standard testing procedure of Piroxicam Injection
1.1 To lay down a procedure for Analysis of Piroxicam Injection.
2.1 This procedure is applicable to the analysis of Analysis of Piroxicam Injection in Quality Control lab
3.1 Q.C- Chemist
4.1 Manager-Quality Assurance
5.2Description: Pour 50ml finish sample in Beaker and observed Visually.
5.3pH: Taken 50ml sample in beaker rinse the pH electrode first with purified water followed by sample dip the electrode in sample and observe the pH.
5.4Volume variation: Measured the volume by syringe and determine the volume variation.
MEMBRANE FILTRATION METHOD
5.5.1 Wipe the sample article individually with 70% IPA solution and keep in a clean S.S trays marked
with Product Name, Batch No. and Lot No, and then transfer the samples to the sterility room
through clean pass box for performing sterility.
5.5.2 Prepare the media tubes (FTM and SCDM) as per the SOP for preparation of culture media
Dispense 100 ml quantity for membrane filtration and for Direct Inoculation Method. Sterilize both
the media at 1210C and 15 psi pressure for 20 minutes as per SOP for Media Sterilization by Autoclaving.
5.5.3 After autoclave Label the tubes with Name of Media, Media Batch No. and pre-incubate the media
tubes at appropriate temperature i.e. SCDM tubes at 20 to 250C whereas FTM tubes at 30 to 350C for 24 – 48 hrs.
before subjecting them for sterility operations.
5.5.4 Autoclave Dress, Scissors, forceps and Filtration unit in a S.S Container at 1210C temperature and 15psi pressure for 30 minutes.
5.5.5 After Sterilization cool the contents and aseptically transfer in a S.S. container to cleaned Pass box.
5.5.6 Transfer the pre incubated sterile media tubes, SCDA plates, sterile swabs, sterilized
Filtration unit, and 0.1% w/v peptone water to the sterility test room through pass box.
5.5.7 Enter in sterility room as per the Entry / Exit procedure for Sterility Room.
5.5.8 Start the LAF as per SOP.
5.5.9 Wipe out all samples to be tested for sterility with 70% filtered IPA solution.
5.5.10Before starting sterility test, expose the SCDA plates as specified locations throughout the testing.
5.5.11Connect the Glass filtration cup holder with the Filtration flask properly with pipe under laminar airflow unit.
5.5.12Check the Manometer reading of working LAF and check the temperature as well as humidity of the sterility room.
5.5.13Switch ON the vacuum pump with the help of switch, present on the wall. Now place 0.45 sterile membrane
filters between filtration cup and receptacle with the help of sterilized forceps and clamped it properly.
5.5.14Wet the membrane filter by adding approx. 15 ml of sterilized Fluid A (0.1% peptone water) to filter holder, and filter the fluid by employing vacuum.
5.5.15Cut the tip of bottle/vial or ampoule with sterile SS blade in front of the gas burner and immediately
transfer the contents of sample to membrane filter. For dry powder or lyophilized container,
add the sterile water 0.1% peptone dissolve and then collect it in sterile flask and immediately transfer
the contents of sample to membrane filter.
5.5.16Immediately filter the solution with the aid of vacuum and wash the membrane three times
with 100 ml of sterilized fluid A (0.1% peptone water).
5.5.17After complete filtration, stop the vacuum pump.
5.5.18Lift the membrane carefully with the help of sterile forceps, aseptically cut the membrane filter
into two halves with sterile SS scissor and transfer one half to FTM and one half to SCDM tubes
by unplugging in front of gas burner only.
5.5.19Label both the tubes with Product name, B. No, Report No.., date of testing, Completion date & Tested by.
5.5.20Simultaneously prepare a negative control by filtering 100 ml of 0.1% peptone water
5.5.21Instead of product sample, cut the membrane into two halves with sterile SS scissor and transfer one half to FTM and one half to SCDM and label both the tubes as Negative control.
5.5.22For media negative control, keep one – one tube of each autoclaved lot of FTM & SCDM un-inoculated.
5.5.23After completion of work transfer all inoculated media through hatch box and then transfers all
the equipment and exposed plates to microbiology analysis section.
5.5.24Incubate the FTM tubes at 300C – 350C and SCDM tubes at 200C – 250C. Incubation period
for terminally sterilized products are 7 days and 14 days for aseptically filled products as per IP.
If the Product is as per USP, BP, incubation period is 14 days for both terminally sterilized
as well as for aseptically filled products.
5.5.25Start the LAF of Biosafety cabinet as per SOP and prepare three positive Control tubes
by inoculating aseptically not more than 100 cfu in FTM tubes with S. aureus, P. aeruginosa
and Clostridium sporogenes. Similarly prepare three SCDM positive control by inoculating
not more than 100 cfu separately with C. albicans, A. Niger, and Bacillus subtilis. Incubate FTM
positive control tubes at 30 – 35ºC & SCDM positive control tubes at 20 – 25ºC.
For bacterial positive controls, incubation period is not more than 3 days & for fungal
positive control, incubation period is not more than 5 days.
5.5.26Bacterial endotoxin test
PROCEDURE FOR TESTING:
5.6 Preparation of Maximum valid dilution:
5.6.1 When the endotoxin limit in the substance or preparation being examined is specified in terms
of weight or units of active drug, the MVD may be calculated by the following formula:
Endotoxin Limit (in EU/ ml /mg) X Potency of product
MVD = ———————————————————————
Labeled sensitivity of lysate in EU /ml ()
Where Potency =Concentration of the drug in mg / ml or unit /ml
5.6.2 When the endotoxin limit in the sample preparation is specified in terms of volume,
the MVD may be calculated by the following formula
Endotoxin Limit (EU/ml)
MVD = —————————————————–
Labeled sensitivity of lysate in EU/ml ()
5.6.3 Dilute the sample MVD/2 times with LAL water when performing the test at MVD. If performing
the test at MVD/2, dilute the sample MVD/4time with LAL water.
5.7 Preparation of E-Coli Control Standard Endotoxin (CSE):
5.7.1 Reconstitute the CSE with appropriate volume (as stated on certificate) of LAL reagent water vortex continuous 30 minutes at the interval of 10minutes (When new bottles reconstitute) & vortex vigorously for five minutes before further dilution
5.8 Preparation of CSE dilution series:
5.8.1 Confirm the labeled sensitivity using at least one vial of each batch/lot of lysate. Prepare a series
of two-fold dilution of the CSE to give concentrations of 2, , 0.5 and 0.25, where is the labeled sensitivity of the lysate in EU per ml.
5.8.2 Perform the test as given as under Procedure on these four standard concentrations in
duplicate or greater in include negative control consisting of water BET.
5.8.3 Calculate the average of the logarithms of the lowest concentration of endotoxin in each series of
dilutions for which a positive result is found. The antilogarithm of this average gives the estimated lysate sensitivity,
which must be greater than or equal to 0.5 and less than or equal to 2.0.
confirm the labeled sensitivity of each new batch of lysate prior to use in the test. Test CSE results are only
valid when the positive water and specimen controls are positive at the 2 & endotoxin concentration.
5.9 Preparation of LAL Reagents:
5.9.1 Test LAL reagent for its sensitivity at the time of receiving. For reconstitution, Collect LAL powder into
the bottom of the vial by tapping on a firm surface, unseal and release the vacuum
by slowly lifting the stopper, avoiding touch contamination. The small amount of LAL on the stopper is insignificant.
5.9.2 Rehydrate the reagent with appropriate volume of (as stated on label and certificate)
LAL reagent water by pipetting directly into the vial immediately before use. Cover the vial with an endotoxin free surface or
the inner side of perafilm when not in immediate use. Gently swirl until LAL dissolves into a colorless solution.
5.10 Test procedure for gel clot:
5.11 Interpretation of results:
5.11.1 Each tube in the gel-clot method is interpreted as either positive or negative. A positive test is defined as formation of a firm gel capable of maintaining its integrity when the test tube is inverted 180°C. A negative test is characterized by the absence of gel or by the formation of a viscous mass, which does not hold when the tube is inverted. Calculate sample endotoxin concentration by the following formula.
Sample Endotoxin Conc. :- Dilution Factor x Sensitivity of l
Conc. of Sample (Potency)
Each ml contains:
Piroxicam BP 20 mg
Benzyl alcohol BP 2% v/v
Ethanol BP 12.6 % v/v
Method of Piroxicam: by liquid chromatography
Buffer: 2.5 ml Orthophoshoric Acid produced to 1000 ml water.
Mobile phase: Buffer : Acetonitrile
30 : 70
Standard preparation: Weight 20mg. Piroxicam diluted to 100 ml volumetric flask with Mobile phase.
Sample preparation: Taken equivalent to 20mg. Piroxicam diluted to 100 ml volumetric flask volumetric flask with Mobile phase.
Flow rate – 1.0ml /mint.
column -C18 (250mm x4.6) 5µm.
Injection Volume -20µl.
Retention time of piroxicam about 3.0 mint
7.0 Method of Ethanol: – by gas chromatography
Standard dilution: – 1 ml alcohol + 1 ml internal standard (I.P.A) dilute up to 100 ml water.
Sample dilution: – 1 ml sample + 1 ml Internal standard (I.P.A) dilute up to 100 ml water.
Chromatographic condition: – column -OV-101 (2.0 m)
Injector -2100 C
Oven – Initial Time 1000 C
Hold time -2 minute at 1000 C
Ramp -1000 C to 2200C at the rate 120 C per minute
Detector – 2200 C
Run time – 20 minute
Injection Volume -2 micro litre.
Retention time about 7-8 minutes Alcohol
Retention time about 9-10 minutes Internal
8.0 Method of Benzyl alcohol: – by gas chromatography
Standard dilution: – 1 ml Benzyl alcohol dilute up to 100 ml volumetric flask with methanol.
Further dilution 2 ml to 10 ml volumetric flask with methanol.
Sample dilution: – 1 ml sample solution (inj. 2 % benzyl alcohol) (I.P.A) dilute to 10 ml volumetric flask with methanol.
Chromatographic condition: – column AT-1000
Injector -2100 C
Oven – 1600 C to 2400 C at 50 C per minutes.
Detector – 2800 C
Injection Volume -2 micro litre.
Retention time about 25 minutes Benzyl alcohol.
|Sr. No.||Abbreviation used.||Full form of abbreviation used.|
|1.0||STP||Standard Testing Procedure.|
|Sr. No.||Reference Title|