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IPQC tests for Parenterals

The document discusses in-process and finished product quality control tests for parenterals. It defines parenterals as sterile preparations intended for administration by injection, infusion, or implantation. It describes various types of parenterals including small volume parenterals like ampoules and vials, as well as large volume parenterals. The document then outlines several important in-process quality control tests that are conducted on parenterals to ensure safety, identity, strength, quality and purity. These include tests like content uniformity, leakage, sterility, bacterial endotoxins and clarity. Specific test methods, acceptance criteria and significance are provided for key tests according to compendial standards.

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IN-PROCESS AND FINISHED PRODUCT QUALITY CONTROL TESTS FOR PARENTERALS Prapti Pattanayak Pharmaceutical Analysis M.Pharm 1st Year 1
CONTENTS 1. Introduction 2. Definition 3. Types Of Parenterals 4. Routes Of Administration 5. Significance Of IPQC Tests 6. IPQC Tests According To IP, BP And USP 2
INTRODUCTION • Derived from Greek words “Para” means “outside” and “enteron” means “intestine”. • Route of administration- mostly intravenous, intramuscular, subcutaneous, intradermal . • Sterile solution or suspension of drug in aqueous or oily vehicle. • Directly administered directly under the skin, veins, muscles or more specialized tissues like spinal cord. • Drug delivery doesn’t utilize alimentary canal for entering into the tissues. 3 Fig 1: Injections, vial, ampoule
DEFINITION • According to IP Preparations that are sterile and intended for administration by injection, infusion or implantation. • According to USP Preparations that includes injections and implanted products that are injected through the skin or other external boundary tissue or implanted within the body to allow direct administration. • According to BP Sterile products that are intended for administration by injection, infusion or implantation into the body. 4
TYPES OF PARENTERALS 1. Small Volume Parenterals (SVPs)  They are given as multiple dose.  Volume- <100ml  Types: Ampoules, vials, dry powders etc. a. Ampoules- These are sealed glass container with an elongated neck that must be broken off. A 5 micron filter needle should be used when drawing the contents of an ampoule into a syringe since glass particles may have fallen inside the ampoule when the top was snapped off. b. Vials- It is made of glass or plastic and sealed with a rubber stopper. Drugs and other additives are packaged as liquid or lyophilized powders. It is designed for both single and multiple dose use. Multiple dose vials contain a preservative to inhibit bacterial contamination. 5 Fig 2: Ampoules Fig 3: Vial
6 c. Dry Powders- These are lyophilized or freeze dried powders that must be reconstituted with some suitable solvent to make a liquid formulation before being withdrawn from the vial. Solvents may be WFI, BWFI, NaCl injection etc. 2. Large Volume Parenterals (LVPs) They are designed to provide fluid (water), calories (dextrose solution) and electrolytes ( saline solution). Volume- > 100ml  4 Types: a. Hyperalimentation solution/ Total Parenteral Nutrition- administration of large amount of nutrients to patients who are unable to take food orally. • Use- administration of life saving drugs to the comatose patients Fig 4: LVPs
7 b. Cardioplegic Solution- it is used for heart surgery to prevent injury to myocardium during reperfusion. • Maintains diastolic arrest. c. Peritoneal Dialysis Solution- It is infused continuously into the abdominal cavity, bathing peritoneum and are then continuously withdrawn. Use- To minimize reperfusion injury resulting from tissue edema. Formulations- Glucose, Antibiotics as prophylactic . d. Irrigating Solution- It is used to irrigate, flush and aid in cleansing body cavities and wounds. Formulation- Normal saline Use- For the treatment of serious wounds infused into blood stream.
ROUTE OF ADMINISTRATION • Angle- 15ﾟ • Dose- 0.1ml • Ex. BCG vaccineINTRADERMAL • Angle- 25ﾟ • Dose- >1litre • Ex. DNS (Dextrose Normal Saline) INTRAVENOUS • Angle- 45ﾟ • Dose- 1-1.3ml • Ex. Insulin SUBCUTANEOUS • Angle- 90ﾟ • Dose- 2-5ml • Ex. Atropine, Haloperidol INTRAMUSCULAR 8
SIGNIFICANCE OF IPQC(In Process Quality Control) • We can remove error from every stage in production and maintain the quality of the final product with the compendial standards as specified in the pharmacopoeia’s. • We can determine the number and types of microorganisms present in the product by sterility testing. • We can ensure the exact quantity of the solution mentioned on the label claim. • We can prevent the contamination of the drug caused due to poor package integrity. • Identification of any crack or leakage present on the container which may hamper the package integrity. 9
IPQC TESTS FOR PARENTERALS CONTENT UNIFORMITY EXTRACTABLE VOLUME PYROGEN TEST STERILITY TEST LEAKAGE TEST BACTERIAL ENDOTOXIN TEST CLARITY TEST 10Fig 5: IPQC tests for Parenterals
CONTENT UNIFORMITY • ACCORDING TO IP(Method) Weight of 10 individual sterile units are noted Content is removed from them and empty individual sterile unit is weighed accurately. Net weight is calculated by substracting empty sterile unit weight from gross weight. 11
Acceptance Criteria (IP) DESCRIPTION RANGES INFERENCE If all individual values are within the range of 85-115% of avg. values and RSD ≤6% Passes If more than 1 individual values OR If any one individual value >85-115% of the avg. value OR >75-125% of the avg. value Fails If one individual value is >85-115% but within the limits 75- 125% of the avg. value RSD >6% Repeat the test using additional 20 containers If in 30 containers, NMT 1 individual value OR None of the units Not exceeds 85-115% of avg. value OR Exceeds 75-125% of avg. value RSD NMT 7.8% Passes 12 Table no. 1
According to BP • Individual contents of the 10 dosage units are taken randomly. TEST DESCRIPTION RANGES INFERENCE TEST A (tab, powders for parenterals administration, opthalmic inserts, suspensions for injection) 1.If each individual contents is 2.If 1 or more than 1 individual content is 3.If 1 individual content 4. If in 30 units, NMT 1 individual b/w 85-115% of avg. contents >75-125% of the avg. value Exceeds 85-115% but within 75-125% limits Exceeds 85-115% of the avg. value and none exceeds 75-125% avg. value Passes Fails Another 20 dosage forms taken randomly Passes TEST B (cap, powders other than for parenterals administration, granules, suppositories, pessaries) 1.If NMT 1 individual content Exceeds 85-115% and none exceeds 75-125% avg. contents Passes 13 Table no. 2
Continue.. 2. If more than 3 individual contents OR If 1 or more individual content 3. If 2 or 3 individual contents 4. If in 30 individual units, NMT 3 individual contents Exceeds 85-115%avg. content OR Exceeds 75-125% avg. content Exceeds 85-115% avg. value but within 75- 125% Exceeds 85-115% and none exceeds 75-125% of avg. contents Fails Randomly 20 dosages forms are added Passes TEST C (Transdermal patches) 1. If avg. content of 10 dosages units is 2. If the individual content of each dosage unit is b/w 90-110% of the content b/w 75-125% of avg. content Passes 14
According to USP • 10 dosage units are taken randomly. DESCRIPTION RANGES INFERENCE If 10 individual units Within 85-115% avg. content and RSD <6% Passes If more than 1 individual value OR If any one individual value Exceeds 85-115% avg. content OR Exceeds 75-125% avg. content Fails Fails If one individual Exceeds 85-115% but with 75-125% avg. content Repeat test with another 20 units randomly If in 30 containers, NMT 1 individual value Exceeds 85-115% and none exceeds 75-125% avg. content value Passes 15 Table no.3
LEAKAGE TEST 16  To test package integrity.  Leakage test is employed to detect incompletely sealed ampoule and vials so that they may be discarded. WHAT IS THE NEED ? • Presence of capillary pores or tiny cracks can cause microbes or other contaminants to enter in the ampoules. • Change in temperature during storage can cause expansion and contraction of the ampoule and thereby causing interchange of its contents if any opening exists. • Leakage test is done by visual inspection, bubble test and dye bath test . A. METHOD ( DYE BATH TEST) TEST CONTAINER IMMERSED IN A DYE BATH UNDER VACUUM AND PRESSURE REMOVED THE CONTAINER FROM DYE BATH AND WASHED CONTAINER IS INSPECTED FOR THE PRESENCE OF DYE EITHER VISUALLY OR BY UV SPECTROSCOPY DYES USED : Mainly methylene blue is used. Other than that green, yellowish-green color are also used. OPTIMIZATION OF DYE TEST: • Addition of surfactant or a low viscosity fluid in the dye. • These will increase the capillary migration of the pores. ADVANTAGES: • Inexpensive • No special equipment required for identification • Only for Qualitative analysis DISADVANTAGES: • Destructive and slow test • Not for Quantitative analysis
17 B. VISUAL INSPECTION • It is the easiest and least sensitive leakage test method . • Used for evaluation of large volume parenterals. • To increase the sensitivity of the method, the visual inspection of the sample container may be coupled with the application of vacuum to make leakage more readily observable. • It is a very simple , inexpensive and operator dependent method. C. BUBBLE TEST • The test package is submerged in liquids. • A differential pressure is applied on the container. • The container is observed for bubbles. • For optimization of leakage test, surfactant(ex. polysorbate 80) added to the liquid along with dark background and high intensity lighting. • Differential positive pressure of 3 psi inside the vial is generated and observation of any leakage using magnifying glass within a maximum test time of 15 minutes. • It can detect leaks as small as 10 mbar-L/sec. • It is also insensitive, operator dependent and qualitative test. Fig 6: Visual Inspection
18 STERILITY TEST  Sterile means free from microorganisms like bacteria, fungi, yeast etc. PRINCIPLE: If the bacteria and fungi are placed in a medium which provides nutrition to the organisms and kept at favorable temperature, the organisms will grow and their presence can be indicated by turbidity in the clear medium. WHAT IS THE NEED? • To detect the presence of viable organisms in the parenterals. • Presence of microbes may contaminate the sterile products.  For sterility testing, aseptic conditions should be maintained. MAJOR FACTORS AFFECTING STERILITY TESTING • The environment in which test is conducted • The quality of culture • Test method • Test procedure • Sample size Fig 7: Sterility testing
19 CULTURE MEDIA USED: There are basically 2 types of culture media: a. Fluid thioglycollate medium b. Soyabean casein digest medium 1. FLUID THIOGLYCOLATE MEDIUM (FTM)  FTM is an excellent medium for the detection of microbial contamination.  It can provide both aerobic and anaerobic environment. COMPOSITIONS OF FTM: a. L- cysteine- it supply carbon and nitrogenous compounds. b. Dextrose- acts as an energy source. c. Yeast extract- act as growth enhancer. d. Sodium chloride- it maintains osmotic equilibrium. e. Sodium thioglycollate- acts as reducing agent which maintains a low oxygen tension by removing molecular oxygen from the environment i.e., it creates anaerobic conditions when it reduces molecular oxygen to water. f. Resazurin- it is a redox indication that turns pink when oxidation increased and colorless when reduction occurred. g. Agar- addition of small amount of agar in medium initiate the growth of small inocula and anaerobes. h. Purified water i. Adjusted to pH 7.1± 0.2  Incubation of the media – 14 days at 30- 35℃ Components Quantity L-Cysteine 0.5g Sodium chloride 2.5g Dextrose monohydrate 5.5g Yeast extract 5.0g Pancreatic digest of casein 15.0g Sodium thioglycollate/thioglycollic acid 0.5g/0.3g Resazurin sodium solution 1.0ml Distilled water q.s to 1000ml pH of medium after sterilization 7.1± 0.2 Table no. 4
20 B. SOYABEAN CASEIN DIGEST MEDIUM  It is used for culture of both fungi and aerobic bacteria. COMPOSITIONS: a.Trypticase soya broth- it is used to grow aerobic bacteria b.Trypticase peptone- it is an animal origin pancreatic digest of casein which provides nutrition. c. Sodium chloride- it maintains osmotic equilibrium d. Dipotassium phosphate- it provides buffering capacity e. Dextrose- provides energy f. Purified water g. Adjusted to pH 7.3± 0.2  Incubation period- 14 days at 20- 25℃ Components Quantity Pancreatic digest of casein 17.0g Papaic digest of soyabean meal 3.0g Sodium chloride 5.0g Dipotassium hydrogen phosphate 2.5g Dextrose monohydrate 2.5g Distilled water q.s. to 1000ml pH of medium after sterilization 7.3± 0.2 Table no. 5
21 STERILITY TESTING METHODS: There are 2 types of sterility testing methods: a. Membrane filtration method b. Direct inoculation method 1. METHOD A (MEMBRANE FILTRATION METHOD) Membrane filtration is preferred for: a. An oil b. An ointment that can be put into solution c. A non bacteriostatic solid not readily soluble in the culture medium d. A soluble powder or a liquid that possesses bacteriostatic or fungistatic properties  It is also used for the positive and negative controls. Medium Test microorganism Incubation Temp. (℃) Duration Type of microorganism Fluid thioglycollate Clostridium sporogenes Staphylococcus aureus Pseudomonas aeruginosa 30-35 30-35 30-35 3days 3days 3days Anaerobic Aerobic Aerobic Soyabean casein digest Asperigillus niger Candida albicans Bacillus subtilis 20-25 20-25 20-25 5days 5days 3days Aerobic Aerobic Aerobic Table no. 6: Strains of microorganisms used in the test Fig 8: Membrane filtration unit
22 TYPES OF FILTERS:  Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions .  Cellulose acetate filters are used for strongly alcoholic solutions. Diluting Fluids (IP, BP): Fluid A: Dissolve 1 g of bacteriological peptone in water to make 1liter, filter/centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20 minutes. Fluid B: If the test sample contains lecithin or oil, use fluid A to each liter, add1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20 minutes. Quantity of parenteral sample Minimum quantity to be used for each culture medium Less than 1ml Total contents of the container 1 ml or more but less than 40 ml Half the contents of the container 40 ml or more but less than 100 ml 20 ml 100 ml or more 10% of the contents of container but not less than 20 ml Antibiotic liquids 1ml Table no.7: Minimum quantity of sample to be used for each culture medium
23 APPARATUS: • Suitable unit consists of a closed reservoir and a receptacle between which a supported membrane of appropriate porosity is placed. • Membrane should has a nominal pore size < 0.45µm and diameter is 50mm(approx.) METHOD OF TEST: • For aqueous preparations, minimum quantity of sample (as mentioned in table no.7) is mixed with diluting fluid A. • Prepare each filter membrane by aseptically transferring a small quantity of fluid A onto the membrane(to moisten it) and filter it. • For each medium to be used, transfer aseptically into 2 separate membrane filter funnels. • Alternatively, transfer aseptically the combined quantities of the preparations under examination in 2 media onto one membrane. • Draw the liquid rapidly through the filter with the aid of vacuum. • If the solution under examination has antimicrobial properties, wash the membrane by filtering through it not less than 3 successive quantities, each of 100ml/filter. • After filtration, aseptically remove the membrane from the holder and transfer the whole membrane or cut it into 2 equal parts. • Transfer one half to each of the 2 suitable media. • Incubate the media not less than 14 days. Observe the containers and if the test specimen is positive before 14 days of incubation, further incubation is not required.
24 • For liquids immiscible with aqueous vehicles, and suspensions, again repeat the method for aqueous preparations but add a sufficient quantity of Fluid A to the pooled sample to achieve rapid filtration. • Sterilize enzyme preparations such as penicillinase or cellulose may be added to Fluid A to dissolve insoluble substances. • If the substance being examined contains lecithin, use fluid B for diluting. 2. METHOD B (DIRECT INOCULATION METHOD) Quantities of sample to be used: The quantity of the substance or preparation under examination to be used for inoculation in the culture media varies according to the quantity in each container which is mentioned in Table no.7. METHOD OF TEST: • For aqueous solutions and suspensions, remove the liquid from the test containers with a sterile pipette or needle or syringe. • Transfer the quantity of the preparation under examination directly into the culture medium so that the volume of preparation under examination is NMT 10% of the volume of medium. • If the preparation under examination has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium. • Incubate the inoculated media for not less than 14 days . Observe the containers and if the test specimen is positive before 14 days of incubation, further incubation is not required.
25 Table no.8 : Minimum number of items to be tested No. of items in a batch Minimum no. of items to be tested Not more than 100 containers 10%/ 4 items whichever is greater More than 100 but not more than 500 containers 10 items For more than 500 2%/ 20 items whichever is less For large volume parenterals 2%/ 10 items whichever is less Advantages of membrane filtration method over direct inoculation method are: 1. Entire contents can be tested which will increase the detection ability of the microorganisms. 2. Organisms present in an oleaginous product can be separated from the product during filtration . 3. It minimize the occurance of false results.
26 PYROGEN TEST  Pyrogen = fever producing organisms  The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination.  It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10ml/kg injected through i.v route within a period of NMT 10 minutes. Test Animals: • Healthy, adult rabbits of either sex, preferably of the same variety. • Weight NLT 1.5kg • Fed on a complete balanced diet • Rabbits should be kept under uniform temperature(± 2℃), with uniform humidity and free from disturbances. Note: 1.Don’t use animals for pyrogen tests more frequently than once every 48 hours. 2.After a pyrogen test, if the rabbit’s temperature has risen 0.6ﾟ or more, then atleast 2 weeks must be allowed to elapse before the animal is used again. Fig 9: Pyrogen testing on rabbit
27 Materials:  All glasswares, syringes and needles must be washed with water for injection and heated in hot air oven at 250ﾟfor 30 minutes or 200ﾟfor 1 hour.  Treat all the diluents and solutions for washing and rinsing of devices in a manner that will they become sterile.  The animal must put in the box 1 hour before the test and remain in them throughout the test.  The room temperature should be within 3ﾟ of that of rabbits living quarters.  Withhold food from the animals overnight and until the test is completed.  withhold water during the test. Recording of Temperature: • Thermometer or thermistor have been caliberated to assure an accuracy of 0.1℃. • Insert the thermometer into the rectum of the test rabbit to a depth of about 5 cm (according to USP- 7.5cm). • If any electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before the injection of the solution being examined and left in position throughout the test. • After a period of time, record the rabbit’s body temperature.
28  SHAM TEST /PRELIMINARY TEST/ RABBIT TEST(IN-VIVO) • If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1-3 days before testing the substance under examination by injecting intravenously into them 10ml/kg of body weight of a pyrogen- free saline solution warmed to about 38.5ﾟ. • Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 1,2,3 hours after injection of the solution being examined. • Any animal showing a temperature variation of 0.6ﾟ or more must not be used in the main test.  MAIN TEST: Carry out the test using a group of 3 rabbits. Preparation of the sample: Dissolve the substance with pyrogen-free saline solution. Warm the liquid under examination to approximately 38.5°C before injection. Method of test: • Record the temperature of each animal 90 minutes before the injection and continue for 3 hours after the injection for every 30 minutes. • Record the "initial temperature" of each rabbit which is the mean of 2 temperatures recorded after 30 minutes. • Rabbits showing a temperature variation greater than 0.2°C between two successive readings in the determination of "initial temperature" should not be used for the test. Do not use any rabbit having a temperature higher than 39.8°C and lower than 38°C.
29 • Inject the solution slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes. • The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. • The difference between the "initial temperature" and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken as its response. When this difference is negative, the result is counted as a zero response. Table no.9: Interpretation of results Pharmacopoeia No. of rabbits in a group Passes if the temp. is not more than Fails if temp. is more than IP 3 8 1.4 3.7 Each rabbit temp. raise should not be more than 0.6℃ BP 3 6 9 12 1.15 2.80 4.45 6.6 2.65 4.30 5.95 6.6 USP 3 8 - 3.3 Each rabbit temp. raise should not be more than 0.6℃
30 BACTERIAL ENDOTOXIN TEST(USP)  It measures the concentration of bacterial endotoxins that may be present in the sample or in the articles to which the test is applied using a lysate derived from amoebocytes of horseshoe crab, Limulus polyphemus.  Other species of horseshoe crab namely Tachypleus gigas, Trachypleus tridentatus.  LAL(Limulus Amoebocyte Lysate) test(IN-VITRO)  Amoebocyte- crab blood cell from which active component is derived.  Lysate – component obtained by separating amoebocyte from plasma and then lysing them. What is endotoxin? • Produced by gram negative bacteria • Outer covering – lipopolysaccharide • Thermostable and water soluble PRINCIPLE: When endotoxin solution is injected to horseshoe crab, the chemical component lysate is secreted by amoebocyte which reacts with the endotoxin and inactivate it. Inactivation of endotoxin by lysate results in formation of gel or turbidity or precipitation. Fig 10: Horseshoe crab
31 METHODS OF ENDOTOXIN TEST: 1. METHOD A: GEL- CLOT LIMIT TEST METHOD 2. METHOD B: SEMI-QUANTITATIVE GEL-CLOT METHOD 3. METHOD C: KINETIC TURBIDIMETRIC METHOD 4. METHOD D: KINETIC CHROMOGENIC METHOD 5. METHOD E: END-POINT CHROMOGENIC METHOD Fig 11: LAL reagent reaction cascade mechanism MECHANISM:  The cascade reaction starts by the presence of a bacterial endotoxin which reacts with lysate, and initially activates factor C i.e., serine protease precursor followed by activation of factor B i.e., serine protease precursor.  Activated Factor B further activates proclotting enzyme into clotting enzyme which hydrolyzes coagulogen into coagulin, forming an insoluble gel.  When (1 → 3)-β-D-glucan (a fungal cell wall component), reacts with lysate, activates factor G.  Activated factor G activates proclotting enzyme and futher forms a gel-clot.
32 METHOD A: GEL-CLOT LIMIT TEST METHOD (Qualitative method) • Prepare the solutions and dilutions with water BET. If necessary, adjust the pH of the solution to 6.0 to 8.0 using sterile 0.1M HCl BET, 0.1M NaOH BET of suitable buffer prepared with water BET. • Prepare the sample solution . Use two positive controls, one having the concentration of 2λ and other is spiked to get the concentration of 2λ.(where λ=0.125 EU/ml) • Add an appropriate volume of negative control (NC), standard CSE solutions in water BET, test solution and positive control(PPC). At regular intervals add an equal volume of the appropriately constituted lysate. Mix it and place it in an incubator. • Incubation should be done at 37º±1º undisturbed for 60±2 minutes. • Remove and examine the receptacles carefully. • A positive reaction is recorded in dilution 2λ when firm gel is formed that retains the integrity when inverted through 180º in one smooth motion. • If no firm gel is formed then it is a negative reaction. Fig 12: After tilting 180ﾟ, if clot retains then test positive and if not then negative CALCULATION: Geometric mean end-point concentration= Antilog(Ʃe/f) Where, Ʃe = sum of the log end-point concentrations of the series of dilutions used f= number of replicate test-tubes
33 METHOD B: SEMI-QUANTITATIVE GEL-CLOT METHOD Preparation of test solutions: Prepare test solutions at concentration of MVD, 0.5 MVD,0.25MVD (Maximum Valid Dilution). [MVD= Endotoxin limit(EU/mg) X concentration of the test solution(mg/ml)/λ] Method: Same as Method A Calculation and interpretation of results • To calculate the endotoxin concentration in the product, determine for the series of test solutions the lowest concentration or the highest dilution giving a positive (+) reaction. • Multiply this dilution with to obtain the endotoxin concentration of the product. • For example, if MVD is equal to 8 and the positive reaction was obtained at 0.25 MVD and λ was equal to 0.125EU/ml, then endotoxin concentration in the test will be, 8x 0.25x 0.125=0.25 EU/ml. • If none of the dilutions of the series gives a positive reaction, the endotoxin concentration will be less than the value obtained by multiplying the lowest diltution factor with λ. • If all the dilutions of the series give a positive reaction, the endotoxin concentration will be more than the value obtained by multiplying the highest dilution factor with λ.
34 METHOD C: KINETIC TURBIDIMETRIC METHOD • Quantitative method • Using CSE( Control Standard Endotoxin), prepare solutions of not less than 3 endotoxin concentrations to get a standard curve. • It is a photometric assay measuring the increase in turbidity caused by reaction of the endotoxin with the lysate. • It is a method measuring either the time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development. METHOD D: KINETIC CHROMOGENIC METHOD • Quantitative method • It is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. • It is a method measuring either the time needed to reach a predetermined absorbance of the reaction mixture or the rate of colour development. Preparation of test solutions: Solution A: Solution of the product under examination at the initial dilution (test solution) Solution B: Test solution spiked with CSE at a concentration at or near the middle of the standard curve (PPC) Solution C: Standard solutions of CSE in water BET covering the linear part of the standard curve Solution D: water BET (NC)
35 Method: Add solution D, followed by solutions C, A, B. Add lysate and carry out the assay . Interpretation of results: The assay is valid only if 1) The standard curve is linear for the range of CSE concentrations used 2) The coefficient of correlation r, is not greater than 0.980; 3) The mean % recovery of the added endotoxin in the positive product control is between 50% and 150%. METHOD E: END POINT CHROMOGENIC METHOD Add solution D, followed by solutions C, A, B. The chromogenic substrate and lysate are added to the solution and incubated for the recommended time. Stop the reaction and measure the absorbance at the specified wavelength in accordance with the instructions of the lysate manufacture. Interpretation of results: The assay is valid only if: 1) The standard curve is linear for the range of CSE concentrations used 2) The coefficient of correlation r, is not less than 0.980; 3) The mean % recovery of the added endotoxin in the positive product control is between 50% and 150%.
36 CLARITY TEST • Clarity is a relative term, its mean a clear solution having a high polish conveys to the observer that the product is of exceptional quality and purity. • Used to check the presence of particulate matter. • The preparations intended for parenteral use should be free from particulate matter and should be clear when inspected visually. • Particulate contamination of injections and infusions consists of extraneous, mobile undissolved particles, other than gas bubbles, unintentionally present in the solutions. • According to USP Pharmacopoeia, for sub-visible particles mainly two methods are employed: 1. Microscopic particle count method 2. Light obscuration particle count method • Visible particles are inspected visually by using clarity test apparatus.
37 For visible particles: Apparatus: viewing station comprising: -a matt black panel of appropriate size held in a vertical position -a non-glare white panel of appropriate size held in a vertical position next to the black panel -an adjustable lamp holder fitted with a suitable shaded, white- light source and with a suitable light diffuser(a viewing illuminator containing two 13W fluorescent tubes, each 525mm in length) For clear glass ampoules, the intensity of illumination is maintained between 2000lux to 3750 lux and higher for colored glass and plastic container. Method: • Remove the labels on the container, wash and dry the outside. • Gently swirl or invert the container ensuring that air bubbles are not introduced and observe for 5 seconds in front of the white panel. • Repeat in front of black panel. • Record the presence of any particles. • Repeat the procedure for further 19 containers. ACCEPTANCE CRITERIA: For liquid preparations, if 1 or more particles are found in more than 1 container, then test fails. Fig 13: clarity testing apparatus
38 For sub-visible particles: Two methods are specified, one involving the counting of particles viewed under the microscope and the other based on the count of particles causing light obscuration. Both methods are applied on small samples. Method 1: MICROSCOPIC PARTICLE COUNT TEST: This method is suitable for revealing the presence of particles the longest axis or effective linear dimension of which is 10m or more.  Mainly used for large volume parenterals. Apparatus: Binocular microscope, filter assembly, membrane filter for retention of particles. Method: • Invert the container of the preparation 20 times in order to mix the contents. • For large volume parenterals, single units should be tested. • For small volume parenterals less than 25 ml in volume, the contents of 10 or more units should be combined in a clean container where the volume of liquid in a container is very small, the test solution may be prepared by mixing the contents of a suitable number of containers and diluting to 25 ml with particle free water. • Small-volume parenterals having a volume of 25ml or more may be tested individually. • Fit the membrane filter on to the membrane filter holder. Transfer the pooled solution or of a single unit, and allowed to stand for a minute and then apply vacuum and filter. • Direct the jet of particle-free water in such a manner as to wash the walls of the funnel. • Remove the membrane filter and allowed to dry in air with a cover on the petri dish. After drying, place the petri dish on the stage of microscope and scan under reflected light.
39 Sample Particle size(µm) equal to or greater than Maximum number of particles Containers with nominal content of more than 100ml(LVP) 10 25 Average in the units tested 12per ml 2 per ml Containers with nominal content of 100ml 10 25 3000 per container 300 per container Less than 100ml(SVP) 10 25 3000 per container 300 per container Table no.10: Limits of microscopic particle count test METHOD 2: LIGHT OBSCURATION PARTICLE COUNT TEST  Not suitable for reduced clarity or increased viscosity such as emulsion, colloids, liposomal preparations.  Mainly used for small volume parenterals. Apparatus: A suitable particle counter based on the principle of light blockage and capable of automatic counting and sizing of particles. Fig 14: microscopic particle count test apparatus
40 METHOD: • Invert the container of the examination 20 times in order to mix the contents. • Wash the outer surface of the container with a jet of particle-free water and remove the closure carefully, without any contamination. • Eliminate the gas bubbles by sonicating. • For LVP, single units should be tested. • For SVP less than 25 ml, the contents of 1o or more units should be combined in a clean container. • SVP having volume more than 25ml may be tested individually. • Count the number of particles equal to or greater than 10µm and 25µm. Calculate the average number of particles. Fig 15: Diagram of light obscuration particle count test
41 Sample Particle size(µm) equal to or greater than Maximum number of particles Containers with nominal content of more than 100ml(LVP) 10 25 Average in the units tested 25per ml 3 per ml Containers with nominal content of 100ml 10 25 6000 per container 600 per container Less than 100ml(SVP) 10 25 6000 per container 600 per container Table no. 11: Limits for light obscuration particle count test
42 EXTRACTABLE VOLUME(USP) • Deliverable volume • To ensure that deliver the volume of dosage form that is mentioned on the label claim. Single dose containers :- METHOD 1: (Where the nominal volume does not exceed 5ml) • Use 6 containers, 5 for the tests and 1 for rinsing the syringe used. • Using a syringe with appropriate capacity, rinse the syringe and withdraw as much as possible the contents of one of the containers reserved for the test and transfer, without emptying the needle, to a dry graduated cylinder of such capacity that the total combined volume to be measured occupies not less than 40% of the nominal volume of the cylinder. • Repeat the procedure until the contents of the 5 containers have been transferred and measure the volume. The average content of the 5 containers is not less than the nominal volume and not more than 115% of the nominal volume. • Alternatively the volume of contents in milliliter can be calculated as mass in grams divided by the density.
43 Volume of the solution Number of containers to be used for the test ≥10ml 1 3-10ml 3 ≤10ml 5 Table no. 12: limits for extractable volume METHOD 2: (Where the nominal volume is more than 5ml) • Transfer the contents of not less than 3 containers separately to dry graduated cylinders such that the volume to be measured occupies not less than 40% of the nominal volume of the cylinder and measure the volume transferred. • The contents of each container are not less than the nominal volume and not more than 110% of the nominal volume.
44 THANK YOU

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IPQC tests for Parenterals

  • 1.
    IN-PROCESS AND FINISHED PRODUCT QUALITYCONTROL TESTS FOR PARENTERALS Prapti Pattanayak Pharmaceutical Analysis M.Pharm 1st Year 1
  • 2.
    CONTENTS 1. Introduction 2. Definition 3.Types Of Parenterals 4. Routes Of Administration 5. Significance Of IPQC Tests 6. IPQC Tests According To IP, BP And USP 2
  • 3.
    INTRODUCTION • Derived fromGreek words “Para” means “outside” and “enteron” means “intestine”. • Route of administration- mostly intravenous, intramuscular, subcutaneous, intradermal . • Sterile solution or suspension of drug in aqueous or oily vehicle. • Directly administered directly under the skin, veins, muscles or more specialized tissues like spinal cord. • Drug delivery doesn’t utilize alimentary canal for entering into the tissues. 3 Fig 1: Injections, vial, ampoule
  • 4.
    DEFINITION • According toIP Preparations that are sterile and intended for administration by injection, infusion or implantation. • According to USP Preparations that includes injections and implanted products that are injected through the skin or other external boundary tissue or implanted within the body to allow direct administration. • According to BP Sterile products that are intended for administration by injection, infusion or implantation into the body. 4
  • 5.
    TYPES OF PARENTERALS 1.Small Volume Parenterals (SVPs)  They are given as multiple dose.  Volume- <100ml  Types: Ampoules, vials, dry powders etc. a. Ampoules- These are sealed glass container with an elongated neck that must be broken off. A 5 micron filter needle should be used when drawing the contents of an ampoule into a syringe since glass particles may have fallen inside the ampoule when the top was snapped off. b. Vials- It is made of glass or plastic and sealed with a rubber stopper. Drugs and other additives are packaged as liquid or lyophilized powders. It is designed for both single and multiple dose use. Multiple dose vials contain a preservative to inhibit bacterial contamination. 5 Fig 2: Ampoules Fig 3: Vial
  • 6.
    6 c. Dry Powders-These are lyophilized or freeze dried powders that must be reconstituted with some suitable solvent to make a liquid formulation before being withdrawn from the vial. Solvents may be WFI, BWFI, NaCl injection etc. 2. Large Volume Parenterals (LVPs) They are designed to provide fluid (water), calories (dextrose solution) and electrolytes ( saline solution). Volume- > 100ml  4 Types: a. Hyperalimentation solution/ Total Parenteral Nutrition- administration of large amount of nutrients to patients who are unable to take food orally. • Use- administration of life saving drugs to the comatose patients Fig 4: LVPs
  • 7.
    7 b. Cardioplegic Solution-it is used for heart surgery to prevent injury to myocardium during reperfusion. • Maintains diastolic arrest. c. Peritoneal Dialysis Solution- It is infused continuously into the abdominal cavity, bathing peritoneum and are then continuously withdrawn. Use- To minimize reperfusion injury resulting from tissue edema. Formulations- Glucose, Antibiotics as prophylactic . d. Irrigating Solution- It is used to irrigate, flush and aid in cleansing body cavities and wounds. Formulation- Normal saline Use- For the treatment of serious wounds infused into blood stream.
  • 8.
    ROUTE OF ADMINISTRATION •Angle- 15ﾟ • Dose- 0.1ml • Ex. BCG vaccineINTRADERMAL • Angle- 25ﾟ • Dose- >1litre • Ex. DNS (Dextrose Normal Saline) INTRAVENOUS • Angle- 45ﾟ • Dose- 1-1.3ml • Ex. Insulin SUBCUTANEOUS • Angle- 90ﾟ • Dose- 2-5ml • Ex. Atropine, Haloperidol INTRAMUSCULAR 8
  • 9.
    SIGNIFICANCE OF IPQC(InProcess Quality Control) • We can remove error from every stage in production and maintain the quality of the final product with the compendial standards as specified in the pharmacopoeia’s. • We can determine the number and types of microorganisms present in the product by sterility testing. • We can ensure the exact quantity of the solution mentioned on the label claim. • We can prevent the contamination of the drug caused due to poor package integrity. • Identification of any crack or leakage present on the container which may hamper the package integrity. 9
  • 10.
  • 11.
    CONTENT UNIFORMITY • ACCORDINGTO IP(Method) Weight of 10 individual sterile units are noted Content is removed from them and empty individual sterile unit is weighed accurately. Net weight is calculated by substracting empty sterile unit weight from gross weight. 11
  • 12.
    Acceptance Criteria (IP) DESCRIPTIONRANGES INFERENCE If all individual values are within the range of 85-115% of avg. values and RSD ≤6% Passes If more than 1 individual values OR If any one individual value >85-115% of the avg. value OR >75-125% of the avg. value Fails If one individual value is >85-115% but within the limits 75- 125% of the avg. value RSD >6% Repeat the test using additional 20 containers If in 30 containers, NMT 1 individual value OR None of the units Not exceeds 85-115% of avg. value OR Exceeds 75-125% of avg. value RSD NMT 7.8% Passes 12 Table no. 1
  • 13.
    According to BP •Individual contents of the 10 dosage units are taken randomly. TEST DESCRIPTION RANGES INFERENCE TEST A (tab, powders for parenterals administration, opthalmic inserts, suspensions for injection) 1.If each individual contents is 2.If 1 or more than 1 individual content is 3.If 1 individual content 4. If in 30 units, NMT 1 individual b/w 85-115% of avg. contents >75-125% of the avg. value Exceeds 85-115% but within 75-125% limits Exceeds 85-115% of the avg. value and none exceeds 75-125% avg. value Passes Fails Another 20 dosage forms taken randomly Passes TEST B (cap, powders other than for parenterals administration, granules, suppositories, pessaries) 1.If NMT 1 individual content Exceeds 85-115% and none exceeds 75-125% avg. contents Passes 13 Table no. 2
  • 14.
    Continue.. 2. If morethan 3 individual contents OR If 1 or more individual content 3. If 2 or 3 individual contents 4. If in 30 individual units, NMT 3 individual contents Exceeds 85-115%avg. content OR Exceeds 75-125% avg. content Exceeds 85-115% avg. value but within 75- 125% Exceeds 85-115% and none exceeds 75-125% of avg. contents Fails Randomly 20 dosages forms are added Passes TEST C (Transdermal patches) 1. If avg. content of 10 dosages units is 2. If the individual content of each dosage unit is b/w 90-110% of the content b/w 75-125% of avg. content Passes 14
  • 15.
    According to USP •10 dosage units are taken randomly. DESCRIPTION RANGES INFERENCE If 10 individual units Within 85-115% avg. content and RSD <6% Passes If more than 1 individual value OR If any one individual value Exceeds 85-115% avg. content OR Exceeds 75-125% avg. content Fails Fails If one individual Exceeds 85-115% but with 75-125% avg. content Repeat test with another 20 units randomly If in 30 containers, NMT 1 individual value Exceeds 85-115% and none exceeds 75-125% avg. content value Passes 15 Table no.3
  • 16.
    LEAKAGE TEST 16  Totest package integrity.  Leakage test is employed to detect incompletely sealed ampoule and vials so that they may be discarded. WHAT IS THE NEED ? • Presence of capillary pores or tiny cracks can cause microbes or other contaminants to enter in the ampoules. • Change in temperature during storage can cause expansion and contraction of the ampoule and thereby causing interchange of its contents if any opening exists. • Leakage test is done by visual inspection, bubble test and dye bath test . A. METHOD ( DYE BATH TEST) TEST CONTAINER IMMERSED IN A DYE BATH UNDER VACUUM AND PRESSURE REMOVED THE CONTAINER FROM DYE BATH AND WASHED CONTAINER IS INSPECTED FOR THE PRESENCE OF DYE EITHER VISUALLY OR BY UV SPECTROSCOPY DYES USED : Mainly methylene blue is used. Other than that green, yellowish-green color are also used. OPTIMIZATION OF DYE TEST: • Addition of surfactant or a low viscosity fluid in the dye. • These will increase the capillary migration of the pores. ADVANTAGES: • Inexpensive • No special equipment required for identification • Only for Qualitative analysis DISADVANTAGES: • Destructive and slow test • Not for Quantitative analysis
  • 17.
    17 B. VISUAL INSPECTION •It is the easiest and least sensitive leakage test method . • Used for evaluation of large volume parenterals. • To increase the sensitivity of the method, the visual inspection of the sample container may be coupled with the application of vacuum to make leakage more readily observable. • It is a very simple , inexpensive and operator dependent method. C. BUBBLE TEST • The test package is submerged in liquids. • A differential pressure is applied on the container. • The container is observed for bubbles. • For optimization of leakage test, surfactant(ex. polysorbate 80) added to the liquid along with dark background and high intensity lighting. • Differential positive pressure of 3 psi inside the vial is generated and observation of any leakage using magnifying glass within a maximum test time of 15 minutes. • It can detect leaks as small as 10 mbar-L/sec. • It is also insensitive, operator dependent and qualitative test. Fig 6: Visual Inspection
  • 18.
    18 STERILITY TEST  Sterilemeans free from microorganisms like bacteria, fungi, yeast etc. PRINCIPLE: If the bacteria and fungi are placed in a medium which provides nutrition to the organisms and kept at favorable temperature, the organisms will grow and their presence can be indicated by turbidity in the clear medium. WHAT IS THE NEED? • To detect the presence of viable organisms in the parenterals. • Presence of microbes may contaminate the sterile products.  For sterility testing, aseptic conditions should be maintained. MAJOR FACTORS AFFECTING STERILITY TESTING • The environment in which test is conducted • The quality of culture • Test method • Test procedure • Sample size Fig 7: Sterility testing
  • 19.
    19 CULTURE MEDIA USED: Thereare basically 2 types of culture media: a. Fluid thioglycollate medium b. Soyabean casein digest medium 1. FLUID THIOGLYCOLATE MEDIUM (FTM)  FTM is an excellent medium for the detection of microbial contamination.  It can provide both aerobic and anaerobic environment. COMPOSITIONS OF FTM: a. L- cysteine- it supply carbon and nitrogenous compounds. b. Dextrose- acts as an energy source. c. Yeast extract- act as growth enhancer. d. Sodium chloride- it maintains osmotic equilibrium. e. Sodium thioglycollate- acts as reducing agent which maintains a low oxygen tension by removing molecular oxygen from the environment i.e., it creates anaerobic conditions when it reduces molecular oxygen to water. f. Resazurin- it is a redox indication that turns pink when oxidation increased and colorless when reduction occurred. g. Agar- addition of small amount of agar in medium initiate the growth of small inocula and anaerobes. h. Purified water i. Adjusted to pH 7.1± 0.2  Incubation of the media – 14 days at 30- 35℃ Components Quantity L-Cysteine 0.5g Sodium chloride 2.5g Dextrose monohydrate 5.5g Yeast extract 5.0g Pancreatic digest of casein 15.0g Sodium thioglycollate/thioglycollic acid 0.5g/0.3g Resazurin sodium solution 1.0ml Distilled water q.s to 1000ml pH of medium after sterilization 7.1± 0.2 Table no. 4
  • 20.
    20 B. SOYABEAN CASEINDIGEST MEDIUM  It is used for culture of both fungi and aerobic bacteria. COMPOSITIONS: a.Trypticase soya broth- it is used to grow aerobic bacteria b.Trypticase peptone- it is an animal origin pancreatic digest of casein which provides nutrition. c. Sodium chloride- it maintains osmotic equilibrium d. Dipotassium phosphate- it provides buffering capacity e. Dextrose- provides energy f. Purified water g. Adjusted to pH 7.3± 0.2  Incubation period- 14 days at 20- 25℃ Components Quantity Pancreatic digest of casein 17.0g Papaic digest of soyabean meal 3.0g Sodium chloride 5.0g Dipotassium hydrogen phosphate 2.5g Dextrose monohydrate 2.5g Distilled water q.s. to 1000ml pH of medium after sterilization 7.3± 0.2 Table no. 5
  • 21.
    21 STERILITY TESTING METHODS: Thereare 2 types of sterility testing methods: a. Membrane filtration method b. Direct inoculation method 1. METHOD A (MEMBRANE FILTRATION METHOD) Membrane filtration is preferred for: a. An oil b. An ointment that can be put into solution c. A non bacteriostatic solid not readily soluble in the culture medium d. A soluble powder or a liquid that possesses bacteriostatic or fungistatic properties  It is also used for the positive and negative controls. Medium Test microorganism Incubation Temp. (℃) Duration Type of microorganism Fluid thioglycollate Clostridium sporogenes Staphylococcus aureus Pseudomonas aeruginosa 30-35 30-35 30-35 3days 3days 3days Anaerobic Aerobic Aerobic Soyabean casein digest Asperigillus niger Candida albicans Bacillus subtilis 20-25 20-25 20-25 5days 5days 3days Aerobic Aerobic Aerobic Table no. 6: Strains of microorganisms used in the test Fig 8: Membrane filtration unit
  • 22.
    22 TYPES OF FILTERS: Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions .  Cellulose acetate filters are used for strongly alcoholic solutions. Diluting Fluids (IP, BP): Fluid A: Dissolve 1 g of bacteriological peptone in water to make 1liter, filter/centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20 minutes. Fluid B: If the test sample contains lecithin or oil, use fluid A to each liter, add1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20 minutes. Quantity of parenteral sample Minimum quantity to be used for each culture medium Less than 1ml Total contents of the container 1 ml or more but less than 40 ml Half the contents of the container 40 ml or more but less than 100 ml 20 ml 100 ml or more 10% of the contents of container but not less than 20 ml Antibiotic liquids 1ml Table no.7: Minimum quantity of sample to be used for each culture medium
  • 23.
    23 APPARATUS: • Suitable unitconsists of a closed reservoir and a receptacle between which a supported membrane of appropriate porosity is placed. • Membrane should has a nominal pore size < 0.45µm and diameter is 50mm(approx.) METHOD OF TEST: • For aqueous preparations, minimum quantity of sample (as mentioned in table no.7) is mixed with diluting fluid A. • Prepare each filter membrane by aseptically transferring a small quantity of fluid A onto the membrane(to moisten it) and filter it. • For each medium to be used, transfer aseptically into 2 separate membrane filter funnels. • Alternatively, transfer aseptically the combined quantities of the preparations under examination in 2 media onto one membrane. • Draw the liquid rapidly through the filter with the aid of vacuum. • If the solution under examination has antimicrobial properties, wash the membrane by filtering through it not less than 3 successive quantities, each of 100ml/filter. • After filtration, aseptically remove the membrane from the holder and transfer the whole membrane or cut it into 2 equal parts. • Transfer one half to each of the 2 suitable media. • Incubate the media not less than 14 days. Observe the containers and if the test specimen is positive before 14 days of incubation, further incubation is not required.
  • 24.
    24 • For liquidsimmiscible with aqueous vehicles, and suspensions, again repeat the method for aqueous preparations but add a sufficient quantity of Fluid A to the pooled sample to achieve rapid filtration. • Sterilize enzyme preparations such as penicillinase or cellulose may be added to Fluid A to dissolve insoluble substances. • If the substance being examined contains lecithin, use fluid B for diluting. 2. METHOD B (DIRECT INOCULATION METHOD) Quantities of sample to be used: The quantity of the substance or preparation under examination to be used for inoculation in the culture media varies according to the quantity in each container which is mentioned in Table no.7. METHOD OF TEST: • For aqueous solutions and suspensions, remove the liquid from the test containers with a sterile pipette or needle or syringe. • Transfer the quantity of the preparation under examination directly into the culture medium so that the volume of preparation under examination is NMT 10% of the volume of medium. • If the preparation under examination has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium. • Incubate the inoculated media for not less than 14 days . Observe the containers and if the test specimen is positive before 14 days of incubation, further incubation is not required.
  • 25.
    25 Table no.8 :Minimum number of items to be tested No. of items in a batch Minimum no. of items to be tested Not more than 100 containers 10%/ 4 items whichever is greater More than 100 but not more than 500 containers 10 items For more than 500 2%/ 20 items whichever is less For large volume parenterals 2%/ 10 items whichever is less Advantages of membrane filtration method over direct inoculation method are: 1. Entire contents can be tested which will increase the detection ability of the microorganisms. 2. Organisms present in an oleaginous product can be separated from the product during filtration . 3. It minimize the occurance of false results.
  • 26.
    26 PYROGEN TEST  Pyrogen= fever producing organisms  The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination.  It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10ml/kg injected through i.v route within a period of NMT 10 minutes. Test Animals: • Healthy, adult rabbits of either sex, preferably of the same variety. • Weight NLT 1.5kg • Fed on a complete balanced diet • Rabbits should be kept under uniform temperature(± 2℃), with uniform humidity and free from disturbances. Note: 1.Don’t use animals for pyrogen tests more frequently than once every 48 hours. 2.After a pyrogen test, if the rabbit’s temperature has risen 0.6ﾟ or more, then atleast 2 weeks must be allowed to elapse before the animal is used again. Fig 9: Pyrogen testing on rabbit
  • 27.
    27 Materials:  All glasswares,syringes and needles must be washed with water for injection and heated in hot air oven at 250ﾟfor 30 minutes or 200ﾟfor 1 hour.  Treat all the diluents and solutions for washing and rinsing of devices in a manner that will they become sterile.  The animal must put in the box 1 hour before the test and remain in them throughout the test.  The room temperature should be within 3ﾟ of that of rabbits living quarters.  Withhold food from the animals overnight and until the test is completed.  withhold water during the test. Recording of Temperature: • Thermometer or thermistor have been caliberated to assure an accuracy of 0.1℃. • Insert the thermometer into the rectum of the test rabbit to a depth of about 5 cm (according to USP- 7.5cm). • If any electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before the injection of the solution being examined and left in position throughout the test. • After a period of time, record the rabbit’s body temperature.
  • 28.
    28  SHAM TEST/PRELIMINARY TEST/ RABBIT TEST(IN-VIVO) • If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1-3 days before testing the substance under examination by injecting intravenously into them 10ml/kg of body weight of a pyrogen- free saline solution warmed to about 38.5ﾟ. • Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 1,2,3 hours after injection of the solution being examined. • Any animal showing a temperature variation of 0.6ﾟ or more must not be used in the main test.  MAIN TEST: Carry out the test using a group of 3 rabbits. Preparation of the sample: Dissolve the substance with pyrogen-free saline solution. Warm the liquid under examination to approximately 38.5°C before injection. Method of test: • Record the temperature of each animal 90 minutes before the injection and continue for 3 hours after the injection for every 30 minutes. • Record the "initial temperature" of each rabbit which is the mean of 2 temperatures recorded after 30 minutes. • Rabbits showing a temperature variation greater than 0.2°C between two successive readings in the determination of "initial temperature" should not be used for the test. Do not use any rabbit having a temperature higher than 39.8°C and lower than 38°C.
  • 29.
    29 • Inject thesolution slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes. • The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. • The difference between the "initial temperature" and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken as its response. When this difference is negative, the result is counted as a zero response. Table no.9: Interpretation of results Pharmacopoeia No. of rabbits in a group Passes if the temp. is not more than Fails if temp. is more than IP 3 8 1.4 3.7 Each rabbit temp. raise should not be more than 0.6℃ BP 3 6 9 12 1.15 2.80 4.45 6.6 2.65 4.30 5.95 6.6 USP 3 8 - 3.3 Each rabbit temp. raise should not be more than 0.6℃
  • 30.
    30 BACTERIAL ENDOTOXIN TEST(USP) It measures the concentration of bacterial endotoxins that may be present in the sample or in the articles to which the test is applied using a lysate derived from amoebocytes of horseshoe crab, Limulus polyphemus.  Other species of horseshoe crab namely Tachypleus gigas, Trachypleus tridentatus.  LAL(Limulus Amoebocyte Lysate) test(IN-VITRO)  Amoebocyte- crab blood cell from which active component is derived.  Lysate – component obtained by separating amoebocyte from plasma and then lysing them. What is endotoxin? • Produced by gram negative bacteria • Outer covering – lipopolysaccharide • Thermostable and water soluble PRINCIPLE: When endotoxin solution is injected to horseshoe crab, the chemical component lysate is secreted by amoebocyte which reacts with the endotoxin and inactivate it. Inactivation of endotoxin by lysate results in formation of gel or turbidity or precipitation. Fig 10: Horseshoe crab
  • 31.
    31 METHODS OF ENDOTOXINTEST: 1. METHOD A: GEL- CLOT LIMIT TEST METHOD 2. METHOD B: SEMI-QUANTITATIVE GEL-CLOT METHOD 3. METHOD C: KINETIC TURBIDIMETRIC METHOD 4. METHOD D: KINETIC CHROMOGENIC METHOD 5. METHOD E: END-POINT CHROMOGENIC METHOD Fig 11: LAL reagent reaction cascade mechanism MECHANISM:  The cascade reaction starts by the presence of a bacterial endotoxin which reacts with lysate, and initially activates factor C i.e., serine protease precursor followed by activation of factor B i.e., serine protease precursor.  Activated Factor B further activates proclotting enzyme into clotting enzyme which hydrolyzes coagulogen into coagulin, forming an insoluble gel.  When (1 → 3)-β-D-glucan (a fungal cell wall component), reacts with lysate, activates factor G.  Activated factor G activates proclotting enzyme and futher forms a gel-clot.
  • 32.
    32 METHOD A: GEL-CLOTLIMIT TEST METHOD (Qualitative method) • Prepare the solutions and dilutions with water BET. If necessary, adjust the pH of the solution to 6.0 to 8.0 using sterile 0.1M HCl BET, 0.1M NaOH BET of suitable buffer prepared with water BET. • Prepare the sample solution . Use two positive controls, one having the concentration of 2λ and other is spiked to get the concentration of 2λ.(where λ=0.125 EU/ml) • Add an appropriate volume of negative control (NC), standard CSE solutions in water BET, test solution and positive control(PPC). At regular intervals add an equal volume of the appropriately constituted lysate. Mix it and place it in an incubator. • Incubation should be done at 37º±1º undisturbed for 60±2 minutes. • Remove and examine the receptacles carefully. • A positive reaction is recorded in dilution 2λ when firm gel is formed that retains the integrity when inverted through 180º in one smooth motion. • If no firm gel is formed then it is a negative reaction. Fig 12: After tilting 180ﾟ, if clot retains then test positive and if not then negative CALCULATION: Geometric mean end-point concentration= Antilog(Ʃe/f) Where, Ʃe = sum of the log end-point concentrations of the series of dilutions used f= number of replicate test-tubes
  • 33.
    33 METHOD B: SEMI-QUANTITATIVEGEL-CLOT METHOD Preparation of test solutions: Prepare test solutions at concentration of MVD, 0.5 MVD,0.25MVD (Maximum Valid Dilution). [MVD= Endotoxin limit(EU/mg) X concentration of the test solution(mg/ml)/λ] Method: Same as Method A Calculation and interpretation of results • To calculate the endotoxin concentration in the product, determine for the series of test solutions the lowest concentration or the highest dilution giving a positive (+) reaction. • Multiply this dilution with to obtain the endotoxin concentration of the product. • For example, if MVD is equal to 8 and the positive reaction was obtained at 0.25 MVD and λ was equal to 0.125EU/ml, then endotoxin concentration in the test will be, 8x 0.25x 0.125=0.25 EU/ml. • If none of the dilutions of the series gives a positive reaction, the endotoxin concentration will be less than the value obtained by multiplying the lowest diltution factor with λ. • If all the dilutions of the series give a positive reaction, the endotoxin concentration will be more than the value obtained by multiplying the highest dilution factor with λ.
  • 34.
    34 METHOD C: KINETICTURBIDIMETRIC METHOD • Quantitative method • Using CSE( Control Standard Endotoxin), prepare solutions of not less than 3 endotoxin concentrations to get a standard curve. • It is a photometric assay measuring the increase in turbidity caused by reaction of the endotoxin with the lysate. • It is a method measuring either the time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development. METHOD D: KINETIC CHROMOGENIC METHOD • Quantitative method • It is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. • It is a method measuring either the time needed to reach a predetermined absorbance of the reaction mixture or the rate of colour development. Preparation of test solutions: Solution A: Solution of the product under examination at the initial dilution (test solution) Solution B: Test solution spiked with CSE at a concentration at or near the middle of the standard curve (PPC) Solution C: Standard solutions of CSE in water BET covering the linear part of the standard curve Solution D: water BET (NC)
  • 35.
    35 Method: Add solution D,followed by solutions C, A, B. Add lysate and carry out the assay . Interpretation of results: The assay is valid only if 1) The standard curve is linear for the range of CSE concentrations used 2) The coefficient of correlation r, is not greater than 0.980; 3) The mean % recovery of the added endotoxin in the positive product control is between 50% and 150%. METHOD E: END POINT CHROMOGENIC METHOD Add solution D, followed by solutions C, A, B. The chromogenic substrate and lysate are added to the solution and incubated for the recommended time. Stop the reaction and measure the absorbance at the specified wavelength in accordance with the instructions of the lysate manufacture. Interpretation of results: The assay is valid only if: 1) The standard curve is linear for the range of CSE concentrations used 2) The coefficient of correlation r, is not less than 0.980; 3) The mean % recovery of the added endotoxin in the positive product control is between 50% and 150%.
  • 36.
    36 CLARITY TEST • Clarityis a relative term, its mean a clear solution having a high polish conveys to the observer that the product is of exceptional quality and purity. • Used to check the presence of particulate matter. • The preparations intended for parenteral use should be free from particulate matter and should be clear when inspected visually. • Particulate contamination of injections and infusions consists of extraneous, mobile undissolved particles, other than gas bubbles, unintentionally present in the solutions. • According to USP Pharmacopoeia, for sub-visible particles mainly two methods are employed: 1. Microscopic particle count method 2. Light obscuration particle count method • Visible particles are inspected visually by using clarity test apparatus.
  • 37.
    37 For visible particles: Apparatus:viewing station comprising: -a matt black panel of appropriate size held in a vertical position -a non-glare white panel of appropriate size held in a vertical position next to the black panel -an adjustable lamp holder fitted with a suitable shaded, white- light source and with a suitable light diffuser(a viewing illuminator containing two 13W fluorescent tubes, each 525mm in length) For clear glass ampoules, the intensity of illumination is maintained between 2000lux to 3750 lux and higher for colored glass and plastic container. Method: • Remove the labels on the container, wash and dry the outside. • Gently swirl or invert the container ensuring that air bubbles are not introduced and observe for 5 seconds in front of the white panel. • Repeat in front of black panel. • Record the presence of any particles. • Repeat the procedure for further 19 containers. ACCEPTANCE CRITERIA: For liquid preparations, if 1 or more particles are found in more than 1 container, then test fails. Fig 13: clarity testing apparatus
  • 38.
    38 For sub-visible particles:Two methods are specified, one involving the counting of particles viewed under the microscope and the other based on the count of particles causing light obscuration. Both methods are applied on small samples. Method 1: MICROSCOPIC PARTICLE COUNT TEST: This method is suitable for revealing the presence of particles the longest axis or effective linear dimension of which is 10m or more.  Mainly used for large volume parenterals. Apparatus: Binocular microscope, filter assembly, membrane filter for retention of particles. Method: • Invert the container of the preparation 20 times in order to mix the contents. • For large volume parenterals, single units should be tested. • For small volume parenterals less than 25 ml in volume, the contents of 10 or more units should be combined in a clean container where the volume of liquid in a container is very small, the test solution may be prepared by mixing the contents of a suitable number of containers and diluting to 25 ml with particle free water. • Small-volume parenterals having a volume of 25ml or more may be tested individually. • Fit the membrane filter on to the membrane filter holder. Transfer the pooled solution or of a single unit, and allowed to stand for a minute and then apply vacuum and filter. • Direct the jet of particle-free water in such a manner as to wash the walls of the funnel. • Remove the membrane filter and allowed to dry in air with a cover on the petri dish. After drying, place the petri dish on the stage of microscope and scan under reflected light.
  • 39.
    39 Sample Particle size(µm) equalto or greater than Maximum number of particles Containers with nominal content of more than 100ml(LVP) 10 25 Average in the units tested 12per ml 2 per ml Containers with nominal content of 100ml 10 25 3000 per container 300 per container Less than 100ml(SVP) 10 25 3000 per container 300 per container Table no.10: Limits of microscopic particle count test METHOD 2: LIGHT OBSCURATION PARTICLE COUNT TEST  Not suitable for reduced clarity or increased viscosity such as emulsion, colloids, liposomal preparations.  Mainly used for small volume parenterals. Apparatus: A suitable particle counter based on the principle of light blockage and capable of automatic counting and sizing of particles. Fig 14: microscopic particle count test apparatus
  • 40.
    40 METHOD: • Invert thecontainer of the examination 20 times in order to mix the contents. • Wash the outer surface of the container with a jet of particle-free water and remove the closure carefully, without any contamination. • Eliminate the gas bubbles by sonicating. • For LVP, single units should be tested. • For SVP less than 25 ml, the contents of 1o or more units should be combined in a clean container. • SVP having volume more than 25ml may be tested individually. • Count the number of particles equal to or greater than 10µm and 25µm. Calculate the average number of particles. Fig 15: Diagram of light obscuration particle count test
  • 41.
    41 Sample Particle size(µm) equalto or greater than Maximum number of particles Containers with nominal content of more than 100ml(LVP) 10 25 Average in the units tested 25per ml 3 per ml Containers with nominal content of 100ml 10 25 6000 per container 600 per container Less than 100ml(SVP) 10 25 6000 per container 600 per container Table no. 11: Limits for light obscuration particle count test
  • 42.
    42 EXTRACTABLE VOLUME(USP) • Deliverablevolume • To ensure that deliver the volume of dosage form that is mentioned on the label claim. Single dose containers :- METHOD 1: (Where the nominal volume does not exceed 5ml) • Use 6 containers, 5 for the tests and 1 for rinsing the syringe used. • Using a syringe with appropriate capacity, rinse the syringe and withdraw as much as possible the contents of one of the containers reserved for the test and transfer, without emptying the needle, to a dry graduated cylinder of such capacity that the total combined volume to be measured occupies not less than 40% of the nominal volume of the cylinder. • Repeat the procedure until the contents of the 5 containers have been transferred and measure the volume. The average content of the 5 containers is not less than the nominal volume and not more than 115% of the nominal volume. • Alternatively the volume of contents in milliliter can be calculated as mass in grams divided by the density.
  • 43.
    43 Volume of thesolution Number of containers to be used for the test ≥10ml 1 3-10ml 3 ≤10ml 5 Table no. 12: limits for extractable volume METHOD 2: (Where the nominal volume is more than 5ml) • Transfer the contents of not less than 3 containers separately to dry graduated cylinders such that the volume to be measured occupies not less than 40% of the nominal volume of the cylinder and measure the volume transferred. • The contents of each container are not less than the nominal volume and not more than 110% of the nominal volume.
  • 44.