D ETER M IN ATIO N O F C LIN D A M Y C IN IN P LA S M A

Vol 37 N o. 1 January 2006 177

C orrespondence: D r K esara N a-B angchang, P harm a-
cology and Toxicology U nit, Faculty of A llied H ealth
S ciences, Tham m asat U niversity (R angsit C am pus)
P aholyothin R oad, P athum thani 12121, Thailand.
Tel: 662-926-9438; Fax: 662-516-5379
E-m ail: nkesara@ hotm ail.com , kesaratm u@ yahoo.
com , nkesara@ tu.ac.th
IN TR O D U C TIO N
C lindam ycin [7(S )-chloro-7-deoxylincom y-
cin] is a lincosam ide antibiotic (Fig 1) w ith pri-
m arily bacteriostatic activity against gram posi-
tive organism s and a w ide range of anaerobic
pathogens as w ell as som e antiprotozoal effi-
cacy. This effect is exerted by its binding to the
50S ribosom al subunit and the consequent in-
hibition of bacterial protein synthesis (B ooth,
2001). A dditionally, clindam ycin is active against
different apicoplexan parasites, including Plas-
modium falciparum. It has been dem onstrated
that clindam ycin targets the prokaryote-like ri-
bosom es of the apicoplast and by this m eans
inhibits self-replication of the organelle (Fichera
and B oos, 1997; K ohler et al, 1997). A s a con-
sequence of this m echanism , clindam ycin dis-
plays a typical delayed kill kinetic effect, the
grow th of parasites being unaffected until the
second replication after drug exposure. U nder
such cond itions, the in vitro grow th of P.
falciparum is inhibited w ith an IC
50
and IC
90
of
approxim ately 25 and 50 nM , respectively.
A num ber of analytical m ethods have been
reported for m easuring clindam ycin in bulk drugs
and form ulations as w ell as in biological fluids
and tissue or cell hom ogenates or organ ex-
tracts. These m ethods involve m icrobiological
A N A LTER N ATIVE H IG H -P ER FO R M A N C E LIQ U ID
C H R O M ATO G R A P H IC M ETH O D FO R D ETER M IN ATIO N
O F C LIN D A M YC IN IN P LA S M A
K N a-B angchang
1
, V B anm airuroi
1
, B K am anikom
1
and D K iod
2
1
P harm acology and Toxicology U nit, Faculty of A llied H ealth S ciences, Tham m asat U niversity
(R angsit C am pus), P athum thani, Thailand;
2
U N IC EF/U N D P /W orld B ank/W H O S pecial P rogram for
R esearch and Training in Tropical D iseases, The W orld H ealth O rganization,
G eneva, S w itzerland
Abstract. A sim ple, sensitive, selective and reproducible m ethod based on a reversed-phase chro-
m atography w as developed for the determ ination of clindam ycin in hum an plasm a. C lindam ycin
w as separated from the internal standard (phenobarbital) on a Luna C 18 colum n (250 x 4.6 m m , 5
m m particle size: P henom enex

, U S A ), w ith retention tim es of 5.6 and 14.2 m inutes, respectively.

U ltraviolet detection w as set at 210 nm . The m obile phase consisted of a solution of 0.02 M
disodium hydrogenphosphate (pH 2.8) and acetonitrile (76:24 v/v), running through the colum n at a
flow rate of 1.0 m l/m in. The chrom atographic analysis w as operated at 25C . S am ple preparation (1
m l plasm a) w as done by a single step liquid-liquid extraction w ith w ater saturated ethylacetate.
C alibration curves in plasm a at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 g/m l w ere
all linear w ith correlation coefficients better than 0.999. The precision of the m ethod based on w ithin-
day repeatability and reproducibility (day-to-day variation) w as below 15% (% coefficient of varia-
tions: % C V) G ood accuracy w as observed for both the intra-day and inter-day assays, as indicated
by the m inim al deviation of m ean values found w ith m easured sam ples from that of the theoretical
values (below +15% ). Lim it of quantification w as accepted as 0.07 g using 1 m l plasm a sam ple.
The m ean recovery for clindam ycin and the internal standard w ere greater than 95% . The m ethod
w as free from interference from fosm idom ycin, including com m only used drugs, antim alarials and
antihelm inthics. The m ethod appears to be robust and has been applied to a pharm acokinetic study
of clindam ycin in a patient w ith m alaria follow ing oral doses of clindam ycin at 10 m g/kg body w eight
given tw ice daily for 7 days.
S O U TH EA S T A S IA N J TR O P M ED P U B LIC H EA LTH
178 Vol 37 N o. 1 January 2006
assay (M etzler et al, 1973; B row n et al, 1981),
spectrophotom etric assay (El-Yazbi and B laih,
1993), radio-im m uno assay (R IA ) (D uckw orth et
al, 1993), gas-liquid chrom atography (G LC )
(O esterling and R ow e, 1970; B row n, 1974; G atti
et al, 1993), and high perform ance liquid chro-
m atography (H P LC ) (B row n, 1978; Landis et al,
1980; La Follette et al, 1988; H ornedo-N unes
et al, 1990; Liu et al, 1997; Fieger-B uschges et
al, 1999; O rw a et al, 1999; S in et al, 2004). M i-
crobiological, spectrophotom etric assays, R IA
and G LC are either non-specific, or tim e-and-
reagent consum ing. The H PLC technique is con-
sidered to be m ost appropriate m ethod for ap-
plication to pharm acokinetic investigation as it
is sensitive, accurate, reproducible and relatively
sim ple. R efractive index (B row n, 1978; Landis
et al, 1980), electrochem ical (H ornedo-N unes et
al, 1990), m ass spectrom etry (Yu et al, 1999;
M artens-Lobenhoffer and B anditt, 2001; C herlet
et al, 2002; R echberger et al, 2003; S in et al,
2004), and U V (Landis et al, 1980; M unson and
K ubiak, 1985; La Follette et al, 1988; Liu et al,
1997; Fieger-B uschges et al, 1999; O rw a et al,
1999) detection have been used in H PLC . H PLC /
M S m etho d s (Yu et al, 1 9 9 9 ; M artens-
Lobenhoffer and B anditt, 2001; C herlet et al,
2002; R echberger et al, 2003) display the high-
est perform ance; how ever, the LC /M S instru-
m ents are not yet readily obtainable in m ost labo-
ratories.
W ith respect to H P LC /U V m ethods, a
straight forw ard H P LC /U V m ethod (La Follette
et al, 1988) using direct injection of plasm a after
a precipitation step w ith acetonitrile has been
reported to be non-reproducible (Liu et al, 1997;
Fieger-B uschges et al, 1999) due to interferences
from plasm a com ponents. The m ethod de-
scribed by Liu et al (1997) is rather sophisticated,
using a couple of colum ns and tw o m obile
phases to extract clindam ycin from hum an
plasm a sam ples. Fieger-B uschges et al (1999)
reported an autom ated m ethod using couple
colum n H P LC after the precipitation of plasm a
proteins w ith saturated am m onium sulfate solu-
tion. R ecently, B atzias et al (2005) reported a
new H P LC /U V m ethod for the quantitative de-
term ination of clindam ycin in dog serum . The
m ethod w as based on d eproteinisation of
sam ples w ith acetonitrile follow ed by extraction
w ith dichlorom ethane. In the present report, w e
describe an alternative m ethod, w hich is rela-
tively sim ple, sensitive, accurate and reproduc-
ible for the determ ination of clindam ycin in bio-
logical fluids. The total run tim e w as less than
18 m inutes. The m ethod w as based on reversed-
phase chrom atography w ith ultraviolet detection.
The m ethod has been applied successfully to
pharm acokinetic studies of clindam ycin w hen
used in com bination w ith fosm idom ycin in a pa-
tient w ith acute uncom plicated falciparum m a-
laria.
M ATER IA LS A N D M ETH O D S
Chemicals
All solvents w ere H PLC grade. O rganic sol-
vents w ere purchased from Fison Scientific Equip-
m ent (B ishop M eadow R oad, Loughborough,
U K ). D isodium hydrogenphosphate w as of ana-
lytical grade, w hich w as obtained from S igm a
C hem ical (St Louis, M O , U SA). U ltrapure analyti-
cal grade Type I w ater (r > 18 M !/cm ) w as pro-
duced by a M illi-Q Plus w ater system (M illipore
C orporation, B edford, M A , U S A ). C lindam ycin
[7(S)-chloro-7-deoxylincom ycin]; Fig 1a) and in-
ternal standard (phenobarbital; Fig 1b) w ere ob-
tained from Sigm a C hem ical (St Louis, M O , U SA).
Standard stock solutions
S tock solutions w ere m ade w ith clinda-
m ycin and the internal standard (phenobarbital).
A ppropriate am ounts of chem icals w ere dis-
solved in distilled w ater in volum etric flasks.
S tock solutions of clindam ycin and internal stan-
dard w ere prepared at a concentration of 1,000
ng/l. The stock solutions w ere further diluted
to m ake w orking solutions at concentrations of
250 and 100 g/m l, for clindam ycin and the in-
ternal standard, respectively. S tandard solutions
w ere stored at -20C until use.
Chromatography
The m ethod w as developed on a chrom ato-
graphic system consisting of a W aters 600 H PLC
solvent D elivery/C ontroller, equipped w ith a
R heodye 7125 injector w ith a 100-m l loop
(R heodyne, B erkeley, C A , U S A ), an ultraviolet
detector (W aters 996 P hotodiode A rray D etec-
D ETER M IN ATIO N O F C LIN D A M Y C IN IN P LA S M A
Vol 37 N o. 1 January 2006 179
tor; M ilford, M A , U S A ), and M illenium 32 S oft-
w are for data integrator. The w avelength w as set
at 210 nm . The separation w as carried out on a
reversed phase colum n Luna C 18 (250 x 4.6
m m , 5 m particle size: P henom enex

, M A ,
U S A ). The elution solvent consisted of a solu-
tion of 0.02 M disodium hydrogenphosphate (pH
2.8) and acetonitrile (46:24, v/v). The chrom ato-
graphic analysis w as operated at 25C . A liquots
of 100 l sam ples or standard solutions w ere
injected onto the colum n w ith a m obile phase at
a flow rate of 1.0 m l/m inute. A ll buffers w ere
vacuum filtered and degassed through 0.2 m
pore size polym eric P TFE filters.
Sample preparation
This procedure w as validated on specim ens
using 1 m l of spiked hum an plasm a. H um an
plasm a w as obtained from healthy subjects, and
stored frozen in aliquots at -20C . To 1 m l
plasm a, 40 m l internal standard w orking solu-
tion (400 g/m l) w as added. A fter thoroughly
m ixing, the sam ple w as extracted tw ice by m e-
chanical tum bling (speed 7, 15 m inutes) w ith 2
x 5 m l w ater saturated ethylacetate. The result-
ing clear organic layer w as separated through
centrifugation at 3,500g for 15 m inutes, and
evaporated to dryness under a gentle nitrogen
stream at 40C . S am ples w ere protected from
light and stored at 4
o
C until injection. For H P LC
injection, sam ples w ere reconstituted w ith 200
l m obile phase, and an aliquot of 100 l w as
injected onto the chrom atographic system .
Calibration curves
Detector linearity. S olutions of clindam ycin in
distilled w ater at concentrations ranging from 0
to 16.0 g/m l w ere injected into the H P LC sys-
tem in order to assess detector linearity. P eak
height w as plotted against the quantity of
clindam ycin injected. C lindam ycin w as linear (r
2
> 0.999) over the concentration range observed.
Plasma. C alibration curves w ere prepared by
replicate analysis of six plasm a sam ples (1 m l
each) spiked w ith varying concentrations of
clindam ycin (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0
m g/m l) and a fixed concentration of the internal
standard (400 g/m l). S am ples w ere analysed
as described previously.
Data analysis. Peak height ratios of clindam ycin/
internal standard w ere calculated. C oncentra-
tions of clindam ycin w ere determ ined by m atch-
ing peak height responses against a calibration
curve of response ratio (height of clindam ycin/
height of internal standard) vs concentration,
obtained from standard sam ple injection. The
internal standard corrected for variation in the
sam ple preparation step used. P eak detection,
peak height integration, peak height ratio calcu-
lation, calibration curve fitting (least square re-
gression w ithout w eighting) and calculation of
sam ple concentrations w ere perform ed by
M illenium 2000 C hrom atograph

softw are.
Method validation
Precision. The precision of the m ethod based
on within-day repeatability w as determ ined by
replicate analysis of six sets of plasm a sam ples
(1 m l each) spiked w ith seven different concen-
trations of clindam ycin (0.25, 0.5, 1.0, 2.0, 4.0,
8.0, 16.0 g/m l). The reproducibility (day-to-day
variation) of the m ethod w as validated using the
sam e concentration range of plasm a as de-
scribed above, but only a single determ ination
Fig 1C hem ical structures of (a) clindam ycin and (b)
internal standard (phenobarbital).
N
N
H
CI
O
OH
OH
OH
O
S
(a)
O
N
N
O
H
H
O
CH
2
CH
3
(b)
S O U TH EA S T A S IA N J TR O P M ED P U B LIC H EA LTH
180 Vol 37 N o. 1 January 2006
of each concentration w as m ade on six different
days. The coefficient of variation (C V) w as cal-
culated from the ratios of standard deviation (SD )
to the m ean and expressed as a percentage.
Accuracy. A ccuracy of the m ethod w as deter-
m ined by replicate analysis of six sets of plasm a
sam ples (1 m l each) at seven different levels of
clindam ycin (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0
g/m l) and com paring the difference betw een
spiked value and that of the actually found (theo-
retical value).
Recovery. The analytical recovery of sam ple
preparation procedure for clindam ycin w as esti-
m ated by com paring the peak heights obtained
from sam ples (1 m l plasm a) prepared as de-
scribed above, w ith those m easured w ith equiva-
lent am ounts of clindam ycin in distilled w ater.
Triplicate analysis w as perform ed at concentra-
tions of 0.25, 2.0, and 8.0 g/m l.
Selectivity. B lank (heparinised) plasm a sam ples
from healthy Thai volunteers w ere tested for in-
terference by endogenous com pounds. The
selectivity of other m ethod w as verified by
checking for interference by fosm idom ycin and
other com m only used drugs: antim alarials
(fosm idom ycin, chloroquine, quinine, m efloquine),
anthelm inthics (albendazole, praziquantel),
paracetam ol and dim enhydrinate after subject-
ing them to sam ple preparation procedures.
Limit of quantification. The lim it of quantifica-
tion (LO Q ) of the assay procedure w as deter-
m ined fro m the lo w est co ncentratio n o f
clindam ycin (in the spiked plasm a sam ple) that
produced a peak height ten tim es the baseline
noise at a sensitivity of -0.2 A in a 1 m l sam ple.
Stability. The stability of clindam ycin w as deter-
m ined by storing spiked plasm a sam ples (1 m l
each at concentrations of 0.25, 2.0, and 8.0 g/
m l; triplicate analysis for each concentration) in
a -20
o
C freezer (S anyo, Japan) for six m onths.
C oncentrations w ere m easured periodically (1,
2, 3 and 6 m onths). For freeze and thaw stabil-
ity, sam ples w ere frozen at -20
o
C for at least 24
hour and thaw ed unassisted at room tem pera-
ture (25
o
C ). W hen com pletely thaw ed, the
sam ples w ere transferred back to the original
freezer and refrozen for at least 24 hour. The pro-
cess w as repeated for three cycles.
Quality control. Q uality control (Q C ) sam ples for
clindam ycin w ere m ade up in plasm a (1 m l) us-
ing a stock solution separate from that used to
prepare the calibration curve, at concentrations
or 0.25, 2.0, and 8.0 g/m l. S am ples w ere
aliquoted into cryovials, and stored frozen at
-20C for use w ith each analytical run. The re-
sults of the Q C sam ples provided the basis of
accepting or rejecting the run. A total of 6 qual-
ity control sam ples (2 for each concentration)
w ere analysed during each run (at the beginning
of each batch of sam ples and the standard
curve). A t least four of the six Q C sam ples had
to be w ithin 20% of their respective nom inal
values. Tw o of the six Q C sam ples could be out-
side the 20% of their respective nom inal value,
but not at the sam e concentration.
Application of the method to biological samples
The m ethod w as applied to the investiga-
tion of the pharm acokinetics of clindam ycin in
plasm a in a patient (from M aeS ot H ospital, Tak
P rovince, Thailand) w ith acute uncom plicated
falciparum m alaria (aged 28 years, w eighing 50
kg) w ho received treatm ent w ith clindam ycin at
a dose of 10 m g/kg body w eight tw ice daily for
7 days, in com bination w ith fosm idom ycin at a
dose of 15 m g/kg body w eight tw ice daily for 7
days. Venous blood sam ples (3 m l) w ere col-
lected in heparin-coated plastic tubes at the fol-
low ing tim e points: 0, 24 (pre-dose), 48 (pre-
dose), 160 (pre-dose), 162, 164, 166, 172, 184,
208, 220, 232, 238, 244, and 250 hours after
the last dose of clindam ycin.
R ES U LTS
Chromatographic separation
A num ber of H P LC chrom atographic sys-
tem s w ere investigated to optim ise the separa-
tion of clindam ycin and the internal standard
(phenobarbital). R etention m aps w ere generated
for both com pounds as a function of stationary
phase (Luna C 18 reversed-phase colum n) and
m obile phase. For, the m obile phase, com posi-
tion of 0.02 M disodium hydrogenphosphate (pH
2.8) and acetonitrile (76:24 v/v) running through
the colum at a flow rate of 1.0 m l/m in gave op-
tim al separation of clindam ycin and internal stan-
dard w ith a 20-m inute run tim e. The retention
D ETER M IN ATIO N O F C LIN D A M Y C IN IN P LA S M A
Vol 37 N o. 1 January 2006 181
tim es (capacity factor) of clindam ycin and the
internal standard w ere approxim ately 5.6, and
14.2 m inutes, respectively.
Sample preparation
The sam ple preparation step used in this
study w as very sim ple as it involved only one
step liquid-liquid extraction. Extraction of plasm a
w ith w ater saturated ethylacetate w as found to
be the m ost optim al condition for sam ple prepa-
ration as it resulted in a clean chrom atogram (Fig
2).
C hrom atogram s of b lank p lasm a and
plasm a spiked w ith clindam ycin at a concentra-
tion of 0.25 g/m l (w ith a fixed concentration of
internal standard of 400 g) are show n in Fig 2a
and 2b.
Calibration curves
Plasm a analysis w as calibrated using a con-
centration range of 0.25-16.0 g/m l. A ll calibra-
tion ranges yielded linear relationships w ith cor-
relation coefficients of 0.999 or better.
Method validation
Precision. Little variation in the clindam ycin as-
says w as observed. C oefficients of variation (C V)
for six analyses at the concentration range ob-
served w ere all below 15% . The intra-assay
(w ithin-day) and inter-assay (day-to-day) varia-
tion for the clindam ycin assay at a concentra-
tion range of 0.25-16.0 g/m l are sum m arised
in Table 1. For intra-day assay validation in
plasm a, the coefficients of variation varied be-
tw een 2.3 and 12.1% . The corresponding val-
ues for inter-day assay validation in plasm a w ere
2.4 and 14.1% .
Accuracy. G ood accuracy w as observed from
both the intra-day and the inter-day assays, as
indicated by the m inim al deviation of m ean val-
ues found w ith m easured sam ples from that of
the theoretical values (actual am ount added). The
intra-assay (w ithin-day) and inter-assay (day-to-
day) accuracy for the clindam ycin assays at a
concentration range of 0.25-16.0 g/m l are
sum m arised in Table 1. For intra-day assay ac-
curacy in plasm a, the m ean deviation from the
theoretical values varied betw een -10.5 and
+1.6% . The corresponding values for inter-day
assay validation in plasm a w ere -9.4 and +0.7.
Recovery. The m ean recoveries for clindam ycin
in plasm a at concentrations of 0.25, 4 and 8 g/
m l, and a internal standard at a concentration
of 400 m g w ere greater than 88% , w ith a m ean
(S D ) of 92.2 (2.5)% . The results reflect the lack
of interference from the sam ple preparation pro-
cedure.
Selectivity. S electivity of the chrom atographic
separation w as dem onstrated by the absence
of interference from endogenous peaks, as w ell
as those from com m only used drugs described
above. Fig 2 (a, b) illustrates typical chrom ato-
gram s for blank plasm a, spiked plasm a w ith
clindam ycin and the internal standard.
Limit of quantification. The lim it of quantifica-
tion (LO Q ) in hum an plasm a for clindam ycin w as
accepted as 0.07 g using 1 m l plasm a.
Fig 2C hrom atogram of (a) blank plasm a, (b) plasm a
spiked w ith 0.25 g/m l clindam ycin and 400 g
internal standard (retention tim es of 5.6 and
14.2 m inutes, respectively).
(a)
(b)
C
l
i
n
d
a
m
y
c
i
n
I
n
t
e
r
n
a
l
s
t
a
n
d
a
r
d
S O U TH EA S T A S IA N J TR O P M ED P U B LIC H EA LTH
182 Vol 37 N o. 1 January 2006
Table 1
S um m ary of assay precision and accuracy
(intra- assay and inter-assay) for clindam ycin
assay in plasm a.
C oncentration
added Intra- Inter- Inter- Inter-
(g/m l) assay assay assay assay
(n=6) (n=6) (n=6) (n=6)
0.25 2.3 5.4 +1.6 -1.6
0.50 4.1 12.9 -2.1 -9.4
1.00 12.1 14.1 -6.8 -5.8
2.00 4.8 6.6 -1.8 -5.3
4.00 3 5.9 -3.7 -3.8
8.00 3.9 9.4 -10.5 -9.3
16.00 5.2 2.4 -2.5 +0.7
a
% D M V = deviation of m ean value from theoretical
value (% )
Precision Accuracy
(% C V) (% D M V)
a
Tim e period C oncentration
(m onths) (g/m l) A ssay 1 A ssay 2 A ssay 3 M ean (S D ) % D EV
a
1 0.25 0.245 0.247 0.244 0.245 (0.001) +1.8
2.0 2.125 2.145 1.980 2.083 (0.090) -4.1
8.0 7.025 8.025 8.125 7.725 +0.6 (3.4)
2 0.25 0.239 0.245 0.246 0.243 (0.003) +2.6
2.0 2.015 1.989 1.890 1.964 (0.065) +1.7
8.0 7.581 7.658 8.012 7.750 (0.229) +3.1
3 0.25 0.247 0.254 0.248 0.249 (0.003) +0.1
2.0 2.015 1.989 1.897 1.967 (0.062) +1.6
8.0 8.569 8.125 7.898 8.197 (0.341) -2.4
6 0.25 0.249 0.247 0.251 0.249 (0.002) +0.4
2.0 2.014 2.156 1.899 2.023 (0.128) -1.1
8.0 7.890 7.991 7.899 7.926 (0.055) +0.9
a
% D EV = deviation of single value from theoretical value (% )
Table 2
S torage stability data of clindam ycin in plasm a at concentrations 0.25, 2.0, and 8.0 g/m l.
(a) Long-term stability at 1, 2, 3 and 6 m onths
C oncentration m easured (g/m l)
C oncentration
added (g/m l) A ssay 1 A ssay 2 A ssay 3 M ean (S D ) % D EV
0.25 0.258 0.249 0.251 0.252 (0.004) -1
2.0 2.012 1.989 1.897 1.966 (0.06) +1.7
8.0 7.890 8.123 8.012 8.008 (0.116) -0.1
a
% D M V = deviation of m ean value from theoretical value (% )
C oncentration m easured (g/m l)
(b) Freeze and thaw stability
Stability
P lasm a sam ples containing clindam ycin at
concentrations of 0.25, 2.0, and 8.0 m g/m l w ere
found to be stable w hen stored at -20C for a
m inim um of six m onths w ithout significant de-
com position of the drug. Freezing and thaw ing
of the spiked sam ples did not appear to affect
the quantification of the analytes (Table 2a). The
m ean deviation (% ) of the m easured concentra-
tions after storage at the observed periods (1,
2, 3 and 6 m onths) varied betw een -4.1 and +
1.7% . Freezing and thaw ing for three succes-
sive cycles did not affect the m easured concen-
trations. M ean deviation from the theoretical val-
ues varied betw een -1.0 and + 1.7% (Table 2b).
Quality control. Analytical values for all the quality
control sam ples (6 sam ples for each run at con-
centrations of 0.25, 2 and 8 g/m l w ere all w ithin
D ETER M IN ATIO N O F C LIN D A M Y C IN IN P LA S M A
Vol 37 N o. 1 January 2006 183
+ 10% of their respective nom inal values.
Application of assay and analysis of specimens
To dem onstrate the clinical applicability of
the m ethod, plasm a concentrations levels of
clindam ycin w ere determ ined in a patient follow -
ing oral doses of clindam ycin at 10 m g/kg body
w eight given tw ice daily for 7 days. A chrom ato-
gram of a plasm a sam ple collected 1 hour after
the last dose of clindam ycin on day 7 of dosing
(spiked w ith 400 g internal standard) is show n
in Fig 3.1. The plasm a concentration-tim e pro-
file for clindam ycin is show n in Fig 4, w hich is in
general agreem ent w ith those previously de-
scribed.
D IS C U S S IO N
W e describe a H P LC assay procedure
based on reversed-phase C 18 chrom atography
w ith ultraviolet detection, for the selective, sen-
sitive, accurate and reproducible quantitative
analysis of clind am ycin in hum an p lasm a
sam ples. Total run tim e w as w ithin 20 m inutes.
The analytical m ethod for the determ ination of
clindam ycin in plasm a established in this study
m eets the criteria (sim plicity, selectivity, accuracy,
good recovery, and high sensitivity) for applica-
tion to routine clinical drug level m onitoring or
pharm acokinetic studies. The advantage of the
m ethod over previously reported ones are its
rapidity, sim plicity and high sensitivity. In addi-
tion, the sam ple preparation procedure is sim ple,
faster and less expensive.
A C K N O W LED G EM EN TS
This investigation received financial support
from the U N IC EF-U N D P /W orld/B ank/W H O S pe-
cial P rogram for R esearch and Training in Tropi-
cal D iseases.
R EFER EN C ES
B atzias G C , D elis G A , K outsoviti-P apadopoulou M . A
new H P LC /U V m ethod for the determ ination of
clind am ycin in d o g b lo o d serum . J Pharm
Biomed Anal 2005; 000-000.
B ooth D W . S m all A nim al C linical P harm acology and
Therapeutics. Philadelphia: W B Saunders, 2001:
170.
B row n LW . G LC determ ination of clindam ycin and re-
lated com pounds. J Pharm Sci 1974; 63: 1597-
600.
B row n LW , B eyer W F. C lindam ycin hydrochloride, in:
B ritain H G (Ed), A nalytical P rofiles of D rug S ub-
stances and Excipients, vol 10, A cadem ic P ress,
N ew York, 1981, 76-91.
B row n LW . H igh-pressure liquid chrom atographic as-
says for clindam ycin, clindam ycin phosphate,
and clindam ycin palm itate. J Pharm Sci 1978;
67: 1254-7.
C herlet M , C roubels S , D e B acker P. D eterm ination of
clindam ycin in anim al plasm a by high-perfor-
m ance liquid chrom atography com bined w ith
electrospray ionization m ass spectrom etry. J
Mass Spectrom. 2002; 37: 848-53.
D uckw orth C , Fisher JF, C arter S A , N ew m an C L,
C ogbum C , N esbit R R . Tissue penetration of
Fig 3C hrom atogram of plasm a sam ple collected at 1
hr after the last dose of clindam ycin on day-7
(spiked w ith 400 m g internal standard).
0 24 48 72 96 120 144 168
Time (hr)
0
2
4
6
8
10
P
l
a
s
m
a

c
o
n
c
e
n
t
r
a
t
i
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(

g
/
m
l
)
Fig 4Plasm a concentration-tim e profile of clindam ycin
in a patient follow ing oral dose of clindam ycin at
10 m g/kg body w eight given tw ice daily for 7
days.
C
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n
d
a
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c
i
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S O U TH EA S T A S IA N J TR O P M ED P U B LIC H EA LTH
184 Vol 37 N o. 1 January 2006
clind am ycin in d iab etic fo o t infectio ns. J
Antimicrob Chemother 1993; 31: 581-4.
El-Yazbi FA , B laih S M . D eterm ination of clinidam ycin in
plasm a by spectrophotom etric m ethod. Analyst
1993; 118: 577-9.
Fichera M E, B oos D S. A Plastid organelle as a drug tar-
get in apicom plexan parasites. Nature 1997; 390:
407-9.
Fieger-B uschges H , S chussler G , Larsim ont V, B lum e
H . D eterm ination of clindam ycin in hum an plasm a
by high-perform ance liquid chrom atography us-
ing coupled colum ns. J Chromatogr B Biomed
Sci Appl 1999; 724: 281-6.
G atti G , Flaherty J, B ubp J, W hite J, B orin M , G am ber-
toglio J. C om parative study of bioavailabilities
and pharm acokinetics of clindam ycin in healthy
volunteers and patients w ith A ID S . Antimicrob
Agents Chemother 1993; 37: 1137-43.
H ornedo-N unes A , G etek TA , K orfm acher F, S im ental
F. D eterm ination of clinidam ycin in plasm a by
high p erform ance liq uid chrom atograp hy. J
Chromatogr 1990; 503: 217-25.
K ohler S , D elw iche C F, D enny P W , et al. A plastid of
probable green algal origin in A picom plexan
parasites. Science 1997; 275: 1485-9.
La Follette G , G am ertoglio J, W hite JA , K nuth D W , Lin
ET. D eterm ination of clindam ycin in plasm a or
serum by high-perform ance liquid chrom atogra-
phy w ith ultraviolet detection. J Chromatogr
1988; 431: 379-88.
Landis JB , G rant M E, N elson S A . D eterm ination of
clincam ycin in pharm aceuticals by high-perfor-
m ance liquid chrom atography using ion-pair for-
m ation. J Chromatogr 1980; 202: 99-106.
Liu C M , C hen YK , Yang TH , H sieh S Y, H ung M H , Lin
ET. H igh-perform ance liquid chrom atographic
determ ination of clindam ycin in hum an plasm a or
serum : application to the bioequivalency study of
clindam ycin phosphate injections. J Chromatogr
B Biomed Sci Appl 1997; 696: 298-302.
M artens-Lobenhoffer J, B anditt P. S ensitive and spe-
cific determ ination of clindam ycin in hum an se-
rum and bone tissue applying liquid chrom atog-
raphy-atm ospheric pressure chem ical ionization-
m ass spectrom etry. J Chromatogr B Biomed Sci
Appl 2001; 755: 143-9.
M etzer C M , D eH aan R ,S chellenberg D , Vandenbosch
W D . C lindam ycin dose-bioavailability relation-
ships. J Pharm Sci 1973; 62: 591-8.
M unson JW , K ubiak EJ. A high-perform ance liquid
chorm atographic assay for clindam ycin phos-
phate and its principal degradation product in
bulk drug and form ulations. J Pharm Biomed
Anal 1985; 3: 523-33.
O esterling TO , R ow e EL. H ydrolysis of lincom ycin-2-
phosphate and clindam ycin-2-phosphate. J
Pharm Sci 1970; 59: 175-9.
O rw a JA , Vand enb em p t K , D ep uyd t S , R oets E ,
H oogm artens J. Liquid chrom atography m ethod
for separation of clindam ycin from related sub-
stances. J Pharm Biomed Anal 1999; 20: 745-52.
R echberger G N , Fauler G , W indischhofer W , K ofeler H ,
E rw a W , Leis H J. Q uantitative analysis o f
clindam ycin in hum an plasm a by liquid chrom a-
tography/electrospray ionisation tandem m ass
spectrom etry using d1-N -ethylclindam ycin as
internal stand ard . Rapid Commun Mass
Spectrom 2003; 17: 135-9.
S in D W , W ong Y, C hun-bong Ip A . Q uantitative analy-
sis of lincom ycin in anim al tissues and bovine m ilk
by liquid chorm atography electrospray ionization
tandem m ass spectrom etry. J Pharm Biomed
Anal 2004; 34: 651-9.
Yu LL, C hao C K , Liao W J, et al. D eterm ination of
clindam ycin in hum an plasm a by liquid chrom a-
tography-eletrospray tandem m ass spectrom -
etry: application to the bioequivalence study of
clindam ycin. J Chromatogr B Biomed Sci Appl
1999; 724: 287-94.