New Approaches for Simultaneous
API and Counterion Analysis
Using Charged Aerosol Detection
Christopher Crafts1, Marc Plante1, Bruce Bailey1, Paul Gamache1,
John Waraska1, Ian Acworth1, and Kannan Srinvasan2
1
ESA−A Dionex Company, Chelmsford, MA, USA; 2Dionex Corporation, Sunnyvale, CA, USA
Abstract
Approximately 50% of all drug molecules used in medicinal therapy are
administered as salts. Salt forms are used to improve the biological and
physiochemical properties of active pharmaceutical ingredients (APIs)
including: aqueous solubility, hygroscopicity, solution pH, melting point,
dissolution rate, chemical stability, crystal form, and mechanical properties. Traditional approaches for salt analysis use several analytical
methods to measure the API and various counterions. Recent innovations
in mixed-mode column technology and universal detection techniques
enable the rapid, sensitive, and simultaneous analysis of an API together with its inorganic or organic counterion. The work in this study
uses HPLC and the Corona® ultra™ detector for the analysis of several
common pharmaceutical salts. A Dionex Acclaim® Trinity™ P1 column,
which combines anion-exchange, cation-exchange, and reversed-phase
functionalities, allows separation of positive and negative counterions, as
well as lipophilic APIs in a single run. Different methods are presented,
including an isocratic approach capable of simultaneously measuring the
API and its counterion in less than 3 min. Figures of merit are reviewed,
including accuracy, precision, linear range, LOD, and LOQ. Comparison
of experimental and theoretical values are within 1%. The ability to test for
ionic impurities of < 0.1% is demonstrated. The combination of the Trinity
column and the Corona ultra detector offers a new, versatile, and powerful
tool for counterion analysis with applications for salt selection, drug
formulation, and drug impurity analysis.
hybrid column technologies. The SeQuant polymeric zwitterionic column
(ZIC®-pHILIC) uses zwitterionic stationary phase on porous polymer particles. The separation is achieved by a hydrophilic interaction mechanism
superimposed on weak electrostatic interactions.3 The Acclaim Trinity P1
column uses silica particles where the inner-pore area is modified with
an organic layer that provides both reversed-phase and anion-exchange
properties. The outer surface is modified with cation-exchange functionality. The Charged Aerosol Detector (CAD®) uses a patented detection
technique described in Figure 1 which is able to achieve near uniform response over a broad spectrum of analytes. The analysis of inorganic ions
involves the use of volatile buffers, such as ammonium acetate or formate,
to create salt particles that can then be charged and detected.
1
10
3
2
8
4
9
6
5
7
Introduction
1. Liquid eluent enters from HPLC system
6. Gas stream passes over corona needle
2. Pneumatic nebulization occurs
Counterions influence an API’s solubility, stability, and hygroscopicity, so
appropriate counterion selection is an important part of drug development
process. The most common pharmaceutical counterions include: chloride,
sodium, sulfate, acetate, phosphate, potassium, maleate, calcium, citrate,
bromide, nitrate, ammonium, tosylate, phosphate, tartrate, and ethylenediamine.1,2 Methods for the analysis of 13 of the 16 most common pharmaceutical salts are demonstrated on the zwitterionic or the nanopolymer silica
3. Small particle droplets enter drying tube
7. Charged gas collides with particles and
transfer charge
4. Large droplets exit to drain
8. High mobility species are removed
5. Dried particles enter mixing chamber
9. Remaining charged particles measured
10. Signal transferred to chromatographic
software
26802
Figure 1. Corona ultra detector workflow.
Simultaneous SEPARATION OF Cations
and Anions
Column:
Mobile Phase:
Flow Rate:
Inj. Volume:
Column Temp.:
Sample Conc.:
Detection:
Filter:
22
Column:
Mobile Phase:
Acclaim Trinity P1 3.0 × 100 mm, 3 µm
Acetonitrile / 20 mM (total)
Ammonium acetate pH 5.2 (60:40)
0.5 mL/min
5 µL
30 °C
~100 µg/mL each
Corona ultra 35 psi nitrogen
Corona
400
mV
Cl–
NO3–
4
2
K+
Na+
Acclaim Trinity P1, 3.0 × 50 mm, 3 µm
A) Acetonitrile
B) Deionized water
C) 200 mM Ammonium acetate pH = 4
Flow Rate:
0.5 mL/min
Inj. Volume:
10 µL
Gradient:
Time (min)
0
2
7
15
%B
35 35
0
0
%C
5
5 90
90
Column Temp.: 30 °C
Sample Conc.: Between 35 and 150 µg/mL each
Filter:
None
5
3
6
1
pA
Peaks:
1. Procaine
2. Choline
3. Tromethamine
4. Sodium
5. Potassium
6. Meglumine
7. Mesylate
8. Maleate
9. Chloride
10. Bromide
11. Iodide
12. Phosphate
13. Malate
14. Tartrate
15. Citrate
16. Sulfate
Br–
7
13
9
8
10 11
14
12
16
15
0
0
2
0
2
4
6
8
Minutes
10
12
14
3
6
Minutes
9
12
15
27132
16
27130
Figure 2. Isocratic analysis on the Acclaim Trinity P1, 3.0 × 100 mm,
3 µm column.
Figure 4. Gradient analysis on the Acclaim Trinity P1, 3.0 × 50 mm,
3 µm column.
Reproducibility and Range
Column:
SeQuant ZIC-p H ILIC 5 µm, 4.6 × 150 mm, 5 µm
Mobile Phase: A) Acetonitrile, isopropanol,
100 mM ammonium acetate, pH = 4.68,
methanol (60:20:15:5)
B) 30 mM Ammonium acetate pH = 4.68,
acetonitrile, isopropanol, methanol (50:25:20:5)
Flow Rate:
0.5 mL/min
Inj. Volume:
10 µL
Gradient:
Time (min) 0
3
24
26
28
29
%B
20
20
70
70
15
20
Column Temp.: 30 °C
Sample Conc.: ~30 µg/mL each
Detection:
Corona ultra 35 psi Nitrogen
Filter:
Corona
Table 1. Intra- and Between-Day
Reproducibility of Five Analytes
ClO4
NO3
–
Cl
pA × (100,000)
–
K+
Na+
% RSD
Day 4
(n = 6)
% RSD
Day 7
(n = 6)
% RSD
All Days
(n = 22)
Nitrate
1.25
1.45
1.00
0.88
2.67
Chloride
0.45
1.33
1.23
0.56
2.55
Sulfate
1.71
0.77
2.21
1.31
4.68
Sodium
1.94
1.35
1.69
1.54
3.30
Potassium
1.16
1.93
1.19
1.39
3.47
Percentage RSD of area averages of anions and cations using the method
described in Figure 3.
PO43–
Li+
% RSD
Day 2
(n = 5)
32
20
–
1.75
% RSD
Day 1
(n = 5)
SO42–
0.25
5
10
15
Minutes
20
25
27131
Figure 3. Gradient analysis on the SeQuant ZIC-pHILIC, 4.6 × 150 mm,
5 µm column.
2
New Approaches for Simultaneous API and Counterion Analysis
Using Charged Aerosol Detection
12,000
Sodium Response Curve
1
Amount (ng on column)
500
mV
Column:
ZIC-p HILIC
Mobile Phase: A) Acetonitrile, isopropanol,100 mM ammonium acetate, pH = 4.68,
methanol (60:20:15:5)
B) 30 mM Ammonium acetate pH = 4.68,
acetonitrile, isopropanol, methanol (50:25:20:5)
Flow Rate:
0.5 mL/min
Inj. Volume: 10 µL
Gradient:
Time (min)
0
3
24
26
28
29
32
%B
20
20
70
70
15
20
20
Sample:
Diclofenac sodium salt, 0.3 µg
Peaks:
1. Diclofenac
2
2. Sodium
0
0
0
0
2
4
6
8
10
Area (pA * Minutes)
12
14
5
10
16
Minutes
15
20
25
27135
27133
Figure 5. Sodium acetate from 5–10,000 ng on column. Three injections, at
each of the 12 concentrations, fit with a polynomial inversed x and y axis
(r 2 = 0.9997) overlaid with the 95% confidence limits.
Figure 7. Chromatogram of a 10 µL injection of diclofenac sodium salt with the
method described in Figure 3.
Fast counterion analysis
API and CounterIons
The simultaneous measurement of an API and its counterion was illustrated using diclofenac sodium salt. Figure 6 shows the analysis on
the Acclaim Trinity P1 column while Figure 7 shows the analysis on the
ZIC-pHILIC column. In this example, the API and counterion elution
order was reversed for the two columns with diclofenac retaining better
on the Trinity column with a shorter run time.
80
Column:
Acclaim Trinity P1, 3.0 × 50 mm, 3 µm
Flow Rate:
1.0 mL/min
Mobile Phase: Acetonitrile/120 mM ammonium acetate
(30 mM total buffer strength) (75:25)
pH varied as shown in figure
Peaks:
1. Naproxen
2. Sodium
pH = 3.8
1
2
PA
0
2
1
pH = 5.2
This approach can be used to measure other APIs with counterions
shown in Figures 2–4 on both the Trinity and ZIC columns.
–60
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
Minutes
27136
Figure 8. Naproxen sodium salt, ~500 ng on column.
80
Column:
Acclaim Trinity P1, 3.0 × 50 mm, 3 µm
Mobile Phase: Acetonitrile/120 mM ammonium acetate pH = 4.8
(30 mM total buffer strength) (75:25)
Flow Rate:
0.8 mL/min
2
Peaks:
1. Sodium
2. Diclofenac
1
pA
Limits of Detection and
0.1% Impurities
–5
0
1
2
3
Minutes
4
5
6
27134
Table 2. LODs for Sodium and Chloride
Using the Corona ultra Detector
ZIC-pHILIC
Trinity P1
Sodium
1.3 ng
0.9 ng
Chloride
1.3 ng
0.2 ng
Figure 6. Diclofenac sodium salt, 1.3 µg on column.
Limits of detection using the methods described in Figures 2 and 3. Limit
defined as signal-to-noise > 3.
3
New Approaches for Simultaneous API and Counterion Analysis
Using Charged Aerosol Detection
Column:
Acclaim Trinity P1,
3.0 × 50 mm, 3 µm
Mobile Phase: Acetonitrile/120 mM
ammonium acetate (75:25) pH = 4.8
(30 mM total buffer strength)
Flow Rate:
0.8 mL/min
10.5
Discussion
Samples: A) Diclofenac sodium unspiked
B) Spiked with 0.1% (w/w) chloride
C) Spiked with 0.15% (w/w) chloride
Peaks:
1. Sodium
2. Chloride
3. Diclofenac
1
The two columns evaluated (ZIC-pHILIC and Trinity P1) both provide
the ability to simultaneously resolve anions and cations as shown in
Figures 2–4. These columns use very different packing materials for
their separations, which lead to different advantages depending on
application requirements.
3
2
C
pA
B
A
0.0
–5.0
0.01
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
27137
Figure 9. Three injections of diclofenac sodium salt, 5.7 µg on column.
Both columns were able to provide good reproducibility data (e.g.,
Table 1, ZIC data; Trinity data not shown) with large dynamic ranges
(e.g., Figure 5, Trinity data; ZIC data not shown).
Table 3. Area Values and Deviation
for Chloride in Diclofenac
Sample
Diclofenac Standard
Diclofenac + 0.1% Cl–
Diclofenac + 0.15% Cl
–
Raw Area
(pA *min)
RSD
(n = 5)
Calculated Chloride
(w/w)
0.0231
13.01%
0.03%
0.1069
2.39
0.14%
0.1318
1.49%
0.17%
Average of the chloride raw area values and the corresponding deviation along
with the calculated weight percent of chloride in dicolfenac sodium samples run as
described in Figure 9.
Column:
ZIC-p H ILIC
Mobile Phase: A) Acetonitrile, isopropanol,100 mM ammonium acetate, pH = 4.68,
methanol (60:20:15:5)
B) 30 mM Ammonium acetate pH = 4.68, acetonitrile, isopropanol,
methanol (50:25:20:5)
Flow Rate:
0.5 mL/min
Inj. Volume:
10 µL
Gradient:
Time (min)
0
3
24
26
28
29
32
%B
20
20
70
70
15
20
20
Peaks:
1. Diclofenac
2. Chloride
3
1
3. Sodium
2
10
0
5
10
Minutes
15
20
25
27138
Figure 10. Chromatogram of a 10 µL injection of diclofenac sodium salt
0.3 µg in acetonitrile/water (80:20) with 0.1% w/w of chloride using ZIC-pHILIC
column and the method described in Figure 3.
4
The focus of the work presented here was the analysis of an API and
counterion on a single platform. This can be achieved using either
columns as demonstrated in Figures 6–7. In these examples, compound
specificity is reversed for the different columns. The API (diclofenac)
is more retained on the Trinity column and the sodium peak elutes
earlier. However, on the ZIC-pHILIC column, the diclofenac elutes very
quickly, close to the void, and the sodium is more retained. Because
the ZIC-pHILIC column uses a HILIC approach, the initial mobile phase
composition needs to contain a high level of organic content, generally
> 70%. For APIs that do not contain strong polar groups and are
retained primarily by reversed-phase properties, the high organic mobile
phase can lead to peaks eluting in or close to the void volume.This
makes the analysis of hydrophobic APIs on the ZIC column difficult.
Figure 8 illustrates the seperation of another common API, naproxen,
and its counterion sodium using the Trinity column. Baseline resolution
can be obtained in less then 1 min. A simple change in the pH from 3.8
to 5.2 determines the elution order of the two major components. One of
the major advantages found with the Trinity column was its ability to run
fast isocratic methods. This resulted in better peak shape and sharper
peaks which leads to lower limits of detection on the Corona ultra, as
shown in Table 2.
mV
40
The ZIC-pHILIC column was found to operate best at a flow rate of
0.5 mL/min with a gradient method. It is possible to run the column
isocratically at flow rates up 1 mL/min. However, long-term stability
was compromised with a loss in peak shape and resolution. This was
possibly due to build-up of trace ionic materials which do not fully elute
under weak isocratic conditions.
Using the improved chromatography discussed here, an experiment
was conducted to determine whether a significant difference between
a diclofenac sodium sample spiked with 0.1% versus a sample spiked
with 0.15% (w/w) chloride could be identified. Figure 9 is an overlay
of chromatograms at each corresponding level. As shown in Table 3,
the calculation of the chloride content with a four-point linear regression curve from 3 to 25 ng chloride on column yielded recoveries of
100 ± 10% of the expected values. The method was sensitive enough to
measure a trace amount of chloride contamination in the injection of the
unspiked standard.
New Approaches for Simultaneous API and Counterion Analysis
Using Charged Aerosol Detection
Conclusion
References
• Analysis using CAD and either the Acclaim Trinity P1 or the
SeQuant ZIC-pHILIC column provided sensitive and reproducible
results for the determination of API counterions.
• The HILIC chemistry and column packing material for the
ZIC-pHILIC column made it necessary to use longer gradient
runs to achieve optimal performance. The need to run under HILIC
conditions make it unsuitable for analyzing hydrophobic APIs.
• The mixed-mode, Nanopolymer Silica Hybrid (NSD) technology
employed in the Trinity P1 column allowed for fast isocratic API
counterion analysis required when screening counterions and in
QC functions.
1. Kumar, L.; Amin, A.; Bansal, A.K. Salt Selection in Drug Development, Pharm Tech. 2008,3 (32), 128–146.
2. Haynes, D.A.; Jones, W., Samuel Motherwell, W.D. Occurrence of
Pharmaceutically Acceptable Anions and Cations in the Cambridge
Structural Database. J. Pharm. Sci. 2005, 94, 2111–2120.
3. Merck website on ZIC-HILIC column http://www.sequant.com.
Acknowledgements
ESA is grateful to Dr. Xiaodong Liu (Dionex) for providing the data on
gradient analysis using the Trinity P1 column.
Trinity and ultra are trademarks and Acclaim, CAD, and Corona are registered trademarks of Dionex Corporation.
ZIC is a registered trademark of Merck SeQuant.
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5
New Approaches for Simultaneous API and Counterion Analysis
©2010 Dionex Corporation
www.dionex.com
Using Charged Aerosol Detection