Taller Ucimed Resumen

2013 Waters Corporation 1

Avances en cromatografa lquida

Teora y prctica

para el anlisis farmacutico

Oscar Cortes Ledezma, Qumico

[email protected]

Adriana Murillo Ramrez, Qumica.

[email protected]


AGRADECIMIENTOS

Dr Jorge Lpez Mora (UCIMED)

Dr Gustavo Senz Garca (UCIMED)

Dra Marisol Flores Campos (UCIMED)

Msc. Jens Voldum (Scanco Tecnologa)

Q.F. Ricardo Martinez (Waters Corporation)

Ing. Jeffry Aparicio (Scanco Tecnologa)

Licda. Vanessa Vagnarello


Contenido

1. Regulaciones y requerimientos de validacin de mtodos

cromatogrficos.

1. Parmetros cromatogrficos y su optimizacin.

2. Seleccin de columnas.

3. Efecto de la fase mvil en el cromatograma.


Regulaciones y requerimientos


"System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done.

The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.

Un SST debe contener:

Para un ensayo:

precisin + otro parmetro mas

Para ensayos de impurezas:

resolucin + precisin + otro parmetro ms

System suitability


Categoras USP

Categora 1: Cuantificacin de componentes

mayoritarios o ingredients activos.

Categora 2: Determinacin de impurezas o

productos de degradacin.

Categora 3: Determinacin de caractersticas de

desempeo.


USP Data Elements Required For Assay Validation

Analytical

Performance

Parameter

Assay Category 1

Assay Category 2Assay

Category 3Quantitative Limit Tests

Accuracy Yes Yes * *

Precision Yes Yes No Yes

Specificity Yes Yes Yes *

LOD No No Yes *

LOQ No Yes No *

Linearity Yes Yes No *

Range Yes Yes * *

Ruggedness Yes Yes Yes Yes

* May be required, depending on the nature of the specific test.


Specificity-Analytical Parameter

Lack of specifity of an analytical proceduremaybe compensated for by other analyticalprocedure

For example, by MS


Parmetros cromatogrficos


Valores lmite segn la USP


K = Capacity FactorA Measure of Retention

1.5

.5 - 1 Vfor k'

1

5.5

.5 - 3 Vfor k' 2

V0

V1

V2V3

TIME .5 1 3 4 6

kn = (tn-t0) / t0

kn = (Vn-V0) / V0


= SelectivityA Measure of Peak Separation

V0

V1

V2V3

TIME .5 1 3 4 6

0V

1V

0V

2V

or

1 Vof k'

2 Vof k'

1.4.5 - 3

.5 - 4 Vfor 3


RS = Resolution

2,2/11,2/1

RR

21

RR

S

12

12

18.1

)(2

1

hh WW

tt

WW

ttR

tR1 tR2

W1 W2

W1/2h,1

W1/2h,2

h1/2))((

1k

k1

4

NRs


Theoretical Plate Number, N

W

W1/2H1/2

H

2

.21

R

R

/

R

W

t

W

2

2

2

545

16

Area

Ht

tN


Impact of Instrument Band Spreading on Small Particle Column Performance

Minimize band spreading from the instrument and the column to improve chromatographic performance

1.7 m on an HPLC 1.7 m on a UPLC


0.020"0.003" 0.040"

Shortest length is best

Smallest

ID is best

Flow Direction

Tubing with a large ID increases the overall system volume and contributes to band spreading

Excessive tubing lengths contribute to band spreading

Decreasing tubing ID and increasing length increases system pressure

System Band Spreading:Effect of Tubing ID and Length


Small Particles Necessitate Low Band Spread Instruments

Band spread is a measure of system dispersionLow band spread ensures separated peaks stay separated

System Band Spread (L) 5 sigma

Shimadzu UFLC 41

Alliance HPLC 29

1260 Infinity 6 L/5 mm bore FC 28

1290 Infinity 1 L/10 mm MaxLight FC (dual column) 23

Original Accela 21

1290 Infinity 1 L/10 mm MaxLight FC (single column) 20

Dionex Ultimate 3000 2.5 L/7 mm FC 17

1260 Infinity 2 L/3 mm bore FC 16

ACQUITY UPLC H-Class with Column Manager 12

ACQUITY UPLC H-Class with Column Heater 9


UPLC Technology & The Fundamental Resolution Equation

))((1k

k1

4

NRs

NRs

Physical Chemical

dp 3X, If N 3x, Rs 1.7x

Sensitivity 1.7X


Constant Column LengthFlow Rate Proportional to Particle Size

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00

4.8 m, 0.2 mL/min, 354 psi

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Theory1.7X Resolution3X Faster1.7X Sensitivity25X Pressure

Reality1.5X Resolution2.6X Faster1.4X Sensitivity22X Pressure

1.7 m, 0.6 mL/min, 7656 psi

2.1 x 50 mm columns Very High System Pressure


Ultra Performance LCTM

Velocidad incrementa volumen de muestras

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

1.

Th

iou

rea -

0.4

30

2.

tolu

en

e -

1.0

34

3.

pro

pylb

en

zen

e -

1.7

42

4.

bu

tylb

en

zen

e -

2.4

13

5.

hexylb

en

zen

e -

5.0

58

No. de componentes: 5

Completa separacin: 6.00 min0.18

0.20

0.22

0.24

1.

Thio

ure

a -

0.0

46

2.

tolu

ene -

0.0

88

3.

pro

pylb

enzene -

0.1

37

4.

buty

lbenzene -

0.1

82

5.

hexylb

enzene -

0.3

60 UPLCTM HPLC

AU

0.00

0.10

0.20

Minutes0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

UPLC

No. de componentes: 5

Completa separacin: : 0.60 min

UPLCTM incremento de velocidad por arriba de 9X


USP Chromatographydefines Allowable adjustments

Adjustments to a USP method may be made to meet system suitability

requirements

Verification tests must be performed after changes

Full re-validation not required

Must use the same L-designation of column

Isocratic hold or dwell volume adjustments are allowed

USP 37 NF 32 S1

- to be official Aug. 1, 2014

- significant changes to Chapter Chromatography


Changes to Effective August 1, 2014

Current (USP36-NF31)

isocratic & gradient isocratic gradient

Particle Size -50%

Column Length 70%

Flow Rate 50% *based on particle size and 50% no changes allowed

Column ID Any allowed Any allowed no changes allowed

Injection Volume Any reduction Any allowed Any allowed

Column Temperature 10% 10% 10%

Mobile Phase pH 0.2 unit 0.2 unit 0.2 unit

VariableAfter Aug 1, 2014 (USP37-NF32 S1)

Allowable Adjustments in USP Chromatography

per constant L/dp or N: -25% to +50% no changes allowed

Flow rate (isocratic): F2 = F1 x [(dc22 x dp1)/(dc1

2 x dp2)]

L/dp = column length (L) to particle size (dp) ratioN = theoretical plate count (for solid-core particles)


30

mm

30

mm

30

mm 30

mm

50

mm

50

mm 50

mm

50

mm

75

mm 75

mm

75

mm

75

mm

10

0 m

m 10

0 m

m

10

0 m

m

10

0 m

m

15

0 m

m

15

0 m

m

15

0 m

m

15

0 m

m

25

0 m

m

25

0 m

m

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

0.005 0.0035 0.0025 0.0017

L/d

p R

atio

particle size (m)

1.7 2.5 3.5 5.0

Moderately Challenging

Easy

ExtremelyDifficult

Difficult

Ratio of Column Length to Particle SizeResolution Capability

50 mm, 1.7 m75 mm, 2.5 m100 mm, 3.5 m150 mm, 5 m

L/dp ~ 30,000


Improve HPLC Productivity Today:Directly Scalable to UPLC Technology

XP 2.5 m


Introducing CORTECS 2.7 m Columns

CORTECS 2.7 m solid-core particles demonstrate an efficiency equivalent to that of 2.2 m fully porous particles and a backpressure equivalent to that of 3.1 m fully porous particles:

CORTECS Particle Size

Equivalent Porous Particles

Efficiency Backpressure

1.6 m 1.3 m 1.8 m

2.7 m 2.2 m 3.1 m

2.22

m 3.1 m2.7 m

Same Efficiency

Same back pressure


High Efficiency at HPLC Backpressures

AU

0.00

0.02

0.04

0.06

0.08

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

1

2 34

Rs 4.9

Rs 6.3

Rs 8.2

Fully Porous C18, 5 mPsi: 1900N peak 4: 10,400

Fully Porous C18, 3.5 mPsi: 2900N peak 4: 17,600

CORTECS C18, 2.7 mPsi: 3200N peak 4: 23,600

1. Estradiol2. Ethinyl estradiol3. Estrone4. Levonorgestrel

Configuration: 4.6 x 150 mm


Seleccin de columnas


Stationary Phase Selectivity:Bonded-Phase [Ligand] and Particle Substrate

Silanol activity and surface charge

Secondary interactions

Affects peak shape and sample loadability

Hydrophobicity

Longer alkyl chain lengths will provide increased retention

Shorter, ionizable ligands will increase polarity

Ligand density

Influences retention and sample loadability

Hydrolytic stability

the number of attachment points to the particle surface can impact

column lifetime


Surface of a Silica Gel Bonded-Phase Packing Material

High Coverage

High LigandDensity

Low Coverage

Low LigandDensity

H

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

OSi

O

O

O

SiO

OO

SiO

O

O

Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

Si

CH3H3C CH3 Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

SiO

O

O

Si

CH3H3C CH3 Si

CH3H3C CH3

HHHHHH H

C8 alkyl chains

Residual silanolsEndcap

Polar analytes are not able to energetically fit between

ligands cant interact with surface or ligands

Si

SiO

O

OSi

O

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

OSi

O

O

O

SiO

OO

SiO

O

O

Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H3C

SiO

O

O

Si

CH3H3C CH3 i

CH3H3C CH3

HHHHHH H

Residual silanol

Endcap

Polar analytes easily interact with surface

and ligands


SiC

CC

CC

CC

CH3

CH3

H3C

OSi

O

O

O

Hydrolysis of a Bonded Phase Material: Monofunctional Ligand

+ HCl

OHSi

O

O

O+

SiC

CC

CC

CC

CH3

CH3

H3C

Cl

OHSi

O

O

O+ Si

CC

CC

CC

CCH3

CH3

H3C

HO

Low pH (hydrolysis of ligand)


Making a Bonded Phase Material: Multifunctional Synthesis

+

+ HCl

C8 Trichlorosilane Ligand

Multi-Point Attachment

ClSi

Cl

ClCH3

OHOH

OHSi

SiSi

O

O

O

O

O O

O

O

Si

O

OHSi

SiSi

CH3

O

O

O

O

O O

O

OH


Effects of High pH Mobile Phases

Surface Modified Silica Particles Hybrid Particles

Complete dissolution of Silica Catastrophic column failure Short lifetimes

Slow rate of surface dissolution Incorporated methyl groups uncovered slows rate of dissolution Longer column lifetimes


Reversed-PhaseColumn Selectivity Chart

(ln [k] acenaphthene)

SunFire C18

YMC-Pack PolymerC18

Hypersil CPS Cyano

YMC-Pack CN

Waters Spherisorb S5 P

Hypersil BDS Phenyl

Nova-Pak Phenyl

YMC-Pack

Phenyl

Hypersil Phenyl

Inertsil Ph-3

YMC-Pack Pro C4

YMCbasic

Symmetry C8

YMC-Pack Pro C8

Nova-Pak

C8

XTerra MS C18Symmetry C18

YMC-Pack Pro C18 Inertsil ODS-3

YMC-Pack ODS-A

Nova-Pak

C18

YMC J'sphere

ODSL80 Nucleosil C18

Waters Spherisorb ODS2

Waters Spherisorb ODS1

Resolve C18

Bondapak C18

YMC-Pack ODSAQ

YMC J'sphere ODSH80

YMC J'sphere ODSM80

Inertsil CN-3

Waters Spherisorb S5CN

Nova-Pak CN HP

SymmetryShield RP8

SymmetryShield RP18

XTerra RP8XTerra RP18

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

(ln

[

] am

itri

pty

lin

e/a

cen

ap

hth

en

e)

XTerra MS C8

Luna C18

(2)ACQUITY

UPLC BEH C18

XTerra Phenyl Luna

Phenyl Hexyl

ChromolithTM

RP-18

Atlantis dC18

Zorbax XDB C18ACT Ace

C18

Zorbax SB C18


Waters column Selectivity Chart

http://www.waters.com/waters/promotionDetail.htm?id=10048475&locale=en_CA


Efecto de la fase mvil en

el cromatograma


Selectivity Differences:Methanol versus Acetonitrile

Selectivity shifts observed.Acetonitrile is a stronger elution solvent.Acetonitrile and methanol can be blended to fine tune separations.

Jenkins, Diehl

XBridge Shield RP18

100% MeOH

100% ACN

Low pH

1:1 ACN:MeOH

1

2

43

56

2

8

79

6

10

12

11

8

9

10 11

10,11

12

128,91 6

7

2

3 4

43

15,7

5


Mobile Phase pH Selectivity:Basic and Neutral Compounds

IA

P

Fl

Fe

D

O

Minutes

0.00 1.20 2.40 3.60 4.80

I

A

P

Fl

Fe

DO

Minutes

0.00 1.20 2.40 3.60 4.80

0.1% formic acid pH 3

Test Probes:I: Imipramine [B]A: Amitriptyline [B]Fl: Flavone [N]O: Octanophenone [N]

ACQUITY UPLC CSH C18

Acetonitrile

0.1% NH4OH pH 10

Observations:

At high pH, bases are in their neutral

(unionized) form, resulting in greater

retention

Neutral compounds are unaffected by pH


Mobile Phase pH Selectivity:Acidic Compounds

IA

P

Fl

Fe

D

O

Minutes

0.00 1.20 2.40 3.60 4.80

I

A

P

Fl

Fe

DO

Minutes

0.00 1.20 2.40 3.60 4.80

Test Probes:P: 1-pyrenesulfonic acid [A]Fe: fenoprofen [A]D: diclofenac [A]Fl: Flavone [N]O: Octanophenone [N]

0.1% formic acid pH 3

0.1% NH4OH pH 10

ACQUITY UPLC CSH C18,

Acetonitrile

Observations:

At low pH, acids are in their neutral

(unionized) form, resulting in greater

retention

Elution order change for acidic compounds


Ion Pair Mechanism


Reference

Exerpts from USP37-NF32 1S (Published)


Allowable adjustments


Changes to Particle Size


Changes to Flow Rate


Changes to Other factors


Example provided

Documents

Taller Ucimed Resumen