ANALYSIS OF INJECTABLES DOSAGE FORMS

PREPARED BY: GUIDED BY:

PAWAR MO. NASIRUDDIN DR.JIGNESH SHAH

M.PHARM SEM 2 (QARA) ASSISTANT PROFESSOR

ROLL NO: 18 DEPARTMENT OF QA

L J INSTITUTE OF PHARMACY

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Content:• Introduction• Characteristics of Parenteral Dosage forms• Properties evaluated For the API• Analytical testing for raw materials• Analysis Testing For Finished Parenteral Products

(a) Chemical Testing

(b) Microbiological Testing• Packaging Components Testing• Process development support• In-process Testing• Release Testing• References

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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.

Administration by injection, subcutaneous, intramuscular, and intravenous ,intra-arterial, intraspinal, routes to achieve local or systemic effects.

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Advantages

Rapid absorption and distribution

high bioavailability

No degradation in the gastrointestinal tract

An ability to be administered to unconscious patient

Major drawback of parenteral delivery is the pain and discomfort associated with needle injection.

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Why quality assurance is needed for parenteral?

Higher tendency for improper administration of injectable products

Administered directly into the human bloodstream

Riskier than oral solid dosage form

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Characteristics of Parenteral dosage forms :

Sterility

Free from pyrogenic contamination and endotoxins

Freedom from particulate matter

Physical, chemical, and microbiological stability

Compatibility

Isotonicity

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Properties evaluated for the API

Colour and odour

Particle size & shape

Melting point and thermal analytical profile

Hygroscopicity

Absorbance spectra

pka of the API

Heat, light, and oxygen sensitivity of the drug substance (API)

Accelerated stability of the drug substance (API)

Impurity profile

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The following steps should be addressed to successfully formulate a parenteral dosage form.

Selection of a suitable vehicle

Selection of formulation excipients

Validated Processing equipment

Final packaging system(container,closure,integrity)

Stability of the finished product

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• Solubility of the drug substance and other critical excipients in water and co-solvent systems and in different buffer system.

• Comparison to generics products.

• 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.

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Pharmaceutical analysis during formulation and process development

• During pre-formulation and formulation stages of parenteral dosage form the physicochemical properties and excipients compatibility to the pharmaceutical active ingredient (API) should be thoroughly evaluated

• The test method requirements are similar to those for oral dosage forms

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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 find out the drug substance from its degradation products and provide an accurate assay for the potency of the drug substance

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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

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• Osmolarity, Appearance, 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 • Bio burden analysis.

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(A)Chemical test

(1) Identification test for API :

By colour development test : • Mixing the test product with a chemical reagent to produce a

characteristic colour.

• E.g. Phenolic compounds react with ferric chloride to produce an intense dark colour.

• If no unique colour reaction test available, a thin-layer chromatography (TLC) Rf value is used.

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By IR spectroscopy:

• Infrared absorption spectrophotometric tests provide meaningful evidence of the identification of many active pharmaceutical ingredients (API).

• The measurement of the infrared absorption of the API in the fingerprint region of the spectrum (3800cm-1 to 700cm-1) can be compared to the known spectra of the API to confirm the identification of sample.

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(2) Potency assay:

By HPLC method:

• An accurately measured equal quantities of the product is diluted with a diluents (normally the mobile phase) and the resulting sample solution is injected into the HPLC.

• In case of emulsion or a suspension ultra-sonication, filtration, etc. must be carried out to dissolve the product to achieve a clear solution.

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• Sample concentration choosen should be such that the peak areas obtained from multiple injections of sample having less than 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 and calculate the concentration of test sample.

• Tailing factor should be Less than 1.5 , capacity factor should be less then 1.Peak shape and retention time important for precision of the assay.

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(3) Determination of degradation products or process related impurities

• Majority by chromatography (HPLC) , UV, Mass spectroscopy, 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.

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• The API peak must be distinguish from impurity and/or degradation peaks along with excipients-related peaks.

I. Chemical degradation• For example, dextrose-related 5 hydroxy methyl furfural (5-HMF)

and related substances are degradation products of dextrose formed primarily during the heat sterilization processes.

• Lactic acid can be determined by titration of excess alkali with 0.1 N hydrochloric acid.

• If the formulation contains dextrose, the titration method for lactic acid cannot be used because treatment of dextrose with alkali results in hydrolysis of dextrose into organic acids.

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II. Enzymatic degradation• Alternate method for the determination of lactic acid is an enzymatic

colorimetric method using the biochemical lactate reagent

Principle: • Conversion of the lactate to pyruvate and hydrogen peroxide by lactate

oxidase. • In the presence of the peroxide formed, peroxidase catalyzes the

oxidative reaction 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

cardio-tonic drug, The interesters of lactic acid are hydrolyzed by overnight heating of the product in an oven at 100 ºC.

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(4) pH:

• pH determinations are performed for release testing of parenteral .

• Commercially available buffer solutions can be used. They 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.

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E.g. : At 25 °C pH 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

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(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.

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(6) Particulate contamination:

• Done by visual inspection(manually).

• Inspecting 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.

• The AQL (accepted quality level) are 0.25-1.0% of the lot size.

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(7)Osmolarity:

• It is a fundamental physical property of solutions related to diffusion of solutes through biological membranes.

• Osmolarity are units of solute concentration expressed as milli-osmols per litre.

• Depends on the number of species in solution. • Apart from the drug, the excipients in solution will also contribute to

Osmolarity of a drug product.• Osmolarity determinations are made using osmo-meter.

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(B)Microbiological testing

1) Sterility test

2) Bacterial endotoxins test(LAL test)

3) Particulate matter testing

4) Bioburden analysis

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Packaging Components Testing

• Mainly three container material Glass/plastic containers Elastomeric closures Plastic bags

o Type I glass o Type II glasso Type III glass o Type iv glass

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TYPE 1 GLASS:

• It is borosilicate glass and is widely used for parenteral products, both in small volume vials ampules or in large volume infusion bottles

TYPE 2 GLASS:

• It is a Treated soda lime glass, that has been de-alkalized, or treated to remove surface alkali.

TYPE 3 GLASS:

• It is a Regular soda lime glass. Used for dry powder and oily solution.

TYPE 4 GLASS:

• It is a General-Purpose Soda-Lime Glass, mainly use for non-parenteral products those intended for topical or oral use.

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• Compendial tests for glass include the powdered glass test, water attack at 121°C.

POWDERED GLASS TEST:• Rinse thoroughly with Purified Water six or more containers selected

at random, and dry them with a current of clean, dry air. • Crush the containers into fragments about 25 mm in size.• Now add methyl red indicator 2 drops, than titrate this powder

contain with 0.020 N sulfuric acid.• Record the volume of 0.020 N sulfuric acid used to neutralize the

extract. The volume does not exceed that indicated in table.

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TYPE GENERAL DESCRIPTION

TYPE OF TEST

LIMITS IN (ml)

I. Highly resistant borosilicate glass

Powdered glass test

1.0

III. Soda lime glass Powdered glass test

8.5

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WATER ATTACK AT 121ºC:• Fill each container to 90% of its overflow capacity with High-Purity

Water.• Empty the contents from 1 or more containers into a 100-mL

graduated cylinder, combining, in the case of smaller containers, the contents of several containers to obtain a volume of 100 ml.

• In the case of smaller container place the pooled specimen in a 250-mL conical flask of resistant glass, add 5 drops of Methyl Red Solution, and titrate, while warm, with 0.020 N sulfuric acid.

• Record the volume of 0.020 N sulfuric acid used.• The volume does not exceed that indicated in table.

TYPE

GENERAL DESCRIPTION

TYPE OF TEST

SIZE IN (ml)

LIMITS IN (ml)

II. Treated soda lime glass

Water attack at 121ºc

≤100 ml>100 ml

0.7 ml0.2 ml

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PLASTIC BAGS:

• Plastic containers are also widely used for packaging parenteral products, such as intravenous (IV) infusion fluid containers, irrigation fluid containers, prefilled disposable syringes, and some administration sets.

• Materials used in the parenteral product market include polyvinyl chloride, cellulosics, polypropylene, and polystyrene.

• Compendial tests for plastic containers include the light transmission test, biological tests for plastics and other polymers.

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ELASTOMERIC CLOSURES:

• This are used in packaging of parenteral products in the form of vial closures, syringe plungers.

• The following criteria should be evaluated to qualify a type of elastomeric material to be used with a parenteral product,

- pressure to puncture,

- biocompatability,

- endotoxin removal, detergent removal,

- break force.

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Process development support

• Analytical method must be available before preformulation studies are started.

• Analytical method should be capable of separating the active and any major degradation product(for stability).

• HPLC has been widely used as the method of choice in recent years

because of its efficiency, applicability for a wide range of chemical compounds, and ease of automation.

• Preliminary validation of the method, which includes accuracy, precision, and linearity.

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• 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 (HPLC).

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In-Process Testing

• Essential part of the manufacturing process.

• It is quick way to confirm that the concentration of the active ingredient in the bulk solution is accurate and within specification limits.

• It is also used to test for homogeneity (uniformity) of the bulk solution by testing samples from top, middle, and bottom portions of the tank.

• UV spectrophotometric method is usually selected.

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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 may interfere with active ingredient. E.g. benzyl alcohol 260 nm.

• In such cases, longer wavelengths (>300 nm) can be used, provided that the drug displays optimum UV absorption at a longer wavelength.

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Release Testing

• Simple identification test is required for parenteral.

• The identification test is typically a colour development test - mixing the test product + chemical reagent to produce a characteristic colour.

• E.g. Phenolic compounds react with ferric chloride to produce an intense dark colour.

• When there is no unique colour 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.

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• The analytical method should be SELECTIVE , SPECIFIC & stability indicating.

• E.g. 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.

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• E.g. In presence of Hydrogen peroxide, peroxidase catalyzes oxidative condensation of chromogenic precursors to produce coloured dye with 540nm (max wave length).

• Lactate in presence of lactate oxidase converts in to pyruvate and hydrogen peroxide.

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Analytical testing for Raw Material

• 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.

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• Relative response factors (RRFs) to the active ingredient can be used to calculate the impurity concentrations.

• RRFs in chromatography and spectroscopy is the ratio between a signal produced by an analyte and the quantity of analyte which produces the signal.

• 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.

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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, sulphate content are performed as part of the release testing of a bulk drug substance (API)

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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 multi-component sample.

• By eliminating the need for sample preparation, the NIR method provides improved method precision over chromatographic methods while equivalent accuracy is maintained.

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Stability – Indicating Methods

• Methods used to monitor the stability of pharmaceutical products must be specific to the major analyte and capable of separating the degradation and impurity peaks.

• The stability-indicating nature of the method can be demonstrated by subjecting the product to forced degradation (usually by heat, acid, alkali, light, and peroxide).

• Conditions (time and temperature) must be controlled so that no more than 20–30% degradation occurs.

• The degraded samples are then analyzed according to the method. • For the method to be stability-indicating, the degradation peaks must

be sufficiently well resolved from the major component so that the specificity and accuracy of the method are not affected.

• Peak purity of the major component must be determined by using a diode-array detector or by using mass spectroscopy.

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References

1. Handbook of modern pharmaceutical analysis by satinder ahuja and stephen scypinski, volume 3 page no 269-303.

2. United state pharmacopoeia.

3. http://www.authorstream.com/Presentation/bhaktiladva-1102970-analysis-of-injectable-dosage-forms

4. http://pharmaexposure.blogspot.in/2011/08/ typesof-glass-used-in-pharmaceutical.html

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