Pharmeuropa 25.

3 | July 2013 101

Reference: PA/PH/Exp. 10A/T (13) 25 ANP
XXXX:2598
LEVOFLOXACIN HEMIHYDRATE
Levooxacinum hemihydricum
C
18
H
20
FN
3
O
4
,H
2
O M
r
370.4
[138199-71-0]
DEFINITION
(3S)-9-Fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-
benzoxazine-6-carboxylic acid hemihydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance: light yellowish-white or slightly yellow powder.
Solubility: sparingly soluble in water, freely soluble in acetic acid, sparingly soluble in methanol,
slightly soluble in ethanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: levooxacin hemihydrate CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than
reference solution GY
6
(2.2.2, Method II).
Dissolve 0.100 g in water R and dilute to 10 mL with the same solvent.
Enantiomeric purity. Liquid chromatography (2.2.29). Prepare the solutions immediately
before use.
Solvent mixture: water for chromatography R, methanol R (50:50 V/V).
Test solution. Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute
to 10.0 mL with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to 100.0 mL with the solvent mixture.
Dilute 1.0 mL of this solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 2.5 mg of ooxacin CRS (levooxacin and impurity A) in the
solvent mixture and dilute to 100.0 mL with the solvent mixture. Dilute 1.0 mL of the solution to
10.0 mL with the test solution.
Column:
size: l = 0.15 m, = 4.6 mm;
stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 m)
(38)
;
temperature: 45 C.
Mobile phase: mix 20 volumes of methanol R and 80 volumes of a solution containing 1.25 g/L
of copper sulfate R, 1.76 g/L of valine R and 7.71 g/L of ammonium acetate R in water for
chromatography R.
Flow rate: 1.0 mL/min.
(38) Atlantis dC18 is suitable.
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102 Pharmeuropa 25.3 | July 2013
Detection: spectrophotometer at 340 nm.
Injection: 10 L.
Run time: twice the retention time of levooxacin.
Relative retention with reference to levooxacin (retention time = about 19 min) : impurity A = about
1.3.
System suitability: reference solution (b) :
resolution: minimum 1.5 between the peaks due to levooxacin and impurity A.
Calculation of percentage content :
for impurity A, use the concentration of levooxacin in reference solution (a).
Limit:
impurity A: maximum 0.3 per cent.
The following chromatogram is shown for information but will not be published in the European
Pharmacopoeia.
1. levooxacin 2. impurity A
Figure 2598.-1. Chromatogram for the test for enantiomeric purity of levooxacin: reference
solution (b)
Related substances. Liquid chromatography (2.2.29). Prepare the solutions immediately
before use.
Solvent mixture: water for chromatography R, methanol R (50:50 V/V).
Test solution. Dissolve 5.0 mg of the substance to be examined in the solvent mixture and dilute
to 10.0 mL with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to 100.0 mL with the solvent mixture.
Dilute 1.0 mL of this solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 10.0 mg of ooxacin impurity E CRS (impurity B and its
enantiomer) and 7.5 mg of ooxacin impurity A CRS (impurity F and its enantiomer) in the solvent
mixture and dilute to 10.0 mL with the solvent mixture. Dilute 1.0 mL of the solution to 100.0 mL
with the solvent mixture. Dilute 1.0 mL of this solution to 10.0 mL with the solvent mixture.
Reference solution (c). Dissolve 2.5 mg of levooxacin for system suitability CRS (containing
impurity C) in 5.0 mL of reference solution (b).
Column:
size: l = 0.15 m, = 4.6 mm;
PA/PH/Exp. 10A/T (13) 25 ANP
Pharmeuropa 25.3 | July 2013 103
stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3 m)
(39)
;
temperature: 40 C.
Mobile phase:
mobile phase A: dissolve 3.45 g of sodium dihydrogen phosphate monohydrate R and 5.0 mL
of triethylamine R in 900 mL of water for chromatography R, adjust to pH 6.0 with phosphoric
acid R and dilute to 1000 mL with water for chromatography R;
mobile phase B: methanol R;
Time
(40)
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 - 6 60 50 40 50
6 - 8 50 50
8 - 28 50 10 50 90
Flow rate: 0.5 mL/min.
Detection: spectrophotometer at 290 nm.
Injection: 20 L.
Identication of impurities: use the chromatogram obtained with reference solution (c) to identify
the peaks due to impurities B, C and F.
Relative retention with reference to levooxacin (retention time = about 11 min) : impurity B = about
0.4; impurity C = about 0.5; impurity F = about 1.2.
System suitability: reference solution (c) :
resolution: minimum 2.0 between impurities B and C;
peak-to-valley ratio: minimum 1.2, where H
p
= height above the baseline of the peak due
to impurity F and H
v
= height above the baseline of the lowest point of the curve separating
this peak from the peak due to levooxacin.
Calculation of percentage contents:
for impurity B, use the concentration of impurity B in reference solution (b) ;
for impurity F, use the concentration of impurity F in reference solution (b) ;
for impurities other than B and F, use the concentration of levooxacin in reference solution (a).
Limits:
impurity B: maximum 0.2 per cent ;
impurity F: maximum 0.15 per cent ;
unspecied impurities: for each impurity, maximum 0.10 per cent ;
total : maximum 0.5 per cent ;
reporting threshold: 0.05 per cent.
(39) Luna C18 is suitable.
(40) D
0
(dwell volume used for development of the method) = 0.7 mL.
PA/PH/Exp. 10A/T (13) 25 ANP
104 Pharmeuropa 25.3 | July 2013
The following chromatogram is shown for information but will not be published in the European
Pharmacopoeia.
1. levooxacin 2. impurity F
Figure 2598.-2. Chromatogram for the test for related substances: solution of levooxacin
and 0.5 g/mL of impurity F
PA/PH/Exp. 10A/T (13) 25 ANP
Pharmeuropa 25.3 | July 2013 105
The following chromatogram is shown for information but will not be published in the European
Pharmacopoeia.
1. impurity B 2. impurity C 3. impurity E 4. impurity D
5. levooxacin 6. unspecied impurity (never
detected)
7. impurity F 8. unspecied impurity
(never detected)
Figure 2598.-3. Chromatogram for the test for related substances: solution of levooxacin
and impurities (0.1 per cent)
Water (2.5.12) : 2.1 per cent to 2.7 per cent, determined on 0.500 g
(41)
.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in 100 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 36.14 mg of C
18
H
20
FN
3
O
4
.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specied impurities: A, B, F.
Other detectable impurities (the following substances would, if present at a sufcient level,
be detected by one or other of the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecied impurities and/or by the general monograph
Substances for pharmaceutical use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for
pharmaceutical use) : C, D, E.
(41) Hydranal Methanol Dry as solvent and Hydranal Composite 5 as titrant are suitable.
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106 Pharmeuropa 25.3 | July 2013
A. (3R)-9-uoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,
4-benzoxazine-6-carboxylic acid,
B. (3S)-9-uoro-3-methyl-7-oxo-10-(piperazin-1-yl)-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-
benzoxazine-6-carboxylic acid,
C. 4-[(3S)-6-carboxy-9-uoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazin-
10-yl]-1-methylpiperazine 1-oxide,
D. (3S)-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-
benzoxazine-6-carboxylic acid,
E. (3S)-9-uoro-3-methyl-10-(4-methylpiperazin-1-yl)-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-
benzoxazin-7-one,
F. (3S)-9,10-diuoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-
carboxylic acid.
PA/PH/Exp. 10A/T (13) 25 ANP