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

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

Ho Tuan Dat

Email: Dat.HoTuan@Redstar-cms.vnTel: +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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* 2D Checkout solution

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Poroshell EC-C183.0 x 150mm, 2.7µm- 1260 Inf II Prime

Eclipse Plus C184.6 x 100mm, 1.8µm- 1260 Inf II Prime

Acta Chemica Scandinavica 49 (1995) 589-598

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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: 30,000Flow: 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

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.

-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.

“horizontal”

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• No Run Time Increase!

Applying Columns With Smaller Particles

gives increased sensitivity

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• Smaller particles give increased peak height

Increase Sensitivity

 2

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 injectionFactor 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>

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

(Same as the Dec 2022 revision)

• Calculated based on the Peak Half Width

• N = 5.54𝑡𝑅

• Calculated based on the Tangent Width of the Peak

• 𝑅𝑆 = 2 x 𝑡𝑊𝑅2 − 𝑡𝑅11+ 𝑊2

• 𝑅𝑆 = 1.18 x 𝑊𝑡𝑅2 − 𝑡𝑅1ℎ1+ 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)

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

(Same as the Dec 2022 revision)

• N = 5.54𝑡𝑅

• Calculated based on the Tangent Width of the Peak

• 𝑅𝑆 = 2 x 𝑡𝑊𝑅2 − 𝑡𝑅11+ 𝑊2

• 𝑅𝑆 = 1.18 x 𝑊𝑡𝑅2 − 𝑡𝑅1ℎ1+ 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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PreviousDec 2022Current (Apr 2023)

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

h5 Times the peak half width based on the reference20 Times the peak half width based on the Blank5 Times the peak half width based on the Blank

EP 11 2.2.46 Chromatographic separation techniquesUSP <621> Chromatography

Current (Jan 2023)Jan 2024

HThe 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

h20 Times the peak half width based on the Blank5 Times the peak half width based on the Blank

S/N ratio = 2𝐻

S/N Ratio Calculation

H = height of the peakh = range of the noise

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• Introduction of Additional Parameters

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

• Newly added

• H = 𝐿

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

-• Newly added

• h = 𝐻

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

-Retention volume of an unretained compound (𝑉0)

• 𝑉0= 𝑡0 x 𝐹 (𝑡0 = retention time of unretained compound, 𝐹 = flow rate)

Total mobile phase volume (𝑉𝑡)

• 𝑉𝑡= 𝑡𝑡 x 𝐹 (𝑡𝑡 = retention time of the smallest compound, 𝐹 = flow rate)

Peak Symmetry

• 𝐴𝑠=𝑊0.052𝑑

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) Vinj2 = Vinj1(L₂ x dc₂²)/(L₁x dc₁²)

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Summary of Allowable Adjustments per USP General Chapter <621> after December 1, 2022

Parameters for System Suitability

Totally Porous to Totally Porous

Isocratic Adjustment

Totally Porous to Totally Porous

Gradient Adjustment

Totally Porous to Superficially Porous

Isocratic Adjustment

Totally Porous to Superficially Porous Gradient Adjustment

Particle Size&Column Length

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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Gradient Adjusted factor (tG2/tG1) -0.4Based on tG2/tG1=(F1/F2) x [(L2 x dc22)/(L1x dc12)]Gradient Conditions

0(3x0.4)=1.21.2+(10 x.0.4)=5.25.2+(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?

𝐶𝑜𝑙𝑢𝑚𝑛 𝑙𝑒𝑛𝑔𝑡ℎ𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒 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

IDLengthParticle sizeTypeFlow rateL/dpDifference

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• What changes when the column specification is modified?

Understanding of the Formular – USP621

TypeI.D.LengthParticle size

Totally porous particle4.6 mm

250 mm5 um

Superficially porous particle2.1 mm

100 mm1.9 um52,632

Flow rate

Injection volumeInjection volumeGradient timetableFlow rate

Gradient timetable

And what else?

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• Brief summary of System and Column selection

Method selection

Legacy MethodSuperficially porous particle

Totally porous particle

4.6 x 250mm, 5um4.6 x 150mm, 2.7um4.6 x 100mm, 1.9um

3.0 x 150mm, 2.7um2.1 x 150mm, 2.7um3.0 x 100mm, 1.9um2.1 x 100mm, 1.9um

4.6 x 100mm, 1.8um3.0 x 100mm, 1.8um2.1 x 100mm, 1.8um

UHPLC

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510152025303540455055606570Retention time [min]

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)

60 %x 3.1

17 %x 6.9

8.3 %x 6.9

When selecting a column, consider the system dwell volume!

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Formular – USP<621>

Gradient timeFlow rate

Injection volume

-To maintain linear velocity as the cross-sectional area decreases, adjust the flow rate

-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

Formular – USP<621>

Gradient timeFlow rate

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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 (VInj) to cross section and reduced zone dilution in

shorter column length L (band broadening)

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Original first gradient step 0-10 min

2.1 x 150 mm column, 3 µm 0.52 mL/min

(increased u)

Col Volume (VC) = 0.52 mL

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𝐶Gradient timeFlow rate

Injection volume

A = 0.2084B = 0.4C = 2.632

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• 2D Checkout

Practice

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• Eclipse Plus C18 vs Poroshell 120 EC-C18

Characteristics of SPP column

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 eachpressure vs linear velocity

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• Phthalate ester

Effectiveness of SPP column in Gradient method

Analytical condition

Time (min)

Resolution 5.14Plates 737620

Resolution 5.56Plates 926475Plates 7756

Plates 8125

Plates 8059

Eclipse Plus C184.6 x 250mm, 5µm- 1260 Inf II Quaternary

When using SPP column, similar performance is observed despite the decrease in L/dp

L/dp = 50,000

L/dp = 37,037

L/dp = 52,632

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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

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

DAD1 C, DAD1B, DAD: Signal B, 275 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI A\GV 1PHARMA\HPLC000001.D)

DAD1 C, DAD1B, DAD: Signal B, 275 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI A\GV 1PHARMA\HPLC000003.D)

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Instrument to Instrument Method Transferability

- Important Parameters

Gradient mixing behavior Pressure x flow rate

Delay volume

Extra column volumeInjection volumeTemperature profileExtra column volume

Data rate

Extra column volumePath-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

minmAU

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• - Example

Dwell volume measurement

GDV = (t50% - 0.5tG)*F

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6.256.506.757.007.257.507.758.008.258.508.759.009.259.509.7510.0010.25Retention time [min]

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

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

• 1260 Infinity II HPLC - Quaternary System vs Prime

Dwell volume measurement

Time (min)

Dwell volume (mL)1Theoretical7.500-20uL → Bypass8.2420.74230uL injection8.5741.074450uL injection8.6171.1175100uL injection8.6641.164

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

1260 Infinity II Prime

6.26.46.66.87.07.27.47.67.88.08.28.48.68.89.09.2Retention time [min]

Dwell volume (mL)1Theoretical7.500-20uL → Bypass7.9950.49530uL injection8.2810.781

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