Practical issues in UV, HPLC analysis, and formulation development of Solid Lipid Nanoparticles

Vijay Kumar E M.Pharm. Pharmaceutics

Email ID: evijay.kumar.phe12@itbhu.ac.in

Presentation outline

• Issue 01: Change in Emax and Valley depth

• Issue 02: RP-HPLC chromatograms peak splitting

• Issue 03: Batch to Batch Variability during replication

Issue 01

Change in Emax and Valley depth (UV-Spectrophotometer)

0

Peak 01

Peak 02

Peak 03

Valley 01Valley 02

200nm 400nm

Abso

rban

ce

(AU

)

Wavelength

Day 03Day 02

Day 01

𝐴l=𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙 .𝑎𝑏𝑠 .l−𝑃𝑢𝑟𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑎𝑏𝑠 .l

Issue 01

1mg/mL

1.2mg/mL

1.4mg/mL

Day 01

Why do peaks attain a new high Emax as the time progresses?

𝐴=𝑎𝑏𝑐

b

Analyte

Day 02

Day 03

Assume analyte has no chromophoric group that absorbs at 300nm and only responsible agent for absorbance is pure solvent.

Organic

𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙.𝑎𝑏𝑠 . l 300=1.47

𝐴l 300=1 .47−1 .50

Day 02𝐴l 300=1 .50−1 .50

𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙.𝑎𝑏𝑠 . l 300=1.50

Day 01

𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙.𝑎𝑏𝑠 . l 300=1.45

𝐴l 300=1 .45−1 .50

Day 03

Why do valley gets even more deep as the time progresses?

Consider the following case 𝐴l=𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙 .𝑎𝑏𝑠 .l−𝑃𝑢𝑟𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑎𝑏𝑠 .l

0

-0.05

300nm

Strategy(s) sought to address the issue

• Store at constant temperature (at 25C) (Temperature ∝ Solubility)

• Adequately tighten the lid of the solution holder

Issue 02

Peak splitting in RP-HPLC chromatograms

AU

-0.002

0.000

0.002

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

AU

0.000

0.005

0.010

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

A

B

A-Chromatogram with split peaks; B-Chromatogram with no split peaks

Strategies sought to address the issue

• Mobile phase ratio manipulation• Solvent effect• Guard column replacement

Negative

Negative

Positive

One factor variation at a time

Representation of elution in normal ODS column (guard column) and simultaneous AU-Time graph

Transverse plane view

Longitudinal plane view

P1

P2

P1=P2

Representation of elution in normal ODS column and simultaneous AU-Time graph

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts P1

P2

P1=P2

Representation of elution in normal ODS column and simultaneous AU-Time graph

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts P1

P2

P1=P2

Representation of elution in normal ODS column and simultaneous AU-Time graph

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts P1

P2

P1=P2

Representation of elution in normal ODS column and simultaneous AU-Time graph

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts P1

P2

P1=P2

Peak splitting in RP-HPLC chromatograms due to guard column malfunction

P1

P2

P1=P2

Peak splitting in RP-HPLC chromatograms due to guard column malfunction

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts

P1

P2

P1>P2

Peak splitting in RP-HPLC chromatograms due to guard column malfunction

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts

P1

P2

P1>P2

Peak splitting in RP-HPLC chromatograms due to guard column malfunction

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts P1

P2

P1>>P2

Peak splitting in RP-HPLC chromatograms due to guard column malfunction

Time

Conc

entr

ation

(mcg

/mL)

/ Ab

sorb

ance

uni

ts

P1

P2

Issue 03

Batch to Batch Variability in particle size and PI during replication

Response Replicate I Replicate II Replicate III

Particle size 291.6 513.9 290.9

PI 0.403 0.359 0.364

Replicate II

287.9

0.412

Temp. =50C

50.00Temp.

Setup for replicate 01 & 03

50.00Temp.

Temp. <<<50C

Setup for replicate 02

• Two unequal volumes of water cannot have same temperature when heated for the same time

25mL250mL

Time of heating process = 30min

A B

After heating for 30min; Temp. of A>>Temp. of B

Depth must be kept constant (crucial at low rotation frequency; found to effect PI)

A clamp

Strategy(s) sought to address the issue

• Minimize the background noise (like Constant temperature maintenance, ultra-sonication duration, depth of homogenizer probe immersion )

Thank you