1
Profª. Drª Márcia Cristina BreitkreitzLaboratório de Pesquisas Farmacêuticas e Quimiometria (LabFarQui)
Instituto de Química/Faculdade de Ciências Farmacêuticas - UNICAMP
[email protected], www.labfarqui.com.br
Design of Experiments (DOE) in the framework of Analytical
Quality by Design (AQbD):
Case studies
Supercritical Fluid Chromatography?
Compared to a liquid: lower
viscosity and higher diffusivity.
Efficient mass transfer;
Faster analysis.
Compared to a gas: better
solubilization capabilities.
Supercritical Fluid:
“Convergence
chromatography”
Rebirth of SFC
New equipments with enhanced technology: UHPSFC or “Modern SFC”
Efficient pressure control (ABPR):
Binary pump: FM: CO2 + MeOH, ACN, EtOH,
IPA, 1-30 %) + water + additives (optional)
Detectability
Repeatability, robustness
Analysis of high molecular mass and
hydrophilic compounds
UPC2 (Waters) - Ultra Performance
Convergence Chromatography.
Packed columns with sub-2µm particles
Compatible with the new equipments;
Diverse polarities:
“NP column” “RP column”
Factors to study:
Design of Experiments (DOE) in SFC
INTERACTIONS!!! Stationary phase chemistry;
Organic modifier (type and %);
Sample diluent;
Additives (type and %);
Pressure;
Temperature;
% Gradient time/slope;
Flow rate.
...
Temperature vs % MeOH Pressure vs % MeOH
6
≠ Antihypertensive drugs - SFC
Mostly basic and polar compounds: Very challenging!
They can be prescribed isolated or associated
(combined theraphy):
The goal was to develop a single and fast
method by SFC for the assay of the main
antihypertensives (multiproduct approach)
7
≠ Antihypertensive drugs - SFC
Factors - 1 0 + 1
NH4HCO2 concentration (mmol L-1) 0 10 20
% Binicial (MeOH:H2O 95:5 v/v) 10 15 20
Temperature (°C) 25 40 55
Pressure (psi/MPa) 1595/11.0 1885/13.0 2175/15.0
MeOH necessary to elute
the polar compounds
Step 01:
Full factorial design (24) with five replicates at the central point to study
the influence of:
NH4HCO2 (additive),
Initial concentration of the modifier - MeOH:H2O 95:5 v/v (% Binitial),
Pressure;
Temperature
What we already knew:
8Fixed factors:
Flow rate: 1,0 mL/min; % Bfinal = 45 % Gradient time: 3.0 minutes; Diluent: Isopropanol; Injection volume: 1,0 μL
Without the Additive:
≠ Antihypertensives drugs - SFC
Retention of Propranol (P)
is increased by decreasing
the % B however peak
shape is worsened
Coelution of Atenolol (A)
and Clortalidone (C) in
every condition without the
additive;
Pressure does not
influence much the overal
results.
9
Separation of atenolol and
chortalidone is achieved by
adding 20 mmol NH4HCO2
When we look at 10 % B
(advisable for retention of
propranolol), there is a huge
baseline instability – specially
for lower pressure values
which impairs the
quantification of propranolol
≠ Antihypertensives drugs - SFC
20 mM of NH4HCO2:
10
≠ Antihypertensive drugs - SFC
And what if we increase temperature?
We solve the
problem of
baseline
instability for
high pressure
values!
11
≠ Antihypertensive drugs - SFC
10 mM AmF is
enough to provide
separation of A
and C;
15 % of modifier is
enough to provide
adequate retention
of propranolol;
T = 40 °C and P =
1885 psi
Is enough to avoid
baseline instability
Retention and peak
shape
Separation
@40 °C
Center points results:
RESULTS OF 5
REPLICATIONS!!!
Always check your repeatability!!
Not critical - to be solved
in the next step!
12
The center point region was
adopted as the most
promissing region for the next
optimization step.
≠ Antihypertensive drugs - SFC
Screening step
acomplished its purpose:
Provide understanding of
the influence of the
factors and guidance
towards the Optimization
step
13
Central Composite Design (CCD) as na Optimization Design;
Temperature and gradient strength are key parameters for the separation;
Pressure and flow rate were included as they influence the fluid density and represent a
fine adjustment of the chromatographic parameters.
≠ Antihypertensive drugs - SFC
Factors - α - 1 0 + 1 + α
Temperature (°C) 15 25 35 45 55
Gradient time (min) 1 2 3 4 5
Pressure (psi/Mpa) 1885/13,0 2030/14,0 2175/15,0 2320/16,0 2465/17,0
Flow rate (mL min-1) 0,80 0,90 1,00 1,10 1,20
Final % B = 50 % to garante the elution of all compounds;
14
↓T ↑Rs L/H↓T ↑Rs A/C
Attention: Crossover here!
≠ Antihypertensive drugs - SFC
Effect of temperature:
15
↓tg ↑Rs A/C
↓tg ↓Rs L/H
≠ Antihypertensive drugs - SFC
Effect of gradient time:
16
≠ Antihypertensive drugs - SFC
Effect of the flow rate:
↓F ↑Rs A/CFlow rate does not affect
separation of H/L!!
17
≠ Antihypertensive drugs - SFC
Effect of the ABPR:
ABPR does not influence the separation of
critical pairs, but influences on the peak
width of propranolol
18
≠ Antihypertensive drugs - SFC
-1
-0,75
-0,5
-0,25
0
0,25
0,5
0,75
1
A B C D AB AC AD BC BD CD A² B² C² D²
Separation of A and C
-1
-0,75
-0,5
-0,25
0
0,25
0,5
0,75
1
A B C D AB AC AD BC BD CD A² B² C² D²
Separation of L and H
-1
-0,75
-0,5
-0,25
0
0,25
0,5
0,75
1
A B C D AB AC AD BC BD CD A² B² C² D²
Propranolol peak width
In red: significant coefficients (and those to support hierarchy). The
bars erros indicate 95 % confidence intervals.
Subtitles:
A = T
B = ABPR
C = tgD = F
The shorter gradient time favors separation of A and C and
reduces the width of P;
The lower temperature favors the separation of L and H;
A higher flow rate improves peak shape of P, although it affects
the separation of A and C.
Coefficients of the models:
19
≠ Antihypertensive drugs - SFCDesign-Expert® Sof twareFactor Coding: ActualSeparation A/C
Design points abov e predicted v alueDesign points below predicted v alue1.0537
1
X1 = A: TemperatureX2 = C: Gradient time
Actual FactorsB: ABPR = 2175D: Flow rate = 1
1
2
3
4
5
15
25
35
45
55
0.98
1
1.02
1.04
1.06
1.08
Se
pa
ratio
n A
/C
A: Temperature (°C)
C: Gradient time (min)
Design-Expert® Sof twareFactor Coding: ActualSeparation L/H
Design points abov e predicted v alue1.07688
0.951447
X1 = A: TemperatureX2 = C: Gradient time
Actual FactorsB: ABPR = 2175D: Flow rate = 1
2
2.5
3
3.5
425
30
35
40
45
0.94
0.96
0.98
1
1.02
1.04
1.06
1.08
Se
pa
ratio
n L
/H
A: Temperature (°C)C: Gradient time (min)
Design-Expert® Sof twareFactor Coding: ActualUSP width P
Design points abov e predicted v alueDesign points below predicted v alue0.0992
0.0521
X1 = C: Gradient timeX2 = D: Flow rate
Actual FactorsA: Temperature = 35B: ABPR = 2175
0.9
0.95
1
1.05
1.12
2.5
3
3.5
4
0.05
0.06
0.07
0.08
0.09
0.1
US
P w
idth
P
C: Gradient time (min) D: Flow rate (mL/min)
Separation of A C
(F = 1.00 mL min-1 and ABPR = 2175 psi)
Separtion of L and H
(F = 1.00 mL min-1 and ABPR = 2175 psi)
Peak width (USP) of P (T = 25 °C and ABPR =
2175 psi)
Tg and Temperature interaction
Temperature quadratic coefficient
Tg quadratic coefficient
≠ Antihypertensive drugs - SFC
(T = 25 °C e ABPR = 2117 psi)
Design Space or Method Operabe Design Region:
Design Expert – Overlay plot Fusion - Desirability
≠ Antihypertensive drugs - SFC
(F = 1,00 mL min-1 e tg = 1,50 min)
Design-Expert® Sof twareFactor Coding: ActualOv erlay Plot
Separation A/C CI LowSeparation L/H CI LowUSP width P CI High
X1 = A: TemperatureX2 = B: ABPR
Actual FactorsC: Gradient time = 1.5D: Flow rate = 1
15 25 35 45 55
1943
2059
2175
2291
2407
2523Overlay Plot
A: Temperature (°C)
B: A
BP
R (
psi)
Separation A/C: 1.042
Separation A/C CI: 1.042
Separation L/H: 1.035
Separation L/H CI: 1.035
USP width P: 0.07USP width P: 0.07
USP width P CI: 0.07
22
≠ Antihypertensive drugs - SFCDesign-Expert® Sof twareFactor Coding: ActualOv erlay Plot
Separation A/C CI LowSeparation L/H CI LowUSP width P CI High
X1 = A: TemperatureX2 = C: Gradient time
Actual FactorsB: ABPR = 2117D: Flow rate = 1
15 25 35 45 55
1
2
3
4
5Overlay Plot
A: Temperature (°C)
C: G
radie
nt tim
e (
min
)
Separation A/C: 1.042Separation A/C CI: 1.042
Separation L/H: 1.035
Separation L/H CI: 1.035
(F = 1.00 mL min-1 ABPR = 2117 psi)
Design-Expert® Sof twareFactor Coding: ActualDesirability
1.000
0.000
X1 = A: TemperatureX2 = C: Gradient time
Actual FactorsB: ABPR = 2117D: Flow rate = 1
15 25 35 45 55
1
2
3
4
5Desirability
A: Temperature (°C)
C: G
radie
nt tim
e (
min
)
0.2
0.4
0.4
0.6
Final conditions:
T = 25 °C
ABPR = 2117 psi (14,6 MPa)
F = 1.00 mL min-1
tg = 1.50 min
D = 0,746
23
VITAMINA NOME GENÉRICOFÓRMULA
MOLECULAR
MASSA
MOLAR (g/mol)pKa LogP ESTRUTURA
A Palmitato de Retinol C36H60O2 524,86 7,04 13,60
D3 Colecalciferol C27H44O 384,64 18,38 7,50
E Acetato de a-tocoferol C31H52O3 472,74 10,80 7,59
B1 Tiamina C12H17N4OS 265,35 15,50 -2,11
B2 Riboflavina C17H20N4O6 376,37 6,97 -1,46
B3 Nicotinamida C6H6N2O 122,13 13,39 -0,45
B6 Cloridrato de Piridoxina C8H11NO3 169,18 9,40 -0,57
B12 Cianocobalamina C63H88CoN14O14P 1355,29 1,84 0,67
CÁcido Ascórbico C6H8O6 176,09 4,36 -1,58
B9 Ácido Fólico C20H23N7O7 441,37 3,37 -1,20
Liposoluble vitamins
(LSV)
Hydrosoluble
vitamins (HSV)
Very different
characteristics!
ACQUITY UPC2 BEH 1.7 µm. 2.1mm x 100mm;
ACQUITY UPC2 HSS C18 SB, 1.8 µm, 2.1mm x 50mm;
ACQUITY UPC2 CSH Fluoro-Fenil, 1.7 µm, 2.1mm x
50mm;
ACQUITY UPC2 Torus 2-PIC (2-Picolilamina) 1.7 µm,
2.1mm x 50mm;
SP Screening:
Retention curves for all SPs, with different
modifiers: MeOH, ACN, isopropanol (IPA);
With and without additives: NH4HCO2, H2O, NaOH,
NH4OH, triethylamine;
≠ Vitamins - SFC
The screening step is a very
flexible step, sometimes there is
no need for a formal DOE.
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Fato
r d
e R
ete
nção
% MeOH
Vitamin A - Metanol
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Fato
r d
e R
ete
nção
% Acetonitrila
Vitamin A - Acetonitrile
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Fato
r d
e R
ete
nção
% 2-Propanol
Vitamina A - 2-Propanol
Adequate retention
only in C18 column for
LSV no matter the
modifier used!
MeOH was selected
due to the higher
chromatographic strength
to elute the HSV;
NH4HCO2 was
important for HSV.
≠ Vitamins - SFC
ACQUITY UPC2 BEH
ACQUITY UPC2 HSS C18 SB
ACQUITY UPC2 CSH Fluoro-Fenil
ACQUITY UPC2 Torus 2-PIC
Retention curves:
Factor Level - Central Point Level +
MeOH at the end of
gradient (%)20 25 30
Pressure (psi) 1500 2000 2500
Temperature (°C) 30 40 50
Responses: Retention factors (to understand the
system); Assymmetry factors; Resolution; Number of
Plates; Area.
Optimization – Central Composite Design: Study of the fluid density:
≠ Vitamins - SFC
Important interaction between
pressure and %B
Retention of LSV: low significance regression: not much affected by the
experimental variables, except the pressure:
≠ Vitamins - SFC
Higher regression significance for HSV!
Temperature Effect
Interaction!
No lack of fit!
≠ Vitamins - SFC
50 °C
DOE tells you when it is not ssible to achieve the a
certain value, no matter what you do!!
30 °C
≠ Vitamins - SFC
Asymmetry:
Area: Unchanged for Vit A, E, D, B2, B3, B6
Significant regression for Vit B1 e Vit C
Vit B1: Not detected in some conditions
Vit C: 30 °C
50 °C
Degradation?
≠ Vitamins - SFC
Number of plates: Important for HSV;
% MeOH and Temperature are the most importante variables:
Vit. C:
30 °C 50 °C
≠ Vitamins - SFC
Desirability: Selected conditions: 30% of MeOH, 30°C. Pressure
does not influence the responses when working with
30 % of MeOH = Subcritical conditions: MP has the
density of a liquid.
Vitamins that never eluted:
(Limitation of the technique)
B12 B9
≠ Vitamins - SFC
1. Vit. E, 2. Vit. A, 3. Vit. D,
4. Vit. B3, 5. Vit. B6, 6. Vit.
B2, 7. Vit. C.
33
≠ Losartan and Amlodipine - HPLC
Development of a stability indicating method for the association of the drugs
Losartan Potassium and Amlodipine Besylate by High Performance Liquid
Chromatography Design of Experiments.
Amlodipine Besylate Losartan Potassium
= Combined theraphy
+
34
Stability indicating method:
RDC 53/2015 (ANVISA): "Validated methods, capable of detecting, over the time,
low concentrations of degradation products and properly
separate all substances present in the sample"
Step 1: Column, modifier and pH screening for both
drugs;
Step 2: Optimization with the degraded sample -
mixture of the main degradation products of the two
drugs!
Forced degradation studies
Potencial Profile
Real Profile
Without
standards
≠ Losartan and Amlodipine - HPLC
35
Columns:
XTerra MS C18 3.5 µm - 3.0x100 mm
XSelect HSS C18 µm 5 µm – 4.6x150 mm
Nova-Pak C18 4 µm – 3.9x150 mm
CORTECS C18+ 2.7 µm – 4.6x150 mm
XSelect CSH Fluoro-Phenyl 3.5 µm – 4.6x150 mm
pH range from 2.5 to 6.0.
Organic modifiers: ACN and MeOH.
Low resolution and high asymmetry for amlodipine peak with both modifiers in
the whole pH range.
pH
≠ Losartan and Amlodipine - HPLC
36
≠ Losartan and Amlodipine - HPLC
37
Zoom
Zoom Zoom
≠ Losartan and Amlodipine - HPLC
38
Amlodipine forced degradation:
Degradation routes: acidic and oxidative.
(Basic to a lesser extent)
Basic degradation (0,1 mol L-1)
Basic degradation (0,5 mol L-1)
Acidic degradation (0,1 mol L-1)
Oxidative degradation (3%)
Control
90% assay
80% assay
The same degradation products were formed
by acidic and oxidative degradation!
≠ Losartan and Amlodipine - HPLC
39
Main degradation route: oxidative
Losartan forced degradation:
Control
Acidic degradation (0,1 mol L-1)
Basic degradation (0,1 mol L-1)
Oxidative degradation (3%)
≠ Losartan and Amlodipine - HPLC
40
“Worst scenario sample”: mix
of samples degraded under
acidic/ basic (Amlodipine) and
oxidative conditions (Losartan).
Acidic degradation (Amlodipine)
Basic degradation (Amlodipine)
Oxidative degradation (Losartan)
≠ Losartan and Amlodipine - HPLC
4141
Central Composite Design:
Factors (-1) (+1) - α + α Responses
pH 3 4 2,5 4,5Peak purity; Tailing
factor; Number of
plates; Resolution
Temperature (°C) 28,8 36,3 25 40
Flow rate (mL min-1) 0,8 1 0,7 1,1
Data treament:
≠ Losartan and Amlodipine - HPLC
42
Source SSQ DF MSQ Fcal P-value Interpretation
Modeo 6,70E+08 9 7,44E+07 26,36 0,0011Significant
A-Flow rate 1,01E+08 1 1,01E+08 36,01 0,0018Significant
B-pH 4,03E+07 1 4,03E+07 14,3 0,0129Significant
C-Temperature 8,19E+07 1 8,19E+07 29,05 0,0030Significant
AB 1,09E+08 1 1,09E+08 38,64 0,0016Significant
AC 4,08E+05 1 4,08E+05 0,14 0,7192Not significant
BC 6,43E+05 1 6,43E+05 0,23 0,6532Not significant
A² 1,29E+07 1 1,29E+07 4,56 0,0857Not significant
B² 3,21E+08 1 3,21E+08 113,89 0,0001Significant
C² 2,74E+07 1 2,74E+07 9,73 0,0263Significant
Residuals 1,41E+07 5 2,82E+06
Lack of fit 5,84E+06 3 1,95E+06 0,47 0,7333Not significant
Pure Error 8,26E+06 2 4,13E+06
Total 6,83E+08 14
The importance of Design
selection
Regression is significant
Model is adequate
Ex: ANOVA - peak purity of losartan:
≠ Losartan and Amlodipine - HPLC
43
T = 28°C 32°C 36°C
Conditions selected for validation within the
DS: Flow rate: 0.95 ml min-1; pH 3.2;
Temperature 28 °C.
≠ Losartan and Amlodipine - HPLC
Peak purity of Losartan: 99.67
Peak Purity of Amlodipine: 99.33
Tailing Factor of Losartan: 1.46
Tailing factor of Amlodipine: 1.39
44 Method was validated according to RDC 166/2017.
0
50000
100000
150000
200000
250000
300000
350000
0 5 10 15 20 25 30 35 40 45
Tempo (min)
Potencial degradation profile –
worst case scenario
≠ Losartan and Amlodipine - HPLC
Very interesting application:
Considering Monte Carlo simulation:
≠ From literature
Average predictions:
Final remarks
Systematic way for method development;
Identification of interactions. Cause and effect
understanding; modelling of the chromatographic
parameters as functions of the experimental variables;
Knowledge of the method: Best performance
conditions, edges of failure. Robustness brought into
development;
Commercial software integrated with the chromatographs:
Recognition by Regulatory Agencies (validation concepts
are being revised!).
DOE in chromatography:
Profª. Márcia Cristina Breitkreitz
Chemistry Institute/Faculty of Pharmaceutical Sciences - UNICAMP
[email protected], www.labfarqui.com.br
Acknowledgments