CHAPTER : 04

ANALYTICAL METHOD VALIDATION

COMMITTEE FOR NON PHARMACOPOEIAL
PRODUCT DEPARTMENT OF DRUG
ADMINISTRATION
NATIONAL MEDICINES LABORATORY

CEFOPERAZONE & SULBACTAM FOR INJECTION

 Analytical Profile No.: CEF SUL 075/076/AP 043

Cefoperazone and Sulbactam for injection contain 90% to 110% of

Cefoperazone and 90% to 110% of /sulbactam of stated amount.

IDENTIFICATION

1.1 Cefoperazone:

In the assay, the principle peak in the chromatogram obtained with the
sample solution should correspond to the peak in the chromatogram
obtained with the reference standard solution of Cefoperazone.

1.2 Sulbactam:

In the assay, the principle peak in the chromatogram obtained with the
sample solution should correspond to the peak in the chromatogram
obtained with the reference standard solution of Sulbactam.
 PH

 pH :4.5-7.0 (25% w/v of sample)

PRINCIPLES OF PH

 In pharmacy practice, pH is a critical variable and a basic

understanding of its principles and measurement is important. It is
vitally important to understand pH and its influence on drug
solubility, stability, and absorption. We will look at each of these
separately over three issues of this newsletter.

 Let's begin with a definition of the term pH. The "p" comes from the
word "power." The "H," of course, is the symbol for "hydrogen."
Together, the term pH means the hydrogen ion exponent. In terms of
the hydrogen ion activity, pH is defined as:
pH = - log10 aH+ or 10-pH = aH+

 pH equals the negative logarithm of the hydrogen ion activity, or the

activity of the hydrogen ion is 10 raised to the exponent -pH. The
latter expression renders the use of the p exponent more obvious.
The activity is the effective concentration of the hydrogen ion in
solution. The difference between effective and actual concentration
decreases as one moves toward more dilute solutions, in which ionic
interaction becomes progressively less important.
 In pure water, hydrogen and hydroxyl ion concentrations are equal at
10-7 M at 25C. This is a neutral or pH 7.0 solution. Since most samples
encountered have less than 1 M H+ or OH-, the extremes of pH 0 for
acids and pH 14 for bases are established; 0-7 is acidic and 7-14 is
basic. Of course, with strong acids or bases, pH values below 0 and
above 14 are possible but infrequently measured.
WATER CONTENT

Water content: NMT 5%

 CLARITY OF THE SOLUTION

Constitute the powder in the vial with 10 ml of water for injection with the
help of a hypodermic needle syringe. The solid dissolves completely,
leaving no visible residue as undissolved matter. The constituted solution is
not significantly less clear than an equal volume of diluents or Purified
water contained in similar vessel and examined similarly. The solution is
essentially free from foreign matter that can be observed on visual
inspection.
 PARTICULATE MATTER
BY LIGHT OBSCURATION PARTICLE COUNTER

≥ 10 µm: NMT 6000 Particles/container

≤25 µm: NMT 600 Particles/container

METHOD 1. LIGHT OBSCURATION PARTICLE COUNT TEST

Use a suitable apparatus based on the principle of light blockage which
allows an automatic determination of the size of particles and the number
of particles according to size. The definition for particle-free water is
provided in Reagent Specifications under Reagents, Indicators and
Solution section. The apparatus is calibrated using dispersions of spherical
particles of known sizes between 10 µm and 25 µm, USP Particle Count
Reference Standard. These standard particles are dispersed in particle-
free water. Care must be taken to avoid aggregation of particles during
dispersion. General precautions The test is carried out under conditions
limiting particulate matter, preferably in a laminar-flow cabinet. Very
carefully wash the glassware and filtration equipment used, except for the
membrane filters, with a warm detergent solution and rinse with
abundant amounts of water to remove all traces of detergent. Immediately
before use, rinse the equipment from top to bottom, outside and then
inside, with particle-free water. Take care not to introduce air bubbles into
the preparation to be examined, especially when fractions of the
preparation are being transferred to the container in which the
determination is to be carried out. In order to check that the environment
is suitable for the test, that the glassware is properly cleaned and that the
water to be used is particle-free, the following test is carried out:
determine the particulate matter in 5 samples of particle-free water, each
of 5 mL, according to the method described below.
If the number of particles of 10 µm or greater size exceeds 25 for the
combined 25 mL, the precautions taken for the test are not sufficient. The
preparatory steps must be repeated until the environment, glassware and
water are suitable for the test. Method Mix the contents of the sample by
slowly inverting the container 20 times successively. If necessary,
cautiously remove the sealing closure. Clean the outer surfaces of the
container opening using a jet of particle-free water and remove the
closure, avoiding any contamination of the contents. Eliminate gas bubbles
by appropriate measures such as allowing to stand for 2 min or sonicating.
For large-volume parenterals, single units are tested. For small-volume
parenterals less than 25 mL in volume, the contents of 10 or more units is
combined in a cleaned container to obtain a volume of not less than 25 mL;
the test solution may be prepared by mixing the contents of a suitable
number of vials and diluting to 25 mL with particle-free water or with an
appropriate particle-free solvent when particle-free water is not suitable.
Small-volume parenterals having a volume of 25 mL or more may be
tested individually. Powders for parenteral use are reconstituted with
particle-free water or with an appropriate particle-free solvent when
particle-free water is not suitable. The number of test specimens must be
adequate to provide a statistically sound assessment. For large-volume
parenterals or for small-volume parenterals having a volume of 25 mL or
more, fewer than 10 units may be tested, based on an appropriate
sampling plan. Remove four portions, each of not less than 5 mL, and count
the number of particles equal to or greater than 10 µm and 25 µm.
Disregard the result obtained for the first portion, and calculate the mean
number of particles for the preparation to be examined. Evaluation For
preparations supplied in containers with a nominal volume of more than
100 mL, apply the criteria of test 1.A. For preparations supplied in
containers with a nominal volume of less than 100 mL, apply the criteria of
test 1.B.
For preparations supplied in containers with a nominal volume of 100 mL,
apply the criteria of test 1.B. If the average number of particles exceeds the
limits, test the preparation by the Microscopic Particle Count Test. Test 1.A
— Solutions for parenteral infusion or solutions for injection supplied in
containers with a nominal content of more than 100 mL. The preparation
complies with the test if the average number of particles present in the
units tested does not exceed 25 per mL equal to or greater than 10 µm and
does not exceed 3 per mL equal to or greater than 25 µm. Test 1.B —
Solutions for parenteral infusion or solutions for injection supplied in
containers with a nominal content of less than 100 mL. The preparation
complies with the test if the average number of particles present in the
units tested does not exceed 6000 per container equal to or greater than
10 µm and does not exceed 600 per container equal to or greater than 25
µm.
 BACTERIAL ENDOTOXIN TEST
Bacterial Endotoxin Test: NMT 0.2EU/mg

The bacterial endotoxins test (BET) is a test to detect or quantify

endotoxins from Gram-negative bacteria using amoebocyte lysate from the
horseshoe crab (Limulus Polyphemus or Tachypleus tridentatus). There
are three methods for this test:

• Method A. The gel-clot technique, which is based on gel formation;

• Method B. The turbid metric technique, based on the development of

turbidity after cleavage of an endogenous substrate;

• Method C. The chromogenic technique, based on the development of color

after cleavage of a synthetic peptide-chromogen complex.

Unless otherwise indicated in the individual monograph proceed by

Method A.

The test is carried out in a manner that avoids endotoxin contamination.

 STERILITY TEST

Sterility test: shows no growth of microorganisms

 Bacteriostasis / Fungistasis Testing

 Sterility Testing
 Direct Transfer
 Closed-System Membrane Filtration
 Media Fill and Growth Promotion
 ASSAY

 Chromatographic Condition :

Column: C18, 150*4.6 mm, 5 μm

Flow rate: 0.8 ml/min
Detector: UV 230nm
Injection
20 μl
volume:
Mobile Phase: Buffer:Acetonitrile (70:30)
Transfer 3.3 ml of Tetrabutyl ammonium hydroxide
Buffer: (40% in water) in 1000ml of water and adjust the pH to
6.6 ± 0.05 with orthophosphoric acid.

 Standard Solution:

Weigh accurately about 42 mg of Cefoperazone Sodium WS and 44

mg of Sulbactam Sodium WS in 20 ml volumetric flask. Dissolve with
mobile phase and make up the volume with same solvent. Dilute 5 ml
of the resulting solution to 25 ml with mobile phase. Filter the
resulting solution through 0.2 μm membrane filter paper.

 Test Preparation:

Weigh twenty units taken randomly, and record their fill weight.
Empty the content of the entire ten containers and mix all the content
and keep the content in the air tight container. Weigh the sample
equivalent to 40 mg of Cefoperazone and transfer into 20 ml
volumetric flask. Dissolve it with mobile phase and make up the
volume with same solvent. Dilute 5 ml of the resulting solution to 25
ml with mobile phase. Filter the resulting solution through 0.2 μm
membrane filter paper.
 Procedure:

Inject the reference solution five/six times and sample solutions. The
test is not valid unless the column efficiency is not less than 2000
theoretical plates, tailing factor is not more than 2.0, the relative
standard deviation for replicate injections is not more than 2.0% and
the resolution between Sulbactam and Cefoperazone is not less than
5. Measure the peak responses. Calculate the content of Cefoperazone
and Sulbactam per tablet.

 Other Tests:

As per pharmacopoieal requirement.