IC12 Quality Assurance and Pharmacopeial Assays

Question 1

What are the Objectives of Pharmaceutical Quality assurance to be met?

Answer 1

1. Ensure the public’s safety
2. Protect against negative publicity
3. Continually increase production efficiency
4. Guarantee compliance with any regulations
5. Importance of quality assurance training

Question 2

What are the differences between Pharmaceutical QA and QC ?

Answer 2

1. Effective QA is proactive while QC testing uncovers quality issues which result in reactive steps to correct the issues.
2. QA is process oriented and focuses on preventing quality issues from arising. QC is product oriented and focusses on the quality of the manufactured products.
3. QA controls overall methods and procedures at the system level. QC controls the parts in the processes that manufacture the products.
4. QA activities is a roadmap for creating high quality products. QC involves verification of the products in post-manufacture stage.
5. QA involves the entire team in the manufacturing facility. QC is conducted by dedicated technical personnel.

Question 3

What are the 5 Key components of Good Manufacturing Practices (cGMP), 5Ps?

Answer 3

5 Key components of Good Manufacturing Practices (cGMP), 5Ps:

1. Primary Materials and Products (good raw materials)
2. Premises and equipments (properly maintained)
3. People (trained)
4. Procedures (using the latest technology and science involved in pharm manufacturing)
5. Processes (documentation of every step to show compliance)

Question 4

Who over looks the quality for medicines?

Answer 4

International Conference on Harmonisation of Technical Requirements For Registration of Pharmaceuticals for Human Use (ICH)

Question 5

What does ICH do?

Answer 5

• organization that standardizes the requirements for medicines regulation throughout the world
• standardizes the validation of analytical procedures and indicates that validation is required for

1. Identification tests
2. Quantitative tests for impurities
3. Limits tests for the control of impurities
4. Quantitative tests of the API, drug products and selected components(s) in the drug product

Question 6

List the sources of impurities exposed to pharmaceutical manufacturing. 5 main ones.

Answer 6

Sources of Impurities

1. Raw Materials
2. Method of Manufacture
   a. Reagents employed in the process
   b. Reagents added to remove other impurities
   i. Chloroform good to remove impurities but need to remove chloroform too
   c. Solvents
   i. Diff grades of solvents provide diff purity
   d. The reaction vessels
   i. When washing can leave behind detergents
3. Atmospheric Contaminants
   a. Dust,CO2 + H2O  carbonic acid changes pH of water
4. Manufacturing Hazards
   a. Particulate contamination (esp.in IV)
   b. Process errors
   c. Cross-contaminations
   d. Microbial contaminations (esp. syrups, solutions)
   e. Packing errors
   f. Can’t use the same facilities to make diff drugs
5. Inadequate Storage
   a. Filth
   b. Chemical Instability
   c. Reaction with container materials
   d. Physical changes
   e. Temperature effects
   f. Humidity (if too high, can cause hydrolysis of drug)

Question 7

What are Limit Tests for?

Answer 7

quantitative (how much is exactly present) or semi- quantitative (as long as impurity in product < impurity in standard) tests designed to identify and control small quantities of impurity which may be present in the drug substance.

Question 8

What is the Limit Tests Comparison Method?

Answer 8

Comparison Methods – A standard containing a definite amount of impurity is set up at the same time and same conditions as the test experiment. The extent of reaction in the experiment is determined by comparison of the test solution and the standard solution.

• The official limits for:
  o chlorides,
  o sulphates,
  o iron, and
  o heavy metals e.g. lead, arsenic are based on this principles
• E.g. Checking for chloride impurities using AgNO3, AgCL is the ppt, if no ppt or turbidity produced then means it meets the standards

Question 9

What are the types of Limit Tests for Quantitative Determinations?

Answer 9

Limit Tests – (B) Quantitative Determinations
Types of tests:

1. Limits of insoluble matter
2. Limits of soluble matters
3. Limits of moisture, volatile matters, and residual solvents
4. Limits of non-volatile matter
5. Loss on ignition
   a. Limits of residue on ignition
   i. Fentanyl Citrate → Residue (impurities) on ignition: NOT > than 0.5%
   b. Ash values
6. Precipitation methods

Question 10

What is the total ash method?
What does High ash value indicate?
For what type of products?

Answer 10

Total Ash method is a measure of the total ash remaining after incineration.
High ash value is an indication of contamination, substitution or carelessness in preparing the crude drugs for marketing Inorganic salts of carbonates, phosphate or silicates of sodium, potassium calcium and magnesium

Question 11

What are the 3 types of Identification Tests in USP?

Answer 11

1. Infrared Absorption test
2. UV absorption test
3. Thin Layer Chromatographic Identification Test

Question 12

What is infrared absorption test used for?

Answer 12

a. **IR for functional group identification

Question 13

How to identify the substance using infrared absorption test?
What is the wavelength range for infrared?

Answer 13

1. Infrared Absorption
   a. IR for functional group identification
   b. Compare the IR spectrum of the test sample and the USP reference standard to seek evidence for identity.
   c. IR absorption relates to stretching and bending of bonds in different functional groups to the structure of the analyte. → will appear as peaks in the spectrum
   d. Fingerprint region (600 - 1400 cm-1) of the IR spectrum
   i. Complex area showing many bonds
   ii. Diff compounds will create diff patterns of trough in this area
   iii. If have same kind of absorbance pattern in the finger print region, then means can identify it

Question 14

How to identify the substance using UV absorption test?
What is the wavelength range for UV?

Answer 14

2. Ultraviolet Absorption
   a. UV absorption measured of a test solution and a standard solution, using a 1cm cell, over 200-400nm.
   b. Compare the UV spectra of the test and standard solution
   c. Determine the absorptivities and or absorbance ratios as indicated in the monograph
   d. The requirements are met if the UV absorption spectra of the test and standard solutions exhibit maxima and minima at the same wavelength and the absorptivities and/or absorbance ratios are within specified limits.

Question 15

How to identify the substance using TLC test?
What is the material of the plate used in TLC?

Answer 15

3. Thin Layer Chromatographic Identification Test
   a. Develop a TLC using silica gel chromatographic plate impregnated with a suitable fluorescing substance.
   b. Apply 10μL of test solution and 10μL of Standard solution prepared from USP reference standard for drug substance to be identified.
   c. Use TLC developing solvent system consisting a mixture of chloroform, methanol, water (180:15:1), unless otherwise stated in the monograph.
   d. The Rf value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution – indicates a positive identity to the Standard reference
   e. Rf = distance travelled by substance/ distance travelled by solvent front
   f. Not as sensitive as HPLC, but some labs don’t have HPLC, so still have TLC

Question 16

What is the Rf formula?

Answer 16

Rf = distance travelled by substance/ distance travelled by solvent front

Question 17

What is Titrimetric Analysis for Pharmaceuticals used for?

Answer 17

Used in pharmacopeial assays:

1. used to estimate the purity of the analyte/ Impurity amt
2. Quantity of API in dosage form (where there are other excipients)
3. CANNOT identify impurities

Question 18

What are the advantages and limitations of using Titrimetric Analysis for Pharmaceuticals?

Answer 18

Advantages:

1. Capable of high degree of precision and accuracy
2. Methods are generally robust
3. Analyses can be automated
4. cheap to perform

Limitations:

1. Method may NOT be selective
2. Time-consuming
3. Require large amounts of sample and reagents

Question 19

What are the types of Titrimetric Analysis in Pharmacopeia?

Answer 19

1. Direct acid/base titration in aqueous phase
2. Indirect titration in aqueous phase
3. Argentometric titration

Question 20

What is a Primary Standards?
What is it for?

Answer 20

Primary Standards

• stable chemical compounds that are available in high purity and that can be used to standardize the standard solutions used in titrations.
• Titrant such as NaOH (secondary standard) standard solution may be standardized against potassium hydrogen phthalate(primary standard), which is available in high purity.
  o Secondary standard – use in the titration in the assay
• Use to determine the correction factor (f)

Question 21

What is the Correction Factor?
When is it usually used?
How to calculate it?

Answer 21

The Correction Factor

• The correction factor (f) is usually used in volumetric analysis to simplify calculations. The factor is calculated as a ratio of (Actual concentration)/(Desired (or nominal)concentration). It tells us how much a given solution differs from the nominal (true) concentration.
• If f < 1, the prepared solution is of lower concentration that what was desired.
• If f > 1, the prepared solution is of higher concentration that what was desired.
• If f = 1, the prepared solution is prepared precisely to the desired concentration.
• Value should always be close to 1
• Find correction factor to calculate the actual concentration (pure) of the substance in the solution
• For example, if f of a 0.5 N H2SO4 solution = 0.96, its actual concentration is 0.5 N × 0.96 = 0.48 N.
  f x N = actual N

Question 22

How to derive the equivalent values?

Answer 22

Derivatization of the Equivalent

CH3COOH + NaOH → CH3COO-Na+ + H2O
1000mL 1N NaOH ≡ H+ ≡ 60.05g CH3COOH

• 60.06g is the MW of CH3COOH

Therefore, 1mL 1N NaOH ≡ 0.06005g CH3COOH
Hence, 1mL 0.1N NaOH = 0.006005g CH3COOH ≡ 6.005mg CH3COOH

Question 23

What is Indirect Titration (Back Titration)?

Answer 23

Indirect Titration (Back Titration)

• Back titrations involve addition (from a pipette) an excess of a standard volumetric solution (VS) to a weighed amount of the analyte, and followed by the determination of the excess unreacted VS.
• VS = a reagent of known concentration
• Not directly titrating the aspirin but the unreacted excess NaOH

Question 24

*When is indirect titration used?

Answer 24

In general, this indirect (back) titration is used for:

1. Volatile substance (e.g. ammonia, volatile oil)
   a. Inaccuracy arises due to loss of substance (vaporise) during direct titration
   b. Thus add in certain amt of reagent that will interact with the volatile substance, then stopper and let the chemical reaction take place
2. Insoluble substances (e.g. CaCO3)
   a. Takes a long time to dissolve
   b. Difficult to detect end point
3. Substances for which a quantitative reaction proceeds rapidly only in excess of a reagent (e.g. lactic acid)
   a. Reaction too slow if do direct titration
4. Substances which require heating with a volumetric reagent during the determination in which a decomposition or loss of the reactants or products would occur in the process (e.g. aspirin)

Question 25

Why is it necessary to do Back Titration with Blank Titration?

Answer 25

• necessary to perform a blank determination in assay which
  o involve heating a liquid containing excess of standard alkali, cooling and back titrate the excess. The heating and cooling may cause apparent changes in the strength of the excess reagent.
  o Sometimes, the standardized reagent will e.g. react with the vessel
  o want to ensure what happens in the blank titration and analyte are the same under the same conditions
  o Therefore, the blank titration will standardize conditions in both the blank and the sample determination.
• If don’t do the blank titration, we will find that whatever we did is inaccurate
• Blank titration – an analysis of sample without the analyte (hard to find) / an analysis without the sample (more common)
• The USP assay for aspirin adopts this approach in the determination of aspirin

Question 26

Back titration Example: Aspirin (can just open to see)

Answer 26

Back titration Example: Aspirin

• This assay involves an alkaline catalysed hydrolysis of ASP with excess NaOH and a back titration to determine the unreacted NaOH in the flask for the analysis of the quantity of ASP.
• From the equation of the hydrolysis of ASP, 2000 mL of 1N (1M) NaOH ≡ 180.16g (1 mole) of Aspirin
• 1 mL of 1N NaOH ≡ 0.09008g of Aspirin
• 1 mL of 0.5N NaOH ≡ 0.04504g of Aspirin ≡ 45.04mg of Aspirin
• The titration involves the use of 0.5N H2SO4 as a titrant to determine the unreacted 0.5N NaOH.
• Each mole of H2SO4 produces 2 moles of H+ which neutralize 2 moles of NaOH
• Therefore, 1 mL 0.5N (0.25M) H2SO4 can neutralize 1 mL 0.5N (0.5M) NaOH
  o Since use same kind of nomality, the volume will be the same
• Therefore the equivalence is such that 1 mL 0.5N H2SO4 ≡ 1mL 0.5N NaOH ≡ 45.04mg Aspirin
  o Since we know the equivalence, no need to know how much NaOH was unreacted, just need to know how much H2SO4 I used to do the titration of analyte and titration in the blank → then find the amt of aspirin
  o Just (H2SO4 in blank titration) - (H2SO4 in analyte titration), then find amt of aspirin
• From the titrant volume determined, the amount of Aspirin can be calculated by using the equivalence
• (Titre of H2SO4 in blank expt) – (Titre of H2SO4 in analyte expt) × fH2SO4 × 45.04mg Aspirin
• Molarity = moles in 1L of solution
• Nomality = no. of reactive species present e.g. 1000mL Of NaOH = 1N | 1000mL of H2SO4 = 2N
• N = m/M = amt of substance in moles

Question 27

What is Argentometric Titration?

Answer 27

Argentometric Titration (Direct)

• Involve the use of Ag ions

Question 28

Example of Argentometric Titration (Direct). Just open to see.

Answer 28

Argentometric Titration (Direct)

• Involve the use of Ag ions
• Quantitative precipitation can be used for volumetric determinations, provided the point at which precipitation is complete can be determined. In argentometric titration, this principle is applied.
  o NaCl + AgNO3 AgCl + NaNO3
• For example, a NaCL solution can be determined by titration with AgNO3 in which AgCL precipitates as AgNO3 VS is added to a solution of NaCL.
• At the end point, the excess silver nitrate reacts with potassium chromate used as the indicator where a red colouration indicates the end point.

Question 29

How to calculate the amt of aminophylline in the argentometric titration test?
Why is an acidic condition needed for this test?

Answer 29

Analysis of Aminophylline An Argentometric Titration

• Aminophylline is a salt of theophylline and ethylenediamine.
• In the argentometric titration, AgNO3 precipitates the theophylline. The filtration process removes the precipitated theophylline, and the filtrate contains the unreacted excess AgNO3 which is determined by titration with ammonium thiocyanate (NH4SCN).
• The blank titration determines the total amount of AgNO3. Therefore, the difference between the blank and the filtrate gives the amount of theophylline present.

Assay of Aminophylline The Chemistry of the Titration

• Excess silver nitrate is titrated against 0.1M NH4SCN
• Titration: AgNO3 (unreacted excess) + NH4SCN → AgSCN + NH4NO3
• Indicator: Ferric ammonium sulfate, FeNH4SO4
• Fe3+ + SCN- → [FeSCN]2+
• Excess AgNO3 reacts with NH4SCN to form AgSCN (white precipitate).
• Once all Ag+ are completed reacted, (the next drop) Fe3+ reacts with SCN-
• Fe3+ + SCN- → [Fe(SCN)]2+ (reddish-brown = colour observed at end
  o point)
• Acidic condition is required to prevent hydrolysis of Fe3+ .
• Based on the given equivalent 1 mL 0.1M AgNO3 ≡ 1 mL 0.1M NH4SCN ≡ 18.02 mg of theophylline;
• Just need to know the volume of NH4SCN used
• The volume of the titrant (NH4SCN) is thus used in the calculation. The difference in the NH4SCN volumes (of blank – experiment) is used to multiply with 18.02 to give the amount of theophylline in milligrams.

Question 30

What are the 3 methods of quantification of HPLC?

Answer 30

• 3 methods of quantification:
  o external standard,
  o internal standard,
  o single point calibration

Question 31

How to get the conc. of substance using analyses based on calibration with an external standard?

Answer 31

1. Analyses based on calibration with an external standard.
   a. **Just read off the scale/graph what the concentration is

See pg 21 of notes

Question 32

When can we use single point calibration?
How to get the conc. of substance using analyses based on a single point calibration?

Answer 32

Analyses based on a single point calibration.

• If recovery is complete, then the peak of the analyte can be compared with a calibration curve of the standard analyte.
• Don’t need to do calibration curve, so accurate that use 1 point to determine the amt/unknown of the analyte

See pg 21 of notes

Question 33

For Analyses using calibration against an internal standard, how should the internal standard be?

Answer 33

Analyses using calibration against an internal standard which

1. Should be closely related in structure to the analyte
2. Should be stable
3. Should be chromatographically resolved from the analyte and any excipients present in the chromatogram of the formulation extract (separate well from analyte, 2 peaks CANNOT overlap)
4. Should elute as close as possible to the analyte with the restrictions above. For a given weight should produce a detector response similar to that produced by the analyte

Question 34

How to get the conc. of substance using analyses based on calibration with an internal standard?

Answer 34

• After getting the results, we need to calculate the peak area ratio and plot a graph of peak area ratio (analyte/internal standard) against the concentration ratio
  f. conc of analyte = (Peak area ratio of analyte/peak area ratio of internal standard) x concentration of internal standard

See pg 22 of notes

Question 35

When should internal standards be used?

Answer 35

Used for TDM (Biological fluids) use plasma for analysis

• When can’t get full recovery means sth happened, thus need to use an internal standard where it’s subjected to the same conditions e.g. binding to plasma proteins
• Thus overcome recovery problem
• Consistently use this method, both internal standard and analyte are subjected to the same conditions, then it’s more consistent then whenever we recover, we can correlate to whatever concentration it should be
• Thus, cannot use external standard

The internal standard is used to improve the precision of quantitative analysis.