PIPERACILLIN Piperacillinum

Piperacillin EUROPEAN PHARMACOPOEIA 5.0
DEFINITION
8-Ethyl-5-oxo-2-(piperazin-1-yl)-5,8-dihydropyrido[2,3d]pyrimidine-6-carboxylic
acid trihydrate.
Content: 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance: pale yellow or yellow, crystalline powder.
Solubility: very slightly soluble in water. It dissolves in
dilute solutions of acids and of alkali hydroxides.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of pipemidic
acid trihydrate.
ASSAY
Dissolve 0.240 g in 50 ml of anhydrous acetic acid R.Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1mlof0.1 M perchloric acid is equivalent to 30.33 mg of
C14H17N5O3. STORAGE
Protected from light.
IMPURITIES
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20 mg of the substance to be A. R1 = H, R2 = OH: 8-ethyl-2-hydroxy-5-oxo-5,8-
examined in 10 ml of the mobile phase and dilute to 20.0 ml dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid,
with the mobile phase. Dilute 1.0 ml of the solution to
B.R1=H,R2=OCH
10.0 ml with the mobile phase.
3: 8-ethyl-2-methoxy-5-oxo-5,8dihydropyrido[2,3-d]pyrimidine-6-carboxylic
acid,
Reference solution (a). Dilute2.0mlofthetestsolutionto
10.0mlwiththemobilephase.Dilute1.0mlofthesolutionC.R1=H,R2=OC2H5:
2-ethoxy-8-ethyl-5-oxo-5,8-
to 100.0 ml with the mobile phase.
dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid,
Reference solution (b). Dissolve10.0mgofethyl
D. R1 = C2H5, R2 = Cl: ethyl 2-chloro-8-ethyl-5-oxo-5,8-
parahydroxybenzoate R in 2.0 ml of the test solution and dihydropyrido[2,3-d]pyrimidine-6-carboxylate,
dilute to 20.0 ml with the mobile phase.
Column:
— size: l =0.15m,Ø=4.6mm,
— stationary phase: octadecylsilyl silica gel for
chromatography R1 (5 µm) with a pore size of 18 nm and
acarbonloadingof13percent.
Mobile phase: mix20volumesofacetonitrile R,
20 volumes of methanol R and 60 volumes of a solution E. R1 = C2H5,R2 = H: ethyl 8-ethyl-5-oxo-2-(piperazin-1-yl)-5,
containing 5.7 g/l of citric acid R and 1.7 g/l of sodium 8-dihydropyrido[2,3-d]pyrimidine-6-carboxylate,
decanesulphonate R.
F. R1 = H, R2 = CO-CH 3: 2-(4-acetylpiperazin-1-yl)-8-ethyl-5-
Flow rate: 0.8ml/min.
oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid
Detection: spectrophotometer at 275 nm.
(acetylpipemidic acid).
Injection: 20µl.
Run time: 2.5 times the retention time of pipemidic acid.
01/2005:1169
System suitability: reference solution (b):
PIPERACILLIN
— resolution: minimum 4.0 between the peaks due to
pipemidic acid and to ethyl parahydroxybenzoate.
Piperacillinum
Limits:
— any impurity: not more than the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent),
— total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent),
— disregard limit: 0.25timestheareaoftheprincipalpeak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
C23H27N5O7S,H2O Mr 535.6
Heavy metals (2.4.8): maximum 20 ppm.
1.0 g complies with limit test C. Prepare the standard using
DEFINITION
2.0 ml of lead standard solution (10 ppm Pb) R.
Piperacillin contains not less than 96.0 per cent and
not more than the equivalent of 101.0 per cent of
Loss on drying (2.2.32): 14.0 per cent to 16.0 per cent,
(2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-
determined on 1.000 g by drying in an oven at 100-105 °C.
yl)carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7
Sulphated ash (2.4.14): maximum 0.1 per cent, determined oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid,
on 1.0 g.
calculated with reference to the anhydrous substance.
2250 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0 Piperacillin
CHARACTERS
A white or almost white powder, slightly soluble in water,
freely soluble in methanol, slightly soluble in ethyl acetate.
IDENTIFICATION
Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
piperacillin CRS.
TESTS
Solution S. Dissolve 2.50 g in sodium carbonate solution R
and dilute to 25 ml with the same solvent.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1).Theabsorbanceof
solution S measured at 430 nm (2.2.25) isnotgreater
than 0.10.
Specific optical rotation (2.2.7). Dissolve 0.250 g in
methanol R and dilute to 25.0 ml with the same solvent. The
specific optical rotation is + 165 to + 175, calculated with
reference to the anhydrous substance.
Related substances. Examine by liquid chromatography
(2.2.29) as prescribed under Assay. Inject 20 µl of reference
solution (b) and elute isocratically with the chosen mobile
phase. Inject 20 µl of test solution (b). Start the elution
isocratically. Immediately after elution of the piperacillin
peak start the following linear gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 - 30 88 → 0 12 → 100 linear gradient
30 - 45 0 → 88 100 → 12 re-equilibration
In the chromatogram obtained with test solution (b), the
area of any peak, apart from the principal peak, is not
greaterthantwicetheareaoftheprincipalpeakinthe
chromatogram obtained with reference solution (b) (2 per
cent). Disregard any peak due to the solvent.
N,N-Dimethylaniline (2.4.26, Method A). Not more than
20 ppm.
Heavy metals (2.4.8). 1.0 g complies with limit test C for
heavy metals (20 ppm). Prepare the standard using 2 ml of
lead standard solution (10 ppm Pb) R.
Water (2.5.12): 2.0 per cent to 4.0 per cent, determined on
0.500 g by the semi-micro determination of water.
ASSAY
Examine by liquid chromatography (2.2.29).
Solvent mixture. Mix 25 volumes of acetonitrile R and
75 volumes of a 31.2 g/l solution of sodium dihydrogen
phosphate R.
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 50.0 ml with
the solvent mixture.
Test solution (b). Prepare the solution immediately before
use. Dissolve 40.0 mg of the substance to be examined in
the solvent mixture and dilute to 20.0 ml with the solvent
mixture.
Reference solution (a). Dissolve25.0mgofpiperacillin CRS
inthesolventmixtureanddiluteto50.0mlwiththesolvent
mixture.
Reference solution (b). Dilute 1.0 ml of reference solution (a)
to 25.0 ml with the solvent mixture.
Reference solution (c). Dissolve10.0mgofpiperacillin CRS
and 10.0 mg of anhydrous ampicillin CRS in the solvent
mixture and dilute to 50.0 ml with the solvent mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a)
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of the
solution to 50.0 ml with the solvent mixture.
The chromatographic procedure may be carried out using:
— a column 0.25 m long and 4.6 mm in internal
diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),
— as mobile phase at a flow rate of 1.0 ml/min a mixture of
88 volumes of mobile phase A and 12 volumes of mobile
phase B:
Mobile phase A. Mix576mlofwater R, 200mlofa
31.2 g/l solution of sodium dihydrogen phosphate R
and 24 ml of an 80 g/l solution of tetrabutylammonium
hydroxide R. If necessary, adjust to pH 5.5 with
dilute phosphoric acid R or dilute sodium hydroxide
solution R; then add 200 ml of acetonitrile R,
Mobile phase B. Mix126mlofwater R, 200 ml of a
31.2 g/l solution of sodium dihydrogen phosphate R
and 24 ml of an 80 g/l solution of tetrabutylammonium
hydroxide R. If necessary, adjust to pH 5.5 with
dilute phosphoric acid R or dilute sodium hydroxide
solution R; then add 650 ml of acetonitrile R,
— as detector a spectrophotometer set at 220 nm.
Inject 20 µl of reference solution (c). The test is not valid
unless in the chromatogram obtained, the resolution
between the peaks corresponding to ampicillin and to
piperacillin is at least 10 (if necessary, adjust the ratio A:B
of the mobile phase) and the mass distribution ratio for
the second peak (piperacillin) is 2.0 to 3.0. Inject 20 µl of
reference solution (d). Adjust the sensitivity of the system to
obtain a peak with a signal-to-noise ratio of at least 3. Inject
reference solution (a) 6 times. The test is not valid unless the
relative standard deviation of the peak area of piperacillin is
at most 1.0 per cent. Inject alternately test solution (a) and
reference solution (a).
IMPURITIES
A. ampicillin,
B. R1 = CO 2H,R2=H:(4S)-2-[carboxy[[(2R)-2-[[(4ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic
acid (penicilloic acids of piperacillin),
C.R1=R2=H:(2RS,4S)-2-[[[(2R)-2-[[(4-ethyl-
2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic
acid (penilloic acids of piperacillin),
F. R1 = CO 2H, R2 = CO-CH 3:(4S)-3-acetyl-2-[carboxy[[(2R)-
2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic
acid (acetylated penicilloic acids of piperacillin),
GeneralNotices(1)applytoallmonographsandothertexts 2251

Piperacillin sodium EUROPEAN PHARMACOPOEIA 5.0
D. (2S,5R,6R)-6-[[(2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-[[(4ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-
2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(piperacillinylampicillin),
E. 1-ethylpiperazine-2,3-dione.
PIPERACILLIN SODIUM
Piperacillinum natricum
01/2005:1168
C 23H 26N 5NaO 7S M r 539.5
DEFINITION
Piperacillin sodium contains not less than 95.0 per cent
and not more than the equivalent of 101.0 per cent of
sodium (2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-
1-yl)carbonyl]amino]-2-phenylacetyl]amino]- 3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate,
calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble
in water and in methanol, practically insoluble in ethyl
acetate.
IDENTIFICATION
A. Dissolve 0.250 g in water R, add 0.5 ml of dilute
hydrochloric acid R and 5 ml of ethyl acetate R; stirand
allow to stand for 10 min in iced water. Filter the crystals
through a small sintered-glass filter (40), applying suction.
Wash with 5 ml of water R and 5 ml of ethyl acetate R
and dry in an oven at 60 °C for 60 min. Examine the
crystals by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
piperacillin CRS.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R
and dilute to 25 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1). The
absorbance of solution S measured at 430 nm (2.2.25) is
not greater than 0.10.
pH (2.2.3). The pH of solution S is 5.0 to 7.0.
Specific optical rotation (2.2.7). Dissolve 0.250 g in water R
and dilute to 25.0 ml with the same solvent. The specific
optical rotation is + 175 to + 190, calculated with reference
to the anhydrous substance.
Related substances. Examinebyliquidchromatography
(2.2.29) as prescribed under Assay. Inject 20 µl of reference
solution (b) and elute isocratically with the chosen mobile
phase. Inject 20 µl of test solution (b). Start the elution
isocratically. Immediately after elution of the piperacillin
peak start the following linear gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0-30 88→ 0 12 → 100 linear gradient
30 - 45 0 → 88 100 → 12 re–equilibration
In the chromatogram obtained with test solution (b), the
area of any peak, apart from the principal peak, is not
greaterthantwicetheareaoftheprincipalpeakinthe
chromatogram obtained with reference solution (b) (2 per
cent). Disregard any peak due to the solvent.
N,N-Dimethylaniline (2.4.26, Method A). Not more than
20 ppm.
Heavy metals (2.4.8). 1.0 g complies with the limit test C for
heavy metals (20 ppm). Prepare the standard using 2 ml of
lead standard solution (10 ppm Pb) R.
Water (2.5.12). Not more than 2.0 per cent, determined on
0.500 g by the semi-micro determination of water.
Bacterial endotoxins (2.6.14): less than 0.07 IU/mg, if
intended for use in the manufacture of parenteral dosage
forms without a further appropriate procedure for the
removal of bacterial endotoxins.
ASSAY
Examine by liquid chromatography (2.2.29).
Solvent mixture. Mix 25 volumes of acetonitrile R and
75 volumes of a 31.2 g/l solution of sodium dihydrogen
phosphate R.
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 50.0 ml with
the solvent mixture.
Test solution (b). Prepare the solution immediately before
use. Dissolve 40.0 mg of the substance to be examined in
thesolventmixtureanddiluteto20.0mlwiththesolvent
mixture.
Reference solution (a). Dissolve 25.0 mg of piperacillin CRS
in the solvent mixture and dilute to 50.0 ml with the solvent
mixture.
Reference solution (b). Dilute 1.0 ml of reference solution (a)
to 25.0 ml with the solvent mixture.
Reference solution (c). Dissolve10.0mgofpiperacillin CRS
and 10.0 mg of anhydrous ampicillin CRS in the solvent
mixture and dilute to 50.0 ml with the solvent mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a)
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of the
solution to 50.0 ml with the solvent mixture.
2252 See the information section on general monographs (cover pages)