analysis of injectable dosage forms

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ANALYSIS OF INJECTABLE DOSAGE FORMS Guided by : Dr. Rina H. Gokani (Associate professor) ( M.Pharm , Ph.D ) 1 Prepared by: Ladva Bhakti J. ( M.Pharm sem II) S.J. Thakkar Pharmacy college, Opp.Drive in cinema,Avadh Road,Opp N.R.I. Bunglow , Kalawad Road,Rajkot.360005 .


2 INTRODUCTION Parenteral preparations are sterile products intended for administration by injection, infusion or implantation into the body. They may be preparations intended for direct parenteral administration or they may be parenteral products for constituting or diluting prior to administration . Sterile drug product, which is presented in the form of solution, suspension, emulsion, or reconstituted lyophilized powder.

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. 3 Rapid absorption and distribution, high bioavailability Zero enzymatic degradation in the gastrointestinal tract An ability to be administered to unconscious patient Advantages Major drawback of parenteral delivery is the pain and discomfort associated with needle injection Administration by injection, subcutaneous, intramuscular, and intravenous ,intrathecal, intraarterial, intraspinal,and intradermal routes to achieve local or systemic effects.

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4 Small-volume parenteral (SVP) Large-volume parenteral (LVP) Lyophilized products. CLASSIFICATION

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Why quality assurance is needed for parenteral? H igher propensity for improper administration of injectable products Administered directly into the human bloodstream R isker than oral solid dosage C ompromised immune systems, among other things. 5

  Characteristics of parenteral dosage forms :

Characteristics of parenteral dosage forms Sterility Free from pyrogenic contamination and endotoxins Freedom from particulate matter Physical, chemical, and microbiological stability Compatibility. Isotonicity 6

Properties evaluated for the API:

Properties evaluated for the API Color and odour Particle size, shape, and crystallinity . Melting point and thermal analytical profile. Hygroscopicity Absorbance spectra 7

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Solubility as a function of pH in a typical range of pH 2–11 pH of the solution, and p Ka of the API Stability profile as a function of solution pH Heat, light, and oxygen sensitivity of the drug substance (API). Accelerated stability of the drug substance (API). Impurity profile. 8

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The following aspects should be addressed to successfully formulate a parenteral dosage form. Selection of a suitable vehicle. Selection of formulation adjuvants . Processing equipment. Final packaging system(container closure integrity). Stability of the finished product. 9

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Solubility of the drug substance and other critical excipients in water and co-solvent systems. Solubility of the drug substance and other critical excipients in different buffer systems. Metal compatibility, filter absorption, and plastic tubing/stopper absorption study. Autoclave study. 10

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11 Comparison to innovator product (for generics) Testing of container and closure components Particle size distribution of suspension or emulsion Water content of the lyophilized products Stability evaluation of the finished product, including sensitivity to light and/or headspace oxygen

Pharmaceutical analysis during formulation and process development:

Pharmaceutical analysis during formulation and process development Pre-formulation and formulation stages. The physicochemical properties and excipients compatibility of the pharmaceutical active ingredient (API) should be thoroughly evaluated The test method requirements are similar to those for oral dosage forms. 12

Difficulties in Analysis during formulation and process development:

Difficulties in Analysis during formulation and process development Solubilization of the drug Formation of salt, pH adjustment using a buffer system, and incorporation of a co-solvent Evaluation of the stability and solubility Analytical method, mostly HPLC is required Method should be able to discern the drug substance from its degradation products and provide an accurate assay for the potency of the drug substance 13


ANALYTICAL TESTING FOR RAW MATERIALS Testing must be performed before they can be used for manufacturing a batch of the product. Assay and ordinary impurity determinations are performed by HPLC methods. When a reference standard for the active ingredient is not available, assay by titration is the method of choice. In the HPLC, main advantage is to have a different sample concentration for the assay and in-process impurity determinations. 14

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Relative response factors (RRFs) to the active ingredient can be used to quantitate impurity concentrations. RRFs can be determined spectrophotometrically by comparing the molar absorptivity of the impurity to that of the active component. RRFs determined by HPLC by comparing peak area. More accurate than those determined by spectrophotometric method. TLC can also used. 15

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Quantitative measurements by densitometry or fluorescence measurements. The spots can also be carefully removed and dissolved in a suitable solvent for spectrophotometric measurement. Residual solvent analysis for drug substances is performed by gas chromatographic methods. Monograph tests such as those for water, residue on ignition, chloride, sulfate content are performed as part of the release testing of a bulk drug substance (API). 16

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The near-infrared (NIR) spectroscopy technique is used to perform raw material, in-process, and finished product testing for quality control. NIR spectral information can be quickly obtained for a single substance or a multicomponent sample . By eliminating the need for sample preparation, the NIR method provides improved method precision over chromatographic methods while equivalent accuracy is maintained. 17

Analytical testing for finished parenteral products :

Analytical testing for finished parenteral products To ensure that each requirements for a parenteral product is properly met. Two type of test generally carried out: (A) Chemical test : Degradation study Identification test for API Potency assay Stability pH 18

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Osmolarity , Apperance,Particle size. distribution for suspensions and emulsions Water content for lyophilized dosage form. Particulate contamination. Integrity of container & closure. (B) Microbiological testing Sterility testing. Bacterial endotoxins testing. Particulate matter testing Bioburden analysis. Closure/ container integrity. 19

(a) Chemical Test :

(a) Chemical Test ( 1) Identification test for API : By color development test : Mixing the test product with a chemical reagent to produce a characteristic color. E.g . Phenolic compounds react with ferric chloride to produce an intense dark color. If no unique color reaction test available, a thin-layer chromatography (TLC) Rf value is used. 20


By HPLC method. An accurately measured aliquot of the product is diluted with a diluent (normally the mobile phase) and the resulting sample solution is injected into the HPLC . In case of emulsion or a suspension ultrasonication , filtration, etc. must be carried out to dissolve the product to achieve a clear solution. (2) Potency assay 21

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Sample concentration choosen should be such that the peak areas obtained from multiple injections of sample are reproducible with min variance ( <2%RSD). A reference std solution should have the same concentration and same diluent as the sample solution is prepared. Prepare the standard plot of API an calculate the concentration of test sample. Tailing factor should LT 1.5 , capacity factor should LT1Peak shape and retention time important for precision of the assay. . 22

(3) Determination of degradation products or process related impurities: :

(3) Determination of degradation products or process related impurities: Majority by chromatography (HPLC) , UV, mass, DSC etc. A concentrated sample solution is commonly used to enhance the sensitivity of the method . Whenever possible a degradation reference standard must be used for the estimation of each known degradation product . 23

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The active peak must be distinguish from impurity and/or degradation peaks along with excipient-related peaks. For example, dextrose-related 5 hydroxymethylfurfural (5-HMF) and related substances are degradation products of dextrose formed primarily during the heat sterilization processes. 24

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HPLC chromatogram of midazolam hydrochloride injection. (Benzyl alcohol and benzaldehyde are excipient -related peaks; desfluoromidazolam is an in-process impurity peak.) 25

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Lactic acid can be determined by back titration of excess alkali with 0.1 N hydrochloric acid. If the formulation contains dextrose, the titration method for lactic acid cannot be used treatment of dextrose with alkali results in hydrolysis of dextrose into organic acids. Alternate method for the determination of lactic acid is an enzymatic colorimetric method using the biochemical lactate reagent. 26

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Principle: Conversion of the lactate to pyruvate and hydrogen peroxide by lactate oxidase . In the presence of the peroxide formed, peroxidase catalyzes the oxidative condensation of chromogenic precursor to produce a colored dye with an absorption maximum at 540 nm. Modification : In the determination of lactic acid in the injection formulation of a cardiotonic drug, The interesters of lactic acid are hydrolyzed by overnight heating of the product in an oven at 100 ◦C. 27

(4) pH :

(4) pH pH determinations are performed routinely for release testing of parenteral . Commercially available buffer solutions can be usedthey are standardized by National Institute of Standards and Technology . Before test sample measurements, the pH meter should be standardized using two buffer solutions that bracket the expected test material pH and whose difference in pH does not exceed 4 units. 28

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E.g. : At 25 o C p H of buffer solution is pH range Potassium tetraoxalate 0.05 – 1.68 Potassium biphthalate 0.05 – 4.01 Equimolal phosphate 0.05 – 6.86 Sodium tetraborate 0.01 - 9.18 29

(5) Stability study::

(5) Stability study: For the determination of shelf life of product . It carried out by subjecting the product to forced degradation usually by heat, acid, alkali, light, and peroxide . Condition (time and temperature) must be controlled so that no more than 20-30% degradation occurs. The degraded samples are then analyzed. 30

(6) Particulate contamination:

(6) Particulate contamination Done by visual inspection(manually ). I nspecting the product against a black and white background using light with an intensity of 100-350 foot-candles at 10 min, distance usually with a magnifying lens (2.5x ) Inspector has normal vision will be able to detect particles in the range of 40-50 µm. 31

(B)Microbiological testing (1) Sterility test: :

(B)Microbiological testing ( 1) Sterility test: Label claim that the contents are sterile. There should be a complete absence of viable microorganism. T wo general methods for sterility test. 1. Direct innoculation method 2. Membrane filtration method 32

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33 FIGURE Schematic diagram of the Millipore MicroStar microbial enumeration system.

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Growth media: 1. Fluid thioglycolate medium(FTM) 2. Alternate fluid thioglycolate medium(AFTM) 3. Soybean-casein digest medium(SCD ) Sampling Generally 10-20 units are randomly sampled from the lot . If the lot size is between 20 to 200 units, then n=10. If the lot size is >200 units, then n=20 . 34

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Time & temperature of incubation: 20-25°C for SCD , 30-35°C for FTM. The incubation period for aseptically filled products is NLT 14 days if the membrane filtration method is used. Interpretation of results: There should be no evidence of microbial growth in the media test containers. If any growth occur, repeat the test with fresh samples and must be carried out with exceptional care to prevent reoccurrence of accidental contamination. 35

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If growth occur in second test, further repeat unless the same organism is found in second test. If the third test is negative, sample passes and if positive, it fails. (2) Bacterial endotoxins test(LAL test) BET measures the concentration of bacterial endotoxins present using a lysate derived from the hemolymph cells or amoebocytes from the horseshoe crab, Limulus polyphemus . 36

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Principle The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or gelation of the mixture. The rate of reaction depends on the concentration of endotoxin , the pH and the temperature. The quantities measured in Endotoxin Units . Mechanism: Intracellular serine protease zymogens in the crab’s blood stream are triggered by presence of bacterial endotoxins that produces coagulin gel clot. 37

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Methods: The following six methods are described in the British Pharmacopoeia 2007 : Method A: Gel clot method: Limit test. Method B: Gel clot method: Semi quantitative method. Method C: Turbidimetric Kinetic method Method D: Chromogenic kinetic method Method E: Chromogenic end- point method Method F: Turbidimetric end point method. 38

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Preparation of test solutions(Preparatory testing) N egative product control(water BET) P ositive water control(control standard endotoxins con. 2 λ ) P ositive water control(contain sample solution ) Procedure: Carry out the in duplicate receptacles.Add to each receptacle an equal volume of constituted lysate . Mix the sample/ lysate mixture gently. Incubate at 37±1 o C temp for 60 ± 2 min. 39

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A positive reaction ( formation of a firm gel) that retains its integrity when inverted through 180 o . Record this result as positive (+). A negative result ( the absence of such a gel or by theformation of a viscous gel) that does not maintain its integrity. Record such a result as negative (-). Interpretation of results : The substance or preparation complies with BET test if the positive product control is positive and the negative controls are negative. 40

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The test is invalid if the positive product control is negative or negative control is positive. If a positive result is found for one of the test duplicates(negative water control) 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. 41

(3) Particulate matter testing::

(3) Particulate matter testing : T wo test approaches (by the USP). L ight obscuration method or the light (laser) scattering method for SVPs The microscopic method for LVPs . ( 1) Light obscuration method A beam of light is passed through the test solution and is measured by a photodiode detector . Particles passing through the light path disrupt the light in a size-proportional manner. 42

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Advantages E ase of use, Enhanced reproducibility Relative ease of method automation. Disadvantages Expensive instrumentation, It does not provide information on the identification of particles Artifacts such as air bubbles can be counted as particles. 43

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(2) The light-scattering method T he behavior of light after it strikes a particle suspended in a clear solution. Measurements of scattered light taken at fixed angles from the direction of the incurrent light beam are proportional to the size of the particle . R apid and can measure particles in as many as six size ranges simultaneously. 44

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(3) Microscopic method This method involves the use of ultraclean membrane filtration equipment. 25 milliliters of test solution is filtered and the membrane is examined by optical microscopy .( 100× magnification). Particles 10 μm and 25 μm are manually sized and counted by highly trained personnel . Used for emulsions, suspensions, and other nonclear fluid injectable products. 45

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Interpretation of Results—Particulate Matter Testing Results of particulate matter tests are interpreted as following: a. For the light obscuration method: ≥10 μm – 6000 per container ≥ 25 μm – 600 per container b. For the microscopic method: ≥ 10 μm – 25 per ml ≥ 25 μm – 3 per ml 46


PACKAGING COMPONENTS TESTING Mainly three container material Glass/plastic containers Elastomeric closures plastic bags. Type I glass Type II and type III glass Type iv 47


PROCESS DEVELOPMENT SUPPORT Analytical method must be available before preformulation . Analytical method should be capable of separating the active and any major degradation product(for stability). Titration and ultraviolet (UV) spectroscopy are not used. HPLC hasbeen widely used as the method of choice. 48

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Preliminary validation of the method, which includes accuracy, precision, and linearity. Specificity is demonstrated by subjecting the product to forced degradation stress studies, such as heat, acid, alkali, light, and peroxide and analyzing the resulting mixture. All degradation peaks must be clearly separated. Peak purity of the analyte peak is evaluated by using a diode-array detector. 49


IN-PROCESS TESTING Essential part of the manufacturing process Way to confirm that the concentration of the active ingredient in the bulk solution is accurate and within specification limits. Used to test for homogeneity UV spectrophotometric method is usually selected. 50

Why uv spectroscopy is selected???:

Why uv spectroscopy is selected??? Time saving compare to HPLC. It does not require elaborate instrument. Easily set up in a manufacturing environment. Excipients in the formulation interfer eg . benzyl alcohol 260 nm 51


RELEASE TESTING Simple identification test is required. The identification test is typically a color development test - mixing the test product + chemical reagent to produce a characteristic color. Eg … phenolic compounds react with ferric chloride to produce an intense dark color. 52

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When there is no unique color reaction test, thin- layer chromatography (TLC) Rf value or an HPLC is used. An HPLC method for the estimation of potency and determination of degradation products is an integral part of release testing. The analytical method should be SELECTIVE , SPECIFIC & stability indicating. 53

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Ex Lactic acid – common excipient used in parenteral is determine by back titration of excess alkali with 0.1N HCL.because dextrose+alkali results in hydrolysis of dextrose to organic acids. The titration is performed after removing active drug in formulation by using chromatography. Alternatively it is done by enzymatic colorimetric using biochemical reagent. 54

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In presence of hybrogen peroxide,peroxidase catalyzes oxidative condensation of chromogenic precausors to produce colored dye with 540nm (max wave length) Lactate in presence of lactate oxidase converts in to pyruvate and hydrogen peroxide. 55

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DOCUMENTATION “It is a systematic & scientific process of collecting & recontrolling In India underschedule ‘U’ & schedule ‘M’ to the drugs and cosmetics Rules that record are manes and path-own they are failing. 1. Batch reconciliation record 2. Environmetal control record 3. Raw material requiring sheet 4. Recovery addition sheet 56

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5. Manufacturing instrunmentation 6. Bottle filling & washing record 7. Leakes test record 8. Finished goods release record 9. Personnel records 10. SOP records. 11. Packaging material record 12. Packaging record 57

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Questions 1 Enlist the analytical tests of parenteral preparation. Discuss BET. 2 Describe particulate matter testing for parenteral preparation. 3 Give characteristics of parenteral dosage forms. Describe requirements for successfully formulated parenteral dosage forms. Discuss Bioburden testing of parenteral preparation. Describe pharmaceutical analysis during 1)Formulation and process development. 2)Finished parenteral preparations. 6 Discuss sterility testin g. 59

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References Handbook of modern pharmaceautical analysis by Satinder Ahuja and Stephen Scypinski, volume 3 United state pharmacopoeia. International Journal of Research in Pharmaceutical and Biomedical Sciences 60

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