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Practical issues in UV, HPLC analysis, and formulation development of Solid Lipid Nanoparticles
Vijay Kumar E M.Pharm. Pharmaceutics
Email ID: [email protected]
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
