3. Synthesis. 4. Methods of Analysis. 4.1 Identification Tests. 4.2 Quantitative Analysis ..... v o l t ammetry ...... L.G. Surhland and A.S. Weisberger, Arch. Intern.
CHLORAMPHENICOL
AbduReah A. M-Badh and Hurneida A. El-Obeid
1. D e s c r i p t i o n 1.1 Nomenclature
1.2
Formulae
1 . 3 Molecular Weight
1 . 4 Elemental Composition 1.5 2.
Appearance, Color, Odor and Taste
Physical Properties 2.1
Melting P o i n t
2.2
Solubility
2.3
Spectral Properties
3.
Synthesis
4.
Methods of A n a l y s i s
4.1
I d e n t i f i c a t i o n Tests
4.2
Q u a n t i t a t i v e Analysis
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15
701
Copyright Q 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
702
5.
Pharmacokinetics 5.1
Absorption and Distribution
5.2
Excretion
5.3
Half-life
5.4 Metabolism Acknowledgement References
CHLORAMPHENICOL
703
1. Description
1.1 Nomenclature 1.1.1 Chemical Names D(-)-threo-2-Dichloroacetamido-l-p-nitro-
phenyl-1,3-propanediol.
D( - ) threo-N-Dichloroacetyl-1-p-nit rophenyl2-amino-1,3-propanediol.
D(-) threo-2,2-Dichloro-N-[B-hydroxy-a(hydroxymethyl)-p-nitrophenethyl]acetamide. D( - ) threo-2,2-Dichloro-N- [ 2-hydroxy-l( hydroxymethyl) -2-( 4-nitrophenyl)ethyl] acetamide. 1.1.2 Generic Names
Chloramphenicol, Chloramfenicol, Chloramphenic o l m , Chloramphenicolo, Laevomycetin. 1.1.3
Trade Names (1) Alficetyn, Ambofen, Amphicol, Anacetin, Aquamycetin, Bemacol, Berlicetin, Biocetin, Biophenicol, Cafenolo, Cebenicol, Chemicetina, Chemyzin, Chlomin, Chloramex, Chloramol, Chloramphenicol-POSY Chlorasol, Chlora-tabs, Chloricol, Chlornitromycin, Chlorocid, Chloromycetin, Chloronitrin, Chloroptic, Chloro-25 Vetag, Chlorsig, Cloramidina, Clorbiotina, Clorof'enicina, Cloromicetin, Cloromycetin, Clorosintex, Comycetin, Cylphenicol, Desphen, Detreomycine, Devamycetin, Dextromycetin, Doctamicina, Duphenicol, Econochlor , Erbaplast , Ert ilen, Farmicetina, Fenicol, Globenicol, Glorous, Halomycetin, Hortfenicol , Isicetin, Ismicetina, Isophenicol, Isopto Fenicol, Kamaver, Kemicetin, Kemicetine, Kloromisin, Labamicol, Leukomycin, Levomycetin, Lomecitina, Loromisin, Mammaphenicol, Pledichol, Micochlorine, Misetin, Mycetin, Mychel, Mycinol, Neocetin, Novochlorocap, Nova-Phenicol, Novophenicol, Oftakloram, Oftalent , Oleomycetin, Opclor, Ophtaphenicol, Ophtochlor, Oralmisetin, Otachron, Otomycin, Otophen, Pantovernil, Paraxin,
CH,- OH I
H,N@ I
VH-CH-NHCOCHCl, OH
O,N-@
YH2 OC 6 H9 7 FH-CH-NHCOCHC1 OH
,
CH OH FH-;H-NH-CCOOH 11 0 OH
o,N@
FH2OH FH-CH-NHCOCH2 OH OH CH, OH FH-CH-NH, I
O,N@
dog.
O,N@
CHO
OH O2N
0
II
H-C-CH,NHCOCHC~,--€
f--+ OH-CH,CH,-NHCOCHCl, 02N@
yH20H ~H-CHNHCOCH2C1 OH
OH
FH2OH
yH2OH
, O,N
yH2 OC 6 H9 7 FH-CH-NHCOCHC12
-00
-@ YH-CH-NHCOCHC1, OH
R a t hepato-
H,N
@-F H - c H - ~ ~ o c H c ~ , OH
0
CH,?HN @ : C H NO C H ~ ,
-
.*
0,N -@:H-CH-NIICOCHCl
CH, OH -@FH-CH-NH, I
OH
4.
yH2 OC 6 H9
OH
0,N Scheme
yH2OH
Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .
7
705
CHLORAMPHENICOL 1.2.3
CAS R e g i s t r y No.
[ 56-75-7 1.2.4
1
Wiswesser Line Notation WNR DYQYIQ MVYGG ( 2 ) .
1.3
Molecular Weight 323.13,
1.4
322.01 ( 2 )
Elemental Composition C 40.88%,
H 3.74%, C 1 2-1.95%,
N
8.67%, 0 24.76%.
1 . 5 Appearance, Color, Odor and T a s t e Fine white t o greyish white o r yellowish white c r y s t a l s , n e e d l e s o r e l o n g a t e d p l a t e s from water o r e t h y l e n e d i c h l o r i d e w i t h very b i t t e r t a s t e . 2.
Physical Properties 2.1
Melting P o i n t
2.2
Solubility S o l u b l e (25') i n water : 2.5 mg/ml , i n propylene g l y c o l : 150.8 mg/ml, v e r y s o l u b l e i n methanol, ethanol, butanol, e t h y l a c e t a t e , acetone. F a i r l y s o l u b l e i n e t h e r , i n s o l u b l e i n benzene, petroleum e t h e r , v e g e t a b l e o i l s . S o l u b i l i t y i n 50% acetamide s o l u t i o n i s 5%. Aqueous s o l u t i o n s are n e u t r a l . N e u t r a l and a c i d s o l u t i o n s are s t a b l e on h e a t i n g .
2.3
Spectral Properties 2.3.1
U l t r a v i o l e t Spectrum The u l t r a v i o l e t a b s o r p t i o n spectrum o f chloramphenicol i n n e u t r a l methanol w a s obtained on a Cary 219 spectrophotometer. The spectrum, shown i n F i g . 1, i s c h a r a c t e r i z e d by a maximum a t 274 nm and a minimum a t 235 nm. The spectrum o f chloramphenicol i n
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
706
w u z 4 m
L
0
In m 4
-
- -
~'
1- * - . * . 220 230 240 250 260 270280290 300 30 320330340 WAVELENGTH Figure 1.
Ultraviolet epectnuP of chlormphenicol in neutral amthmol.
707
CHLORAMPHENICOL
water showed a maximum a t 278 nm (E 1%, 1 cm 2 9 8 ) ; i n 0.1N NaOH, a maximum a t 276 nm (E l%, 1 cm 2 0 0 ) ; i n 0.1N H SO4, a maximum at 278 nm ( E 1%, 1 cm 284) 73). 2.3.2
I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum o f chloramp h e n i c o l o b t a i n e d from a potassium bromide d i s p e r s i o n i s shown i n F i g u r e 2. The spectrum w a s recorded on a Pye Unicam SP 1025 i n f r a r e d spectrophotometer. The c h a r a c t e r i s t i c bands o f t h e spectrum w i t h t h e a s s i g n ments are l i s t e d below: Frequency ( cm-l)
Assignment
3230, 3520
Broad H-bonded OH and NH stretch
3100
Aromatic C-H s t r e t c h
1700, 1570
C = 0 s t r e t c h amide 1
1530, 1360
N
1070
C
-
850
C
-N
band, amide I1 band. 0
s t r e t c h (ArN02)
0 s t r e t c h (primary
alcohol)
s t r e t c h (ArN02)
The p r i n c i p a l peaks as r e p o r t e d by Clarke ( 3 ) are 1682, 1061 and 1351 or 1526 cm'l
2.3.3
I
H-Nuclear Magnetic Resonance Spectroscopy
H ' NMR s p e c t r a o f chloramphenicol i n DMSO-d6 ( F i g u r e 3 ) and i n DFlso-dg i n t h e p r e s e n c e of D20 ( F i g u r e 4) are o b t a i n e d on a Varian-T6OA NMR s p e c t r o m e t e r . The band assignments are r e f e r e n c e d t o TMS and are l i s t e d below:
f
WAVEL NGTH Urn4
01
-
5
3500 3WO 2500 2000
WAVENUMBER
Figure 1.
7
6
1800
1600
1400
8
9
10 11 12 U 14151 i
$'
1200
lo00
Infrared e p e c t w of chloramphenicol from IEBr dime.
800
100 90 80 70 60 50 40
62
30 20 10 0
CHLORAMPHENICOL
709 0
100
500
m
TM!
,......... 8.0
1.
7.0
Ii#uro 3.
500
.
.
.
6.0 'H-NMR
400
.
.
.
,
5.0
.
.
.
.
...........
I
co
PPM ( 6 )
30
.
.
2.0
.
a
.
1.0
. . . *
0
apectrum of chloramphenicol i n DWO-de.
0
100
300
patcr
TM!
I
'
, . . - . . " . . ~ . . . . . . ' . . . . ' . . . ' ' . . . . ' . . . . . . .
8.0
7.0
6.0
50
GO PfW ( 8 )
3.0
2.0
Figure 4. lH NHU spectrum of chloramphenicol in WSO-d, and D1O.
1.0
0
710
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
Chemical s h i f t *
(6)
Proton Assignment
Multiplicity
No. of
Protons
a.
3.43
b.
3.47
a.
3.97
b.
3.98
a.
4.93
b.
Exchanged
a. b.
5.12 ) ) 5.08 )
Triplet
Cg-OH
1
a.
5.97
Doublet
CH-OK
1
b.
Exchanged
-
-
a.
6.47 )
b.
6.40 )
a.
7.60 )
1 )
1
b.
7.58
a.
8.15 ) 1 ) )
b.
8.13
a.
8.25
b.
Exchanged
M u l tiplet
-CH20H
2
M u l tiplet
Cg-N-
1
Triplet
-CH2-Og
1
-
-
-
-cocgc1
Singlet
Doublet
OpN
q
Doublet
a, i n DMSO-d6 ;
1
2
2
-H Doublet
-NH
b , i n DMSO-d6
1
-
~
*
-
+ D20.
711
CHLORAMPHENICOL
2.3.4
1 3 C Nuclear Magnetic Resonance ( 1 3 C NMR)
The 1 3 C NMR s p e c t r a of chloramphenicol a r e obtained i n DMS-d6, containing a drop of CDC13, a t ambient temperature with 'Hdecoupling (Figure 5 ) and off-resonance (Figure 6 ) . The s p e c t r a are recorded using TMS as i n t e r n a l standard on a JEOL FXlOO MHz instrument. The chemical s h i f t s , m u l t i p l i c i t i e s and s p e c t r a l assignments a r e given below: Chemical s h i f t
(6)
Multiplicity
56.71
Doublet
60.18
Triplet
66.35
Doublet
68.93
Doublet
122.76
Doublet
127.17
Doublet
146.31
Singlet
151.12
Singlet
163.33
Singlet
Carbon assi nment* c2
c3
c21
c31
c4'
*Refer t o s t r u c t u r e i n Figure 5 f o r carbon numbering. 2.3.5
Mass Spectrum
The combined gas-chromatographic/massspectrophotometric technique w a s used f o r t h e i d e n t i f i c a t i o n and a n a l y s i s o f chloramphenic o l i n aqueous s o l u t i o n s ( 4 ) and f o r chloramphenicol and i t s metabolites i n animal t i s s u e s and body f l u i d s ( 5 , 6 ) . Becker et a1 ( 7 ) and Krueger ( 8 ) s t u d i e d t h e s p e c t r a of nonv o l a t i l e substances by f i s s i o n fragment
712
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
NH c"0 6H CI, 4N
3
Figure 5.
Proton decoupled 13C lsyR rpwtrwn of chlorunphoniool in DYBO-dg + a drop of CDClg.
DMSO
Figure 6. Off-resonance 13C NMR spectrum of chlorsmphenicol In DYBO-dg + a drop of CDC13.
CHLORAMPHENICOL
713
d e s o r p t i o n mass spectrometry. In t h e s p e c t r a o f chloramphenicol b o t h quasimolec u l a r i o n s are p r e s e n t e d w i t h t h e i r t y p i c a l two-chlorine i s o t o p i c p a t t e r n . The p o s i t i v e i o n s p e c t r a show v a r i o u s d i f f e r e n t subgroups. Although t h e r e i s v e r y l i t t l e f r a g m e n t a t i o n i n t h e n e g a t i v e i o n spectrum, a s t r o n g peak group arises n e a r m/e 152. E l spectrum o f chloramphenicol w a s shown (2) t o p o s s e s s t h e f o l l o w i n g peaks: 153(100%),
60(99%), 70(85%) 15508%), 170(70%), 106(46%), 77(40%).
P r e s e n t e d i n F i g u r e 7 i s t h e 70 e V e l e c t r o n impact ( E I ) mass spectrum o f chloramphenicol o b t a i n e d on Varian MAT 311 mass s p e c t r o m e t e r u s i n g i o n s o u r c e p r e s s u r e o f 10-6 T o r r , i o n s o u r c e t e m p e r a t u r e of 18OoC and a n emission c u r r e n t o f 300 PA. No molecular i o n i s d e t e c t e d and t h e spectrum i s dominated by m / e 153 i o n (base peak) r e s u l t i n g from t h e l o s s o f 02NC6H4CHO and H20. A proposed mechanism o f f r a g m e n t a t i o n and t h e mass/ charge r a t i o s o f t h e major fragments i s given i n Scheme 1. The chemical i o n i z a t i o n (CI) spectrum ( F i g u r e 8) w i t h methane g a s as a r e a g e n t i s o b t a i n e d on a Finnigan 4000 mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy o f 100 eV, i o n s o u r c e p r e s s u r e o f 0.3 T o r r , i o n s o u r c e t e m p e r a t u r e o f 150°C and emission c u r r e n t of 300 PA. The spectrum shows no p a r e n t molecular i o n b u t a pronounced peak r e s u l t i n g from t h e l o s s o f water (MH+-18) c o n s t i t u t e t h e b a s e peak a t m / e = 305. A quasi-molecular i o n ( M + 1) i s a l s o prominent. Two peaks a p p e a r i n g at m / e = 351 and m / e 363 are a t t r i b u t e d t o t h e t r a n s f e r o f c a r b o c a t i o n s from t h e c a r r i e r g a s . The mass s p e c t r a l assignment o f t h e prominent i o n s under CI c o n d i t i o n s i s g i v e n i n Table I .
lW.0-
-
€0
a.0-
70
.
170
Sl
-
1W.O-
-
a.0-
200
220
Figure 7.
2w
2 40
280
300
320
Yaee spectrum of chlorunphenicol (EI).
SO
I
MIL
2M
200
Figure 8 .
$00
PO
YO
960
300
LOQ
420
Haem epectrum of chloramphenicol (CI).
440
28416.
02N@
$5
CH20H YH-CH I - NH-C-CHClp OH m/e
322 (0%)
--*
[
02N@
CH2 OH I FH-CH - NH-C E
3
+-+ 02N-@!€$H ,
OH
+I
-NHC=g 0 -HCH20H
m/e 239
m/e 239
NHCOCHC12
I
1
m/e 152
CH,= CHNHCOCH = C1+ m/e 118 CH, = CH-NH-C +
0
m/e 77
1
+
CH, = CH-NH-C = O
Scheme 1. Mechanism of chloramphenicol fragmentation.
m/e 70
J
CH = C1
+
1
c1 m/e 83
Scheme I (continued) CH2 OH ~
02N
1
+.
y-&-NH-COCHClp
d
0
2
.
9
+
-2P+
CH-OH
HOCH,CH = NH-CO-CC1,
U
+
OH
+
0,N
CH = OH
+
m / e 152
HOCH2CH =
i
NH2
m / e 60 m / e 106
+
r
L
OH
m / e 291
J
+ O=C=C
/
\
c1 c1
717
CHLORAMPHENICOL Table 1. Mass S p e c t r a l Assignments of Chloramphenicol Using C I with Methane as Reagent Gas.
m/e
3.
Species
363
[M + C3H51+
351
[M + C2H51
32 3
MH+
305
[MH
- H20]+
287
[MH
-
HCl]'
27 5
[MH
-
(H20
+
+ CH2 =
O)]'
Synthesis a.
The a n t i b i o t i c w a s i n i t i a l l y i s o l a t e d from c u l t u r e s o f v a r i o u s Streptomyces s t r a i n s . The s t r u c t u r e s i m p l i c i t y of chloramphenicol made it amenable t o p r e p a r a t i o n by t o t a l s y n t h e s i s both i n t h e l a b o r a t o r y and on commercial s c a l e . One method o f s y n t h e s i s involves a base-catalysed condensation of benzaldehyde with n i t r o e t h a n o l t o a f f o r d t h e a l d o l product as a mixture of stereoisomers (Scheme 2 ) . C a t a l y t i c r e d u c t i o n g i v e s an aminodiol whose threo-isomer i s s e p a r a t e d and resolved i n t o t h e o p t i c a l isomers. The ( - ) isomer i s t r e a t e d with d i c h l o r o a c e t y l c h l o r i d e followed by treatment w i t h base t o remove t h e 0a c y l a t e d products t o a f f o r d t h e amide. The hydroxyl groups are t h e n p r o t e c t e d by means of a c e t i c anhydr i d e . The product i s n i t r a t e d t o produce t h e pn i t r o d e r i v a t i v e . Removal of t h e p r o t e c t i n g groups i s achieved by treatment with a base t o g i v e chloramphenicol ( 9 ) .
b.
'H-Chloramphenicol and i t s erythro-diastereomer with high s p e c i f i c a c t i v i t i e s were prepared by o x i d a t i o n of t h e nonlabeled a n t i b i o t i c t o i t s 0x0 d e r i v a t i v e (Scheme 3 ) , which upon reduction with 3 H - s o d i ~borohydride w a s converted t o t h e corresponding d i a s t e r e o mers. The diastereomers were s e p a r a t e d by HPLC. 2HChloramphenicol diastereomers can b e s y n t h e s i s e d s i m i l a r l y using 2H-sodium borohydride ( 1 0 ) .
X"
8 u I N
0"
!3-E I
8-3
8 x" u 1
x
I
V-Z
8-3
6
X
0
+
I
B-3
718
0
k
O2N
a
CH2OH YH-CH-NHC-CHC12 I
I
I
OH
It
0
Oxidation
-4 c ED
0
C - CH
Scheme 3
.
Synthesis of labeled chloramphenicols.
-
NH
-
It C
-
CHC12
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
720
4.
Methods of Analysis
4.1
I d e n t i f i c a t i o n Tests Dissolve about 10 mg i n 1 ml of alcohol (50%), add 3 m l o f a 1% of calcium c h l o r i d e and 50 mg o f zinc powder, heat on a water b a t h for 1 0 minutes, cool and f i l t e r ; t o t h e f i l t r a t e add 100 mg of anhydrous sodium a c e t a t e and 2 drops o f benzoyl c h l o r i d e , shake f o r 1 minute and t h e n add 0.5 ml of f e r r i c c h l o r i d e s o l u t i o n and 3 ml o f d i l u t e hydrochloric a c i d and mix; a reddish-violet or purple color i s produced. No such c o l o r i s produced when t h e t e s t i s repeated without zinc powder (11). To 5 ml of 0.1% s o l u t i o n add a few drops of s i l v e r n i t r a t e s o l u t i o n , no p r e c i p i t a t e i s produced. Heat about 50 m g with 2 ml of alcoholic potassium hydroxide s o l u t i o n on a water bath for 1 5 minutes, add a s m a l l q u a n t i t y o f decolorizing charcoal, shake, f i l t e r and t o t h e f i l t r a t e add s i l v e r n i t r a t e s o l u t i o n ; a white p r e c i p i t a t e i s produced which i s i n s o l u b l e i n n i t r i c a c i d but s o l u b l e a f t e r washing with water, i n d i l u t e ammonia s o l u t i o n . (11)
.
Dissolve about 1 0 mg i n 2 m l of alcohol ( 5 0 % ) add 4.5 m l o f d i l u t e s u l f u r i c a c i d and about 50 mg zinc powder, allow t o stand f o r 10 minutes and decant t h e supernatant l i q u i d ; cool t h e supernatant l i q u i d i n i c e ; add 0.5 ml sodium n i t r i t e s o l u t i o n , allow t o stand f o r 2 minutes and t h e n add 1 gm of urea followed by 1 ml of 2-naphthol s o l u t i o n and 2 ml of sodium hydrox i d e s o l u t i o n ; a red c o l o r i s produced (11). The n i t r o group i s reduced t o an amino group by zinc-HC1 and t h e amine i s caused t o r e a c t with dimethylaminobenzaldehyde , t h e r e s u l t i n g Schif f base gives a colored s a l t i n a c i d medium. The method can be applied as a spot t e s t . Chloramphenicol i n ointments i s e x t r a c t e d i n t o 96% ethanol. Riboflavine does not i n t e r f e r e i n t h e d e t e c t i o n o f chloramphencol (12).
CHLORAMPHENICOL
721
An orange-red c o l o r i s produced and ammonia i s evolved when chloramphenicol i s heated with 50% NaOH s o l u t i o n ( 3 ) . Chloramphenicol g i v e s a p o s i t i v e r e a c t i o n t o Fujiwara's T e s t ( 3 ) as follows: a l i t t l e s o l i d o r drop of a t e s t s o l u t i o n i s added t o a mixt u r e of p y r i d i n e (1m l ) and 20% NaOH s o l u t i o n The mixture i s heated on a b o i l i n g (2 ml) water b a t h f o r 3-5 minutes with vigorous p e r i o d i c shaking. A c o n t r o l t e s t must be c a r r i e d o u t . A red c o l o r appears i n t h e p y r i d i n e l a y e r .
.
Ammonium molybdate t e s t for micro q u a n t i t i e s g i v e s f a i n t b l u e color ( 3 ) . 4.2
Q u a n t i t a t i v e Analysis 4.2.1
B i o l o g i c a l Methods 4.2.1.1
Microbiological Methods Chloramphenicol bioassay have been r e p o r t e d by Bannatyne and Cheung ( 1 3 ) . The a u t h o r s described an accurate p l a t e diffusion bioassay f o r t h e drug, i n which t h e f a s t r e p l i c a t i n g Beneckea n a t r i e g e n s and 1.5% s a l t a g a r are used. Zone of i n h i b i t i o n were w e l l defined a f t e r 3 hours and t h e l i m i t of s e n s i t i v i t y o f t h e method w a s around 2 Llg/ml.
Fabiansson and Rut egaerd ( 1 4 ) have reviewed t h e b i o l o g i c a l methods i n c u r r e n t use f o r t h e d e t e c t i o n of a n t i b i o t i c r e s i d u e s i n s l a u g h t e r anim a l s and reported a modified method i n which t h e c o n d i t i o n s f o r t h e c o n t r o l were standardized. The standardized c o n d i t i o n s i n c l u d e t h e use of a s p o r u l a t i n g organism, B a c i l l u s s u b t i l i s , an inoculum s i z e of 0 . 5 x 105 spores/ml medium, add 5 m l o f medium (pH 6.0) p e r p l a t e . A preincubation d i f f u s i o n t i m e of
722
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID 1 hour at room temperature i s recommended before incubat ion.
Horng and KO (15) have reported systematic a n a l y s i s of a n t i b i o t i c s via agar g e l electrophoresis and antimicrobial spectrum of methodology. A n t i b i o t i c s are detected i n food and drugs, by a method which employed both agar g e l electrophoresis and antimicrobial spectrum. Horng e t a 1 (16) have also reported systematic a n a l y s i s of a n t i b i o t i c s via agar g e l electrophoresis and antimicrobial spectrum-candidacy f o r detecting residual a n t i b i o t i c s i n foods. The method i s a modification of t h e method of Horng and KO (15) t o increase i t s r e s o l u t i o n power and s e n s i t i v i t y . Modification included t h e amount of agar g e l and t h e height of t h e t e s t organism s t r i p and t h e width of sample s l i t and enabled t h e a n a l y s i s of a n t i b i o t i c s at food residue concentrations (1 pg/ml).
4.2.1.2
Enzymatic Methods Smith and Smith (17) have reported an improved enzymic assay of chloramphenicol. R-factor-incoded chloramphenicol a c e t y l t r a n s e f e r a s e , from an Escherichia & mutant t h a t was highly r e s i s t a n t t o chloramphenic o l was p a r t l y p u r i f i e d and used f o r t h e assay. Only a n t i b i o t i c a l l y a c t i v e chloramphenicol i s attached by t h e enzyme. C r y s t a l l i n e chloramphenicol i s d r i e d t o constant weight a t 60" and dissolved i n serum, and 5 0 - ~ 1portions (as standards) a r e a r e s t o r e d at -70'. Serum samples and standards a r e heated a t 60° f o r 1 5 minutes bef re addition of 1 0 pl t o 50 p1 of ['&I acetylcoenzyme A
CHLORAMPHENICOL
723 s o l u t i o n (pH 7.8) and 25 p 1 o f enzyme source ( 0 . 1 spectrophotomet r i c u n i t ) . A f t e r incubation a t 37' f o r 60 minutes, t h e d i a c e t y l a t e d product i s s e l e c t i v e l y absorbed on micropore f i l t e r s and t h e n assayed by s c i n t i l l a t i o n counting
.
Robison e t a1 (18) have developed a s i m p l i f i e d radio-enzymatic assay f o r chloramphenicol , by elminat i n g t h e need for cumbersone e x t r a c t i o n procedure. A f t e r t h e a c e t y l a t i o n of chloramphenicol w i t h 14C-labeled a c e t y l CoA i n t h e presence of chloramphenicol ac e t y l t r a n s f e r a s e t h e r e a c t i o n mixture w a s added t o a toluene-based s c i n t i l l a t i o n f l u i d . Since 14C-ac e t y l a t ed c h l o r amphenicol i s more s o l u b l e t h a n 14C-labeled a c e t y l CoA i n t o l u e n e , t h e radioa c t i v e product could be counted directly. Detection and q u a n t i t a t i o n of chloramphenicol by competitive enzymel i n k e d immunoassay w a s r e p o r t e d by Campbell e t a1 ( 1 9 ) . The a s s a y f o r t h e drug i n meat involves competit i v e i n h i b i t i o n , by free chloramphenic o l i n t h e sample, of t h e binding of s p e c i f i c r a b b i t antibody t o solidphase-bound chloramphenicol. The antibody not d i s p l a c e d w a s measured by using a commercially a v a i l a b l e enzyme-linked a n t i - r a b b i t 1 gm prep r a t ion and added s u b s t r a t e Enzyme a c t i v i t y , measured spectrophotometric a l l y , w a s inversely proportional t o t h e c o n c e n t r a t i o n of chloramphenic o l i n t h e sample.
.
4.2.2
Chemical Methods 4.2.2.1
T i t r i m e t r i c Methods Navik and Polyakova ( 2 0 ) have r e p o r t e d t h e a n a l y s i s of some multi-
724
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID component w a t er-alcohol mixtures where aqueous ethanol s o l u t i o n of chloramphenicol was determined by a tit rimet r i c method. El-Sebai e t a 1 (21) have described new i n t e r n a l i n d i c a t o r s f o r t h e determination of primary aromatic amines (chloramphenicol y i e l d s an amino group on reduction with zinc dust and hydrochloric a c i d ) . Various diazo- compounds were synthes i z e d and t e s t e d a s an i n d i c a t o r s i n t h e t i t r a t i o n of such amines with NaN02 solution. Talegaonkar e t a1 (22) have described an a l k a l i m e t r i c determination of chloramphenicol i n dimethylformamide. A solution of t h e drug i n dimethylformamide i s t i t r a t e d with sodium methoxide s o l u t i o n i n benzene-methanol 4:1, with 2 drops 1% 2-nitroaniline s o l u t i o n i n benzene a s i n d i c a t o r , t h e color change i s from yellow t o red. The r e a c t i o n involved i n t h e t i t r a t i o n i s r e p l a c e ment of one chlorine atom i n t h e drug by a methoxy group. Koka (23) has described an iodimetric method f o r t h e determination of chloramphenicol i n some medicinal mixtures. The sample i s boiled with sodium hydroxide s o l u t i o n , cooled, d i l u t e d , t r e a t e d with 0.1N iodine, s e t a s i d e for 1 0 t o 1 5 minutes i n t h e dark, and then t r e a t e d with potassium iodide s o l u t i o n and d i l u t e sulphuric acid, and t h e l i b e r a t e d iodine i s t i t r a t e d w i t h sodium thiosulphate s o l u t i o n using starch a s indicator.
-
Koka and Koltun (24) have reported another iodimetric method f o r t h e determination of chloramphenicol i n
CHLORAMPHENICOL
725 some o t h e r medicinal forms.
4.2.2.2
Polarographic Methods Chloramphenicol [ i n milk] w a s d e t e r mined by Fossdal and Jacobsen ( 2 5 ) p o l a r o g r a p h i c a l l y . The e l e c t r o n r e d u c t i o n o f t h e drug w a s s t u d i e d by polarography of 0.5 mM s o l u t i o n of v a r i o u s e l e c t r o l y t e s and of 1.7 mM t o ~.IM s o l u t i o n i n 0.5M-acetate b u f f e r a t pH 4.7 and by c y c l i c v o l t ammetry chronopot ent iomet r y and coulometry; t h e r e a c t i o n s involved a r e discussed. A well-defined polarographic wave was obtained which even i n t h e presence of 50% of milk i s a n a l y t i c a l l y u s e f u l over t h e range of 0.3 t o 60 pg of a n t i b i o t i c per m l . Chloramphenicol and it h y d r o l y s i s product 2 amino-1-( p-nitropheny1)1,3-propanediol were determined p o l a r o g r a p h i c a l l y i n pharmaceutical formulations ( 2 6 ) a f t e r s e p a r a t i o n by t h i n - l a y e r chromatography on s i l i c a g e l GF254 using a 4 : l : l nbut anol-acet i c ac id-wat e r or 2 :2 :4 ac et one-benz ene-pet roleum e t h e r solvent mixtures. The s e p a r a t i o n of t h e above two compounds by highp r e s s u r e l i q u i d chromatography (Perkin-Elmer r e v e r s e phase C l 8 column with 45:55:1 methanol-watera c e t i c a c i d o r 30:70:1 isopropanolwater-acetic a c i d ) made p o s s i b l e simultaneous determination o f t h e above two compounds. Polak e t a1 ( 2 7 ) have determined chloramphenicol i n body f l u i d s by d i f f e r e n t i a l p u l s e polarography Reduction of t h e drug a t a droppingmercury e l e c t r o d e a t -0.4 V ( v s t h e s . c . e ) a t pH 4.5 ( B r i t t o n Robinson b u f f e r ) i s used f o r t h e determination
.
726
ABDULLAH A. AL-BADR AND'HUMEIDA A. EL-OBEID of t h e drug i n such samples a f t e r p r e c i p i t a t i o n of p r o t e i n s w i t h a c e t o n i t r i l e or methanol. The c o e f f i c i e n t of v a r i a t i o n w a s 4% f o r determination of 1 5 pg/ml of chloramphenicol i n serum. Convent i o n a l d.c. plarography i s s u i t a b l e f o r determining t h e drug only i n urine. 4.2.2.3
Colorimetric Methods Several colorimetric methods f o r t h e determination of chloramphenicol have been reported i n t h e l i t e r a t u r e . Plourde and Braun (28) have described a colorimetric procedure f o r t h e determination of t h e drug i n t a b l e t s and capsules. For t a b l e t s , powder t h e m a t e r i a l and dissolve a sample containing 20 mg of chlorphenicol i n 50 ml of 1,2-dichloroethane a t 60°, cool and d i l u t e t o 100 m l w i t h dichloroethane. F i l t e r t h e s o l u t i o n and r e j e c t t h e f i r s t few m l s . For s o f t capsules, s e c t i o n longitudinaly, and dissolve i n dichloroethane (150 ml) at 60°, cool and d i l u t e t o 250 ml w i t h dichloroethane. F i l t e r , r e j e c t t h e first few m l s . and d i l u t e an a l i quot containing 20 mg of chloramphenicol t o 100 ml w i t h dichloroethane. To t h i s d i l u t e s o l u t i o n (4.5 m l ) add dichloroethane ( 6 . 5 r n l ) and 'piperidine-8-hydroxyquinoline vanadate' reagent ( 4 m l ) and l e a v e f o r 30 minutes a t room temperature. Extract excess reagent w i t h M-NaOH ( 1 0 m l ) for 30 seconds, s e t aside f o r 30 seconds and f i l t e r t h e organic phase over Na2S04 i n t o 1 m l of 5% dichloroacetic a c i d s o l u t i o n i n a c e t i c a c i d then measure t h e extinction of the r e s u l t i n g blue s o l u t i o n a t 625 nm within 2.5 hours.
CHLORAMPHENICOL
727 A c o l o r i m e t r i c method using pdimethylaminobenzaldehyde i s desc r i b e d ( 2 9 ) f o r t h e determination of chloramphenicol and o t h e r compounds. I n an a c i d medium p-dimethylaminobenzaldehyde produced r e a c t i o n prod u c t s varying i n c o l o r from yellow t o deep r e d . The r e a c t i o n products showed maximud absorption spectrum peaks between 425 and 450 nm. Ivakhnenko et a1 ( 3 0 ) described a procedure f o r absorbtiometric d e t e r mination of chloramphenicol. D i l u t e t h e r e d u c t i o n product of 0.5 gm of chloramphenicol t o 100 ml. To 1 ml of t h e s o l u t i o n add 5 ml of N-HC1, 2 ml of 0.01M - NaNO and, a f t e r 4 t o 5 minutes, 5 m l of 0.3% s o l u t i o n of a diaminoacridine reagent ( e t h a c r i d i n e l a c t a t e or p r o f l a v i n e ) ; after a f u r t h e r 2 minutes d i l u t e t h e s o l u t i o n t o 50 ml and measure t h e e x t i n c t i o n of t h e r e s u l t i n g diazocompound a t 508 nm for t h e f i r s t reagent o r a t $84 nm for t h e o t h e r a g a i n s t water. The determination of chloramphenicol i n pharmaceuticals ( s u p p o s i t o r i e s and coated t a b l e t s ) have been r e p o r t e d by Cieszynski e t a1 ( 3 1 ) using colorimetry. The product of t h e r e d u c t i o n of chloramphenicol r e a c t s w i t h guaiacol i n a l k a l i n e medium (pH 9.6) t o form a b l u e complex, which i s s t a b l e f o r up t o 5 hours, with measurement of t h e e x t i n c t i o n , a t 610 nm, 30 minutes a f t e r t h e s o l u t i o n s a r e mixed. Przyborowski ( 3 2 ) described a method for t h e determination of t h e drug and i t s p a l m i t a t e i n pharmaceuticals. The method involves t h e h y d r o l y s i s of chloramphenicol by NaOH i n a medium containing hydroxylammonium c h l o r i d e , and r e a c t i o n of t h e result-
728
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID ing 2,2-dichloroacetohydroxamic a c i d with Fe3+. The coloured complex produced i s determined by spectrophotometry at 505 nm. A modified method f o r t h e determination of chloramphenicol palmitate and t h e contents of chloramphenicol and chloramphenicol palmitate i n s e v e r a l preparation i s a l s o given. Krezk and Lechniak (33) have reported t h e a p p l i c a t i o n of copper (11) t o t h e colorimetric determinat i o n of chloramphenicol i n ointments. The method i s based on t h e formation of a complex a f t e r mixing methanolic s o l u t i o n s o f chloramphenicol, copper (11) and methanol; t h e p r e c i p i t a t e of Cu(OH)2 i s f i l t e r e d and t h e absorbance o f t h e f i l t r a t e i s measured a t 550 nm. The composition o f t h e complex corresponds t o copper: chloramphenicol molar r a t i o = 1:2. Catechol and iodine have r e c e n t l y been used f o r t h e spectrophotometric determination of aromatic m i n e s ( 34). The method, involves mixing 15 m l of potassium a c i d p h t h a l a t e buffer s o l u t i o n (pH 3.1) , 1 m l of aqueous 0.1% catechol, 1 ml of 0.01Niodine and 1.5 ml of t h e m i n e s o l u t i o n , d i l u t i o n of t h e mixture t o 25 m l with water, and, after 5 t o 30 minutes (depending on t h e m i n e ) , spectrophotometry a t 500 t o 520 nm ( v s a reagent blank). A modification of t h i s procedure i s described f o r compounds t h a t y i e l d primary arylamino-groups on reduction (chloramphenicol)
.
Yang ( 3 5 ) has described a simult aneous determination of chloram-
phenicol and i t s metabolite D-threo2-amino-1-( 4-nitrophenyl )propane-l,3d i o l i n i n j e c t i o n s . A 1 0 m l sample
729
CHLORAMPHENICOL
i s d i l u t e d t o 25 ml w i t h anhydrous ethanol and a p o r t i o n i s sampled f o r s p e c t r o p o l a r i m e t r i c determinat i o n of t h e s p e c i f i c o p t i c a l r o t a t i o n a t 418 and 589 nm for t h e drug, and i t s m e t a b o l i t e s , r e s p e c t i v e l y . Divakar e t a1 ( 3 6 ) have used b r u c i n e and sodium metaperiodate f o r t h e c o l o r i m e t r i c e s t i m a t i o n of chloramphenicol. A 10-ml p o r t i o n o f t h e t e s t s o l u t i o n was b o i l e d under r e f l u x f o r 45 minutes w i t h 1 0 ml of 2M-HC1, and t h e excess of H C 1 was removed i n vaccu. The r e s i d u e was d i s s o l v e d i n 20 m l of w a r m water, and t h e s o l u t i o n was d i l u t e d t o 50 m l with water. This s o l u t i o n i n t e s t tubes w a s then t r e a t e d with 3 ml of 5 mM-brucine, 1 . 5 m l of 5 mM-NaIO4 and 2 m l of 2.3 M-H2S04 and t h e s o l u t i o n w a s d i l u t e d t o 1 0 m l with water. The t e s t t u b e s were shaken and heated on a boiling-water b a t h f o r 15 minutes. A f t e r cooling t h e cont e n t s of each t u b e were d i l u t e d t o 25 m l w i t h water and t h e absorbance of t h e s o l u t i o n w a s measured at 500 nm. 4.2.2.4
U l t r a v i o l e t Spectrophotometric Methods Chloramphenicol w a s determined i n pharmaceutical p r e p a r a t i o n s containing b o r i c a c i d , g l y c e r i n , sodium c h l o r i d e , zinc s u l p h a t e , belladonna e x t r a c t , s t r e p t o c i d , glucose, or r esorc i n o l by u l t r a v i o l e t spectrophotometric a n a l y s i s a t 278 or 315 nm. The drug w a s determined w i t h a r e l a t i v e e r r o r of f 3.46 ( 3 7 ) . Buryak ( 3 8 ) r e p o r t e d t h e a n a l y s i s of multicomponent medicines w i t h t h e a i d of a computer. The sample i s
730
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID b o i l e d f o r 5 minutes w i t h 95% ethanol, and t h e e x t r a c t i s f i l t e r e d and d i l u t e d as necessary with ethan o l . The absorbance i s measured a t various wavelengths between 226 and 330 nm vs ethanol. The concentr a t i o n of individual components are c a l c u l a t e d from a matrix of equations r e l a t i n g t h e t o t a l absorbance t o t h e sum of t h e p a r t i a l absorbances of t h e components. The method i s s u i t a b l e f o r lanolin-based ointment s containing chloramphenicol. 4.2.2.5
I n f r a r e d Spectrophotometric Methods I n f r a r e d spectroscopy had been used for t h e determination o f t h e a n t i b i o t i c s t a b i l i t y ( 3 9 ) . The e f f e c t of e x t e r n a l f a c t o r s such as h e a t , a c i d i t y and hydrolysis on c r y s t a l l i n e a n t i b i o t i c s was examined spectroscopically. Chloramphenicol r e s i s t e d dry heat at 80' f o r 24 hrs, but was unstable at 100' f o r 2 h r s . Namigohar e t a1 (40) reported an i n f r a r e d spectrophotomet r i c determinat ion of chloramphenicol. The drug was e x t r a c t e d from capsules, creams and eye drops, with ethanol or e t h y l acetate-chloroform and chloramphenicol palmitate was extracted with chloroform. Chloramphenicol and chloramphenicol palmit a t e were then determined by i n f r a r e d spectrophotometry, i n KBr and i n chloroform, r e s p e c t i v e l y .
4.2.2.6
Proton Magnetic Resonance Spectrometric Methods Chloramphenicol i n pharmaceutical preparation has been determined using proton magnetic resonance spectrometry (41). The drug was dissolved i n dimethyl sulfoxide containing
731
CHLORAMPHENICOL
maleic a c i d as i n t e r n a l standard. The n.m.r spectrum o f t h e s o l u t i o n was recorded, and t h e peaks f o r t h e aromatic protons o f chloramphenicol at 7.6 and 7.5 ppm and t h e v i n y l i c protons of maleic a c i d a t 6.25 ppm were i n t e g r a t e d ; t h e amount of chloramphenicol w a s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and t h e known amount of maleic a c i d . For t e n samples c o n t a i n i n g 100 t o 150 mg of pure chloramphenicol, t h e average recovery w a s 100.22% ( standard d e v i a t i o n 1.37%). The method has been a p p l i e d t o t h e a n a l y s i s of commercial capsules and o r a l suspens i o n of chloramphenicol p a l m i t a t e ; it i s r a p i d and simple and can a l s o b e used t o check t h e p u r i t y of t h e drug.
Mass Spectrometric Methods
4.2.2.7
Mass spectrometric methods have been described f o r t h e a n a l y s i s of chloramphenicol i n aqueous s o l u t i o n s (4) and i n animal t i s s u e s and body f l u i d s ( 5,6 ,52)
.
4.2.3
Chromatographic Methods A multitude of t h i n l a y e r , paper, column, gas and l i q u i d chromatographic methods have been developed f o r t h e d e t e c t i o n and d e t e r mination of chloramphenicol i n pharmaceutical formulations, b i o l o g i c a l f l u i d s and animal t i ssues
.
4.2.3.1
Thin Layer, Paper and Column Chromatography Various t h i n l a y e r , paper and column chromatographic methods used for t h e a n a l y s i s of chloramphenicol a r e o u t l i n e d i n Table 2.
732
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
4.2.3.2 Gas Chrmatographic Methods Gas chromatographic methods have been used for the determination of chloramphenicol in dosage forms and biological fluids and tissues. Table 3 summarises some of these methods.
4.2.3.3
High Performance Liquid Chromatography-(HPLC) HPLC has been extensively usea f o r the determination of chloramphenicol in pharmaceutical formulations and biological fluids as well as for the detection and determination of the drug residues in animal tissues. Some of these methods are outlined in Table 4.
Table 2.
Thin Layer, Paper and Column Chromatographic Methods for t h e A n a l y s i s o f Chloramphenicol.
Support
Solvent System
Detect i o n
Ref.
Silanized s i l i c a g e l (reverse phase)
Mixture of s o l v e n t s e .g . Dioxane , a c e t o n e , i s o p r o p y l a l c o h o l , methanol, t e t r a h y d r o f u r a n or e t h y l methyl k e t o n e w i t h c i t r a t e - p h o s p h a t e b u f f e r (pH 3, 5 or 7 ) .
-
42
Silica gel
C H C l -methanol-2.5% a q . NH3 (60:6:1)
Spray with SnC12 solut i o n , h e a t t o llO'for 7 min and s p r a y w i t h 4-dimethylaminobenzaldehyde
43
3
.
S i l u t o l W 254 Sheet
Ethyl ether.
Fluorescence quenching
44
S i l i c a gel
CH C 1 - e t h y l a c e t a t e ( 1 : b ) . 2 2
15% SnC12 i n aq. H C 1 , t h e n W.
46
High p e r f o r mance TLC cont a c t spotter
Chloroform-heptane-methanol ( 4 :2: 1)
W a t 280 nm.
46
S i l i c a g e l GF 254
( 4 :1:1)
n-Butanol-acetic
acid-water
Acetone-benzene,
petroleum e t h e r ( 2 : 2 : 4 )
or
26
Table 2 (Continued) Support
Solvent System
Detect i o n
Ref.
Whatman No. 1 Paper
2.5% Acetic a c i d i n butanol water ( 2 2 : 3 ) .
4-Dimethylaminobenzaldehyde
47
17.5 cm x 2 cm
Ethanol-ethylacetate-aq. NH3 (50: 50:l)
Biological assay
48
column of neutral alumina
.
735
(u In
m
Ln
Table 4.
6,
Ref.
Column
Mobile Phase
Detect or
Micropak CN
Hexane-CH C 1 -methanol. 2 2
W at 254 nm
A r e v e r s e phase column
A c i d i f i e d ethanol-water
w
55
DVB-MCL-0
Methanol-ammonia.
w
56
w
57
W a t 254 m
58
RP-2 (10 4 0
HPLC Methods f o r t h e Determination of Chloramphenicol
m)
0.01M K2HP04 -methanol ( 29 :21 )
.
4.5).
P a r t i s i l - 1 0 ODs
50 mM KH2P04 (pH
1.1 Bondapak phenyl
0.05M H P O - a c e t o n i t r i l e 3 4
1-1 Bondapak C18
20% A c e t o n i t r i l e i n 0.05M N a a c e t a t e b u f f e r (pH 5.3).
W at 278 nm
60
Sep-Pak C18
Ethyl e t h e r and e t h a n o l .
w
61
Nucleosil C18
Wat er-methanol
W at 278 and 350 nm
62
W a t 276 nm
63
w
64
( 5 Fun).
Hypersil H
ODS
5
1-1 Bondapak C
18
( 7 :3 )
(3:l)
.
Wat e r - a c e t o n i t r i l e - a c e t a t e b u f f e r (80:20:1). Aqueous methanol
.
59
Table
4.
( Continued)
Column
Mobile Phase
Detector
Ref.
A c e t o n i t r i l e - p h o s p h a t e b u f f e r (1:3 )
W a t 278
52
RP-18
35 t o 40% a q . methanol c o n t a i n i n g 1 0 0 mg/L o f K2HP04.
w
45
Radial-PAK c18 w i t h RCSS GuardPAK precolumn.
Methanol-0.75% a c e t i c a c i d ( 3 : 7 ) a d j u s t e d t o p H 5.5 w i t h t r i e t h y l a m i n e .
W at 280 nm
65
c18 Varian Micropak MC H ( 1 0 urn)
Phosphate b u f f e r (pH 3.25)-methanola c e t o n i t r i l e (27:9:4).
w
66
Perkin-Elmer Reverse Phase c18 column.
Methanol-water-acetic a c i d ( 4 5 : 5 5 : l ) o r Isopropyl alcohol-water-acetic a c i d (30:70:1).
Polarographic
26
1 ~ - Bondapak C18
Wat er-methanol-acet i c a c i d
w
67
1-1 Bondapak C
4
w
4
18
at 278 nm
a t 280
738
5.
ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID Pharmacokinet i c s
5.1 Absorption and D i s t r i b u t i o n Oral doses o f 1 gm chloramphenicol produce peak l e v e l s of 10-20 pg/ml at 2 t o 4 h r . (68, 69). The serum l e v e l peak following o r a l a d m i n i s t r a t i o n i s approximately t h e same as t h a t o b t a i n e d following I V a d m i n i s t r a t i o n , although peak l e v e l s are reached slower by t h e former r o u t e . Yogev e t a1 ( 7 0 ) conducted a s t u d y i n which 39 c h i l d r e n w i t h H i n f l u e n s a e m e n i n g i t i s were t r e a t e d f o r 5 days w i t h o r a l chloramphenicol. All p a t i e n t s responded w e l l t o t h e r a p y and no r e l a s p s e developed. The r o u t e of a d m i n i s t r a t i o n had l i t t l e impact on t h e p e d i a t r i c p a t i e n t s t r e a t e d and t h e serum l e v e l s a f t e r o r a l a d m i n i s t r a t i o n w a s equal t o o r g r e a t e r t h a n I V f o r m u l a t i o n s . Chloramphenicol p a l m i t a t e administ e r e d t o c h i l d r e n between t h e age o f 2 months and 14 y e a r s o r a l l y every 6 h r i n doses o f 60-70 mg/kg/day r e s u l t e d i n a serum c o n c e n t r a t i o n ( a t s t e a d y s t a t e ) r a n g i n g from 1 5 . 5 t o 29.0 pg/ml w i t h a mean o f 20.2 ug/ml a f t e r 90 min from a d m i n i s t r a t i o n (71). Chloramphenicol s u c c i n a t e a d m i n i s t e r e d every 6 h r by IV r o u t e t o 18 c h i l d r e n between t h e age o f 2 months and 14 y e a r s i n doses o f 60 t o 109 mg/kg/day r e s u l t e d i n a serum c o n c e n t r a t i o n ( a t s t e a d y state) ranging from 1 2 . 5 t o 43.1 pg/ml a f t e r 90 min. from administr a t i o n (71). Lower blood l e v e l s are produced by 1M chloramphenicol sodium s u c c i n a t e t h a n by i d e n t i c a l I V doses ( 7 2 ) and about 50% t h e serum l e v e l o b t a i n e d a f t e r i d e n t i c a l doses given by o r a l r o u t e (5-6 g m / m l ) ( 6 9 ) . Oral a d m i n i s t r a t i o n o f chloramphenicol r e s u l t e d i n about 75-90% a b s o r p t i o n w i t h peak l e v e l s o c c u r i n g 0 . 5 t o 2 h r f o l l o w i n g a d m i n i s t r a t i o n (73, 7 4 ) . The p a l m i t a t e ester, however, must be hydrol y s e d b e f o r e a b s o r p t i o n . Hydrolysis may be inadeq u a t e i n newborns, i n f a n t s and c h i l d r e n and absorpt i o n delayed and u n r e l i a b l e ( ~ 5 ~ 7 6 )Chloramphenicol . b a s e a d m i n i s t e r e d o r a l l y produces peak serum l e v e l s equivalent t o o r higher than I V administration (77, 78). Peak l e v e l s o f 10-13 pg/ml are o b t a i n e d i n about 2 h r a f t e r t h e a d m i n i s t r a t i o n o f 1 gm o r a l d o s e , and s u s t a i n e d a d m i n i s t r a t i o n every 6 h r provides cumulative e f f e c t w i t h somewhat h i g h e r peak l e v e l s (68,79). Chloramphenicol sodium s u c c i n a t e , u s i n g 1 gm I V dose, produces similar blood pe& l e v e l s
CHLORAMPHENICOL
739
o n l y o c c u r i n g immediately. Blood l e v e l s o f t h e same o r d e r are o b t a i n e d i n c h i l d r e n w i t h an e q u i v a l e n t s i n g l e o r a l or I V dose ( 7 9 ) . I n newborn i n f a n t s 2 , h , or 1 2 h r a f t e r chloramphenicol s u c c i n a t e administr a t i o n ( 1 2 . 5 mg/kg, I V ) serum chloramphenicol concentr a t i o n s were < 1 0 mg/L a t each t i m e s t u d i e d . A f t e r l o a d i n g dose o f 20 mg drug/kg t h e mean serum drug c o n c e n t r a t i o n s were h i g h e r i n i n f a n t s I 2 day old t h a n i n i n f a n t s 2 3 days old ( 8 0 ) . The e f f e c t of dosing methods on chloramphenicol a b s o r p t i o n was s t u d i e d (81) u s i n g young y e l l o w t a i l s . The a b s o r p t i o n i s found t o b e g r e a t e r i n p r p o r t i o n t o t h e i n c r e a s e i n t h e drug c o n t e n t i n t h e f e e d . Of s e v e r a l d i e t s t e s t e d with varying concentration of t h e drug, a d i e t c o n t a i n i n g 50% o f t h e drug and 0.2% of a b i n d e r produced t h e h i g h e s t drug l e v e l i n t h e fish. The a b s o r p t i o n o f chloramphenicol w a s s t u d i e d ( 8 2 ) i n 6 normal v o l u n t e e r s by v a r i o u s r o u t e s of administr a t i o n . Peak serum l e v e l s were maximum w i t h t h e o r a l r o u t e o f a d m i n i s t r a t i o n . With t h e 1M r o u t e t h e average peak l e v e l w a s o n l y 70% of t h a t of t h e o r a l r o u t e . The a b s o r p t i o n of t h e drug w a s minimum and v a r i a b l e a f t e r r e c t a l a d m i n i s t r a t i o n . Animal s t u d i e s were conducted for improving t h e r e c t a l a b s o r p t i o n of chloramphenicol ( 8 3 ) . The drug r e c t a l a b s o r p t i o n was found t o b e s t r o n g l y species-dependent. In r a b b i t s t h e drug a b s o r p t i o n w a s g r e a t l y i n c r e a s e d a f t e r r e c t a l a d m i n i s t r a t i o n o f chloramphenicol erythromycin m i x t u r e and chloramphenicol - oleandomycin m i x t u r e , compared t o a b s o r p t i o n a f t e r administr a t i o n of o t h e r chloramphenicol m i x t u r e o f t h e drug e s t e r s . The i n c r e a s e i n a b s o r p t i o n w a s not observed i n people, r a t s and g u i n e a p i g s . Chloramphenicol e s t e r s e . g . chloramphenicol p a l m i t a t e must be hydrol y s e d by p a n c r e a t i c l i p a s e s i n t h e duodenum b e f o r e a b s o r p t i o n t a k e s p l a c e . Accordingly t h e r a t e o f h y d r o l y s i s o f t h e p a l m i t a t e i s a major f a c t o r i n determining t h e u l t i m a t e blood l e v e l s achieved. That t h e a b s o r p t i o n o f t h e p a l m i t a t e i s slower has been shown by Weiss e t a1 ( 7 5 ) i n t h e i r s t u d i e s i n newborn i n f a n t s . I n o l d e r c h i l d r e n , it i s a l s o r e p o r t e d (76), up t o 50% o f an a d m i n i s t e r e d dose o f p a l m i t a t e may be l o s t i n t h e f e c e s . The dose of t h e p a l m i t a t e must be h i g h e r t h a n w i t h t h e c r y s t a l l i n e chloramphenicol base (100-200 mg/kg/day) This i s i n agreement w i t h t h e r e s u l t s o f e a r l i e r s t u d i e s (84, 8 5 ) .
.
740
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID The slow h y d r o l y s i s r e s u l t i n g i n lower blood l e v e l s r e p o r t e d by some i n v e s t i g a t o r s i s due t o t h e s e p a r a t e polymorphic s t a t e s i n which t h e p a l m i t a t e can e x i s t . One c r y s t a l l i n e form i s s u b s t a n t i a l l y more hydrolysed t h a n t h e o t h e r and t h e blood l e v e l s observed are d i r e c t l y r e l a t e d t o t h e p r o p o r t i o n o f t h a t form which i s p r e s e n t i n t h e p r e p a r a t i o n ( 8 6 ) . Subsequent t o t h e s e r e p o r t s , however, Park-Davis Company has i n d i c a t e d t h a t t h e l e s s h y d r o l y s a b l e polymorph of t h e p a l m i t a t e h a s been removed from t h e preparat i o n and t h e a b s o r p t i o n o f chloramphenicol from t h e p a l m i t a t e i s now complete and r e l i a b l e , producing blood l e v e l s e q u i v a l e n t t o I V a d m i n i s t r a t i o n ( 7 8 ) . A p r o s p e c t i v e , randomized e v a l u a t i o n o f o r a l chloramp h e n i c o l a d m i n i s t r a t i o n f o r completion of t h e r a p y o f H i n f l u e n z a e t y p e b m e n i n g i t i s i s conducted (87) i n 44 c h i l d r e n : 21 p a t i e n t s r e c e i v e d t h e drug o r a l l y a f t e r t h e second day of t h e r a p y , t h e remainder c o n t i nued t o r e c e i v e t h e drug v i a I V r o u t e . There was e q u i v a l e n t b i o a v a i l a b i l i t y o f chloramphenicol. I n 43 p a t i e n t s t h e r e s o l u t i o n o f c l i n i c a l m a n i f e s t a t i o n s and CSF a b n o r m a l i t i e s of m e n i n g i t i s w a s e q u i v a l e n t with b o t h r o u t e s . These f i n d i n g s i n d i c a t e t h a t higher t h a n normal doses of chloramphenicol p a l m i t a t e a r e not n e c e s s a r y . A 6 h o u r l y devided dosage o f 1 0 0 mg/kd/day w i l l produce blood l e v e l s e q u i v a l e n t t o t h o s e o b t a i n e d by I V o r o r a l a d m i n i s t r a t i o n o f t h e base. Adequate CSF l e v e l w i l l be maintained s i n c e c o n c e n t r a t i o n s o f chloramphenicol i n t h e CSF r e a c h l e v e l s as high as 50% of t h a t o b t a i n e d i n t h e blood and a r e w e l l above t h e M I C f o r H i n f l u e n z a e . The a b s o r p t i o n o f chloramphenicol a f t e r o r a l a d m i n i s t r a t i o n i n s e v e r e l y malnourished c h i l d r e n was found t o be e r r a t i c . T h i s r o u t e should b e avoided i n such patients (88). The b i o a v a i l a b i l i t y o f t h e o r a l chloramphenicol p a l m i t a t e salt w a s i n v e s t i g a t e d ( 7 1 ) and compared t o t h a t o f I V s u c c i n a t e s a l t i n 18 c h i l d r e n , age 2 months t o 14 y e a r s . The b i o a v a i l a b i l i t y of t h e o r a l dose w a s found t o b e g r e a t e r t h a n t h e I V p r e p a r a t i o n . The r e l a t i v e b i o a v a i l a b i l i t y o f t h e s u c c i n a t e compared t o t h e p a l m i t a t e w a s 7 0 % . T h i s can be explained by t h e prominent i n t e r p a t i e n t v a r i a t i o n i n t h e e x t e n t of h y d r o l y s i s of t h e I V s u c c i n a t e s a l t t o t h e b i o l o g i c a l l y a c t i v e chloramphenicol when compared t o t h e o r a l s a l t form. I n t h i s s t u d y a mean of 36% o f t h e a d m i n i s t e r e d I V dose w a s e x c r e t e d
CHLORAMPHENICOL
74 1
unchanged i n u r i n e . T h i s v a l u e probably a c c o u n t s f o r t h e 30% r e d u c t i o n i n b i o a v a i l a b i l i t y of t h e I V dosage form when compared t o t h e o r a l form which i s more completely hydrolysed t o t h e a c t i v e form i n t h e GIT
.
Simultaneous a d m i n i s t r a t i o n o f v i t a m i n s w i t h t h e a n t i b i o t i c has been found t o reduce chloramphenicol blood l e v e l s ( 8 9 ) . Chloramphenicol i s widely d i s t r i b u t e d i n t h e body w i t h t h e r a p e u t i c l e v e l s o c c u r i n g i n most body c a v i t i e s , t h e eye and CSF. Chloramphenicol i s 60 t o 80% p r o t e i n bound ( 7 3 ) . Other s t u d i e s , however, suggest t h a t it i s about 36% p r o t e i n bound ( 9 0 ) . There i s no l i t e r a t u r e r e p o r t on complete pharmacokin e t i c p r o f i l e o f chloramphenicol i n t h e c e r e b r o s p i n a l f l u i d . Although many s t u d i e s have documented t h e achievement o f t h e r a p e u t i c l e v e l s i n t h e CSF, o n l y one has measured s e q u e n t i a l CSF l e v e l s over an e n t i r e dosing p e r i o d ( 9 1 ) . A male a d u l t p a t i e n t w i t h H i n f l u e n z a e m e n i n g i t i s r e c e i v e d o r a l chloramphenicol 1 2 . 5 mg/kg every 6 h r . Serum and CSF l e v e l s were measured a f t e r t h e 7 t h dose t o e n s u r e s t e a d y s t a t e k i n e t i c s . The CSF l e v e l s were 5.4 pg/ml a t 0 h r , 5.7 at 30 min, 6 . 3 a t 1 h r , 7.4 a t 2 h r , 7 . 2 a t 3 h r and 7.9 a t 6 h r . The mean CSF/serum r a t i o w a s 36%. R e s u l t s o f i s o l a t e d experiments i s i n c l u d e d i n Table 5. The r e p o r t e d results emphasise t h e consid.erable i n d i v i d u a l v a r i a t i o n s , probably due t o v a r y i n g d e g r e e s o f miningeal inflammation, t h e drug a p p e a r s t o produce v e r y good CSF l e v e l s when s u f f i c i e n t doses are a d m i n i s t e r e d . With I V a d m i n i s t r a t i o n t h e CSF/serum r a t i o s range from 22.5-99%, w i t h s t e a d y s t a t e CSF l e v e l s from 4-23.3 pg/ml. Oral dosages produce s i m i lar and p o s s i b l y h i g h e r CSF l e v e l s w i t h CSF/serum r a t i o from 20-60% and CSF l e v e l s o f 4-32 pg/ml. A comparative s t u d y ( 1 0 0 ) o f t h e s t e a d y - s t a t e CSF l e v e l s a f t e r I V or o r a l doses of 1 0 0 ug/kg/day o f chloramphenicol i n 1 4 p a t i e n t s showed t h a t serum l e v e l s a f t e r I V a d m i n i s t r a t i o n occured a t 45 min. With mean corresponding t o CSF l e v e l s of 4.2 pg/ml. Peak serum l e v e l s r e s u l t i n g from o r a l dosing occured
Table
5.
Dose (mg/kg/D)
7 7 9 50
4
;P
- 15
- 15 - 45
28 - 66 50 - 68 18 - 26
Summary of CSF Levels Reported i n I s o l a t e d Determinations
Dosage form Oral
Rectal Or& ( s i n g l e ) Or& ( m u l t i p l e ) I M (single)
0
4 - 32
32
128
0
2 - 3.6 12.2 - 34 1 - 42 5 . 1 - 23
20- 64
4 8
32- 40
* CSF/Serum r a t i o (%)
Ref.
0
92
6 - 20 20-40 25 0
93
24.8 - 74.4
25-42 0 - 39 o - 28 6 - 27
3 - 21.8
50
94
IM
4.- 10.3 o - 16.7 0 - 4.2 3.2 - 9.9
66
O r a1
2-
50
IV
14
25
56
95
25 - 35
IV
mean
14.1 - 54.4
50 - 87
96
unknown
18 - 72
8.9
23.3 +/- 7.7
"Through Drugdex, Microfiche System, Micromedex, I n c . , Englewood, Colorado, U . S . A .
Table 5 (Continued)
... Serum l e v e l (U d m l )
CSF/Serum ratio (%)
eak a f t e r 3 hr) 11.5(peak a f t e r 3 hr) (Ventricular fluid)
1 6 . 5 ( peak a f t e r
22.5
2 O ( peak a f t e r 30 m i n )
57.5
97
IV
4 - 18
8
45 - 99
98
100
IV
5.5 - 1 3
10
29.5
23 - 85
99
100
IV Oral
mean peak 1 5 a t 45 min. mean peak 81.5 a t 2-3 hr.
mean 65
100
mean 4.2 (1 - 7 . 5 ) mean 6.6 (1.5 - 1 1 . 5 )
Dosge (mg/kg/D)
Dosage form
60
IV
75
4(p
30 m i n )
~
- 25 (neonates) 40 - 100
12.5 -4 0
-
40
Ref.
(Older
c h i 1d r en )
-
mean 60
100
ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID
744
a t 2-3 h r w i t h CSF l e v e l s o f 6.6 pg/ml. Four premature i n f a n t s under 5,500 gm were t r e a t e d w i t h p a r e n t e r a l chloramphenicol f o r c e n t r a l nervous system i n f e c t i o n due t o organisms r e s i s t a n t t o t h e p e n i c i l l i n s . Serum, c e r e b r o s p i n a l f l u i d (CSF) and v e n t r i c u l a r f l u i d c o n c e n t r a t i o n of t h e drug were measured f r e q u e n t l y d u r i n g t h e r a p y and were used t o m a i n t a i n drug dosages i n t h e safe and t h e r a p e u t i c range. Concentration of t h e drug i n t h e lumbar CSF and v e n t r i c u l a r f l u i d had a mean o f 23.3 vg/ml, c o n s i s t e n t l y g r e a t e r t h a n 45% of peak serum l e v e l s ( 9 6 ) . The d a t a show t h a t chloramphenicol e n t e r s t h e CSF i n b o t h v e n t r i c u l a r and lumbar r e g i o n s i n t h e r a p e u t i c c o n c e n t r a t ' o n s when a d m i n i s t e r e d I . V . The d i s t r i b u t i o n of ltC-labeled D( -)-threo-chloramphenic o l was s t u d i e d ( 1 0 1 ) i n newborn p i g s by whole-body The amount o f r a d i o a c t i v i t y i n t h e autoradiography l u n g , l i v e r , a d r e n a l c o r t e x , kidney, myocardium, Pancreas, t h y r o i d , s p l e e n and s k e l e t a l muscles w a s higher t h a n t h a t i n t h e blood s h o r t t i m e a f t e r t h e i n j e c t i o n and remained h i g h e r upto 8 r . A f t e r 4 and 8 h r t h e b r a i n c o n c e n t r a t i o n o f 1CC was a l s o h i g h e r t h a n t h a t o f t h e blood. I n t h e bone marrow, however, t h e c o n c e n t r a t i o n d i d not r e a c h t h a t o f t h e blood d u r i n g t h e whole experiment. I n t h e organs > 90% of t h e r a d i o a c t i v i t y was r e p r e s e n t e d by unchanged chloramphenicol; t h e e x c r e t o r y o r g a n s , t h y r o i d s and a d r e n a l s being e x c e p t i o n s . I n t h e s t u d y (81) u s i n g c u l t u r e s y e l l o w t a i l , S e r i o l a q u i n q u e r a d i d a , t h e d i s t r i b u t i o n o f chloramphenicol f o l l o w s t h e o r d e r : l i v e r > muscle > b l o o d . The volume o f d i s t r i b u t i o n f o r chloramphenicol was r e p o r t e d t o b e about 40 l i t r e s ( 7 4 ) .
.
5.2
Excretion Chloramphenicol i s r e p o r t e d t o b e 5 - 15% e x c r e t e d unchanged (73,102) w i t h r e p o r t e d r e n a l c l e a r a n c e o f 13-36 ml/min ( 7 3 ) . Renal l e v e l s may b e i n a d e q u a t e t o treat urinary t r a c t i n f e c t i o n s e s p e c i a l l y i n t h e presence o f moderately t o s e v e r e l y impaired r e n a l f u n c t i o n ( 1 0 3 ) . Some nom-al p a t i e n t s and p a t i e n t s w i t h impaired r e n a l f u n c t i o n e x h i b i t impaired f r e e drug e l i n i n a t i o n ( 1 0 4 ) . The recovery o f f r e e drug from t h e u r i n e i s d i r e c t l y p r o p o r t i o n a l t o c r e a t i n i n e c l e a r a n c e . With c r e a t i n i n e c l e a r a n c e s o f l e s s t h a n
CHLORAMPHENICOL
745
20 ml/min, l e s s t h a n 1% o f t h e a d m i n i s t e r e d doses t h a t are recovered i n t h e i n e r t i n a c t i v e form. With c r e a t i n i n e c l e a r a n c e s of < 40 m l / m i n , u r i n a r y c o n c e n t r a t i o n s o f t h e drug a r e g e n e r a l l y not h i g h enough t o t r e a t s u s c e p t i b l e organisms ( 1 0 5 ) . The s t e a d y s t a t e k i n e t i c s o f t h e o r a l p a l m i t a t e vers’is t h e I V s u c c i n a t e s a l t w a s s t u d i e d (71). No s i g n i f i c a n t c o r r e l a t i o n between t h e dose of chloramphenic o l s u c c i n a t e and serum c o n c e n t r a t i o n s o f ”free” chloramphenicol o r t h e average u r i n a r y c o n c e n t r a t i o n were found. However, t h e average u r i n a r y concentrat i o n and serum c o n c e n t r a t i o n of o r a l chloramphenicol p a l m i t a t e c o r r e l a t e s w e l l w i t h dose i n d i c a t i n g more complete and p r e d i c t a b l e h y d r o l y s i s . V a r i a b l e f r a c t i o n s of t h e dose ( a mean of 36%) w a s e x c r e t e d i n u r i n e unchanged and w a s t h e r e f o r e n o t b i o a v a i l a b l e i n a c t i v e form. Both v a r i a b l e h y d r o l y s i s and r e n a l e l i m i n a t i o n o f t h e nonhydrolyzed chloramphenicol s u c c i n a t e seems t o reduce t h e b i o a v a i l a b i l i t y of t h e a n t i b i o t i c and a p p e a r s t o c o n t r i b u t e s u b s t a n t i a l l y t o t h e wide v a r i a t i o n s i n serum c o n c e n t r a t i o n s produced f o l l o w i n g an I V dose. I n premature i n f a n t s , a n i n c r e a s e d b i o a v a i l a b i l i t y o f chloramphenicol was a result o f decreased r a t e of clearance of t h e succinate salt causing a g r e a t e r f r a c t i o n o f t h e s a l t dose t o be hydrolysed t o chloramphenicol (106). With t h e d a t a c u r r e n t l y availa b l e , chloramphenicol i s not a d v i s e d d u r i n g t h e b r e a s t f e e d i n g p e r i o d . Chloramphenicol i s e x c r e t e d i n t o breast m i l k , i n some i n s t a n c e s i n c o n c e n t r a t i o n s which are 50% of blood l e v e l s ( 1 0 7 ) . S i n g l e 1 gm o r a l doses produce peak milk l e v e l s at 3 h r , which are u n d e t e c t a b l e a t 6 h r ( 1 0 8 ) . S i n g l e dose of 500 mg o r a l l y given every 6 h r produced serum l e v e l s of 0.98 - 3.5 pg/ml i n b r e a s t milk ( 1 0 9 ) . Most i n f a n t s do not have developed h e p a t i c c o n j u g a t i o n system f o r g l u c u r o n i d a t i o n which could r e s u l t i n t o x i c i t y . Although chloramphenicol milk l e v e l s a r e not s u f f i c i e n t t o induce t h e grey-baby syndrome, t o x i c i t y t o t h e bone marrow may occur ( 1 1 0 ) . Toxic e f f e c t s i n i n f a n t s had been r e p o r t e d (111) d u r i n g t h e b r e a s t feeding period.
5.3
Half-Life I n normal, o t h e r w i s e h e a l t h y a d u l t s , t h e h a l f - l i f e o f chloramphenicol r a n g e s from 1.6 - 3 . 3 h r w i t h an
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
746
average v a l u e o f 2.7 h r (102,112). I n r e n a l and l i v e r f a i l u r e s t h e h a l f - l i f e i s a p p r e c i a b l y prolonged. I n i n f a n t s and c h i l d r e n between t h e age o f 1 month and 11 y e a r s , a mean apparent h a l f - l i f e w a s r e p o r t e d (113) t o b e 5.94 h r . A n a l y s i s o f v a r i a n c e r e v e a l e d a sample v a r i a n c e o f 21.85 i n c h i l d r e n l e s s t h a n 4 months o f age compared t o 5.87 i n c h i l d r e n g r e a t e r t h a n 4 months o f age r e f l e c t i n g a h i g h l y v a r i a b l e h a l f - l i f e i n t h e younger i n f a n t s . A s t u d y ( 1 1 4 ) o f t h e pharmacokinet i c parameters o f chloramphenicol s u c c i n a t e i n i n f a n t s and young c h i l d r e n r e v e a l e d t h a t h a l f - l i f e o f serum chloramphenicol s u c c i n a t e d i d not c o r r e l a t e with t h e h a l f - l i f e o f serum chloramphenicol. Chloramphenicol s u c c i n a t e i s l o s t i n t o t h e u r i n e i n s i g n i f i c a n t q u a n t i t y and t h i s u r i n a r y loss must b e t a k e n i n t o account i n t h e e s t i m a t i o n o f chloramphenic o l pharmacokinetic parameters. One d u r a t i o n o f i n f u s i o n o f chloramphenicol s u c c i n a t e does not a f f e c t t h e amount e x c r e t e d i n t h e u r i n e .
5.4
Metabolism I n e a r l y s t u d i e s by Glazko e t a1 (73,115,116) d a t a on t h e f a t e o f chloramphenicol i n d i f f e r e n t s p e c i e s were produced. A major m e t a b o l i c r o u t e i n v o l v i n g g l u c u r o c o n j u g a t i o n as w e l l as t h e r e d u c t i o n o f t h e n i t r o group by t h e i n t e s t i n a l f l o r a and conjugat i o n o f t h e r e s u l t i n g m i n e s were d e s c r i b e d i n r a t s , guinea p i g and dog. I n man, 90% o f a s i n g l e dose o f o f t h e drug a p p e a r s i n t h e u r i n e w i t h i n 24 h r , c h i e f l y as chloramphenicol-3-glucuronide ( 1 1 7 ) . The l i v e r i s t h e main s i t e o f g l u c u r o n i d a t i o n . With t h e development o f more s e n s i t i v e a n a l y t i c a l t o o l s o t h e r m e t a b o l i t e s of chloramphenicol have been sugggest ed (Scheme 4 ) . I n t h e i r s t u d y o f chloramphenicol metabolism i n i s o l a t e d rat h e p a t o c y t e s S i l i c i a n o e t a1 (118) chloramphenicol-3-glucuronide w a s found t o b e t h e major m e t a b o l i t e t o g e t h e r w i t h a minor m e t a b o l i t e b e l i e v e d t o b e D( -) threo-2-amino-l-( p-nitropheny1)1,3-propanediol, The formation o f t h e 3-glucuronide w a s l i n e a r w i t h r e s p e c t t o b o t h t h e c e l l concentrat i o n and t o t h e t i m e o f t h e f i r s t hour o f i n c u b a t i o n . The Ic, and Vmax v a l u e s f o r t h e g l u c u r o n i d a t i o n o f chloramphenicol were 6.4 x 10-6 M and 420 pmol/min/l08 c e l l s r e s p e c t i v e l y . The k i n e t i c s o f t h e glucuronidat i o n r e a c t i o n i n r a t hepatocytes suggest a low hepat i c e x t r a c t i o n r a t i o o f chloramphenicol ,
CH,- OH I
H,N@ I
VH-CH-NHCOCHCl, OH
O,N-@
YH2 OC 6 H9 7 FH-CH-NHCOCHC1 OH
,
CH OH FH-;H-NH-CCOOH 11 0 OH
o,N@
FH2OH FH-CH-NHCOCH2 OH OH CH, OH FH-CH-NH, I
O,N@
dog.
O,N@
CHO
OH O2N
0
II
H-C-CH,NHCOCHC~,--€
f--+ OH-CH,CH,-NHCOCHCl, 02N@
yH20H ~H-CHNHCOCH2C1 OH
OH
FH2OH
yH2OH
, O,N
yH2 OC 6 H9 7 FH-CH-NHCOCHC12
-00
-@ YH-CH-NHCOCHC1, OH
R a t hepato-
H,N
@-F H - c H - ~ ~ o c H c ~ , OH
0
CH,?HN @ : C H NO C H ~ ,
-
.*
0,N -@:H-CH-NIICOCHCl
CH, OH -@FH-CH-NH, I
OH
4.
yH2 OC 6 H9
OH
0,N Scheme
yH2OH
Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .
7
748
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID The u s e o f chloramphenicol h a s been shown t o cause bone marrow d e p r e s s i o n . T h i s t o x i c i t y i s u s u a l l y r e v e r s i b l e i f t h e drug i s d i s c o n t i n u e d , b u t i n r a r e c a s e s (1 i n 20,000) p a t i e n t s develop a p l a s t i c anemia a bone marrow d i s e a s e which i s o f t e n i r r e v e r s i b l e and f a t a l ( 1 1 9 ) . Many mechanisms have been suggested (120-126) t o account f o r t h i s chloramphenicol-induced t o x i c i t y , but have not been unequivocally e s t a b l i s h e d . T h i s rare i n c i d e n c e o f chloramphenicol t o x i c i t y suggested t o Pohl and Krishna (127) t h a t a minor a c t i v e m e t a b o l i t e may be involved i n i t s i n d u c t i o n . Using a cytochrome P-450 enzyme system i n l i v e r rnicrosomes o f r a t s , t h e y s t u d i e d t h e mechanism o f t h e metabolic a c t i v a t i o n o f chloramphenicol by measuring t h e c o v a l e n t b i n d i n t o microsomal p r o t e i n s of s p e c i f i c a l l y l a b e l l e d [l C ] and [ 3 H ] d e r i v a t i v e s o f chloramphenicol. The l a c k o f b i n d i n g o f d i c h l o r o a c e t i c a c i d , chloramphenicol base (2-amino-l-( pnitrophenyl)-1,3-propanediol), and t h e acetamido and t r i f l u o r o a c e t amido d e r i v a t i v e s of chloramphenicol i n d i c a t e s t h a t t h e dichloroacetamido group is required f o r a c t i v a t i o n . The b i n d i n g o f dichloroacetamide support t h i s c o n c l u s i o n . Moreover, t h e C-H bond o f t h e dichloromethyl carbon of chloramphenicol a p p e a r s t o be broken i n t h e a c t i v a t i o n p r o c e s s s i n c e t h e hydrogen i s l o s t i n c o v a l e n t b i n d i n g . Accordingly, a mechanism (Scheme 5 ) i s proposed i n which chloramp h e n i c o l i s a c t i v a t e d by h y d r o x y l a t i o n o f t h e dichloracetamido group followed by spontaneous deh y d r o c h l o r i n a t i o n t o a n oxamyl c h l o r i d e which a c y l a t e s microsomal p r o t e i n s . R e c e n t l y , it has been shown t h a t cytochrome P-450 i s t h e predominant p r o t e i n i s l i v e r microsomes t h a t i s a c y l a t e d by t h e oxamyl c h l o r i d e ( 1 2 8 ) . Moreover , t h e c o v a l e n t l y modified cytochrome P-450 a p p e a r s t o b e i r r e v e r s i b l y i n a c t i v a t e d as a mixed-function o x i d a s e ( 1 2 9 , 1 3 0 ) .
,f
Morris e t a1 ( 1 3 1 ) have f u r t h e r c h a r a c t e r i z e d t h e o x i d a t i v e metabolism o f chloramphenicol and have found a new pathway f o r t h e o x i d a t i v e metabolism of chloramphenicol i n a d d i t i o n t o t h e o x i d a t i v e dehalog e n a t i o n r e a c t i o n o u t l i n e d above. According t o t h e s e a u t h o r s , when chloramphenicol was incubated w i t h r a t l i v e r microsomes, four p r e v i o u s l y u n i d e n t i f i e d metab o l i t e s were d e t e c t e d and i d e n t i f i e d . They i n c l u d e chloramphenicol aldehyde, p-nitrobenzyl a l c o h o l , N(2-oxoethyl) d i c h l o r o a c e t a m i d e , and N-( 2-hydroxyethyl)
0,@TI
a
CH, OH Nucleophil i c fH-CH-NHCOCONu-P I 0, N OH
H:
P r o t e i n ( P-Nu)
O,N
-
-@
OH Reduction Scheme 5.
OH
CH,OH OH I I CH-CH- NHCO-C-C1 I I OH c1 P-450, NADPH, CHzOH 1-
02NaFH-!!H OH
I 02N@
CH, OH I
OH p H C 1
1 ozN*EH2
CH, OH CH I -
CHO +
-
NHCOCHCl,
Retroaldol condensat i o n
OHC-CH2NHCOCHC12
+ -*
Reduct i o n
HO-CH2CH2NHCOCHC1,
Mechanism o f chloramphenicol o x i d a t i v e pathways by r a t l i v e r microsomes (127, 131).
750
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID dichloroacetamide. The formation of t h e s e metabol i t e s was dependent upon t h e presence of NADPH and 02 and w a s uninhibited when SKF' 5258 or CO/O2 (8 : 2, v / v ) were present i n t h e r e a c t i o n mixture. Moreover, t h e metabolites were formed by l i v e r microsomes from phenobarbital-treated rats but not by microsomes from untreated rats or rats t r e a t e d with 6-naphthoflavone. The formation of t h e s e metab o l i t e s i s c o n s i s t e n t with a mechanism t h a t involves an i n i t i a l oxidation of chloramphenicol t o chloramphenicol aldehyde by cytochrome P-450. The metabolite, being a 0-hydroxyaldehyde, can chemically undergo a retro-aldol cleavage t o p-nitrobenzaldehyde and N( 2-oxoethyl)dichloroacetamide. Enzymatic reduction of t h e s e aldehyde intermediates would y i e l d p-nitrobenzyl alcohol and N-( 2-hydroxyethyl) dichloroacetamide, r e s p e c t i v e l y (Scheme 5 ) . I n t h e above study by Pohl and Krishna (127) it w a s observed t h a t only a 58% decrease i n covalent binding of chloramphenicol metabolites t o microsomal p r o t e i n occurred when r e a c t i o n s were conducted i n an atmosphere of nitrogen. A t t h e time, it was f e l t t h a t t h e reason t h e covalent binding w a s not decreased t o an even g r e a t e r extent w a s because of i n s u f f i c i e n t deoxygenat i o n of t h e incubation mixtures. I n a f u r t h e r study by Morris et a1 (132), however, it i s shown t h a t a t low oxygen t e n s i o n , chloramphenicol i s a c t i v a t e d by reductive dechlorination pathways of metabolism. Thus, when chloramphenicol was incubated with rat l i v e r microsomes anaerobically, it was metabolized predominently t o deschloro-chloramphenicol and products t h a t become i r r e v e r s i b l y bound t o microsomal p r o t e i n . Cytochrome P-450 induced by phenobarbital appeared t o c a t a l y s e t h e s e r e a c t i o n s 'most e f f e c t i v e l y . Glutathione increased t h e formation of deschlorochloramphenicol by 13%and decreased t h e amount of i r r e v e r s i b l y bound product by 18%. Only small amount of t h e nitroaromatic-reduced product, chloramphenicol m i n e , w a s detected by HPLC. The r e s u l t s a r e consist e n t with t h e drug being biotransformed and a c t i v a t e d by cytochrome P-450 anaerobically through predominently reductive dechlorination. A proposed mechanism f o r t h e reductive dechlorination i s shown i n Scheme 6. Bories e t a1 (133) developed a simple and ion-pair reverse phase high performance l i q u i d chromatographic separations combined with s e l e c t i v e e x t r a c t i o n i n
L-450,
Fe ++
pc1-
Covalent b i n d i n g 4 . -
Scheme
6.
NHZCOCH 02N@CH-CH-CH20H I I OH
A proposed mechanism for t h e r e d u c t i v e d e c h l o r i n a t i o n o f c h l o r m p h e n i c o l by rat l i v e r microsomes ( 1 3 2 ) .
75 2
ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID order t o achieve an improved a n a l y t i c a l t o o l for chloramphenicol metabolic p r o f i l i n g . Q u a n t i t a t i o n w a s achieved by summation of r a d i o a c t i v i t y values f o r f r a c t i o n s belonging t o t h e same peak. Metabol i t e s of chloramphenicol were i d e n t i f i e d by electronimpact and chemical i o n i z a t i o n mass spectrometry. The study l e a d t o t h e confirmation 'of previously suggested chloramphenicol metabolites as well as t h e i d e n t i f i c a t i o n of new metabolites.
Acknowledgement t h e authors would l i k e t o thank M r . Tanvir A . Butt for typing this manuscript.
753
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