SlideShare a Scribd company logo

Pyrogen testing 112070804005

1 of 48
GUIDED BY :                        PRESENTED BY :
Mr. Anand K. Patel                 PARTH PATEL
                                   M.Pharm Sem-1
                                   Roll no: 5



        DEPARTMENT OF QUALITY ASSURANCE
   APMC COLLEGE OF PHARMACEUTICAL EDUCATION &
             RESEACH, HIMMATNAGAR
Pyrogen   Testing
  Rabbit test (sham test)
  LAL Test (Bacterial Endotoxin Test)
Depyrogenation Techniques
  Physically removal of Pyrogens
  Inactivation of Pyrogens
Comparison of Pyrogen Test of IP, BP, USP.
Pyrogen testing  112070804005
A. Rabbit test (sham test)
B. LAL Test (Bacterial Endotoxin Test)
The test involves measurement of the
 rise in body temperature of rabbits following
 the intravenous injection of a sterile solution of
 the substance being examined.
It is designed for products that can be tolerated
 by the test rabbit in a dose not exceeding 10 ml
 per kg injected intravenously within a period
 of not more than 10 minutes.
Selection   of animal
Materials:

Heated    in oven at 250ºc for 30 min & 200ºc for
 1 hour.
Diluents   & Solutions Should be sterile &
 Pyrogen free.

Recommended

Pyrogen testing
Pyrogen testingPyrogen testing
Pyrogen testingnilesh1208
 
Pyrogen teat(lal test)
Pyrogen teat(lal test)Pyrogen teat(lal test)
Pyrogen teat(lal test)MdIrfanUddin2
 
Expt. 9 Test for pyrogens ( rabbit method)
Expt. 9 Test for pyrogens ( rabbit method)Expt. 9 Test for pyrogens ( rabbit method)
Expt. 9 Test for pyrogens ( rabbit method)VISHALJADHAV100
 
Expt. 7 Effect of saline purgative on frog intestine
Expt. 7 Effect of saline purgative on frog intestineExpt. 7 Effect of saline purgative on frog intestine
Expt. 7 Effect of saline purgative on frog intestineVISHALJADHAV100
 
Microbiological assay of antibiotics
Microbiological assay of antibioticsMicrobiological assay of antibiotics
Microbiological assay of antibioticsmonnask
 
Bioassay of Digitalis, d-tubocurarine , Oxytocin
Bioassay of Digitalis, d-tubocurarine , OxytocinBioassay of Digitalis, d-tubocurarine , Oxytocin
Bioassay of Digitalis, d-tubocurarine , OxytocinHeena Parveen
 

More Related Content

What's hot

Evaluation of ophthalmic preparation
Evaluation of ophthalmic preparationEvaluation of ophthalmic preparation
Evaluation of ophthalmic preparationSuneal Saini
 
Sterility testing of Pharmaceutical Products
Sterility testing of Pharmaceutical ProductsSterility testing of Pharmaceutical Products
Sterility testing of Pharmaceutical ProductsARUNGOPALAKRISHNAN18
 
Quality control test for packaging material ,qc test for glass,metal,rubber
Quality control test for packaging material ,qc test for glass,metal,rubberQuality control test for packaging material ,qc test for glass,metal,rubber
Quality control test for packaging material ,qc test for glass,metal,rubberKunalPatel257
 
quality control test for soft gelatin capsule and minim per gram factor
quality control test for soft gelatin capsule and minim per gram factorquality control test for soft gelatin capsule and minim per gram factor
quality control test for soft gelatin capsule and minim per gram factorSUJIT DAS
 
Dissolution Testing Apparatus
Dissolution Testing ApparatusDissolution Testing Apparatus
Dissolution Testing ApparatusSourav Kar
 
Pyrogen testing as per IP, BP & USP
Pyrogen testing as per IP, BP & USPPyrogen testing as per IP, BP & USP
Pyrogen testing as per IP, BP & USPbhandarisaurav
 
Expt 5 three point bioassay
Expt 5 three point bioassayExpt 5 three point bioassay
Expt 5 three point bioassayMirzaAnwarBaig1
 
Medicinal chemistry of Antifungal agents
Medicinal chemistry of Antifungal agentsMedicinal chemistry of Antifungal agents
Medicinal chemistry of Antifungal agentsGanesh Mote
 
LIQUID ORALS INDUSTRIAL PHARMACY
LIQUID ORALS INDUSTRIAL PHARMACYLIQUID ORALS INDUSTRIAL PHARMACY
LIQUID ORALS INDUSTRIAL PHARMACYRACHIT KUMAR GUPTA
 
Expt. 6 Bioassay of histamine using guinea pig ileum by matching method
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodExpt. 6 Bioassay of histamine using guinea pig ileum by matching method
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
 
Antibiotics - Medicinal chemistry
Antibiotics - Medicinal chemistry Antibiotics - Medicinal chemistry
Antibiotics - Medicinal chemistry kencha swathi
 
Spectroscopy in pharmacognosy
Spectroscopy in pharmacognosySpectroscopy in pharmacognosy
Spectroscopy in pharmacognosySurbhiSharma196
 
Absorption of drugs from non per os extravascular administration
Absorption of drugs from non per os extravascular administrationAbsorption of drugs from non per os extravascular administration
Absorption of drugs from non per os extravascular administrationSuvarta Maru
 
Bioassay of oxytocin
Bioassay of oxytocinBioassay of oxytocin
Bioassay of oxytocinTrisha Das
 

What's hot (20)

Designing of aseptic area
Designing of aseptic areaDesigning of aseptic area
Designing of aseptic area
 
Evaluation of ophthalmic preparation
Evaluation of ophthalmic preparationEvaluation of ophthalmic preparation
Evaluation of ophthalmic preparation
 
Sterility testing of Pharmaceutical Products
Sterility testing of Pharmaceutical ProductsSterility testing of Pharmaceutical Products
Sterility testing of Pharmaceutical Products
 
Quality control test for packaging material ,qc test for glass,metal,rubber
Quality control test for packaging material ,qc test for glass,metal,rubberQuality control test for packaging material ,qc test for glass,metal,rubber
Quality control test for packaging material ,qc test for glass,metal,rubber
 
Bioassay
BioassayBioassay
Bioassay
 
Microbiological Assay
Microbiological AssayMicrobiological Assay
Microbiological Assay
 
Bioassay of Digitalis
Bioassay of DigitalisBioassay of Digitalis
Bioassay of Digitalis
 
Bio assays of insulin
Bio assays of insulinBio assays of insulin
Bio assays of insulin
 
quality control test for soft gelatin capsule and minim per gram factor
quality control test for soft gelatin capsule and minim per gram factorquality control test for soft gelatin capsule and minim per gram factor
quality control test for soft gelatin capsule and minim per gram factor
 
Dissolution Testing Apparatus
Dissolution Testing ApparatusDissolution Testing Apparatus
Dissolution Testing Apparatus
 
Pyrogen testing as per IP, BP & USP
Pyrogen testing as per IP, BP & USPPyrogen testing as per IP, BP & USP
Pyrogen testing as per IP, BP & USP
 
Expt 5 three point bioassay
Expt 5 three point bioassayExpt 5 three point bioassay
Expt 5 three point bioassay
 
Medicinal chemistry of Antifungal agents
Medicinal chemistry of Antifungal agentsMedicinal chemistry of Antifungal agents
Medicinal chemistry of Antifungal agents
 
LIQUID ORALS INDUSTRIAL PHARMACY
LIQUID ORALS INDUSTRIAL PHARMACYLIQUID ORALS INDUSTRIAL PHARMACY
LIQUID ORALS INDUSTRIAL PHARMACY
 
Expt. 6 Bioassay of histamine using guinea pig ileum by matching method
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodExpt. 6 Bioassay of histamine using guinea pig ileum by matching method
Expt. 6 Bioassay of histamine using guinea pig ileum by matching method
 
PYROGEN TESTING
PYROGEN TESTINGPYROGEN TESTING
PYROGEN TESTING
 
Antibiotics - Medicinal chemistry
Antibiotics - Medicinal chemistry Antibiotics - Medicinal chemistry
Antibiotics - Medicinal chemistry
 
Spectroscopy in pharmacognosy
Spectroscopy in pharmacognosySpectroscopy in pharmacognosy
Spectroscopy in pharmacognosy
 
Absorption of drugs from non per os extravascular administration
Absorption of drugs from non per os extravascular administrationAbsorption of drugs from non per os extravascular administration
Absorption of drugs from non per os extravascular administration
 
Bioassay of oxytocin
Bioassay of oxytocinBioassay of oxytocin
Bioassay of oxytocin
 

Similar to Pyrogen testing 112070804005

Qc for sterile pharmaceutical product
Qc for sterile pharmaceutical productQc for sterile pharmaceutical product
Qc for sterile pharmaceutical productAniket Gholap
 
Water test methods
Water test methodsWater test methods
Water test methodsSoumen Basu
 
Chemical Tests - PDW final 2.11.2014
Chemical Tests - PDW final 2.11.2014Chemical Tests - PDW final 2.11.2014
Chemical Tests - PDW final 2.11.2014SATISH KUMAR
 
PROTEOMICS -DTNB ASSAY
PROTEOMICS -DTNB ASSAYPROTEOMICS -DTNB ASSAY
PROTEOMICS -DTNB ASSAYNidhi Puranik
 
Measuring pKas, logP and Solubility by Automated titration
Measuring pKas, logP and Solubility by Automated titrationMeasuring pKas, logP and Solubility by Automated titration
Measuring pKas, logP and Solubility by Automated titrationJon Mole
 
Western Blot Guide
Western Blot GuideWestern Blot Guide
Western Blot GuideElabscience
 
microbial assay antibiotics, vitamins, amino acids
microbial assay antibiotics,  vitamins,  amino acidsmicrobial assay antibiotics,  vitamins,  amino acids
microbial assay antibiotics, vitamins, amino acidsMicroShamim
 
Western blotting protocol & troubleshooting
Western blotting protocol & troubleshootingWestern blotting protocol & troubleshooting
Western blotting protocol & troubleshootingJames Waita
 
Bioassay 112070804012
Bioassay 112070804012Bioassay 112070804012
Bioassay 112070804012Patel Parth
 
Covid 19 spike protein elisa kit 20220223143226
Covid 19 spike protein elisa kit 20220223143226Covid 19 spike protein elisa kit 20220223143226
Covid 19 spike protein elisa kit 20220223143226Annieliu694961
 
Human DLL4(delta like protein 4) elisa kit
Human DLL4(delta like protein 4) elisa kitHuman DLL4(delta like protein 4) elisa kit
Human DLL4(delta like protein 4) elisa kitziyiChen18
 
Human S100B(protein S100 B) elisa kit
Human S100B(protein S100 B) elisa kitHuman S100B(protein S100 B) elisa kit
Human S100B(protein S100 B) elisa kitziyiChen18
 
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdf
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdfFY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdf
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdffeiyue lottie
 
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docx
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docxSynthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docx
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docxssuserf9c51d
 

Similar to Pyrogen testing 112070804005 (20)

Qc for sterile pharmaceutical product
Qc for sterile pharmaceutical productQc for sterile pharmaceutical product
Qc for sterile pharmaceutical product
 
BET.pptx
BET.pptxBET.pptx
BET.pptx
 
Lab discussion
Lab discussionLab discussion
Lab discussion
 
Water test methods
Water test methodsWater test methods
Water test methods
 
Chemical Tests - PDW final 2.11.2014
Chemical Tests - PDW final 2.11.2014Chemical Tests - PDW final 2.11.2014
Chemical Tests - PDW final 2.11.2014
 
PROTEOMICS -DTNB ASSAY
PROTEOMICS -DTNB ASSAYPROTEOMICS -DTNB ASSAY
PROTEOMICS -DTNB ASSAY
 
Aseptic Process Technology.pptx
Aseptic Process Technology.pptxAseptic Process Technology.pptx
Aseptic Process Technology.pptx
 
Sterility testing
Sterility testingSterility testing
Sterility testing
 
Measuring pKas, logP and Solubility by Automated titration
Measuring pKas, logP and Solubility by Automated titrationMeasuring pKas, logP and Solubility by Automated titration
Measuring pKas, logP and Solubility by Automated titration
 
Western Blot Guide
Western Blot GuideWestern Blot Guide
Western Blot Guide
 
microbial assay antibiotics, vitamins, amino acids
microbial assay antibiotics,  vitamins,  amino acidsmicrobial assay antibiotics,  vitamins,  amino acids
microbial assay antibiotics, vitamins, amino acids
 
DNA Extraction & PCR protocol
DNA Extraction & PCR protocolDNA Extraction & PCR protocol
DNA Extraction & PCR protocol
 
Western Blotting Protocol & Troubleshooting
Western Blotting Protocol & TroubleshootingWestern Blotting Protocol & Troubleshooting
Western Blotting Protocol & Troubleshooting
 
Western blotting protocol & troubleshooting
Western blotting protocol & troubleshootingWestern blotting protocol & troubleshooting
Western blotting protocol & troubleshooting
 
Bioassay 112070804012
Bioassay 112070804012Bioassay 112070804012
Bioassay 112070804012
 
Covid 19 spike protein elisa kit 20220223143226
Covid 19 spike protein elisa kit 20220223143226Covid 19 spike protein elisa kit 20220223143226
Covid 19 spike protein elisa kit 20220223143226
 
Human DLL4(delta like protein 4) elisa kit
Human DLL4(delta like protein 4) elisa kitHuman DLL4(delta like protein 4) elisa kit
Human DLL4(delta like protein 4) elisa kit
 
Human S100B(protein S100 B) elisa kit
Human S100B(protein S100 B) elisa kitHuman S100B(protein S100 B) elisa kit
Human S100B(protein S100 B) elisa kit
 
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdf
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdfFY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdf
FY-ECH4770 Chicken Total bile acide (TBA) ELISA Kit .pdf
 
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docx
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docxSynthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docx
Synthesis of E, E-dibenzalacetone (E, E-DBA) Lab ReportN.docx
 

More from Patel Parth

Automated analysis by yatin sankharva copy
Automated analysis by yatin sankharva   copyAutomated analysis by yatin sankharva   copy
Automated analysis by yatin sankharva copyPatel Parth
 
Automated analysis 112070804013
Automated analysis 112070804013Automated analysis 112070804013
Automated analysis 112070804013Patel Parth
 
Anda registration in us eu
Anda registration in us  euAnda registration in us  eu
Anda registration in us euPatel Parth
 
Analytical tech in pre formulation 112070804009
Analytical tech in pre formulation 112070804009Analytical tech in pre formulation 112070804009
Analytical tech in pre formulation 112070804009Patel Parth
 
Analysis of solid oral
Analysis of solid oralAnalysis of solid oral
Analysis of solid oralPatel Parth
 
Analysis of solid oral dosage forms 112070804010
Analysis of solid oral dosage forms 112070804010Analysis of solid oral dosage forms 112070804010
Analysis of solid oral dosage forms 112070804010Patel Parth
 
Analysis of parenteral dosage forms bjl final seminar
Analysis of parenteral dosage forms bjl final seminarAnalysis of parenteral dosage forms bjl final seminar
Analysis of parenteral dosage forms bjl final seminarPatel Parth
 
Analysis of data (pratik)
Analysis of data (pratik)Analysis of data (pratik)
Analysis of data (pratik)Patel Parth
 
Analysis of cosmetics 112070804018
Analysis of cosmetics 112070804018Analysis of cosmetics 112070804018
Analysis of cosmetics 112070804018Patel Parth
 
A seminar on applications of various analytical technique
A seminar on applications of various analytical techniqueA seminar on applications of various analytical technique
A seminar on applications of various analytical techniquePatel Parth
 
23117 copy of oral solid dosage forms
23117 copy of oral solid dosage forms23117 copy of oral solid dosage forms
23117 copy of oral solid dosage formsPatel Parth
 
4016 solid state analysis
4016 solid state analysis4016 solid state analysis
4016 solid state analysisPatel Parth
 
4003 regulatory aspect_of_bulk,pharmaceutical,biotech
4003 regulatory aspect_of_bulk,pharmaceutical,biotech4003 regulatory aspect_of_bulk,pharmaceutical,biotech
4003 regulatory aspect_of_bulk,pharmaceutical,biotechPatel Parth
 
A.a sequence analysis 112070804002
A.a sequence analysis 112070804002A.a sequence analysis 112070804002
A.a sequence analysis 112070804002Patel Parth
 
Sterility testing 112070804014
Sterility testing 112070804014Sterility testing 112070804014
Sterility testing 112070804014Patel Parth
 
Ria 112070804007
Ria 112070804007Ria 112070804007
Ria 112070804007Patel Parth
 
Qc lab 112070804001
Qc lab 112070804001Qc lab 112070804001
Qc lab 112070804001Patel Parth
 

More from Patel Parth (20)

Automated analysis by yatin sankharva copy
Automated analysis by yatin sankharva   copyAutomated analysis by yatin sankharva   copy
Automated analysis by yatin sankharva copy
 
Automated analysis 112070804013
Automated analysis 112070804013Automated analysis 112070804013
Automated analysis 112070804013
 
Anda registration in us eu
Anda registration in us  euAnda registration in us  eu
Anda registration in us eu
 
Anda ppt
Anda pptAnda ppt
Anda ppt
 
Analytical tech in pre formulation 112070804009
Analytical tech in pre formulation 112070804009Analytical tech in pre formulation 112070804009
Analytical tech in pre formulation 112070804009
 
Analysis of solid oral
Analysis of solid oralAnalysis of solid oral
Analysis of solid oral
 
Analysis of solid oral dosage forms 112070804010
Analysis of solid oral dosage forms 112070804010Analysis of solid oral dosage forms 112070804010
Analysis of solid oral dosage forms 112070804010
 
Analysis of parenteral dosage forms bjl final seminar
Analysis of parenteral dosage forms bjl final seminarAnalysis of parenteral dosage forms bjl final seminar
Analysis of parenteral dosage forms bjl final seminar
 
Analysis of data (pratik)
Analysis of data (pratik)Analysis of data (pratik)
Analysis of data (pratik)
 
Analysis of cosmetics 112070804018
Analysis of cosmetics 112070804018Analysis of cosmetics 112070804018
Analysis of cosmetics 112070804018
 
Agencies dhwani
Agencies dhwaniAgencies dhwani
Agencies dhwani
 
A seminar on applications of various analytical technique
A seminar on applications of various analytical techniqueA seminar on applications of various analytical technique
A seminar on applications of various analytical technique
 
23117 copy of oral solid dosage forms
23117 copy of oral solid dosage forms23117 copy of oral solid dosage forms
23117 copy of oral solid dosage forms
 
4016 solid state analysis
4016 solid state analysis4016 solid state analysis
4016 solid state analysis
 
4003 regulatory aspect_of_bulk,pharmaceutical,biotech
4003 regulatory aspect_of_bulk,pharmaceutical,biotech4003 regulatory aspect_of_bulk,pharmaceutical,biotech
4003 regulatory aspect_of_bulk,pharmaceutical,biotech
 
A.a sequence analysis 112070804002
A.a sequence analysis 112070804002A.a sequence analysis 112070804002
A.a sequence analysis 112070804002
 
Chi square test
Chi square testChi square test
Chi square test
 
Sterility testing 112070804014
Sterility testing 112070804014Sterility testing 112070804014
Sterility testing 112070804014
 
Ria 112070804007
Ria 112070804007Ria 112070804007
Ria 112070804007
 
Qc lab 112070804001
Qc lab 112070804001Qc lab 112070804001
Qc lab 112070804001
 

Pyrogen testing 112070804005

  • 1. GUIDED BY : PRESENTED BY : Mr. Anand K. Patel PARTH PATEL M.Pharm Sem-1 Roll no: 5 DEPARTMENT OF QUALITY ASSURANCE APMC COLLEGE OF PHARMACEUTICAL EDUCATION & RESEACH, HIMMATNAGAR
  • 2. Pyrogen Testing  Rabbit test (sham test)  LAL Test (Bacterial Endotoxin Test) Depyrogenation Techniques  Physically removal of Pyrogens  Inactivation of Pyrogens Comparison of Pyrogen Test of IP, BP, USP.
  • 4. A. Rabbit test (sham test) B. LAL Test (Bacterial Endotoxin Test)
  • 5. The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance being examined. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 minutes.
  • 6. Selection of animal Materials: Heated in oven at 250ºc for 30 min & 200ºc for 1 hour. Diluents & Solutions Should be sterile & Pyrogen free.
  • 7. Recording of Temperature Thermometer or thermistor. Insert the thermometer or temperature sensing probe into the rectum of the test rabbit to a depth of about 5-cm. The depth of insertion is constant for any one rabbit in any one test.
  • 8.  PRELIMINARY TEST: To the rabbit Inject intravenously 10 ml of Pyrogen-free saline solution. Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6oC or more must not be used in the main test.
  • 9.  Preparation of the sample  Procedure: 1. Carry out the test using a group of three rabbits. 2. Record the temperature of each animal. Not more than 40 minutes immediately preceding the injection of the test dose, 3. Record the "initial temperature" of each rabbit.( dT Sh’d not >0.2) 4. Do not use any rabbit having a temperature higher than 39.8oC and lower than 38oC . 5. Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes 9
  • 10. 6. The amount of sample to be injected varies according to the preparation being examined and is prescribed in the individual monograph. 7. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Records the temperature of each animal at half-hourly intervals for 3 hours after the injection. 8. The difference between the "initial temperature" and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken to be its response. When this difference is negative, the results is counted as a zero response.
  • 11. If the responses of the group of three rabbits in which individual response is less than 0.6 oC , the preparation being examined passes the test. If the response of any rabbit is 0.6 oC or more, or if the sum of the response of the three rabbits exceeds 1.4oC continue the test using five other rabbits. If not more than three of the eight rabbits show individual responses of 0.6oC or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7oC , the preparation being examined passes the test.
  • 12.  It can be replicated and demonstrate the production of fever in humans  Detects all kinds of injectable Pyrogen unlike LAL test.  DISADVANTAGE:  Time consuming  Expensive Procedure  It is passfail test than assay  It cannot be used to test certain drugs that depresses the fever  Tolerance to certain class of drugs can develop in rabbits & also biological variations are observed
  • 13.  LAL test = Limulus Amebocyte Lysate Test  Test also referred by USP, IP, BP as BET test : Bacterial Endotoxin Test
  • 14. The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or gel. The rate of reaction depends on the concentration of endotoxin , the pH and the temperature. The reaction requires the presence of certain bivalent cations , a proclotting enzyme system and clottable protein all of which are provided by the lysate.
  • 15. Mechanism of Action: Endotoxin 1-3-β- D- Glucan Factor C ----> Factor G ----> Factor C* Factor G* Factor B ----> Factor B* PRECLOTTING ENZYME ----> CLOTTING ENZYME COAGULOGEN ----> COAGULIN 15
  • 17. The following six methods are described in the I.P. 2007 and B.P. 2007 1) Method A: Gel clot method: Limit test 2) Method B: Gel clot method: Semi quantitative test. 3) Method C: Turbidimetric Kinetic method 4) Method D: Chromogenic kinetic method 5) Method E: Chromogenic end- point method 6) Method F: Turbidimetric end point method.  the final decision is made based upon Method A , Unless otherwise indicated in the monograph:
  • 18. Depyrogenate all glassware  Time and temperature is required 30 minutes and 250 0C  Plastic apparatus- which is free from detectable endotoxins 1) Water BET 2) 0.1M Hydrochloric acid BET 3) 0.1M Sodium hydroxide BET 4) Tris-chloride buffer pH 7.4 BET.  Dissolve 0.6057 g of tris -(hydroxymethyl) methylamine in 30 ml of water BET, add 0.33 ml of hydrochloric acid, dilute to 100 ml with water BET and mix. It gives a negative result under the conditions of the test.
  • 19. 5) Lysate: A lysate of amebocytes from the horseshoe crab, Limulus polyphemus, reconstituted as stated on the label. For each batch, confirm the stated sensitivity as prescribed under Sensitivity of the lysate. 6) Lysate solution 7) Reference Standard Endotoxin 8) The standard endotoxin stock solution  Endotoxin is expressed in International Units (IU)  1 IU=1 EU (ENDOTOXIN UNIT).
  • 20. 9) STANDARD ENDOTOXIN SOLUTIONS 10) PREPARATION OF THE TEST SOLUTIONS Preparation of test solution by dissolving or diluting active substance Adjust the pH of test solution(about 6 to 8) pH adjust with use of acid, base and buffer Buffer must be validated to be free of detectable endotoxin
  • 21. The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at which the endotoxin limit can be determined. MVD = endotoxin limit × conc. of test solution λ λ = the labeled lysate sensitivity in the gel clot, turbidimetric or chromogenic technique.
  • 22. The Endotoxin limit= K/M K = Threshold pyrogenic dose of endotoxin per kg of body mass in single hour period. M = Maximum dose of product per kg of body mass in a single hour period  The value of K is 5.0 EU/kg for injectable preparations except for those administered intrathecally and is 0.2 EU/kg for intrathecal preparations.
  • 23.  The gel-clot technique allows detection or quantification of endotoxins and is based on clotting of the lysate in the presence of endotoxins.  The concentration of endotoxins required to cause the lysate to clot under standard conditions is the labelled lysate sensitivity  To ensure both the precision and validity of the test, confirm the labelled lysate sensitivity and perform the test for interfering factors as described under Preparatory testing
  • 24. SENSITIVITY OF THE LYSATE Confirm in 4 replicates the labelled lysate sensitivity(λ) Prepare standard solutions of at least 4 concentrations equivalent to 2λ , λ , 0.5λ and 0.25λ from stock by diluting with water for BET Mix lysate solution with standard in each tube.(1:1) Incubate the reaction mixture at 37 ± 1 °C for 60 ± 2 min (avoiding vibration) Invert it through approximately 180° in one smooth motion.
  • 25. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The end-point is the last positive result in the series of decreasing concentrations of endotoxin. Calculate Geometric mean end-point concentration = Where, ∑e = Sum of the log end-point concentrations. f = number of replicates.  The geometric mean end-point concentration is the measured sensitivity of the lysate solution (IU/ml).  If this is not less than 0.5λ and not more than 2λ, the labelled sensitivity is confirmed
  • 26. Solution Endotoxin Diluents Dilution Initial Number conc./sol. to factor endotoxin of which conc. replicate endotoxin is added A None/Test - - - 4 (Sol. Of Prepa.) solution B 2λ/Test Test 1 2λ 4 (Test for solution solution 2 1λ 4 Interference) 4 0.5λ 4 8 0.25λ 4 C 2λ/Water for Water for 1 2λ 2 (+ ve control) BET BET 2 1λ 2 4 0.5λ 2 8 0.25λ 2 D None/Water for - - - 2 (- ve control) BET 26
  • 27. Prepare solutions A, B, C and D as shown in Table and use the test solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described under Confirmation of the labelled lysate sensitivity.  The geometric mean end-point concentrations of solutions B and C are determined. The test is not valid unless all replicates of solutions A and D show no reaction and the result of solution C confirms the labelled lysate sensitivity
  • 28. If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the solution interferes with the test  If the preparation being examined interferes with the test at a dilution less than the MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD  Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis or heat treatment.
  • 29. Solution Endotoxin conc./solution to No. of which endotoxin is added Replicates A None/Test solution 2 B 2λ / Test solution 2 C 2 λ/water for BET 2 D None/Water for BET 2  Prepare solutions A,B,C, and D as shown in table, and perform the test on these solutions by following the procedure described in Confirmation of the labeled lysate sensitivity
  • 30.  Prepare solution A and Solution B (Positive Product control) using a dilution not greater than the MVD and treatments Test for interfering Factors. Interpretation:  Ifa positive result is found for one of the test duplicates and a negative result for the other, the test may be repeated as described above. The results of the retest should be interpreted as for the initial test.  The substance or preparation being examined meets the requirements of the test if the concentration of endotoxin is less than the endotoxin limit stated in the monograph.
  • 31. Solution Endotoxin Diluents Dilution Initial Number conc. /solution factor endotoxin of to which conc. replicate endotoxin is added A None/Test Water for 1 - 2 solution BET 2 - 2 4 - 2 8 - 2 B 2λ/Test solution - 1 2λ 2 (+ve control) C 2λ/Water for Water for 1 2λ 2 BET BET 2 1λ 2 4 0.5λ 2 8 0.25λ 2 D None/Water for - - - 2
  • 32.  The test is not valid unless the following three conditions are met: 1. Both replicates of solution D(negative control) are negative. 2. Both replicates of solution B(positive control) are positive 3. The geometric mean end-point concentration of solution C is in the range of 0.5 λ to 2 λ.  To determine the endotoxin concentration of solution A. calculate the end-point concentration for each replicates series of dilutions by multiplying each end-point dilution factor by λ
  • 33.  The endotoxin concentration in the test solution is the geometric mean end point concentration of the replicates, if the test is conducted with a diluted test solution calculate the concentration of endotoxin in the original solution by multiplying the results by dilution factor.  If none of the dilution of the test solution is positive in a valid test, record endotoxin concentration as less than λ .  If all dilutions are positive, the endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied by λ
  • 34.  The preparation meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph.
  • 35. 1. Turbidimetric technique (Method C and F)  This technique is a photometric test to measure the increase in turbidity,  Based on the test principle employed classified as,  The end –point turnidimetric test (F)  The kinetic- turbidimetric test (C)
  • 36. 2.CHROMOGENIC TECHNIQUES (METHODS D &E)  This techniques is used to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate.  Depending on the principle employed , this technique is classified as  End- point chromogenic test (E)  The kinetic- chromogenic test.(D)
  • 37. As the use of LAL test became more & more prevalent, the FDA decided that single standardize document was needed to govern all FDA regulated products that were subject to LAL testing,  An FDA task force was formed by representatives from 1) Center for drug evolution & Research. 2) Center for biological evolution & Research. 3) Center for devices & Radiologic health. 4) Center for veterinary medicine.
  • 38. Three basic requirements i. The LAL reagent used in cell validation in process and end product LAL tests must be licensed by CBER ii. The product manufacturer must perform an initial Qualification of their LAL test laboratory PERSONNEL iii.Inhibition & enhancement tests must be performed on test products do not interfere with the enzymatic LAL endotoxin reaction.
  • 39. A. Inhibition  Occurs when the test recovers less endotoxin Causes: • Chemical nature of the drug product or excipients • Factors that negatively effect serine protease enzyme reaction such as High or low pH • Oxidants or antioxidants • Proteolytic agents • High heat • Chelating agents • Inadequately dispersed purified endotoxin
  • 40. B. Enhancement:  Occurs if more Endotoxins recovered than expected Causes:  Chemical nature of the product  Endotoxin contamination present in product  Surfactants by increasing surface area of endotoxin.
  • 41.  ADVANTAGE OF LAL TEST  In vitro test ,  More sensitive  Easier to perform.  Can give Quantitative results.  Less time consuming.  Less expensive.  LIMITATION OF LAL TEST  Specific for gram negative pyrogens only.  Clotting enzyme is heat labile, pH sensitive.  Possible interference Problems.
  • 42. 1) Pharmaceuticals:  Parenteral dosage form  Large volume Parenterals  Small volume Parenterals 2) Biologicals  In blood products & plasma fractions  Vaccines 3)Medical Device  Nebulizers used in Respiratory therapy 4)Diagnosis of disease caused by Gram –negative bacteria 5) In food & drinking water 6) Others: For validation of dry heat sterilization
  • 43. INVITRO PYROGEN TEST (IPT):  This test exploits the reaction monocytes/macrophages for detection of pyrogens .  Human whole blood taken from healthy volunteers is incubated in presence of test sample, pyrogenic contamination initiate the release of “the endogenous pyrogen”  Interlukin 1-β determined by ELISA after incubation.
  • 44. (A)Removal of Pyrogens by physical Methods (1) Dilution (2) Ultra-filtration (3) Reverse osmosis (4) Distillation (5) Adsorption on Charcoal (6) Column Chromatography (7) Charge Modified Media & Electrostatic Attraction (8) Hydrophobic attraction to hydrophobic medium
  • 45. (1) Dry heat sterilization (2) Moist heat sterilization (3) Use of dilute acids & Bases (4) Oxidation (5) Alkylation
  • 46. Number of Maximum total peak Minimum total peak Rabbits response (°C) to pass response (°C) to fail the the test test USP BP USP BP 3 1.4 1.15 1.4 2.65 6 - 2.80 - 4.30 8 3.7 - 3.7 - 9 - 4.45 - 5.95 12 - 6.60 - 6.60
  • 47.  Encyclopedia of Pharmaceutical Technology, Volume:13,By James Swarbick & James Boylan.  Indian Pharmacopoeia 2007,Vol.-1,Appendice-1.  British Pharmacopoeia 2007.  United state pharmacopoeia.  Remington( The science and practice of pharmacy), Volume-I , Page no.562,832.