An Evaluation of a Multi-Mode Stationary Phase for
HPLC Separations Using a Multi-Component Test Mix
Denise Logue1, Dave Lugo1, Cesar Ramirez-Molina1 and Dave Mallett2
1Translational Discovery, EpiNova DPU, II CEDD, GSK & 2R CEDD DMPK, GSK

Introduction

Results & Discussion

Within Translational Discovery, rapid sample analysis is paramount, withminimal time available for method development. To aid the turnaround, theuse of a generic HPLC column applied to multiple applications isadvantageous. The suitability of different HPLC columns for genericpp
can be comparedpusingg a standard test mix.
applications

A 5 minute chromatographic method was selected for the separation of thetest mix on the 5cm Scherzo SM-C18TM column (Figure 1). The test mix wasthen analysed on an Ascentis Express C18 column (Figure 2), and an Agilent
Zorbax SB-CN column (Figure 3). In each figure, 10 chromatograms havebeen overlayed to highlight the reproducibility of the separation method. Itwas observed that separation of the 10 compounds was possible using theScherzo SM-C18. However, when using a standard C18 column the 10compounds were not fully resolved.

C18 columns used in reverse phase chromatography are largely limited tonon-polar compounds; this can be problematic for drug metabolites as theseare often ionic. A mixture containing both acidic and basic analytes will often
require different columns and/or gradients for separation.1
A novel stationary phase has been proposed for this type of separation. TheScherzo SM
SM-C18
C18TM column1 is claimed to be the “World’s
World s first multi
multi-mode
mode
octadecyl silane (ODS) column.” Containing ionic, cationic and ODSligands, the column is designed to perform in normal and reverse phasemodes in addition to the separation of ionic analytes.1

1. Nicotinic Acid
2. Tryptophan
3. Bromoguanosine
4. Labetalol
5. Aspirin
6. Fexofenadine
7. Reserpine
8. SB243213A
9 Ibuprofen
9.
10. Penicillin

Time0.0

Figure 1: 10 overlayed
chromatograms of a typical test
mix separation on the 5cm
Scherzo SM-C18 column%B00.8

5

3.8

100

4.01004.20

5.00

Separation of all 10 compounds
using the Scherzo SM-C18TMcolumn. Note: nicotinic acid isnot well resolved from thesolvent front.

18Objective

76
954

2

evaluation of the applicability of the Scherzo SM
SM-C18
C18TM for generic HPLC
separations in a DMPK laboratory.

Methods
A pool of 30 common test mix compounds were preselected from literaturedata. From this pool, 10 representative compounds were selected to form
the final test mix (Table 1), based on their chemical properties.

1. Solvent Front/Nicotinic Acid
2. Tryptophan
3. Bromoguanosine
4/5. Aspirin/Labetalol
6. Fexofenadine
7. Reserpine
10. Penicillin
8. SB243213
9. Ibuprofen

1

Table 1: Chemical Properties of the test mix

CompoundNicotinic Acid
Tryptophan
Bromoguanosine
Labetalol
Aspirin
Fexofenadine
Reserpine
SB243213A
Ibuprofen
Penicillin V

4/5

A/B Impurities(Identified in stock
solutions of multiple
compounds)
TimeAcid
Zwitterion
Base
Zwitterion
Acid
Zwitterion
Base
Base
Weak Acid
Zwitterion

0.000.8

5

3.8

100

4.0

100

4.2

0

5.00

7
926B

A

3

H2O + 0.1% (v/v) formicacid (pH 2)

MeCN + 0.1% (v/v)
formic acid (pH 5)

Buffer Only

H2O + 10mM NH4COOH(pH 6)

90% MeCN (aq) + 10mM
NH4COOH (pH 6)

Buffer + Acid

H2O + 10mM NH4COOH +0.1% ((v/v)) formic acid(pH 3)

90% MeCN (aq) + 10mM
NH4COOH + 0.1% ((v/v))
formic acid (pH 4)

No Additives

H2O Only (pH 6)

MeCN Only (pH 5)

Results Observed
Best overall separation – one peak
present for each compound.
Poor resolution of nicotinic acid fromsolvent front. Co-elution of SB243213A,
penicillin & ibuprofen. Peak broadeningobserved.
Poor resolution of nicotinic acid fromsolvent front. Co-elution of SB243213A,
penicillin & ibuprofen.
Compounds elute in same order as
observed for generic mobile phaseconditions. Poor resolution of nicotinicacid from solvent front.

Figure 2: 10 overlayed
chromatograms of a typical test
mix separation on the 5cm
Ascentis Express C18 column

1. Nicotinic Acid
2. Tryptophan
3. Bromoguanosine
4. Labetalol
5. Aspirin
6. Fexofenadine
7. Reserpine
8. SB243213
9. Ibuprofen
10.Penicillin

72

Separation of the 10compounds on the standard
C18 column used in the DMPK
laboratory. Note: All 10compounds are not fullyresolved.

18Time
0.0%B
00.85

6.090

6.490

7.40909.005310469Figure 4: A typical test mix separation on the 10cm Scherzo SM-C18 column using a 9 minute gradient2. Tryptophan
3. Bromoguanosine
5. Aspirin
4. Labetalol (Split Peak)
6/10. Fexofenadine/Penicillin
7/8. Reserpine/SB243213A
9. Ibuprofen

Separation of the test mix was optimised on a Waters Acquity UPLCTM
system coupled to a photodiode array UV Detector. Data was captured inthe 210 – 350nm range. A generic 1 minute separation method wasinitially used, with the 10 test mix compounds being added sequentially tothe analyte mixture. The gradient was modified as necessary to achieve
the optimum resolution for all 10 compounds.

7/8
6/10
9(Mobile phase A was HPLC water containing 0.1% (v/v) formic acid;
Mobile phase B was acetonitrile containing 0.1% (v/v) formic acid).

%B

100.4
-1.3
-2.27
2.7
1.4
5.6
3.2
5.3
3.6
1.4

Generic (Acid)

8MW (g) Classification cLogP
123.1
203.2
362.14328.4
180.2
501.66608.7
428.42204.1350.39

Table 2: Summary of Experiments Conducted Under Different Mobile Phase Conditions

Conditions Mobile Phase A Mobile Phase B

Additional work on a 10cm Scherzo SM-C18TM column was carried out. As
initial data looked promising, the HPLC method was optimised and extended
to 9 minutes and separation of the 10 compounds was improved (Figure 4).
Nicotinic acid had been separated from the solvent front, compared to the5cm Scherzo SM-C18TM column (Figure 1 vs. Figure 4).

103• To develop a chemically diverse test mix; and utilise this tool in theMobile phase composition was investigated by using different combinationsof ammonium formate and formic acid additives. The results of theseexperiments are summarised in Table 2. No significant improvements weremade to the original separation, therefore, all further experiments wereperformed under generic mobile phase conditions.

5243

References:
1. Imtakt Technical Report TR03A, ‘The world’s first multi-mode ODS column’, Imtakt Corp, Japan, 2009

Time
0.0%B
00.8

53.8

100

4.01004.205.00Figure 3: 10 overlayed
chromatograms of a typical test
mix separation on the 5cmAgilent Zorbax SB-CN column
A typical cyano column is notcapable of separating all 10compounds under thesegeneric separation conditions.

Conclusions & Further Work
Separation of the test mix was optimised on the 5cm and 10cm Scherzo SMC18TM columns, using a generic gradient and typical DMPK mobile phases.
The SM-C18TM is more effective in separating a mixture of analytechemistries, than a standard C18 column and a cyano column. This has thepotential for rapidpp analysis
yof samples
pwhere compounds
pwith different
physicochemical properties are quantified in addition to the analyte ofinterest. Work is currently ongoing to test the column robustness by LCMS/MS analysis of the test mix in blood, and to identify further possible drug
metabolism applications for the Scherzo SM-C18TM column.
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