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

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.
2

3. .
Administration by injection, subcutaneous, intramuscular,
and intravenous ,intrathecal, intraarterial, intraspinal,and
intradermal routes to achieve local or systemic effects.
Advantages
Rapid absorption and distribution, high bioavailability
 Zero enzymatic 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
3

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

5. Why quality assurance is
needed for parenteral?
Higher propensity for improper administration of
injectable products
Administered directly into the human bloodstream
Risker than oral solid dosage
Compromised immune systems, among other things.
5

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

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

8. • Solubility as a function of pH in a typical range of pH 2–
11 pH of the solution, and pKa 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

9. 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

10. • 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

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
11

12. 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

13. 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

14. 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

15. • 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

16. • 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

17. • 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

18. 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

19. 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

20. (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

21. (2) Potency assay
• 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.
21

22. • 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

23. (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

24. • 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

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

26.  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

27.  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

28. (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

29. • E.g. : At 25oC 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
29

30. (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

31. (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.
31

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

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

34. 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

35.  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

36. • 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

37.  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

38. • 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

39. Preparation of test solutions(Preparatory testing)
– Negative product control(water BET)
– Positive water control(control standard endotoxins con.
2 λ)
– Positive 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±1o C temp for 60 ± 2 min.
39

40. A positive reaction ( formation of a firm gel) that retains its
integrity when inverted through 180o . 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

41. • 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

42. (3) Particulate matter testing:
• Two test approaches (by the USP).
– Light 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

43. • Advantages
(1) Ease of use,
(2) Enhanced reproducibility
(3) Relative ease of method automation.
• Disadvantages
(1) Expensive instrumentation,
(2) It does not provide information on the identification
of particles
(3) Artifacts such as air bubbles can be counted as
particles.
43

44. (2) The light-scattering method
• The 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.
• Rapid and can measure particles in as many as six size
ranges simultaneously.
44

45. (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

46. • 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

47. 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

48. 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

49.  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

50. 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

51. 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

52. 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

53. 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

54. • 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

55. • 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

56. 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

57. 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

58. .
RECENTLY DEVELOPED ANALYTICAL METHOD
58

59. 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.
4 Discuss Bioburden testing of parenteral preparation.
5 Describe pharmaceutical analysis during
1)Formulation and process development.
2)Finished parenteral preparations.
6 Discuss sterility testing.
59

60. References
1. Handbook of modern pharmaceautical analysis
by Satinder Ahuja and Stephen Scypinski,
volume 3
2. United state pharmacopoeia.
3. International Journal of Research in
Pharmaceutical and Biomedical Sciences
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61. 61