Sesi 2 Petra Lewis MERCK

Pharmaceutical
impurity profiling
by HPLC
Petra Lewits, Global Product Manager HPLC Columns

Relevant Parameters Drug Product
Connections
Impurity Profile Assay

Quantitative and qualitative investigation of Quantitative determination of
Degradation products the API in reference to the
label claim
Microbial
Purity Stability
For oral dosage forms: Short-term stability testing
total aerobic microbial count (Formulation Development)
total combined yeast and molds
Stability testing acc. to ICH
For parenteral dosage forms: and forced degradation
sterility experiments
Endotoxins
Disintegration & Appearance & physical

Dissolution Characteriatics
For solid oral dosage forms to simulate the Solids: Appearance, Size, Hardness,
physiological dissolution Friability
Liquids: particulate contamination,
Osmolality, pH
2 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt
Relevant Parameters Drug Product
Connections
Impurity Profile Assay

Quantitative and qualitative investigation of Quantitative determination of
Degradation products the API in reference to the
label claim
Microbial
Purity Stability
For oral dosage forms: Short-term stability testing
total aerobic microbial count (Formulation Development)
total combined yeast and molds
Stability testing acc. to ICH
For parenteral dosage forms: and forced degradation
sterility experiments
Endotoxins
Disintegration & Appearance & physical

Dissolution Characteriatics
For solid oral dosage forms to simulate the Soils: Appearance, Size, Hardness,
physiological dissolution Friability
Liquids: particulate contamination,
Osmolality, pH
From Discovery to QC
Small Molecule Drug Development Workflow
MANUFACTURE
• Filtration, Chromatography
• Sterility testing, bioburden,
environmental monitoring
• HPLC analysis
• HPLC Analysis
• Reference materials
• Reference
Materials • Impurity testing
• Impurity
testing
• Cell Analysis
• Cell Analysis • GC analysis
• MultiScreen Filter • Sterility testing, bioburden,
• MultiScreen • MultiScreen Filter
Plates environmental monitoring
Filter Plates Plates
• Life Science • Media Fill Release Testing/
• Life Science • Life Science
Research Tools Dehydrated Culture Media
Research Research Tools • Sterility testing,
Tools • Chemical bioburden, • HPLC analysis
• Chemical synthesis
synthesis environmental
• Chemical • HPLC analysis • Reference materials
Synthesis monitoring
• Bioanalysis
• GMP-grade raw • Impurity testing
materials
• HPLC analysis
• Sterility testing
• Reference
• Life Science • HPLC analysis
materials
Research Tools • Reference materials
• HPLC Analysis
• Impurity testing
• Reference
materials
• Bioanalysis

Definition
Impurities
According to ICH guidelines on impurities in new drug products, identification

of impurities below 0.1% level, is not considered to be necessary, unless
potential impurities are expected to be unusually potent or toxic.
Following terms are used by various regulatory bodies and ICH to describe the impurities:
1. Intermediate
2. Penultimate intermediate
3. By-products
4. Transformation products
5. Interaction products
6. Related products
7. Degradation products

Regulatory guidelines
Impurities
▪ ICH guidelines “stability testing of new drug substances and products"- Q1A
▪ ICH guidelines “Impurities in New Drug Substances”- Q3A
▪ ICH guidelines “Impurities in New Drug Products”- Q3B
▪ ICH guidelines “Impurities: Guidelines for residual solvents”- Q3C
▪ US-FDA guidelines “NDAs -Impurities in New Drug Substances”
▪ US-FDA guidelines “ANDAs – Impurities in New Drug Substances”
▪ Australian regulatory guideline for prescription medicines, Therapeutic
Governance Authority (TGA), Australia

United States Pharmacopeia
HPLC Method requirements
Any analytical procedure submitted should be Full validation is required for purity, assay and dissolution
described in sufficient detail, includes: methods (HPLC, UV) :
➢ Specificity – all components resolved
Preparation of mobile phase
➢ Linearity – min 5 concentrations linear response (assay
Chromatographic condition: 80-120%)
 Column: type (e.g., C18 or C8), dimension (length, ➢ Accuracy – API/FPP spiked with known imp. LOQ-150%
inner diameter), particle size (10μm, 5 μm) ➢ Repeatability (syst. /method) – n>5, RSD<1 assay,
<5% rel. sub.
 Detector: wavelength
➢ Intermediate precision – repeatability different days,
 Injection volume analyst, inst.
 column T ➢ LOD/LOQ (not required for assay, dissolution)
 flow rate ➢ Robustness (recommended) – pH, buf. comp., T effects
Elution procedure: isocratic or gradient elution Validation Verification
Performance
<1225> <1226>
Preparation of standards and samples Accuracy Yes No
Operation procedure: sequence of injections Precision Yes Maybe
Specificity Yes Yes
System suitability testing (SST) and criteria LOD No No
LOQ Yes Yes
Calculations
Linearity Yes No
Range Yes No

United States Pharmacopeia
HPLC System Suitability
The various components of the equipment employed must be qualified and be capable of achieving the performance required
to conduct the test or assay. The system suitability tests represent an integral part of the analytical procedure and
are used to ensure adequate performance of the chromatographic system. The following requirements are to be
fulfilled, in addition to any other system suitability criteria stated in the monograph. When specific requirements are stated
in the monograph, they supersede the requirements mentioned in General chapter 621
▪ Number of theoretical plates (N): column efficiency ≥ 2000
▪ Retention factor (mass distribution ratio)
▪ System repeatability: When a relative standard deviation requirement is specified in an individual monograph, if the requirement is 2.0 or
less the calculation is based on data from five replicate injections of the analyte, if the requirement is more than 2.0% data from six replicate
injections are used.
▪ Precision:
❖ Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5, >2% n=6 Compliance with the system suitability
❖ Impurities: in general, RSD ≤ 5% at the limit level, up to 10% or higher at LOQ, n ≥ 6 criteria is required throughout the
chromatographic procedure. No sample
▪ Signal to noise ratio: analysis is acceptable unless the suitability
❖ Detection limit: signal-to-noise ratios are 2:1 or 3:1. of the system has been demonstrated
❖ Quantitation limit: typically acceptable signal-to-noise ratio is 10:1.
▪ Tailing factor/peak asymmetry: Unless otherwise stated, in a test or assay, the symmetry factor (tailing factor) of the peak used for
quantification is 0.8–1.8.
▪ Resolution (R): >2

USP43-NF38
Fingolimod Hydrochloride HPLC Assay and Impurity Profiling
Column: Purospher® STAR RP-18 Endcapped (3μm) Hibar® RT 150x3.0 mm
Mobile Phase A: 0.1% Phosphoric Acid in H2O
Mobile Phase B: Acetonitrile
Gradient: Time (min) A (%) B (%)
0 80 20
20 5 95
23 5 95
23.1 80 20
33 80 20
Flow rate: 0.8 mL/min
Column Temperature : 40°C
Injection volume: 5 μL
Autosampler Temperature : 10°C
Detection: UV @ 215 nm (DAD)
Pressure: 135-300 bar
Diluent: Mobile Phase A:B (50:50)
Test solution: Dissolve 15 mg of Fingolimod Hydrochloride CRS in 25 mL diluent (0.6
mg/mL).
System suitability solution: Dissolve 15.0 mg of USP Fingolimod for System Suitability
using 25 mL diluent (0.6 mg/mL).
Standard solution: Dilute 1.0 mL of the test solution to 100.0 mL using mobile phase
further dilute 1.0 mL of this solution to 10.0 mL using the mobile phase (0.003 mg/mL).

USP43-NF38 – Chromatographic Results

USP43-NF38 – Chromatographic Results
Chromatographic Data & Specificity (System Suitability Solution)
Repeatability (System Suitability Solution)



USP41-NF36
Organic Impurity Profiling Method for Desvenlafaxine
Extended-Release Tablets
Experimental Conditions SST Stock 0.25 mg/mL of USP Venlafaxine Hydrochloride RS and 0.1 mg/mL of USP
Solution Desvenlafaxine Related Compound A RS prepared as follows. Transfer
Column Purospher® STAR RP-18 endcapped (5µm) 250x4.6 mm suitable amounts of USP Venlafaxine Hydrochloride RS and USP
Detection UV @ 226 nm (analytical flow cell; 10 µL) Desvenlafaxine Related Compound A RS to an appropriate volumetric flask
and add 65% of the total flask volume of Diluent. Dilute with Diluent to
Injection volume 20 µL volume.
Flow rate 1 mL/min
SST Solution 500 μg/mL of USP Desvenlafaxine RS, 2.5 μg/mL of USP Venlafaxine
Column: 35°C Hydrochloride RS, and 1 μg/mL of USP Desvenlafaxine Related Compound A
Temperature RS prepared as follows. Transfer 50 mg of USP Desvenlafaxine RS to a 100-
Autosampler: 20°C
mL volumetric flask. Add 4 mL of citric acid and 20 mL of acetonitrile.
Pressure drop 90-155 bar (1305-2248 psi) Sonicate to dissolve and then add 30 mL of sodium chloride and sonicate
Buffer 5.6 g/L of sodium perchlorate in water for another 5 min. Then add 1 mL of SST stock solution and dilute with
sodium chloride to volume.
Mobile Phase A: Buffer : Acetonitrile (90:10)
B: Acetonitrile Standard 1 μg/mL (1 ppm) of USP Desvenlafaxine RS in sodium chloride solution,
Diluent Mix water and acetonitrile 50:50 (v/v) Solution filter with 0.45-μm pore size. Discard 2 mL and use the filtrate.
Sensitivity 0.25 μg/mL of USP Desvenlafaxine RS from Standard solution in sodium
Gradient see table
Solution chloride passed through a suitable filter of NLT 0.45-μm pore size. Discard
NLT 2 mL and use the filtrate.
Time (min) A(%) B(%)
Test Solution Nominally 500 μg/mL of desvenlafaxine from Tablets prepared as follows.
0 80 20
Transfer a portion of powder from NLT 20 Tablets, equivalent to 125.0 mg
5 80 20 of desvenlafaxine, to a 250-mL volumetric flask. Add 50 mL of acetonitrile
35 60 40 and sonicate for about 5 min. Add 10 mL of citric acid and sonicate for
about 5 min more with occasional shaking. Add 100 mL of sodium chloride
40 20 80
and sonicate for about 20 min with intermittent shaking. Dilute with sodium
42 80 20 chloride to volume. Pass through a filter of 0.45-μm pore size, discard NLT
50 80 20 2 mL, and use the filtrate.

USP41-NF36
Chromatographic Data
Desvenlafaxine
Retention Theoretical Tailing

Peak Compound Time (min) Plates Factor
1 Desvenlafaxine 8.0 16145 1.1
System Suitability Solution Sample Solution
Retention
Time Tailing
Peak Compound (min) Resolution Factor RRT
1 Desvenlafaxine 8.0 - 1.5 1.0

Related
2 16.2 24.9 1.1 2.0
compound A
3 Venlafaxine 17.7 4.4 1.1 2.2

USP41-NF36
Validation and verification
1. Specificity: Inject solution and determine the 3. Linearity, LOD & LOQ
retention time of desired analyte in presence of other
Concentration
component like impurities and excipient. Desvenlaflaxine
(μg/mL)
Compound RT (min) 0.25 19085
Desvenlafaxi 0.5 34351
1 8.0
ne
1 72931
Related
2 16.2 5 352585
compound A
12.5 874866
3 Venlafaxine 17.7
25 1748644
40 2838462
2. Standard Repeatability (25 ppm) 50 3553944
STD 1 69251 60 4242529
STD 2 69500 75 5268328
STD 3 68967 LOD (ppm) 0.07
STD 4 69301 LOQ (ppm) 0.22
STD 5 69126
Mean 69229
Standard Deviation 198.97
RSD (%) 0.3
All acceptance criteria fully met


USP41-NF36
Space for improvement?
Purospher® STAR RP-18 endcapped (5µm) 250x4.6 mm
Speed
Cost

From HPLC to UHPLC
Ramipril and Related Substances
USP36 –NF31 monograph method for ramipril related compounds
Column: Purospher® STAR RP-18 endcapped (5μm) Hibar® RT 250x4.6 mm Purospher ® STAR RP-18 endcapped (2μm) Hibar ® HR 100x2.1 mm
Injection: 10 μL 2 μL
Cell: 10 μL 2.5 μL (Use 0.1 mm tubing)
Flow Rate: 1.0 mL/min Time saving: 72% 0.3 mL/min
Pressure Drop: 61 to 74 Bar (884 to 1073 psi) 196 to 164 Bar (2827 to 2378 psi)
Solvent saving: 92%
The resolution, R, between ramipril related

compound A and ramipril (not less than 3.0)
The relative retention time between ramipril
related compound A (ramipril RS A), ramipril
and ramipril related compound B (ramipril RS B)
The tailing factor for the ramipril peak (between
0.8 and 2.0).
The application using HPLC conditions also meet
the retention time requirement for ramipril
16 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt 

Allowed changes as per USP Chapter 621 from Dec. 2022
Isocratic Gradient
Column length & particle May be modified, provided that the ratio of the column length (L) to the particle size (dp) remains constant or in the
size (FPP to FPP) range between -25% to +50% of the prescribed L/dp ratio.
Column length & particle Other combinations of L and dp can be used, Other combinations of L and dp can be used provided that
size (FPP to SPP or provided that the plate number (N) is within -25% the ratio (tR/Wh)2 is within -25% to +50%, relative to the
monolithic) to +50%, relative to the prescribed column. These prescribed column for all the peaks used to determine the
changes are acceptable, provided that system system suitability parameters. These changes are acceptable
suitability criteria are fulfilled, and selectivity and provided system suitability criteria are fulfilled, and
elution order of the specified impurities to be selectivity and elution order of the specified impurities to be
controlled are demonstrated to be equivalent. controlled are demonstrated to be equivalent.
Inner diameter In absence of a change in particle size and/or length, the internal diameter of the column may be adjusted.
Flow rate When particle size is changed, the flow rate requires Flow rate recalculation using formula.
adjustment, but column performance should not
drop by more than 20%; when column dimension
does not change ±50% change is permitted
pH ± 0.2 units
UV wavelength No adjustment permitted No adjustment permitted
Buffer salts ± 10%
concentration
Mobile phase ± 30% relative or ± 10% absolute whichever is ± 15 % retention window, SST must be met
composition smaller
Column temperature ± 100C ± 50C
Injection volume Equation from FPP to FPP. May not be applicable to

changes from TPP columns to SPP columns.
From HPLC to UHPLC
Cell: 10 μL Adjustment of column length 2.5 μL (Use 0.1 mm tubing)
Flow Rate: 1.0 mL/min 0.3 mL/min
Monograph: [L/dp = 50000]
Pressure Drop: 61 to 74 Bar (884 to 1073 psi) 196 to 164 Bar (2827 to 2378 psi)
New: [L/dp = 50000]

Isocratic Gradient
pH ± 0.2 units
Buffer salts ± 10%
concentration
composition smaller

Liquid Chromatography: Gradient Elution
Adjustment of Chromatographic Conditions
C) Column dimensions (internal diameter):

• If there is no change in particle size and/or length of the column, the internal diameter of the column may be adjusted
• Caution is necessary when the adjustment results in smaller peak volumes, due to a smaller particle size or a smaller internal diameter, a
situation that may require adjustments to minimise extra-column band broadening by factors such as instrument connections, detector
cell volume and sampling rate, and injection volume
• When the particle size is changed, the flow rate requires adjustment, because smaller-particle columns will require higher linear velocities
for the same performance (as measured by reduced plate height). Flow rate is adjusted for changes in column diameter and particle size
using the following equation:
F1 = monograph flow rate (mL/min)

F2 = adjusted flow rate (mL/min)
dc1 = monograph column id (mm)
dc2 = column id to be used (mm)
dp1 = monograph particle size (mm)
dp2 = particle size to be used (mm)
• 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
• Since the gradient volume is the gradient time (tG) multiplied by the flow rate (F), the gradient time for each gradient segment must be
adjusted to maintain a constant ratio of the gradient volume to the column volume (expressed as L × dc2)

From HPLC to UHPLC
Cell: 10 μL 2.5 μL (Use 0.1 mm tubing) Injection Volume

Flow Rate: 1.0 mL/min 0.3 mL/min
Pressure Drop: 61 to 74 Bar (884 to 1073 psi) 196 to 164 Bar (2827 to 2378 psi) [0.83 µL]
Adjustment of column I.D.
Flow rate f2 = f1 x (d2)2 / (d1)2
2.1² / 4.6² x 1 = 0.21 mL/min
2.1² x 5
x 1 = 0.52 mL/min

4.6² x 2

Validation and Verification
Test method Required action Minimal parameters to evaluate
Must use an already established „fully Verification Precision

validated method“ (e.g. compendial, Bias (including matrix variations)
official) that includes analytical Possibly linearity
characteristics
An established fully validated method Verification/Matrix extension Precision
but using different/new matrix Possible partial or full revalidation Bias (including matrix variations)
Objective is to ensure the new matrix has
not introduced new sources of error.
Published but no compendial/official Partial validation Precision
method (e.g. journal) that may or may Bias (including matrix variations)
not include analytical characteristics Ruggedness
Linearity
New method inhouse development Validation Selectivity/Specificity
Linearity
Accuracy
Precision
Limit of detection (LOD)
Limit of quantification (LOQ)
Robustness

HPLC Stationary Phases
Chromatographic base materials
Fully porous silica particles (FPP)
▪ Discovery® /Ascentis®
▪ Scalable from Micro-LC to Preparative ▪ Purospher® STAR / SeQuant®
▪ Full loadability
Fused-Core®
Superficially porous silica particles (SPP)
▪ Ascentis Express®
▪ BIOshell®
▪ Analytical scale Micro /UHPLC / HPLC
▪ Highest efficiency (Resolution)
Macropores
Monolithic silica 2µm
▪ Chromolith®
▪ Chromolith® WP
1.5µm
▪ Scalable from Micro-LC to Semi-Preparative
1.15µm
▪ Outstanding matrix tolerance / lifetime 120Å
Mesopores
300Å
Fully porous polymeric particles ▪ SeQuant® ZIC® pHILIC

▪ apHera
▪ Outstanding pH stability (pH 0-14)
Fully porous Carbon particles (PGC) ▪ SupelTM Carbon

▪ Analytical – unique retention mechanism
▪ Outstanding pH stability (pH 0-14) and
temperature stability (250°C)
Isocratic Gradient
pH ± 0.2 units
Buffer salts ± 10%
concentration
composition smaller

Particle size distribution
3000
Particle size
distribution 2500
Fused-Core particle size distribution
Average = 2.77 µm;
2000 standard deviation = 6% of mean
1500 Particle size distribution of a typical commercial

totally porous packing
Fully porous Particle Average = 3.78 µm;
1000 standard deviation = 19% of mean
500
Amount
Fused Core Particle 0
Particle Diameter, µm
-500
1 2 3 4 5 6 7

Influence of particle size distribution on Eddy diffusion
Eddy
diffusion
Poorly-ordered HPLC packed bed
Fully porous Particle
Highly-ordered packed bed Flow
Fused Core Particle

Influence of diffusion path
Diffusion
path/Mass
Transfer
Common Fully Porous C18, 3 µm
72.5 % ACN 2
N = 140,600 N/m, N = 21,090 N/col.
Pressure = approx. 4,000 psi
1 4
3
1. p-Hydroxy ethylbenzene
2. Napthalene
3. p-Xylene
4. Biphenyl 0 2 4
2
Ascentis Express C18, 2.7 µm
65 % ACN
N = 237,700 N/m, N = 35,655 N/col.
Pressure = approx. 4,000 psi 1
4
0 2 4
Column: 150 x 4.6 mm; Mobile phase: ACN/water; Flow rate: 1.5 mL/min; Injection: 2.0 mL; Detection: 220 nm

Influence of Particle size on resolution
Ascentis C18 Ascentis C18 Ascentis C18 Fused-Core C18

N = 5,700 N = 15,199 N = 23,411 N = 35,271
Peak H60 = 23.35 Peak H
60 = 36.38 Peak60 H = 45.26 Peak H = 59.16
Rs = 1.42 Rs = 2.32 Rs = 2.84 Rs = 3.63
P = 58bar P = 115bar P = 153bar P = 316 bar
10 µm 3 µm 2.7µm
5 µm
40 40 40 Columns: All C18, All 150 x

4.6mm
Flow: 1.8mL/min
Mobile Phase: 70/30 or 62/38
ACN/Water
3mm Detection: 254nm
Temp: 30°C
Injection Vol: 5mL
Analytes:
20 20 20 1. o-Xylene (0.04mg/mL)
2. p-Xylene (0.01mg/mL)
0 0 0
2.0 3.0 2.0 3.0 2.0 3.0

Antiviral / Therapeutic Drug Development
Lopinavir – Assay Method (EP)
Column Ascentis® Express C18 (2.7µm) 150x4.6 mm
Detection UV = 215 nm (micro flow cell; 1.4 µL/7mm)
Mobile phase A Acetonitrile/phosphate buffer solution 45/55 (v/v)
Phosphate buffer Dissolve 0.9 g of dipotassium hydrogen phosphate and 2.7 g of potassium dihydrogen
phosphate in
900 mL of water and mix well. Adjust to pH 6.0 with phosphoric acid, dilute to 1000 mL
with water and filter.
Solvent mixture Acetonitrile/water 50/50 (v/v)
Test solution (a) Dissolve 50.0 mg of the substance to be examines in the solvent mixture and dilute to
100 mL with the solvent mixture.
Test solution (b) Dilute 5.0 mL of the test solution (a) to 100 mL with the solvent mixture.
Reference solution (a) Dissolve 50.0 mg of Lopinavir CRS in the solvent mixture and dilute to 100 mL with the
solvent mixture.
Dilute 5 mL of this solution to 100 mL with the solvent mixture.
Injection: 12 µL
Flow Rate: 1.0 mL/min
Temperature: 50 °C
Pressure Drop: 153 bar (2219 psi)
No. Compound Retention Time (min) Tailing Factor
1 t0 void volume 1.1
2 Lopinavir CRS 16.2 0.97
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/small-molecule-hplc/lopinavir-european-
pharmacopeia-hplc-assay-method

Maximum Resolution and Speed
Increased Speed without Loss of Resolution
HPLC in Standard Configuration
N = 25,900
o ECV ~35 µL
FPP C18 o Standard flow cell, 14 µL 0.5 sec. response time
250 mm x 4.6 mm I.D., 5 µm o Standard length and ID tubing (0.007” ID x 750 mm)
1 mL/min
Rs = 6.5 L/dp = 250/0.005 = 50,000

For -25-50%, L/dp can be 37,500-77,500
N = 25,900
0 12 For -25-50%, N can be 19,425-38,850
Time, min
HPLC in Ultra-Low ECV Configuration

N = 24,500 o ECV ~10 µL
o Semi-micro flow cell, 5 µL 0.5 sec. response time
o Reduced length and ID tubing (0.005” ID x 460 mm)
Ascentis® Express C18

100 mm x 4.6 mm I.D., 2.7 µm
2 mL/min
L/dp = 100/0.0027 = 37,037 = criteria not met,
but is met for plates
N = 24,500
Rs = 6.3
6 times faster run time!
33 Time,
KGaA,min
0 ©2023 HPLC Impurity profiling - Petra Lewits, Merck Darmstadt
2
USP Monograph for Itraconazole
• Itraconazole is an antifungal medication
FPP C18, 3 µm, 100 mm x 4.6 mm I.D. • Suitability Requirements
10 µL, 1.5 mL/min, 30 C • Tailing factor: NMT 2.0
Gradient: 20−50% B in 12 min • RSD: NMT 2.0%
Back pressure: 242 bar
L/dp = 100/0.003 = 33,333

For -25-50%, L/dp can be 25,000-50,000
Ascentis® Express C18, 2.7 µm, 100 mm x 4.6 mm I.D.

10 µL, 1.5 mL/min, 30 C
Gradient: 20−50% B in 12 min
Back pressure: 290 bar
L/dp = 100/0.0027 = 37,037 Monograph Ascentis® Express C18

Column
Tailing Factor Pass Pass
RSD Pass Pass

USP Monograph for Estradiol – Modified
Ascentis® Express C18, 2.7 µm

100 mm x 1.5 mm I.D.
Original Method Modified Method
Column 300 x 3.9 mm 100 x 1.5 mm
Flow Rate (mL/min) 1 0.2
1. Ethyl Paraben Time (min) 8 1.5

2. Estradiol
3. Estrone Volume (mL) 8 0.3
L/dp = 300/0.01 = 30,000

For -25-50%, L/dp can be 22,500-45,000
L/dp = 100/0.0027 = 37,037

Column material selection
Matrix tolerance vs. Efficiency
FPP
SPP
10 µm
Monolith
Particles
Pressure (bar)
Monolith
Matrix tolerance
5 µm 5µm
3 µm
2.7µm
2µm
<2 µm
100 000 N/m 150 000 N/m 200 000 N/m 300 000 N/m
Separation efficiency
Column material selection Loadability
Matrix tolerance vs. Efficiency +++
FPP
SPP +
10 µm
Monolith ++
Particles
Pressure (bar)
Monolith
Matrix tolerance
5 µm 5µm
3 µm
2.7µm
2µm
<2 µm
100 000 N/m 150 000 N/m 200 000 N/m 300 000 N/m
Separation efficiency
Chromatographic Performance
Comparison Monolithic vs. FPP vs. SPP
Silica monolith Fully porous silica particle (FPP) Superficially porous silica particle (SPP)
Chromolith® HighResolution RP-18e C18, 3 µm C18, 2.7 µm
Pressure = 36 bar Pressure = 146 bar Pressure = 170 bar
Chromatographic Conditions
Column: Silica monolith: Chromolith® HighResolution RP-18e 100-4.6 mm
FPP: PurospherTM STAR RP18e 3 µm, 100-4.6mm
SPP: Ascentis® Express C18, 2.7µm 100-4.6 mm
Mobile Phase: A: 100% acetonitrile
B: 20 mM Phosphate buffer pH 4.5
Gradient: Time/min %A %B
0 20 80
12.0 80 20 Sample: 1. Ascorbic acid
Flow rate: 1.0 mL/min 2. 4-Hydroxybenzoic acid
Detection: UV 230 nm 3. Benzoic acid
Detection cell: Standard 11 µL 4. Sorbic acid
Temperature: 22 °C 5. Methyl 4-hydroxy benzoic acid
Injection volume: 2.0 µL 6. Methyl 4-hydroxy benzoic acid
7. Methyl 4-hydroxy benzoic acid

Monolithic Silica
Bi-modal pore structure
Mesopores:
Mesopores form a fine porous structure
with a large uniform surface area on which
adsorption takes place, thus enabling high-
performance chromatographic separation.
[130 Å; 150 Å; 300 Å]
Macropores:
The large Macropores allow rapid flow of the
mobile phase at very low back pressure with
maximum robustness and selectivity.
[1.15 µm; 1.5 µm, 2 µm]
Pore volume: 1.0 mL/g

Total porosity: >80 %
Surface area: 300 m2/g

Concerns on changes?
Some questions to USP
It is stated that it is not allowed to change chromatographic *”The chapter does not say it is not
support – does that mean that it is not allowed to change allowed. Only indicates that the
particulate material to monolithic even if both materials
are made of pure silica?
support should be similar to the one
Is it possible to change from type A silica to Type B silica prescribed. All adjustments require
material? verification before implementation.”
MERCK
How above-described situation changes *”As soon as we have data showing column equivalency, we
if Ph method mentioning that column can add to the monograph that monolith columns can be used.
is packed with 5µm particles (and We need to have data demonstrating column equivalency
monoliths has no particles)? for the particular method.”
If Ph method is mentioning 150 cm *”The user needs to verify in a case-by-case approach if two
column, for whatever reason is it columns in series is equivalent to the column mentioned in
allowed to use 10 and 5 cm the monograph. If data is shared with USP we can add this
columns coupled together instead? information in the particular monograph”
What kind of test users must perform in order to implement *”The elution order must be the same
such change? and system suitability requirements
must be met.”
*Horacio N. Pappa, CQE, Ph. D., Senior Director – General Chapters, Global Science and Standards Division, Email: hp@usp.org
& Margareth R. C. Marques, M.Sc., Ph.D. Sr. Principal Scientist

Spironolactone ASSAY (USP38-NF33)
Increase flow rate – faster result
Column:
Injection:
Chromolith® HighResolution RP-18 endcapped 150x4.6 mm
20 μL
Isocratic:

Detection:
Cell:
UV, 230 nm
11 μL
“when column dimension does not change
Flow Rate: 1.0 mL/min [A] and 1.5 mL/min [B] ±50% change is permitted”
Mobile Phase: Water and methanol 40:60 (v/v)
Temperature: Ambient
Diluent: Acetonitrile/Water 1:1 (v/v)
Sample Solution: 0.5 mg/mL (500 ppm) of Spironolactone RS in diluent
Pressure Drop: 67 bar (972 psi) [A] and 110 bar (1595 psi) [B]
A B

From Particulate to Monolithic Column
Amlodipine Besylate and Related Substances (USP36-NF31)
Performance criteria to be met:
For the purpose of identification, the relative retention times (RRT) are about
0.2 for benzene sulfonate,
0.5 for amlodipine impurity A*, and
1.0 for amlodipine.
The resolution, R, between amlodipine impurity A and amlodipine is not less than 4.5.
*Amlodipine impurity A is 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methylpyridine-3,5-dicarboxylate
Chromatographic Conditions particulate column Chromatographic Conditions monolithic column

Column: Purospher® STAR RP-18 endcapped (5µm) Hibar® RT 150x4.6 mm Column: Chromolith® High Resolution RP-18 endcapped 100x4.6 mm
Injection: 10 µL Injection: 10 µL
Detection: Shimadzu Prominence, UV 237 nm Detection: Shimadzu Prominence, UV 237 nm
Cell: 10 µL Cell: 10 µL
Flow Rate: 1.0 mL/min Flow Rate: 1.0 mL/min
Mobile Mobile
Phase: Acetonitrile:Methanol:Buffer 15:35:50 (v/v) Phase: Acetonitrile:Methanol:Buffer 15:35:50 (v/v)
Buffer: Add 7.0 ml of triethylamine in 1000 mL Milli-Q water, mix Buffer: Add 7.0 ml of triethylamine in 1000 mL Milli-Q water, mix
and adjust pH to 3.0 with orthophosphoric acid. Sonicate. and adjust pH to 3.0 with orthophosphoric acid. Sonicate.
Temperature: 25 °C Temperature: 25 °C
Diluent Mobile phase
Standard:
Standard:
Weigh 10 mg of Amlodipine Besylate in 20 mL volumetric flask.
Weigh 10 mg of Amlodipine Besylate in 20 mL volumetric flask.
Add about 5.0 mL diluent and sonicate it. Dilute it up to the mark with the same.
Add about 5.0 mL diluent and sonicate it. Dilute it up to the mark with the same. Further
Further dilute 1.0 ml to 100 ml with diluent.
dilute 1.0 ml to 100 ml with diluent.
Resolution
Solution: Weigh 5.0 mg of Amlodipine Besylate in 5.0 mL volumetric flask. Add 5.0 mL Resolution Solution: Weigh 5.0 mg of Amlodipine Besylate in 5.0 mL volumetric flask. Add 5.0 mL hydrogen
hydrogen peroxide. Heat at 70°C for 45 min in water bath. peroxide. Heat at 70°C for 45 min in water bath.
Sample: Crush 20 tablets. Weigh 50mg equivalent powder in 50 mL Sample: Crush 20 tablets. Weigh 50mg equivalent powder in 50 mL
volumetric flask. Add about 30 ml diluent and sonicate it volumetric flask. Add about 30 ml diluent and sonicate it
for 20 min. Dilute it up to the mark with the same. for 20 min. Dilute it up to the mark with the same.
From Particulate to Monolithic Column
Amlodipine Besylate and Related Substances (USP)
Purospher® STAR RP-18 endcapped (5 µm) Chromolith® HighResolution RP-18 endcapped

150x4.6 mm column 100x4.6 mm column *
250
150
Impurity A
200
120
Intensity (mV)
150
Intensity (mV)
90
Impurity A
100
60
50
30
0 0
0 5 10 15 20 0 2 4 6 8 10
Retention time (min) Retention time (min)
Chromatographic Data : Chromatographic Data :

No. Compound Retention Time RRT Resolution Theoretical No. Compound Retention Time RRT Resolution Theoretical
(min) plates (min) plates
1 Amlodipine Impurity A 6.0 0.5 - 3966 1 Amlodipine Impurity A 3.1 0.5 - 11163
2 Amlodipine Besylate 13.1 1.0 12.5 5026 2 Amlodipine Besylate 5.7 1.0 16.1 12438
* no specific particle size is mentioned in the current monograph - thus any type of column backbone can be used without additional verification!

Monolithic Silica Columns
UHPLC-like performance at low column backpressure
Monolithic HighResolution RP-18e, FPP C18, 1.7 µm,
Column: Chromolith® HighResolution RP-18e 50-2 mm 50-2.1 mm
50-2 mm Pressure = 74 bar Pressure = 476 bar
FPP C18, 1.7 µm, 50-2.1 mm
Mobile phase: A: 100% acetonitrile
B: 20 mM Phosphate buffer, pH 4.5
Gradient: Time/min %A %B
0 40 60
0.1 40 60
2.0 60 40
Flow rate: 600 µL/min
Pressure: 74 / 476 bar
Detection: DAD 20Hz UV, 220 nm
Detector cell: LightPipe 10 mm
Temperature: 23 °C
Injection volume: 0.2 µL
Sample: Indoprofen 5.0 mg
Ketoprofen 5.9 mg
Fenoprofen 5.2 mg
Fluorbiprofen 3.4mg
Ibuprofen 6.1mg
dissolved in 100 mL A/B 50/50

Chromolith® HighResolution RP-18e 2 mm I.D.
Diclofenac sodium in Gel - Stability test
The Stability test was performed to demonstrate the resistance to column clogging rep. matrix-tolerance and stability of a
monolithic silica column in comparison to a 1.7 µm fully porous particulate (FPP) column which is typically used in UHPLC
applications. Both columns have been treated the same way with frequent overloading to stress both columns to a maximum.
Both columns were tested with an RP-Test after stressing the columns 10 times. No pre-columns were used for this test.
Experimental Conditions for Stability test
®
Column_1: Chromolith HighResolution RP-18 endcapped 100 x 2 mm
Column_2: FPP Competitor column (hybrid silica C18 1.7 µm, 100 x 2.1 mm)
Mobile phase A: Water
Mobile Phase B: Methanol
Flow Rate: 200 µL/min for Chromolith; 221 µL/min for FPP 1.7 µm column (due to laminar flow conditions (2.0 mm I.D. for monolithic / 2.1 mm I.D. for FPP)
Isocratic: A/B 35/65 (v/v)
Temperature: Ambient
Injection volume: 3 times 1 µL; 10 times 10 µL; 3 times 1 µL; 10 times 10 µL etc.
Detection: UV @254 nm
Sample preparation: Shake a quantity of the gel containing 50 mg of diclofenac sodium with 50 mL acetone for 10 minutes, filter and evaporate the filtrate to dryness under a gentle
nitrogen flow. Dissolve the residue in 100 mL of a mixture of 40 volumes of water and 60 volumes of methanol, dilute 1 volume of this solution to 10 volumes with
the mobile phase and filter through a glass fiber filter.

Chromolith® HighResolution RP-18e 2 mm I.D.
Diclofenac sodium in Gel - Stability test
Column clogged Column backpressure / Injektionsvolumen
1400
1200
column backpressure/bar
1000
800
600 Chromolith HighResolution

BEH column
400
200
0
0 1000 2000 3000 4000 5000 6000 7000 8000
Injection volume/µL
Stability test data on Chromolith HighResolution RP-18 endcapped 100 - 2 mm I.D. column and FPP 1.7 µm Hybrid silica column from competitor 100 –
2.1 mm I.D. Data points with 1 µL injection were used for measurement.
The 1.7 µm FPP column clogged after 520 injection volumes.
The monolithic Chromolith® HighResolution column started to clog after 7000 injection volumes.

Greener Mobile Phases?
Eluent properties
Low viscosity of solvents Transmittance or absorbance
 low back pressure  can significantly decrease sensitivity
 high flow rate  large differences in the UV range (190 to 300 nm)
 enhanced mass transfer -> fast analysis  buffers or additives can also affect transmittance

Monolithic Silica Columns
Enable greener mobile Phases
Bisphenol A in Olive oil
Chromatographic Data [Olive oil spiked with 1.25 µg/mL BPA] Experimental Conditions
®
Column Chromolith HighResolution RP-18e 100x2 mm
Olive oil spiked Detection Dionex Ultimate 3000 FLD; @ = Ex 275 nm Em 305 nm
2.E-01 Mobile phase A Water
Mobile Phase B Ethanol

Intensity (counts)
2.E-01
Isocratic A/B 51/49 (v/v)
2
1.E-01 Flow Rate: 190 µL/min
5.E-02 Pressure Drop: 95 bar (1378 psi)
1 Temperature: 30 °C
0.E+00
Injection 1 µL
volume:
-5.E-02
0 1 2 3 4 5 Dilue Ethanol
nt
Retention Time (minutes)
sample solution Weigh 2.0 g of olive oil into a 15 mL screw-cap test tube, add 1
spiked mL of standard solution, seal with screw cap and shake for 30
No. Compound Retention Time (min) S/N Area (Counts) Tailing Factor
(1.25 µg/mL sec. Add 5 mL of diluent seal with screw cap and shake for 1 min.
1 t0 void volume 0.8 BPA) Sonicate the solution for 15 min. at room temperature, centrifuge
at 5000 rev/min for 25 min. Take an aliquot of the supernatant
2 Bisphenol A 2.5 17.4 120337 1.3 and pass it through a 0.45 µm PTFE filter into a HPLC vial.

Stability and Reproducibility of Chromolith® columns

HPLC Columns – small molecules
Selection by molecule
Hydrophobic Hydrophilic
Too much
Poor Peak Shape Aromatic
Isomers retention Retention too short or inadequate Separation on RP-18
(Basic Compounds) compounds
on C18
Closely Polar compounds

related Pi-Pi Interactions with high water HILIC
compounds content
Ascentis® Express
RP- F5 Phenyl- ES- Si

C30 C18 C8 Bihenyl AQ C18 OH 5
Amide (PFP) Hexyl Cyano HILIC
Chromolith®
C18e C8e
Diol Cyano Amino Si
/ (HR) / (HR)
Purospher®STAR
Discovery®
RP- F5 Diol Amino Si ZIC
C18 C8 Phenyl Cyano
Ascentis® Amide (PFP)
SeQuant®

Separation of polar compounds
What if RP-Chromatography fails? Column C-18e (5µm)
Gradient
Eluent A: 25 mM K2HPO4 /
N KH2PO4 buffer/ 10 mM TBAHS
NH (Tetrabutylammonium-
320 hydrogensulfate) pH 6.2
N Creatinine Column: ZIC-HILIC
300 O Eluent B: 80 % Acetonitrile / 20
H Mobile Phase A: 5 mM
280 % 5 mM TBAHS
260 ammonium acetate
240
0.1% formic acid (pH 4)
220
Mobile Phase B: MeCN
0.1% formic acid
200
Intensity,
Intensity, cps
Gradient: 5% A to 95% A
180
in15 min.
160
Inject. Vol.: 20 µL,
Reversed Phase C 18 tR = t0
cps
140
Flow: 0.6 mL/min
120
100 HILIC tR = 8.2 min
80
60
40
20
0
2 4 6 8 10 12 14 16 18 20 22 24
Time, min
Reversed phase
HILIC
Separation of polar compounds
Comparison of HILIC columns
k U – absolute retention
α (CH2) – degree of
hydrophobicity
α (OH) – degree of
hydrophilicity
α (V/A) – separation factor

for configurational isomers
α (2dG/3dG) – separation
factor for positional isomers
α (AX) – degree of anion

exchange interactions
α (CX) – degree of cation

exchange interactions
α (Tb/Tp) – acidic-basic
nature of the stationary
phase
Y. Kawachi et al., J. Chromatogr. A 1218 (2011) 5903-5919.

Metformin and Related Impurities
Column: SeQuant® ZIC®-cHILIC (3μm, 100Å) 150x4.6 mm
Injection: 5 μL
Detection: UV 218 nm
Cell: 8 μl
Flow Rate: 1.5 mL/min
Mobile Phase: Buffer: Dissolve 4.62 g of ammonium acetate in 1000 ml water (60 mM).
Adjust buffer to pH 5 using glacial acetic acid. Mix Acetonitrile and Buffer 90:10 (v/v)
Temperature: 30 °C
Sample: 5000 ppm metformin & 1 ppm of each impurity: A, C and melamine &
5 ppm of impurity B in mobile phase
Pressure Drop: 105 bar (1522 psi)

Supelco columns for small and large molecules
A wide range of column technologies to meet customer needs
Purospher STAR (120 Å)

− Optimal Reproducibility Ascentis Express (90, 160 Å)
− Outstanding Peak Symmetry BIOshell (160, 400, 1000 Å)
− Stable from pH 1.5 - 10.5 (RP-18) − Fast separations with very high efficiency
− Wide range of phases
− Available in micro format
Ascentis (100 Å) / Discovery
− Wide range of phase chemistries
SeQuant (100, 200 Å)

− Orthogonal selectivity to RP for separation
of polar compounds
− zwitterionic functionality designed for HILIC
mode
− Highly suitable for LC/MS use
Supel™ Carbon
− Unique retention mechanism
Discovery BIO (300 Å) − Enhanced separation of polar
− Three choices for reverse phase mode
compounds
− Full pH stability
Chromolith (150 Å) − Unsurpassed temperature stability
Astec CHIROBIOTIC™ Chiral columns Chromolith WP (300 Å)
- Chiral separations − Rugged and robust technology
− Analysis of matrix-rich samples
− Low back pressure
− Increased column lifetime
− Available in micro format

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Pharmaceutical

Petra Lewits, Global Product Manager HPLC Columns

Impurity Profile Assay

Disintegration & Appearance & physical

Impurity Profile Assay

Disintegration & Appearance & physical

4 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

According to ICH guidelines on impurities in new drug products, identification

5 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

6 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

7 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

▪ Retention factor (mass distribution ratio)

▪ Resolution (R): >2

8 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

9 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

10 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Repeatability (System Suitability Solution)

12 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Retention Theoretical Tailing

1 Desvenlafaxine 8.0 16145 1.1

System Suitability Solution Sample Solution

1 Desvenlafaxine 8.0 - 1.5 1.0

13 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

STD 1 69251 60 4242529

STD 2 69500 75 5268328

STD 3 68967 LOD (ppm) 0.07

STD 4 69301 LOQ (ppm) 0.22

14 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Purospher® STAR RP-18 endcapped (5µm) 250x4.6 mm

15 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

The resolution, R, between ramipril related

16 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt 

Injection volume Equation from FPP to FPP. May not be applicable to

18 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Injection volume Equation from FPP to FPP. May not be applicable to

C) Column dimensions (internal diameter):

F1 = monograph flow rate (mL/min)

20 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Adjustment of column I.D.

Flow rate f2 = f1 x (d2)2 / (d1)2

2.1² / 4.6² x 1 = 0.21 mL/min

21 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Test method Required action Minimal parameters to evaluate

Must use an already established „fully Verification Precision

22 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Fully porous polymeric particles ▪ SeQuant® ZIC® pHILIC

Fully porous Carbon particles (PGC) ▪ SupelTM Carbon

Injection volume Equation from FPP to FPP. May not be applicable to

1500 Particle size distribution of a typical commercial

Fused Core Particle 0

25 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Fully porous Particle

Highly-ordered packed bed Flow

Fused Core Particle

26 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

27 ©2023 HPLC Impurity profiling - Petra Lewits, Merck KGaA, Darmstadt

Ascentis C18 Ascentis C18 Ascentis C18 Fused-Core C18

40 40 40 Columns: All C18, All 150 x

2.0 3.0 2.0 3.0 2.0 3.0