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[ APPLICATION NOTE ]
WATERS SOLUTIONS
Alliance™ HPLC System
ACQUITY UPLC H-Class PLUS System
Empower™ 3 Chromatography Data Software
KEYWORDS
Quetiapine fumarate, column scaling,
method modernization, dwell volume,
USP, life cycle management
APPLICATION BENEFITS
■
■
The Waters™ Columns Calculator enables
users to geometrically scale methods
■
■
Improved throughput is obtained by
scaling HPLC columns to those with
smaller particle sizes and shorter
column lengths
■
■
The quetiapine fumarate impurities
method run time was reduced by 51%
using a 2.5 µm column with a UHPLC
system and 75% using a 1.7 µm column
with a UPLC™ system
■
■
Equivalent chromatographic and
quantitative performance was achieved
for the quetiapine fumarate impurities
method across all separations
INTRODUCTION
Pharmaceutical companies often follow compendial high performance
liquid chromatography (HPLC) methods for the analysis of raw materials
and finished products. However, modernization of older HPLC methods,
which can include scaling or transfer1 of a method to new column or LC
technologies should be considered as part of pharmaceutical lifecycle
management.2 The successful scaling of a method requires the proper
adjustment of various method parameters including column particle size
and dimension, flow rate, injection volume, and gradient timing.
In this study, the USP impurity monograph for quetiapine fumarate3 will
be scaled to smaller particle sized columns using the Waters Columns
Calculator. The scaled methods will then be compared to the original
HPLC method to ensure no loss of chromatographic or quantitative
performance. The scaled methods provide decreased run times and solvent
consumption while providing equivalent chromatographic performance.
Gradient Method Scaling for Life Cycle Management of a USP
Impurities Method
Amanda B. Dlugasch, Jennifer Simeone, and Patricia R. McConville
Waters Corporation, Milford, MA, USA
Figure 1. Chemical structures of quetiapine fumarate, and its impurities,
quetiapine related compound B, quetiapine related compound G, and
quetiapine desethoxy.
Gradient Method Scaling for Life Cycle Management of a USP Impurities Method
[ APPLICATION NOTE ]
[ APPLICATION NOT
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EXPERIMENTAL
Sample description
The quetiapine fumarate standard (catalog#: 1592704), and the quetiapine system suitability standard (catalog#: 1592715) were
purchased from the United States Pharmacopeia. The unknown quetiapine fumarate sample was purchased from Alibaba.com.
All solutions were prepared to the designated concentrations per the USP monograph. The system suitability and the standard solutions
were prepared in the diluent comprised of Solution A: Solution B (86:14). The unknown sample solution was prepared in Solution A.
The concentrations of the solutions are 1.0 mg/mL for the system suitability solution, 0.001 mg/mL for the standard solution,
and 1.0 mg/mL for the unknown sample solution.
Method conditions
Mobile phase:
Solution A:
Acetonitrile and buffer (25:75)
Solution B: Acetonitrile
Buffer:
3.1 g/L of ammonium acetate in water.
2 mL of 25% ammonium hydroxide
was added to each 1 liter of solution.
The final pH is not less than (NLT) 9.2
PDA wavelength:
250 nm at 4.8 nm resolution
Gradient table:
Instrument gradient time
Gradient composition
HPLC
(min)
UHPLC
(min)
UPLC
(min)
Solution A
(%)
Solution B
(%)
0.0
0.0
0.0
100
0.0
25.0
11.90
6.07
100
0.0
60.0
28.57
14.57
29.3
70.7
60.1
28.62
14.6
100
0.0
68.0
32.38
16.51
100
0.0
70.0
34.0
17.00
100
0.0
LC systems and conditions
HPLC system:
Alliance e2695 Separations Module
with 100 µL syringe, 2998 PDA Detector
and CH-30 equipped with the passive
column preheater
Column:
XBridge BEH C
8, 3.5 µm,
4.6 mm × 150 mm (p/n: 186003055)
Sample temp.:
4 °C
Column temp.:
45 °C
Injection volume:
20.0 µL
Flow rate:
1.500 mL/min
Pre-injection volume: NA
Run time:
70 minutes
UHPLC system:
ACQUITY Arc (path 2) with active
solvent preheating (CH-30A)
and 2998 PDA Detector
Column:
XBridge BEH C
8 XP, 2.5 µm,
3.0 mm × 100 mm (p/n: 186006047)
Sample temp.:
4 °C
Column temp.:
45 °C
Injection volume:
5.7 µL
Flow rate:
0.893 mL/min
Pre-injection volume: 388 µL
Run time:
34 minutes
UPLC system:
ACQUITY UPLC H-Class PLUS with
active solvent preheating (CH-30A),
50 µL extension loop and
ACQUITY UPLC PDA Detector
Column:
ACQUITY UPLC BEH C
8, 1.7 µm,
2.1 mm × 75 mm (p/n: 186005606)
Sample temp.:
4 °C
Column temp.:
45 °C
Injection volume:
2.1 µL
Flow rate:
0.644 mL/min
Pre-injection volume: 285 µL
Run time:
17 minutes
Data management
Empower 3 Chromatography Data Software, FR 4
[ APPLICATION NOTE ]
3
Gradient Method Scaling for Life Cycle Management of a USP Impurities Method
RESULTS AND DISCUSSION
METHODOLOGY
The quetiapine fumarate impurities USP method was first analyzed on the Alliance HPLC System using the described monograph
conditions.3 Performance was evaluated based on the system suitability requirements as outlined in the monograph, which
include resolution, tailing, and RSD for peak retention time and area. The column dimensions and method conditions were then
geometrically scaled to columns with smaller particles.4
The first step in method scaling is to select the column dimensions and particle size. The column selected should maintain the
L/dp ratio, where L is the length of the column and dp is the diameter of the particle size. The L/dp ratio is critical to maintain the
resolving power of the column.5
Once the appropriate column length and particle size are determined, the adjusted flow rate can be calculated. This ensures the
same linear velocity is maintained from the original method to the scaled method. The modified flow rate is based on the internal
diameter of the columns, the particle size of the columns, and the original flow rate using the following equation:
F
2 = F1 × (dp1/dc1) / (dp2/dc2)
where F
1 and F2 are the flow rates (mL/min) for the original and scaled method, respectively; dp1 and dp2 are the diameters of the
particle sizes (µm) of the original and scaled methods, respectively, and dc
1 and dc2 are the column diameters (mm) for the original
and scaled method, respectively.6
In scaling methods, it is also important to adjust the injection volume to maintain sensitivity, linearity, etc. Thus, the injection
volume needs to be adjusted with column volumes using the following equation:
V
inj2 = Vinj1 × (V02/V01)
where V
inj1 and Vinj2 are the injection volumes for the original and scaled methods, respectively, and V01 and V02 are the column void
volumes for the original and scaled methods, respectively.6
To maintain the separation, the gradient step must be kept constant in terms of column volumes. To do this, the column volumes
must be calculated for the original method and then preserved for the scaled method. The number of column volumes determined
for each segment is calculated as follows:
CV = ( F × T) / V
0
where CV is equal to column volumes, F is the flow rate (mL/min), T is the segment duration (minutes), and V
0 is the column void
volume (mL).6 Since the void volume and flow rate are constant, the time duration of each gradient step in the scaled method can
be calculated based on the required column volume.
[ APPLICATION NOTE ]
4
Gradient Method Scaling for Life Cycle Management of a USP Impurities Method
Geometrically scaling a gradient method can
seem challenging, but there is a tool to assist
users in completing all of the necessary method
adjustments.7 The Waters Columns Calculator
determines the flow rate, the injection volume,
as well as the timing for each gradient step.
Once a user enters in the required information
(column dimensions, particle size, original
method gradient table, etc.) the scaled method
conditions are automatically calculated.
(Figure 2 and Figure 3).
In order to preserve the original HPLC column
L/dp ratio the column dimensions and particle
size were scaled to a UHPLC column with a
2.5 µm particle size and 3.0 mm × 100 mm
column dimensions. The UHPLC column L/dp
ratio decreased by 7% from the HPLC column.
The Waters Columns Calculator (Figure 2) scaled
the flow rate to 0.893 mL/min and the injection
volume to 5.7 µL for the UHPLC method.
The Waters Columns Calculator was also used
to scale the quetiapine fumarate impurity method
to a UPLC column with 1.7 µm particle size and
2.1 mm × 75 mm dimensions (Figure 3). The
scaled column dimensions resulted in an L/dp
ratio increase of 3%, a flow rate of 0.644 mL/min
and the injection volume of 2.1 µL.
The dwell volume, or gradient delay volume, is the
volume between the point of solvent mixing and
the head of the column. Since the dwell volume
is effectively an isocratic hold at the beginning
of a gradient, it can affect selectivity, resolution,
and retention in method scaling. Therefore, when
scaling methods, the dwell volume is often kept
constant in terms of column volumes. In fact,
this is part of the method scaling parameters
determined using the Waters Columns Calculator.
To account for the dwell volume differences, the
values for the two LC systems were determined8
and entered into the Waters Columns Calculator.
The dwell volume in terms of column volume
varied for the Alliance HPLC System, the
ACQUITY UHPLC Arc System, and the ACQUITY
UPLC H-Class PLUS System. Thus, when scaling
from the Alliance HPLC System (and column),
“pre-injection volumes” were required for both
the UHPLC scaled method (388 µL) and the
UPLC method (285 µL).
Original conditions
Scaled UHPLC conditions
Figure 2. Waters Columns Calculator. HPLC method conditions calculated to UHPLC
method conditions.
Figure 3. Waters Columns Calculator. HPLC method conditions calculated to UPLC
method conditions.
Original conditions
Scaled UPLC conditions
[ APPLICATION NOTE ]
5
Gradient Method Scaling for Life Cycle Management of a USP Impurities Method
Resolution
(peak 1 and 2)
Resolution
(peak 3 and 4)
Quetiapine
tailing
Quetiapine
area
%RSD
Quetiapine
retention
time
%RSD
Run
time
(min)
Solvent
consumption
per sample
(mL)
Alliance HPLC
14.0
7.0
1.03
1.24
0.04
70
105
ACQUITY Arc UHPLC
13.2
6.7
0.95
0.57
0.02
34
30
ACQUITY UPLC
H-Class PLUS
11.2
5.4
1.04
0.65
0.02
17
11
SCALING OF A GRADIENT METHOD FROM HPLC TO UHPLC AND UPLC
To evaluate the performance of the scaled methods, the results were compared to the original HPLC method run on
the Alliance HPLC System. Additionally, an unknown sample was analyzed on each of the three systems to determine
the quantitative reproducibility of the scaled methods.
The original HPLC method as well as the two scaled methods all show similar chromatographic performance (Table 1)
in terms of resolution, tailing, and peak area and retention time RSDs. Chromatograms of the system suitability solution
and the unknown sample solution are shown in Figure 4 and 5, respectively.
Table 1. Comparison of the results obtained on the Alliance HPLC System, the ACQUITY Arc UHPLC System, and the ACQUITY UPLC H-Class PLUS Systems.
Also included is the run time and solvent consumption for each method.
Figure 4. Quetiapine fumarate system suitability solution run on the Alliance HPLC System
(3.5 µm particle column), the ACQUITY Arc UHPLC System (2.5 µm particle column),
and the ACQUITY UPLC H-Class PLUS System (1.7 µm particle column). Peak identification:
1: quetiapine related compound G, 2: quetiapine related compound B, 3: quetiapine desethoxy,
and 4: quetiapine.
Figure 5. Comparison of the unknown sample solution analyzed on the Alliance HPLC System
(3.5 µm particle column), the ACQUITY Arc UHPLC System (2.5 µm particle column), and the
ACQUITY UPLC H-Class PLUS System (1.7 µm particle column). Peak identification:
1: quetiapine desethoxy, 2: quetiapine, and 3: unknown impurity.
ACQUITY UPLC H-Class PLUS System
ACQUITY Arc UHPLC System
ACQUITY HPLC System
ACQUITY UPLC H-Class PLUS
ACQUITY Arc UHPLC
Alliance HPLC
Scaling the original HPLC method to a smaller
particle column significantly decreased the
run time and solvent consumption. Scaling the
original method to a 2.5 µm column decreased
the run time from 70 minutes to 34 minutes (51%),
and decreased the solvent usage by 71%.
Further scaling the method to a 1.7 µm column
was able to decrease the run time from
70 minutes to 17 minutes (75%) and reduce
the solvent usage by 89%.
To evaluate the quantitative reproducibility of
the methods, an unknown sample was analyzed.
The standard solution and the unknown sample
solution data were used to calculate the percent
of impurity for each peak in the unknown sample
as follows:
Result = (r
u /rs) × (Cs/Cu ) × (1/F) × 100
where r
u is the peak response of each impurity
from the sample solution, r
s is the peak response
of quetiapine from the standard solution, C
s is
the concentration of USP quetiapine fumarate
standard in the standard solution (mg/mL), C
u is
the concentration of quetiapine fumarate in the
sample solution (mg/mL) and F is the relative
response factor for the impurity peak provided
in the monograph.3
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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Waters, The Science of What’s Possible, ACQUITY, Alliance, Arc, Empower, UPLC, and XBridge are
trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2019 Waters Corporation. Produced in the U.S.A. June 2019 720006577EN AG-PDF
Two impurity peaks were found in the unknown sample, quetiapine desthoxy
and an unknown impurity. The calculated percent for each impurity as well as
the total amount of impurities in the unknown sample can be found in Table 2.
All methods provided equivalent impurity amounts for the unknown sample.
Unknown sample
Quetiapine
desethoxy
Unknown
impurity
Total
impurities
Alliance HPLC System
0.12%
0.08%
0.22%
ACQUITY Arc UHPLC System
0.09%
0.06%
0.17%
ACQUITY UPLC
H-Class PLUS System
0.10%
0.07%
0.19%
Table 2. Calculated impurity results obtained for all three methods on the three different
LC systems.
Whenever a method is adjusted, producing consistent reliable results is
a critical factor. Scaling the USP quetiapine fumarate impurities method
across the different LC systems produced equivalent quantification of
impurities contained within a sample of API.
CONCLUSIONS
It is possible to scale traditional HPLC methods to columns with a
smaller particle size in order to significantly decrease run time and
solvent consumption while still providing the same chromatographic
and quantitative performance. This was demonstrated by scaling a USP
monograph which uses a gradient elution using the Waters Columns
Calculator. The scaled method conditions reduced the original run time
by 51% for the 2.5 µm column and 75% for the 1.7 µm column. The scaled
methods maintained similar chromatographic performance in terms of
resolution, peak tailing, and retention time and peak area RSD. Additionally,
quantitative results for impurities contained in the API sample were
consistent regardless of which method was used.
References
1. Fountain, Kenneth. Transferring Compendial HPLC
Methods to UPLC Technology for Routine Generic
Drug Analysis. Application Note. 720004251en, 2012.
2. Guidance for Industry Q10 Pharmaceutical Quality
System. ICH, 2008.
3. Official Monographs, Quetiapine Fumarate USP 40
NF35 S1, United States Pharmacopeia and National
Formulary (USP 40-NF35 S1) Baltimore, MD: United
Book Press, Inc.; 2017. p. 5939.
4. Neue Uwe D., McCabe, Doug, Ramesh, Vijaya,
Pappa, Horacio, DeMuth Jim. Transfer of HPLC
Procedures to Suitable Columns of Reduced
Dimensions. Pharmacopeial Forum 2009 Nov–Dec;
35(6):1622.
5. Swann, Thomas. Nguyen, Jennifer M. USP Method
Modernization Using “Equivalent L/dp” and
“Equivalent N” Allowed Changes with CORTECS
C8 and CORTECS C8 Columns. Application Note.
720005666en, 2016.
6. Columns Calculator Online Help. Waters Columns
Calculator, version 2.0.
7. Waters Corporation, Application Solutions.
Transferring Compendial HPLC Methods to
UPLC Technology. Application Notebook.
720004313en, 2013.
8. Hong, Paula. McConville, Patricia R. Dwell Volume
and Extra-Column Volume: What Are They and
How Do They Impact Method Transfer. White Paper.
720005723en, 2018.
