Application Note
Pharma & Biopharma
Author
William J. Long
Agilent Technologies, Inc.
Abstract
This application note develops a method for salicylic acid drug substance and
drug products analysis using HPLC equipment and superficially porous columns.
Superficially porous HPLC columns such as 2.7 µm Agilent InfinityLab Poroshell 120
show comparable efficiency to sub 2 µm particles with about half the backpressure.
Columns packed with these materials can be used in older HPLC instruments or
in new, higher-pressure instruments for high resolution in longer column format.
Additionally, since they use larger 2 µm frits commonly found in 5 µm columns, extra
sample preparation is not necessary.1
Several stationary phases are evaluated with simple mobile phase additives on
short 50 mm columns. Using five or six isocratic experiments, a retention model
is built using Optichrom LC.2 Multivariate optimization is performed within the
model (resolution: 2, minimum time, pressure under 400 bar). Isocratic and gradient
methods are considered.
Modernization of USP Salicylic
Acid HPLC Analysis Using Agilent
InfinityLab Poroshell 120 Columns
2
Introduction
Superficially porous particle LC
columns are a popular tool in liquid
chromatography. Superficially porous
particle columns generate high efficiency
at lower pressure, relative to their totally
porous particle column counterparts.
This is primarily due to a shorter mass
transfer distance and substantially
narrower particle size distribution of the
particles in the column.1 The current
trend with superficially porous particles
is reducing particle size for further
efficiency improvements. The higher
efficiency can be used to speed up
analyses or improve results by increasing
resolution and sensitivity.
As part of an effort to modernize USP
monographs, modern HPLC and UHPLC
methods are sought for analysis of
active ingredients and impurities to
replace older nonchromatographic
methods such as titration. Salicylic
acid is an over-the-counter (OTC) active
occurring in hundreds of products in
dozens of delivery forms. Given the
market complexity, public standards
need to contain chromatograph options
to accommodate the wide variety of
products and not create compliance
issues.
Several stationary phases are evaluated
with simple mobile phase additives
on short 50 mm columns. Using five
isocratic experiments, a retention model
is built using Optichrom LC.2 Multivariate
optimization is performed, within the
model (resolution: 2, minimum time,
pressure under 400 bar). Isocratic and
gradient methods are considered.
Experimental
An Agilent 1260 Infinity LC was used
in this work. The system was used in
standard configuration, and is listed in
Table 1. Salicylic acid and gentisic acid
were purchased from Sigma-Aldrich
(St. Louis, MO, USA); 4-hydroxyiopthallic
acid (4-HIPA, salicylic acid
impurity B) and 2-hydroxyhippuric acid
were purchased from Acros Organics
(Thermo Fisher Scientific, Geel, Belgium).
Phenol was purchased from EM Science.
These compounds were prepared at
0.5 mg/mL in water except for salicylic
acid, which was prepared at 1 mg/mL.
Figure 1 presents structures of these
compounds. Agilent InfinityLab Poroshell
120 Phenyl-Hexyl (p/n 699975-312)
and Agilent Poroshell 120 SB-Aq
(p/n 699975-314) 2.7 μm, 3 × 50 mm
columns, as well as other columns, were
evaluated. Trifluoracetic acid (TFA),
acetic acid, and formic acid were
purchased from Sigma-Aldrich. Methanol
and tetrahydrofuran (GC/GPC grade)
were purchased from Honeywell (Burdick
and Jackson, Muskegon, MI, USA). A
Milli-Q system (Millipore, Burlington, MA,
USA) provided 18 MΩ·cm water that was
passed through 0.2 µm filter. Samples
of commercially available corn removal
pads were examined.
Table 1. Instrument configuration details.
Parameter
Value
Column
Agilent InfinityLab Poroshell 120 SB-Aq, 3 × 50 mm, 2.7 µm, part number 699975-314
Agilent InfinityLab Poroshell 120 Phenyl-Hexyl, 3 × 50 mm, 2.7 µm, part number 699975-312
Mobile Phase
Methanol: 0.1% TFA in water (varies)
Flow Rate
0.63 mL/min
Column Temperature
35 °C
Injection Volume
1 µL
Degasser
Agilent 1260 Infinity micro degasser (G1379B)
Pump
Agilent 1260 Infinity binary pump (G1312B)
Autosampler
Agilent 1260 high performance autosampler (G1367C)
Column Oven
Agilent 1290 Infinity thermostatted column compartment (G1316C)
Detector
Agilent 1260 Infinity II diode array detector WR (G7115A)
Figure 1. Structures of salicylic acid and related compounds.
OH
O
HO
OH
Phenol
Chemical formula: C
6H6O
Molecular weight (Da): 94.11
[108-95-2] pK
a= 10.01
4-Hydroxybenzoic acid
Chemical formula: C
7H6O3
Molecular weight (Da): 138.12
[99-96-7] pK
a= 4.48
OH
OH
O
HO
OH
OH
O
Salicylic acid
Chemical formula: C
7H6O3
Molecular weight (Da): 138.12
[69-72-7] pK
a= 2.98
Gentisic acid
Chemical formula: C
7H6O4
Molecular weight (Da): 154.12
[490-79-9] pK
a= 3.54
O
OH
N
H
O
OH
OH
OH
O
HO
O
4-Hydroxyisophthalic acid
Chemical formula: C
8H6O5
Molecular weight (Da): 182.13
[636-46-4] pK
a= 3.4
2-Hydroxyhippuric acid
Chemical formula: C
9H9NO4
Molecular weight (Da): 195.17
[487-54-7] pK
a= 3.54
3
Results and discussion
Several stationary phases were
evaluated with simple mobile phase
additives used to control pH. Data from
experimental chromatographic runs
were acquired and used to build an
empirical retention model. Optichrom
LC optimizes the remaining variables, as
experimental data are required to build
a retention model. These are collected
after selecting a single column and after
selecting and fixing the weak (A) and
strong (B) mobile-phase components,
pH, and temperature. The software is
only programmed for binary mobile
phase optimization; however, these can
contain blends of solvents, modifiers, or
buffers. The various experimental log k
values for each solute are regressed
against %B. As few as two values of
%B may be used, but we generally use
five different %B values and regress
using a quadratic model. The regression
coefficients are later used to predict k
values of each peak as a function of %B.
Individual standards were run after
enough equilibration at 5, 10, 15, 20, and
25% methanol. The data was entered in
the Optichrom LC spreadsheet, and using
the solver function of Excel, the best
isocratic and gradient solutions for each
column and condition were determined.
Based on this study, isocratic conditions
consisting of 15% methanol/85% water
with 0.1% TFA was chosen. A gradient
after the analysis was initially proposed
to remove excess excipient material
from the column but was not needed for
this work. It may be re-added if column
lifetime and pressure increase become
an issue.
The best isocratic separations found
used InfinityLab Poroshell 120 SB-Aq
and Poroshell 120 Phenyl-Hexyl columns
with 0.1% TFA in water with methanol.
Separations using formic acid or acetic
acid suffered from lower resolution,
unresolved peaks, or poor peak shape.
While these mobile phase additives are
easier to use with MS, the availability of
inexpensive standards and desire for
low-cost analysis limit the need for MS.
A plot of selectivity versus %B was
developed and appears in Figure 2B
for the InfinityLab Poroshell 120 SB-Aq
TFA/MeOH separation. The plot shows
a flat region between 15 and 25%
methanol. Use of the higher methanol
concentration would speed up the
separation, but may also require a faster
data collection speed. To allow the use
of a wider range of detectors, the lower
organic mobile phase was used.
Figure 2. Agilent InfinityLab Poroshell 120 Phenyl-Hexyl and SB-Aq retention model data Log k versus
%B (Optichrom LC fit data). (A) InfinityLab Poroshell 120 Phenyl-Hexyl: 0.1% TFA in water/methanol;
(B) InfinityLab Poroshell 120 SB-Aq: 0.1% TFA in water/methanol; (C) InfinityLab Poroshell 120 SB-Aq: 0.1%
acetic acid in water/methanol; (D) InfinityLab Poroshell 120 SB-Aq: 0.1% formic acid in water/methanol.
Salicylic acid
Gentisic acid
4HBA
Phenol
2-HHA
4-HIA
Salicylic acid
Gentisic acid
2HBA
Phenol
2-HHA
HIPA
Salicylic acid
Gentisic acid
2HBA
Phenol
2-HHA
HIPA
Salicylic acid
Gentisic acid
2HBA
Phenol
2-HHA
HIPA
A
C
B
D
0.0
0.5
1.0
1.5
2.0
2.5
0
10
20
30
%B
Log
k
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
0
10
20
30
%B
Log
k
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
10
20
30
%B
Log
k
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
10
20
30
%B
Log
k
4
Figure 4 shows a comparison of the
optimized method using the InfinityLab
Poroshell SB-Aq and Phenyl-Hexyl
columns. As the modeling predicted, the
InfinityLab Poroshell 120 SB-Aq column
delivered better Rs ≥ 2 resolution for all
peak pairs (minimum Rs of 2.17 between
2HBA/GA, and Rs = 2.76 for HIPA/SA).
The Phenyl-Hexyl separation at the same
isocratic conditions shows a different
elution order, although with salicylic acid
eluting last in both cases. In addition, at
these conditions, a minimum Rs of 0.89
for 2-HBA/HIPA and 1.70 for HIPA/GA
was determined. The retention time for
SA on the SB-Aq column is roughly half
that on the Phenyl-Hexyl column, leading
to shorter analysis times.
1
2
3
4
5
6
7
8
9
mAU
0
50
100
150
200
250
300
350
0.483
1.321
2.054
2.270
3.884
4.411
1
2
3
4
5
6
7
8
9
mAU
0
100
200
300
400
0.535
2.000 2.089
2.270
2.997
9.472
Rs = 2.17
Rs = 2.76
Rs = 0.89
Rs = 1.70
1. Phenol
2. 2HBA
3. GA
4. 2HAA
5. HIPA
6. Salicylic acid
1
5
4
2
3
6
6
5
2
3
1
4
Agilent InfinityLab Poroshell 120 SB-Aq, 2.7 µm (p/n 699975-314)
15% water with 0.1% TFA/85% MeOH, 25 °C, 0.63 mL/min
Agilent InfinityLab Poroshell 120 Phenyl-Hexyl, 2.7 µm (p/n 699975-312)
15% water with 0.1% TFA/85% MeOH, 25 °C, 0.63 mL/min
Time (min)
Time (min)
0.63 mL/min 15% water with 0.1% TFA/85% methanol, 2 µL injection, 35 °C
Figure 4. A comparison of the optimized isocratic method using the Agilent InfinityLab Poroshell SB-Aq and Phenyl-Hexyl columns (0.63 mL/min 15% water
with 0.1% TFA/85% methanol, 2 µL injection, 35 °C).
Figure 3. Resolution map (minimum α versus %B plot).
1
1.05
1.1
1.15
1.2
1.25
1.3
0
5
10
15
20
25
30
α (minimum
)
% B
Window Diagram
5
Using the Poroshell 120 SB-Aq method
conditions, a calibration curve was
developed. It was found to be linear
across the range of interest.
Analysis of real samples was attempted.
Corn removal pads were purchased from
a local pharmacy; these pads contained
40% salicylic acid in a synthetic rubber
matrix. The rubber pads were carefully
peeled from the bandage before
extraction. Before extraction, the pad
should be examined for inert bandage
material. Extracting the pads in 100%
water yielded inferior results, with only
low levels found. Because a synthetic
rubber is used in the pad, 2 mL THF was
added as a first step in the extraction
with dramatic improvement in extraction.
The analyses are very consistent. In the
case of the water extraction, sample
3 was found floating on the top of the
extraction vessel, with samples 1 and 2
completely submerged. In every case,
the water extraction results yielded very
low recovery. However, with the addition
of THF, better extraction was found, but
a calculated recovery was nearly 100%.
It is important to carefully scrape off
any adhesive or backing so as not to
dilute the sample with inert material.
No additional optimization of extraction
time was carried out (due to lack of
samples), but the 45-minute extraction
after the initial THF soak could possibly
be reduced.
Figure 5. Salicylic acid calibration curve.
0
0
1,000
2,000
3,000
4,000
5,000
6,000
Area
Concentration (mg/mL)
Linearity
y = 1,734.2 × 3.4148
R² = 0.9999
Average area
Linear (average area)
2
1
3
4
Table 2. Summary of extraction study. Two pads: extracted with
25 mL water; sonicated for 60 minutes. Recovery based on two pads
weighing 50 mg consisting of 40 wt % SA.
Injection
Area
Concentration
(mg/mL)
% Recovery
Sample 1, Injection1
57.59
0.9957
4.98
Sample 1, Injection 2
52.43
0.9215
4.60
Sample 2, Injection1
51.94
0.9145
4.58
Sample 2, Injection 2
51.07
0.9020
4.50
Sample 3, Injection1
38.21
0.7170
3.58
Sample 3, Injection 2
38.07
0.7150
3.56
Table 3. Summary of extraction study. Two pads: extracted with
2 mL THF; soaked for 15 minutes; 23 mL water added; sonicated
for 45 minutes. Agilent InfinityLab Poroshell 120 SB-Aq, 2.7 µm
(p/n 699975-314) 15% MeOH/85% water with 0.1% TFA, 25 °C,
0.63 mL/min.
Injection
Area
Concentration
(mg/mL)
% Recovery
Sample 1, Injection1
1372.36
19.90
99.5
Sample 1, Injection 2
1366.53
19.82
99.1
Sample 2, Injection1
1369.58
19.86
99.32
Sample 2, Injection 2
1339.30
19.42
97.14
Sample 3, Injection1
1352.50
19.62
98.1
Sample 3, Injection 2
1358.63
19.71
98.52
www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Printed in the USA, January 7, 2020
5994-1658EN
DE.4515162037
Conclusion
A fast and low-cost isocratic method
for the analysis of an OTC medicine is
presented. The method is demonstrated
on an Agilent 1260 Infinity LC and run
at approximately 170 bar (2,470 psi)
at 35 °C. Chromatographic run time
is approximately five minutes. The
extraction procedure as demonstrated
takes approximately 60 minutes, but
multiple samples could be extracted
simultaneously. An optimized extraction
procedure could be faster.
Thank you to The Proctor & Gamble
Company for allowing use of
Optichrom LC.
References
1. Gratzfield-Hugsen, A.; Naegele, E.,
Maximizing Efficiency Using Agilent
Poroshell 120 Columns. Agilent
Technologies application note,
publication number 5990-5602EN,
2016.
2. Chester, T. L. Business-Objective-
Directed, Constraint-Based
Multivariate Optimization
of High-Performance Liquid
Chromatography Operational
Parameters. J. Chromatogr. A 2003,
1016(2), 181–193.
