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Sections in This Document

Purpose ....................................................................................................................................2

Scope .......................................................................................................................................3

Responsibility............................................................................................................................3

Background...............................................................................................................................4

FDA Resource Links.................................................................................................................6

Pharmaceutical Organization Resource Links..........................................................................6

Reading Material (Most can be accessed through the FDA Library)........................................7

Procedure .................................................................................................................................8

8.1. Methodology...................................................................................................................8

8.1.1. Introduction ......................................................................................................8

8.2. Methodology Sources ....................................................................................................8

8.2.1. Official Compendia...........................................................................................8

8.2.2. Petition Methods (NDAs and ANDAs)..............................................................9

8.2.3. AOAC Official Methods ..................................................................................10

8.2.4. Pharmacopeial Forum....................................................................................10

8.2.5. Compliance Programs ...................................................................................10

8.2.6. Code of Federal Regulations .........................................................................10

8.2.7. Journals and Other Literature ........................................................................10

8.2.8. Questions.......................................................................................................11

8.3. Pharmaceutical Chemistry ...........................................................................................11

8.3.1. Pharmaceutical Products Overview ...............................................................12

8.3.2. Questions.......................................................................................................13

8.4. Instrumentation and Technique....................................................................................14

8.4.1. Basic Analytical Techniques ..........................................................................14

8.4.2. Ultraviolet-Visible Spectroscopy (USP <857>) and Ultraviolet-Visible 

Spectroscopy-Theory and Practice (USP <1857>)........................................16

8.4.3. Mid-Infrared Spectroscopy (USP <854>) and Mid-Infrared Spectroscopy-Theory 

and Practice (USP <1854>)...........................................................................17

8.4.4. Optical Rotation/Polarimeter (USP <781>)....................................................19

8.4.5. Chromatographic Techniques........................................................................20

8.4.6. Column Chromatography (USP <621>).........................................................20

8.4.7. Thin Layer Chromatography (TLC) (USP <621> and <201>)........................21
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8.4.8. Gas Chromatography (GC) (USP <621>)......................................................22

8.4.9. High Performance Liquid Chromatography (HPLC) (USP <621>).................24

8.4.10. Other Specialized Techniques .......................................................................27

8.4.11. Dissolution (USP <711>) ...............................................................................27

8.4.12. Advanced Techniques ...................................................................................29

8.4.13. Inspections/Investigations..............................................................................29

Glossary/Definitions................................................................................................................30

Records ..................................................................................................................................30

Document History ...................................................................................................................31

Change History.......................................................................................................................31

Attachments............................................................................................................................34

Attachment A - Exercise: Practice with Samples....................................................................35

Attachment B - Proficiency Samples ......................................................................................40

Attachment C - Statistical Analysis.........................................................................................42

Attachment D - Answer Key....................................................................................................45

Attachment E - Chromatography Chart ..................................................................................63

Purpose

The drug analysis training program provides preliminary training in basic 

technical areas. The Office of Training, Education and Development (OTED) 

offers New Hire Laboratory Analyst Training curriculum (Bingo card) which 

specifies sections of the ORA Lab Manual as part of the New Hire Curriculum. 

Described are drug analyses for new hire training and suggested on-the-job 

training samples of selected techniques. Additional techniques and training 

exercises are included that are occasionally used in drug analysis.

The Center for Drugs and Evaluation Research (CDER) is responsible for 

establishing the inspectional and analytical guidelines associated with the FDA

drug analysis programs.

This training chapter introduces the trainee to regulatory pharmaceutical 

analyses in FDA, analytical separation techniques, instrumentation, and the 

resolution to typical problems a trainee will encounter with pharmaceutical 

analyses. The trainee should discuss each exercise in advance with the 

trainer. If a laboratory does not perform analyses in that area, that portion of 

the training can be abbreviated or eliminated.
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The training is separated into modules using defined pharmaceuticals. Before 

beginning the sample analysis, the trainer should consider and discuss topics 

that will cultivate a thoughtful and responsible approach to each assignment. 

Topics may include the following: 

A. Why was the sample collected? (Assume it was not a training sample.) 

Does the sample match the description on the collection report?

B. Criteria for method selection. What care is taken in selecting an 

analytical method? Is the method the correct one? Is the method valid?

C. Correct sample handling. Is there a microbiological analysis associated 

with this sample that may necessitate the microbiologists to handle the 

sample first?

D. Is there enough sample for all the regulatory tests? For an additional 

and check analysis? For an exhibit, if needed?

E. Does the sample have an expiration date that would impact the validity 

of test results?

F. What regulatory action might result from the analysis of the sample? 

When the trainee has completed each exercise, the analysis, worksheet 

preparation, and answers to the questions should be discussed thoroughly. 

The analyst also shows that they are proficient in the technique and 

instrumentation.

Scope

The incumbent is a regulatory scientist in an FDA/ORA field laboratory and is 

expected to bring full professional competence in his/her discipline to bear in 

carrying out analyses and interpreting the significance of test results. The 

incumbent is assigned scientific analyses on a wide range of samples 

including those that are difficult, complex, or unusual.

These responsibilities support the FDA and it mission to support public health 

through the regulation of products and manufacturers under the jurisdiction of 

the Federal Food, Drug, and Cosmetic Act (FD&C Act) and Related Acts.

Responsibility

A. Trainer

1. Coordinates training with Supervisor(s) and Trainee.

2. Works with trainees to ensure completion of all training needed to 

meet their regulatory responsibilities.
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B. Trainee

1. Completes required training within specified timeframes.

2. Reports training received. 

3. Submits documentation for training to supervisor.

C. Supervisor

A. Implements and reviews training records.

D. Quality Management

1. Maintains employee competencies matrix records for staff.

Background

The Food and Drug Act of June 30,1906 prohibited adulteration and 

misbranding of drugs in interstate commerce. Following an adverse ruling 

(United States v. Johnson) by the Supreme Court of the United States in 1911, 

Congress passed the Sherley Amendment the same year, which added to the 

Act a prohibition against claims of curative or therapeutic effects being placed 

on the package label with intent to defraud the purchaser. The burden of proof 

of adequacy of claims, however, was on the government. Prior testing of drugs 

for safety was not addressed. 

In early 1938, the Wheeler Lea Amendment to the Federal Trade Commission 

(FTC) Act clarified the jurisdiction of advertising between the FTC and the 

FDA. The FTC reserved control of drug advertising. The weakening of FDA 

authority, in conjunction with the “Elixir of Sulfanilamide” drug related disaster, 

paved the way for the passage of a completely new law.

The "Elixir of Sulfanilamide" tragedy of 1937 killed 107 people due to the use 

of the solvent diethylene glycol in the product. Since the law did not require 

prior testing of drugs for safety, there was no way to anticipate, or prevent, the 

marketing of this lethal mixture.

The Federal Food, Drug, and Cosmetic Act (FD&C Act) was passed on June 

25, 1938. The new law prohibited the marketing of "new drugs," (a term 

defined in the FD&C Act), unless the new drug had been tested and found to 

be safe for use under the prescribed conditions. The new law also required the 

active drug ingredient names and amounts be declared and defined labeling 

be found on habit forming drugs. For false or fraudulent drug claims, the 

requirement that intent be demonstrated was dropped.

The Durham Humphrey Amendment of 1951 defined the term "prescription 

drug"; these drugs could only be dispensed with a legal prescription. Over the 
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counter (OTC) drugs were to bear labeling that contained directions for use 

and warnings against misuse.

In 1962, the Thalidomide drug disaster, which demonstrated the danger of 

incomplete drug testing prior to clinical trials, ensured the passage of sweeping 

drug control legislation. Thalidomide, a sedative taken during pregnancy, was 

found to cause the deformity known as phocomelia in children. The drug had 

never been approved for commercial sale in the United States. The use of this 

drug during pregnancy caused nine children in the United States to be born 

with this deformity. The tragedy had much greater proportions in Europe 

where thalidomide had been widely distributed. 

Congress passed the Kefauver Harris Drug Amendments on October 10, 

1962, which answered the need for tighter controls over drugs. These 

amendments included the following changes:

A. FDA was authorized to establish current good manufacturing practices 

(cGMPs) for drugs.

B. Drug manufacturers were required to register annually.

C. A drug is proven to be safe and effective by the manufacturer. A new 

drug could no longer be marketed prior to FDA approval based on 

convincing evidence of the drug's safety and effectiveness.

D. Reasons were listed for which a previously approved drug could be 

removed from the market.

E. New controls were placed on experimental and investigational drugs.

F. Regulation of advertising of prescription drugs was returned to FDA.

The Drug Listing Act of 1972 amended the FD&C Act so that drug 

establishments that are engaged in the manufacturing, preparation, 

propagation, compounding, or processing of a drug are required to register 

their establishments and list all their commercially marketed drug products with 

the FDA. Giving the agency means to readily determine which drugs were 

being manufactured and commercially distributed.

In 2012, an outbreak of fungal meningitis linked to a contaminated 

compounded drug product resulted in the loss of 64 lives and caused more 

than 751 illnesses. In response, Congress enacted the 2013 Drug Quality and 

Security Act (DQSA) that insures greater regulatory oversight of facilities 

creating compounded drugs. Among other provisions, it outlines steps for an 

electronic and interoperable system to identify and trace certain prescription 

drugs throughout the U.S.
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FDA Resource Links

A. Office of Medical Products and Specialty Lab Operations (OMPSLO) - 

Home (sharepoint.com)

B. Milestones in U.S. Food and Drug Law

C. Laws Enforced by FDA 

D. Chapter V of the Federal Food, Drug, and Cosmetic Act 

(sharepoint.com). 

E. CFR - Code of Federal Regulations Title 21 (fda.gov) Pts. 31, 200-499; 

Pts. 210-Current Good Manufacturing Practice in Manufacturing, 

Processing, Packing, or Holding of Drugs in General and Pt. 211-

Current Good Manufacturing Practice for Finished Pharmaceuticals. 

Washington, DC: U.S. Government Printing Office.

F. U.S. Food and Drug Administration, Office of Regulatory Affairs, Office 

of Enforcement Guidance and Procedures 

G. U.S. Food and Drug Manual of Compliance Policy Guides, Chapter 4, 

Subsection 400, Human Drugs. 

H. U.S. Food and Drug Administration, Office of Regulatory Affairs, Office 

of Enforcement Investigations Operations Manual | FDA

I. U.S. Food and Drug Administration, Office of Regulatory Affairs, Center 

for Drug Evaluation and Research Drug Compliance Program Manual , 

Sections 46, 52, and 56

J. Official Methods of Analysis of AOAC International (current ed.)

K. Library - Home Page (sharepoint.com) provides access for FDA staff to 

e-journals, FDC Reports, and WebLERN site that includes the USP 

current version with monographs, American Drug Index, Physician’s 

Drug Reference, Drug Facts and Comparisons, and other useful 

information.

Pharmaceutical Organization Resource Links 

A. International Federation of Pharmaceutical Manufacturers Associations 

(https://www.ifpma.org/) is a channel to exchange information within 

the international industry and various international organizations.

B. International Council for Harmonisation of Technical Requirements for 

Pharmaceuticals for Human Use (ICH) Quality Guidelines

C. United States Pharmacopeia and National Formulary 

https://www.uspnf.com/
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D. The Merck Index, An Encyclopedia of Chemicals, Drugs, and 

Biologicals, Merck & Co., Inc., Whitehouse Station, NJ. (Most recent 

edition). Network access provided to FDA staff by FDA Library

Reading Material (Most can be accessed through the FDA Library)

A. Chapter 1 of Bane's "Principles of Regulatory Drug Analysis" (See 

Section 9K) and Part 1 of "A Chemist's Guide to Regulatory Drug 

Analysis" (See Section 9L).

B. Remington's pharmaceutical sciences (current ed.). Easton, PA: Mack 

Publishing Company.

C. Banes, D. (1966). Principles of regulatory drug analysis (chap. 1). 

Arlington, VA: Association of Official Analytical Chemists.

D. Banes, D. (1974). A chemist's guide to regulatory drug analysis (Pt.1). 

Arlington, VA: Association of Official Analytical Chemists

E. Chatten, L. G. (1969). Pharmaceutical chemistry (Vol. 1, pp. 66-67; Vol. 

2, pp. 62-125). New York: Marcel Dekker.

F. Nilsen, C. L. (1998). The QC laboratory chemist: plain and simple. 

Buffalo Grove, IL: Interpharm Press, Inc.

G. Gringauz, A. (1997). Introduction to medicinal chemistry, how drugs act 

and why. New York: Wiley-VCH.

H. Ansel, H. C., Allen, L.V., Popovich, N. G. (1999). Pharmaceutical 

dosage forms and drug delivery systems (7th ed.). Philadelphia: 

Lippincott, Williams & Wilkins.

I. Fowlis, I. A. (1995). Gas chromatography, analytical chemistry by open 

learning (2nd ed.). New York: John Wiley & Sons. or other version

J. Linsay, S. (1987). High performance liquid chromatography, analytical 

chemistry by open learning. New York: John Wiley & Sons. or other 

version

K. Denny, R. C., Sinclair, R. (1987). Visible and ultraviolet spectroscopy, 

analytical chemistry by open learning. New York: John Wiley & Sons. or 

other version

L. Snyder and Kirkland (1979). Introduction to modern liquid 

chromatography (2nd ed.) New York: Wiley Interscience.

M. Wilson, C. O. et al. (2004). Wilson& Gsivold’s Textbook of Organic 

Medicinal and Pharmaceutical Chemistry.
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N. Rouessac and Rouessace (2000) Chemical Analysis:Modern 

Instrumentation Methods and Techniques.

O. George, B., McIntyre, P. (1987). Infrared spectroscopy analytical 

chemistry by open learning. New York: John Wiley & Sons.

P. Moffat, Anthony C et al, 2004, Clarke’s Analysis of Drug and Poisons, 

Third Edition, 2 volume set

Q. Ahuja, S., and Scypinski, 2001, Handbook of Modern Pharmaceutical 

Analysis, Academic Press San Diego CA

R. Safety Data Sheets, Toxicology Reports, and other safety information.

Procedure

8.1. Methodology 

8.1.1. Introduction

Modern pharmaceutical analysis has evolved from partitioning an active 

ingredient into a volatile solvent and performing a titration or gravimetric 

analysis, to techniques that include almost every known analytical procedure.

Spectrophotometry is performed in the three most accessible regions of the 

spectrum - the ultraviolet, visible, and infrared. Atomic absorption, 

fluorescence, flame emission, and mass spectrometry (GC/MS and LC/MS) 

are also used. Pharmaceutical analysis now relies heavily on chromatographic 

techniques, especially high performance, or pressure liquid chromatography 

(HPLC), since these techniques provide rapid, accurate, qualitative, as well as 

quantitative analysis. These techniques are also used to detect degradants 

and contaminants. 

This section serves not only to review the methods, but more importantly, to 

understand where methods can be obtained for analyzing assigned drug 

samples. The analyst always considers two questions before beginning an 

analysis. Is the method the correct one? Is the method valid? This assignment 

will set the groundwork for answers to these questions.

8.2. Methodology Sources 

8.2.1. Official Compendia

An "official compendium" is one that is officially cited in the FD&C. The 

United States Pharmacopeia (USP) (now combined with the National 

Formulary (NF)) is the major official compendium. The Homeopathic 

Pharmacopoeia is seldom used and is relatively obsolete. ORA Lab 
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Manual, Volume IV, Section 1.3 Analytical Methods provides additional 

information on official compendia.

The USP-NF, published annually, is cited in the Food Drug and Cosmetic 

Act, 201, (g)(1), 201(j), 501(b).

The USP-NF contains valuable information. Analysts should become 

familiar with the location of the following sections found in the Table of 

Contents: “General Notices”, “Monographs”, “General Chapters”, 

“Reagents”,” Tables”, and the sections of the NF. The General Chapters 

contains general tests and assay information, scientific principles and 

theory, as well as drug regulations and laws.

The Monographs section, comprising a major part of the USP-NF, serves 

as a definition and as a standard for a given drug substance, drug product, 

or drug ingredient. It is recommended that the analyst read the ‘General 

Notices and Requirements’ and several monographs noting their 

organization and content.

8.2.2. Petition Methods (NDAs and ANDAs) 

Each New Drug Application (NDA) (PAC 46.832) or Abbreviated New Drug 

Application (ANDA) (PAC 52.832) includes a method for analysis of the 

final dosage form and in many cases the Active Pharmaceutical Ingredient 

(API). This is the control method used by the manufacturer, and it has been 

shown to be usable for regulatory purposes after review by the FDA. 

Methods in manufacturer’s Applications and Petitions that have been 

approved have “official” status. These include New Drug Applications 

(NDA), Abbreviated New Drug Applications (ANDA), New Animal Drug 

Applications (NADA), Food Additive Petitions (FAP), and Pesticide 

Petitions (PP).

All material in NDA and ANDA submissions are confidential and subject to 

the protection required by the FD&C Act, sections 301(j) and 303(a) (5). 

The analyst may not cite NDA or ANDA methods in a publication or discuss 

them with persons outside FDA. The documents require secure storage; 

the analysts are to be careful in how they handle the documents. Further 

document security and confidentiality information can be found in the 

following: The Supervisory Staff Manual Guides, FDA F:2280.2, "Physical 

Security in Field Activities," Part 8, "Document Security", and 21 CFR Part 

31.14 (See Section 9B), "Confidentiality of Data and Information in a New 

Drug Application (NDA) File." 

For more information about the Method Validation samples refer to 

Compliance Program CP7346.832 (most current version) and “Supplement 

to CP7346.832 Pre-approval Inspections/Investigations, 7/8/96.”
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8.2.3. AOAC Official Methods

The AOAC INTERNATIONAL (AOAC) Official Methods of Analysis of the 

AOAC (see Section 9 I) is specified in the Code of Federal Regulations, 21 

CFR Part 2.19. Supplements, containing a cumulative index between 

editions, are published annually. 

Sections to review in the AOAC include the following: “Definitions of Terms 

and Explanatory Notes,” page xv, “Microchemical Methods,” Chapters 18–

22, Drugs I-V, and “Vitamins and Other Nutrients”. During the review, note 

the presentation style and content found in these segments. 

8.2.4. Pharmacopeial Forum

The Pharmacopeial Forum is a periodical issued six times a year by the 

United States Pharmacopeial Convention. It contains proposed new methods, updated lists of official reference standards and general comments on 

matters of interest in drug analysis and on use of the compendium.

8.2.5. Compliance Programs

Many Drug Compliance Programs, especially surveillance sample 

programs (PAC 56008) contain analytical directions, methods, or 

modification of methods to analyze products. The analyst needs to review 

the compliance program prior to analysis. 

8.2.6. Code of Federal Regulations

Title 21 of the Code of Federal Regulations (CFR) provides labeling 

requirements for many pharmaceutical products. The CFR requirements 

frequently include official methodology to analyze the products. An example 

is “ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN 

USE” found in 21 CFR 331.

8.2.7. Journals and Other Literature

When methods are not located in the conventional sources mentioned 

above, there are applicable procedures found in the literature. 

The Laboratory Information Bulletin (LIB) is a popular source of unofficial 

methodology in FDA published by the Office of Regulatory Science. The 

LIBs can aid with methods and resolution to problems the analyst may 

encounter with a method. Consult the LIB cumulative index to see if 

another FDA analyst has already solved the problem. If there is a related 

LIB method, it may be advantageous to discuss the problem with the 

author.
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The Journal of Pharmaceutical Sciences (J. Pharm. Sci.) and the Journal of 

AOAC INTERNATIONAL (J. AOAC Int.) are useful references. An annual 

index is found in the last journal for the year. 

International Pharmaceutical Abstracts, which contains abstracts from the 

two journals found above, is an excellent key to the entire pharmaceutical 

literature.

The Analytical Profiles of Drug Substances/Analytical Profiles of Drug 

Substances and Excipients, containing a series of monographs found in 

over thirty volumes, is another excellent reference. The first set of twenty 

volumes is edited by Klaus Florey of Squibb Institute; the remaining 

volumes are edited by Harry G. Brittain of Ohmeda Pharmaceutical 

Products Division, Inc., and published by Academic Press. A table of 

contents and cumulative index is included in each volume. 

Other useful materials for completing the exercises that follow are given in 

Section 9M-AA.

8.2.8. Questions

A. What are the five most significant items of information in the USP 

"General Notices"? Compare the "General Notices" with the introductory 

chapter in the Official Methods.

B. Using the current USP, find the Dissolution procedure for Ascorbic Acid 

Tablets and how does it differ from most other Dissolution procedures? 

C. Where in the USP is the listing for column types used in HPLC and 

GLC? How are they designated?

D. Locate the USP Reference Standards in the USP. What information is 

included here? What important information is missing and where is it 

found?

E. Obtain an NDA method and review it as one might review a research 

paper. Is it complete, specific, and unambiguous? Does it meet the 

requirements of the Code of Federal Regulations? 

F. What does the term “Official Compendium” mean? Name the three 

major types of “official” methods used by FDA. 

If a non-official method is used and the sample is violative, the check analysis 

must be performed using an official method or a Center approved method.

8.3. Pharmaceutical Chemistry 

The exercises that follow are presented as modules providing examples of the 

techniques needed to analyze a variety of pharmaceuticals in a variety of 
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dosage forms according to the associated Program Assignment Code (PAC). 

All exercises and topic discussions should be documented, approved, and the 

records placed in the trainee’s training file.

8.3.1. Pharmaceutical Products Overview 

The following outline serves as a guide in developing and customizing a drug 

training program by delineating pertinent knowledge areas and concepts with 

which a drug chemist needs to be familiar.

USP < > refers to the General Chapters – General Tests and Assays, 

General Requirements for Tests and Assays found in the back of the USP-NF.

A. Modern Pharmacology

B. How a New Drug is developed 

C. Standards

1. Working 

2. USP Reference Standards (USP <11>)

3. NIST

4. Other Pharmacopeial Standards

D. Controlled Substance--DEA Schedule

Schedule I, II, III, IV, V (USP 23-NF18, (1995), <1076> 'Controlled 

Substances Act Regulations') or earlier editions of USP-NF. Note: Later 

editions of USP-NF do not contain this section; however, the information 

is available in 21 CFR Part 1308: Schedule of Controlled Substances

E. How Drugs Work - drugs and receptors

F. Overview of the Chemistry of Organic Pharmaceuticals

1. Basic drugs and their salts

2. Acidic drugs and their salts

3. Neutral drugs 

G. Drug Formulations (USP <1151> Pharmaceutical Dosage Forms) 

1. Tablets (immediate and extended release)

2. Capsules (soft and hard gelatin)

3. Liquid Products (solutions, elixirs)

4. Topical Products (creams, ointments, gels, lotions)

5. Injections (single and multidose)

6. Transdermal patches and implants
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7. Other products (inhalation, aerosols, suppositories as listed in USP)

H. Types of Pharmaceuticals

1. Analgesics

2. Antianxiety - sedative, hypnotic

3. Antiemetic

4. Local anesthetics

5. Antibiotics

6. Antiviral

7. Other

I. Excipient Performance (USP <1059>)

1. Bulking materials and fillers

2. Lubricants

3. Colorants

J. Drug Programs and Sample Types in FDA

1. Compliance Samples: PAC 56002

2. Surveillance Samples: PAC 56008A, L

3. New Drug Applications (NDA): PAC 46832

4. Abbreviated New Drug Applications (ANDA): PAC 52832

5. Consumer Complaints: PAC 56R801

6. Shelf-Life Extension Program: PAC 88D_ _ _

7. Import Samples: PAC 56008H & J

8. Method Validation Samples other than NDA/ANDAs 

9. Other Program Samples: PAC 56021, 56021A & B, 56022A,

61003, 63001

8.3.2. Questions

A. When would you use a USP standard and when would you use a 

working standard? What is needed for working standards that is not 

needed for USP standards and why?

B. What is the procedure in the laboratory for obtaining USP standards? 

NIST standards? Controlled drug standards? 
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C. Describe the differences between an immediate release tablet/capsule, 

an extended-release tablet/capsule and a delayed release tablet. How 

would someone classify transdermal patches and implants?

D. What items would likely be found in compressed tablets and what are 

their purpose? In capsules? Name at least three items.

E. Describe where to find the PAC code for a product. 

8.4. Instrumentation and Technique

8.4.1. Basic Analytical Techniques

A. Objective

To review and use very basic analytical techniques and equipment (based 

on availability) routinely used in the laboratory (listed below). The student is 

expected to read and become familiar with the following techniques as 

described in the USP. The trainer is expected to provide explanations and 

devise simple exercises for the student to practice the techniques.

1. Volumetric Apparatus (care, usage, and Class A terminology) (USP 

<31>) and Specific Gravity (USP <841>)

2. Balances and Weights (general, analytical, micro analytical) (USP 

<41> and USP <1251>)

3. Thermometers (mercury, alcohol, thermocouple) (USP<1118>)

4. pH (calibration and use) (USP <791>)

5. Melting Range or Temperature (USP <741>) 

6. Gravimetric: Residue on Ignition (USP <281>) and Readily 

Carbonizable Substances Test (USP <271>)

7. Titrations (aqueous and non-aqueous, pH, potentiometric) (USP 

<541>)

8. Water Determination (KF Titration) (USP <921>) and Loss on Drying

(USP <731>)

9. Identification - Organic Nitrogenous Bases (USP <181>)

10.Uniformity of Dosage Units (USP <905>)

11.Dissolution (USP <711>) Apparatus 1 and 2

12.Drug Release (USP <724>)

13.Tablet Friability (USP <1216>), Tablet Breaking Force (USP

<1217>)
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B. Exercises

Note: These tests may be performed in conjunction with other sub-sections 

of this section and need not be performed in order.

1. For the following exercises use product #1. ‘Acetaminophen (API)’ 

identified in Attachment A.

a) Melting Range or Temperature (USP <741>), part 1.2

b) Gravimetric: Residue on Ignition (USP <281>) and Readily 

Carbonizable Substances Test (USP <271>), parts 1.4 and 1.8

c) Water Determination (Method I, USP <921>), part 1.3

d) Limit Test, part 1.5

e) Organic Impurities (USP <621> and <1086>), part 1.6

2. For the following exercises use product #2. ‘Ascorbic Acid Tablets’ 

identified in Attachment A. 

a) Titrations (aqueous) Assay part 2.1

b) Dissolution Test, Procedure for a Pooled Sample <711> part 2.3

c) Uniformity of Dosage (USP <905> [Weight Variation]), part 2.4

d) Identification Tests A, B, C, part 2.2

3. For the following exercises use product #8. ‘Dextrose’ identified in 

Attachment A.

a) Water Determination (Method III) USP <921>, part 8.5

b) Acidity - Titration (aqueous – limit test), part 8.4

c) Identification – precipitate, part 8.1

d) Color of Solution - part 8.2

4. For the following exercise use product #7 ‘Saccharin’ identified in 

Attachment A.

a) Titration (aqueous) – Assay part 7.10

C. Questions

1. What other standard techniques can be used for determining Melting 

Range and why are these used? What standards are used for 

calibration of the Apparatus? 

2. Why are limit and qualitative tests added to USP monographs? 

Compare the limit tests of the Acetaminophen product with those 
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found in the Dextrose (including those not run). Which tests use the 

same method?

3. Both the Acetaminophen product and Dextrose have a method for 

Water. What is the difference between the two methods? Can 

Acetaminophen method be used for Dextrose? Can the Dextrose 

method be used for Acetaminophen? Explain the answers.

8.4.2. Ultraviolet-Visible Spectroscopy (USP <857>) and Ultraviolet-Visible 

Spectroscopy-Theory and Practice (USP <1857>)

A. Objective

To learn or review essentials of quantitative UV/VIS spectrophotometry 

and to operate spectrophotometers in the laboratory for drug analysis 

(topics outlined below). The student is expected to read and become 

familiar with the following techniques as described in the USP or other 

literature, or through discussion with the trainer. The trainer is expected 

to provide explanations and devise simple exercises for the student to 

practice the techniques.

1. Theory

a) Essentials of spectrophotometry

i.Ultraviolet Range

ii.Visible Range

iii.Near Infrared Range

b) Linearity

c) Limitations

d) Effects of solvent, pH, and concentration

2. Instrumentation

a) Components:

i. Source - Tungsten-halogen, Deuterium

ii. Monochromator – Single path, dual monochromator

iii. Sample compartment - cuvettes (silica, glass, plastic)

iv. Detector 

• Photomultiplier

• Diode and Diode Array 

b) Calibration Procedures

c) Data Collection and Handling

i. Data Collection Systems

ii. Evaluating Spectra and Data

iii. Qualitative 
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iv. Quantitative

v. Data Storage

B. Exercises

1. Run holmium oxide filter and compare wavelengths obtained with 

listed values.

2. Run potassium dichromate solutions and compare to listed values.

3. Using several standard solutions from the drug exercise below, 

check the linearity of the instrument in a range of 0.1 AU to 2.0 AU.

4. For the following exercises use product 1, ‘Acetaminophen (API)’ 

identified in Attachment A.

5. Identification Test B, UV <197U>, part 1.1

6. Assay – UV, part 1.8

C. Questions

1. Define: absorbance, absorptivity, molar absorptivity.

2. What is the typical cell size, specimen concentration and 

absorbance range used in the analysis of a substance in the UV or 

visible range?

3. What do the expressions "similar preparation" and “similar solution" 

indicate (as used in tests and assays involving spectrophotometry in 

the USP)?

4. What do the expressions "concomitantly determine" and 

"concomitantly measured" indicate (as used in tests and assays 

involving spectrophotometry in the USP)?

5. Good practice demands that comparisons be made at the 

wavelength at which peak absorption occurs. What difference in nm 

for the wavelength specified in the USP monograph is considered 

acceptable? 

8.4.3. Mid-Infrared Spectroscopy (USP <854>) and Mid-Infrared 

Spectroscopy-Theory and Practice (USP <1854>)

A. Objective

To review the theory, instrumentation, and proper techniques for FTIR 

sample preparation and analysis in the drug laboratory (topics outlined 

below). The student is expected to read and become familiar with the 

following techniques as described in the USP, other literature, or through 
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discussion with the trainer. The trainer is expected to provide explanations 

and devise simple exercises for the student to practice the techniques.

1. Theory

a) IR radiation

b) Bonds

c) Vibrations

d) Resultant peaks

2. Instrumentation

a) Fourier Transform

3. Sampling Techniques

a) KBr pellets

b) Liquid cell

c) Salt plates

d) ATR

4. FTIR instrument operation – Software, Programs, Macros

a) Calibration

b) Collect data

c) Overlay

d) Library Search

e) Quantitation

5. Inspectional Aspects

a) Calibration

b) Quality of Spectra

c) Accuracy

B. Exercises

1. Scan the IR spectrum of a thin (1.5 mil) film of standard polystyrene 

film and compare the major peaks (see Figure 1) with the IUPAC 

standard polystyrene spectrum.
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 Figure 1 Major peaks for comparison exercise #1

3026.27 cm -1 + 2 cm -1 

2849.9 cm -1 + 2 cm -1 

1601.0 cm -1 + 2 cm -1 

1027.7 cm -1 + 2 cm -1 

906.5 cm -1 + 2 cm -1 

2. For the following exercises use product 1. ‘Acetaminophen (API)’ 

identified in Appendix 1.

a) Identification Test A, Infrared <197K>, part 1.1

3. Use the library function of the FTIR, if present, to compare spectra 

with that in the library database. 

C. Questions

1. What is FTIR?

2. What is the advantage of an FTIR spectrometer (interferometer) 

over a conventional (dispersive) spectrometer (with a prism or 

grating monochromator)?

3. What is the purpose of the interferometer in the FTIR spectrometer?

4. List the types of sample preparation techniques used for analysis 

with an FTIR spectrometer and conventional/dispersive 

spectrometer and explain when they would be used.

8.4.4. Optical Rotation/Polarimeter (USP <781>)

A. Objective

To review the theory, instrumentation and proper techniques for optical 

rotation sample preparation and analysis in the drug laboratory (topics 

outlined below). The student is expected to read and become familiar with 

the following techniques as described in the USP, other literature, or 

through discussion with the trainer. The trainer is expected to provide 

explanations and devise simple exercises for the student to practice the 

techniques.

1. Theory

2. Instrumentation

3. Sampling Techniques 
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4. Operation of Instrument

B. Exercises

1. Check instrument calibration. 

2. For the following exercise use product 8. ‘Dextrose’, identified in 

Attachment A.

a) Dextrose - Assay (use procedure for Dextrose Injection), part 

8.13

C. Questions

1. Describe polarimetry and the types of products for which it is 

used.

2. What is the purpose of the solid phase calibration cell and how is 

it used?

3. What is the general equation used in polarimetry and how does 

temperature effect polarimeter readings?

8.4.5. Chromatographic Techniques

Chromatography is a physical method of separation in which the components 

to be separated are distributed between two phases, one of which is stationary 

(stationary phase) while the other (the mobile phase) moves in a definite 

direction. The ultimate limits in efficiency, for all chromatography (and all 

separations), are set by the rates of mass transport within the system. A 

chromatographic system typically consists of several components.

The student is expected to read and become familiar with the following 

techniques as described in the USP, other literature, or through discussion 

with the trainer. The trainer is expected to provide explanations and devise 

simple exercises for the student to practice the techniques.

8.4.6. Column Chromatography (USP <621>)

A. Objective

To acquaint the analyst with the underlying principles of column 

chromatography as a basis of all chromatographic techniques used for drug 

analysis. 

1. Theory

2. Column Types

a) Celite or Cellulose packed in Glass

b) Solid Phase Extraction Columns (SPE) – Sep-Pak
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c) Gel

d) Ion Exchange

3. Sampling Techniques

B. Exercises (Do one of the two depending on equipment located in 

the laboratory)

1. See Laboratory Information Bulletin (LIB) # 3357 and # 2571. 

Prepare a glass column packed with Solka-Floc material. Separate a 

color sample product such as grape juice or a mixed standard using 

the column and method cited. Collect each color and determine their 

identity using UV-Vis Spectrophotometry.

2. See LIB # 3420 and # 2610. Separate a color sample product such 

as grape juice or a mixed standard using the column and method 

cited. Collect each color and determine their identity using UV-Vis 

Spectrophotometry.

C. Questions

1. When and why would someone use column chromatography?

2. (To be done at completion of each chromatography section.) 

Prepare a chart, comparing and relating each of the techniques used 

in chromatography for the following: column materials, separation 

theory, phases, equipment needed, ease of use, accuracy, sampling 

and sampling techniques, automation, accuracy, and quantitation.

8.4.7. Thin Layer Chromatography (TLC) (USP <621> and <201>)

A. Objective

To acquaint the analyst with the underlying principles of Thin Layer 

Chromatography, (a form of planar chromatography) and the equipment 

and techniques used for presumptive identification/semi-quantitation of 

drug samples.

1. Theory

a) Definitions of Terms 

i. Adsorbent 

ii. Solvent front 

iii. Rf value 

iv. Origin 

v. Eluent
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2. Types

a) Silica Gel

i. Binders

ii. Fluorescent Additives

b) Cellulose

c) High Performance (HPTLC)

3. Sampling Techniques

4. Application Techniques

B. Exercises

1. For the following exercise use product 5. ‘Acetaminophen Oral 

Solution’ identified in Attachment A.

a) Identification Test B. <201>, part 5.1

2. Spot above sample or standard at several orders of magnitude of 

concentration. For example, 1ul, 2ul, 5ul, 10ul, 20ul, 50ul, 100 ul, 

200ul, and 500ul.

C. Questions

1. TLC is a qualitative method. How can someone use this technique 

as a semi-quantitative tool? As a quantitative tool?

2. The exercise uses UV light as a visualizing tool. What other 

visualizing tools are commonly used for TLC?

3. In exercise 2 above, what was the smallest spot seen? In the larger 

spots, were other ‘breakdown’ or ‘related substances’ spots seen in 

the chromatogram? If found, can the quantity be estimated based 

on the size of the smaller standard spots?

4. (To be done at completion of each chromatography section.) 

Prepare a chart, comparing and relating each of the techniques used 

in chromatography for the following: column materials, separation 

theory, phases, equipment needed, ease of use, accuracy, 

sampling, and sampling techniques, automation, accuracy, and 

quantitation.

8.4.8. Gas Chromatography (GC) (USP <621>)

A. Objective

To present theory, instrumentation, parameters, and techniques for GC 

pharmaceutical analysis (topics outlined below). 
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1. Theory/Applications

2. Instrument Components

a) Columns

i. Packed Columns

ii. Capillary Columns

b) Gases

c) Temperatures

i. Isothermal

ii. Temperature Program

d) Injection

i. Packed

ii. Split/Splitless

e) Detectors

i. FID

ii. ECD

iii. Nitrogen/Phosphorus

iv. MS

v. Other

f) Data Handling

g) Calculation Techniques

3. Sampling Techniques

a) Direct Injection Techniques

b) Autoinjection 

c) Headspace Sampling

4. Operation

5. Quality Assurance (System Suitability)

B. Exercises

1. For the following exercises use product 5. ‘Acetaminophen Oral 

Solution’ identified in Attachment A.

a) Alcohol Content (if alcohol not already present spike at a 5% 

concentration) Method 2 <611> (packed column GC analysis), 

part 5.3 or Method 11 b <611> (capillary column).

2. For the following exercises use either product 

a) Product 10 ’Glycerin’ - Organic Impurities Procedure 1: Related 

Compounds [Capillary column GC analysis], part 10.3
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b) or include product 7. ‘Saccharin’ Limit of Toluenesulfonamides -

[Capillary column GC analysis], part 7.6

3. Calculate the results using both area and peak height calculations 

(electronically or manually) and compare the results.

C. Questions

1. What type of products can be tested by GC? What products cannot 

be tested? Describe a GC method that can analyze products that 

normally cannot be tested by a GC method.

2. Describe how a Flame Ionization Detector (FID) works. Describe at 

least three other GC detectors commonly used. Which detector is 

similar to FID and how is it used?

3. Describe capillary GC and how it differs from packed column GC. 

What are the advantages and disadvantages of each technique? 

4. When is it appropriate to use temperature programming? What are 

the advantages of using temperature programming?

5. GC commonly uses four different gases. Air, Nitrogen, Helium and 

Hydrogen. What is the purpose of each gas, how is it used and at 

what flow rates?

6. (To be done at completion of each chromatography section.) 

Prepare a chart, comparing and relating each of the techniques used 

in chromatography for the following: column materials, separation 

theory, phases, equipment needed, ease of use, accuracy, 

sampling, and sampling techniques, automation, accuracy, and 

quantitation.

8.4.9. High Performance Liquid Chromatography (HPLC) (USP <621>)

A. Objective

To present theory, instrumentation, parameters, and techniques for HPLC 

pharmaceutical analyses (outline below). HPLC has become the most 

common and important analytical tool in the modern drug laboratory. 

1. Theory/Application 

2. Instrument Components

a) Columns

i. Normal Phase Columns

ii. Reverse Phase Columns

iii. Ion Exchange Columns
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iv. Guard Columns

v. FAST Columns

vi. Specialty Columns

vii. USP Classifications

b) Column Parameters

i. Packings

ii. Size (both column and packing)

iii. Dimensions

c) Mobile Solvents

i. Aqueous and non-aqueous

ii. Buffers

iii. PIC and ion-pair modifiers

iv. Solvent Programming - Gradient

d) Injection

i. Techniques

ii. Auto injection

e) Solvent Delivery (Pumps)

i. Isocratic

ii. Solvent Program

f) Detectors

i. UV

ii. Electrochemical

iii. MS

iv. Other Specialty

g) Data 

i. Integration 

ii. Data Protection

3. Sampling Techniques

a) The need for clean samples and their preparation 

4. Quality Assurance (System Suitability)

B. Exercises

1. For the following exercises use product 5. ‘Acetaminophen Oral 

Solution’ identified in Attachment A.

a) Assay. MeOH/H2O, Simple Isocratic Systems, part 5.4

b) HPLC Identification Test A, part 5.1

c) pH <791> for product, part 5.2
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2. For the following exercises use product 4. ‘Acetaminophen and 

Caffeine Tablets’ identified in Attachment A. 

a) Assay, HPLC multi-component with IS, part 4.4

b) HPLC Identification, part 4.1

c) Dissolution and Uniformity of Dosage Units (HPLC), parts 4.2 

and 4.3. (optional)

3. For the following exercises use product 3. ‘Aspirin Tablets’ identified 

in Attachment A.

a) Assay, HPLC ion-pair system, part 3.5

b) Limit of Free Salicylic Acid, HPLC, part 3.4

c) Uniformity of Dosage Units (HPLC), Identification test A, 

(optional)

d) Identification Test B, part 3.1

4. For the following exercises use product 9. ‘Naltrexone Hydrochloride 

Tablets’ identified in Attachment A.

a) Assay, HPLC gradient system, part 9.4

b) Identification, part 9.1 

c) Optional – Uniformity of Dosage Units, part 9.3

C. Questions

1. Describe a typical ‘basic’ HPLC system and the purpose of each 

component.

2. What is the difference between normal phase and reverse phase? 

List at least three column types for each phase.

3. For the chromatograms obtained in exercise 2 ‘Acetaminophen and 

Caffeine Tablet’ calculate the following for each of the peaks: 

Retention Time, Retention Volume, Relative Retention Time to IS, 

Capacity Factor, Resolution, Tailing, Theoretical Plates, Height 

Equivalent Theoretical Plate, Peak Widths at base, half height, and 

5% height. Also calculate one set of sample results using both peak 

area and peak height calculations. Are the results different? If so, 

explain why differences may result.

4. What is the purpose of the internal standard used in exercise 2? 

Calculate one set of sample results without using the internal 

standard. Are the results different? If so, explain why differences 

may result. 
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5. When and why would gradient elution be used in HPLC? What is the 

effect of temperature on HPLC? Would temperature programming 

such as found in GC be effective for HPLC?

6. (To be done at completion of each chromatography section.) 

Prepare a chart, comparing and relating each of the techniques used 

in chromatography for the following: column materials, separation 

theory, phases, equipment needed, ease of use, accuracy, sampling 

and sampling techniques, automation, accuracy, and quantitation.

8.4.10. Other Specialized Techniques

There are many specialized techniques used in pharmaceutical chemistry 

analysis. Except for dissolution, most of these techniques are unique to the 

specialty laboratory or have not found wide recognition and thus are not usable 

for a basic training outline. All these techniques would be considered in 

advanced drug training. These other techniques will be mentioned by category 

only and could be expanded if the training laboratory has a need for this area 

of analytical techniques. 

8.4.11. Dissolution (USP <711>)

A. Objective

Dissolution is a technique commonly employed for tablet and capsule-type 

drug products. Modifications of the technique allow its use for other dosage 

forms such as patches, creams, and ointments. USP Apparatus 1 and 2 

are covered in this section since these are the apparatus found in most 

drug laboratories.

1. Theory

2. Instrumentation

a) USP Apparatus 1 (basket)

b) USP Apparatus 2 (paddle)

c) Other USP Apparatus (discussion only)

3. Calibration

a) Mechanical Calibration

i. Temperature

ii. Rotation Speed

iii. Vibration

iv. Height (positioning)

v. Wobble

vi. Centering
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vii. Level

b) USP Performance Verification Test

4. Operation – Sampling

a) Single Time period

b) Profile Sampling

c) Extended-Release Products

5. Auto sampling Techniques

B. Exercises

1. Perform the dissolution performance verification test in accordance 

with the current USP procedure.

2. For the following exercise use product #3. ‘Aspirin Tablets’ identified 

in Attachment A.

a) Dissolution, Apparatus 1 @ 50 rpm – 500 mL pH 4.5 buffer – UV 

determination, part 3.2.

3. For the following exercise use product #4. ‘Acetaminophen and 

Caffeine Tablets’ identified in Attachment A.

a) Dissolution, Apparatus 2 @ 100 rpm – 900 mL water – HPLC 

determination, part 4.2.

4. For the following exercise use product #2. ‘Ascorbic Acid Tablets’ 

identified in Attachment A.

a) Dissolution, Apparatus 2 @ 50 rpm – 900 mL water ‘Procedure 

for pooled sample’ – titration determination, part 2.3.

C. Questions

1. What parameters need to be checked and corrected before 

dissolution is run? Describe the effect that each would have on an 

analysis. 

2. Exercise 2 demonstrates a profile analysis. When would this 

technique be used? For what type of products could this technique 

be used? Find a monograph in USP that uses a profile type analysis.

3. Describe the reason and procedure for removing air from the 

dissolution media. 

4. Why not use water for all dissolution media and have constant 

paddle/basket rotation speed for all determinations?

5. Compare in-vitro dissolution results with in-vivo clinical studies? 

Explain the answer. 
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8.4.12. Advanced Techniques

To be developed as advanced training on an as-needed basis: (USP <___> 

identifies sections found in General Chapters, General Tests, and Assays 

found in the USP).

A. Particulate Matter in Injections (USP <788>)

B. Capillary Electrophoresis (CE) (USP <1053>)

C. Ion Chromatography (USP <1065>>)

D. Atomic Absorption Spectrophotometry (AA) (USP <852>)

E. Fluorescence Spectroscopy (USP <853> and <1853>) 

F. GC/Mass Spectrometry (USP <621> and <736>)

G. LC/Mass Spectrometry (USP <621> and <736>)

H. Nuclear Magnetic Resonance (NMR) (USP <761>)

I. Refractive Index (USP <831>)

J. Differential Scanning Calorimetry (DSC) and Thermal Gravimetric 

Analysis (TGA) (USP <891>)

K. Polarography (USP <801>)

L. Immunological Test Methods (USP <1106>)

M. Osmolarity (USP <785>)

N. Dissolution Apparatus III to VII (USP <711> and <724>)

O. Drug Release (Extended Release, Delayed Release and Transdermal 

Systems) (USP <724>)

P. Guidance and Procedures for characterization of biotechnology-derived 

articles by CE, Biotechnology-Derived Articles – Isoelectric Focusing 

(USP <1053> and <1054>)

Q. Inhalation and Nasal Drug Products: Aerosols, Sprays, and PowdersPerformance Quality Tests (USP <601>)

R. Plasma Spectrochemistry (USP <730>)

8.4.13. Inspections/Investigations

All analysts are to participate in at least one inspection prior to attending the 

FDA sponsored FDA Law and Evidence Development Course. In addition, 

analysts in drug specialty laboratories are provided specialized training in the 

inspection of pharmaceutical laboratories as defined in Current Good 

Manufacturing Practices (cGMPs) under 21CFR Parts 210 and 211. This 
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training program is developed according to the needs of the laboratory. 

Analysts should become familiar with sections of the Investigations Operations 

Manual (IOM) that involve preparing for and conducting team inspections. An 

example of training is as follows:

A. Types of Inspections

1. GMP

2. Pre-Approval

3. Post Approval

4. Contract Laboratory

5. Private Laboratory (Import work)

6. Clinical Laboratory

7. Foreign Inspections

B. Preparing for Team Inspections

C. How to start an inspection (482)

D. What to look for in the Laboratory

1. FDA Inspection Guides

E. Evidence Development

1. Documentation and Affidavits

2. Photography

F. Prepare a 483

G. Prepare an EIR

Glossary/Definitions

None

Records

None
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Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.3 R 11/03/2005 LMEB LMEB

1.4 R 01/17/2008 LMEB LMEB

1.5 R 02/02/2010 LMEB LMEB

1.6 R 02/05/2012 LMEB LMEB

1.7 R 02/13/2014 LMEB LMEB

02 R 05/05/2020 LMEB LMEB

03 R Refer to 

QMiS

ORS, Office of Medical Products & 

Specialty Laboratory Operations 

representatives

LMEB

* - D: Draft, I: Initial, R: Revision

Change History

Revision 

# Change

1.3

Table of Contents – Added 3.6 Appendix 2; added 3.8

Revised Section 3.1, 3.2 B., 3.3 B. 5., 3.4.1, 3.4.2.1 A., B. & C., 3.4.2.2, 3.4.2.2 I. B., 

3.4.2.2 II. B., 3.4.2.2 III., 3.4.2.3 III. B. & IV. B., 3.4.2.4 I. A. & II., 3.4.3, 3.5, 3.6 

Appendix 1 & 3; added 3.6 Appendix 2.

1.4

3.1, 3rd para, last sentence – Deleted “altogether”

3.2, 3rd para - Changed “diethyl” to “diethylene”

3.3 A. 1st para – added “or pressure” after high performance

3.3 B. 1., 4th para – revised

3.3 B. 6. – revised

3.3 B. 8. – updated 5th web address

3.3 C. 2. – revised

3.4.2.3 B. 3. – deleted

3.4.2.3 III. C. 2. – added (FID)

3.4.2.4 I. C. 3. – revised 5th sentence

3.5 – deleted 1st para

3.7 – revised 3.3.2, 3.4.2.2. (I) 1. & 5., 3.4.2.2 (II) 4., 3.4.2.3. (III) 1. & 4., 3.4.2.3. (IV) 

2., 3.4.2.4 (I).1.
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Revision 

# Change

1.5

3.3.B. – updated web links in 5., 7., & 8.; added last sentence to 8.

3.4.2.4 I. A. III. B. – revised

3.4.2.4 I. B. 1. – revised

3.4.2.4 I. B. 2. - deleted

3.5 – updated web links

Footer – updated web link

1.6

3.3 B. 2. – changed “testing” to “review” in second sentence

3.3 C. 5. – deleted

3.4 – added last sentence

3.4.2 I. A. – added USP reference to bullet 7; deleted bullet 10; added bullets 13 and 

14

3.4.2.I. B. 2. – deleted Disintegration Test from bullet 2

3.4.2.I. B. 3. – “6” changed to “8” in bullets

3.4.2.2 II. A. III. – added D. and E.

3.4.2.3.III B. 1. – changed “Suspension” to “Solution” in 1.; added Method 11 b to 

bullet

3.4.2.3 III B. 2. – revised 

3.4.2.3 IV. A. 2. – added 7.; added “Gradient” to C.4.

3.4.2.3.IV. B.1. – “Suspensions” changed to “Solution”; B change to A, part 5.1 in 

second bullet; 5.1 deleted in third bullet

3.4.2.4. I. C – deleted 2.

3.4.2.4.II. 16. – changed VIII to VII

Appendix 1 I. – deleted 2.3; changed Apparatus 2 to Apparatus 1 in 3.2; changed 

Suspension to Solution in 5. ; changed 6.1 B. to HPLC; deleted 6.6, 6.7, and 6.8; 7.1 

changed to FTIR; 7.6 spelling corrected and changed to FID; deleted 7.10; added 

“loss on” to 8.5; added <281> to 8.6; 8.12 changed to color from limit test; 9.2 

changed from UV to HPLC; added 10. 

Appendix 2 I. – added 4. and changed GC product and test method

3.7 Answer Key 3.3 3.- answer revised and deleted 4., 5., and 6.

3.7 Answer Key 3.4.1 1. – first and second sentence revised 

3.7 Answer Key 3.4.2.1 – 3. deleted sentences 3., 4., 5., and the last sentence

3.7 Answer Key 3.4.2.4. (I) –deleted 2.

1.7 Header – Division of Field Science changed to Office of Regulatory Science

7. – Division of Field Science changed to Office of Regulatory Science
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Revision 

# Change

02

Document revised to current format guidance -numbering 

All references revised accordingly

Under Introduction:

ORA U revised to OTED 

Add sample match to sample description in collection report

Add sample expiry 

Add DQSA information

Pharmaceutical Products Overview 

Removed Biotechnology Developed Drugs Reference

Revised Fillers Reference; added Excipient Performance

Revised Shelf-Life PAC; added 88D

Deleted - “to find” PAC

Revised Thermometers; add (USP<1118>)

Revised titrations; added (USP<541>)

Water Determination Added (USP<731>)

Deleted Limit Tests (USP<231>) and all subsequent references

Revised Ultraviolet/Visible Spectrophotometry (USP <857>) and ULTRAVIOLETVISIBLE SPECTROSCOPY—THEORY AND PRACTICE (USP <1857>)

Revised. Fourier Transform Infrared Spectrophotometry (FTIR) [USP <854>] and MIDINFRARED SPECTROSCOPY—THEORY AND PRACTICE (USP <1854>)

Removed and revised Specific rotation reference 

Exercise B revised; added Identification Test B <201>, part 1.1

Aspirin Uniformity of Dosage, ID A, ID B revised for clarity

Advanced Techniques 

Added Particulate Matter (USP <788>)

Revised CE reference; added (USP<1053>)

Revised AA reference; added (USP<852>)

Revised Fluorimetry reference; added (Florescence Spectroscopy (USP<853> and 

<1853>)

Deleted Electrophoresis

Deleted Immunoassays; added Immunological Test Methods (USP <1106>)

Deleted Analytical Biotechnology using CE, HPLC, Isoelectric focusing. (USP <1047>)

Add Plasma Spectrochemistry (<730>)

Section 3.5 References renamed as Section 9 Supporting Documents

Training Sample Section References updated

Delete and update Training Sample Section references to reflect template numbering 

changes throughout the document

03 Format and spelling errors corrected. USP titles confirmed or added. Minor additions added 

are highlighted within document. References updated. 
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Attachments

List of Attachments

Attachment A - Exercise: Practice with Samples ........................................................35

Attachment B - Proficiency Samples...........................................................................40

Attachment C - Statistical Analysis .............................................................................42

Attachment D - Answer Key ........................................................................................45

Attachment E - Chromatography Chart.......................................................................63
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Attachment A - Exercise: Practice with Samples

Training Samples

Training samples may vary and depend on what is available in the laboratory. The training 

samples, using USP products whenever possible, also demonstrate the trainee’s ability and 

proficiency in performing regulatory tests. The following were used for the above training 

program and are examples of products that have been used successfully for training. 

However, other products may be used that demonstrate the same level of training. Specific 

tests may not apply based on current laboratory practice or revised monograph 

requirements. Underlined parts of the following products were not used in the exercises but 

may be added to complete a full product analysis. Full worksheets are to be generated for 

each product analyzed. Italicized items 1, 4 & 7 are also proficiency samples. (* also 

identified as a proficiency test in Appendix 2).

USP < > refers to the General Chapters – General Tests and Assays, General 

Requirements for Tests and Assays found in the back of the USP-NF

I. Training Samples

1. Acetaminophen (API) – USP, current edition, {Section 6.3.1.2.A, 6.3.2.1.2D, 

6.3.2.2.2B}

1.1 Identification

Test A: Infrared <197K> *

Test B: UV <197U>

Test C: TLC <201> *

1.2 Melting Range <741>

1.3 Water Method 1 <921> KF Titration *

1.4 Residue on Ignition <281>

1.5 4-Aminophenol in Acetaminophen-Containing Drug Products (USP 

<227>) - limit test; intra-monograph test applicable for HPLC (USP 

<621>)

1.6 Organic Impurities (USP <621> and <1086>) HPLC.

1.7 Assay – UV *
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2. Ascorbic Acid Tablets – USP, current edition, {Section 6.3.1.2B, 6.3.5.1.2D}

2.1 Assay – titration

2.2 Identification – Test B

Test A – spot test

Test B – color test

Test C – color test

2.3 Dissolution, Procedure for Pooled Sample <711>

2.4 Uniformity of Dosage (Weight Variation)

3. Aspirin Tablets – USP, current edition {Section 6.3.4.4.2C, 6.3.5.1.2B}

3.1 Identification

Test A – retention time

Test B – IR <197K>

3.2 Dissolution <711> Apparatus 1 – UV Det

3.3 Uniformity of Dosage Units <905>

3.4 Limit of Free Salicylic Acid – HPLC

3.5 Assay – HPLC with ion-pair reagent

4. Acetaminophen and Caffeine Tablets – USP, current edition, {Section 

6.3.4.4.2B, 6.3.5.1.2C}

4.1 Identification - HPLC

4.2 Dissolution <711> Apparatus 2 - HPLC * 

4.3 Uniformity of Dosage Units <905> HPLC

4.4 Assay – HPLC two component with IS *
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5. Acetaminophen Oral Solution – USP, current edition, {Section 6.3.4.2.2A, 

6.3.4.3.2A, 6.3.4.4.2A}

5.1 Identification – FTIR <197K>

A. HPLC

B. TLC <201>

5.2 pH <791>

5.3 Alcohol Content (if present) Method II <611> 

(If alcohol is not present spike at 5% ethanol)

5.4 Assay – HPLC <621>

6. Caffeine API – USP, current edition 

 6.1 Identification

 A. Infrared Absorption <197M>

 B. HPLC

 6.2 Melting Range <741>

 6.3 Water, Method III <921>

 6.4 Residue on Ignition <281>

 6.5 Organic Impurities - HPLC

 6.6 Assay – HPLC <621>

7. Saccharin – NF, current edition, {Section 6.3.1.2.D, Section 6.3.4.3.2B2} 

7.1 Identification – FTIR

7.2 Melting Range <741>

7.3 Loss on Drying <731>

7.4 Readily Carbonizable Substances <271>

7.5 Residue on Ignition <281>
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7.6 Toluene sulfonamides – column and gas chromatography (FID)

7.7 Benzoic and salicylic acids – chemical test

7.8 Color of solution

7.10 Assay – titration * 

8. Dextrose – USP, current edition, {Section 6.3.1.2.C, 6.3.3.3.2B}

8.1 Identification – color precipitate

8.2 Color of Solution – limits test

8.3 Specific Rotation <781S>

8.4 Acidity – titration

8.5 Water Method III <921> (loss on drying)

8.6 Residue on Ignition <281>

8.7 Chloride <221> - limit test

8.8 Sulfate <221> - limit test

8.9 Arsenic Method I <211> - limit test

8.11 Dextrin – limit test

8.12 Soluble starch, sulfites – color

8.13 Assay - Optical Rotation (Use current USP procedure for Dextrose 

Injection).

9. Naltrexone Hydrochloride Tablets – USP, current edition, {Section 6.3.4.4.2D}

9.1 Identification - HPLC

9.2 Dissolution <711> Apparatus 2 – HPLC Determination

9.3 Uniformity of Dosage Units – HPLC gradient

9.4 Assay – HPLC gradient
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10. Glycerin – USP, current edition, {Section 6.3.4.3.2B, 3.4.2.3 IVB}

10.1 Identification –

A. FTIR

B. Limit of Diethylene Glycol and Ethylene Glycol (GC-FID)

10.2 Assay – Titration

10.3 Impurities

Inorganic Impurities

Organic Impurities –

Procedure 1 Related Compounds (GC-FID)

Procedure 2 – Limit of Chlorinated Compounds

Procedure 3 – Fatty Acids and Esters

10.4 Specific Gravity <841>

10.5 Water Determination <921>

11. Ascorbic Acid API – USP, current edition, {Section 6.3.1.2B, 6.3.5.2.3D}

 11.1 Optical Rotation – <781S>

II. Additional Training Samples as needed:

Additional training samples may be added or substituted to demonstrate and reinforce 

the analytical procedures listed in the training program.
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Attachment B - Proficiency Samples

Proficiency Training Samples for new hires:

The following is designed to show proficiency in 7 technologies commonly used in drug 

analysis. An analyst should demonstrate the ability to successfully analyze these as a 

minimum requirement. 

Each analyst will complete the following tests to demonstrate proficiency for each of the 

techniques outlined below with the identified sample.

1. Acetaminophen API – ID by FT-IR & TLC, ID/Assay by UV/Vis, and Water (Karl 

Fischer Titration) (Item 1, Part II Training Samples below for specific information).

2. Acetaminophen & Caffeine Tablets – Assay by HPLC and Dissolution with HPLC 

detection (Item 4, Part II Training Samples below for specific information. Quantify for 

both acetaminophen & caffeine). 

3. Saccharin – Assay by Titration (Item 7, Part II Training Samples below for specific 

information).

4. Glycerin – Organic Impurities by GC (Item 10, Part II Training Samples below for 

specific information).

Technology, Product, and Method
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USP and NF ( ) refers to the Test in the Official Monographs for the listed product.

II. Additional Proficiency Samples as needed:

Additional proficiency samples may be added or substituted. The products need to include 

the above ‘Test Type’ and show proficiency in those techniques. 
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Attachment C - Statistical Analysis

Analytical results observed in the laboratory are to be accurate and reliable based upon 

statistical principles and by the current Good Manufacturing Practices. ORA LAB Manual 

Vol. III Section 4, Basic Statistics and Data Presentation, covers statistical analysis in detail 

and should be reviewed. The following information is supplemental to LAB Manual, Vol. III. 

sec. 4 and is provided as a summary and a guide.

A. Definitions

Some of the definitions are from the ICH Validation Procedures definitions, which appeared 

in the Federal Register #60, March 1, 1995, p. 11260.

1) Number of Significant Places: The total number of digits, not counting leading 

zeros, from the first non-zero digit, to the last digit known with a reasonable 

confidence.

2) Error: Difference between the expected and the encountered value of some 

variable.

3) Incertitude: Lack of availability of information about a reading caused by the 

impossibility of reading closer than a certain readout limit. For example, a 

digital readout of 22.57, with no digits beyond the second decimal, has 

incertitude of +/- 0.01. 

4) Standard Deviation, σ = √[Σ(xi-xaverage)

2

/(n-1) . See Precision. 

5) RSD: Relative standard deviation. The standard deviation divided by the 

average; usually expressed as percent by multiplying it by 100.

6) Accuracy: The closeness of agreement between the value which is accepted 

(either as a conventional true value or as an accepted reference value) and the 

value found.

7) Precision: The closeness of agreement between a series of measurements 

obtained from multiple sampling under the same conditions. Usually expressed 

as standard deviation.

8) Repeatability: Precision measured in the same laboratory and under the same 

operating conditions, over a short period of time.
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9) Intermediate precision: Precision within the same laboratory but under different 

conditions, analysts, and equipment.

10)Reproducibility: Precision between different laboratories, such as in 

collaborative studies.

11)Robustness: The ability of a procedure to remain unaffected by small but 

deliberate variations in method parameters. It is an indication of reliability 

during normal usage.

B. Using Significant Digits (see MAN-000048 ORA LAB Manual, Vol. III, Sec. 4.3.2 

Significant Figures) 

Normally, the confidence in the value of a number decreases from the first to the last 

digit. Thus, if a volume measurement gives 10.567 mL, we may be quite confident that 

the value is close to 10 mL but less confident about the decimal "5", still less about the 

"6" and not sure at all about the "7". In this case, the digit 7 is called the least significant 

digit (LSD). The number 10.567 is said to have 5 significant digits. The number of 

significant digits is obtained counting all digits that give reasonable confidence, not 

including leading zeros. For example, 0.0031 has two significant digits. 

The rules of significant digits are rules of data communication. Only report the digits in 

which there is confidence. The non-significant ones are eliminated according to the rules 

outlined below. Data in process are kept with an extra significant figure and rounded to 

the correct significant figures for the final answer. A good rule is using five digits 

throughout but reporting at the end with the correct number of significant digits. 

Note: Do not confuse significant digits, with significant places. The latter depend on the 

units used and the concept should be avoided in this context. For example, 12.53 cm = 

125.3 mm. Both have four significant digits, but different number of significant places 

(two and one respectively).

C. Performing Rounding (see MAN-000048 ORA LAB Manual, Vol. III, Sec. 4.3.1 

Rounding of Reported Data) 

The procedure (see USP General Notices 7.20. Rounding Rules) for rejecting the digits 

beyond the least significant digit, LSD, is the following:

1. Establish the position of the LSD.
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2. If the digit to the right of the LSD is less than 5, eliminate all remaining digits after the 

LSD.

3. If the next digit is larger than or equal to 5, increase the LSD by one and eliminate all 

the digits beyond the LSD.

Remember: The calculations are always retaining an extra significant figure over the 

significant ones. Rounding should be done only at the end to the correct significant figures.

D. Using Average and Standard Deviation (see MAN-000048 ORA LAB Manual, Vol. III, 

Sec. 4.2.2 Error and Deviation; Mean and Standard Deviation)

The average and standard deviation are estimates after the fact. This means that several

measurements are first made and then their statistical properties are obtained from the 

analysis of the set of measurements. Calculations: 

Average = Σ(xi)/n, Sum of all data divided by the number of data

Variance = Σ((xi-xaverage)2/(n-1) The number n-1 is the number of data 

 minus one. 

Standard deviation, σ = √[Σ(xi-xaverage)2/(n-1)]

%RSD = Relative Standard Deviation = (standard deviation)/(average)*100
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Attachment D - Answer Key

6.2.1 Methodology Sources

A. What are the five most significant items of information in the USP "General 

Notices?" Compare the "General Notices" with the introductory chapter in the 

Official Methods. This is a subjective answer, but the answer could include the 

following: The use of "Official" and "Official Article”; Significant Figures and 

Tolerances; Reference Standards/Reagents; Tests and Assays; Preservation, 

Packaging, Storage and Labeling (especially storage temperature); Weights and 

Measures; and Concentrations. The “Guide to General Chapters" contains 

information regarding procedures used throughout the USP; the General Notices 

serves as a general guidance document.

B. Using the current USP, find the Dissolution procedure for Ascorbic Acid 

Tablets and how does it differ from most other Dissolution procedures? 

Ascorbic Acid Tablets monograph uses a pooled sample procedure where equal 

volumes of each vessel are pooled, and the one-pooled solution is assayed. The 

assay procedure is performed on the solution from each vessel for most Dissolution 

procedures. 

C. Where in the USP are the listings for column types used in HPLC and GLC? 

How are they designated? There is a separate section “Chromatographic Columns”. 

This lists both the column used for each general chapter or monograph and the

column types for both HPLC and GLC. HPLC is listed as packings with a letter 

designation beginning with ‘L’. GLC is listed as phases with a letter designation 

beginning with ‘G’.

D. What does the term “Official Compendium” mean? Name the three major types 

of “official” methods used by FDA. "The term 'official compendium' means the 

official United States Pharmacopeia, official Homeopathic Pharmacopeia of the 

United States, official National Formulary, or any supplement to any of them.” See 

Section 9F Chapter II Definitions Sec. 201 (j), three major types of “official” methods 

used by FDA are the (1) United States Pharmacopeia, (2) AOAC Official Methods. (3) 

FDA approved New Drug Application or Abbreviated New Drug Application methods.

6.3.2 Pharmaceutical Products Overview

A. When would you use a USP standard and when would you use a working 

standard? What is needed for working standards that is not needed for USP 

standards and why? The USP standard when available is to be used for the 
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analysis of all USP products. Working standards are used for products and dosage 

forms not found in the USP or when an USP standard is not available. The purity and 

identity of all working standards are to be verified before use to assure that the 

standard is sufficiently pure, and it is the correct material. The standard purity is to be 

used in the assay calculations. The USP standard is assumed to be 100% unless 

otherwise stated on the label. 

B. What is the procedure in the laboratory for obtaining USP standards? NIST 

standards? Controlled drug standards? The answer to this question is dependent 

on the District and Laboratory policy. The trainee is to describe this procedure and 

reference the Laboratory’s SOP.

C. Describe the differences between an immediate release tablet/capsule, an 

extended-release tablet/capsule and a delayed release tablet. How would one 

classify transdermal patches and implants? Immediate release tablet and 

capsules release the active ingredient within a small period of time, typically less than 

30 minutes. Extended-Release tablets release the active ingredient at a sustained 

and controlled release rate over a period of time. Typically, extended-release tablets 

and capsules release their ingredient with time periods of 8 hours, 12 hours, 16 

hours, and 24 hours. Delayed Release tablets release the pharmaceutical dosage 

after a set time. These are frequently enteric coated to prevent release in the 

stomach thus release the dosage in the intestinal track. Transdermal patches and 

implants would be classified as extended-release products but release the dosage 

over a much greater period of time such as weeks or months.

D. What items would likely be found in compressed tablets and what is their 

purpose? In capsules? Name at least three items.

a. Active Ingredient – supplies pharmacological dose: any drug product.

b. Diluents (Fillers) – provide bulk: lactose, calcium phosphate, microcrystalline 

cellulose.

c. Binders – impart cohesive properties: starch, povidone (PVP).

d. Lubricants – reduce mechanical friction and prevent tablets from sticking to 

punches and dies: magnesium stearate.

e. Glidants (Flow Agents) – promotes free flowing characteristics: colloidal silicon 

dioxide, talc.

f. Disintegrants – enable tablets to break apart in aqueous environments: starch, 

crospovidone (PVP-XL), croscarmellose, Na starch glycolate.

g. Capsule – holder for active and filler materials: gelatin. 

E. Describe where you would find the PAC code for a product. All FDA collected 

samples appear in FACTS, which lists the PAC codes and the type of sample. 

PACs for the various programs are in the Compliance Program Guidance Manual 
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(CPGM). The CPGM frequently provides additional sample information including 

methodology to be used for the products being analyzed.

 

6.4.1 C Basic Analytical Techniques

1. What other standard technique can be used for determining the Melting Range 

and why are these used? What standards are used for calibration of the 

Apparatus? A USP Reference Standard or National Formulary Reference Standard 

corresponding to an authentic specimen of the substance being tested is added in 

equal parts to the substance until an intimate mixture is obtained. Agreement of 

observations on the authentic and the mixture provides reliable evidence of chemical 

identity. USP Melting Point Reference Standards are used for calibration of the 

apparatus. There are six such standards: Acetanilide, Caffeine, Phenacetin, 

Sulfanilamide, Sulfapyridine and Vanillin. The one that melts nearest the expected 

melting temperature of the substance under test is used.

2. Why are limit and qualitative tests added to USP monographs? Compare the 

limit tests of the Acetaminophen product with those found in the Dextrose 

(including those not run). Which tests use the same method? USP monographs 

serve to define the identity, strength, quality, and purity of the substances for which 

they are written. Limit and qualitative tests are part of that determination. Tests for the 

presence of foreign substances and impurities are provided to limit such substances 

to amounts that are unobjectionable. Both monographs use the same tests for 

residue on ignition <281>, . Acetaminophen also has limit tests for water 

(<921>Method I), Limit tests for Organic Impurities (HPLC) - sulfide, free paminophenol, and p-chloroacetanilide. Dextrose has limit tests for water 

(<921>Method I), dextrin and soluble starch sulfites.

3. Both the Acetaminophen product and Caffeine have a method for Water. What 

is the difference between the two methods? Can the Acetaminophen method 

be used for Caffeine? Can the Dextrose method be used for Acetaminophen? 

Explain the answers. The method used for water determination in Acetaminophen is 

titrimetric. There is a quantitative reaction of water with an anhydrous solution of 

sulfur dioxide and iodine in the presence of a buffer, which reacts with hydrogen ions 

(Karl Fischer Reagent – sulfur dioxide & iodine dissolved in pyridine & methanol). A 

gravimetric method is used for water determination in Caffeine. Caffeine is dried at 80 

degrees for 4 hours. The hydrous form loses NMT 8.5% of its weight. The anhydrous 

form loses not more than 0.5% of its weight. The method for Acetaminophen and 

Caffeine are not interchangeable because one must account for the water of 

hydration.
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6.4.2.C Ultraviolet-Visible Spectroscopy (USP <857>) and Ultraviolet-Visible 

Spectroscopy-Theory and Practice (USP <1857>)

1. Define: absorbance, absorptivity, molar absorptivity. 

(See USP<857> for more information.)

Beers Law supplies the terms: log10 (1/T) = A = abc

• Absorbance: [A] is the logarithm, to the base 10, of the reciprocal of the 

transmittance (T). 

• Absorptivity: [a] – the quotient of the absorbance (A) divided by the product of the 

concentration of the substance (c) expressed in grams per liter, and the 

absorption path length (b) in cm.

• Molar Absorptivity: [Symbol like a rounded capital E] -- the quotient of the 

absorbance (A) divided by the product of the concentration, expressed in moles 

per liter, of the substance and the absorption path length in cm. It is also the 

product of the absorptivity (a) and the molecular weight of the substance.

2. What is the typical cell size, specimen concentration and absorbance range used 

in the analysis of a substance in the UV or visible range?

• Typical cell size (path) is 1 cm.

• For many pharmaceutical substances ‘concentrations of about 10 ug of the 

specimen per ml often will produce absorbances of 0.2 to 0.8 in the ultraviolet or 

the visible region.’ Ref. USP <857>.

3. What do the expressions "similar preparation" and "similar solution" indicate (as 

used in tests and assays involving spectrophotometry in the USP)? The 

expressions "similar preparation" and "similar solution” indicate that the reference 

specimen, generally a USP Reference Standard, is to be prepared and observed in a 

manner identical for all practical purposes to that of the test specimen. Ref. USP 

<857>.

4. What do the expressions "concomitantly determine" and "concomitantly 

measured," indicate (as used in tests and assays involving spectrophotometry in 

the USP)? The expressions "concomitantly determine" and "concomitantly measured" 

indicate that the absorbances of both the solution containing the test specimen and the 

solution containing the reference specimen, relative to the corresponding test blank, 

are to be measured in immediate succession. Ref. USP <857>.
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5. Good practice demands that comparisons be made at the wavelength at which 

peak absorption occurs. What difference in nm for the wavelength specified in 

the USP monograph is considered acceptable? If the difference is more than +/- 1 

nm from the specified wavelength, re-calibration of the instrument may be indicated. 

Ref. USP <857>.

6.4.3.C Mid-Infrared Spectroscopy (USP <854>) and Mid-Infrared Spectroscopy-Theory 

and Practice (USP <1854>)

1. What is FTIR? A computerized process in which the source of energy is sent through an 

interferometer (instead of a monochromator) and onto a sample which is irradiated. An 

interferogram (intensity vs. time spectrum) is obtained, which has the characteristics of a 

spectrum. A computer mathematically converts the (intensity vs. time) spectrum (fourier 

transform) into an (intensity vs. frequency) fourier transform infrared spectrum.

2. What is the advantage of an FTIR spectrometer (interferometer) over a 

conventional (dispersive) spectrometer (with a prism or grating monochromator)?

3. What is the purpose of the interferometer in the FTIR spectrometer? The 

interferometer replaces the conventional monochromator so that energy throughput is 

inherently greater (no slits) and all wavelengths present reach the detector during the 

entire time needed to observe a spectrum. The interferometer is coupled to a detector 

and a computer that reduces the data to an ordinary spectral curve faster.
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4. List the types of sample preparation techniques used for analysis with an FTIR 

spectrometer and conventional/dispersive spectrometer and explain when they 

would be used. 

• Mulls formed by grinding the compound and dispersing it in mineral oil (nujol). The 

oil/sample mixture is then placed between salt plates and pressed together.

o Poor solubility in a useful solvent and saving of time in sampling handling.

• Gases and vapors in gas sample cells produce spectra, which differ fundamentally 

from those observed in a condensed state (solids, liquids, solutions).

o Sample is to be in a gaseous state that can be transferred to a gas sample 

cell.

• Solutions by dissolving the compound (liquid or solid) in a solvent and placing it in 

a sealed liquid cell.

o Sealed cell is perhaps the simplest technique to use for liquids with 

viscosities less than or similar to water.

• KBr discs (pellets) prepared using a press.

o A dried powdered or crystalline sample is ground with KBr.

• Attenuated Total Reflectance (ATR) 

o A solid sample place in situ on diamond/crystal center.

6.4.4.C Optical Rotation/Polarimeter (USP <781>

1. Describe polarimetry and the types of products for which it is used. Polarimetry is 

the measurement of optical rotation. Many pharmaceutical substances are optically 

active in the sense that they rotate an incident plane of polarized light so that the 

transmitted light emerges at a measurable angle to the plane of the incident light. Ref. 

USP<781>. Pharmaceutical products that show a dextrorotatory, or (+) optical isomers, 

and levorotatory, or (-) optical isomer. The products that show such optical rotatory 

power are chiral. 

2. What is the purpose of the solid phase calibration cell and how is it used? The 

solid phase calibration cell is used for checking the calibration of the polarimeter. This 

calibrator consists of a plate of quartz mounted in a holder perpendicular to the light 

path. The calibrator standard readings are traceable to NIST. The cell replaces the 

need to prepare Dextrose and Sucrose solutions that also can be used for calibration. 

The solid phase calibration cell is stabilized to a temperature of 25oC and placed in the 

instruments cell holder. The degree of rotation of the cell can be read directly on the 

instruments readout system.
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3. What is the general equation used in polarimetry and how does temperature effect 

polarimeter readings? The general equation for polarimetry is [α]

tλ where ‘α’ is 

specific rotation at ‘t’ temperature λ wavelength, which equals 100a/lc where ‘a’ is the 

observer rotation in degrees, ‘l’ is the path length in decimeters and ‘c’ is the 

concentration in grams per 100 ml. Temperature is in the equation and has a direct 

effect on the measured specific rotation. Temperatures need to be maintained within 

0.5oC of the stated value in the USP for the calculations to be valid for a measured 

product.

6.4.6 C Column Chromatography (USP <621>)

1. When and why would someone use column chromatography? Column 

chromatography offers a wide choice of stationary phases and is useful for the 

separation of individual compounds, in quantity, from mixtures." Ref. USP <621>. This 

technique is used for color analysis and when large quantities of purified materials are 

needed.

2. (To be done at the completion of each chromatography section.) Prepare a chart, 

comparing and relating each of techniques used in chromatography for the 

following: column materials, separation theory, phases, equipment needed, ease 

of use, accuracy, sampling and sampling techniques, automation, accuracy, and 

quantitation. See Attachment E

6.4.7 C Thin Layer Chromatography (TLC) (USP <621> and <201>)

1. TLC is a qualitative method. How can someone use this technique as a semiquantitative tool? As a quantitative tool? A visual comparison of the size of the spots 

may serve for semi-quantitative estimation. Quantitative measurements are possible by 

means of densitometry, fluorescence, and fluorescence quenching; or the spots may be 

carefully removed from the plate, followed by elution with an effective solvent and 

spectrophotometric measurement. Ref. USP <621>.

2. The exercise uses UV light as a visualizing tool. What other visualizing tools are 

commonly used for TLC? The spots produced by paper, or thin-layer chromatography 

may be located by: (1) direct inspection if the compounds are visible under white or 

either short-wavelength (254 nm) or long-wavelength (360 nm) UV light; (2) inspection in 
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white or UV light after treatment with reagents that will make the spots visible. Ref. USP 

<621>.

3. In exercise 2 above, what was the smallest spot seen? In the larger spots, were 

other ‘breakdown’ or ‘related substances’ spots seen in the chromatogram? If 

found, can the quantity be estimated based on the size of the smaller standard 

spots? (Result from experiment reported here.) Most of the USP monographs that use 

the TLC technique for chromatographic purity call for preparing a reference solution with 

a known concentration, usually a low concentration. For example, if a standard of about 

2ug is prepared and a breakdown spot is observed, an analyst can estimate the quantity 

as “NMT 2 ug” if the Rf and the intensity of the secondary spot are close to the values of 

the reference standard solution.

4. (To be done at completion of each chromatography section.) Prepare a chart, 

comparing and relating each of techniques used in chromatography for the 

following: column materials, separation theory, phases, equipment needed, ease 

of use, accuracy, sampling and sampling techniques, automation, accuracy, and 

quantitation. See Attachment E

6.4.8 C Gas Chromatography (GC) (USP <621>)

1. What type of products can be tested by GC? What products cannot be tested? 

Describe a GC method that can analyze products that normally cannot be tested 

by a GC method. GC is a chromatographic technique that can be used on any 

pharmaceutical product that contains volatile organic compounds of interest. Typical 

tests may include assay, impurity testing and organic volatile impurity analysis. The 

product is to be volatile above the lower temperature limit of the stationary phase and 

below the upper limit of the stationary phase. Products which are not volatile organic 

compounds cannot be tested without further treatment. For non-volatile compounds (for 

example sugars) and thermally labile compounds (for example antibiotics) derivatization 

can be used to transform normally non-volatile or thermally unstable compounds to a 

volatile compound usable for GC. 

2. Describe how a Flame Ionization Detector works. Describe at least three other GC 

detectors commonly used. Which detector is similar to FID and how is it used?

• An FID detector consists of an air/hydrogen flame and a collector electrode. Ions 

collected produce an electrical signal.
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• Electron-Capture detector (contains a radioactive source of ionized radiation) is 

very sensitive for compounds containing halogens and nitro groups. 

• Thermal Conductivity detector (employs a heated wire placed in the carrier gas 

stream) is used for all volatile compounds but has very low sensitivity. This is 

sometimes considered to be the universal detector since it can detect most 

volatile compounds regardless of structure.

• Nitogen-Phosphorus or alkali flame-ionization detectors (contains a thermionic 

source such as alkali-metal salt or rubidium glass bead) are very sensitive for 

organic nitrogen or phosphorus compounds but have low sensitivity for other 

hydrocarbons.

• Mass Spectrometer detector is used to identify ion fragments of the parent 

molecule. This is a very sensitive detector that can give molecular structural 

information for the compound of interest.

• The Nitrogen Phosphorus Detector is a variant of the FID. If the same sample and 

column are used with an FID and NPD, the same chromatogram will be produced 

except the NPD will ignore the hydrocarbon solvent and other non-nitrogen 

containing components and produce a much simpler chromatogram. Used for 

nitrogen containing drugs. 

3. Describe capillary GC and how it differs from packed column GC. What are the 

advantages and disadvantages of each technique? 

• Capillary GC columns are long (25 - 50 meters), thin (0.18 to .53mm ID) fused 

silica columns with higher separation efficiency (more theoretical plates) and 

superior resolution than that of a packed GC column (typically 2 meters long and 

1⁄4 inch diameter).

• Advantages are their high efficiency and sturdiness. They are easy to handle and 

do not break as easily as glass columns. The capillary column is widely used for 

complex mixtures because of their superior resolution.

• The disadvantage of capillary GC is that the columns can become overloaded, 

which affects the chromatography, and it needs a modified inlet or injection port. 

Capillary columns are also generally more expensive.

4. When is it appropriate to use temperature programming? What are the advantages 

of using temperature programming? If there is a wide range of boiling points, the 

analyst needs to increase the column temperature over time in order to elute the high 

boiling point compounds of interest. Besides decreasing the time needed for analysis, 

temperature programming provides improved peak shapes, and because later peaks 

are now taller and sharper, it provides better detection.
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5. GC commonly uses four different gases. Air, Nitrogen, Helium and Hydrogen. 

What is the purpose of each gas, how is it used and at what flow rates?

• Air is typically used as the oxidant to support the flame in an FID detector. Typical 

flows are 350 – 450 mls/min. 

• Nitrogen and Helium are typically used as carrier gases. The type of carrier gas 

used is dependent on the detector used. For capillary and megabore columns, 

typical flows are 0.5 – 10mls/min, for packed columns 8 – 40 mls/min. Nitrogen 

and helium can also be used as make-up gas in an FID detector when using 

capillary columns. Make-up gas + column flow in an FID detector typically should 

be 25- 30mls/min.

• Hydrogen is typically used as the fuel to support the flame in an FID detector. 

Typical flow rates are 30 ml/min. It is also sometimes used as a carrier gas due of 

its high efficiency in temperature programming as per the van Deemter curve.

6. (To be done at completion of each chromatography section.) Prepare a chart, 

comparing and relating each of techniques used in chromatography for the 

following: column materials, separation theory, phases, equipment needed, ease 

of use, accuracy, sampling and sampling techniques, automation, accuracy, and 

quantitation. See Attachment E

6.4.9 C High Performance Liquid Chromatography (HPLC) (USP <621>)

1. Describe a typical ‘basic’ HPLC system and the purpose of each component. 

A typical liquid chromatograph consists of:

• Reservoir - to contain the mobile phase.

• Solvent Delivery or Pumping System – causes the mobile phase to flow through 

the system at a specified flow rate. Single or multi-pump designs can be

purchased.

• Injector - to introduce the sample into the mobile phase. Autosamplers are 

commonly used.

• Column - component in which the separation is achieved by different mechanisms 

such as partition, adsorption, or ion-exchange of compounds in the test solution 

between the mobile and stationary phases.

• Detector – detects or measures compounds as they elute from a column. 

Common detectors include Single Wavelength UV, Diode Array/UV, Refractive 

Index and Fluorescence.

• Data collection device - to receive and store detector output – usually a computer 

or integrator. 
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2. What is the difference between normal phase and reverse phase? List at least 

three column types for each phase.

• Normal phase: polar stationary phases and nonpolar mobile phases

• Reverse phase: non-polar stationary phase and polar mobile phase. 

• Examples of column type for each phase:

Reverse Phase: C18, C12, C8, C4, C2, C1, Phenyl

Normal Phase: Silica, CN, NH2, PAC, Diol, Alumina

3. For the chromatograms obtained in exercise 2 ‘Acetaminophen and Caffeine 

Tablet’ calculate the following for each of the peaks: Retention Time, Retention 

Volume, Relative Retention Time to IS, Capacity Factor, Resolution, Tailing, 

Theoretical Plates, Height Equivalent Theoretical Plate, Peak Widths at base, half 

height, and 5% height. Also calculate one set of sample results using both peak 

area and peak height calculations. Are the results different? If so, explain why 

differences may result. 

(Formulas and calculations to be shown) Example given below: (Student is to perform 

exercise using information obtained from experiment.)

HPLC Exercise

Note: Electronic integrators perform most of the following calculations in automated systems. 

However, it is important to verify if the formula used by the system conforms to the USP

Figure 2 Chromatographic separation of two substances

Va = 0.70 cm V1 = 3.35 cm Values are not at 

scale. This is for 

example purpose 

only.
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In which Va and V1 are the retention volumes for the non-retained component and the 

compound under test, respectively. And t1 and t2 are the retention times measured from time 

of injection to time of elution of peak maximum.

8.31

min8.0

min0.3

1 1 ' =−= =−

a

t

t

k USP <621>

Figure 3 Capacity Factor Calculation (k1)
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Express k' in terms of the amount of mobile phase it takes to elute each compound off the 

column using the retention volumes.

8.3

70.0

70.035.3 1 ' = − =

−

=

cm

cm cm

Va

V Va

k 

Relative Retention Time (RR) of Peak 2 with respect to Peak 1

3.2

min0.3

min0.7

1

2 == =

t

t

RR

Figure 2: Interpretation of chromatograms

6.30 cm

3.35 cm

12

) 12 (2

WW

tt

R

+

−

= (The retention times and the widths have the same units)

2.4

7.07.0

)35.330.6(2

=

+

− = cm cm R

Note: The USP provides another formula to determine resolution and the number of theoretical 

plates that are convenient when electronic integrators are used. Refer to USP <621>

0.7 cm

0.7 cm
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2.1

20.5

0.12

2

%5 = × == cm

cm

f

W

T

Tailing Factor (T) Calculation

 

Number of Theoretical Plates (N)

Values from Figure 2 Interpretation of chromatograms

4.366

2

7.0

35.3

16

2

= 16 =  =



 



 



 





cm

cm

W

t

N

Note: The USP provides another formula to be used with the value of Wh/2, the peak width at 

half-height. There is also in the USP another convenient formula to be used when electronic 

integrators are used.

Calculations using peak areas and peak heights 

"Peak areas and peak heights are usually proportional to the quantity of compound eluting. 

These are commonly measured by electronic integrators but may be determined by more 

classical approaches. Peak areas are generally used but may be less accurate if peak 

interference occurs. Ref. USP <621>. 

4. What is the purpose of the internal standard used in exercise 2? Calculate one set 

of sample results without using the internal standard. Are the results different? If 

so, explain why differences may result. 

A major source of error in quantitative comparison is irreproducibility in the amount of 

sample injected, notably when manual injections are made with a syringe, in the case of 

GC methods. In these cases, the effects of variability can be minimized by addition of an 

internal standard. Today’s automatic injectors greatly improve the reproducibility of 

sample injections and reduce the need for internal standards. Ref. USP <621>.

Figure 3 Asymmetrical chromatograph
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Following, a theoretical calculation example using an internal standard:

Injection Area of Analyte Area Internal 

Standard

Response Ratio 

(Analyte/Internal STD)

STD 38.14009 35.60928 1.07107

Inj1 37.02482 36.19063 1.02305

Inj2 29.65308 27.93705 1.06142

STD 38.72269 34.96597 1.10744

Average of STD Bracket: 1.07274

Concentration of the STD (C): 10.143 mg in 50 mL volumetric flask: 0.20286 mg/mL

Label Claim of the product: 5 mg

Dilution Factor: 100

Sample weight: 0.36135 mg

Average tablet weight (ATW): 0.09059 mg

To determine the quantity, in mg, of the portions of tablet taken:





 



















 =

Sample Weight

ATW

100

s R

uR

mg C

Where Ru and Rs are the ratios of the peak responses of the corresponding analyte 

and internal standard peaks obtained from the Assay preparation and the Standard 

preparation, respectively.

Example for injection 1: mg 85.4 mg

0.36135

0.09059

07274.1

02305.1 ×= 20286.0100  =



 









 





Injection 1: Percent of label claim: 4.85/5 = 97.0%

Example for injection 2: mg 03.5 mg

0.36135

0.09059

07274.1

06142.1 ×= 20286.0100  =



 









 





Injection 2: Percent of label claim: 5.03/5 = 100.6%

Without using the internal standard...
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We used peak responses instead of the ratios:

mg 90.4 mg

0.36135

0.09059

43139.38

02482.37 ×= 20286.0100  =



 









 





Percent of label claim: 4.90/5 = 98.0%

mg 92.3 mg

0.36135

0.09059

43139.38

65308.29 ×= 20286.0100  =



 









 





Percent of label claim: 3.92/5 = 78.4%

THERE IS A DIFFERENCE!

Sample and standard solutions that do NOT contain an internal standard CANNOT 

compensate for variations in injection systems, injection volumes and 

sample/standard solutions.

5. When and why would someone use gradient elution in HPLC? What is the effect 

of temperature on HPLC? Would temperature programming such as found in GC 

be effective for HPLC? Ref. USP <621>.

• "The technique of continuously changing the solvent composition during the 

chromatographic run is called gradient elution or solvent programming. It is 

sometimes used to chromatograph complex mixtures of components differing 

greatly in their capacity factors. By increasing the mobile phase strength over time 

during the chromatographic separation the separation time between components 

is decreased."

• "Retention time in all LC separations generally decreases with increase in 

temperature. Another effect is the increase of the column efficiency. At elevated 

temperatures, viscosity of liquids decreases, and the diffusion coefficient 

increases. However, temperature effects in HPLC are not as significant as in gas 

chromatography. Volatile solvents cannot be allowed to rise to higher 

temperatures, and the high temperature may influence the stability of the attached 

bonded ligands on the adsorbent surface. The common temperature range for 

HPLC is from ambient temperature up to 60 or 70 degrees C.

• Temperature programming is not effective for HPLC.

6. (To be done at completion of each chromatography section.) Prepare a chart, 

comparing and relating each of techniques used in chromatography for the 

following: column materials, separation theory, phases, equipment needed, ease 

of use, accuracy, sampling and sampling techniques, automation, accuracy, and 

quantitation. See Attachment E
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6.4.11 C Dissolution (USP <711>)

1. What are the parameters that need be checked and corrected before dissolution 

can be run? Describe what effect each would have on an analysis.

• Temperature is to be at 37° C +/- 0.5°C (unless otherwise specified).

o Temperatures higher or lower can affect the rate the product goes into 

solution

• Rotation speed of paddle or basket.

o The faster the speed the faster the product is likely to go into solution.

• Paddle or basket height or positioning.

o Both are specified in the USP as 25 +/- 2mm from bottom of paddle/basket 

to interior vessel bottom. The distance can have a great affect for paddles 

which relates to the distance from the product. The affect is minimal for the 

basket where the product is within the basket. 

• Other Physical Parameters (level, wobble or runout, centering, vibration, verticality 

or perpendicularity).

o If these parameters are not correct, they can alter the dissolution rates 

generally resulting in erroneous results.

• The vessel also needs to be checked physically for proper dimensions, 

cleanliness, irregularities and for micro-fractures or scratches in the glass.

o Improper dimensions and irregularities can cause turbulence and 

interferences.

• Each method in the USP is developed using a specified set of parameters. The 

‘Q’ value and general limits of each (extended release) product are established 

using the parameters listed. If any parameter is not correct, then the Dissolution 

analysis is not valid.

2. Exercise 2 demonstrates a profile analysis. When would this technique be used? 

For what type of products could this technique be used? Look in the USP and list 

a monograph that uses a profile type analysis. Use this technique to determine the 

full-time range in which a tablet or capsule dissolves. This is frequently used to develop 

dissolution methods and determine the optimal time that the ‘Q’ value is established. A 

modified form of the profile technique is used for controlled release products, where a 

sample solution is drawn at set times over an extended period. Frequently used for 6, 8, 

12, 16 and 24 hr. extended-release capsules and tablets. Also, Transdermal Delivery 

Systems use modified procedures that determine a profile of a product over a period of 

time. The USP <724> describes several methods and acceptance tables for ExtendedRelease, Delayed Release and Transdermal Delivery Systems. Products include 

Clorpheniramine Maleate Extended-Release Capsules, Aspirin Extended-Release

Tablets, and Nicotine Transdermal Systems.
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3. Describe the reason and procedure for removing air from the dissolution media? 

Dissolved gases can cause bubbles to form, which may change the results of the test. 

Ref. USP <711>. One method of de-aeration is as follows: Heat the medium, while 

stirring gently to about 41°C. Immediately filter under vacuum using a filter having a 

porosity of 0.45 um or less. Provide vigorous and continuous stirring of the filtrate under 

vacuum for 5 minutes. Media must be de-aerated immediately before use. Gently 

transfer the medium directly to the vessel. Do not introduce air into the medium. Other 

validated de-aeration techniques for removal of dissolved gases may be used. Ref. USP 

<711>. There are also commercial apparatus that use a vacuum to draw the medium 

through a fine pinhole into a large holding vessel.

4. Why not use water for all dissolution media and have constant paddle/basket 

rotation speed for all determinations? Many drugs are insoluble or have limited 

solubility in water. Buffers dilute acids and surfactants are routinely used for Dissolution 

medium. They increase dissolution rates and stabilize the pH of resulting solutions. Test 

conditions such as pH and surface tension can vary depending on the source of water 

and may change during the dissolution test itself, due to the influence of the active and 

inactive ingredients. The paddle/basket speeds can affect the rate of dissolution, 

generally the faster the speed the faster the dissolution. Methods have been developed 

to optimize the paddle/basket speeds so that a satisfactory ‘Q’ value is obtained within a 

specified time. 

5. Can one compare in-vitro dissolution results with in-vivo clinical studies? Explain 

the answer. Yes. In general, an in-vitro, in-vivo (IVIV) correlation has been recognized. It 

is the goal of the pharmaceutical scientist to find a relationship between an in-vitro 

characteristic of a dosage form and its in-vivo performance. An effort to connect 

dissolution and pharmacokinetics is often referred to as “in vitro-in vivo correlation” 

(IVIVC) analysis. Numerous IVIVC studies can be found in the literature. Controlled 

release products, rather than immediate release products, are the focused in the IVIVC 

literature. Since only products with dissolution rate-limited absorption (and 

with complete complete absorption) can be expected to exhibit a slope of one and zero 

intercept (Y=mX) mX). The relationship is not as well defined for immediate release 

products but depending on the in-vitro release rate and the in-vivo adsorption rate, the 

two methods are generally recognized as satisfactory for comparison.
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Document Number:

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Revision #: 03

Revised:

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Title: 

ORA Lab Manual Vol. IV Section 3 - Drug Chemistry Analysis (IV-03) Page 63 of 63

For the most current and official copy, check QMiS.

Attachment E - Chromatography Chart