The relationship between serum clozapine concentrations and hematological parameters by a validated mass spectrometric method

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

The

relationship

between

serum

clozapine

concentrations

and

hematological

parameters

by

a

validated

mass

spectrometric

method

Karam

Mazin

Kamil

Gharab

a

_,

_Duygu

_Eryavuz

_Onmaz

a,∗

_,

_Sedat

_Abusoglu

a

_,

Memduha

Aydin

b

_,

_Abdullah

_Sivrikaya

a

_,

_Oguzhan

_Tok

a

_,

_Gulsum

_Abusoglu

c

_,

_Ali

_Unlu

a a_Department_of_{Biochemistry,}_Selcuk_University,_Faculty_of_Medicine,_Konya,_Turkey

b_Department_of_Psychiatry,_Selcuk_University,_Faculty_of_Medicine,_Konya,_Turkey

c_Department_of_Medical_Laboratory_Techniques,_Selcuk_University,_Vocational_School_of_Health,_Konya,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24September2019 Receivedinrevisedform 10December2019 Accepted18December2019 Availableonline20December2019 Keywords:

Therapeuticdrugmonitoring Clozapine

Tandemmassspectrometry Sideeffects

a

b

s

t

r

a

c

t

Objective:Clozapineisoneofthemosteffectivedrugsforresistantschizophrenia,butitsseveremetabolic andhematologicalsideeffectslimittheuseofclozapine.Ithasbeenreportedthatclozapineblood concen-trationsshouldbemaintainedbetween350−600ng/mL.Ouraimwastodevelopadeterminationmethod forclozapineanditsmainmetabolitesnorclozapineandclozapine-N-oxide,toperformvalidationstudies andtoinvestigatethechangeofvariousbiochemicalparametersinpatientsusingclozapine.

Methods:Aliquidchromatography-tandemmassspectrometry (LC–MS/MS)method wasdeveloped andvalidatedforclozapinemeasurement.Thus,bloodsampleswerecollectedfrom38patientswith schizophreniaand32healthyvolunteers.Biochemicalandhematologicalparametersweremeasuredby Beckman-CoulterAU5800(BeckmanCoulter,Brea,USA)andBeckmanCoulterLH780analyzer(Beckman Coulter,Miami,FL,USA),respectively.HormonelevelswereanalyzedusingCobas6000analyzer(Roche Diagnostics,Germany).

Results:TheLC MS/MSmethodwaslinearbetween1.22−2500ng/mL(r2₌_0.9971)_for_clozapine._The retentiontimesofclozapine,norclozapineandclozapine-N-oxidewere0.92,0.89and0.95,respectively. Bloodglucose(GLU)(p=0.025),lowdensitylipoprotein(LDL-cholesterol)(p=0.015),triglyseride(TG) (p=0.042)andtotalcholesterol(TC)(p=0.024)levelswerehigher;hemoglobin(HGB)(0.015),mean corpuscularhemoglobin(MCH)(0.036),redbloodcellcount(RBC)(0.020),neutrophil(NEU)(0.034),and platelet(PLT)(P=0.005)levelswerelowerintheclozapinegroup.

Conclusions:ThisLC–MS/MSmethodwasrapid,simple,cost-effectiveandsuitablefortheroutine cloza-pinemonitoring.Furthermore,norclozapineandclozapine-N-oxidewerealsodetermined.Monitoring ofmetabolicandhematologicalparameterswithclozapinelevelsisveryimportant.However,the limi-tationsofthestudywerethatthemethodwasnotvalidatedfornorclozapineandclozapine-N-oxide,so thevalidationparameterswerenotevaluatedforthesetwometabolites.

Abbreviations:ALT,alanineaminotransferase;AST,aspartateaminotransferase;CLSI,TheClinical&LaboratoryStandardsInstitute;CRE,creatinine;ELISA,enzyme-linked immunosorbentassay;FDA,foodandDrugAdministration;fT3,freeT3;fT4,freeT4;GC–MS,gaschromatography-massspectrometry;GC–MS/MS,gas chromatography-tandemmassspectrometry;GLU,glucose;HDL-cholesterol,highdensitylipoprotein;HGB,hemoglobin;HPLC,high-performanceliquidchromatography;K,potasium; K2-EDTA,dipotassiumethylenediaminetetraaceticacid;LC–MS,liquidchromatography-massspectrometry;LC–MS/MS,liquidchromatography-tandemmassspectrometry; LDL-cholesterol,lowdensitylipoprotein;LYM,lymphocyte;MCH,meancorpuscularhemoglobin;MCV,meancorpuscularvolume;MPV,meanplateletvolume;Na,sodium; NEU,neutrophil;PRL,prolactin;RBC,redbloodcellcount;TC,totalcholesterol;TG,triglyseride;TSH,thyroidstimulatinghormone;WBC,whitebloodcellcount.

∗ Correspondingauthorat:BiochemistryDepartment,SelcukUniversity,FacultyofMedicine,AlaaddinKeykubatCampus,42075,Selcuklu,Konya,Turkey. E-mailaddress:duygueryavuz@hotmail.com(D.E.Onmaz).

https://doi.org/10.1016/j.jpba.2019.113056

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Clozapine, a derivative of tricyclic dibenzodiazepine, is ﬁrst

atypicalantipsychoticdrugintroducedtoclinicalpsychiatry[1].It

isasecondgenerationagentusedinthetreatmentofschizophrenia

orschizoaffectivedisordersresistanttootherantypsycoticsaswell

asschizophreniapatientswithsuicidaltendency[2].Itissuperior

fromconventionalantipsychoticsbecauseitdoesn’traiseprolactin

(PRL)levels,andleadstolessextrapyramidalsymptoms,especially

tardivedyskinesia[3].Furthermore,inthestudynamedastheCost

UtilityoftheLatestAntipsychoticDrugs(CUtLASS2),itwasstated

thattreatmentefﬁcacyofclozapinewashigherthanotheratypic

antypsycotics[4]. Despitealltheseadvantages,there are

differ-entsideeffectsthatlimittheuseofclozapine.Itcancauserare,

serioushematologicalsideeffectssuchasagranulocytosis,

throm-bocytopeniaandanemia[5].Agranulocytosis,aserioussideeffect

in1–2%ofpatientstreatedwithclozapine,limitedtheuseofthe

drugandrequiredthefollow-upofleukocytesandabsolute

neu-trophilscountsbyroutinebloodtests[6].Clozapinehasvarious

metabolicadverse effectssuchasincreaseofblood glucoseand

lipidelevels[7].Atthesametime,sideeffectssuchas

cardiotoxi-city,hepatotoxicity,hypersalivationlimittheuseofclozapine[8].

Consideringitspharmacodynamicproperties,ithasawiderange

ofreceptorbindingcapacity.Clozapinehasahigherantagonistic

effectforD1andD4receptorsthanD2andalsoantagonizes

sero-tonin5-HT2,muscarinic,histamineand␣-adrenergicreceptors[9].

Itisalmostcompletelyabsorbedwhentakenorallyandits

bioavail-abilityvariesbetween60–70%dependingontheﬁrstpasseffect.

Theeliminationhalf-lifeisabout14hatsteady-stateconditions.

ItismetabolizedbythehepaticcytochromeP450enzymesystem

totwomajormetabolitesknownasnorclozapine,active

metabo-lite,andclozapine-N-oxide[10].Clozapineismetabolizedmainly

byCYP1A2attherapeuticconcentrations,whereasitis

metabo-lizedmainlybyCYP3A4athighconcentrations[11].80%ofthe

doseiseliminatedasa metaboliteinurine(50%)andfeces(30

%)[12].Itwasdemonstratedthatthepharmacokineticsof

cloza-pinevaryconsiderably intra-and inter-individualduringrutine

theurapeuticdrugmonitoring[13].GeneticpolymorphismsinCYP

enzymesmayexplainsomedifferencesinclozapinemetabolism.

However,non-geneticfactors(hormones,diseases, age,

medica-tion,smoking) canalso modifyCYP activity and thereby affect

drugmetabolism,leadingtovariationsinserumlevelsofclozapine

[14].Consequently,serumclozapineconcentrationsvarywidely

among individuals. However, clinical response is highly

corre-latedwithserum clozapineconcentrations. Recentstudieshave

reportedthat clozapineserum concentrations should be

main-tainedbetween350and600ng/mlinordertoachieveaneffective

clinicalresponseandavoidseriousadverseeffects[15].Clozapine

drugmonitoringisveryimportanttopreventdrug-relatedadverse

effects,tooptimizedosage,toevaluatepatients’compliancewith

treatment,and topreventdrug-druganddrug-diet interactions

[16].

Up to date, clozapine levels were measured with various

methods such as HPLC-UV, HPLC-DAD, gas

chromatography-mass spectrometry (GC–MS), gas chromatography-tandem

mass spectrometry (GC–MS/MS), capillary

electrophore-sis however, these methods are very laborious, require

long pretreatment steps, are time consuming and have

low selectivity, speciﬁcity and accuracy. LC–MS/MS

has high sensitivity, speciﬁcity, selectivity and

cost-effective.

OuraimofthisstudybydevelopingLC–MS/MSdetermination

methodforclozapineanditsmainmetabolites, wasperforming

validationstudiesforclozapinemeasurementandtoinvestigate

thechangeof variousbiochemicalparameters inpatientsusing

clozapine.

2.1. Patients

ThisstudywascarriedoutinKonyaSelcukUniversityFacultyof

MedicinebetweenFebruaryandDecember2018.38patients

diag-nosedwithschizophreniaorschizoaffectivedisorderaccordingto

DSM-IVcriteriaand32controlswereenrolledtothestudy.Patients

includedinthestudyweretreatedwith100(n=15),300(n=4),500

(n=3),600(n=3)and900(n=13)mgclozapinedailyforatleast4

months.Patientswereexcludedfromthisstudyiftheyhad1)no

responsetoclozapinetreatment;2)intoleranttoclozapine

treat-ment;3)usedadifferentantipsychoticinadditiontoclozapine;4)

usedlipid-loweringagent,antidiabetic,betablockers;5)reported

disease suchas cardiovascular, thyroid, hypertension, diabetes,

adrenal,hepatic.Thecontrolgroupconsistedof32healthy

vol-unteerswithoutanychronicorpsychiatricdisease.Themeanages

ofclozapineandcontrolgroupwere40.94±10.15,40.09±1.67,

respectively.Thisstudywasapprovedbytheethicscommitteeof

SelcukUniversity.(Number:2017/308,Date:01/11/2017).

2.2. Laboratorymeasurements

Bloodsamplesweretakenintotubeswithgelandtubes

contain-ingdipotassiumethylenediaminetetraaceticacid(K2-EDTA)after

12hoffasting.Hematologicalparameters includinghemoglobin

(HGB),meancorpuscularhemoglobin(MCH),redbloodcellcount

(RBC), mean corpuscular volume (MCV), mean platelet volume

(MPV),whitebloodcellcount(WBC),neutrophil(NEU)and

lym-phocyte (LYM)counts weremeasured fromcomplete blood by

Beckman Coulter LH 780 analyzer (Beckman Coulter, Miami,

FL, USA). Serum total cholesterol (TC), low density

lipopro-tein(LDL-cholesterol),highdensitylipoprotein(HDL-cholesterol),

triglyseride(TG),creatinine(CRE),glucose(GLU),aspartate

amino-transferase(AST), alanineaminotransferase (ALT), sodium(Na),

potasium(K),urealevelswereanalyzedwithBeckman-CoulterAU

5800(BeckmanCoulter,Brea,USA)accordingtothemanufacturer’s

instructions.Thyroidstimulatinghormone(TSH),freeT4(fT4),free

T3(fT3), vitamin B12and PRLlevelsweremesured using

elec-trochemiluminescence method (Roche Diagnostics, Cobas 6000

analyzere601module,Germany).

2.3. LC–MS/MSanalysis

2.3.1. Chemicalsandreagents

Clozapine(CASNumber:5786-21-0),acetonitrile(CASNumber:

75-05-8),high-performanceliquidchromatography(HPLC)grade

water(CASNumber:7732)-18-5),formicacid(CASNumber:

64-18-6),sodiumhydroxide(CASNumber:1310-73-2),ethylacetate

(CASNumber:141-78-6),Acetaminophen(CASNumber:103-90-2)

wereobtainedfromSigmaAldrich(St.Louis,MO,USA).

2.3.2. Instrumentationandconditions

ShimadzuHPLCsystem(Kyoto,Japan)consistedofapump

(LC-20AD),anautomaticsampler(SIL-20ACHT)andaunitforonline

degasser(DGU-20A3).API3200triplequadrupolemass

spectrom-eterequippedwithanelectrosprayionizationinterfacewasused

(AppliedBiosystems/MDSSciex)asdetector.

SeparationwascarriedoutusingaPhenomenexC18HPLC

col-umn(50mmx4.6mm,partno:00B-4041-E0).Themobilephase

Awascontaining0.1%formicacidandHPLCgradewaterandthe

mobilephaseBwascontaining0.1%formicacidandacetonitrile.

Theﬂowratewas1mL/minandgradientconditionswere0.1min

50%B,1min50%B,2min100%B,3min100%B,4min50%

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Fig.1. ThechromatogramoftheblankserumsamplespikedwiththeanalytesattheLLOQconcentrations. Representativechromatogramsofblankserumsamplespikedwith1.22ng/mLclozapine.

Table1

Precisionandaccuracyresultsofclozapine.

Intra-day Inter-day

QC Concentration(ng/mL) Mean(ng/mL) Accuracy% Precision(CV%) Mean(ng/mL) Accuracy% Precision(CV%)

LLOQ 1.22 1.3 106.39 6.48 1.38 113.11 7.75 LQC 3.66 3.62 98.96 5.16 3.55 96.99 6 MQC1 1250 1272.4 101.79 3.53 1256 100.48 5.78 MQC2 1875 1930 102.93 3.1 1920 102.4 5.56 HQC 2500 2374 94.96 2.24 2464 98.56 2.96 Table2

Clozapinerecovery%andmatrixeffectresults.

Recovery Matrixeffect

Concentration(ng/mL) 625 312 156 625 312 156

Results(%) 97.76 97.11 102.10 4.24 −6.1 −8.5

Table3

Clozapinestabilitystudybias%results.

Frozen(-20◦_C)_for₄₅_day _Freeze-thaw_stability

Concentrations(ng/mL) 15.Day(%) 30.Day(%) 45.Day(%) 2.(%) 3.(%) 4.(%)

625 −1.5 −5.3 −10.4 −0.7 −1.7 −4.4

250 −2.3 −5.7 −11.2 −1.5 −9.2 −15.9

andtheinjectionvolumewasadjusted40␮l.Totalruntimewas

determined5min.

TheQ1toQ3iontransitionsweredeterminedas327.1/270.1,

152.1/65.1, 313/192.2 and 343/192.2 for clozapine, internal

standard (acetaminophen),norclozapine and clozapine-N-oxide,

respectively.Declustering, entrance, collisioncellexit potential,

collisionenergy,ionsprayvoltage,sourcetemperature,curtain,ion

source(GS1)andionsource(GS2)gasvalueswereadjustedto50,

10,4,35,5000V,350O_C,_20,_40,₆₀_psi,_{respectively.}

2.3.3. Samplepreparation

250␮Loftheinternalstandardwasaddedto250␮Lstandard

solutionorserumsamples.1mLofNaOH(pH11–12)wasadded

toprovidealkaliconditions.Serumsampleswereextractedwith

1.5mLofethylacetate(30svortexed).Themixturewascentrifuged

at3500×rpmfor5min.Aftercentrifugation,theorganiclayerwas

taken into glasstubes and evaporated undernitrogen gas.The

residuewasdissolvedin200␮Lacetonitrile-water(50:50;v:v%)

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Fig.2.Representativechromatogramsof(a)blankserum,(b)internalstandardand(c)theclozapineat625ng/mLinserumwithlow(leftpanel)andhigh(rightpanel) olanzapinelevels.

2.3.4. Methodvalidation

ThevalidationstudywascarriedoutinaccordancewithCLSI

(ClinicalandLaboratoryStandardsInstitute)andFDA(Food and

DrugAdministration)protocols[17,18].Linearity,accuracy,

selec-tivity,precision,recovery,parameterswereevaluatedatthisstudy.

2.4. Statisticalanalysis

StatisticalanalysiswascarriedoutbyusingSPSSstatistical

soft-warepackageversion21.0,EPEvaluatorRelease8versionandExcel

(2010),p<0.05wasconsideredasstatisticallysigniﬁcant.

3. Results

3.1. LC–MS/MSanalysis

3.1.1. Methodvalidationofclozapine

Thecalibrationstandardswerepreparedbyspikingworking

solution(10% of total serumvolume) toblankserum and

lin-earity study was performed with these spiked samples in the

concentration range of 0.076–2500ng/mL and each

concentra-tion level was analyzed in duplicate. Results were evaluated

by linear regression analysis. The LC–MS/MS method was

lin-earbetween1.22−2500ng/mL.Correlationcoefﬁcient,slopeand

interceptparametersweredeterminedas0.9971,0.9969and0,

respectively.

Limitofdetectionwascalculatedbasedonasignaltonoiseratio

of3.Limitofdetectionwasdeterminedas0.076ng/mL.Thelowest

concentrationlevelatwhichthesignaltonoiseratiowas

approxi-mately10withacceptableaccuracyandprecisionwasdetermined

asthelowerlimitofquantiﬁcation(LLOQ).TheCLSIguideline

rec-ommendstheuseofdatafrom20replicatesofaspikedsample

fromatleast5differentrunsovera5-daytimeperiodforLLOQ.

LLOQhaveaprecisionoflessthanorequaltoacoefﬁcientof

vari-ation(CV)of20 %and anaccuracyof80–120%ofthenominal

analyteconcentration.LLOQwasdeterminedas1.22ng/mL.The

intraandinter-dayprecisionandaccuracyatLLOQconcentration

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Table4

Clozapineinterferencestudybias%results.

D1 D2 D3 D4 D5

625ng/mLbias% −2.14 −2.70 −2.72 −3.98 −4.16

312.5ng/mLbias% −1.24 −1.80 −1.12 −2.56 −4.56

D1:0%,D2:5%,D3:50%,D4:75%,D5:100%interferant(olanzapine)level.

valuewasacceptedtobe1.22ng/mL,sincetheconcentrationlevel

wherethesignal-to-noiseratiowas≥10withacceptableaccuracy

andaccuracywas1.22ng /mL.Thechromatogramoftheblank

serumsamplespikedwiththeanalytesattheLLOQconcentrations

demonstratedinFig.1.

Accuracyandprecisionweredeterminedanalyzingsixsamples

perlevelattheLLOQ,low,medium1,medium2andhighQC

sam-ples(1.22,3.66,1250,1875,2500ng/mL)for5consecutivedays.

Accuracyiscalculatedasapercentageofthemeasuredvalueto

theexpectedvalue,andtheprecisionresultsareexpressedasCV%.

ResultsofprecisionandaccuracystudieswereexpressedinTable1.

Theacceptabilitycriterionforaccuracyand precisionexceptfor

LLOQisthatthedeviationvalueislessthan15%.Theacceptability

criteriaforaccuracyandprecisionatLLOQisupto20%.The

accu-racyandprecisiondeviationlevelsarelowerthan15%foralllow,

medium1,medium2andhighqualitycontrollevels,andlessthan

20%forLLOQ.Theresultsshowedverygoodaccuracyandaccuracy.

Toevaluatetheefﬁciencyoftheextractionprocess,samples

werepreparedat threedifferentconcentrationlevels(156, 312

and625ng/mL)andrecoverystudywasperformed.Recoverystudy

wascarriedoutbycomparingthemeasurementresultsofextracted

andunextractedsamples.Thematrixeffectstudywasperformed

according to the proceduredescribed by Chambers et al. [19].

Resultsofrecoveryand matrixeffectstudieswerepresentedin

Table2.

Stability studies were performed at two different (250 and

625ng/mL)concentrationlevelstoassessthestabilityof

clozap-ineinserumat-20◦Candafterfreeze-thawcycles.Resultswere

presentedinTable3.

Carry-overstudywasperformedbyanalyzingconsecutivelow

andhighlevelstandards.Themeanandstandarddeviationvalues

ofgroupswerecalculatedwithEPEvaluatorRelease8program.The

carry-overvaluewasdeterminedas-21.4ng/mL.

Theselectivitystudywasperformedbyanalyzingsixdifferent

humanblankserum samplesandinvestigate potential

interfer-encesofolanzapin.Thechromatogramsofblankserum,internal

spikedserumandanalytespikedserumwerecomparedatlowand

higholanzapinelevels(Fig.2).

Thesechromatogramsdemonstratethatthismethodwas

selec-tive for clozapine. Furthermore, samples containing 312.5 and

625ng/mL clozapine in six different individual sources of the

appropriateblankserummatrixforeachconcentrationlevelwere

preparedthen0%,25%,50%,75%,100%olanzapinewasaddedto

eachsample.Theresultsoftheinterferencestudieswereexpressed

asbias%inTable4.

3.1.2. LC–MS/MSmeasurementofclozapinelevels

Serum samples of 38 patients using clozapine were

ana-lyzed by LC–MS/MS. Clozapine serum levels altered between

42.90–1785ng/mL. Serum clozapine and norclozapine levels

were measured as 594.90±492.90 and 220.33±182.55ng/mL,

respectively. The median (min-max) serum concentrations of

patientsusing clozapineat dosesof100, 300,500, 600,900mg

dailyweredeterminedas137.85(42.90–760.5),336(186–292.5),

451.5(426–549), 844.5(784.5–944.5), 1078.5(603–1785) ng/mL,

respectively.Achromatogramofclozapineanditsmain

metabo-litesisshowninFig.3.

Table5

.Demographicandlaboratoryparametersofallgroups.

Clozapinegroup Controlgroup p

Age 40.94±10.16 40.10±8.67 0.640

Gender(F/M) 21/17 18/14

ALT 17(5–77)U/L 17(8–28)U/L 0.339

AST 21(13–40)U/L 18.5(12–34)U/L 0.136

GLU 93(76–343)mg/dL 89(75–98)mg/dL 0.025 5.2(4.2–19)mmol/L 4.94(4.2–5.4)mmol/L Urea 24.26±8.48mg/dL 27.81±7.43mg/dL 0.060 4.0±1.41mmol/L 4.63±1.24mmol/L CRE 0.76±0.16mg/dL 0.77±0.15mg/dL 0.880 0.07±0.01mmol/L 0.06±0.01mmol/L TG 147.5(40–493)mg/dL 97.5(75–98)mg/dL 0.042 1.7(0.5–5.6)mmol/L 1.1(0.8–1.1)mmol/L HDL 42(30–71)mg/dL 45(27–67)mg/dL 0.065 1.08(0.8–1.8)mmol/L 1.2(0.7–1.7)mmol/L LDL 142(57–187)mg/dL 122(94–157)mg/dL 0.015 3.7(1.5–4.8)mmol/L 3.2(2.4–4.1)mmol/L TC 218.63±36.24mg/dL 200.56±27.45mg/dL 0.024 5.7±0.94mmol/L 5.2±0.91mmol/L Na 139(136–143)mmol/L 138.5(134–143)mmol/L 0.226 K 4.30(3.86–14.11)mmol/L 4.34(3.82–5.53)mmol/L 0.641 PRL 14.67(4.39–138)␮g/L 12.31(4.74–80.46)␮g/L 0.869 B12 344.1(207–686)ng/L 311.3(176–601)pg/mL 0.596 TSH 1.55(0.28–13.71)mU/L 1.8(0.59–4.88)mU/L 0.612 fT3 2.95±0.46ng/L 3.08±0.38pg/mL 0.19 4.53±0.71pmol/L 4.73±0.58pmol/L fT4 1.27(0.61–1.8)ng/dL 1.26(0.83–1.93)ng/dL 0.804 16.7(7.85–23.16)pmol/L 16.21(10.6–24.8)pmol/L RBC 4.76±0.45106_/␮L _5.02_±_0.45₁₀6_/␮L _0.020 4.76±0.451012_/L _5.02_±_0.45₁₀12_/L MCV 89.24±4.71fL 86.10±3.34fL 0.002 MCH 21.95±1.79pg 30.80±1.48pg 0.036 1.36±0.11fmol 1.91±0.09fmol PLT 212.59±84.55k/␮L 265.47±62.34k/␮L 0.005 212.59±84.55109_/L _265.47_±_62.34₁₀9_/L MPV 8.2(6.7–12.5)fL 8.8(8.1–10.5)fL 0.003 WBC 7.25(3.6–108)k/␮L 7.1(4.3–11.6)103_/␮L _0.581 7.25(3.6–108)109_/L _{7.1(4.3–11.6)}₁₀9_/L HGB 14.12±1.16g/dL 14.75±0.89g/dL 0.015 141.2±11.6g/L 147.5±8.9g/L NEU 3.84(2.22–7.5)k/␮L 4.79(2.46–7.0)103_/␮L _0.034 3.84(2.22–7.5)109_/L _{4.79(2.46–7.0)}₁₀9_/L LYM 1.99±0.54k/␮L 2.24±0.81103_/␮L _0.140 1.99±0.54109_/L _2.24_±_0.81₁₀9_/L

3.2. Biochemicalmeasurementresults

38 patients used clozapine and 32 heathy volunteers were

includedthisstudy.Themeanageofclozapineandcontrolgroups

was40.94±10.15,40.09±1.67,andtherewasnostatistically

sig-niﬁcant difference between themean ages of the two groups.

Whenthetwogroupswerecomparedstatistically,itwasshown

thatGLU(p=0.025),TG(p=0.042),LDL-cholesterol(p=0.015),TC

(p=0.024),MCV(0.002)levelswerehigherintheclozapinegroup

comparedtothecontrolgroupandRBC(p=0.020),MCH(0.036),

HGB(0.015),PLT(0.005),NEU(0.034)levelswerelowerthanthe

control group.Furthermore, PLT(179.18±84.70, 239.45±76.03

k/␮L [179.18±84.70, 239.45±76.03 109_/L]; _p₌_0.027) _and _fT3

(2.74±0.36, 3.12±0.47ng/mL [4.21±0.55, 4.79±0.72pmol/L];

p=0.010)levelswerestatisticallysigniﬁcantlowerinpatientswith

serum clozapineconcentrations above 600ng/mL than patients

withserumclozapineconcentrationsbelow600ng/mL.All

demo-graphicﬁndingsand laboratorymeasurementsarepresentedin

Table5.

Furthermore,itwasshowedthatserumclozapinelevelswere

positively correlated with TC (r=0.335, p=0.040)

concentra-tion and negatively correlated withHGB (r=−0.342, p=0.025),

PLT (r=−0.362, p=0.025), NEU (r=−0.385, p=0.017) and fT3

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Fig.3.Chromatogramforclozapine(1530ng/mL),norclozapine(310ng/mL)andclozapine-N-oxideintheserumsample.

4. Discussion

Primaryoutcomewastovalidateamassspectrometricmethod

forclinicaluseand toﬁndout themetabolicchanges and

rela-tionship with serum clozapine concentrations. Clozapine is an

atypicalantipsychoticthatiscommonlyusedinthetreatmentof

schizophreniaandiseffectiveagainstbothnegativeandpositive

symptomsofschizophrenia.Clozapineissuperiortoconventional

antipsychoticsbecauseit causesless extrapyramidal symptoms

anddoesn’taffectprolactinlevels,butitcancausesevere

hema-tological,metabolic sideeffects that limit the useofclozapine.

Atthesametime, theraupeuticdrugmonitoringisessential for

clozapine due to the fact that clozapine levels vary

consider-ablyamongindividualsandthefrequencyofseriousdrug-related

adverseeffects increaseswithincreasingdrugblood

concentra-tion.Variousstudies [20,21], haveshown that theincidence of

adverse effects increases when serum clozapine concentration

exceeds750ng/mL.Therefore,monitoringofclozapinelevelswith

metabolicandhematologicalparametersinpatientsusing

cloza-pineisextremelyimportant.DifferentLC–MS/MSmethodswere

developedforclozapinemeasurement.

Wohlfarthetal.[22]developedamethodforthedetermination

ofclozapineanditsmetabolitesbyQTraptandemmass

spectrom-eter,andthismethodperformedchromatographicseperationin

15min.Ourmethodcanperformseperationinamuchshortertime

(5min).AravagiriM.andMarderSR[23]performedtandemmass

analysisusing500␮Lofsampleinthepretreatmentsteps,250␮L

sampleissufﬁcientforourmethod.Inthemethoddevelopedby

AravagiriM.andMarderSRwasusedahighvolume(7mL)mixture

oforganicsolventsconsistingofethylacetate,methylenechloride

andpentaneinthepretreatmentsteps.Itwasusedonlyoneorganic

solvent(ethylacetate)inasmallervolume(1.5mL)during

extrac-tioninourmethod.Itsvortexingtimeduringextractionis10min

whileinourmethod30sissufﬁcient.Inthemethoddevelopedby

AravagiriM.andMarderSR,CV%valuesforclozapinevarybetween

3.2%–12.5%andinourmethoditvariesbetween2.24%–7.75%.Its

extractionrecoveryis84%andourrecoveryisover97%.In

sum-mary,ourmethodishighlyadvantageousinthatitrequiresasmall

amountofsample,isaneconomical,practicalmethod,andhashigh

accuracyand recovery.However,theLLOQ value(1ng/mL)and

retentiontime(<3min)forclozapineweresimilartoourmethod

(1.22ng/mLand<1.5min,respectively).

Domingues et al. [24] reported LC–MS/MSmethods for the

determination of various drugs including clozapine and this

methodwas linear in the 1.5−1550ng/mL concentration range

andcorrelationcoefﬁcientwashigherthan0.99forclozapine.Our

methodwaslinearbetween1.22−2500ng/mLandcorrelation

coef-ﬁcientwashigherthan0.99.

Niederlaenderetal.[25]developedLC–MS/MSmethodbyusing

on-linesolidphaseextractiontodetermineclozapinelevels,

quan-titationanddetectionlimitswerereportedas0.15and50ng/mL,

respectively.Ourlimitofdetectionandquantitationvalueswere

0.076and1.22ng/mL,respectivelyfurthermoreourmethodis

sim-pleandcost-effective.

Raoetal.[26]determinedintra-dayprecisionCVsas4.6%and

for 325ng/mL and 2.1 %for 607ng/mL. Between-day CVswere

expressedas6.7 %for 325ng/mL and 4.1 %for607ng/mL. Our

resultsareconsistentwiththesevalues.

The extraction recovery was determined between

97.11%–101.13%,theextractionstepisverysimpleandsufﬁcient.

Anotherimportantaspectofourstudywastheinvestigation

ofthechangeofvariousbiochemicalparametersinpatientsusing

clozapine.Variousstudieshavereportedthatclozapinetreatment

isassociatedwithincreasedserumlipidandGLUlevelsandmay

causemetabolicdisorderssuchasweightgain,hyperlipidemiaand

hyperglycemia.Lindenmayeretal.[27]reportedincreasedserum

GLUandTClevelsinpatientsusingclozapine.Idonijeetal.[28]

reportedincreasedLDL-cholesterol,TC,TGlevelsandreduced

HDL-cholesterollevelsinclozapinegroupcomparedwithcontrolgroup.

(7)

lev-elswerehigherintheclozapinegroupthaninthecontrolgroupand

therewasapositivecorrelationbetweenclozapineblood

concen-trationandTClevels.

Alsoinourstudy,serumfT3levelswerenegativelycorrelated

withserumclozapinelevels.Clozapinemightaffectthyroid

func-tions. Thus metabolic disturbances in patientstaking this drug

might berelated withaltered thyroid function. Althoughthese

resultswereobservedwithlimitednumberofpatientsinthisstudy,

thisissuemustberesearchedbyanalyzingalargescaleparticipiant

study.

Althoughneutropeniaisawell-knownhematologicalsideeffect

ofclozapine,differenthematologicaladverseeffectssuchas

throm-bocytopenia andanemia due toclozapinetreatmenthavebeen

reportedinvariousstudies[29,30].WefoundthatNEU,HGB,RBC,

MCHlevelswerelowerinpatientsusingclozapinecomparedto

controlgroup,andtherewasanegativecorrelationbetween

cloza-pinebloodconcentrationandHGB,NEUandPLTlevels.

5. Conclusions

TheLC–MS/MSmethodisveryfast,simpleandcost-effective.

Ithashighaccuracy,sensitivityand speciﬁcitythusit hasideal

validationproperties.Itcanalsodetectnorclozapineand

clozapine-N-oxideinserum.Ourmethodissuitableforroutinedruglevel

monitoringofclozapine.Inaddition,ourstudyiscomprehensive

anduniqueinthatitevaluatestherelationshipbetween

clozap-inelevelsandvariousclinicalparameters.Regularmonitoringof

biochemicaland hematological parametersaswellas clozapine

druglevelisextremelyimportant..However,thelimitationsofthe

studywerethatthemethodwasnotvalidatedfornorclozapineand

clozapine-N-oxide,sothevalidationparameterswerenot

evalu-atedforthesetwometabolite,lackofCYPpolymorphismstudiesof

ourpopulationandthereisaneedforfurtherstudiestoclarifythe

variationinclozapinebloodconcentrationsbetweenindividuals.

CRediTauthorshipcontributionstatement

Karam MazinKamilGharab: Validation, Investigation,Data

curation,Software. DuyguEryavuzOnmaz: Methodology,

Vali-dation,Investigation,Writing-originaldraft,Writing-review&

editing. Sedat Abusoglu:Methodology, Investigation, Writing

-review&editing.MemduhaAydin:Investigation,Project

adminis-tration.AbdullahSivrikaya:Writing-review&editing.Oguzhan

Tok:Validation,Software.GulsumAbusoglu:Validation,Software.

AliUnlu:Conceptualization,Projectadministration.

DeclarationofCompetingInterest

None.

Acknowledgment

This work was supported by Selcuk University Scientiﬁc

Research Projects Coordinator (grant number 17202068).The

authorsthanktheSelcukUniversityforfundingthisproject.

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