Validation of a Particle Size Determination Method
for Nano-particles by Dynamic Laser Light Scattering
AAPS Annual Meeting 2010
J. Brunotte, M. Darter, J. Vaughn and V. Kulkarni
RESULTS
DPT Laboratories, San Antonio, Texas
0.500
4.0
R2 = 0.9982
0.250
3.0
0.200
0.150
2.0
0.100
1.0
0.050
0.0
1
2
3
4
5
6
Fig. 1: Measured diameters of standard particles are plotted against
the certified values. A good correlation (R2 >0.999) is seen for the
particles in size range of 60 nm to 350 nm and R2 of 0.998 for range
of 60 nm to 5 µm. However, large deviation (≥10%) from the standard
value is observed for the large particles.
Standard
Particles of
100 nm
Robustness
71
70
69
68
67
66
65
64
63
62
10
15
20
25
Concentration of KCI (mM)
Robustness
2.50
Average
Minimum
Median
Maximum
SD
% RSD
Z
Average
(nm)
Polydispersity
Index
104
103.3
103.8
102.7
103.9
103.5
104.4
102.6
104.3
104
103.9
104.2
103.4
103.6
102.6
103.2
103.5
103.8
103.6
102.6
103.7
104.4
0.55
0.53
0.006
0.003
0.015
0.002
0.006
0.007
0.006
0.011
0.032
0.015
0.011
0.017
0.005
0.013
0.011
0.004
0.012
0.016
0.011
0.002
0.011
0.032
0.01
66.60
0.050
0
1
2
3
4
5
6
7
8
9
10
11
12
60
300
40
200
20
0
500.0
0.150
400.0
300.0
0.100
200.0
0.050
100.0
1
2
3
4
5
6
7
8
9
10
11
12
0.000
0
0
System
Temperature (°C): 20.0
Duration Used (s): 70
Count Rate (kcps): 230.9
Measurement Position (mm): 0.85
Cell Description: Disposable sizing cuvette
Attenuator: 2
Results
Results
Peak 1:
Peak 2:
Peak 3:
315.8
0.070
0.924
Good
% Intensity
100.0
0.0
0.0
Width (d.nm)
92.41
0.000
0.000
Z Average (d.nm):
Pdl:
Intercept:
Result Quality:
Mean
Intensity
Peak (nm)
% Intensity
89.8
8.5
1.7
Width (d.nm)
240.4
21.55
4.240
10
8
6
4
1
10
100
1000
10000
Robustness
350.00
Measurement
of Intralipid®
300.00
250.00
Average
SD
% RSD
200.00
150.00
10
15
20
25
30
Temperature (°C)
DPT 10% emulsion
Intralipid 20%
Fig. 4: Particle size (Z average) for Intralipid® and the proprietary
nano-emulsion were measured at different temperatures. Data
show that, for both products, particle size is not influenced by the
sample temperature in the range of 15-30oC.
35
Analyst I
Analyst II
Particle Size (nm)
295.8
312.4
296.7
316.2
300.9
315.7
295.8
314.6
296.7
314.7
300.9
313.8
297.8
314.6
2.4
1.4
0.8
0.4
Pooled
Average
306.2
Pooled SD
% RSD
8.97
2.93
1
2
3
4
5
6
7
8
9
10
11
12
Attenuator
Fig. 8: Trend of polydispersity index and particle size with the
attenuator (inversely related to the sample concentration) for a
nano-emulsion.
40
500.0
20
0
0
1
2
3
4
5
6
7
8
9
10
11
12
0.0
Attenuator
Fig. 9: The trend shows that for the nano-emulsion, with increasing
attenuator, transmittance of light increases while turbidity decreases.
Both transmittance and turbidity reach equilibrium for attenuators 6-11.
MATERIALS AND METHODS
1
10
100
1000
10000
Size (d.nm)
Record 344: 20% Intralipid IV Emulsion
Fig. 11: A very concentrated sample of Intralipid® showed multiple
peaks with high polydispersity index (0.45) at attenuator of 2, suggesting
that the sample concentration is too high for accurate size analysis.
System
Temperature (°C): 25.0
Duration Used (s): 20
Count Rate (kcps): 237.0
Measurement Position (mm): 4.65
Cell Description: Disposable sizing cuvette
Attenuator: 11
System
Temperature (°C): 25.0
Duration Used (s): 20
Count Rate (kcps): 78.3
Measurement Position (mm): 1.25
Cell Description: Disposable sizing cuvette
Attenuator: 11
Results
Z Average (d.nm):
Pdl:
Intercept:
Result Quality:
390.9
Peak 1:
0.203
Peak 2:
0.864
Peak 3:
Refer to quality report
Size (d.nm)
353.0
2528
0.000
% Intensity
90.0
9.0
0.0
Width (d.nm)
76.95
183.5
0.000
Results
Z Average (d.nm):
Pdl:
Intercept:
Result Quality:
Size (d.nm)
2478
0.000
0.000
3933
Peak 1:
0.984
Peak 2:
0.124
Peak 3:
Refer to quality report
Size Distribution by Intensity
% Intensity
100.0
0.0
0.0
Width (d.nm)
210.9
0.000
0.000
DISCUSSION
Specificity of the method was evaluated by using different types of particles, including nano-emulsions and suspended particles. Attenuation
index data from the Zetasizer was correlated to the turbidity of the sample and transmittance at the visual wavelength range of 600-700 nm.
The studies indicated that the size of standard nano-particles determined by dynamic light scattering technique showed out-of-specificationhigh results in the dispersant without a strong electrolyte, whereas in the presence of an electrolyte the size data passed the specifications,
suggesting that the presence of small amounts of strong electrolyte is essential for accurate size measurements. This observation is consistent
with previously reported data.7 Results obtained for standard particles >1 µm showed >10% deviation from certified value, indicating that
additional sample manipulation may be required, including density matching.8
Size Distribution by Intensity
20
50
15
40
10
5
CONCLUSIONS
30
20
10
100
1000
10000
0
1
10
100
16
Dv10
0.800
0.750
Results
Dv50
Dv90
Z Average from Nano-S
0.700
10
0.650
8
0.600
6
0.550
4
0.500
2
0.450
0
0.400
1200
0
200
400
600
800
1000
Concentration (µg/mL)
Fig. 14: Comparison of size distribution data obtained from
Mastersizer and that from the Nano-S for Kaolin.
Fig. 13: A sample of deionized water showed polydispersity index of 0.98
and attenuator of 11, suggesting that when attenuator of 11 is reached,
sample dilution is too high to accurately measure the size.
System
Temperature (°C): 20.0
Duration Used (s): 70
Count Rate (kcps): 197.7
Measurement Position (mm): 4.65
Cell Description: Disposable sizing cuvette
Attenuator: 7
Specificity
12
10000
Record 882: di water 1
Fig. 12: A very dilute sample of Intralipid® showed two peaks and high
polydispersity index (0.2) at attenuator of 11, suggesting that the sample
concentration is too dilute for accurate size analysis.
14
1000
Size (d.nm)
Record 850: 1.0 ppm IL Suspension 2
Table 2: Particle size (Z average, in nm) data for Intralipid® obtained by
two analysts is shown.
Fig. 3: Effect of measurement duration on 2 µm standard particles.
The error bars indicate variation of ±1 standard deviation in the
measured diameters.
0
140.0
1000.0
5. Kaolin Colloidal Powder (hydrated aluminum silicate), USP (Mineral and Pigment Solutions Inc., South Plainfield, NJ).
Z Average (d.nm):
Pdl:
Intercept:
Result Quality:
636.0
Peak 1:
0.560
Peak 2:
0.887
Peak 3:
Refer to quality report
Size (d.nm)
305.7
2617
0.000
% Intensity
66.0
33.0
0.0
40
30
20
10
0
10
100
Data showed that measured value of diameters of standard particles was linear in the range of 60 nm to 350 nm; however, for particles
of ≥1 µm, deviation of >10% from the certified value is observed, suggesting that the method is not suitable for large particles.
The concentration of the particles in suspension is critical for accurate measurement of size by dynamic light scattering.
A sample of pure deionized water showed the presence of particles, suggesting that it is critical to review other parameters, including
polydispersity index and attenuation index, when interpreting the data obtained from the instrument.
The dynamic light scattering technique is not suitable for the particles that have a tendency to settle. For the product comprised of
particles of >1 µm, it is important to study effect of “measurement duration” during method development.
The study indicates that particle size determination method using a dynamic laser light scattering technique can be validated in
accordance with the FDA guidelines.
REFERENCES
50
1
•
•
•
•
•
Width (d.nm)
47.37
113.6
0.000
Size Distribution by Intensity
Intensity (%)
Measurement Duration (s)
0.000
60
4
0
Fig. 10: At an appropriate dilution, Intralipid® showed a single sharp
peak with low polydispersity index (0.07) and attenuator of 8.
Intermediate Precision
70
0.020
1500.0
4. Intralipid® 20% (Baxter Healthcare Corp., Deerfield, IL) and Proprietary Nano-emulsion.
Size (d.nm)
60
145.0
80
6
Record 853: 50 ppm Intralipid 2
1
50
0.040
2000.0
3. Suspensions of Standard Particles of 60 nm to 5 µm diameter (Thermo Scientific Inc., Fremont, CA).
Size (d.nm)
2.00
40
0.060
100
8
0
30
150.0
2500.0
120
2
2
2.10
20
0.080
Turbidity
2. Turbidimeter, 2100N (Hach Company, Loveland, CO) and UV Spectrophotometer (UV-1700, Pharma Spec, Shimadzu Scientific Instruments,
Columbia, MD).
10
12
200
155.0
0.100
Transmittance
1. Particle Size Analyzer: Zetasizer Nano S (ZEN 1600) and Mastersizer 2000 (both from Malvern Instruments, Westborough, MA).
Size Distribution by Intensity
14
0
Size (d.nm)
479.5
71.68
22.25
Peak 1:
Peak 2:
Peak 3:
277.1
0.452
0.743
Good
Size Distribution by Intensity
16
150
Fig. 7: The graph shows dependence of polydispersity index (PDI)
and turbidity on the concentration of the sample of Intralipid.®
Fig. 6: The trend shows that for Intralipid® with increasing attenuator,
transmittance of light increases while turbidity decreases. Both
transmittance and turbidity reach equilibrium for attenuators 9-11.
Size (d.nm)
344.3
0.000
0.000
100
0.0
250
Particle Size
Concentration of Sample (µg/mL)
System
Temperature (°C): 25.0
Duration Used (s): 20
Count Rate (kcps): 332.9
Measurement Position (mm): 4.65
Cell Description: Disposable sizing cuvette
Attenuator: 8
Z Average (d.nm):
Pdl:
Intercept:
Result Quality:
50
Attenuator
Fig. 5: Trend of polydispersity index and particle size with the
attenuator (inversely related to the sample concentration) for
Intralipid.®
104.7
104.1
104.1
103.5
104.8
104.7
105.1
103.8
104.2
104.3
104.4
104.4
104.7
103.9
103.9
103.9
104.9
105.6
104.4
103.5
104.4
105.6
0.53
0.51
Turbidity
100
0
250.0
0.200
PDI
Particle Size (nm)
400
PDI
10
10
0.120
Turbidity (NTU)
500
80
Attenuator
2.20
0
600.0
160.0
Transmittance at 700 nm (% T)
270.0
2.30
1.90
0.140
Polydispersity Index (PDI)
0.100
% Transmittance
Polydispersity Index (PDI)
290.0
2.40
100.00
Particle Size (nm)
0.150
Particle Size (µm) Distribution (Mastersizer)
Particle Diameter, Z Average (µm)
Fig. 2: Influence of an electrolyte in the media (KCl) on the
measured diameters for standard latex particles of 60 nm.
Particle Diameter, Z Average (nm)
0.200
100
Z Average (µm) from Nano-S
Particle Diameter, Z Average (nm)
72
5
310.0
Table 1: Data given in the Table shows particle size (Z average, nm),
polydispersity index and mean intensity peak (nm) for 100 nm standard
particles. Measurements were made on triplicate sample preparations
with six replicate measurements per sample preparation. Average,
median, standard deviation (SD), percent relative standard deviation
(% RSD), minimum and maximum values determined from the 18
measurements are shown in the Table.
Certified Diameters of Standard Particles (µm)
0
0.250
600
Turbidity
Intensity (%)
R = 0.9994
2
0
330.0
Intensity (%)
0.350
0.000
0.300
Intensity (%)
5.0
Measured Diameters, Z Average (µm)
Measured Diameters, Z Average (µm)
0.400
0.300
350.0
0.350
Reproducibility
Linearity
370.0
Intensity (%)
In order to evaluate specificity, samples of emulsions and dispersed solid particles were characterized using both DLS and laser diffraction
techniques. Robustness was evaluated by monitoring the effects of dispersant electrolyte concentration, sample concentration,
measurement duration and temperature.6 Linearity and reproducibility were determined using certified sizing standards, and
intermediate precision was evaluated using a second analyst. LOD was determined by sample concentration and correlating it with
attenuation index, turbidity and transmittance.
Size (nm)
0.400
0.000
700.0
PDI
Transmittance at 700 nm (% T)
The transfer of methods for size determination from a method development laboratory to a quality group necessitates validation in
accordance with FDA regulations.4 The current study focuses on method development and validation of a size determination method
for drug products comprised of nano-particles by DLS and focuses on the following validation parameters: 1) Linearity, 2) Robustness,
3) Specificity, 4) Reproducibility, 5) Intermediate Precision and 6) Limit of Detection.5
0.250
700
Limit of Detection
Limit of Detection
Limit of Detection
120
390.0
0.450
Polydispersity Index (PDI)
Analysis of particle size is of paramount importance in pharmaceutical product development. Although there are several methods for
size characterization of particles, light scattering and optical microscopy methods are typically employed for particle size analysis in
pharmaceutical product development and quality control activities.1,2 Because the light scattering technique for size measurement
is rapid, reproducible, easy to use and suitable for both wet and dry particles, and because the software is compliant with regulatory
requirements of quality control/assurance laboratories, it is a preferred technique in pharmaceutical product development and stability
studies. For wet particles smaller than 1 µm, dynamic laser light scattering (DLS), also known as photon correlation spectroscopy (PCS),
is commonly used.3
RESULTS
Limit of Detection
Limit of Detection
Turbidity (NTU)
INTRODUCTION
1.866.CALL.DPT
W W W. D P T L A B S. C O M
1000
10000
Size (d.nm)
Record 45: 100 ppm Kaolin
Fig. 15: A sample of 100 ppm Kaolin showed multiple peaks and high
polydispersity index of 0.560.
1.
2.
3.
4.
5.
6.
7.
8.
Jillavenkatesa, A., Dapkunas, S.J., and Lum, L-S.H. “Particle Size Characterization” NIST Special Publication 960-1; U.S. Department of
Commerce, U.S. Government Printing Office: Washington, D.C., 2001; pp. 1-2.
Meijer, N., Abbes, H., and Hansen, W.G. Particle size distribution and dispersion of oil-in-water emulsions; an application of light
microscopy. American Lab. 33(8), 28-31(2001).
Burgess, D.J., Duffy, E., Etzler, F., and Hickey, A.J. “Particle Size Analysis: AAPS Workshop Report, Cosponsored by the Food and Drug
Administration and the United States Pharmacopeia” AAPS J. 6(3), 1-12 (2004).
FDA Draft CMC Guidance for Industry. “Analytical Procedures and Methods Validation” Section XI, Part F, August (2000).
USP General Chapter <429> “Light Diffraction Measurement of Particle Size” Pharmacopeial Forum 28(4), 1293-1298 (2002).
“Method Validation for Laser Diffraction Measurements” Malvern Application Note MRK671-01.
“The Measurement of Latex Standards by Photon Correlation Spectroscopy” Malvern Application Note MRK111-01.
“Measuring NIST Traceable Standards Using Dynamic Light Scattering (DLS)” Malvern Application Note MRK481-01.
Turbidity (NTU)
Poster# W4110