Đao Tạo Hplc 20240517 - Usp 621 & Chuyển Đổi Phương Pháp - Version 2.Pdf

2022 revision EP 11 Edition 2.2.46 Agilent OpenLab CDS 2.2 ~ 2.7 • Calculated based on the Peak Half Width • N = 5.54 ?? ?ℎ2 • Calculated based on the Peak Half Width • N = 5.54 ?? ?ℎ2 •

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Modernization of HPLC Method: USP <621> & Transfer method

Ho Tuan Dat

Email: Dat.HoTuan@Redstar-cms.vn

Tel: +84.356.170.539

Application team

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1 HPLC method modernization

3 Transfer method between systems.

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Cases 1: Transfers from HPLC to UPLC

Let's plan the method conversion to UPLC !

Oh no! UPLC easily clogs columns, requires frequent

maintenance, and the columns degrade quicklyBut they say it has high productivity, solvent-saving, overall still saving more than 1 billion a year

Oh no! Full adherence to the method is required,

regulations do not allow otherwise

USP 621 allows for changes within limits

Cases 2: Transfers between the same systems

Cases 2: Transfers between the different systems (model, brands)

We will invest in expansion, purchasing an additional Agilent UPLC 1290

Oh no, we already have one from brand X, converting the method is difficult and time-consuming Let's just buy one similar to the old system, boss!

I like Agilent Don't worry They have ISET technology Just give them a call!

We will build a new factory and invest in a similar HPLC system

You prepare to transfer and train the method for the new factory!

Yes, boss, but it will take a long time to transfer the method!

"Don't worry, I bought more ISET options They said only need 01 for transfers."

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Understanding HPLC method modernization

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2 x10

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

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 3.2

Solvent consumption

* 2D Checkout solution

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• Solution stability of rosuvastatin under acidic dissolution media

Solution State Stability

Latin American Journal of Pharmacy - 32 (10) - 2013

APIs with poor solution stability should be tested quickly

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• Comparative dissolution test

Solution State Stability

Total run time = {(Reference solution (Blank, STD 1 ~ 5) 6 vial) x 3 set + (Reference drug + Test drug) x 12 EA x

12 time point} x 4 medium x 10 min

= {6 x 3 + 2 x 12 x 12} x 4 x 10 min

= 12,240 min = 204 hrs

L/dp: 30,000 Flow: 1.0 mL/min, Run time: 10 min

L/dp: 27,778 (-7%) Flow: 0.525 mL/min, Run time: 1.2 min

204 Hours Mobile phase 12.24 L

24.5 Hours Mobile phase 771 mL

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• The effect of decreasing particle size

Understanding Van Deemter Equation

*Wikipedia

How does it work in the Modern HPLC Method?

solid core columns and shortening analyte diffusion path

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Superficially Porous Particle Column (SPP)

USP officially allows the use of SPP columns.

USP<621>

- Solid core particle with thin porous shell coating

- Improved column efficiency and separation performance

- Reduced diffusion path within the porous shell leads to faster mass transfer and higher resolution

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• Van Deemter Plots vs System Pressure

Effects Of Smaller Particles

• Small particles lead to lower HETP, and therefore higher separation efficiency.

• Small particles lead to narrower, higher peaks.

• For smaller particles the separation efficiency suffers less when increasing flow rate.

But: Smaller particles generate higher back pressure.

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• No Run Time Increase!

Applying Columns With Smaller Particles

min

mAU

02040

gives increased sensitivity

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• Smaller particles give increased peak height

Increase Sensitivity

 2

max

N d

V

c A

c

i i

1200 µl/min; 10 sec peak w baseline = 200 µl; 10 µl injection

600 µl/min ; 2 sec peak w baseline = 20 µl; 2 µl injection Factor 2 sensitivity

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Understanding USP (621)

& Transfer HPLC to UPLC

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• Chages in Key parameters (Harmonization with EP)

USP Changes to Chapter <621>

Parameter USP <621> (Before Dec 2022) USP <621> (Apr 2023)

(Same as the Dec 2022 revision)

EP 11 Edition 2.2.46 (Agilent OpenLab CDS 2.2 ~ 2.7)

• Calculated based on the Peak Half Width

• N = 5.54 𝑡𝑅

𝑊ℎ2

• N = 5.54 𝑡𝑅

𝑊ℎ2

• Plate Number (N) and Resolution (Rs) have been harmonized to use the same calculation methods as USP and EP

• With the revised USP calculation method based on the existing EP calculation method, it is now easier to apply the changes in CDS.

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• Chages in Key parameters (Harmonization with EP)

Parameter USP <621> (Before Dec 2022) USP <621> (Apr 2023)

(Same as the Dec 2022 revision)

EP 11 Edition 2.2.46 (Agilent OpenLab CDS 2.2 ~ 2.7)

• N = 5.54 𝑡𝑅

𝑊ℎ2

• N = 5.54 𝑡𝑅

𝑊ℎ2

• Plate Number (N) and Resolution (Rs) have been harmonized to use the same calculation methods as USP and EP

• With the revised USP calculation method based on the existing EP calculation method, it is now easier to apply the changes in CDS.

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• Header Text: Peak Resolution EP → Peak Resolution USP

• Value: Peak_Resolution_EP

Applying the Updated Resolution and Plate Number in the Report

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Previous Dec 2022 Current (Apr 2023)

H

The maximum of the peak to the extrapolated baseline

of the signal - At least 5 times the width at half-height

based on the reference

of the signal - At least 20 times the width at half-height based on the reference

of the signal - At least 5 times the width at half-height based on the reference

h 5 Times the peak half width based on the reference 20 Times the peak half width based on the Blank 5 Times the peak half width based on the Blank

EP 11 2.2.46 Chromatographic separation techniques

USP <621> Chromatography

Current (Jan 2023) Jan 2024

H The maximum of the peak to the extrapolated baseline of the signal - At least 20

times the width at half-height based on the reference

The maximum of the peak to the extrapolated baseline of the signal - At least 5 times

the width at half-height based on the reference

h 20 Times the peak half width based on the Blank 5 Times the peak half width based on the Blank

S/N ratio = 2𝐻

ℎ

S/N Ratio Calculation

H = height of the peak

h = range of the noise

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• Introduction of Additional Parameters

Parameter USP <621> (Previous) USP <621> (Current)

• Newly added

• H = 𝐿

𝑁 (L = Column length, N = Plates Number)

-• Newly added

• h = 𝐻

𝑑𝑝 (𝐻 = Plates height, 𝑑 𝑝 = particle diameter)

-Retention volume of an unretained compound (𝑉 0 )

• 𝑉 0 = 𝑡 0 x 𝐹 (𝑡 0 = retention time of unretained compound, 𝐹 = flow rate)

Total mobile phase volume (𝑉 𝑡 )

• 𝑉 𝑡 = 𝑡 𝑡 x 𝐹 (𝑡 𝑡 = retention time of the smallest compound, 𝐹 = flow rate)

Peak Symmetry

• 𝐴 𝑠 = 𝑊0.05

2𝑑

W0.05 = width of the peak at one-twentieth of the peak height

d = distance between the perpendicular dropped from the peak maximum and

the leading edge of the peak at 1/20 of the peak height

• Known as the asymmetry factor or tailing factor (OpenLab CDS)

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• USP general chapter 621 and all relevant USP monographs are legally binding for validated methods.

• New Regulatory Limits

USP <621>

Allowable Adjustments of Chromatographic Conditions

USP <621> August 1st 2014 (USP37-NF32) USP <621> December 1st 2022

L/dp = column length (L) to particle size (dp) ratio

(1) USP Flow Rate (isocratic) : F₂ = F₁ x [(dc₂² x dp₁)/(dc₁² x dp₂)]

(2) V inj2 = V inj1 (L₂ x dc₂²)/(L₁x dc₁²)

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Summary of Allowable Adjustments per USP

General Chapter <621> after December 1, 2022

Totally Porous to Superficially Porous

Isocratic Adjustment

Totally Porous to Superficially Porous Gradient Adjustment

Ratio of Components in Mobile

Phase

Minor component (≤50%): ±30% relative, but cannot exceed ±10% absolute; may only adjust 1 minor component in ternary mixtures

The principal peak(s) elute(s) within ±15% of the retention time(s) obtained with the original conditions; this requirement does not apply when the column dimensions are changed The composition of the mobile phase and the gradient are such that the first peaks are sufficiently retained, and the last peaks are eluted

Minor component (≤50%): ±30% relative, but cannot exceed ±10% absolute; may only adjust 1 minor component in ternary mixtures

The principal peak(s) elute(s) within ±15% of the retention time(s) obtained with the original conditions; this requirement does not apply when the column dimensions are changed The composition of the mobile phase and the gradient are such that the first peaks are sufficiently retained, and the last peaks are eluted

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Variable Original Adjusted Comments

Gradient Adjusted factor (t G2 /t G1 ) - 0.4 Based on t G2 /t G1 =(F 1 /F 2 ) x [(L 2 x dc 2 2 )/(L 1 x dc 1 2 )] Gradient Conditions

30 30 70 30

0 3 13 16

0 (3x0.4)=1.2 1.2+(10 x.0.4)=5.2 5.2+(3x0.4)=6.4

2.5x time saving! (9.6min saved) 2.5x solvent saving! (27.6mL solvent saved)

• New Regulatory Limits –Example

USP <621>

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What is L/dp?

𝐿

𝐶𝑜𝑙𝑢𝑚𝑛 𝑙𝑒𝑛𝑔𝑡ℎ 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑠𝑖𝑧𝑒

e.g 4.6 x 250mm, 5µm → 250,000 / 5 = 50,000

L/dp ratio: -25% ~ 50%

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What column can be changed to?

• Column list that can be changed from 4.6mm ID column

ID Length Particle size Type Flow rate L/dp Difference

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• What changes when the column specification is modified?

Understanding of the Formular – USP621

Flow rate Injection volume

Injection volume Gradient timetable

Flow rate Gradient timetable

And what else?

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• Brief summary of System and Column selection

Method selection

Legacy Method

Superficially porous particle

Totally porous particle

4.6 x 250mm, 5um 4.6 x 150mm, 2.7um

4.6 x 100mm, 1.9um

3.0 x 150mm, 2.7um 2.1 x 150mm, 2.7um 3.0 x 100mm, 1.9um 2.1 x 100mm, 1.9um

4.6 x 100mm, 1.8um 3.0 x 100mm, 1.8um 2.1 x 100mm, 1.8um

UHPLC

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2D Checkout - Eclipse Plus C18 2.1 x 100mm, 1.8um_2D Checkout.amx | DAD1A,Sig=254,4 Ref=off | 2023-04-04 15-33-39+09-00-02.dx

What happens if system performance is insufficient?

2D Checkout - Eclipse Plus C18 2.1 x 100mm, 1.8um_2D Checkout.amx | PMP1A,Pressure | 2023-04-04 15-33-39+09-00-02.dx

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System Dwell Volume and Column Void Volume

volume (mL)

Injection volume (µL)

Flow rate (mL)

1290 H (Bypass)

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- To increase the efficiency of particle size, increase the flow rate

- Adjust the injection volume considering column capacity

- To obtain similar peak height (Signal to noise ratio)

- Adjust the gradient time considering the decreased column length and increased flow rate

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• Let’s simplify the formular

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Scaling Flow Rate To New Column Dimension

Target flow rate (F) adapting linear velocity (if particle size changes)

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Scale injection volume (V Inj ) to cross section and reduced zone dilution in

shorter column length L (band broadening)

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52 0 52 0

5 1 10

1 1 2

C

C G

G

V

V F

F t

2.1 x 150 mm column, 3 µm 0.52 mL/min

(increased u)

Col Volume (VC) = 0.52 mL

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𝐶

Gradient time Flow rate

Injection volume

A = 0.2084 B = 0.4 C = 2.632

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• 2D Checkout

Practice

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• Eclipse Plus C18 vs Poroshell 120 EC-C18

0 1 2 3 4 5

0 1 2 3 4 5 6

0.9 1.0 1.1 1.2 1.3

0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4

2D Chechout - Poroshell EC-C18 2.1 x 100mm, 1.9um_2D Checkout.amx | DAD1A,Sig=254.0,4.0 Ref=off | 2023-04-05 09-27-21+09-00-02.dx

Totally porous particle

Superficially porous particle

* 2D Checkout solution

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Full Scalability

Traditional ZORBAX chemistries are aligned with InfinityLab Poroshell chemistries to offer

simplified method transfer from fully porous particles to superficially porous particle columns.

Aligned Chemistry

ZORBAX Eclipse Plus C18

ZORBAX Eclipse Plus C8

ZORBAX Eclipse Plus Phenyl-Hexyl

InfinityLab Poroshell 120 EC-C18

InfinityLab Poroshell 120 EC-C8

InfinityLab Poroshell 120 Phenyl-Hexyl

InfinityLab Poroshell 120 SB-C18

InfinityLab Poroshell 120 SB-C8

InfinityLab Poroshell 120 SB-Aq

InfinityLab Poroshell 120 Bonus-RP

InfinityLab Poroshell 120 EC-CN

InfinityLab Poroshell 120 HILIC

Method Transferability Across Product Families

For more information on method transfer: Technical Overview 5990-6588EN

66 compounds two solvents (MeOH, ACN)

at 3 pH values each pressure vs linear velocity

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• Phthalate ester

Effectiveness of SPP column in Gradient method

Analytical condition

Time (min)

Resolution 5.14 Plates 737620

Resolution 5.56 Plates 926475

Plates 7756

Plates 8125

Plates 8059

Eclipse Plus C18 4.6 x 250mm, 5µm

- 1260 Inf II Quaternary

Poroshell EC-C18 3.0 x 100mm, 2.7µm

- 1260 Inf II Prime

Poroshell EC-C18 3.0 x 100mm, 1.9µm

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Transfer method between systems

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The same method with two different LC Systems

- Impact of delay volume and mixing behavior

min

mAU

050100150200 DAD1 B, Sig=275,4 Ref=400,40 (B:\RIC\AGI RIC DATA\1 PHARMA METOCLOPRAMIDE\WAD PHARMA\XBRIDGE-2000004.D)

The result:

• Difference in RT and Resolution

• One peak is missing!

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Instrument to Instrument Method Transferability

- Important Parameters

Pump

Sampler

Column Thermostat Detector

Retention Time Resolution Sensitivity

Retention Time Resolution

Resolution Retention Time Sensitivity

Delay volume Gradient mixing behavior Pressure x flow rate

Delay volume Extra column volume Injection volume

Temperature profile Extra column volume

Data rate Extra column volume Path-length

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InfinityLab LC Series

Gradient Delay Volume

• Affects or Results in:

• an isocratic hold step at the beginning of every gradient

• sharpness of the gradient

• required equilibration time and therefore total cycle time

• Early eluting peaks are more

affected than later eluting peaks

• System Design

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• Mixing Behavior Differences

0 0.5 1 1.5 2 2.5 3 3.5 0

Mixing Behaviour

1100 GDV

1290 GDV

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6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.25 9.50 9.75 10.00 10.25

Retention time [min]

0 10 20 30 40 50 60 70 80 90

Water 0uL injection | DAD1A,Sig=265,4 Ref=off Water 0uL injection | PMP1D,Solvent Ratio B

Water 0uL injection - valve bypass | DAD1A,Sig=265,4 Ref=off Water 0uL injection - valve bypass | PMP1D,Solvent Ratio B

• 1260 Infinity II HPLC - Quaternary System vs Prime

Dwell volume measurement

Time (min)

Dwell volume (mL)

Water 0uL injection | DAD1A,Sig=265,4 Ref=off Water 0uL injection | PMP1F,Solvent Ratio D

Water 0uL injection - valve bypass | DAD1A,Sig=265,4 Ref=off Water 0uL injection - valve bypass | PMP1F,Solvent Ratio D

Dwell volume (mL)

1 Theoretical 7.500

-2 0uL → Bypass 7.995 0.495

3 0uL injection 8.281 0.781

2 3

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Water 0uL injection | DAD1A,Sig=265.0,4.0 Ref=off Water 0uL injection | PMP1E,Solvent Ratio B

Water 0uL injection - valve bypass | DAD1A,Sig=265.0,4.0 Ref=off Water 0uL injection - valve bypass | PMP1E,Solvent Ratio B

6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.25 9.50 9.75 10.00 10.25

0 10 20 30 40 50 60 70 80 90

• 1260 Infinity II HPLC - Quaternary System vs 1290 Infinity II UHPLC

Dwell volume measurement

Time (min)

Dwell volume (mL)

6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.25 9.50 9.75 10.00 10.25

0 10 20 30 40 50 60 70 80 90

Dwell volume (mL)

1 Theoretical 7.500

-2 0uL → Bypass 7.610 0.110

3 0uL injection 7.715 0.215

Tiêu đề	Modernization of HPLC Method: USP <621> & Transfer method
Tác giả	Ho Tuan Dat
Trường học	Red Star Vietnam Company Limited – CMS Branch
Chuyên ngành	HPLC
Thể loại	Presentation
Năm xuất bản	2024

Định dạng
Số trang	80
Dung lượng	8,02 MB