Bioanalytical Development and Validation of Liquid Chromatographic

7/25/2019 Bioanalytical Development and Validation of Liquid Chromatographic

1/24

Bioanalytical development and validation of liquid chromatographictandem mass

spectrometric methods for the quantification of total and free cefazolin in human plasma and

cord blood

a b s t r a c t

Objectives:

Cefazolin is a commonly prescribed -lactam antibiotic for prophylais against s!in

infections follo"ing surgery# including caesarean sections$ %ssessment of maternal and

neonatal eposure is important for correlating drug concentrations to clinical outcomes$ &hus#

bioanalytical methods for the quantification of both total and free cefazolin in maternal

plasma and cord blood can assist in the comprehensive evaluation of cefazolin eposure$

'esign and methods:

(pecimen preparation for the measurement of total cefazolin "as performed via protein

precipitation "ith acetonitrile containing the internal standard cloacillin$ )ltrafiltration "as

used to isolate free cefazolin$ *rocessed samples "ere analyzed on a *relude (*+C system

coupled to a &( ,triple quadrupole antage mass spectrometer$ .ethods "ere validated

follo"ing /'% bioanalytical guidelines$

0esults:

&he analytical measuring ranges of these methods "ere 1$23231 mg4m+ and 1$12323

mg4m+ for total and free drug# respectively$ Calibration curves "ere generated using 54 6

"eighted linear regression analysis$ &otal cefazolin demonstrated inter- and intra-assay

precision of 7 618 at the ++O, and 7 55$68 at other levels$ /ree cefazolin demonstrated

inter-and intra-assay precision of 7 53$98 at the ++O, and 7 56$8 at other levels#

respectively$ %ccuracy ;8'


2/24

1. ?ntroductionCephalosporin antibiotics have become one of the mostcommonly prescribed classes

of -lactams due to their broad spectrum of anti- bacterial activity# lo" toicity

profile# and ease of administration A5#6$ Consequently# cephalosporins have been

utilized for the treatment of a number of soft tissue and s!in infections in both

perioperative and post-surgical settings# particularly as prophylactic agents A5#9$

Cephalosporins are semi-synthetic compounds initially derived from the fungus

Cephalosporium acremonium A$ (tructurally# these drugs are comprised of a -

lactam moiety fused to a#-dihydro-6D-5#-thiazinering A5$ &here are several

generations of cephalosporin antibiotics# and family members are stratfied based on

their anti-bacterial activity and route of administration$

Cefazolin# a first-generation cephalosporin# is "idely used to manage s!in infections#

and has also sho"n therapeutic efficacy in the treatment of pulmonary infections and

methicillin-susceptible (taphylococcusaureus AE#3$ +i!e other first-generation

cephalosporins# cefazolin is effective intreating Fram-positive bacteria# but does not

elicit equally effective bactericidal effects against Fram-negative bacteria A#>$

.oreover# cefazolin has been administered prophylactically to prevent post-surgical

s!in infections# including those incurred during cesarean deliveries A51#55$ Cesarean

delivery is the primary ris! factor for postpartum maternal infections caused by Fram-

positive bacteria# and studies have demonstrated that the infection rate in a post-

cesarean setting can be as high as 38 A56$ Gotably# the prophylactic administration

of cefazolin prior to s!in incision reduced post-cesarean morbidity as "ell asendometritis# and has been recommended by the %merican College of Obstetrics and

Fynecology as a perioperative prophylactic agent during cesarean deliveries A555$

Hhile the majority of the drug is predominantly protein bound by albumin ;I>18

bound=# cefazolin elicits its antimicrobial effects in the unbound#or free# form A525$

&hus# determination of both total and free cefazolin concentrations may be helpful in

better characterizing the pharmaco!inetics of the cephalosporin in a variety of clinical

scenarios# including in a post-cesarean setting$ Cefazolin quantification in the mother#

as "ell as the neonate# can provide clarity on neonatal drug eposure# as "ell as its

potential impact on post-delivery out comes# including the prevention of neonatalsepsis A5E$ Geonatal drug eposure measurement has been assessed in many matrices

including urine# blood# meconium# hair# and umbilical cord blood$ )mbilical cord

blood is a convenient matri to assess neonatal eposure because it should reflect

recent changes in neonatal eposure at the time of delivery "ithout having to perform

a venipuncture$

Cefazolin quantification has been previously reporte dusing high performance liquid

chromatographic ;D*+C= A5361 or liquid chromatographictandem masss

pectrometric ;+C.(4.(= A6569 approaches$ Do"ever# none of the a for

ementioned methods have etensively evaluated the acceptability of cord blood as amatri for cefazolin determination$Hhile several methods provide strategies for


3/24

directly quantifying free drug concentration# only the methods published by

Jhangetal$ A65 and 'ouglasetal$ A6 utilized tandem mass spectrometry as

ananalytical detector A5361#66$ Dere "e present a comprehensive method

describing the development and validation of +C.(4.( methods for the

quantification of total and free cefazolin via ultrafiltration in plasma and cord blood$&he method use salo" volume of specimen for determination# requiring less than 691

m+ for determination of both total cefazolin and free drug "ith in a sample$

2. .aterials and methods6$5$ Chemicals

Cefazolin ;>E8purity= as its sodium salt# its structural analogue cloacillin

;>28purity= as its hydrated sodium salt ;/ig$ 5=# and ethylene diamine tetra acetic acid

;1#6211# and

23#111 mg4m+ in 5:5 acetonitrile : "ater$ Hor!ing solutions for the preparation of ,C

materials "ere diluted into K611


4/24

mg4m+ "as prepared by "eighing standard material and diluting volumetrically# and

subsequent "or!ing stoc! solutions "ere prepared as previously described$ &hese

"or!ing solutions "ere used to prepare ,C materials in plasma ultra filtrate at final

concentrations of 1$123 ;++O,=# 1$52 ;lo" ,C=# 5$3 ;mid ,C=# and 25 ;high ,C=

mg4m+$ ?nternal standard solutions "ere prepared in D*+C grade acetonitrile for totalcefazolin determination and D*+C grade "ater for free cefazolin determination at

final concentrations of >$6 mg4m+ and5$3 mg4m+# respectively# by diluting a 5$3

mg4m+ stoc! solution that "as prepared by "eighing cloacillin and diluting it

volumetrically using 918 acetonitrile$

6$$ (ample preparation

6$$5$ &otal cefazolin

&otal cefazolin concentrations in plasma and cord blood "ere determined after

samples had been subjected to protein precipitation$ &"enty microliters of calibratoror

,C "as diluted 5 : 91 "ith acetonitrile at ambient temperature containing >$6 mg4m+

cloacillinina /isherbrand 5$9 m+ polypropylene micro centrifuge tube ;Haltham#

.%M final cloacillin concentration of >$1 mg4m+=$ &his miture "as vorteed for 9

min using a (cientific ?ndustries orte Fenie 6 ;Bohemia# Ge"Nor!=$ %fter miing#

samples "ere centrifuged at 5E#>65 g for 9 min using an


5/24

;Corning (*?G-P)/ 61 m+ capacity 51 !.H cut off concentration devices=$ *lasma

"as added to spin devices and centrifuged at 529 g for 1 min using a &hermo

(cientific Qouan C2 is "inging buc!et centrifuge ;(an Qose# C%=$ )ltrafiltrate "as

pooled from collections and used in the preparation of the calibrators and ,C

materials$

6$9$ ?nstrument and acquisition parameters

Chromatographic separation "as performed on the &hermo (cientific *relude (*+C

system ;(an Qose# C%= operating in laminar only mode ;+P=$ (pecimens "ere

maintained at 2 5C in a C&C*%+ autosampler ;Carrboro# GC=$ Cefazolin and its

structural analog "ere chromatographically separated using a *henomene Kinete

C3 ;91I6$5 mm6# 5$E mm particle size= column ;&orrance# C%= maintained at 21C$

&he mobile phase system consisted of "ater containing 68 acetonitrile and 1$58

formic acid ;mobile phase %# .*%= and a cetonitrile containing 1$58 formic acid

;mobile phase B# .*B=$ % third solvent consisting of 29:29:51 acetonitrile :

isopropanol : acetone "as introduced to the analytical column during the "ash step$

&o reduce carry over#51 + of 5 m. e= and 2E-51 m4z ;C< : 59e=# respectively$

6$$ 'ata evaluation

Pcalibur 6$6 soft"are ;&hermo (cientific= "as used for data acquisition and

processing$ Calibration curves "ere generated using linear regression "ith 546

"eighting and pea! areas generated after Faussian smoothing using the Fenesis

integration algorithm$.icrosoft Office


6/24

Chromatographic conditions for the determination of cefazolin and free cefazolin$

Coefficient of variation ;8 C= is defined belo"#"here S is the standard deviation

and is the average concentration observed$

8 '


7/24

Concentrations determined "ere those generated from the calibration curve performed on

each run$

$6$ Carryover

Carryover "as determined by alternating injections of lo" ;+= concentration calibrator

;either 1$23 or 1$123 mg4m+ for cefazolin or free cefazolin# respectively= and high ;D=

concentration calibrator ;either 251 or 25 mg4m+ for cefazolin or free cefazolin#

respectively= and comparing the mean concentration of the lo" calibrators injected prior

to high calibrator to those injected after$ (pecifically#calibrators "ere injected in the order

: +5# +6# +# D5# D6# +2# D# D2# +9# +# +E# +3# D9# D# +># DE# D3# +51# D># D51#

and +55$ %cceptability criteria "ere mean post-high injection levels L(' greater than

pre-high injection levels$ &hese acceptability criteria are consistent "ith C+(? protocol


8/24

.atri effects "ere evaluated by there covery ratio of post- etracted:

unetractedM recovery efficiency "as evaluated by there covery ratio of pre-

etracted: post-etractedM processing efficiency "as evaluated by there covery

ratio of pre-etracted: unetracted$

$9$ Cord blood comparison

&he described +C.(4.( method "as also used to assess drug quantification of

total cefazolin in cord blood and free cefazolinin cord blood ultrafiltrate#

respectively$ %cceptability of quantifying drug concentrations in cordblood from

an adult human plasma ;non-cord blood= calibration curve "as determined by

calculating 8'?/ of lo"# mid# and high ,C levels as compared to adult plasma

,C material analyzed on the same calibration curve$ Cefazolin "or!ing solutions

for the preparation of ,C materials for total and free cefazolin "ere used to

generate equivalent concentration materials in both cord blood plasma and plasma

ultrafiltrate$ Cord blood plasma ultrafiltrate "as prepared using the same protocolfor determination of free cefazolin: briefly# Corning ultrafiltration devices "ere

implemented for the filtration of 611 m+ sample$ % pool of ultrafiltrate "as

generated for the preparation of ,C materials$ Cord blood materials "ere prepared

at the same lo"# mid# and high ,C concentrations as prepared for total and free

cefazolin determination$ %cceptability limits "ere defined as 7U598'?/ from

material prepared in human plasma or human plasma ultrafiltrateM this

acceptability threshold has been implemented for stability-challenged samples#

and "as thus applied "hen comparing plasma and cord blood results$

2$ 0esults and discussion

2$5$+iquid chromatographictandem mass spectrometric ;+C.(4.(= parameters

Cefazolin "as chromatographically separated from matri components under a

gradient elution profile "ith a *henomene Kinete C3 column ;&able5 and /ig$ 6=$

)sing the described method# cefazolin and cloacill in eluted at 5$9 and 6$1 min#

respectively$ &he *henomene Kinete C3 column is a sub-6 mm porous particle

column that lend sit self to clinical applications because of fast# sharp pea!s and

moderate bac! pressure A6>$ 'ue to the lac! of availability of isotopically labeled

cefazolin# a -lactam structural analogue# cloacillin# "as used as an internal standard$Other methods described for cefazolin analys is also used structural analogues as

internal standards A669$ %cetonitrile "as chosen as mobile phase Basit

demonstrated higher ionization efficiency than methanol ;data not sho"n=$ &he

addition of


9/24

method$ &he strong "ash solvent "as introduced using a second binary pump "ith as

"itching valve and a tee into the analytical column$

Optimized ionization source and mass spectrometry parameters for cefazolin and

cloacillin "ere determined by manually adjusting conditions during direct infusion$

Both compounds "ere analyzed in their protonated A. V DVforms$ (elected reaction

monitoring ;(0.= mode ion transitions "ere selected on the basis of signal intensity

and imprecision during suitability eperiments$ .oreover# though the molecular

formula of these fragment ions "as not confirmed "ith labeling studies# their masses

indicate they are not products of common losses ;condensation# decarboylation# etc$=

and may contain the tetrazole ring#"hich is the characteristic 06moiety for cefazolin$

Chromatograms generated from qualifier ion transitions "ere evaluated on the basis

of their pea! shape$ %pplication of qualifier transitions is especially important "hen

utilizing a structural analogue as an internal standard for quantitative mass

spectrometry$ One major consequence of a structural analogue as an internal standardis that it often does not co-elute "ith the analyte$ *otential interferences may cause

ion suppression and significantly affect the accuracy of results$

&he method published by 'ouglasetal et al$ used in vivo microdialysis to determine

free cefazolin concentrations# "hich is impractical for routine clinical testing A6$

&he method published by Jhangetal et al$ presented a relatively comprehensive

validation for determining total and free cefazolin in human plasma# but did not

provide details on comparative studies in cord blood# nor did it provide details on

?mproving analytical selectivity through the introduction of a qualifier ion transitionA65$


10/24

/ig 6$ 0epresentative chromatograms for ;%= cefazolin ++O, lo" ,C and blan!

palasma and ;B= cloacillin internal standard and blan! plasma$

/ig $ 0epresentative chromatograms of carryover oprimization sequences$

Chromatograms from ;%= are prior to optimization$ Chromatograms from ;B= are

post-optimization$

2$6$ *recision# accuracy and calibration curve analysis

?ntra - and inter-assay precision and accuracy for total cefazolin and free cefazolin

"ere determined follo"ing there commendations of the /'% guide lines as previously

described ;&able6=$ &otal cefazolin intra-assay precision ranged from $68 to

61$18C at the ++O,$ ?ntra- assay accuracy as 8'$58 at the ++O,$ ?nter-

assay accuracy as 8'8$ /ree cefazolin intra-assay

precision ranged from E$68 to 5E$38 at the ++O,$ /ree cefazolin intra-assay

accuracy ranged from I$E8 to $28$ /ree cefazolin inter-assay precision ranged

from 9$E8 to 53$98 and inter-assay deviation ranged from I5$>8 to 6$98$ %ll

observed results "ere acceptable for all ,C levels$ &he %.0s of the assays "ere

1$23231 mg4m+ for total cefazolin and 1$12323 mg4m+ for free cefazolin ;/ig$ 2

and data not sho"n=$ &he %.0s are sufficiently dynamic and encompass typical

cefazolin concentrations observed follo"ing standard dosing# as total cefazolin

plasma concentrations typically achieve levels I5$2525 mg4m+ A66$ &he %.0 "as

epanded I-fold beyond this limits to account for unepected over-dosing# under-

dosing# as "ell as studies that adjust the timing of dosing$ (tandard dosing protocolsinclude56 g cefazolin t$i$d$ for treatment of infections or 56 g single dose for

prophylais during surgery A6#5#6$ &he analytical measuring range of unbound

cefazolin spans 5 order of magnitude less than the analytical measuring range for total

cefazolinM this is consistent "ith plasma protein binding resulting in free

concentrations 518 of total concentrations A$ Both assays utilized standard curves

constructed using 546 "eighted linear regressions of the pea! area ratio of cefazolin

and cloacillin relative to the cefazolin concentration of calibrators$ &he standard

curve intercepts and linear coefficients "ere used to determine total cefazolin and free

cefazolin concentrations in quality control materials and un!no"n specimens$ &he

average correlation coefficient 06 for regression fittings "as W 1$>> ;/ig$ 2=$ &he

upper limit of quantification "as determined by observing the highest concentration

that provided a linear calibration curve# in this method that concentration ;231 or 23

mg4m+ for total cefazolin and free cefazolin= provided an analytical measuring range

of three orders of magnitude$ &he upper end of the calibration curve does appear to

slightly under-recover# as reflected by the high ,C bias of IE$E8# but this small value

"as still "ith in the acceptability criterion defined by /'% Bioanalytical .ethod

alidation guidelines$ %doption of an isotopically labeled internal standard of

cefazolin may mitigate this bias$

Table2 Inter-assay and intra-assay precision and accuracy


11/24

Fig. 4. Representative calibration curves for total cefazolin analysis.

The y-axis of !" is the pea# area ratio observed bet$een cefazolin

and cloxacillin %uantifier transitions. The y-axis of &" is the

calculated concentration using linear regression.

2$$ Carryover

Carryover analysis "as conducted according to C+(? guidelines by evaluating the

determined average concentration of lo" concentration standards injected in

alternating series "ith high concentration standards A6E$ Overcoming issues

"ithcarryover "as critical during method development because of the broad dynamic

range of the method$ Carryover "as not noticed "hen observing analogous signal of

the cloacill in internal standard# suggesting potential accumulation of cefazolin in the

(*+C system at high concentrations$ Based on these observations# astringent "ash of

29:29:51 acetonitrile : 6-propanol : acetone "as introduced during the "ash step as

"ell as introducing


12/24

;7 U 598 '?/=$ Do"ever# the freezetha" cycle data all indicate negative differences#

"hich suggest some instability of the analyte to sustain freetha" cycles# leading to

degradation$

2$9$ (electivity and matri effects

Go pea!s "ere observed near the retention times for cefazolin or cloacillin in blan!

plasma ;/ig$ 6=$ .oreover# si independent lots of human plasma "ere evaluated in

the process and sho"ed no interfering pea!s ;data not sho"n=$ .atri effects "ere

evaluated by comparing pea! area signals generated from cefazolin spi!ed into "ater

;unetracted= and processed according to the protocol# cefazolin spi!ed into plasma

and processed according to protocol ;pre-etracted=# or spi!ed into blan! plasma

etract ;post-etracted=$ .atri effects are determined by comparing the apparent

signal from post-etracted material to unetracted material$ &here covery efficiency

for matri effects at lo"# mid# and high ,C level sranged 33$951$8 for cefazolin

and >5$E>3$>8 for cloacillin ;&able 2=$ % teach ,C level# there is less than 518

difference in overall matri effects bet"een cefazolin and cloacillin# further

supporting the use of cloacillin as an internal standard$ 0ecovery efficiency "as

determined by comparing the pea! area intensities of pre-etracted material to post-

etracted material$ &hese recoveries ranged from E2$8 to 3E$>8 for cefazolin and

515$28 to 51E$8 for cloacillin$ ?t is epected for recovery of cefazolin to be less

than that of cloacillin as cefazolin must be isolated from the plasma matri# "hereas

the internal standard is in solvent$ *rocessing efficiency "as evaluated by comparing

pre-etracted material to un-etracted material$ &here coveries ranged from 3$E8 to

>$E8 for cefazolin and >8 to 518 for cloacillin$ &he overall processing

efficiency for cefazolin reflects the minimal matri effects seen in addition to the

decreased recovery from plasma$ Do"ever# as there are no relative matri effects and

cefazolin concentrations are determined by the pea! area ratio of drug to internal

standard# the method may be used for cefazolin quantification$

2$$ Cord blood comparison

?n lieu of full validation by preparing calibration and quality control material sets in

cord blood#"e validated its acceptability as a specimen matri by evaluating recovery

of cefazolin spi!ed directly into cord blood or cord blood ultrafiltrate$ &heseconcentrations "ere determined using calibration curves generated using total

cefazolin calibrators in human plasma or free cefazolin calibrators in human plasma

ultrafiltrate for total cefazolin and free cefazolin# respectively$ &he values determined

from the cord blood plasma quality control materials "ere compared to the values

determined from plasma and plasma ultrafiltrate$ &he difference from the t"o values

"as calculated and a 8'?/ "as determined# "ith an acceptability criteria of 7U598$

&he relative recovery for cefazolinin cord blood and cord blood ultrafiltrate ranged

I2$>8 to >$58 for total cefazolin and I5$28 to I55$>8 for free cefazolin ;&able9=$

&hese values "ere "ithin our limit of acceptability of rU598'?/$ &hese resultssuggest that cord blood plasma and cord blood plasma ultrafiltrate may be analyzed


13/24

along side plasma and plasma ultrafiltrate specimens using the a for ementioned

conditions described in the protocol$ &his is the first demonstration verifying

acceptability for analysis of both total and free cefazolinin plasma and plasma

ultrafiltrat eas "ell as cord blood plasma and cord blood plasma ultrafiltrate$

&he described +C.(4.( methods facilitate the quantification of total and free

cefazolin in both adult and cord blood plasma$ &hese methods ta!e advantage of

convenient ultrafiltration techniques for separating the free drug from plasma

proteins$ &he methods are fast at 9 min per analysis and only require61 l of plasma

for determination of total cefazolin and 611 l of plasma for determination of free

cefazolin$ Do"ever# there are some limitations to the described methods$ &he steps

required to achieve satisfactory carryover performance "ith additional "ash steps that

could be prohibitive if the D*+C does not easily facilitate introduction of strong "ash

solvent during an analysis$ %nother limitation is the necessity of consuming a nultra

filtration device for assessing free cefazolin concentrations$ &he free cefazolin methoduses standard ultrafiltration condition store move plasma proteins# but the calibrators

and quality control materials "ere prepared directly in plasma ultrafiltrate$ ?deally# a

method "ould utilize "ell-characterized materials of !no"n free drug concentrationas

calibrators and quality controls$ %dditionally# alternative methods to ultrafiltration#

such as rapid equilibrium dialysis# may reduce the amount of technician time required

for analysis$ .oreover# these method shave not been compared to a reference

laboratory due to a lac! of testing availability$ +astly# "e have not evaluated the

described methods in a patient cohortM this "ill be performed in forthcoming studies$

&able 2

&otal cefazolin matri effects studies describing recoveries for the analyte cefazolin

;C/J= and cloacillin ;C+P=$

2$ Conclusions

% rugged +C.(4.( method for the quantification of total and free cefazolin in both

adult and cord blood plasma has been developed and validated$

%c!no"ledgments

&his "or! "as supported in part by the &hermo /isher (cientific through are search

contract and placement of instrumentation$


14/24

penge'bangan bioanalitis dan validasi cair #ro'atogra(-tande' 'etode

spe#tro'etri 'assa untu# #uanti(#asi total dan bebas cefazolin dala'

plas'a 'anusia dan darah tali

abstra#

tu)uan*+efazolin adalah ,-la#ta' antibioti# u'u'nya diresep#an untu#

pro(la#sis terhadap infe#si #ulit setelah operasi ter'asu# operasi caesar.

enilaian paparan ibu dan bayi penting untu# 'enghubung#an #onsentrasi

obat untu# hasil #linis. /engan de'i#ian 'etode bioanaliti# untu#

#uanti(#asi bai# total dan bebas cefazolin dala' plas'a dan su'su' ibu

darah dapat 'e'bantu dala' evaluasi 'enyeluruh paparan cefazolin.

/esain dan 'etode*

persiapan spesi'en untu# pengu#uran total cefazolin dila#u#an 'elalui

presipitasi protein dengan asetonitril 'engandung cloxacillin standar

internal. 0ltra(ltrasi diguna#an untu# 'engisolasi cefazolin gratis. sa'peldiproses dianalisis pada siste' + relude digabung#an #e

spe#tro'eter 'assa T 3triple %uadrupole antage. 5etode yang

divalidasi pedo'an bioanalitis F/! beri#ut.

hasil*

Rentang pengu#uran analisis dari 'etode ini adalah 46-467 'g 8 ' dan

7746-46 'g 8 ' untu# obat bebas dan total 'asing-'asing. #urva

#alibrasi yang dihasil#an 'engguna#an 1 8 x2 analisis regresi linear

terti'bang. Total cefazolin 'enun)u##an antar dan intra-assay presisi 9

27: di ;3 dan 9 112: pada ting#at lain. cefazolin bebas 'enun)u##an

inter-dan intra-assay presisi 9 16" a#u'ulasi efe# 'atri#s pe'ulihan

dan stabilitas studi )uga diteri'a berdasar#an re#o'endasi F/!.

elan)utnya itu 'enun)u##an bah$a sa'pel disiap#an dala' darah tali

dapat secara a#urat diu#ur dari #urva #alibrasi plas'a de$asa dengan

pe'ulihan 9 ?1: /IF dan 9 11?: /IF total dan bebas cefazolin 'asing-

'asing.

#esi'pulan*

'etode +-5 8 5 yang di)elas#an 'e'ung#in#an untu# pengu#uran

total dan bebas cefazolin di #edua plas'a dan darah tali pusat. @ 271/T! plas'a$ith solusi saha'

yang tepat. Konsentrasi 'encer'in#an #uantitas cefazolin gara' natriu'

dala' larutan. Total volu'e organi# dita'bah#an #e plas'a adalah L2:.

#ontrol #ualitas 3+" bahan disusun 'engguna#an solusi be#er)a disiap#an

dari independen berat dari solusi yang diguna#an untu# 'enyiap#anstandar #alibrasi. solusi be#er)a untu# persiapan bahan 3+ disiap#an pada

#onsentrasi 1?72477 dan 46.777 'g 8 ' dala' 1* 1 asetonitril* air.

&e#er)a solusi untu# persiapan bahan 3+ diencer#an dala' K2>/T!

plas'a pada #onsentrasi a#hir 746 batas ba$ah dari #uanti(#asi ;3"

14 3+ rendah" 167 pertengahan 3+" dan 417 tinggi 3+" 'g 8 '.

+efazolin solusi be#er)a untu# penyusunan standar #alibrasi disiap#an

untu# penentuan cefazolin gratis dengan a$alnya 'e'persiap#an

asolution 1?77 'g 8 ' cefazolin di 1* 1 asetonitril* air dengan

'eni'bang bahan standar dan 'enipis#an volu'etrically. solusi be#er)a

disiap#an pada #onsentrasi a#hir 71? 'g 8 ' 1? 'g 8 ' dan 1?7 'g 8' 'elalui pengenceran serial. standar #alibrasi yang preparedat

#onsentrasi a#hir 7746 7.7?


17/24

D+ #elas total cefazolin te#ad dan air #elas D+ untu# cefazolin

penentuan gratis pada #onsentrasi a#hir ?2 'g 8 ' and1.6 'g 8 '

'asing-'asing dengan cara pengenceran 16 'g 8 ' larutan sto# yang

disiap#an dengan 'eni'bang cloxacillin dan 'enca'purnya

volu'etrically 'engguna#an


18/24

bahan 3+.

2.


19/24

punca# dengan #onsentrasi #alibrator.

: /IF dide(nisi#an di ba$ah ini di 'ana x Referensi adalah rata-rata

diu#ur #onsentrasi bahan 3+ dala' #ondisi referensi dan x /iobati adalah

#onsentrasi rata-rata diu#ur untu# ting#at 'ateri 3+ yang sa'a

diperla#u#an berbeda.

: 5>: > dan: R> disa)i#an sebagai pe'ulihan relatif di 'ana x1adalah intensitas punca# daerah berte#ad rata-rata untu# pengobatan

referensi untu# #o'ponen cefazolin o rcloxacillin" ditentu#an dari bahan

3+ dan x2 adalah rata-rata daerah pera$atan tes intensitas untu# bahan

3+. 5isalnya dala' 'enentu#an efe# 'atri#s cefazolin x1 adalah

intensitas rata-rata luas untu# cefazolin dala' persiapan tere#stra#si dan

x2 adalah intensitas rata-rata luas untu# cefazolin dala' bahan pasca

die#stra#si.

C. 5etode validasi

5etode +-5 8 5 telah divalidasi berdasar#an F/! edo'an untu#

Industri* re#o'endasi &ioanalytical alidasi 5etode A2=B. 'etri# validasiter'asu# intra dan inter-assay presisi dan a#urasi linearitas stabilitas

a#u'ulasi dan efe# 'atri#s. ara'eter ini sepenuhnya ditandai total

cefazolin dala' plas'a 'anusia. cefazolin gratis divalidasi di ultra(ltrate

plas'a 'anusia dengan 'engatasi presisi a#urasi dan a#u'ulasi. darah

tali dievaluasi dengan 'engu#ur pe'ulihan darah tali cefazolinin dan

#abel ultra(ltrate darah dibubuhi cefazolin dari #urva #alibrasi yang

disiap#an di non-#abel plas'a darah.

C.1. resisi dan a#urasi

Intra-assay presisi ditentu#an dengan in)e#si ulangan n S =" total ataubebas bahan cefazolin 3+ pada e'pat ting#at. /ia'ati berarti / dan:

+ dinilai pada setiap ting#at. The ;3 dide(nisi#an sebagai #alibrator

terendah pada #urva #alibrasi dan sesuai dengan re#o'endasi F/!

'enghasil#an a#urasi dan presisi #riteria 9 27: /> dari #onsentrasi

teoritis dan #etida#tepatan dari 9 27: +. Target untu# ting#at atas ;3

yang 9 1 dari #onsentrasi teoritis dan #etida#tepatan dari 91


20/24

41 'g 8 ' untu# cefazolin atau cefazolin gratis 'asing-'asing" dan

'e'banding#an #onsentrasi rata-rata dari #alibrator rendah disunti##an

sebelu' #alibrator tinggi #e disunti##an setelah. ecara #husus #alibrator

disunti# dala' urutan* 1 2 C D1 D2 4 DC D4 17-!2 A2EB.

elain itu /urin analisis groutine sunti#an pelarut ! 71: asa' for'at"

diin)e#si setelah penyunti#an dari #alibrator #onsentrasi tertinggi. Kriteria

peneri'aan adalah pasca-in)e#si sinyal #osong diu#ur sebagai daerah

punca# #ro'atogra( L27: dari sinyal yang dia'ati untu# #alibrator

#onsentrasi terendah.

C.C. tabilitas

tudi stabilitas dila#u#an untu# #eseluruhan analisis cefazolin. Rendah

'enengah dan ting#at 3+ yang tinggi 'en)adi sasaran stabilitas 'atri#sin)e#si stabilitas sa'pel 'atri#s dan freeze-tha$ stabilitas. In)e#si

'atri#s percobaan stabilitas dila#u#an oleh e#stra# 3+ ulang 'enganalisis

dipertahan#an pada 4+ e'pat hari setelah persiapan dengan #alibrator

ulang 'enganalisis dan bahan 3+ 'engu#ur atas dasar #urva #alibrasi

baru dan dibanding#an dengan nilai-nilai asli. a'pel 'atri#s percobaan

stabilitas dila#u#an dengan 'enganalisis bahan #ontrol #ualitas setelah

in#ubasi sela'a 24 )a' pada suhu #a'ar. Freeze-tha$ percobaan

stabilitas dila#u#an dengan 'e'banding#an baru dibanding#an bahan

#ontrol #ualitas untu# 'ateri yang telah 'en)alani n S 4 si#lus be#u-

'encair 'engguna#an freezer dipertahan#an pada 27+. percobaanstabilitas saha' dila#u#an di 'ana solusi saha' diin#ubasi pada suhu

#a'ar 4+ dan 27+ sela'a C 'inggu. eneri'aan ditentu#an sebagai L

1fe# 5atrix pada

ionisasi dinilai dengan 'engguna#an 'etode yang sebelu'nya di)elas#an

oleh 5atusze$s#i dan re#an A26B 'engguna#an set bahan 3+ disiap#an

pada rendah 'enengah dan #onsentrasi tinggi. &ahan-bahan inidisiap#an untu# analisis dala' tiga set independen* tere#stra#si pra-

die#stra#si dan pasca-die#stra#si. ecara sing#at sa'pel tere#stra#si

disusun 'engguna#an proto#ol di)elas#an tapi 'engganti 5! 71: asa'

for'at" di te'pat plas'a sa'pel pra-die#stra#si 'engandung #adar 3+

'elon)a# 'en)adi plas'a dari ena' banya# individu dan satu set pasca-

die#stra# yang 'elibat#an pena'bahan solusi spi#ing #e pasca die#stra#

'atri#s plas'a dari tersebut ena' banya# independen. Konsentrasi (nal

di rendah 'enengah dan tinggi 3+s dipertahan#an di se'ua tiga set.

Hilai daerah punca# ba#u yang dihasil#an oleh analisis ini dibanding#an

dengan 'enghitung efe# 'atri#s e(siensi pe'ulihan dan e(siensipengolahan. Ku'pulan individu: Ri$ayat Didup )uga ditentu#an. >fe#


21/24

5atrix dievaluasi oleh rasio covery ada dari pasca die#stra#* tere#stra#siM

e(siensi pe'ulihan dievaluasi dengan rasio covery ada pra-die#stra#si*

pasca-die#stra#siM e(siensi pengolahan dievaluasi dengan rasio covery

ada pra-die#stra#si* tere#stra#si.

C.


22/24

ecessity untu# 'encapai rentang pengu#uran analitis tepat dina'is

!5R" 'enga#ibat#an 'odi(#asi beri#utnya dengan 'etode +. elarut

$ash #uat diper#enal#an 'engguna#an po'pa biner #edua dengan #atup

sebagai 'e'pesona#an dan tee #e dala' #olo' analitis.

/iopti'al#an su'ber ionisasi dan para'eter spe#tro'etri 'assa untu#

cefazolin dan cloxacillin ditentu#an dengan 'enyesuai#an #ondisi 'anualsela'a infus langsung. Kedua senya$a dianalisis di A5 U DB terprotonasi

'ere#a U bentu#. Rea#si yang dipilih pe'antauan 'odus R5" transisi

ion yang dipilih atas dasar intensitas sinyal dan #etida#tepatan sela'a

percobaan #esesuaian. elain itu 'es#ipun ru'us 'ole#ul dari ion-ion

frag'en tida# di#on(r'asi dengan studi pelabelan 'assa 'ere#a

'enun)u##an 'ere#a bu#an produ# dari #erugian u'u' #ondensasi

de#arbo#silasi dll" dan 'ung#in berisi cincin tetrazol yang 'erupa#an

bagian R2 #ara#teristi# untu# cefazolin . Kro'atogra' yang dihasil#an

dari transisi ion #uali(#asi dievaluasi atas dasar bentu# punca# 'ere#a.

enerapan transisi #uali(#asi sangat penting #eti#a 'engguna#an analogstru#tural sebagai standar internal untu# spe#tro'etri 'assa #uantitatif.

alah satu #onse#uensi uta'a dari analog stru#tural sebagai standar

internal adalah bah$a hal itu sering tida# #o-elusi dengan analit. otensi

gangguan dapat 'enyebab#an pene#anan ion dan secara signi(#an

'e'pengaruhi #ea#uratan hasil.

5etode yang diterbit#an oleh /ouglasetal et al. diguna#an in vivo

'icrodialysis untu# 'enentu#an #onsentrasi cefazolin gratis yang tida#

pra#tis untu# pengu)ian #linis rutin A2CB. 5etode yang diterbit#an oleh

Jhangetal et al. disa)i#an validasi yang relatif #o'prehensif untu#

'enentu#an cefazolin total dan bebas dala' plas'a 'anusia tetapi tida#'e'beri#an rincian tentang studi banding dala' darah tali pusat )uga

tida# 'e'beri#an rincian tentang

5ening#at#an sele#tivitas analitis 'elalui pengenalan transisi ion

#uali(#asi A21B. >valuasi transisi ion #uali(#asi 'ena'bah spesi(sitas

analisis ta'bahan untu# 'etode spe#tro'etri 'assa #arena gangguan

'ung#in 'e'ili#i e(siensi diferensial untu# 'enghasil#an sinyal untu#

transisi diVerention. +ord darah se'entara tida# 'atri#s independen

divalidasi dala' hal presisi dan a#urasi penelitian untu# 'enilai setiap

interferents potensial pada #uanti(#asi obat dan untu# lebih 'enciri#an

#e#asaran #eseluruhan 'etode analisis. 5etode ini divalidasi'engguna#an garis panduan F/! 5etode &ioanalytical alidasi #arena itu

adalah satu set 'apan pedo'an coco# untu# studi far'a#o#ineti# yang

a#an 'en)adi apli#asi uta'a untu# pengu)ian tersebut.

0ltra(ltrasi dipilih untu# 'engisolasi fra#si bebas dari cefazolin #arena

cepat relatif 'urah untu# 'enerap#an pada s#ala studi yang terbatas

dan hanya re%uiresa centrifuge 'i#ro yang u'u'nya tersedia dala'

penelitian #linis laboratory.The standar internal dita'bah#an #e

ultra(ltrate dari diproses 'aterial #arena pena'bahan bahan sebelu'

(ltrasi a#an 'engganggu #esei'bangan siste'. !saresult #a'i

'engguna#an #ondisi (ltrasi standar teruta'a yang dirancang untu#'enyaring albu'in untu# sa'pel fra#si bebas dari cefazolin


23/24

'engguna#an ultra(ltrasi. The topicin u'u' telah dievaluasi dala'

literatur #hususnya dala' penerapan fenitoin penentuan gratis dengan

i''unoassay beri#ut ultra(ltrasi AC7B.

#ro'atogra' a'bar 2. er$a#ilan !" cefazolin ;3 rendah palas'a

3+ dan #osong dan &" internal standar cloxacillin dan plas'a #osong.

#ro'atogra' a'bar C. er$a#ilan dari urutan opri'ization a#u'ulasi.Kro'atogra' dari !" yang sebelu' opti'asi. Kro'atogra' dari &"

adalah pasca-opti'asi.

4.2. resisi a#urasi dan analisis #urva #alibrasi

Intra - presisi dan antar-assay dan a#urasi total cefazolin dan cefazolin

gratis ditentu#an beri#ut ada pu)ian dari garis panduan F/! seperti

di)elas#an sebelu'nya Tabel 2". Total cefazolin intra-assay presisi ber#isar

dari =2: 'en)adi 277: + di ;3 itu. Intra a#urasi assay sebagai:

/> ber#isar antara G4.C: 'en)adi 146:. Total cefazolin antar-assay

presisi ber#isar dari =2: 'en)adi 1?1: di ;3 itu. !ntar a#urasi assay

sebagai: /> ber#isar antara GE.E: 'en)adi 11?:. ratis cefazolinintra-assay presisi ber#isar antara E2: sa'pai 1E6: di ;3 itu. ratis

cefazolin a#urasi intra-assay ber#isar antara G=.E: 'en)adi C4:. ratis

cefazolin antar-assay presisi ber#isar dari


24/24

Table2 Inter-assay dan intra-assay presisi dan a#urasi

!ra. 4. #urva #alibrasi er$a#ilan total analisis cefazolin. N-su'bu !"

adalah rasio luas punca# dia'ati antara cefazolin dan cloxacillin %uanti(er

transisi. N-su'bu &" adalah #onsentrasi dihitung 'engguna#an regresi

linear.

4.C. 5enopanganalisis a#u'ulasi dila#u#an sesuai dengan pedo'an +I dengan

'engevaluasi #onsentrasi rata-rata ditentu#an standar #onsentrasi rendah

disunti##an di bola# seri dengan standar #onsentrasi tinggi A2EB.

5engatasi 'asalah $ithcarryover #ritis sela'a penge'bangan 'etode

#arena rentang dina'is yang luas dari 'etode ini. uncuran tida# 'elihat

#eti#a 'enga'ati sinyal analog dari cloxacill di internal standard

'enun)u##an potensi a#u'ulasi cefazolin dala' siste' + pada

#onsentrasi tinggi.

4.4. tabilitas

4.

Bioanalytical Development and Validation of Liquid Chromatographic

Documents