+
+
already described. About 0.4 g of a light yellow powder was obtained.
An aqueous solution of this material was purified as already described
on a 2-g C18 silica column. The final product was a very white
amorphous powder with HPLC purity in the 75-85% range.
Reductive Hydrolysis with DithioerythritolsOne gram of ceftiofur
sodium was dissolved in 20 mL of water in a 50-mL glass stoppered
centrifuge tube. Ten milliliters of an aqueous solution of 4% each of
dithioerythritol and triethylamine was added. The mixture became
cloudy and thick. The tube was immersed in a water bath at 45-50
°C and heated until clear (15-20 min). The pH of the clear solution
was adjusted to 2.5-3.0 with cold 20% (v/v) H3PO4 and cooled at -10
°C. The precipitate was washed free of furoic acid, dithioerythritol,
and inorganic salts with cold 0.2% (v/v) acetic acid. It was then
lyophilized from water to obtain an amorphous white powder.
P r ep a r a tion of Desfu r oyl Ceftiofu r Dim er : Hyd r olysis of
Ceftiofu r Sod iu m F ollow ed by Oxid a tion w ith H2O2sCeftiofur
sodium was hydrolyzed to desfuroyl ceftiofur according to the
procedure just described. The pH of the hydrolyzate was adjusted to
6 with 20% (v/v) H3PO4 while stirring under a blanket of N2. Four
milliliters of cold 10% (v/v) H2O2 was added and allowed to stand for
∼1 h or until the dimerization was complete. If the solution was too
thick, it was diluted with water. The pH was then adjusted to 2.5-
3.0 with cold 20% (v/v) H3PO4. The precipitate was separated by
centrifugation. The furoic acid and inorganic salts were removed by
two to three washings with cold 0.2% (v/v) acetic acid followed by
centrifugation. The precipitate was then suspended in water and
lyophilized. The final product was a white amorphous powder that
produced a single peak when analyzed by gradient elution HPLC,
with an area percentage purity of over 90%.
Table 1
sProton NMR Chemical Shift Assignment for Ceftiofur Sodium
and the Major Hydrolysis Products
Chemical Shifts, ppm
assignments
Ceftiofur
II
IV
V
a (Aromatic doublet)
b (Aromatic doublet)
c (Aromatic doublet)
d (Aromatic singlet)
e (CHN)
f (CHN)
g (OCH3)
h (1/2 CH2AB)
h (1/2 CH2AB)
i (1/2 CH2AB)
i (1/2 CH2AB)
Solvent
7.74
6.62
7.33
6.95
5.78
5.17
3.97
3.33
3.64
3.91
4.20
D2O
Absent
Absent
Absent
7.06
5.80
5.26
4.03
3.35
3.56
3.57
Absent
Absent
Absent
6.8
5.94
5.16
Absent
Absent
Absent
7.00
5.80
5.20
4.03
3.53
3.80
3.70
3.96
3.93a
3.87a
3.74a
3.33a
CD3OD
3.74
D2O
4.12
CD3OD
a These are unassigned peaks.
Preparation of Desfuroyl ThiolactonesAbout 1 g of ceftiofur sodium
was hydrolyzed to desfuroyl ceftiofur according to any of the proce-
dures already described. To an aqueous suspension of the hydroly-
zate, 45 mL of cold 4.5 N methanolic HCl was added in small portions
with mixing. This mixture was allowed to stand overnight under a
blanket of N2. Seventy-five milliliters of water was added and
extracted with 2 × 50 mL of ethyl acetate and 1 × 50 mL of CH2Cl2.
The organic washes were discarded. The aqueous phase was trans-
ferred to a beaker and the pH was adjusted to 4.2 with solid NaHCO3.
The white precipitate was cooled, filtered, and washed with a small
quantity of ice cold water and dried under reduced pressure. About
0.3 g of an off-white powder was obtained, and gradient elution HPLC
showed it to be essentially one component.
P r ep a r a tion of th e Oxygen An a log of Desfu r oyl Ceftiofu r
(Desa cetyl Cefota xim e)sThis compound was conveniently prepared
by base hydrolysis of cefotaxime. To 0.5 g of cefotaxime suspended
in 80 mL of CH2Cl2, 30 mL of 0.1 N KOH containing 0.05%
tetrasodium EDTA was added, mixed gently, and allowed to stand
until the hydrolysis was complete (∼1 h). The aqueous phase was
separated, and the pH was adjusted to 5.6 with 1 N HCl and with
0.1 N HCl as the pH approached 5.6. The desacetyl cefotaxime that
was in solution at this pH was lyophilized to yield a white powder.
An aqueous solution of this material was charged onto a 2-g C18 silica
column and eluted with cold deaerated water. The eluant was
collected in 2-mL fractions that were monitored by HPLC for purity.
The fractions containing most of the compound were combined and
lyophilized. The product was a white amorphous powder.
to 19% (v/v) organic phase for 43 min. The column was then flushed
with 90% (v/v) organic phase.
Structural AnalysissProton nuclear magnetic resonance (NMR)
data was aquired on a Varian XL 300 FT NMR with solvents as listed
in Table 1. The infrared (IR) spectra were recorded on a Digilab model
FTS15E spectrophotometer after making KBR pellets of the respective
compound. Thermospray mass spectra were obtained on a Finnigan
4600 quadrupole mass spectrometer equiped with a Finnigan ther-
mospray interface using a 30:70 methanol:0.05 ammonium acetate
mobile phase. Fast-atom bombardment (FAB) mass spectra were
obtained on a Finnigan MAT CH5-DF instrument with a xenon atom
beam.
P r ep a r a tion of Desfu r oyl Ceftiofu r sHydrolysis in Saturated
Potassium Chloride SolutionsFifty milliliters of saturated KCl
containing 0.5 g of tetrasodium EDTA and 0.4 g sodium bisulfite was
deaerated by sonication and cooled to 4 °C. One gram of ceftiofur
sodium was added and dispersed by sonication. The mixture was
again cooled to near 0 °C and stirred with a magnetic stirring bar
under a N2 atmosphere. To this solution, 3 mL of cold deaerated
22.5% (w/v) KOH solution containing 0.5% (w/v) tetrasodium EDTA
was added in a dropwise manner. After a 1-h incubation under N2
at 0 °C, all of the ceftiofur sodium was hydrolyzed, as determined by
HPLC analysis. The pH of the hydrolyzate was then adjusted to 2.5
with cold 20% (v/v) H3PO4. An off-white precipitate was obtained.
The suspension was cooled in an acetone-dry ice bath to coagulate
the precipitate and then centrifuged in 50-mL tubes, and the
supernatant was discarded. The precipitate was washed twice with
cold deaerated 0.2% (v/v) acetic acid and once with cold deaerated
water. The cake was suspended in ∼30-40 mL of cold deaerated
water and lyophilized. About 0.4 g of a light yellow powder was
obtained. This material was purified on a 2-g C18 silica column eluted
with cold deaerated water. The effluent, collected in 2-mL fractions,
was monitored by HPLC for purity. The fractions containing the least
amount of impurities were combined and lyophilized. The product
was a white amorphous powder with HPLC purity in the 75-85%
range.
P r ep a r a tion of Th iofu r oic Acid a s th e Th ioester sOne hun-
dred milligrams of ceftiofur sodium was dissolved in 2 mL of 0.01 M
NaHCO3 in a stoppered test tube and heated on a steam bath for 2
h. The generated thiofuroic acid was derivatized to its methylester
by adding 0.3 mL of methyl iodide in 1 mL of ethyl acetate and
allowing to stand overnight. The lower phase, which appeared to be
a semisolid, was separated by decantation and partitioned with 3 mL
hexane. The hexane phase was evaporated under N2. HPLC and
TLC of the residue showed a single component.
Hydrolysis of a Suspension in Methylene ChloridesAbout 1 g of
ceftiofur sodium or ceftiofur hydrochloride was suspended in 90 mL
of methylene chloride. Thirty milliliters of 1 N KOH saturated with
KCl and also containing 0.5% (w/v) disodium EDTA was added. The
suspension was gently mixed and left at room temperature under a
blanket of N2 for 0.5 to 1 h. When the reaction was complete, as
monitored by HPLC, the aqueous phase was separated and adjusted
to pH 2.5-3.0 with 20% (v/v) H3PO4. The suspension was cooled in
an acetone-dry ice bath to coagulate the precipitate. The precipitate
was washed with 0.2% (v/v) acetic acid and water and lyophilized as
Results and Discussion
Ceftiofur sodium undergoes extensive degradation in alka-
line and acid solutions, producing a large number of com-
pounds as seen by HPLC with UV detection. Figures 1A and
1B are chromatograms of alkaline- and acid-degraded solu-
tions of ceftiofur sodium, respectively.
Metabolic studies of ceftiofur sodium in cattle indicate that
it is immediately metabolized to a more polar compound,
390 / Journal of Pharmaceutical Sciences
Vol. 86, No. 3, March 1997