Gradient Method Scaling for Life Cycle Management of a USP Impurities Method

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[ APPLICATION NOTE ]

WATERS SOLUTIONS
Alliance™ HPLC System

ACQUITY™ Arc™ UHPLC System

ACQUITY UPLC H-Class PLUS System

Waters Columns Calculator

XBridge™ Columns

ACQUITY UPLC Columns

Empower™ 3 Chromatography Data Software

KEYWORDS
Quetiapine fumarate, column scaling, 
method modernization, dwell volume,  
USP, life cycle management

APPLICATION BENEFITS

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The Waters™ Columns Calculator enables 
users to geometrically scale methods

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Improved throughput is obtained by 
scaling HPLC columns to those with 
smaller particle sizes and shorter  
column lengths

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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

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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

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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 ]

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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

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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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©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.