Compendial HPLC Practices

Behnam Davani
USP Consultant

Last Update: January 2018

Horacio Pappa
CM-621-02

Disclaimer

Because USP text and publications may have legal implications in the U.S. and elsewhere, their
language must stand on its own. The USP shall not provide an official ex post facto interpretation
to one party, thereby placing other parties without that interpretation at a possible disadvantage.
The requirements shall be uniformly and equally available to all parties.

In addition, USP shall not provide an official opinion as to whether a particular article does or does
not comply with compendial requirements, except as part of an established USP verification or
other conformity assessment program that is conducted separately from and independent of USP's
standard-setting activities.

Certain commercial equipment, instruments or materials may be identified in this presentation to

specify adequately the experimental procedure. Such identification does not imply approval,
endorsement, or certification by USP of a particular brand or product, nor does it imply that the
equipment, instrument or material is necessarily the best available for the purpose or that any
other brand or product was judged to be unsatisfactory or inadequate.

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Behnam Davani

USP Affiliation: USP Consultant (Former USP employee 1999 – July 2017)
Title: Consultant
Company: Independent Consultant
Education: Ph.D. in Analytical Chemistry
MBA in Management

Dr. Behnam Davani is currently an Independent Consultant. He has more than 28 years of experience in industry,
Instructor Photo compendial/regulatory science and training. He has extensive experience as faculty for the development and
teaching of several compendial and cGMP courses both domestically and internationally for USP Global Education
Program. Dr. Davani is also editor and author of a recent book: "Pharmaceutical Analysis for Small Molecules",
published in August 2017.

Prior to this position, he was the Principal Scientific Liaison between the USP Science Division and the USP
Education Department. He was responsible for scientific management of the development and teaching of USP
Education courses for stakeholders worldwide. Additional responsibilities included:
• Faculty for several Pharmacopeial Education course topics worldwide (China, India, Russia, Korea, Europe, Latin America and Middle East).
These courses included validation/verification/transfer of analytical procedures, impurities in drug substances and drug products, compendial
HPLC, residual solvents, spectroscopy and stability studies for pharmaceutical products.
• Providing scientific support to the USP International Regulatory Affairs Department and Global Public Health Department for outreach and
training for international regulatory agencies.
Prior to this position, Dr. Davani was Director of the Chemical Medicine Department at USP. In this capacity, he provided scientific leadership and
training to a team of US and internationally based staff, responsible for the development and modernization of USP monographs for low molecular
weight pharmaceuticals
Prior to joining USP in 1999, Dr. Davani worked in industry (Sigma-Aldrich, MRI Global) for more than ten years in various technical management
positions. He is a member of the American Chemical Society and the American Association of Pharmaceutical Scientists.

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Outline

Theory

Equipment

System Suitability

Adjustments to the Chromatographic System

Harmonization

Validation of Compendial Procedures

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 Compendial HPLC Practices
Theory

Chromatography

Chromatography is a separation technique

Components of a mixture are distributed between two phases, one is stationary
and the other mobile.

The stationary phase may be a solid or a liquid supported on a solid.

The mobile phase may be gaseous or liquid or supercritical fluid.

The separation may be based on adsorption, partition, or ion exchange; or it
may be based on differences in molecules, size, mass, or volume.

USP 36–NF 31 <621> 6

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Chromatography

Types of Chromatographic tests in USP

– Paper--useful for identification, convenient and simple
– Thin-layer--useful for identification, convenient and simple
– Column--useful for the separation of individual compounds from mixtures
– Gas--requires more elaborate instrumentation, useful for identification and quantitation
including low level impurities
– HPLC--requires more elaborate instrumentation, useful for identification and quantitation
including low level impurities

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HPLC

HPLC-High Performance (Pressure) Liquid Chromatography

– Definition: Separation Technique based on a solid stationary phase and a liquid mobile phase
– Separation Mechanisms: partition, adsorption, ion-exchange or molecular size depending on
the type of stationary phase used
– Useful for drugs, nonvolatile or thermally unstable compounds

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Solvent Polarities

Common solvents in order of increasing polarity:

– Pentane
– Hexane, Heptane, Isooctane
– Toluene
– Isopropyl alcohol
– Tetrahydrofuran
– Chloroform
– Acetone, methanol
– Acetonitrile
– Water
Reference: L.R. Snyder, J. Chromatogr., 92, 223 (1974); J Chromatogr. Sci., 16, 223 (1978)
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Normal Phase HPLC

Polar stationary phase and a non-polar mobile phase

Typical column: L3

Typical mobile phase: n-Hexane

Example: Anthralin Assay

Column: L3

Mobile Phase: n-hexane, dichloromethane, and glacial acetic acid (82: 12: 6)

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Reversed Phase HPLC

Non polar stationary phase with a polar mobile phase

Typical columns used are L1 (C18) and L7 (C8)

Typical mobile phase include a mixture of aqueous buffer with an organic
solvent or water and an organic solvent

Example: Acetaminophen Capsules Assay

Column: L1

Mobile Phase: water and methanol (3:1)

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Ion Pair Chromatography

Ionized or ionizable sample components (charged)

Ion Pair reagent (opposite charge) add to the mobile phase to form neutral
complex (ion-pair) with the sample component

Separate using a reversed phase column

The more hydrophobic the pairing agent the greater the retention on the column

Solute RP Ion-Paring Reagent

Anionic (Acid) Tetraalkylammonium cation

Cationic (Base) Alkyl Sulfonate Anion

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Ion Pair Chromatography

Example: Tripelennamine Hydrochloride

Column: L7

Mobile Phase: 530 mL methanol, 1.0 mL N,N-dimethyloctylamine, 430 mL of
Ion-pair solution adjust to
pH 3.0

Ion-pair solution: 29 mM sodium 1-octanesulfonate solution

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Ion-Exchange Chromatography

Stationary phase synthetic organic resins

Separation affected by pH, temperature, ionic concentration, organic modifiers,
etc.

Increasing the ionic concentration causes competition for active sites which
displaces solutes

Vary pH to get differences in the charge of various sample components. Charge
depends on pH of solvent and pKa of ionizable group

Useful for peptides, proteins, oligonucleotides, carbohydrates, etc.

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Ion-Exchange Chromatography

Cation exchange resins contain negatively charged active sites to separate

basic substances like amines

Typical columns used L6, L17, L22, L19, L34

Example: Lactitol Assay

Column: L34

Mobile Phase: water

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Ion-Exchange Chromatography

Anion exchange resins contain positively charged active sites for separation of
negatively charged groups such as phosphate and carboxylate

Typical columns used are L12, L23, L31

Example: Cinoxacin Assay

Column: L12

Mobile Phase: Dilute 100.0 mL of Sodium borate solution and 0.426 g of sodium
sulfate with water to 1000mL

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Size Exclusion Chromatography

Gel or Porous stationary phase

Separation by size

Solute exchange between mobile phase in and outside of pores

Too large to enter pores = unretained

Increased retention with smaller size molecules

Pore size range of stationary phase determines size range of separated
components

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Size Exclusion Chromatography

Gel permeation chromatography: nonaqueous mobile phase

Gel filtration chromatography: aqueous mobile phase

Typical columns used L25, L33, L37, L54

Example: Dextran 1 Molecular weight distribution and average molecular weight

Column: L54

Mobile Phase: sodium chloride solution 2.9 g/L

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Hydrodynamics of Chromatography

Peak width is dictated by the HETP (Height Equivalent to a Theoretical Plate)

HETP will depends on the mobile phase hydrodynamics, the stationary phase
and the properties of the mixture to be separated

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The Van Deemter Equation

H= A · dp + B/µ + C · de2 · µ

Eddy Diffusion
Longitudinal Diffusion
Multi-path Effect Mass Transfer (Kinetics)
dp = Diameter of particle
µ = Linear velocity de = “Effective particle
Also related to the quality size” (In the case of fully
of the packing of the porous particles this is dp)
column

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Van Deemter: “A” Term

H = A·dp + B/µ + C·de2·µ

Eddy Diffusion or Multi-Path-Effect

Desired:
• Smaller particles
• Narrow particle size distribution
with uniform shape
• Optimized packing

Dispersion due to eddy diffusion or multi-path effect

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Van Deemter: “B” Term

H= A · dp + B/µ + C · de2 · µ
Longitudinal Diffusion

For HPLC
Longitudinal diffusion inside the Zero linear velocity
column is negligible with higher
linear velocities and independent of
w1/2
particle size
6

Very low linear velocity tR

w1/2
5 6

At or above optimum linear velocity tR

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Van Deemter: “C” Term

H = A·dp + B/µ + C·de2·µ

Mass Transfer Kinetics

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The Van Deemter Equation

H= A · dp + B/µ + C · de2 · µ

Eddy Diffusion
Longitudinal Diffusion
Multi-path Effect Mass Transfer (Kinetics)
dp = Diameter of particle
µ = Linear velocity de = “Effective particle
Also related to the quality size” (In the case of fully
of the packing of the porous particles this is dp)
column
Plate Height (H)

C
B
A
Mobile phase velocity (µ)
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Resolution Equation

N  α - 1  k 
RS = •  • 
4  α   1 + k 
Efficiency Selectivity Retention

N = Theoretical plates
α = t’R2/t’R1
k = tR-to/to

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Retention

N  α - 1  k 
RS = •  • 1 + k 
4  α   
Efficiency Selectivity Retention
Term Term Term

Varies with mobile phase composition

Optimal 2 ≤ k ≤ 10

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Selectivity

Related to peak separation

Depends on stationary phase, solvent, temperature, etc.

High α

N  α - 1  k 
RS = • •
4  α   1 + k 

Efficiency Selectivity Retention
Term Term Term Low α

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Efficiency

Low N
N  α - 1  k 
RS = •  • 1 + k 
4  α   
Efficiency Selectivity Retention
Term Term Term
High N

It dictates the peak width

Depends mainly on the column length and particle size

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Compendial HPLC Practices
Equipment
HPLC Equipment Components

Pump

Injector

Column

Detector

Data Collection/Analysis Device

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Pumping Systems

Deliver Mobile phase at a constant rate with minimal fluctuations

Operating pressure up to 6000 psi (400 bar)

Inert to corrosive mobile phase components

Programmable to vary the range of Mobile phase components--Gradient or
Isocratic Mixing
– High pressure mixing
– Low pressure mixing

Most common-dual piston reciprocating pump

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Pumping Systems

Isocratic run--the mobile phase composition is kept the same throughout the run
– Pre-mix the mobile phase
– Mix mobile phase by pump

Gradient run--changing the mobile phase composition during the

chromatographic run

Gradient useful for complex mixtures with different capacity factors to reduce
analysis time

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High Pressure Mixing System

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Low Pressure Mixing System

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Dead Volume, Dwell Volume, etc.

Dead Volume: Volume of mobile phase inside the column

Extra-column Volume: All the volume accessible to the analyte outside the
column

Dwell Volume: Only apply to gradients. Represents the volume of mobile phase
from the point where the gradient is formed to the column inlet. It is critical
during method transfer

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Dead Volume, Dwell Volume, etc.

Dwell volume determination (Eu. Ph.)

• Gradient: Water / 0.1% acetone in water

• Gradient duration (tG) 20’
• Detection 265 nm
• Without column

tD

tD=t0.5 – 0.5tG

Dwell Volume (D)= tD x F

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Injector

Dissolved solute is injected into the mobile phase

Manually
– Six port two position valve.
– Position 1 flow moves from pump to column and bypasses injector loop
– Position 2 flow moves from pump through injector loop to column

Automatically by autosampler
– Useful for quality control and high throughput screening
– May need to change syringe or loop based on injection amount
– May need to check precision and linearity, sample carryover, wash solvent, vial filling-minimum
and maximum
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Injector

Want sample to enter column as a tight band or plug

Avoid excess tubing (length and diameter) between injector and column and
between column and detector and connectors with dead volume which may
cause the sample band to become broader before entering the column

Band broadening increases peak widths which decreases column efficiency and
decreases resolution

Band broadening can also occur as the sample passes through the column

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Columns

Monograph specifies packing category: L1, L2, L3…

Reagents, Indicators, and Solutions—Chromatographic Columns defines
packing categories

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Columns (cont)

Different brands of columns in the same category may behave differently in a

particular method:
– Behavior depends on:
• Carbon load
• Surface area
• Endcapping
• Particle shape
• Particle size
• Effect of surface silanols

Variations may also occur with different lots of the same brand

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Solid Support Types

Silica
– Type A
– Type B

Polymers

Hybrids

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Silica Materials

Structurally strong

Easy-to-modify surface

pH stability (pH 1.5 – 8 or 10)

High efficiency

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Type A Silica

Old type (Irregular or spherical)

– Low amount of bonded or associated silanols

– High metal content (>1000 ppm)

• Metals activate silanols, inducing ionization

• Metals chelate acids, interact ionically with bases

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Type B Silica

Newer Type

– High amount of bonded or associated silanols

– Ultra-high purity (>99.9%)

– Low metal content (<50 ppm)

– Better peak shape for acids and bases

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Silica Bonded Phase – pH stability

Low pH Ligand Hydrolysis High pH Silica Dissolution

H+
H+ OH- OH-
H+ OH-
H H
O O O O
O Si O Si O O Si O Si O

O O O O

Silica Surface w/ Alkyl Chain Silica Surface w/ Alkyl Chain

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Polymeric Particles

Extended pH stability (1-14)

Chemically inert

Poor mechanical strength
– Lower efficiency
– Less reproducible

Very hydrophobic

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Efficiency Comparison - Polymer vs. Silica

Polymer
L21 5µm 150x4.6
N = 1,163
2
3
1 4

2 4 6 8 10

Silica
L1 5µm 150x4.6 N = 18,992 Isocratic:80:20 ACN/Water
3 Flow: 1mL/min
2
• Uracil
• Acetophenone
• Toluene
1 • Acenaphthalene

2 4 6 8 10
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Hybrid Particles

Mixture of Silica and Polymer

– Mixed throughout
– Surface coated

Higher mechanical stability

Efficiency more similar to Silica

pH stability 1-12

Easier to modify, more stationary
phases available

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Efficiency Comparisons

2 Silica L1 5µm 150 x 4.6 mm

N = 84,573 p/m

1 Mobile phase: 50:50 Acetonitrile/

3 4 20 mM NaH2PO4, pH 2.5
5
Flow rate: 1.0 mL/min
Detection: 230 nm
Components: 1. Ethylparaben
2. Naproxen
4 6 8 10 12 14
3. Diflunisal
Time (min)
4. Indomethacin
2 5. Ibuprofen
Hybrid L1 5µm 150 x 4.6 mm
N = 80,480 p/m
1
3
4 5

2 4 6 8 10 12 14
Time (min) 50
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Effect of Particle Size at 30°C

28 1 .9 u 3 0 C
2 .5 u 3 0 C
26
3u30C
24 5u30C
22

20
Plate Height µm

18

16

14 5µm
12

10
3µm
8
2.5µm
6
1.9µm
4
0 2 4 6
L in e a r V e lo c ity ( m m /s e c )
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General Theory

L

Performance Increase N =
hd p

N  α − 1  k 

Resolution Increase R =   
4  α  k + 1 

φη L µ

Backpressure Increase ∆P = 2
µ = linear velocity d p
L = column length
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Fully Porous Particles

Standard Pressures =
Conventional HPLC

5 µm 3 µm
10 µm
Operating Pressure up to 400 bar (6,000 psi)

Ultra-High Pressure =
UHPLC
sub-2 µm
Pressure 400-1000 bar (15,000 psi)

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Monolithic Columns

Não é possív el exibir esta imagem.

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Core Shell Columns

Non-porous silica core with thin porous shell

Thin shell reduces the diffusion path length

Result: ultra-fast kinetics of diffusion

Efficiencies similar to sub 2 µm fully porous particles

Back pressure similar to 3 µm

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Columns

What brand of column was used for a particular test?

− If you have access to USP–NF on-line scroll to the end of the monograph containing the test of
interest and click on the link under the heading Chromatographic Column
• You can also find Chromatographic Columns on-line database on the USP website at:
http://www.usp.org/resources/chromatographic-columns

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USP–NF Online

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www.uspchromcolumns.com

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www.uspchromcolumns.com

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Briefing in PF

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Columns

Column Equivalency Compendial Tool:

http://apps.usp.org/app/USPNF/columns.html

An interactive tool that allows users to find possible alternative HPLC columns to
the column used to develop and validate a chromatographic procedure.

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Column Characterization

Two approaches cited by USP:

– USP database: NIST SRM 870 mix
– PQRI/Column equivalence database

Other theories exist – None is 100%

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SRM 870

Void Marker:
– Unretained volume required to calculate retention factor

Methylene Selectivity:
– Differentiation based on differences in hydrophobicity

Efficiency (N):
– Toluene or Ethylbenzene

Peak Asymmetry:
– Chelating Agent: indicator of metals in silica

Silanol Activity:
– Active base that interacts with free silanols
– Peak shape is a measure of column deactivation

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SRM 870 Conditions

Columns: L1 10 µm 250 x 4.6 mm Type B

L1 10 µm 250 x 4.6 mm Type A
Mobile phase: Isocratic with 80/20 MeOH / 20 mM KH2PO4 buffer at pH
7.0 for 25 min
Flow rate: 1.0 mL/min
Injection Vol.: 2.0 µL
Detection: 254 nm

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SRM 870: Type B

Peak Name
1 Uracil
mAU

2 Toluene
Efficiency 3 Ethyl Benzene
200

Void + 4 Quinizarin
Marker Metal Chelator
Methylene 5 Amitriptyline
150
1 Selectivity
4
100
2 3 Silanol Activity

50
5

0 5 10 15 20

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Metal Chelation

Si O H
O O

Si O O

3+ HO
Al Chelation

HO
Si O
CO O H
O
HO O C
Si
O
Si O H

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SRM 870: Type A

Quinizarin
Peak Name

1 Uracil
200 2 Toluene
3 Ethyl Benzene
150
High Metal Content 4 Quinizarin
1 Silica 5 Amitriptyline
100

2 3
50

5
4
0

0 5 10 15 20

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PQRI Database

Proposed by Synder/Dolan

Columns are evaluated for:
– Hydrophobicity (H)
– Steric interaction (S)
– Hydrogen-bond acidity (A)
– Hydrogen-bond basicity (B)
– Ion-exchange capacity (C)

Two pH choices - Low 2.8 or High 7.0

Similarity given by an F rating:
– F < 3 are likely to give equivalent results

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PQRI Database Cont.

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Column Maintenance

Follow manufacturer’s recommendations

In general, for reversed phase columns, routinely flush with strong mobile phase
component. Flush buffers with water/organic mixture in the same ratio as the
mobile phase, and store columns in water/organic mixture such as methanol or
acetonitrile and water (1:1)

Use a log book

Use the reference chromatogram and standard conditions to check column
performance

Segregate columns by use (mobile phase, product, test, etc.)

Use an inline filter
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Column Maintenance: Use of Guard Column

“In LC procedures, a guard column may be used with the following

requirements, unless otherwise is indicated in the individual monograph:

the length of the guard column must be NMT 15% of the length of the analytical column,
(a)
and

(b)the packing material should be the same as the analytical column (e.g., silica) and contain
the same bonded phase (e.g., C18).

In any case, all system suitability requirements specified in the official

procedure must be met with the guard column installed”
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Standard and Sample Preparation

The dilution scheme can be changed, but the final concentration of the analyte
can not change more than 10% -- see General Notices

Glassware sizes, weights, volumes, concentrations can be changed

Errors associated to dilutions and weighing (see USP <31> and <41>)

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Detector

Spectrophotometric
– Variable
• Single wavelength
• Dual wavelength
– Multiwavelength

• Diode array

Refractive Index

Fluorometric

Electrochemical

MS
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Quantitation

During quantitation, disregard peaks caused by solvents and reagents or arising

from the mobile phase or the sample matrix.

In the linear range, peak areas and peak heights are usually proportional to the
quantity of compound eluting. The peak areas and peak heights 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.

In tests for impurities for both the External Standard Method, when a dilution of
the sample solution is used for comparison, and the Normalization Procedure,
any correction factors indicated in the monograph are applied (e.g., when the
relative response factor is outside the range 0.8–1.2).

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Quantitation

When the impurity test prescribes the total of impurities or there is a quantitative
determination of an impurity, choice of an appropriate threshold setting and
appropriate conditions for the integration of the peak areas is important. In such
tests the limit at or below which a peak is disregarded is generally 0.05%. Thus,
the threshold setting of the data collection system corresponds to at least half of
this limit. Integrate the peak area of any impurity that is not completely
separated from the principal peak, preferably by valley-to-valley extrapolation
(tangential skim).

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Methods of Integration

A C

B D

Bicking, M. LCGC NORTH AMERICA VOLUME 24 NUMBER 4 APRIL 2006

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Periodic Instrument Check for HPLC

Pump (solvent delivery system)

– Flow rate accuracy
– Gradient composition accuracy

Injector
– Volume precision
– Carry-over
– Thermostating accuracy and precision (Auto sampler)

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Periodic Instrument Check for HPLC (cont.)

Detector
– Linearity
– Wavelength accuracy
– Noise and drift
– Sensitivity (Florescence)
– Signal stability (Electrochemical)

Column compartment (Oven or cooling device)

– Temperature accuracy

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Compendial HPLC Practices
System Suitability
System Suitability

…used to verify that the chromatographic system is adequate for the intended
analysis.

The Chromatographic system includes:

– Equipment
– Electronics
– Analytical Operations
– Samples

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Parameters

Resolution

Efficiency

Precision

Tailing

Retention Factor (Capacity Factor)

Signal to Noise ratio

Peak to valley ratio

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Resolution

Resolution, Rs, ensure that closely eluting compounds are

resolved from each other

2 ( t Rb − t Ra ) 1 . 18 ( t Rb − t Ra )
R = R =
Wa + Wb W 0 .5 a + W 0 .5 b

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Resolution

Function of column efficiency (N), the separation factor, and the capacity factor,
k

Measure of the resolving power of the system

Generally, not less than 2.0

Most closely eluting pair

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Resolution

R = 1.0 R = 1.5 R = 2.0

FDA (CDER), Reviewer Guidance: Validation of Chromatographic Methods, Nov. 1994

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Separation as a Function of Rs

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Column Efficiency

2
 t 
2
 
N = 16  R   t
N = 5 . 54  R

W  Wh 
 2 

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Column Efficiency

Method Peak width Factor

Inflection point 60.3% of peak height 4.000

Half-height 50% of peak height 5.545

4 Sigma method 13.4 of peak height 16.00

5 sigma method 4.4% of peak height 24.00

Intersection of tangents with

Tangent method 16.00
baseline

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Column Efficiency

Only one peak of interest

Measure of peak sharpness

Detection of trace components

Generally, not less than 2000 (HPLC)

Isocratic/isothermal system

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Repeatability

USP: Data from five replicate injections of the analyte are used to calculate the
relative standard deviation if the requirement is 2.0% or less; data from six
replicate injections are used if the relative standard deviation requirement is
more than 2.0%

Number of individuals injections

3 4 5 6
Upper limit Maximum permitted relative standard deviation
2.0 0.41 0.59 0.73 0.85
2.5 0.52 0.74 0.92 1.06
3.0 0.62 0.89 1.10 1.27

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Asymmetry Factor (Tailing)

(a + b) b'
T = A =
2a a' T A
1.0 1.0

1.2 1.3

1.4 1.6

1.6 1.9

a’ 1.8 2.2
b’ 10 % of Peak Height

a b 5 % of Peak Height

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Retention Factor (or Capacity Factor, k)

Measure of where the peak of interest is located with respect to the void volume,
i.e., elution time of the non-retained components

Generally, not less than 2

k = (tR - tM)/ tM

tR: retention time of the analyte

tM: retention time of a non-retained component

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Signal to Noise Ratio (S/N)

S/N = 2H/h

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Peak-to-Valley Ratio

p /v = H p H v

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System Suitability USP <621>

Unless otherwise directed in the monograph, system suitability parameters are

determined from the analyte peak.

Measured values of Rr or RF or tR for the sample substance do not deviate from
the values obtained for the reference compound and mixture by more than the
statistically determined reliability estimates from replicate assays of the
reference compound.

Relative retention times may be provided in monographs for informational
purposes only to aid in peak identification. There are no acceptance criteria
applied to relative retention times.

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System Suitability USP <621>

Make injections of the appropriate preparation(s) as required in order to

demonstrate adequate system suitability (as described in the Chromatographic
system section of the method in a monograph) throughout the run.

Whenever there is a significant change in the chromatographic system
(equipment, mobile phase component, or other components) or in a critical
reagent, system suitability is to be reestablished.

No sample analysis is acceptable unless the suitability of the system has been
demonstrated

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Compendial HPLC Practices
Adjustments to the Chromatographic System
Adjustments to the Chromatographic System

USP <621> provides allowable adjustments to:

– Mobile phase composition
– Mobile phase pH
– Buffer concentration
– Column temperature
– Injection volume
– UV-VIS detector wavelength
– Particle size
– Column length
– Column inner diameter
– Flow rate
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Adjustments: Isocratic vs. Gradient

“Adjustments to the composition of the mobile phase in gradient elution may

cause changes in selectivity and are not recommended. If adjustments are
necessary, change in column packing (maintaining the same chemistry), the
duration of an initial isocratic hold (when prescribed), and/or dwell volume
adjustments are allowed. Additional allowances for gradient adjustment are
noted in the text below.”

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Adjustments: Isocratic vs. Gradient

Adjustment Isocratic Gradient

pH of Mobile Phase Allowed Allowed

Concentration of Salts in Buffer Allowed Allowed
Allowed Not allowed
Ratio of Components in Mobile Phase

Column dimensions (length, diameter) Allowed Not allowed

Particle size Allowed Not allowed
Flow rate Allowed Not allowed
Injection volume Allowed Allowed
Column temperature Allowed Allowed

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Adjustments to the Chromatographic System

Must show improvement in chromatography

Cannot be made where there is column failure or system malfunction

Adjustment may require verification

Consider multiple adjustments carefully

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Adjustments to the Chromatographic System

Adjustments made to the chromatographic system in order to meet system

suitability requirements cannot be incorporated into the method.

Changes made to the chromatographic system because of the use of columns

with different particle sizes can be incorporated into the method. (example,
changing from HPLC to UHPLC).

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Mobile Phase Composition

Allowable Adjustment:
– Minor component (≤50%) ± 30% relative
– Adjustment cannot exceed ± 10% absolute
– Final concentration cannot be reduced to zero
– Changes to gradient methods are not recommended

Effect:
– Solvent strength can increase or decrease retention
– Gradient must be scaled if column dimensions change

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Mobile Phase Composition

Example (binary)

Specified ratio of 50:50
– 30% of 50 is 15% absolute (too much),
– Maximum allowable change is 10%
– Allowable Adjustments: 40:60 to 60:40

Specified ratio of 2:98

– 30% of 2 is 0.6% absolute
– Well within Max 10%
– Allowable Adjustments: 1.4:98.6 to 2.6:97.4
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Mobile Phase Composition

Example: Ternary Mixtures

Specified ratio 60:35:5 (A:B:C)
– Component B:
• 30% of 35 is 10.5% absolute, exceeds max 10%
• Adjusted only within the range of 25% to 45% (± 10%)
– Component C:
• 30% of 5 is 1.5% absolute
– Component A:
• Sufficient quantity used to give a total of 100%

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Mobile Phase pH

Allowable Adjustment:
– pH can be changed ± 0.2 units

Effect
– pH scale is logarithmic, 1 unit =10X increase
– Low pH can protonate acidic compounds (silanols)
– High pH can neutralize basic compounds
– Ensure pH keeps the drug in one state
• Avoid pH = pKa

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Performance at Different pH

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Buffer Concentration

Allowable Adjustment:
– Buffer concentration ± 10%
– Provided permitted pH variation is also met

Effect
– If buffer concentration is insufficient, pH changes may occur depending on injection
– Concentration is most critical at pH extremes of that buffer

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Column Temperature

Allowable Adjustment:
– Column temperature ± 10°C

Effect
– Temperature can cause shifts in retention time
– Ambient Temp. is relative, it should be controlled
– Higher Temp. decreases mobile phase viscosity
• Improves column efficiency
• Lower column back pressure
• Decrease analysis time

– These changes may affect resolution

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Changing Temperature

Method: Nitroaromatics

Mobile phase: 55:45 MeOH/Water hold 2 min

to 75:25 in 22 min

Flow rate: 1.0 mL/min

Columns used: C18, 250 x 4.6 mm, 4 µm

Temperature: 25 °, 30 °, 35 °

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Column Temperature

3
Temperature: 25 °C

13
7 8
1 5
2 14

4 6 9 10 11 12

10 12 14 16 18

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Column Temperature

Elution Order
Co-Elution
Change Temperature: 30 °C

5,7
2,3
Decreased
1 8 Retention
6 13

14
4 9 10 11
12

8 10 12 14

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Column Temperature

Co-Elution

Elution Order
5,6,7 Temperature: 35 °C
Change

3 Co-Elution
Decreased
Retention
13
8 11,12
1 2
14

4 9 10

18 10 12 14

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Injection Volume

Allowable Adjustment:
– The injection volume can be adjusted as far as it is consistent with accepted precision, linearity, and
detection limits.
– Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction
in N and resolution.
– Applies to both gradient and isocratic separations.

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UV/VIS Detector Wavelength

Allowable Adjustment:
– No deviation permitted
– If detector is suspected
• Verify detector wavelength ±3 nm
• Use manufacture or other validated protocol

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Particle Size

For isocratic separations, the particle size and/or the length of the column may
be modified provided that the ratio of the column length (L ) to the particle size
(dp ) remains constant or into the range between - 25% to +50% of the
prescribed L /dp ratio.

Alternatively (as for the application of particle-size adjustment to superficially

porous particles), other combinations of L and dp can be used provided that the
number of theoretical plates (N ) is within -25% to +50%, relative to the
prescribed column.

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Particle Size

Flow Rate: When the particle size is changed, the flow rate may require
adjustment:
F2 = F1 (dc22 dp1) / (dc12 dp2)

Where F1 and F2 are the flow rates for the original and modified conditions,
respectively; dc1 and dc2 are the respective column diameters, and dp1 and dp2 are
the particle sizes.

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Particle Size

dc 22 × dp 1
F 2 = F1
dc 12 × dp 2
Relative Values
L, mm dc, mm dp, µm L/dp F N Pressure Time
250 4.6 10 25,000 0.5 0.8 0.2 3.3
150 4.6 5 30,000 1.0 1.0 1.0 1.0
150 2.1 5 30,000 0.2 1.0 1.0 1.0
100 4.6 3.5 28,600 1.4 1.0 1.9 0.5
100 2.1 3.5 28,600 0.3 1.0 1.9 0.5
75 4.6 2.5 30,000 2.0 1.0 4.0 0.3
75 2.1 2.5 30,000 0.4 1.0 4.0 0.3
50 4.6 1.7 29,400 2.9 1.0 8.5 0.1

Transfer of HPLC Procedures to Suitable Columns of Reduced Dimensions and Particle Sizes. Pharm. Forum 35(6), www.usppf.com
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Particle Size

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Column ID

Allowable Adjustment:
– Can be adjusted provided that linear velocity is kept constant

F2 = F1 (dc22 dp1) / (dc12 dp2)

Effect
– Solvent savings
– Reduced band broadening (sharper peaks)
– Increased resolution (sharper peaks)

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Column ID

Method: Atenolol Impurity

Mobile phase: Isocratic: 100% of 2.0g of Sodium Octanesulphonate + 0.8g of Tetrabutyl

Ammonium Hydrogen Sulphate in 2L of 360ml Methanol + 40ml of
THF+1600ml of 3.4g of potassium Dihydrogen Phosphate in 2L of water
(pH adjusted to 3.0 using Phosphoric acid)

Flow rate: 0.8 & 0.36 mL/min (scaled for linear velocity)

Columns used: L1 5µm 150 x 4.6* mm

L1 5µm 150 x 3.0* mm

System suitability: Rs = Impurities I & J > 1.4

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 Column ID
mAU

B
8 Rs =3.81
6
4.6 mm
J Solvent Usage: 28 mL
4

2 I F
G
0

5 10 15 20 25 30 35

Rs =3.75
mAU
B
J
8

2 X Sensitivity 3.0 mm
6 Solvent Usage: 12.6 mL
4
I
2
F
G
0

5 10 15 20 25 30 35
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Changing Column ID

Summary: Decrease column ID by 35%:

1. Retention time: About the same

2. Solvent Usage: Decreased by 57%

3. Sensitivity: ~ 2X Increase

4. System suitability: Retained Rs > 1.4 from excipients

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Flow Rate

Allowable Adjustment:
– Flow rate ± 50%
– If changing particle size and/or ID scale to maintain linear flow velocity
– Scaled flow can then be adjusted ± 50%

Benefit
– Increasing flow is the easiest way to reduce run time
– Isocractic: 2 X Flow = ½ Run Time
– Resolution decreases, but not as dramatically
– Faster linear velocity is required with smaller particles
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Changing Flow Rate

Method: Estradiol

Mobile phase: Water / Acetonitrile (45:55)

Flow rate: 1.0 and 1.5 mL/min

Columns used: L1 5µm 250 x 4.6 mm

System suitability: Rs; Estradiol and Estrone > 2.0

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 Changing Flow Rate

1
mAU
1.0 mL/min
350
1. Ethyl Paraben (IS)
300
2. Estradiol
250
3. Estrone 2
200 Rs = 7.11
150 tR = 8.15 min
100

50 3
0

1 2 3 4 5 6 7 8 9 min

1
1.5 mL/min

2 Rs = 6.15
tR = 5.56 min
3

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Changing Flow Rate

Summary: Increasing flow rate 50%

1. Retention time: Reduced from 8.15 to 5.56 min

32% reduction

2. Resolution: Reduced from 7.11 to 6.15

13.5% reduction

3. System suitability: Maintained Rs > 2.0

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Questions (True/False)

A. The acceptable variation for relative retention time is about +/-10%.

B. p/v ratio is the preferred term for calculation of peak resolution.

C. The repeatability guideline in USP <621> needs to be followed in all cases

because it is a mandatory general chapter.

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Questions (True/False)

D. The repeatability criteria in USP <621> should are used for the drug product
only if this criteria is not in the monograph.

E. Adjustments to the chromatographic system is only allowed for isocratic

procedures.

F. Adjustments to the chromatography system is allowed to provide flexibility for

the column failure.

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Compendial HPLC Practices

Harmonization
Pharmacopoeial Discussion Group (PDG)

USP has participated since PDG’s inception in 1989

Informal body of representatives from
– European Pharmacopoeia (EDQM)
– Japanese Pharmacopoeia (MHLW)
– United States Pharmacopeia (non-governmental)
– WHO (observer since 2001)

Linked to ICH (until 2011)

Focused on harmonizing general chapters and excipient monographs

Steady but limited progress (Retrospective Harmonization)
– 29 out of 36 General Chapters
– 46 out of 62 Excipients

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PDG Stages of Harmonization

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Liquid Chromatography: Isocratic Elution

Column parameters and flow rate

Stationary phase: no change of the physico-chemical characteristics of the

stationary phase permitted,

Column dimensions:
– The particle size and/or length of the column may be modified provided that the ratio of the
column length (L) to the particle size (dp) remains constant or in the range between -25 per
cent to +50 per cent of the prescribed L/dp ratio.. Further adjustments in method conditions
(mobile phase, temperature, pH, etc.) may be required, within the permitted ranges described
under System Suitability and Adjustment of chromatographic conditions in this chapter.

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Liquid Chromatography: Isocratic Elution

When the particle size is changed, the flow rate may require
adjustment, because smaller-particle columns will require higher
linear velocities for the same performance (as measured by reduced
plate height). Flow rate changes for both the change in column
diameter and particle size can be made by:

F2 = F1 (dc22 dp1) / (dc12 dp2)

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Liquid Chromatography: Isocratic Elution

Injection volume
When the column dimensions are changed, injection volume adjustment may be guided by,
Vinj,2 = Vinj,1 (L2 dc,22) / (L1 dc,12)
Vinj,1 = original injection volume
Vinj,2 = new injection volume
L1 = original column length
L2 = new column length
dc,1 = original column internal diameter
dc,2 = new column internal diameter: ,
Independent of changing column dimensions, Injection volume may be varied provided System Suitability criteria
remain within their established acceptability limits. When Injection volume is decreased, special attention should be
given to (limit of) detection and repeatability of the peak(s) to be determined. An increase is permitted provided in
particular linearity and separation of the peak(s) to be determined remain satisfactory
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Liquid Chromatography: Gradient Elution

A change in column dimensions, and thus in column volume, impacts the gradient volume which
controls selectivity. Gradients are adjusted to the column volume by changing the gradient volume in
proportion to the column volume. This applies to every gradient segment volume. Because the
gradient volume is the gradient time, tG, multiplied by the flow rate, F, the gradient time for each
gradient segment needs to be adjusted to maintain a constant ratio of the gradient volume to the
column volume (expressed as L x dc2). Thus, the new gradient time, tG2 can be calculated from the
original gradient time, tG1, the flow rate(s), and the column dimensions as follows:
tG2 = tG1 x (F1 / F2) [(L2 x dc22) / (L1 x dc12)]

Thus, the change in conditions for gradient elution requires three steps:
– (1) Adjust the column length and particle size according to L/dp,
– (2) Adjust the flow rate for changes in particle size and column diameter, and
– (3) Adjust the gradient time of each segment for changes in column length, diameter and flow rate. The
example below illustrates this process.

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Liquid Chromatography: Gradient Elution

Variable Original Adjusted Conditions Comment

Conditions
Column length (L) in mm 150 100 user choice
Column diameter (dc) in mm 4.6 2.1 user choice
Particle size (dp) in µm 5 3 user choice
L/dp 30.0 33.3 (1)
Flow rate in mL/min 2.0 0.7 (2)
Gradient adjustment factor 0.4 (3)
Gradient conditions
%B Time (min) Time (min)
30 0 0
30 3 (3x0.4)=1.2
70 13 [1.2+(10x0.4)]=5.2
30 16 [5.2+(3x0.4)]=6.4

(1) 11% increase within allowed L/dp change of -25% to +50%

(2) calculated using F2= F1 [(dc22 x dp1) / (dc12 x dp2)]
(3) calculated using tG2 = tG1 x (F1 / F2) [(L2 x dc22) / (L1 x dc12)]
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Liquid Chromatography: Gradient Elution

Injection volume

When the column dimensions are changed, injection volume adjustment may be
guided by,

Vinj,2 = Vinj,1 (L2 dc,22) / (L1 dc,12)

Independent of changing column dimensions, injection volume may be varied

provided System Suitability criteria remain within their established acceptability
limits. When Injection volume is decreased, special attention should be given to
(limit of) detection and repeatability of the peak(s) to be determined. An
increase is permitted provided in particular linearity and separation of the peak(s)
to be determined remain satisfactory
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Harmonization – Next Steps

New proposal published in PF 43(5)

Comments are being collected and summarized

Comments will be sent to Eur. Pharm. (coordinator)

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Sources of information

www.usppf.com

www.chromatographyonline.com

www.uspchromcolumns.com

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 Click to edit
Validation
master title style

Definitions of Validation: USP

USP <1225>:
– “Validation of an analytical procedure is the process by which it is established, by laboratory
studies, that the performance characteristics of the procedure meet the requirements for the
intended analytical applications.”

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Validation Characteristics

Accuracy

Precision

Specificity

Detection Limit

Quantitation Limit

Linearity

Range
Not part of the formal validation study:

Robustness

Sample solution stability
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USP <1225> Validation of Compendial Procedures

Performance Category I Category II Category III Category IV

Characteristic
Quant Limit
Test

Accuracy Yes Yes * * No

Precision Yes Yes No Yes No

Specificity Yes Yes Yes * Yes

Detection Limit No No Yes * No

Quantitation Limit No Yes No * No

Linearity Yes Yes No * No

Range Yes Yes * * No

* May be required, depending on the nature of the specific test

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ICH Q2 (R1)

Performance Assay Impurities Identification

Characteristic
Quant Limit Test

Accuracy Yes Yes No No

Precision
Repeatability Yes Yes No No
Interm. Precision Yes (1) Yes (1) No No

Specificity (2) Yes Yes Yes Yes

LOD No No (3) Yes No

LOQ No Yes No No

Linearity Yes Yes No No

Range Yes Yes No No

(1) in cases where reproducibility has been performed, intermediate precision is not needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting procedure(s)
(3) may be needed in some cases
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Specificity

Definition – The method’s ability to specifically measure the analyte in the

presence of the components in the analytical matrix

Impurities, degradation products and/or excipients are available.

When impurities, degradation products or excipients are not available:

– Compare the results with another well-characterised analytical method
– Perform an artificial degradation under drastic conditions (light, heat, acids y bases, etc.)

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Specificity

Required for the validation of

– Identification Tests(category IV)
– Impurity Tests (category II)
– Assay (category I)
– Performance Tests (category III)

In the pharmacopeial analysis, the lack of specificity of a method can be supplemented

with another to obtain the desired total level of selectivity

IUPAC, AOAC prefer the term selectivity, reserving the term specificity for fully selective
procedures
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Specificity

Peak purity

Peak shape analysis

Diode array detection

HPLC-MS

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Forced Degradations

In solution and/or in solid phase

The conditions are more severe than in an accelerated stability study

One Lot

Mechanisms of thermolysis, hydrolysis, oxidation and photolysis in the drug and

product

PDA or MS Detectors are necessary for validation, not for QC

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Forced Degradations

Guidelines for submitting samples and analytical data for methods validation,
FDA, 1987.

Drug substances:
– Heat (50°C)
– Light
– Hydrolysis: acid (HCl 0.1 N) / base (NaOH 0.1 N)
– Oxidation (H2O2 3%)

Drug Products
– Heat
– Light
– Humidity (85%)

Other conditions may be necessary

Exposure time, temperature and concentration can be adjusted to obtain an
appropriate degradation of the sample (10% to 15%)
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Linearity

Definition – The ability of a method to produce results that are directly proportional
to the concentration of the sample at a given interval

5 levels of concentration. Different dilutions of an stock solution or separate

weights of the component

3 samples per level of concentration

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Linearity

Calibration model Requirements

Single-point calibration
Multiple-point calibration (linear, unweighted)
Multiple point calibration (linear, weighted)
• Linear response function
• Negligible systematic error
• Homosedasticity
• Linear response function
• Homosedasticity
• Linear response function

Multiple point calibration (non-linear) • Continuous response function

Area normalization • For main peak
‒ Linear response function
‒ Negligible systematic error
‒ Homosedasticity
• For impurities
‒ Linear response function
‒ Negligible systematic error
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Linearity

y = a + bx
 [( x − x )( y − y ]
i
i i
b=
 (x − x)i
i
2

a = y − bx
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Coefficient of Correlation

 [( x − x )( y
i
i i − y )]
r=
 2  
 
 i
( xi − x )  
 i
( y i − y ) 

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Confidence Intervals

(y i
i − yˆ i ) 2
Sy x =
n−2

Sy x
b ± t(n-2) Sb
Sb =
 i
( xi − x ) 2 a ± t(n-2) Sa

 i
x i2
Sa = Sy
n  ( xi − x ) 2
x

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Residue Analysis

20
15
10
Residuals

5
0
-5
-10
-15
-20
0 5 10 15 20 25 30 35
Cc (mg/ml)

Check that the graph has a random pattern.

A trend indicates a failure in the chosen model.

An increase or decrease in residue dispersion indicates that the data is not homoscedastic.

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Residue Analysis

Normal

Pattern Increasing trend

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Residue Analysis

Weighted Linear Regression

– The homoscedasticity is a prerequisite for the common lineal regression

– In LC-UV it is generally assumed that this occurs within a range of one order of magnitude

– In greater concentration ranges it would probably violate this assumption

– Ifrequired to quantify a large range of concentrations (more than two orders of magnitude) the
lack of homoscedasticity should be compensated by a weighted linear regression
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Impurity Analysis – Various Types

Area normalization

Individual impurity is calculated as follows:

(Areaimp/Areatotal) 100

Useful in the initial stages of development. Does not require a reference standard

Requires linearity over a wide range

If the concentration is reduce to maintain linearity, impurity can not be detected

Assume the response factors are similar

Accuracy evaluation is not required

Example: Dousa M., Gibala P, Pekarek T – New approach of validation using internal
normalization technique for quantification of related substances in raw materials, intermediates
and pharmaceutical substances by HPLC. J. Pharm. Biom. Anal. 114(2015): 133 – 138.
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Impurity Analysis – Various Types

“High-Low”

A concentrated sample solution compared against a diluted sample solution

Removes limitation of the previous method with respect to linearity

The peaks are compared in a limited concentration range

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Impurity Analysis – Various Types

External Standard

The concentration is determined from a calibration curve

Uses a reference standard or assumes that the response factors are equal

The single-point calibration is appropriate if the y-intercept is not significant

Uses a small linear range

Improve sensitivity

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Accuracy

Definition - The difference between results obtained by the method and the actual
value.

It is calculated by determining the % recovery of the method

Cobs × 100
R=
Cad

Three replicates at three levels

– 80% -120% for the principal component
– 50% - 150% for impurities

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Accuracy

Drug substance
– Analysis of reference material

Drug product
– Analysis of synthetic mixtures

Impurities
– Analysis of samples spiked with known amounts of impurities

Compare the result with a second well-characterized method

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Recovery Test

40
y = 0.9989x + 0.3272
R² = 0.9974 Amount added (mg) Amound detected (mg) Recovered
24.35 24.70 101.4%
35
25.15 25.41 101.0%
25.15 25.17 100.1%
observed

30.04 30.79 102.5%

30
29.74 30.18 101.5%
30.14 30.38 100.8%
25 35.33 35.70 101.0%
34.63 34.56 99.8%
Ecuacion de la rect a
36.73 37.01 100.8%
20 rado = a + ( b x Añadido)
Encont Mean: 101.0%
20 25 b= 30 35
0.9989
added SD: 0.7945
a= 0.3272

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Recovery Test – USP <1225>

Assessment of accuracy can be accomplished in a variety of ways,

including evaluating the recovery of the analyte (percent recovery) across the
range of the assay, or evaluating the linearity of the relationship between
estimated and actual concentrations. The statistically preferred criterion is that
the confidence interval for the slope be contained in an interval around 1.0, or
alternatively, that the slope be close to 1.0. In either case, the interval or the
definition of closeness should be specified in the validation protocol. The
acceptance criterion will depend on the assay and its variability and on the
product. Setting an acceptance criterion based on the lack of statistical
significance of the test of the null hypothesis that the slope is 1.0 is not an
acceptable approach.

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Precision

Definition - The degree of agreement between results when a procedure is

repeatedly applied to a homogenous sample

Repeatability, Intermediate Precision, Reproducibility

Calculation of the SD or %RSD

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Precision

Repeatability: is precision under the same operating conditions for a short

period of time. It is determined by a minimum of 9 measurements within the
given range of the procedure (3 concentrations/ 3 replications) or a minimum of
6 replications at 100%

Intermediate Precision: indicates intra-laboratory variations; different days,

different analysts, different equipment

Reproducibility: indicates inter-laboratory variations

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Range

Definition - The difference between the highest and lowest concentrations of

sample (includes these two values) that can be determined with acceptable
precision, accuracy and linearity.

Assay of Macro-components 80-120%

Uniformity of Dosage Units 70-130%

Dissolution Test ± 20%

Quantification of Impurities 50%-120%

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Range

Response

Linearity and Accuracy

Precision

Range

Concentration

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Detection/Quantification Limits

Detection Limit – The minimum concentration of analyte that can be detected

under the established experimental conditions

Quantification Limit – The lower concentration of analyte that can be

determined with acceptable precision and accuracy

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Detection/Quantification Limits

Q3A(R2): Impurities in New Drug Substances

Maximum Daily Reporting Identification Quantification

Dose Threshold Threshold Threshold
</= 2g/día 0.05% 0.1% o 1.0 mg per 0.15% o 1.0 mg per
day (whichever is day (whichever is
lower) lower)
> 2 g/día 0.03% 0.05% 0.05%

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Detection/Quantification Limits

Q3B(R2): Impurities in New Drug Products

Maximum Daily Dose Threshold
Reporting Threshold
≤1g 0.1%
>1g 0.05%
Identification Threshold
< 1 mg 1.0% o 5 μg TDI, whichever is lower
1 mg - 10 mg 0.5% o 20 μg TDI, whichever is lower
>10 mg - 2 g 0.2% o 2 mg TDI, whichever is lower
>2g 0.10%
Qualification Threshold
< 10 mg 1.0% o 50 μg TDI, whichever is lower
10 mg - 100 mg 0.5% o 200 μg TDI, whichever is lower
>100 mg - 2 g 0.2% o 3 mg TDI, whichever is lower
>2g 0.15%
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Basic Approaches to Determination

ICH Q2 lists the following

Visual

Signal-to-Noise ratio

Blank standard deviation and regression line

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Visual

I can see it!!

ICH Q2 – determined by the analysis of samples with known concentrations of
analyte and by establishing
– The minimum level at which the analyte can be reliably detected (LOD)
– The minimum level at which the analyte can be quantified with acceptable accuracy and
precision (LOQ)

Seems more appropriate for LOD

Subject to analyst interpretation

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Signal-to-Noise Ratio

Q2 and USP <1225> -- Compare

measured signals from samples
with known low concentrations of
10:1
analyte with those of blank
samples

Requirements
– S/N > 3 (or 2) for LOD
3:1
– S/N > 10 for LOQ

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Signal-to-Noise Ratio

h: range of the background noise in a

chromatogram obtained after injection
or application of a blank, observed
over a distance equal to 5 times the
width at half-height of the peak in the
chromatogram obtained with the
prescribed reference solution and, if
possible, situated equally around the
place where this peak would be found
S/N = 2H/h

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Detection/Quantification Limits

Based on the standard deviation of the response:

LOD= 3.3 S /b
LOQ= 10 S /b
S = Standard deviation of the response
b= Slope of the calibration curve

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Basis for this Approach

+10
SD

5%
Blank
+3.3 Sigma
5% +10 Sigma +3.3
SD

blank response

LOD LOQ

The 3.3 and 10 SD values are intended to separate the distribution of

responses at LOD and LOQ from the distribution of blank samples

Dividing by the slope, b, converts differences in y (response) axis to differences

in x (concentration) axis
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Detection/Quantification Limits

Options for S:

SD of blank responses
– Analyze various blanks and calculate the SD

Residual SD from regression line

 i i
(
i
y − y
ˆ ) 2

Sy x =
n−2

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USP <1225> Recommendation

Whatever method is used, the detection [quantitation] limit should be

subsequently validated by the analysis of a suitable number of samples known
to be near, or prepared at, the detection [quantitation] limit

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 Example

Concentration (μg/ml) Peak Area LOD = 3.3 * 0.00033149/0.2920791

0.0100994 0.003108
0.0252486 0.008015
0.0504972
0.0757458
0.015540
0.022415
= 0.0037
0.1009944 0.029510
0.2524860 0.074235
LOQ = 10 * 0.00033149/0.2920791
Source: Table 3-11 of Ermer & Miller

SUMMARY OUTPUT
= 0.0113
Regression Statistics
Multiple R 0.99993354
R Square 0.99986709
Adjusted R Square 0.99983386
Standard Error 0.00033149
Observations 6
Note: “Standard Error” is Residual SD
ANOVA
df SS MS F Significance F
Regression 1 0.003306581 0.003307 30090.49 6.62516E-09
Residual 4 4.39552E-07 1.1E-07
Total 5 0.003307021

Coefficients Standard Error t Stat P-value Lower 95% Upper 95%

Intercept 0.0003969 0.00019801 2.004458 0.115527 -0.000152862 0.0009467
Concentration (μg/ml) 0.2920791 0.001683782 173.4661 6.63E-09 0.287404158 0.296754

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Robustness

Definition – A measure of a method’s ability to accept small but deliberate

variations in the parameters of the method

Steps to Consider
– Design and execution of the experiment
– Calculating the effects
– Statistical/graphical analysis of the effects
– Relevant conclusions and eventual modifications of the method

Changes in: pH, column, mobile phase, etc.

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Robustness

Experimental Design

One factor at a time

Factorial Designs
– Full Factorial
– Fractional Factorial
– Plackett-Burman

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Full Factorial Design (2k Runs)

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Fractional Factorial Design – 5 Factors

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Placket - Burman

7 factors (A-G) 2 levels

Exp. Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8

Eff = ( y+ -  y-)/4
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Example – multi-variate

Table A. Factor levels used in the design

Nominal Lower Upper Variation

Factor Value Level (-1) Level (+1) (%)
Gradient Profile 1 0 2 0
Column Temp. (°C) 40 38 42 5
Buffer Conc. (mM) 40 36 44 10
Buffer pH 5 4.8 5.2 4
Wavelength (nm) 446 441 451 1
Triethylamine (%) 0.23 0.21 0.25 8
Dimethylformamide (%) 10 9.5 10.5 5

Romero, R. et. al. - LC-GC, 20(1): 72 - 80, 2002

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Example – multi-variate

Table B. Experimental design for robustness test

RESPONSE VARIABLES
FACTOR Phenylethylamine Tyramine
Buffer Buffer Wave- TEA Peak Peak tr Unadj.
Run Gradient Temp (°C) Conc. pH length (%) DMF (%) Area* Height* (min) tr (min) R
1 +1 +1 +1 +1 +1 +1 +1 2,365 221.4 18.331 1.1121 1.624
2 +1 +1 -1 -1 +1 -1 -1 2,376 226.0 18.370 1.1108 1.673
3 +1 -1 +1 +1 -1 -1 -1 2,305 226.1 18.671 1.1087 1.745
4 +1 -1 -1 -1 -1 +1 +1 2,330 225.1 18.524 1.1106 1.641
5 -1 +1 +1 -1 -1 +1 -1 2,378 221.8 18.504 1.1101 1.693
6 -1 +1 -1 +1 -1 -1 +1 2,384 224.3 18.561 1.1101 1.736
7 -1 -1 +1 -1 +1 -1 +1 2,448 235.0 18.548 1.1132 1.783
8 -1 -1 -1 +1 +1 +1 -1 2,384 234.7 18.853 1.1071 1.787
9 0 0 0 0 0 0 0 2,357 221.2 18.648 1.1086 1.653
10 0 0 0 0 0 0 0 2,311 218.4 18.639 1.1092 1.689
11 0 0 0 0 0 0 0 2,312 219.9 18.667 1.1088 1.733
* Arbitraty Units

Romero, R. et. al. - LC-GC, 20(1): 72 - 80, 2002

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Definitions for Verification: USP

USP <1225>: “…users of analytical methods described in the USP–NF are not
required to validate accuracy and reliability of these methods, but merely verify
their suitability under actual conditions of use.”

USP <1226>: “Verification consists of assessing selected analytical performance

characteristics, such as those that are described in USP <1225>, to generate
appropriate, relevant data rather than repeating the validation process.”

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Definitions for Verification: FDA

Guide to Inspections of Pharmaceutical Quality Control Laboratories:

“Methods appearing in the USP are considered validated and they are
considered validated if part of an approved ANDA”

“For compendial methods firms must demonstrate that the method works under
the actual conditions of use.”

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Concepts

Validation: Challenges the analytical method using a well defined sample

Verification: Challenges the analytical environment using a well defined method

– Analyst
– Instrument
– Reagents
– Matrix

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USP <1226> Summary

The intent of this chapter is to provide general information to laboratories on the

verification of compendial procedures that are being performed for the first time
to yield acceptable results utilizing the laboratories’ personnel, equipment, and
reagents

Not intended for retroactive application to already successfully established

laboratory procedures

Verification consists of assessing selected Analytical Performance

Characteristics, such as those which are described in USP chapter <1225>, to
generate appropriate, relevant data rather than repeating the validation process

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A Risk-based Approach

“Verification requirements should be based on an assessment of the complexity of

both the procedure and the material to which the procedure is applied.”

“The degree and extent of the verification process may depend on the level of
training and experience of the user, on the type of procedure and its associated
equipment or instrumentation, on the specific procedural steps, and on which
article(s) are being tested.”

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Common Practices in the Industry

System suitability requirements

Specificity –
– Peak purity

– Matrix

– Accuracy

Accuracy: Recovery in the specification range

Precision: Repeatability in the specification range

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Common Practices in the Industry (cont)

LOD: Verification of detection at 50% of the specification

LOQ: Verification of quantitation at 50% of the specification

Usually not needed: Linearity, Range, and Robustness

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Common Practices in the Industry

Other details to be considered

– Columns vs. cartridges

– Mobile phase preparation

– Gradient and dead volume

– Sample solution preparation

– Sample solution stability

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USP <621> Chromatography – Decision Tree

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