HPLC & Bioequivalence study

Dr. Dipesh Raj Panday

Department of Clinical Pharmacology & Therapeutics

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What is HPLC?

• High-performance Liquid Chromatography• High-pressure Liquid Chromatography• What is Chromatography?

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What is Chromatography?

• The term chromatography is derived from the two Greek words: ‘Chroma’-Colour, and ‘Graphein’-Write)

• So, literally it means ‘Colour Writing’.• First time when it was discovered, it was used

in separation of coloured plant pigments.

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Chromatography

• Actually, Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction (mobile).

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Principle in Chromatography

• Lets consider two immiscible liquids.– Water (hydrophilic)– Cooking oil (lipophilic or hydrophobic)

• Both have ability to dissove sugar separately.• Separately,

– 100ml oil dissolves 10mg sugar.– 100ml water dissoves 20mg sugar.

• Mix 100ml water containing 20mg sugar with 100ml sugar-free oil.

• There will be redistribution of sugar in mixture.504/14/2023

• Between two immiscible phases, the way in which an analyte distributes or the ratio of their distribution coefficient (Kd) is a constant at a given condition.

Principle in Chromatography contd..

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• Here,Kwater/ Koil =20/10=2• Solving, 100ml water contains 13.33mg sugar

and 100ml oil will contain 6.67mg sugar. 04/14/2023

Advantageous Modification In Relation to Orhrodox Chromatography?

• High pressure generated via pump accounts for better performance or better resolving power.

• Therefore, HPLC is better for identifying, quantifying and purifying the individual components of the mixture.

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Reverse-Phase HPLC

• Most widely used used HPLC mode.• As opposed to normal mode, here mobile

phase is polar (hydrophilic) and stationary phase is non-polar or lipophilic.

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HPLC parts-schematic diagram

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Measurement of plasma level of a drug with the help of HPLC

• Before HPLC actually begins to measure plama level of any drug whose level is to be monitored , we calibrate or validate its measurement.

• We take 1. a blank plasma from any normal individual &2. pure drug of study (which is very costly) – and artificially prepare plasma of different concentration

around the therapeutic blood level.

• We inject different concentration of such artificially prepared solution of plasma in the HPLC machine and obtain the chromatograms.

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We measure area of Curve at 1µg/ml

We measure area of curve at 2µg/ml

Chromatogram when injecting 1µg/ml

Chromatogram when injecting 2µg/ml

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End product of HPLC-Chromatogram.

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• This is a chromatogram, pictorial record of detector response as a function of amount of analyte.

• The time at which a specific analyte elutes (emerges from the column) is called the retention time. The retention time measured under particular conditions is considered as an identifying characteristic of a given analyte.

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Decoding Calibration curve

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Decoding Calibration curve -a journey from area of drug in

chromatogram to plasma level

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Using Calibration curve

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Using Calibration curve

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Using Calibration curve

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Using Calibration curve

Bioequivalence study

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Design of bioequivalence study

25Standard Two-sequence, Two-period Crossover Design04/14/2023

What is the use of HPLC in Bioequivalence study.

• HPLC machine is used in to measure serial plasma level of of the drug in bioequivalence study.

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Dru

g p

lasm

a le

vel

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Dru

g p

lasm

a le

vel

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Dru

g p

lasm

a le

vel

Cm

ax

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Dru

g p

lasm

a le

vel

Cm

ax

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Dru

g p

lasm

a le

vel

Cm

ax

Tmax

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Dru

g p

lasm

a le

vel

Cm

ax

Tmax

AUC

Bioavailabilty curves of Test drug and Reference drug

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0 10 20 30 40 50 60 70 800.00

0.20

0.40

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1.00

1.20

1.40

1.60

VOLUNTEER NO 1

Reference

Test

TIME (H)

PLA

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Derivation of required parameters from Bioavailability Curve

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0 10 20 30 40 50 60 70 800.00

0.20

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VOLUNTEER NO 1

Test

TIME (H)

PLA

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0 10 20 30 40 50 60 70 800.00

0.20

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1.00

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VOLUNTEER NO 1

Test

TIME (H)

PLA

SM

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ON

CE

NT

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MC

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Cmax

Derivation of required parameters from Bioavailability Curve

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0 10 20 30 40 50 60 70 800.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

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VOLUNTEER NO 1

Test

TIME (H)

PLA

SM

A C

ON

CE

NT

RA

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MC

G/M

L)

Tmax

Cmax

Derivation of required parameters from Bioavailability Curve

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0 10 20 30 40 50 60 70 800.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

VOLUNTEER NO 1

Test

TIME (H)

PLA

SM

A C

ON

CE

NT

RA

TIO

N (

MC

G/M

L)

Tmax

Cmax

AUC

Derivation of required parameters from Bioavailability Curve

When will the two drug be bioequivalent?

• To claim bioequivalence, 2 of the above 3 parameters should coincide within + 20% in the same volunteer between taking reference or test drug.

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Thank you

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