STAR LABORATORIES

PENCIN - LA INJECTION 100ml

(penicillin G and Dihydrostreptomycin injection)
Specifications and analytical control procedure
Doc.# STR/QC/SAP- V-F-139 Status 01 Issue date 26.4.16
Page1of 4 rev. date 26.4.2019

PENCIN - LA INJECTION 100ml

(Finished product D.R. No.063626)
Product Name: PENCIN - LA INJECTION
Generic Name: penicillin G and Dihydrostreptomycin injection suspension
Composition:
Each ml contains:
Benzathine penicillin G (BP)..............................................................................................100,000 IU
Procaine penicillin G (BP)..................................................................................................150,000 IU
Dihydrostreptomycin sulfate eq to Dihydrostreptomycin base (BP. Vet) ....................200mg

Ref: Star specs

Sr.# TESTS SPECIFICATIONS
1. White to off white injectable suspension, free from particulate
matter. filled in amber glass vial, closed with rubber closure,
Description sealed with tear off seal, neatly labeled and packed in a unit
carton.
2. Penicillin G Should be identified
Identification Dihydrostreptomycin Sulphate Should be identified
3 PH 5.0 to 8.0
4 Average volume filled per
100ml to 102ml
vial
5 Assay 45.0 – 55.0 % of the stated amount
(Contents of Total (Stated amount 0.2339gm Penicillin G Procaine + Penicillin
Penicillin G) G Benzathine/mll )
6 Endotoxin Test NMT 83.3 E.U/ml
7
Sterility Tests Should be Sterile
8 Benzathine Penicillin G 90,000 i.u / ml to 110,000 iu / ml
9 Procaine Penicillin G 135,000 i.u / ml to 165000 i.u / ml
10
Assay 180mg to 220mg / ml
(Contents of
90.0 – 110.0% of the stated amount
Dihydrostreptomycin )

Prepared by: Reviewed by: Approved by:

Analyst Asist. QCM Manager Quality Control
MS. Sadaf Hafeez Mr.Ayyaz Ali Mohammad Shakeel

Signature Signature Signature

 STAR LABORATORIES

PENCIN - LA INJECTION 100ml

(penicillin G and Dihydrostreptomycin injection)
Specifications and analytical control procedure
Doc.# STR/QC/SAP- V-F-139 Status 01 Issue date 26.4.16
Page2of 4 rev. date 26.4.2019

1-Description :
White to off white injectable suspension, free from particulate matter. filled in amber glass vial, closed
with rubber closure, sealed with tear off seal, neatly labeled and packed in a unit carton.
2-Identification :
complies as per method followed in assay preparation
3-PH:Measure the pH on calibrated PH meter, which should be in range 5.0 to 8.0
4- Average volume filled per vial:
Collect all 5 vials, individually take up the contents of each vial, measure in ml in a calibrated cylinder, It should be
in range as 100 ml to 102 ml.
5-Assay (Contents of Total Penicillin G):
Take 1.0ml of injectable suspension (eq. to 115.96 mg of total penicillin G) and dilute to 100ml in water. Take 2 ml
from the in 250 ml stoppered titration flask and add 5 ml of 1 N Sodium Hydroxide Solution. .Add 6 ml of 1N
Hydrochloric Acid and 10 ml of 0.01 N Iodine Solution. Cover, shake well and allow to stand in the dark for 25
minutes. Titrate with 0.01 N Sodium Thiosulphate Solution using Starch Solution as an indicator. End point will be
colourless. Proceed blank without sample same as above mentioned procedure. Each ml of 0.01N Iodine Solution
vs is equivalent to 0.003344 gm of Penicillin G.
x x 0.003344 x 100 = %
0.00232 of the stated amount
6-BACTERIAL ENDOTOXIN TEST:
Sensitivity 0.125EU/ml
Preparation of dilutions
AS is,1/333, 1/666 MVD is 1/666.4
+ve control std. bacterial Endotoxins 2λ
-ve control TAL water ( Endotoxin free water )
6.1 Procedure:-
(for single test vial)
Reconstitute the TAL/LAL reagent ampoules with 0.1ml of TAL water, mark/identify each ampoule with relative
dilution serial. Add 0.1ml of each of prepared dilution in to reaction ampoule. In +ve control add 2λ of std. bacterial
Endotoxin, in –ve control add 0.1ml of TAL water, mix gently and keep at 37 oC ± 1oC for 60 minutes ± 2 minutes.
Any gel formation indicates the presence of Endotoxin of 0.125EU/ml or more than 0.125EU/ml.
For multitest vial, reconstitute the TAL/LAL reagent vial as per instructions mentioned on vial
Calculation Reciprocal of dilution x sensitivity = EU/ml
Limit: NMT: 83.3 E.U/ml
7-STERILITY TEST:
7.1- Sampling
Take the number of units as 2% or 20 ampoule /vials or whichever is less of the whole one batch randomly from
different trolleys and different trays after the autoclave process is complete.
7.2- Apparatus
7.2.1-Enough media bottles containing sterilized medium solution properly Stoppered.
7.2.2-Glass Filtration Assemblies washed and sterilized (at 121 C˚ for 30 min).
7.2.3-SS filtration flask chain attached with vacuum line through collection Tank (of filtered liquid).
7.2.4-Forceps and scissors sterilized wrapped in aluminum foil (at 121 C˚ for 30 minutes).
7.2.5-Sterilized (at 121˚C for 30 minutes) parachute overhaul with cap, sterile Gloves.
7.2.6-Spirit lamp, marker for identification on media bottles.
7.2.7-Incubators set at temperature 32.5˚C±2.5˚C and 22.5˚C±2.5˚C.
7.2.8-Room temperature should not exceed 25˚C, differential pressure should not be less than 0.10 inches of water
gauge in sterility test room (i-e LAF room) to maintain the integrity of sterile room.
7.3-Preparation of Fluid Thioglycollate Medium
 STAR LABORATORIES

PENCIN - LA INJECTION 100ml

(penicillin G and Dihydrostreptomycin injection)
Specifications and analytical control procedure
Doc.# STR/QC/SAP- V-F-139 Status 01 Issue date 26.4.16
Page3of 4 rev. date 26.4.2019
Take 29.5 gm of Fluid Thioglycollate Medium (already available in market) in glass flask, add slowly distilled water
with Continuous shaking so as to avoid lump formation, and make the volume up to 1000ml with distilled water, boil
to dissolve the contents
Completely. Cool to approximate 25˚C (Check the pH of solution and adjust if necessary using 1N NaOH so that It
could be in range 7.1±0.2 after sterilization). Pour this solution aseptically in media bottles enough to dip the filter
piece (0.45um), stopper the Bottles immediately and sterilized by autoclaving at 121C˚ for 15 minutes If more than
20% of media is pink (due to oxidation) reheat for 10 minutes don’t reheat more than once. Use heat indicator to
assure temperature level in autoclave.
7.4-Preparation of Soybean-Casein Digest Medium (Tryptone Soya Broth)
Take 30 gm of Soybean-Casein Digest Medium (already available in market) in glass flask, add slowly distilled water
with continuous shaking so as to avoid lump formation, and make the volume up to 1000ml with distilled, boil to
dissolve the contents completely. Cool to approximate 25C˚ (check the pH of solution and adjust if necessary using
1N NaOH so that it could be in range 7.1+0.2 after sterilization). Pour this solution aseptically in media bottles
enough to dip the filter piece(0.45um),stopper the bottles immediately and sterilized by autoclaving at 121˚C for 15
minutes if more than 20% of media is pink (due to oxidation) reheat for10 min. Don’t reheat more than once. Use
heat indicator to assure temperature level in autoclave.
7.5-Procedure
Take all autoclaved items into first buffer room, clean and wipe out side of all items with swab soaked in 70% IPA or
96% ethyl alcohol. Take off your shoes, enter in to second buffer room, shoes, clean your hands with 70% IPA or
96% ethyl alcohol. wear sterile overhaul, cap, and shoes, mark each of the Medias bottles with product name, B#,
date of test start and media identification as FT (for fluid Thioglycollate medium) and SD (for
soybean-casein digest medium). Putt off the UV light of laminar air flow (LAF), put on the tube light and HEPA filter
fan. Wear the sterilized gloves; insert the sintered portion of filtration
assembly in to the hole of series of SS filtration stand. Put on the spirit lamp; take out the filter paper (0.45micron)
with sterile forceps and put it on to the sintered part of filtration assembly. Keep over it the cup of filtration assembly
fix it with clamp (already cleaned with 70% IPA).Pour the contents of ampoule/vial in to the cup of filtration
assembly. Run the vacuum pump till the whole contents are filtered. In case of antibiotic wash the membrane with
FLUID-A (Peptic Digest Solution) to rinse the traces of antibiotic. At least use 500ml/membrane FLUID-A for rinsing
antibiotic. Stop the pump take off the clamp as well as the cup of Filtration assembly. Cut the filter paper in to two
parts using sterile scissors. Put one part in to FT media bottle and second one in to SD media bottle already
identified as per product name, B#, test date start, and FT or SD for media identification. Proceed negative control
and positive control along with each autoclave process of prepared media. Incubate the media bottles in their
relevant incubation chambers
(SD media bottles at 22.5˚C +2.5˚C, FT media bottles at 32.5˚C+2.5˚C) at least for fourteen days and record the
observation daily. Ref1&Ref2-USP28/2254, BP2009 A400
INTERPRETATION OF RESULTS:
Each day record the observation, examine the media for macroscopic evidence of growth, Sterility test passes if all
media bottles(FT and SD) including –ve controls are clear with out any turbidity or haziness and +ve controls show
turbidity or haziness .If the evidence of microbial growth is found, the product to be examined does not comply the
sterility test. If the material being tested renders the turbid and the presence or absence of microbial growth cannot
be easily determined by visual examination medium after 14 days of beginning of incubation, transfer a portion (not
less than 1ml) of medium to fresh bottles of same medium and then incubate the original and transfer, bottles for not
less than 14 days. If no evidence of microbial growth is found, the product to be examined complies the sterility test.
If the evidence of microbial growth is found, the product to be examined doest not comply the sterility test.
The test may be considered invalid only if one or more of the following conditions are fulfilled.
A: The data of microbiological monitoring of the sterility testing facility Show a fault.
B: A review of testing procedure used during the test in question reveals a Fault.
C: A microbial growth is found in negative controls.
D: After determination of identity of microorganisms isolated from the Growth of this species (or these species) may
be described unequivocally to fault with respect to material and or the technique used in conducting the sterility test
procedure. If the test is declared invalid, it is repeated with the same number of units as in original
test. If no evidence of microbial growth is found in the repeat test, the product to be examined complies the sterility test if the
evidence of microbial growth is found in the repeat test, the product to be examined does not comply the sterility test.
 STAR LABORATORIES

PENCIN - LA INJECTION 100ml

(penicillin G and Dihydrostreptomycin injection)
Specifications and analytical control procedure
Doc.# STR/QC/SAP- V-F-139 Status 01 Issue date 26.4.16
Page4of 4 rev. date 26.4.2019

8-Determined quantity ( of Benzathine Penicillin G)

X 100,,000 i.u = i.u/ml

50.0
9-Determined quantity (of Procaine Penicillin G )

X 150,000 i.u = i.u/ml

50.0

10- Contents of DihydroStreptomycin :

Standard Preparation:
Weigh accurately DihydroStreptomycin Sulphate, (ref std/w std) eq. to 100 mg of DihydroStreptomycin in
volumetric flask,and make the volume upto 50 ml with Distilled water (2mg/ml)
Take 1.0 ml in 25ml volumetric flask; add 1 ml of Nitroprusside reagent ( equal volume of 10 % NaOH, 10 %
potassium ferricyanide and 10 % Nitroprusside solutions) and stay for 10 minutes at room temperature.

Sample Preparation:
Take 1ml of injectable suspension( eq. to 200 mg of DihydroStreptomycin ) in 100 ml volumetric flask,and make
up the volume with Distilled water (2mg/ml)
Take 1.0 ml in 25ml volumetric flask; add 1 ml of Nitroprusside reagent ( equal volume of 10 % NaOH, 10 %
potassium ferricyanide and 10 % Nitroprusside solutions) and stay for 10 minutes at room temperature.
Measure the absorption of sample and standard preparations at 490 nm on U.V Spectrophotometer and Calculate
the contents of Dihydrostreptomycin.

Abs of sample x 100 = %

Abs of standard of the stated amount

Determined quantity (of DihydroStreptomycin )

X 200 = mg/ml
100