QUALITY MANAGEMENT – CLASS NOTES BY ZUBIA

ASSAYS
Types of Assays
1. Biological Assay: This involves using living organisms or tissues to determine the potency or
concentration of a substance.
E.g.: Testing the effectiveness of a drug by observing its effects on a living organism or tissue.
2. Chemical Assay: This type of assay focuses on the chemical composition of a substance,
determining its quality and quantity.
E.g.: Using HPLC to analyze and quantify the chemical components of a sample.
3. Pharmaceutical Assay: This refers to tests conducted according to the official standards
outlined in pharmacopeias, such as the United States Pharmacopeia (USP).
E.g: Performing tests specified by the USP to ensure the quality, purity, and potency of
pharmaceutical products.
4. Immunological Assay: This involves the use of antibodies and antigens to detect and quantify
substances, typically used in medical and biological research.
E.g.: Enzyme-Linked Immunosorbent Assay (ELISA) is a common immunological assay that
detects the presence of a specific substance (antigen) in a sample using antibodies.
5. Biochemical Assay: This type of assay involves analyzing the chemical processes within living
cells or organisms.
E.g.: Bioassays can be considered a form of biochemical assay, where the response of a living
organism is measured to determine the concentration or potency of a substance. For instance,
testing the response of a rabbit to detect endotoxins. (Pyrogen Test).
In this case, the response of a living organism (bioassay) is combined with the detection of a
chemical substance (endotoxin, a component of bacterial cell walls). The key lies in the
emphasis on living systems in bioassays and the chemical composition in chemical assays.
 List of Potential Problems or Considerations Associated with
Bioassays, especially when comparing them to Chemical Assays:
a. Environmental Factors Accuracy: Bioassays may be influenced by environmental factors such
as temperature, humidity, and other conditions that can affect living organisms. Chemical
assays, on the other hand, are often less susceptible to such environmental variations.
Consideration: Controlling environmental factors is crucial in bioassays to ensure accurate and
reproducible results.
b. Sample Size: Bioassays may require larger sample sizes, especially when using living
organisms, which can be logistically challenging and costly.
Consideration: It's essential to consider practical constraints and the ethical treatment of living
organisms when determining sample sizes for bioassays.
c. Mathematics as Pseudoscience: This might be a subjective statement. However, it's
important to recognize that both bioassays and chemical assays rely on statistical analyses, and
proper statistical methods are essential for drawing reliable conclusions.
Consideration: Good statistical practices are critical in both types of assays to ensure the
validity and reliability of results.
d. Specific Animals Requirement: Bioassays often require specific organisms or tissues, which
may raise ethical concerns and limit the availability of suitable subjects.
Consideration: Ethical considerations and alternative methods (such as cell cultures or in vitro
assays) should be explored to minimize the use of animals whenever possible.
e. Different Statistical Approaches: The statistical analysis in bioassays may differ from that in
chemical assays due to the complex responses of living organisms.
Consideration: Understanding and applying appropriate statistical methods tailored to the
nature of bioassays are crucial for accurate interpretation of results.
g. Franz Diffusion Cell and In Vitro Relationships: The Franz diffusion cell is commonly used for
in vitro studies, providing controlled conditions for studying the permeation of substances
through membranes.
Consideration: While in vitro assays offer controlled environments, the challenge lies in
extrapolating these results to predict in vivo responses accurately.
h. Chemical Assay vs. Bioassay Statistical Results: The statement suggests that chemical assays
may yield more reliable or consistent statistical results compared to bioassays.
Consideration: The choice between chemical and bioassays often depends on the specific goals
of the analysis, the nature of the substance being tested, and the regulatory requirements.
 BIOASSAY VS CHEMICAL ASSAY
PARAMETER BIOASSAY CHEMICAL ASSAY
Control Group: Typically involves a control group that is May not always involve a
subjected to the vehicle or a placebo to separate control group since
compare with the group exposed to the test the focus is on analyzing the
substance. chemical composition directly.
Number of Often requires a larger number of samples, Can sometimes be conducted
Samples: especially when using living organisms, with smaller sample sizes,
which can be logistically challenging. depending on the analytical
method.

Statistical Analysis: Statistical analysis in bioassays can be Statistical analysis is typically

complex due to the variability in living more straightforward and
systems, and different statistical approaches follows established methods for
may be needed. quantifying chemical
components.
Standard Sample It may be challenging to use a standard Relies on standard samples and
Animal sample animal due to factors such as genetic reference materials for
Availability: variations, ethical concerns, and availability. calibration and comparison.

Reproducibility: Reproducibility can be challenging due to the Generally, more reproducible,

complexity of living organisms, leading to as chemical reactions and
variations in responses even under measurements are subject to
seemingly identical conditions. less variability.

Temperature and Sensitivity to environmental variations, Less affected by environmental

Environmental including temperature differences, can factors, providing more control
Variations: impact the results. over experimental conditions.

Model Complexity The use of living organisms may raise ethical Generally, involves less ethical
and Ethical Issues: concerns, and the complexity of animal concerns, especially when using
models can limit experimental design. inanimate samples or chemical
reagents.

Time of Effect The time taken for an effect to occur may Results are often obtained more
vary, introducing additional challenges in rapidly, as they involve direct
experimental design and monitoring. chemical analyses.

Individual Living organisms exhibit individual variations Variations are more predictable
Variations: in responses, adding complexity to data and can be controlled through
interpretation. standardized procedures.
Model Requires the development of specific Focuses on direct analysis
Development: models, which may be time-consuming and without the need for complex
may not always accurately represent human models, providing more
conditions. immediate results.
 Vitamin D– BIOASSAY
The biological assay of vitamin D comprises the recording and interpretation of observations on groups
of rats maintained on specified dietary regimens throughout specified periods of their lives whereby the
biological response to the preparation under assay is compared with the response to USP Vitamin D
Capsules RS. USP Reference Standards 11 — USP Cholecalciferol RS.

Method Type: Biological assay for Vitamin D.

Subjects: Groups of rats.

Observations: Record and interpret observations.

Dietary Regimens: Rats on specified diets for defined life periods.

Comparison: Compare biological response to the tested substance with USP Vitamin D Capsules RS.

Reference Standards: Utilize USP Reference Standards 11, specifically USP Cholecalciferol RS.

Preliminary Period
“Throughout the preliminary period in the life of a rat, which is not longer than 30 days and extends
from birth to the first day of the depletion period, maintain litters of rats under the immediate
supervision of, or according to the directions of, the individual responsible for the assay. During the
preliminary period, use a dietary regimen that provides for normal development but is limited in its
content of vitamin D, so that when placed upon the Rachitogenic Diet in the depletion period the rats
develop rickets. At the end of the preliminary period, reject any rat that weighs less than 44 g or more
than 60 g, or that shows evidence of injury, disease, or anatomical abnormality.”

Period Duration: Up to 30 days from birth to the start of the depletion period.

Supervision: Rats under direct supervision or as per assay director's instructions.

Diet in Preliminary Period: Supports normal development but low in vitamin D.

Goal: Rats develop rickets on the Rachitogenic Diet in the depletion period.

Criteria for Rejection (End of Preliminary Period):

 Rats weighing under 44g or over 60g.

 Rats with evidence of injury, disease, or anatomical abnormalities.

Key Point: Preliminary period sets the stage for vitamin D assay, ensuring controlled conditions and
proper development for subsequent testing on the Rachitogenic Diet.
 Depletion Period
Through the depletion period, which extends from the end of the preliminary period to the first day of
the assay period, provide each rat ad libitum with the Rachitogenic Diet and water, and allow access to
no other food or dietary supplement.

Period Duration: From the end of the preliminary period to the start of the assay period.

Diet in Depletion Period:

 Rachitogenic Diet provided ad libitum (rats have unrestricted access to the specified
Rachitogenic Diet, allowing them to eat freely).
 Rats have access to water.
 No other food or dietary supplements allowed during this period.

Key Point: Depletion period involves sustained exposure to the Rachitogenic Diet and water, with strict
restrictions on other food or dietary supplements, ensuring a controlled environment for the vitamin D
assay.

Rachitogenic Diet
The Rachitogenic Diet consists of a uniform mixture of the following ingredients in the proportions
shown in the accompanying table. Rachitogenic Diet: (Ingredient - Parts by weight).

Whole yellow corn, ground – 76; Wheat gluten, ground – 20; Calcium carbonate – 3; Sodium chloride –
1. When a chemical analysis of the entire ration shows a Ca : P ratio of less than 4:1 or more than 5:1,
the proportion of calcium carbonate may be varied to bring the adjusted ratio to a uniform level within
this range.

Composition of Rachitogenic Diet:

Whole yellow corn, ground: 76 parts by weight.

Wheat gluten, ground: 20 parts by weight.

Calcium carbonate: 3 parts by weight.

Sodium chloride: 1 part by weight.

Ca:P Ratio Adjustment: Check the chemical analysis of the entire ration.

If Ca:P ratio is less than 4:1 or more than 5:1, adjust calcium carbonate proportion.

Ensure the adjusted ratio falls within the uniform range of 4:1 to 5:1.

Key Point: Rachitogenic Diet is a specific mixture with controlled proportions of ingredients, and
adjustments are made to maintain a consistent calcium-to-phosphorus (Ca:P) ratio within the desired
range during the vitamin D assay.
 Assigning Rats to Groups for Assay Period
Systematic approach to assigning rats to groups, ensuring representation, and maintaining balance for
accurate vitamin D assay results.

1. Litter (group of baby rats born to the same mother at the same time) Suitability for Assay Period:

“Consider a litter suitable for the assay period when individual rats in the litter show evidence of rickets
such as enlarged joints and a distinctive wobbly, rachitic gait, provided that the depletion period is not
less than 19 or more than 25 days.”

 Evidence of rickets in individual rats (enlarged joints, distinctive wobbly gait).

 Depletion period not less than 19 or more than 25 days.

2. Rickets Confirmation Methods:

“The presence of rickets may be established also from the width of the rachitic metaphysis upon X-ray
examination or by applying the Line Test (described below) to a leg bone of one member of each litter.”

 Clinical signs or X-ray examination (width of rachitic metaphysis).

 Line Test application to a leg bone.

3. Weight Recording and Group Assignment:

“Record the weight of each rat, and assign it to a group, in which each rat will be fed a specified dose of
the Reference Standard or of an assay sample that is under examination for its vitamin D potency. For
each assay sample provide one or more assay groups and not less than two standard groups. The two
standard groups may be used for the concurrent assay of more than one assay sample.”

 Record weight of each rat.

 Assign rats to groups based on a specified dose of Reference Standard or assay sample for
vitamin D potency examination.
 Provide at least two standard groups and one or more assay groups for each assay sample.

4. Assignment Design:

“Within an interval not exceeding 30 days, complete the assignment of rats to groups according to a
design that divides litters among the groups, to achieve a complete balance. For complete balance,
whereby each litter is represented equally in every group, use 7 or more litters containing at least as
many depleted rats as there are groups.”

 Complete assignment within 30 days.

 Achieve complete balance by dividing litters among groups.
 Use 7 or more litters with depleted rats equal to or more than the number of groups.

5. Balancing Method:

“From a given litter, assign one rat, selected at random, to each group on the same day. If a litter
contains twice as many rats as there are groups, assign a second series of rats similarly.”

 Assign one rat per group from each litter selected at random.
  If a litter has twice as many rats as groups, assign a second series of rats similarly.

6. Weight Balance:

“The last one or two litters to be assigned may be allotted to groups so that at the start of the assay
period the average body weight of any completed groups will not differ by more than 8 g from that of
any other group.”

 Last one or two litters assigned to ensure the average body weight difference is not more than
8g among completed groups at the start of the assay period.

Assay Doses
Methodical selection of dosage levels, ensuring ratio criteria, and consideration of factors like
calcification expectations and appropriate pre-feeding measures for accurate vitamin D assay results.

USP Cholecalciferol RS Dosage Selection:

“Select two dosage levels of the USP Cholecalciferol RS, spaced so that the ratio of the larger to the
smaller dose is not less than 1.5 or more than 2.5.”

 Number of Doses: Choose two dosage levels of USP Cholecalciferol RS.

 Ratio Requirement: Ensure the ratio of the larger to the smaller dose falls between 1.5 and 2.5.

Sample Dosage Levels:

“Select one or two dosage levels based upon a single assumed potency for each sample. The dosage
levels of the sample are equivalent to those of the standard or to a mid-level equal to the square root of
the product of the two dosage levels of the standard.”

 Selection Basis: Choose one or two dosage levels for each sample.
 Assumed Potency Basis: Determine dosage levels based on a single assumed potency for each
sample.
 Euivalence to Standard: Set sample dosage levels equivalent to the standard or a mid-level
calculated as the square root of the product of the two dosage levels of the standard.

Calcification Expectation in Rats:

“Select dosage levels such that, when fed to rachitic rats, they are expected to produce degrees of
calcification within the range specified under the test of data acceptability.”

 Purpose: Dosage levels for both standard and samples should induce calcification in rachitic rats.
 Acceptable Range: Calcification levels must fall within the specified range under the test of data
acceptability.

Pre-feeding Considerations:

“Before feeding, the Reference Standard and/or sample may be diluted with cottonseed oil, provided
that not more than 0.2 mL is fed on any one day. Store the oil solutions in well-closed bottles, protected
from light, at a temperature not exceeding 10 , and use within 5 weeks..”
  Dilution: Reference Standard and/or sample can be diluted with cottonseed oil before feeding.
 Limit per Day: Not more than 0.2 mL should be fed on any given day.
 Storage: Store oil solutions in well-closed bottles, protected from light, at a temperature not
exceeding 10°C.
 Expiration: Use the diluted solutions within 5 weeks.

Group Assignment:

“Assign one group of rats to each dosage level of the standard and of the one or more samples.”

 Rat Group Assignment: Assign one group of rats to each dosage level of the standard and each
sample.

ASSAY PERIOD
“During the assay period, which extends from the end of the depletion period for a fixed interval of 7 to
10 days, cage each rat individually and provide it ad libitum with the Rachitogenic Diet and water. Supply
a Rachitogenic Diet prepared from the same lots of ingredients to all rats. On the first and on the third
(or fourth) day of the assay period, feed each rat one-half of its total assigned dose. Throughout the
assay period, maintain as uniform environmental conditions as possible for all rats, and exclude
exposure to antirachitic radiations. At the end of a fixed period of 7 to 10 days, weigh and kill each rat.
From those rats that do not weigh less at the end than at the start of the assay period and that have
consumed each assigned dose within 24 hours of the time it was fed, dissect out one or more leg bones
for examination by the Line Test.”

Period Duration: Assay period extends from the end of the depletion period for 7 to 10 days.

Housing and Diet:

 Individual Caging: Each rat is housed individually during the assay period.
 Diet Provided: Rats are given ad libitum access to the Rachitogenic Diet and water.
 Uniform Diet: All rats receive Rachitogenic Diet prepared from the same lots of ingredients.

Dosage Administration: On the first and third (or fourth) day of the assay period, each rat is fed one-half
of its total assigned dose.

Environmental Conditions:

 Uniformity: Maintain as uniform environmental conditions as possible for all rats throughout
the assay period.
 Exclusion: Exclude exposure to antirachitic radiations during the assay period.

Endpoint Procedures:

 Weighing and Killing: At the end of the fixed 7 to 10 days, weigh and euthanize each rat.
 Selection Criteria: Select rats that do not weigh less at the end than at the start and have
consumed each assigned dose within 24 hours.
  Bone Examination: Dissect out one or more leg bones from selected rats for examination by the
Line Test.

Rigorous procedures during the assay period, including controlled diet, dosage administration,
environmental conditions, and careful endpoint assessments, are critical for accurate vitamin D assay
results.

LINE TEST
“Remove the proximal end of a tibia or the distal end of a radius, and clean adhering tissue from it, in
any one assay using the same bone from all animals. With a clean, sharp blade cut a median,
longitudinal section through the juncture of the epiphysis and diaphysis at the same place on each bone.
Rinse both sections in purified water, immerse immediately in silver nitrate solution (1 in 50) for 1
minute, and rinse again in purified water. Expose the cut surface of bone, in water, to daylight or
another source of actinic light until the calcified areas develop a clearly defined stain without marked
discoloration of the uncalcified areas. The staining procedure may be modified to differentiate more
clearly between calcified and uncalcified areas. Score the degree of calcification of the rachitic
metaphysis in each rat, according to a scale that allows the average response to be plotted as a straight
line against the logarithm of the dose.”

Bone Selection:

 Bone Type: Tibia or radius.

 Consistency: Same bone used from all animals in one assay.

Preparation Steps:

 Proximal/Distal End Removal: Remove the proximal end of a tibia or the distal end of a radius.
 Tissue Cleaning: Clean adhering tissue from the selected bone.
 Uniform Sectioning: Cut a median, longitudinal section through the juncture of the epiphysis and
diaphysis at the same place on each bone.

Staining Process:

 Rinse and Clean: Rinse sections in purified water after cutting.

 Silver Nitrate Solution: Immerse sections in silver nitrate solution (1 in 50) for 1 minute.
 Rinse Again: Rinse sections again in purified water.
 Exposure to Light: Expose the cut surface of the bone to daylight or actinic light until calcified
areas develop a defined stain without marked discoloration of uncalcified areas.
 Optional Modification: Staining procedure can be modified for clearer differentiation between
calcified and uncalcified areas.

Scoring Process:

 Calcification Assessment: Score the degree of calcification of the rachitic metaphysis in each rat.
 Scoring Scale: Use a scale allowing the average response to be plotted as a straight line against
the logarithm of the dose.
 ACCEPTABILTIY
“Observations are acceptable for use in calculation of the potency only from those groups in which two-
thirds or more but not less than 7 rats show calcification at least as great as the lowest level and not
greater than the highest level. If the average score of the standard group on the high dosage level is not
greater than the average score of the standard group on the low dosage level, discard the results, and
repeat the assay. If an assay sample is represented solely by assay groups that are not acceptable for
measuring vitamin D potency and in each of which the average score is less than the average score of
the standard group on the low dosage level or more than the average score of the standard group on
the high dosage level, its assayed content of vitamin D is respectively less than that represented by the
low dose or more than that represented by the high dose of the Reference Standard.”

1. Observation Inclusion Criteria:

 Calcification Requirement: Acceptable observations for potency calculation are limited

to groups where two-thirds or more, but not less than 7 rats, exhibit calcification.

 Calcification Level Range: Calcification levels should be at least as great as the lowest
level and not greater than the highest level.

2. Standard Group Comparison:

 Comparison Basis: Assess the average score of the standard group on the high dosage
level against that on the low dosage level.

 Acceptability Condition: If the high dosage level average score is not greater than the
low dosage level average score, discard results, and repeat the assay.

3. Assay Sample Assessment:

 Acceptability Condition: If an assay sample is solely represented by unacceptable assay

groups:

 Average score in each group is less than the low dosage level standard group.

 Or, average score in each group is more than the high dosage level standard
group.

4. Implications for Assay Sample Content:

 Inferior Content: If average scores are consistently less, the assayed content of vitamin
D is less than that represented by the low dose of the Reference Standard.

 Excessive Content: If average scores are consistently more, the assayed content of
vitamin D is more than that represented by the high dose of the Reference Standard.

Key Point: Acceptability criteria ensure reliable observations for vitamin D potency calculation, with
specific conditions for standard group comparisons and implications for assay sample content.
 CALCULATIONS
1. Data Tabulation:

 Scores (y): Tabulate scores, listing each litter in a separate row with treatment groups in
columns.

 Omission Criteria: Omit groups that do not meet the Acceptability test.

2. Equalization of Observations:

 Equalize Observation Numbers: Ensure an equal number of observations in acceptable

groups.

 Disregard Non-Represented Litters: Disregard results from litters not equally

represented in the groups.

3. Total Scores Calculation:

 Total Scores (T1 and T2): Sum the f scores for each treatment group, designating totals
as T1 and T2 for low and high dosage levels, respectively.

4. Slope (b) Computation:

 Equation: Compute the slope (b) using the equation: b = (ST2 – ST1) / ifh¢.

 Parameters: i is the logarithm of the ratio of high dose to low dose, and h¢ is the
number of preparations represented at both dosage levels.

5. Logarithm of Relative Potency:

 Equation: Compute the logarithm of the relative potency using the equation:
Log(Relative Potency) = (bar(y)U - bar(y)S) / (Tb - Ta).

 Parameters: bar(y)U and bar(y)S are mean scores for the assay sample and Reference
Standard, respectively. Tb and Ta are defined parameters.

6. Conversion to Relative Potency:

 Antilogarithm: Convert each observed M ¢ (logarithm of relative potency) to its

antilogarithm.

 Result: Obtain the relative potency of the sample.

7. Assayed Content Calculation:

 Multiplication: Multiply the relative potency by the assumed potency of the assay oil in
Units per g.

 Result: Obtain the assayed content of vitamin D in USP Units per g.

 INSULIN ASSAY
1. Insulin Activity and Blood Glucose:

 Insulin's primary activity is the rapid reduction of blood glucose levels.

 Biologic assay based on this blood glucose decrease has been a key method since the early
clinical use of insulin.

2. Evolution of Assay Methods:

 Over time, physicochemical methods like liquid chromatography have been developed for
quantitative measurement of insulin potency.
 These methods are practical and sophisticated, providing accurate results for insulin and its
products.

3. Bioidentity Assessment Challenges:

 Physicochemical methods do not assess the bioidentity of insulin and its products.

4. Importance of Rabbit Blood Sugar Method:

 The chapter emphasizes the Rabbit Blood Sugar Method as a crucial test.
 This method is specifically included for assessing the bioidentity of insulin and insulin products in
rabbits.

5. Applications of Rabbit Blood Sugar Method:

 Used for determining the potency of Insulin Reference Standards.

 Employed in validating the stability of new insulin preparations.
 Applied to determine specific activities of insulin analogs.

6. Compendial Test Enhancement:

 The Rabbit Blood Sugar Method complements physicochemical methods, enhancing the overall
accuracy and precision of insulin testing.

7. Call for Bioidentity Test in Monographs:

 The chapter advocates for the inclusion of the Rabbit Blood Sugar Method in appropriate
monographs.
 Underlines the importance of bioidentity assessment for insulin and insulin products.

The discussion highlights the historical use of blood glucose decrease for insulin activity assessment, the
evolution of quantitative methods, the challenge of bioidentity assessment, the significance of the
Rabbit Blood Sugar Method, and its applications in insulin testing and validation.
 ASSAY METHOD
1. Diluent Composition:

 Prepare the aqueous solution with 0.1%–0.25% (w/v) cresol or phenol, 1.4%–1.8% (w/v)
glycerin, and hydrochloric acid to achieve pH 2.5–3.5, unless stated otherwise.

2. Standard Stock Solution:

 Prepare solution containing 40 USP Insulin Units/mL of USP Insulin RS of appropriate species
in Diluent, with pH 2.5–3.5, unless directed otherwise.
 For insulin of mixed bovine & porcine species, prepare a solution containing 34.8 USP Insulin
Beef Units/ml & 5.2 USP Insulin Pork Units/mL in Diluent and have pH btw 2.5-3.5
 Storage recommendation: Cold place, protected from freezing, use within 6 months.

3. Standard Solutions:

 Prepare dilutions of Standard stock solution in Diluent to create two solutions: 1.0 USP
Insulin Unit/mL (Standard solution 1) and 2.0 USP Insulin Units/mL (Standard solution 2).

4. Sample Stock Solution:

 Similar to Standard stock solution but uses the preparation under test instead of USP Insulin
RS, with about 40 USP Insulin Units/mL.

5. Sample Solutions:

 Prepare dilutions of Sample stock solution in Diluent to create two solutions: 1.0 USP Insulin
Unit/mL (Sample solution 1) and 2.0 USP Insulin Units/mL (Sample solution 2).
 pH adjustment to 2.5–3.5 for neutral insulin injection.

6. Injection Doses:

 Selection of injection doses based on trial or experience, typically between 0.30 and 0.50
mL.
 Equal volumes for Standard and Sample solutions for each animal.

Preparation of Animal:
7. Animal Selection and Care:

 Choose healthy rabbits weighing at least 1.8 kg.

 Acclimate rabbits in the laboratory for a minimum of 1 week on a consistent diet with
continuous access to water before use in the assay.
 Analysis:
8. Grouping and Feeding Protocol:

 Divide rabbits into four equal groups, with a preference for no less than six rabbits in each
group.
 Provide each rabbit with a defined amount of food 20 hours before the assay, ensuring
consumption within 6 hours.
 Maintain the same feeding schedule before each test day.
 Withhold all food during the assay until after the final blood specimen is taken.

9. Injection Procedure:

 Handle rabbits with care to prevent undue excitement.

 Administer subcutaneous injections based on the design outlined in Table 1.
 Ensure the second injection is given on the day after the first injection or no more than 1
week later.
 Maintain a consistent time interval between the first and second injections for all rabbits.

10. Injection Design (Table 1):

 Group 1: First injection - Standard solution 2, Second injection - Sample solution 1. (2.1)
 Group 2: First injection - Standard solution 1, Second injection - Sample solution 2. (1.2)
 Group 3: First injection - Sample solution 2, Second injection - Standard solution 1. (2.1)
 Group 4: First injection - Sample solution 1, Second injection - Standard solution 2. (1.2)

1. Blood Sampling:

 Obtain suitable blood specimens from marginal ear veins at 1 hour ± 5 minutes and 2.5
hours ± 5 minutes after injection.

 Effective blood collection can also be performed from the central auricular artery.

2. Dextrose Determination:

 Determine dextrose content in blood specimens using a procedure adapted for

automated analysis.

The following procedure maybe used:

i. Anticoagulant Solution:
a. Prepare an anticoagulant solution by dissolving 1 g of edetate sodium and 200 mg of
sodium fluoride in 1 L of water and mix.
ii. Dextrose Standard Preparations:
a. Transfer known concentrations of USP Dextrose RS to suitable vessels.
b. Dilute quantitatively and stepwise with Anticoagulant solution (1:9) to create a
range of Dextrose standard preparations.
 c. Aim for concentrations between 20 and 100 mg per 100 mL, matching the
concentrations in rabbit blood samples.
iii. Sample Preparations:
a. Pipet 0.1 mL of each blood sample into separate vessels.
b. Add 0.9 mL of Anticoagulant solution to each vessel.

iv. Analysis Procedure:

a. Subject Sample preparations to dialysis across a semipermeable membrane for a
sufficient time so that dextrose passes.
b. Allow dextrose to pass through the membrane into a saline TS solution containing
glucose oxidase, horseradish peroxidase, 3-methyl-2-benzothiazolinone hydrazone
hydrochloride TS, and N,N-dimethylaniline.
c. Determine absorbances of Sample preparations at 600 nm using a recording
colorimeter.
d. Similarly, determine absorbances of Dextrose standard preparations at the start and
end of each run.

Calculation and Analysis:

1. Individual Response Calculation:

 Calculate the response of each rabbit to each injection by summing the two blood sugar
values.

 Subtract responses disregarding the chronological order to obtain individual differences

(y) as shown in Table 2.

 When the data for one or more rabbits are missing in an assay, do not use the
confidence interval formulas given here, but seek statistical help.

 The data can still be analyzed with proper analysis of variance (ANOVA). ANOVA is a
statistical method used to analyze the differences among group means in a sample. It
can handle situations where data points are missing, and statistical experts can apply
techniques to address such missing data in a way that doesn't compromise the validity
of the analysis.

2. Total Response (T) Calculation:

 When the number of rabbits (f), carried though the assay is the same in each group,
total the y’s in each group and compute:

 Ta=−T1+T2+T3−T4

 Tb=T1+T2+T3+T4

3. Logarithm of Relative Potency of the Test Dilutions (M′):

 Calculate M′ using the formula: M′=0.301Ta/Tb.

 4. Potency Calculation:

 The potency of the injection in USP Units/mg equals the antilog of (logR+M′), where:

 R = Vs/Vu

 Vs = number of USP Units/mL of the Standard solution

 Vu = number of mg/mL of insulin of the corresponding Sample solution

5. Confidence Interval Determination:

 Use Fieller’s Theorem to determine the 95% confidence interval for the log-relative
potency.

 If the confidence interval is more than 0.082, which corresponds at P = 0.95 to

confidence limits of about ±10% of the computed potency, repeat the assay until the
combined data of two or more assays, redetermined as described in Combination of
Independent Assays, meet the acceptable limit.

6. Repetition of Assay:

 If needed, repeat the assay until the combined data of two or more assays meet the
acceptable limit.

 Redetermine the combined data as described in Combination of Independent Assays.

Table 2:

 Group-wise presentation of differences (y), total responses (T), and standard deviations of
differences (S).

Appendix - Fieller’s Theorem:

 Provides the formula for calculating the 95% confidence interval for the ratio with specific
parameters and conditions.
 Bioidentity Test
Proceed as directed in Rabbit Blood Sugar Method—Quantitative with the following modifications.

1. Procedure Modification:

 Divide rabbits into four equal groups of two rabbits each.

2. Calculation Modification:

 Proceed with the calculation as directed in the Rabbit Blood Sugar Method—
Quantitative, but do not determine the confidence interval of the log-relative potency,
M′.

3. Interpretation:

 If the potency value obtained is at least 15 USP Units/mg, the Bioidentity Test
requirement is met.

 If the potency value is less than 15 USP Units/mg, repeat the test using eight more
rabbits.

 If the average potency of the two sets of tests is at least 15 USP Units/mg, the
requirement of the test is met.

ADDITIONAL REQUIREMENTS

Change to read:

• USP REFERENCE STANDARDS:

 USP Dextrose RS
 USP Insulin Aspart
 USP Insulin Glargine RS
 USP Insulin Human RS
 USP Insulin Lispro RS
 USP Insulin Pork RS
 USP DIGITALIS
1. Source of Digitalis:

 Digitalis is derived from the dried leaf of Digitalis purpurea Linné, belonging to the
Scrophulariaceae family.

2. Potency:

 The potency of Digitalis is standardized (when assayed as directed) so that 100 mg is

equivalent to a minimum of (NLT) 1 USP Digitalis Unit.

3. Dispensing Form:

 When prescribed, Powdered Digitalis is the recommended form for dispensing.

4. Packaging, Storage, and Labeling:

 Digitalis should be preserved in containers that prevent moisture absorption.

 Digitalis labeled specifically for use in glycoside manufacture is exempt from certain
moisture and storage requirements.

5. USP Reference Standards:

 USP Digitalis RS (Reference Standard) is available for ensuring quality and consistency.

Quality Standards of Digitalis

1. Acid-Insoluble Ash (561): The acid-insoluble ash content in Digitalis should not exceed 5.0%.

2. Foreign Organic Matter (561): The presence of stems, browned leaves, flowers, and other
foreign organic matter in Digitalis should not exceed 2.0%.

3. Water Content (Method III, Procedure for Articles of Botanical Origin 921): The water content
in Digitalis, determined by Method III (Procedure for Articles of Botanical Origin 921), should not
exceed 6.0%.

4. Organic Volatile Impurities (Method IV): Digitalis must meet the requirements specified for
organic volatile impurities according to Method IV (467).

5. Residual Solvents (467): Quality Standard: Digitalis must meet the requirements specified for
residual solvents according to Method IV (467).
 Assay Procedure
1. Standard Preparation:

This summary outlines the steps involved in preparing the standard for the assay of USP Digitalis,
including weighing, adding a specific menstruum, shaking, centrifuging, and preserving the tincture
for subsequent use within a specified timeframe.

 Weigh the contents of 1 container of USP Digitalis RS to the nearest mg, either in the
original container or in a weighing bottle.
 Transfer the weighed content to a dry, hard-glass, glass-stoppered container or centrifuge
tube (at least 50 mL capacity).
 Complete the weighing within 5 minutes after opening the ampul.
 Add a menstruum consisting of 4 volumes of alcohol and 1 volume of water, ensuring a total
volume of menstruum corresponding to 10 mL for each gram of powder.
 Grease the upper third of the stopper lightly with petrolatum. And insert the stopper.
 Shake the mixture for 24 ± 2 hours at 25 ± 5 degree by mechanical means, ensuring
continuous contact of solid material with the liquid phase.
 Immediately transfer, if necessary, to a centrifuge tube, centrifuge, and decant the
supernatant tincture.
 Transfer the tincture into a dry, hard-glass bottle with a tight closure.
 Preserve under refrigeration.
 Use within 30 days.

2. Assay Preparation:

Digitalis Sample:

 Transfer about 5 g of finely powdered Digitalis, accurately weighed, to a hard-glass, glass-

stoppered container or centrifuge tube (at least 50 mL capacity).
 Follow the Standard preparation procedure, starting with "Add a menstruum."
o Add a menstruum consisting of 4 volumes of alcohol and 1 volume of water,
ensuring a total volume of menstruum corresponding to 10 mL for each gram of
powder.
o Grease the upper third of the stopper lightly with petrolatum. And insert the
stopper.
o Shake the mixture for 24 ± 2 hours at 25 ± 5 degree by mechanical means, ensuring
continuous contact of solid material with the liquid phase.
o Immediately transfer, if necessary, to a centrifuge tube, centrifuge, and decant the
supernatant tincture.
o Transfer the tincture into a dry, hard-glass bottle with a tight closure.
 Preserve the prepared solution under refrigeration.
 Use within 30 days.
3. Pigeons for Assay:

i. Selection of Pigeons:

 Use adult pigeons free from gross evidence of disease or emaciation.

 Ensure pigeons have weights such that the heaviest weighs less than twice the weight of the
lightest.
 Divide pigeons into groups with similar breeds and weights.
 Ensure that the average weight of the group assigned to the Standard preparation does not
differ by more than 30% from the group assigned to the preparation to be assayed.

ii. Preparation for Assay in Pigeons:

 Withhold food, but not water, during the period 16 to 28 hours prior to use.
 Lightly anesthetize the pigeon with ether preparatory to injection.
 Immobilize the pigeon.
 Expose an alar vein and cannulate it with a suitable cannula.
 Maintain anesthesia during cannulation at a level where pain is absent, pupillary and
corneal reflexes are present, and voluntary musculature is not relaxed beyond permitting
occasional voluntary movement.

4. Preparation of Test Dilutions:

On the Day of Assay:

 Dilute portions of the Standard preparation and the Assay preparation with isotonic sodium
chloride solution.
 Ensure the estimated fatal dose for each dilution is 15 mL per kg of body weight.

5. Injection of Test Dilutions:

Arrangement for Injection:

 Use suitable means, such as a small-bore buret calibrated to 0.05 mL.

 Start the injection after ensuring the absence of air bubbles from the injection
apparatus.
 Infuse a volume of the test dilution equivalent to 1 mL per kg of body weight within a
few seconds.
 Repeat the dose at 5-minute intervals until the pigeon dies of cardiac arrest.

6. Assay Criteria:

 Use not less than 6 pigeons for the Standard Preparation and not less than 6 pigeons for the Assay
Preparation.
 If the average number of doses for a given dilution is less than 13 or greater than 19, or if the larger
exceeds the smaller in the same assay by more than 4 doses, consider these data as preliminary.
 Use them as a guide and repeat with a fresh, higher, or lower dilution.

Assay Completion: Complete the assay within 30 days to comply with the preservation period for the
Standard preparation and Assay preparation.
 Calculation of Potency
1. Tabulation and Averaging:

 Tabulate and average the number of doses of the Standard preparation, designating the
average bar(Z)s.

 Obtain the corresponding average bar(Z)u, for the Assay preparation.

2. Compute Potency:

 Compute the potency in USP Digitalis Units per mL (i.e. per 100 mg) of the Assay
preparation using the formula:

Potency = bar(Z)s R / bar(Z)u

where R equals Vs/ Vu, in which

 Vs is the number of USP Digitalis Units per mL of Standard preparation

dilution,
 Vu is the volume, in mL, of Assay preparation per mL of dilution.

3. Compute Confidence Interval (L):

 Compute the confidence interval using Equation (31) under Confidence Intervals for
Individual Assays in Design and Analysis of Biological Assays 111.

 If L exceeds 0.30, repeat the assay or inject more pigeons with one or both preparations
until the confidence interval is 0.30 or less.

4. Potency Evaluation:

 The potency of Digitalis, calculated from that of the Assay preparation, is satisfactory if
the result is not less than 0.85 USP Digitalis Unit per 100 mg.

5. Additional Information:

 One USP Digitalis Unit represents the potency of 100 mg of USP Digitalis RS.
 Belladonna Extract (to read only)
Assay Procedure:

1. pH 9.5 Phosphate Buffer:

 Dissolve 34.8 g of dibasic potassium phosphate in 900 mL of water.

 Adjust to pH 9.5 electrometrically using 3 N hydrochloric acid or sodium hydroxide.

2. Internal Standard Solution:

 Dissolve about 40 mg of USP Homatropine Hydrobromide RS in about 25 mL of dilute

sulfuric acid (1 in 350) in a 50-mL volumetric flask.
 Add the same dilute acid to volume and mix. Prepare fresh on the day of use.

3. Standard Preparation:

 Dissolve about 10 mg of USP Scopolamine Hydrobromide RS in about 5 mL of dilute sulfuric

acid (1 in 350) in a 10-mL volumetric flask (Solution A).
 Dissolve about 20 mg of USP Atropine Sulfate RS in about 25 mL of dilute sulfuric acid (1 in
350) in a 50-mL volumetric flask.
 Add 2.0 mL of Solution A, mix, add dilute sulfuric acid (1 in 350) to volume, and mix. Prepare
fresh on the day of use.

4. Extraction Blank:

 Place about 10 mL of dilute sulfuric acid (1 in 350) in a 60-mL separator.

 Proceed as directed under Assay preparation, starting with "then add 15 mL of chloroform."
 The blank chromatogram should contain no significant interferences at the locus of
atropine, scopolamine, or homatropine.

5. Assay Preparation:

 Weigh accurately about 0.5 g of Extract, transfer to a 125-mL conical flask, and add 40 mL of
dilute sulfuric acid (1 in 350).
 Heat to a temperature not above 45°C, stir to hasten solution, filter through filter paper into
a 100-mL volumetric flask.
 Wash flask and filter with two 20-mL portions of warmed dilute sulfuric acid (1 in 350),
collect washings in the flask.
 Add dilute sulfuric acid (1 in 350) to volume, mix, pipet 10 mL into a 60-mL separator.
 Add 1.0 mL of Internal standard solution, then add 15 mL of chloroform, shake, allow layers
to separate, and discard the chloroform layer.
 Add another 15 mL of chloroform, extract again, discard chloroform phase.
 Add 15 mL of pH 9.5 Phosphate buffer and 1 N sodium hydroxide to yield a final pH between
9.0 and 9.5.
 Add 15 mL of chloroform, shake, allow layers to separate, filter organic phase through
sodium sulfate into a suitable container.
 Extract again with two 15-mL portions of chloroform, collecting clarified organic phase.
  Wash sodium sulfate and funnel tip with 5 mL of chloroform.
 Evaporate combined organic phases under reduced pressure, at a temperature below 45°C.
 Add 1 mL of chloroform, mix to dissolve alkaloids, wetting sides of the container.

6. Standard Curve:

 Prepare three Standard solutions using Standard preparation.

 Pipet into three separate 60-mL separators 1.0-, 2.0-, and 3.0-mL portions, respectively, of
Standard preparation.
 Add 9.0, 8.0, and 7.0 mL, respectively, of dilute sulfuric acid (1 in 350).
 Proceed as directed under Assay preparation, starting with "add 1.0 mL of Internal standard
solution."

7. Chromatographic System:

 Use a gas chromatograph with a 1.2-m × 4-mm glass column packed with 3% G3 on S1AB.
 Maintain the column at about 215°C, injection port and detector block at about 240°C.
 Use dry helium as a carrier gas at a flow rate of about 65 mL per minute.

8. System Suitability:

 Chromatograph six to ten injections of the solution and assess system suitability based on
specified criteria.

9. Procedure:

 Inject a portion (about 5 µL) of each Standard solution into the gas chromatograph with a
flame-ionization detector.
 Measure peak areas for atropine (aA), homatropine (aH), and scopolamine (aS).
 Calculate ratios AA and AS (aA / aH and aS / aH).
 Plot Standard curves for RA and RS against amounts of atropine and scopolamine.
 Inject a portion of the Assay preparation into the chromatograph, obtain chromatogram
area ratios, and calculate the area ratios.
 Record quantities of atropine and scopolamine in the volume of specimen taken from the
Standard curve.
 Add the quantities and multiply by 10 to obtain the weight of alkaloids in the portion of
Extract taken.