendly with the vicar, the poor man's violent death naturally shocked and upset her greatly. Moreover, the heartless comments which Mallien the cynic was more than likely to make, assuredly would add to Dorinda's distress. By the time he reached the station, Rupert had explained away to his own satisfaction the unusual emotion of the girl.

True to his promise Carrington arrived by the midday train and hopped out onto the platform as lively as a cricket. In gray flannels, a straw hat and brown shoes, the barrister looked handsome, wellbred and very much alive. The sight of his keen face and intelligent

dark eyes comforted Hendle, as he knew that Carrington, if anyone, would be helpful in the matter of the vicar's mysterious murder.

"Here you are and here I am, Hendle," cried the new arrival briskly, as he gave up his ticket and walked out of the station along with the Squire. "I say, old chap, you're worrying considerably over this will business. There's a drawn, tired look on your face, which shows that you haven't slept a wink."

"Well, I didn't have a particularly restful night," admitted the other with a sigh. "And what has happened this morning doesn't help to make me feel any happier, Carrington."

"Eh, what?" the barrister stopped. "Then Leigh has found the will and----"

"Leigh is dead," Hendle informed him abruptly.

"Dead!" Carrington stared. "Dead! What are you talking about?"

"About what has happened," replied the other heavily. "Leigh was found dead in his study this morning."

Carrington looked at Hendle doubtfully. "You're pulling my leg," he said, in a disbelieving tone.

"I don't pull people's legs over such a serious matter. I tell you positively that the vicar is dead. All the village is in commotion."

"Dead!" repeated Carrington once more as they moved on toward Barship. "The unexpected has happened with a vengeance. Well, well, he wasn't young, and looked like a delicate man, who would pop off at any moment."

"This death has nothing to do with delicacy, Carrington. Leigh has been murdered."

"Oh, Lord!" Man of the world as he was, Carrington received a shock. "Poor old chap. Murdered! What a beastly thing to happen! Who murdered him?"

"No one knows. The police are looking into the matter now. He was found dead in his study at seven this morning, and there is a wound on the right temple. So far, the only conclusion arrived at is that some one tried to rob the house, and, being discovered, struck Leigh down."

"I can't see that there was anything in the house worth a burglar committing such a crime for," remarked Carrington, taking off his hat.

"There wasn't. No one in the village would have attempted a burglary, since Leigh was known to be very poor. Besides, Leigh was too popular for anyone to hurt him. But a stranger----"

"Ah," broke in Carrington swiftly, "a stranger. Has any stranger been seen hovering about the Vicarage?"

"No. Kensit, our village policeman, was on his rounds as usual last night, but declares that he saw no one."

"But some tramp----"

"No tramps have been hanging about the village of late."

Carrington looked puzzled. "It seems to be a mystery. At what time was the poor chap murdered?"

"No one knows. But Dr. Tollart thinks the blow was struck about eleven o'clock last night."

"Has the weapon been found?" "No!"

"Did that housekeeper hear any noise?"

"No! Nothing was known of the murder until she found her master dead near his writing table. The Inspector has been sent for to Tarhaven and will be here shortly. Indeed, I expect he is here now. He will take charge of the house and look into the matter."

"Humph!" remarked the barrister thoughtfully. "As I said before, it seems to be a mystery. This Inspector will take charge of all Leigh's books and papers, I suppose."

"Yes. Why not?"

"Oh, I am not saying against his handling them. But the will----"

"The will. Yes?"

"Can't you see, Hendle. If this Inspector looks through the papers left by Leigh, which he probably will, he is bound to come across that hundred-year-old testament you mentioned yesterday."

Rupert winced. "I expect he will, unless poor Leigh has so carefully mislaid it that it cannot be found. But what if he does?"

"Well, then all the fat will be on the fire," said Carrington with an air of finality.

"I suppose you mean that the will must be made public. Why not? If it is a legitimate document, Mallien must get the money, and if it isn't, my position remains unchanged. In any case, whether Leigh lived or died, what he discovered would have to be shown all round."

"Quite so. But you didn't want it to be shown all round until you looked into the matter privately along with me," argued Carrington, quickly.

"True enough. I should like to have seen the document before Mallien became aware that it existed. However, as things stand, the will is bound to be found, and Mallien is bound to know. We must thresh out the matter openly straightway, and I shall do my best to avoid trouble."

"I don't see how you can avoid it, Hendle. Mallien is not the man to let a chance of getting a fortune go."

"I am sure he isn't," retorted the Squire positively. "And he is certain to make things as disagreeable for me as possible. But if I surrender the property, should the will prove to be legal, I don't see that he can worry me."

"You will lose everything," warned the barrister, significantly.

"Unfortunately, yes."

"Including Miss Mallien."

"I suppose so," admitted the Squire reluctantly. "Even if she remains true to me, as I am sure she will, I can't ask her to marry me on nothing a year."

There was silence for a few minutes as the two men walked into the village, and it was Carrington who spoke first. "I'm awfully sorry for you, old man."

"I'm rather sorry for myself. However, what must be must be, so there's no more to be said. By the way, Dorinda told me that Leigh had made me his executor. I never knew that he had, until she told me."

"Leigh took your friendship for granted, it seems. Who inherits?"

"I don't know. His sole relative is a sea captain, somewhere in Australia. I have heard him speak of the young fellow a cousin of

sorts--as the last of the Leighs. There isn't much to leave in the way of property."

"So you are executor," murmured Carrington thoughtfully. "In that case, you will have the handling of the papers, and may be able to get possession of the will before the Inspector lays hands on it."

"What good will that do?" asked Hendle, irritably.

"You can suppress the will."

"I shouldn't think of doing such a thing."

"You'll lose all if the will proves to be genuine," Carrington warned him.

"Then I must lose all."

"That's quixotic."

"So you said yesterday. But I mean to be honest." And again there was silence, Carrington secretly considering his friend an honorable ass.

CHAPTER VIII

MALLIEN SPEAKS

Anxious to help Rupert, and, at his friend's request, Carrington remained at The Big House until the inquest was over, and the burial

of the murdered man took place. Both he and the Squire could do little save watch the course of events, as neither of them wished to say anything about the missing will, and neither could suggest any reason why the crime should have been committed. And, indeed, the police were equally unable to solve the problem, since the murder, on the face of it, appeared to be purposeless and the assassin could not be discovered. Inspector Lawson, of Tarhaven, did his best to find a clue, but from first to last was unsuccessful. He did not even know where to look for one, and when the inquest was held, had absolutely no evidence to place before the Coroner and jury. Leigh's murderer had come out of the night and had gone into the night; but why he had come to commit so dastardly a crime, and whither he had gone after achieving his aim, it was wholly impossible to say. The affair was unpleasant, mysterious and uncanny.

Pursuant to the opinion of Dr. Tollart, proceedings in connection with the death were hurried on as speedily as possible. The weather was certainly amazingly hot, as for weeks a powerful sun had been blazing in a cloudless blue sky. The gardens glowed with manycolored flowers, but the growing crops were parched for want of rain, and everywhere in the district people were complaining of the shortage of water. Under the circumstances, and because nothing relevant to the assassin could be discovered, Tollart's advice seemed to be very sensible. Therefore the inquest was held at The Hendle Arms on the day after Mrs. Jabber had discovered her master's corpse, and on that same afternoon the body was placed in the family vault of the Leighs. The trouble had happened so suddenly, the proceedings had been carried through so swiftly, that everything in connection therewith was over and done with before people had time to wholly realize what had taken place.

With regard to the inquest, that necessary function was dispatched very quickly. There was little to be done and little to be said, as no new details were forthcoming concerning the dreadful event. The jury inspected the body at the Vicarage, and then went on to The Hendle Arms to hear what could be said about the matter. Several

reporters from London journals were present, but the interest in the case was more local than general, as there was nothing in it likely to cause a sensation. The general opinion was that some burglar had entered the ill-guarded Vicarage, and that the parson had been struck down while trying to capture the thief. But, as nothing was missing from the house, many scouted this idea, and ascribed the death to a deeper cause. But what that cause might be, this minority were unable to say. Nor did the evidence procurable tend to lighten the darkness which shrouded the crime.

Mrs. Jabber, more respectably dressed than usual, and even more voluble, gave her evidence with many tears and sighs. The old woman had been deeply attached to the vicar, and could not understand why he should have met with so terrible and unexpected a death. She deposed to going to bed at ten o'clock as usual, after taking into the study a glass of milk for her master.

"And there I left him, as happy as a trout in a pond," cried Mrs. Jabber, with tears running down her face, "busy with his books as usual; he, enjoying them the more after having been to see Mrs. Patter, as I'm glad to say is getting better, though it's more nor she deserves, her being such a gossip, and----"

Here the witness was checked by the Coroner, on the ground that she was dealing with matters irrelevant to the inquiry. "Did Mr. Leigh expect anyone to visit him on that night?"

"Lord, bless you, no, sir, and if he did, he wouldn't have mentioned it to me."

"You retired at ten o'clock?"

"Me and Jabber, yes, sir, both being tired with the heat and the day's work."

"And you saw nothing of Mr. Leigh until seven the next morning?"

"Not even the nose of him, sir, and I heard no noise, me being a heavy sleeper as Jabber is, although I don't snore, say what he likes."

In fact Mrs. Jabber's statement did nothing to solve the mystery. She admitted that the bolts and bars at the Vicarage were not what they should be, considering the lonely position of the house. "But, Lord bless you, sir, there ain't never been no trouble with thieves and robbers nohow, as there wasn't anything to tempt them."

"Then you don't think that a burglar----"

"No, I don't, sir. There's nothing missing."

Mrs. Jabber stuck to her tale, and what she said was corroborated by her husband, a meek, trembling little man, wholly dominated by his stronger-minded wife. He had gone to bed at ten o'clock; he had heard nothing during the night likely to arouse his suspicions, and the first news he had of the murder was from his wife, when she stumbled on the dead body at seven in the morning. "And then I went and told Kensit all about it," finished Mr. Jabber with a very white face, evidently afraid lest he should be accused of committing the crime.

Tollart, who was just as red-faced, but much more sober than usual, stated that he had been called in by the village constable within an hour after the body had been discovered. Mr. Leigh had been struck on the right temple by some heavy instrument--probably a bludgeon--and the blow, taken in connection with his weak heart, must have caused death instantaneously. The certificate of death was worded to that effect. Leigh was a patient of his, and had never been very strong, added to which, his mode of life had weakened him considerably. On the whole, the shabby, disreputable doctor, knowing that the eyes of his little world were on him, gave his evidence very clearly and resolutely, so that he created a good impression. There was no question as to the cause of death after

Tollart's statement, even though his coupling of heart disease and a blow seemed rather muddled. No one in the village had expected Leigh to live to any considerable age, owing to his delicate appearance, so it was quite certain that the violent assault had killed him. It would have been a wonder to many had he survived the blow.

For no very apparent reason Hendle was called, but all that he could say brought nothing to light. He related how Leigh had dined with him, and how he had called at the Vicarage next day while on his way to London. So far as the witness knew, Leigh was in good health and spirits. "The announcement of his death came as a shock to me," finished Rupert.

"Had he any enemies?" questioned the Coroner.

"Not to my knowledge. A more amiable man never existed."

"Do you know anything of his past life?"

"Only that he had been vicar here ever since I was a child, and was devoted to books and to archæology. With the exception of his parishioners, myself and Mr. Mallien and his daughter, I don't think he ever saw anyone. He was wholly wrapped up in his books."

"Then there was nothing in his past life which suggests any reason why this crime should have been committed?"

"Certainly not, so far as I know."

Inspector Lawson and Kensit, the village policeman, gave what sparse evidence they could. The latter declared that while on his rounds on the night of the murder he had met no one and had seen nothing suspicious when he passed the gate of the Vicarage. At the hour when the crime was said by Dr. Tollart to have been committed, witness was on the other side of the village. Lawson deposed that

no weapon had been found, that no evidence of any intruder had been discovered.

"I understood that the study was in a state of disorder," said the Coroner.

"I gather from many sources that the study was always in a state of disorder," retorted the Inspector.

Kensit, recalled, said that he did not think that the study was even more untidy than usual. Everything was turned upside down--books and papers, "Just as if some one had been searching for something," declared the witness.

"Then you think that the murderer killed the vicar, and then looked about to find something, which he wished to get, and for the possession of which he committed the crime?"

Kensit hesitated. "I am not prepared to go that far," he remarked, after a pause. "All I can say is that I gained some such impression."

When this speech was made, Rupert glanced at Carrington and Carrington looked at Rupert. The same idea struck them simultaneously, that the murderer might have been searching for the will of John Hendle. But then the existence of that document was known only to the dead man, to the barrister and to the Squire. Rupert had been fast asleep when the crime was committed, and Carrington had been in London, so, of course, neither of the two could have had anything to do with the matter. Still, it seemed strange that the books and papers of the deceased should have been messed up. If search had not been made for the will in question, for what had the mysterious murderer been looking? This question both the young men asked themselves, and asked each other when the inquest was over.

It came to an end very speedily. The Coroner could only direct the attention of the jury to the facts laid before them, and did not offer

any opinion, as indeed he could not. The jury brought in a verdict of "Willful murder against some person or persons unknown," which was all that could be done. Then the meeting broke up, the reporters slipped away with their loaded notebooks, grumbling at the dullness of the matter, and the crowd of villagers dispersed to wonder, for the hundredth time, who could have killed their amiable and kindly natured vicar.

"The beast who murdered Leigh could not have been looking for that will."

It was Hendle who spoke, as he walked back to The Big House with Carrington. The barrister shrugged his shoulders and replied, "I had the same idea when that policeman made his statement, and I saw you look at me. I agree with you, although it is strange that the books and papers should have been turned upside down. But only you and I know of----"

"Of course, of course," broke in the Squire quickly, "and, as I was in bed, and you in London, of course we had nothing to do with the matter."

"Did you tell anyone else about the will?"

"No. I never mentioned it to a soul."

"Good. I shouldn't if I were you."

Carrington's tone was so significant that the Squire turned on him in a sharp, inquiring way. "What do you mean?"

"I mean that if anyone knew about the existence of John Hendle's will, and what it meant to you, it is possible that on you suspicion might rest."

"What rubbish!" said Rupert uncomfortably. "I was in bed and asleep at the time the crime was committed."

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