practical issues in uv, hplc analysis and formulation development

Practical issues in UV, RP-HPLC analysis, and Formulation development

Vijay Kumar Ekambaram M.Pharm. Pharmaceutics

e-mail 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 400nmWavelength

Day 03Day 02

Day 01

Issue 01

1mg/mL

1.2mg/mL

1.4mg/mL

Day 01

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

b

Analyte

Day 02

Day 03

Assume analyte has no chromophoric group that absorbs at 300nm (mid point of valley for the assumed analyte) and only responsible agent for absorbance is pure solvent.

Day 02

Day 01

Day 03

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

Consider the following case (below are the raw data)

0

-0.05

300nm

Strategy(s) sought to address the issue

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

• 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

P1

P2

P1=P2

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

Time

P1

P2

P1=P2

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

Time

P1

P2

P1=P2

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

Time

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

P1

P2

P1>P2

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

Time

P1

P2

P1>P2

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

Time

P1

P2

P1>>P2

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

Time

P1

P2

• A difference in pressure in the assumed inner and outer circle would suffice to theoretically explain the issue and look for solution.

Issue 03

Batch to Batch Variability in particle size and PI during replication (solid lipid nanoparticiles)

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

• Hot homogenization method was adopted. In this case the method followed demands to keep the temp. at 50C after addition of Aq. Phase to Org. Phase.

• However, due to lower temperature in the experimental bowl in replicate 2 setup, the lipid solidifies in less time thereby hindering/resisting easy size reduction and homogenization by the homogenizer giving relatively higher particle size range

• e.g. to explain why temp. difference between exp. bowl and indicator bowl.

• 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