Improving the Analysis of Herbal Medicinal Products by Fused Core™ particle technology and evaluation of different

Liquid Chromatography Stationary Phase ChemistriesDr. Frank Michel

frank.michel@sial.com

sigma-aldrich.com

AOAC Symposium „Quality Control of Botanicals, TCM, Herbal Food Supplements

and Herbal Medicinal Products“,Nürnberg, 10.10. – 12.10.2011

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Content

•Herbal Medicinal Products – what are the challenges?•Fused Core technology•Selectivity as best approach to solve separation challenges

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Challenges in QC, in-process control and stability testing of Herbal Medicinal Products

Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003370.pdf). Guideline on declaration of herbal substances and herbal preparations in herbal medicinal products/traditional herbal medicinal products (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003272.pdf).

Constituent with known therapeutic activityConstituents which are generally accepted to contribute substantially to the therapeutic activityActive MarkerGenerally accepted to contribute to the therapeutic activity but are not responsible for the full therapeutic effectAnalytical MarkerCharacteristic constituents that serve sole analytical purposes

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Minutes0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00

Challenges in QC, in-process control and stability testing of Herbal Medicinal Products

Osthol

0 20 40 60 80 90Time [min]

Angelica root extract

Stat. Phase:Reprosil-Pur ODS-3, 150 x 4,6 mm, 3 µmMob. Phase: Wasser/TFA/ACN/MeOH,GradientFlow rate:1,0 ml/minTemperature:45 °CDetection:323 nmHPLC system:Waters Alliance 2695

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Challenges in QC, in-process control and stability testing of Herbal Medicinal Products

Herbal Medicinal Product Acontaining Angelica root extract

0 20 40 60 80 90Time [min]

Stat. Phase:Reprosil-Pur ODS-3, 150 x 4,6 mm, 3 µmMob. Phase: Wasser/TFA/ACN/MeOH,GradientFlow rate:1,0 ml/minTemperature:45 °CDetection:323 nmHPLC system:Waters Alliance 2695

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Improvements in analysis of Herbal MedicinalProducts by Fused CoreTM technology

silib

in a

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,120

silib

in b

- 30

,812

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0,030

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0,060

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0,080

0,00 5,00 10,00 15,00 20,00 25,00 30,00

Herbal MedicinalProduct B

Stat. Phase:Ascentis Express C18,100 x 2.1 mm (2.7 µm)Mob. Phase: Water/TFA/ACN/MeOH,GradientFlow rate:0,4 ml/minTemp.: Det.:30 °C 287 nmHPLC system:Waters Acquity UPLC

0 10 20 30Time [min]

• D. Steinmann, M. Ganzera, J PharmBiomed Analysis, 55/4 (2011) 744-757

• E.R. Badman et al., J. Chromatogr. B 878 (2010) 2307–2313

• A. Abrahim et al., Journal of Pharmaceuticaland Biomedical Analysis 51 (2010) 131–137

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•Innovative approach in HPLC by Dr. J. J. Kirkland in 2006•Porous silica with high capacity on solid, non-porous core•Highly pure silica • 2.7 µm silica particle

• 1.7 µm solid core

• 0.5 µm porous SiO2 layer

• 90 Å pore size

• Very narrow particle size distribution

• C18, C8, HILIC, RP-Amide, Phenyl-Hexyl, Penta-fluorophenyl, Peptide

0.5 µm

1.7 µm2.7 µm

Fused-Core technology

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•Innovative approach in HPLC by Dr. J. J. Kirkland in 2006•Porous silica with high capacity on solid, non-porous core•Highly pure silica • 2.7 µm silica particle

• 1.7 µm solid core

• 0.5 µm porous SiO2 layer

• 90 Å pore size

• Very narrow particle size distribution

• C18, C8, HILIC, RP-Amide, Phenyl-Hexyl, Penta-fluorophenyl, Peptide

0.5 µm

1.7 µm2.7 µm

Fused-Core technology

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Fused-Core technology provides higher efficiency

0.5 µm

1.7 µm2.7 µm

Diffusionpathway 1.5 µm

The shorter diffusion pathway facilitates the mass transfer!

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-500

0

500

1000

1500

2000

2500

3000

1 2 3 4 5 6 7

Particle Diameter, µm

Am

ount

Fused-Core particle size distribution Average = 2.77 µm; standard deviation = 6% of mean

Particle size distribution of a typical commercial totally porous packingAverage = 3.78 µm; standard deviation = 19% of mean

Fused-Core technology provides higher efficiency

Narrow particlesize distribution!

•Bed uniformity (better A term)•Larger frits (column lifetime)

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HET

P (

m)

1 2 3 4 5

35.00

30.00

25.00

20.00

15.00

10.00

5.00

2 4 6 8 10 12

16,000

14,000

12,000

10,000

8,000

6,000

4,000

2,000Pr

essu

re (p

si)

2.7 µm Ascentis Express

H = A + B/u + Cu P =1000FLr2dp

2P =

3 µm

1.7 µm

2.7 µm FC

*50x4.6 mm columns, 55/45 ACN/water

1-2 mL/min

Linear velocity of the mobile phase (mm/sec)

Comparison of pressure and efficiency

Linear velocity of the mobile phase (mm/sec)

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Three Factors Control HPLC and UHPLC Resolution

Rs = kk+1

α-1α4

• •

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5K 10K 15K 20K 25K

0 5 10 15 20 25

N

k

Nk

Res

olut

ion

(R)

Zhao, J.H., P.W. Carr., Anal. Chem. 71(14) (1999) 2623-2632

k = (tR – t0)/t0

α = k2/k1

N = 16 (tR/w)2 or

N = 5.5 (tR/w)2

All factors are important, but selectivity is considered the most powerful term.

N

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Selectivity Variables in Reversed-Phase *

•Continuous variables (solvent):• type (organic, water)• pH (especially ionizable solutes)• additives (type and concentration)• temperature• solvent strength

•Discontinuous variable (column):• Stat. type (phase and substrate)

* Excerpted with permission from John Dolan, 2009 Minnesota Chromatography Forum Spring Symposium; adapted by R. Henry.

More predictable(modeling software available)

Less predictable(screening required)

Some analysts may spend too much time here “force feeding” C18 columns.

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RP-Amide

C18

Phenyl

1. Butylbenzene2. o-Terphenyl3. Triphenylene, 4. Amylbenzene

F5

0 2 4 6 8 10 12 14 16 18

24 3

1

21

3 4

4

21

3

21 3

4

2134

C8

Col. Dim.: 15x4.6 5um65% ACN/35% Water1.5mL/min, 220nm, 5uL

Van der Waals interactions are the main interaction responsible for the separation of hydrophobic molecules or moieties. Amylbenzene retention is directly related to the strength of the van der waals interactions of a phase. Even with strictly hydrocarbon molecules, there are selectivity variations based on such phenomena as shape selectivity.

Retentivity

Selectivity by different stationary phases

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Alkyl Bonded Phases (C18 and C8)

• C18 reagents are large and can leave some silanols unreacted (ca. 50%).

• C8 reagents are smaller and provide better silanol coverage

• At pH >4, silanols can ionize and add cation-exchange character.

OH O

Si

O

Si

OH

R

Free silanol

“Endcapping” reagent

Primary phase reagent

Hydrophobic selectivity

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Alkyl Amide Bonded Phases (abbreviated: RP-Amide)

• Possible Solute Shielding(basic solutes)- alkyl phases with embedded polar groups (EPG) have been reported to correlate much better with logP data than C18 columns due their higher base deactivation1.

• Possible H-bonding with solutes that are good H-bond donors (acids, etc.)

OH O

Si

O

Si

OH

NC=O

RPrimary phase reagent

Endcappingreagent

Amide carbonyl is also a strong H-bond acceptor

Free silanol

Shielding and H-bonding

Hydrophobic

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Phenyl Bonded Phases

OH O

Si

O

Si

OH

Free silanol

“Endcapping” reagent

Primary phase reagent

• Phenyl is a Lewis base or electron donor; π-πinteraction can occur with solutes that are deficient in electrons (Lewis acids).

• Due to the rigid nature of the aromatic ring, solute shape can dictate selectivity (how closely solutes can approach the ring).

π base

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Pentafluorophenyl (F5) Bonded Phases

OH O

Si

O

Si

OH

Free silanol

“Endcapping” reagent

Primary phase reagent

F

F

F

F

F• PFP is a Lewis acid or

electron acceptor; π-πinteraction can occur with solutes that are rich in electrons (Lewis bases).

• Due to the rigid aromatic ring, solute shape can dictate selectivity.

• Dipolar (possible H-bond) interactions can occur

π acid and dipole (H-bond)

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Classification by Possible Chemical Interactionsa

ModerateStrong (Rigid)

Strong AcceptorStrongModerate

AcceptorModerateF5 or PFP

Very weakWeakNoModerateStrong AcceptorStrongRP-

Amide

ModerateNoWeakStrongWeak Acceptor

Light to ModerateCyano

WeakStrong (Rigid)

Strong DonorWeakWeak

AcceptorStrongPhenyl

WeakNoNoNoWeakStrongC8

ModerateNoNoNoWeakVery StrongC18

IonicbStericbπ-πDipolarH-BondingHydrophobicBondedPhase

a. Using Euerby2 variation of Snyder-Dolan-Carr Hydrophobic Subtraction Model3.

b. Steric and Ionic probe data are not very helpful in predicting or interpreting steroid selectivity results; however, they are always underlying factors with silica bonded phases.

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Columns with same or similar phases should show high correlation: C18 vs C8

R2 = 0.97

13mM ammonium acetate, 30:70 H20:ACN, pH 6.91,50 solutes (acid, base, neutral)

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Columns with different phases should show orthogonality: C18 vs F5 (PFP)

R2 = 0.63

13mM ammonium acetate, 30:70 H20:ACN, pH 6.91,50 solutes (acid, base, neutral)

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Experimental Design

•Five different stationary phases on Fused Core particles• Ascentis Express C18• Ascentis Express C8• Ascentis Express RP-Amide• Ascentis Express F5 (Pentafluorophenyl)• Ascentis Express Phenyl-Hexyl

•have been tested with two mobile phases consisting of either• Methanol/water• Acetonitrile/water

•as organic modifier on different herbal drugs such as Withania, Panax Ginseng and Milk Thistle.

237.0 8.0 9.0 10.0

Time (min)

10.0 11.0Time (min)

Whitania potential marker compounds, C18

methanol/waterpeak qty = 7min resl = 1.28

acetonitrile/waterpeak qty = 7min resl = 0.76

•Withanone•Withanolide A•Withanolide B•Withaferin A•12-Deoxywithastra-monolide•Withanoside IV•Withanoside V

Ascentis Express C18

246.0 7.0 8.0 9.0

Time (min)

10.0 11.0 12.0Time (min)

Whitania potential marker compounds, PFPmethanol/waterpeak qty = 6min resl = 1.40

acetonitrile/waterpeak qty = 7min resl = 2.76

•Withanone•Withanolide A•Withanolide B•Withaferin A•12-Deoxywithastra-monolide•Withanoside IV•Withanoside V

Ascentis Express F5

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Withania results –Sorted by peak quantity then min. resolution

phase organic peaks min reslF5 acetonitrile 7 2,76Phenyl-Hexyl acetonitrile 7 2,01Phenyl-Hexyl methanol 7 1,36C18 methanol 7 1,28C8 acetonitrile 7 0,98C18 acetonitrile 7 0,76RP-Amide acetonitrile 7 0,46C8 methanol 6 2,55F5 methanol 6 1,40RP-Amide methanol 5 0,90

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Panax ginseng results –Sorted by peak quantity then min. resolution

phase organic peak qty min reslC18 methanol 7 0,99C8 methanol 7 0,94Phenyl-Hexyl methanol 7 0,77F5 methanol 7 0,49RP-Amide methanol 6 1,63RP-Amide acetonitrile 6 1,39C8 acetonitrile 6 1,24C18 acetonitrile 6 0,71F5 acetonitrile 6 0,58Phenyl-Hexyl acetonitrile 5 1,94

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Extract of Chinese Panax Ginseng

Column: Ascentis Express C18, 150 x 4.6 mmMob. Phase A: water Mob. Phase B: acetonitrileGradient: 0-1.5 min at 25% B, 1.5-13.5 min to 85% B, 13.5-14.5 min at 85% BFlow rate: 1.5 mL/min Temp.: 60 oCDet.: 205 nm Injection: 10 µLSample: extract of Chinese Ginseng with water/ethanol (50:50, v/v)

Gin

seno

side

Re

Gin

seno

side

Rg1

Gin

seno

side

Rd

Gin

seno

side

Rf

Gin

seno

side

Rc

Gin

seno

side

Rb2

Gin

seno

side

Rb1

0 2Time (min)

64 8

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Milk Thistle (Silybum marianum) results –Sorted by peak quantity then min. resolution

phase organic peak qty min reslC18 methanol 9 -F5 methanol 8 -C18 acetonitrile 8 -Phenyl-Hexyl methanol 7 -Phenyl-Hexyl acetonitrile 7 -F5 acetonitrile 7 -C8 methanol 7 -C8 acetonitrile 7 -RP-Amide methanol 6 -RP-Amide acetonitrile 6 -

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Milk Thistle (Silybum marianum) –Markers (red) and Herbal Medicinal Product (black)

0 10 20Time (min)

taxi

folin

sily

chris

tinap

igen

7-gl

ucos

ide

sily

dian

in

quer

citri

n

silib

inA

silib

inB

isos

ilibin

Ais

osilib

inB

Milk Thistle Xtra 250 mg, extracted w/ water:ethanol 50:50

Column: AscentisExpress C18, 100 x 3 mmA: MeOHB: Water + 0,1 % FAGradient: 0-3 min at 35% B, 3-13 min to 45% B, 13-15 min at 45% BFlow rate: 0.6 mL/min. Temp.: 35 oCDet.: 254 nm

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Conclusions

•Fused Core particle technology provides a good base for separation of highly complex mixtures

•Selectivity by initial screening of orthogonal stationary and mobile phases results highest resolution• Selectivity is most strongly impacted by different

stationary phases•The approach of initial column screening provides fast and reliable separations

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Thank you!

•Dave S. Bell•Hugh Cramer

•Richard A. Henry