Analyzing Active Pharmaceutical Ingredients with AccuSizer

Various dosage forms such as oral, parenteral, inhalation, topical, ophthalmic and suppository can be used to introduce an API to the patient. Tablets, suspensions and solutions come under oral dosage forms. The particle size in suspensions and tablets is vital for a number of reasons including process characteristics such as dissolution rate^1,2, content uniformity^3,4 and powder flow. When it comes to content uniformity, some large particles can cause a dose to surpass safe limits which can be detrimental to patient health.

A number of techniques are used to determine the particle size of active pharmaceutical ingredients (APIs) for oral dosage forms. These include sieves, microscopy, particle counting and laser diffraction techniques. Among these, microscopy is the most direct measurement and offers shape information. Sieves are typically employed for larger particle sizes (>50 µm) during powder analysis. Laser diffraction is considered to be the most common method, because it is repeatable, fast and covers a wide dynamic range. Counting techniques essentially have higher resolution and can give quantitative concentration results.

Single Particle Optical Sizing (SPOS) is an advanced, high resolution particle counter and particle size analyzer. Particles in liquid suspension flow via a photozone, where they interact with a laser light source through extinction and/or scattering (Figure 1). The scattering/extinction by the particle is related to size and concentration of particles through the use of a calibration curve and a pulse height analyzer. The result produced is the particle size distribution and concentration of the particles in suspension.

Figure 1. SPOS technique.

As stated before, laser diffraction is a standard particle size analysis technique, which is used in a wide range of industries, including the pharmaceutical industry (Figure 2). From Figure 2, it is seen that particles flow through a cell (4) that is illuminated by one or more laser light sources (1). Interactions between laser and particle produce scattered light collected on many angles and multiple detectors (6-7). The light scattering angles and particle size have an inversely proportional relationship; smaller particles scatter at higher angles and larger particles scatter at low angles. The scattered light is converted to a particle size distribution using proprietary algorithms based on either Mie or Fraunhofer theory. While the use of Mie theory can create more accurate results at smaller particle sizes (< 20 µm), it needs accurate refractive index (RI) values for the dispersed phase (the particles).

Figure 2. Laser diffraction technique.

Both laser diffraction and SPOS techniques were used in this study to determine the particle size distribution of an API powder suspended in liquid. The API suspension was then spiked with 50 µm polystyrene latex (PSL) particles in order to compare the sensitivity of both techniques against a second population outside from the main distribution.

Materials

A powdered form of Aripiprazole was used as the API in this analysis. The sample was inspected using the Malvern Mastersizer laser diffraction analyzer, dynamic range 0.2-2000 µm with the HydroS liquid sampler and the Entegris AccuSizer A7000 AD SPOS system with the LE-400 sensor, dynamic range 0.5 – 400 µm. The powder was then damped and dispersed using Igepal CA-630 (Octylphenoxy poly(ethyleneoxy)ethanol), a non-ionic surfactant, Sigma Aldrich product number I3021. This was followed by testing the AccuSizer with a 49.5 + 0.7 µm PSL standard from calibration kit part number 075DT0F, lot no. RA06B-N from Micro Measurement Laboratories. Next, the Mastersizer was tested with the help of a 50 µm PSL standard from Thermo Fisher cat no. 4250A, lot no. 44795, mean size = 49.5 + 0.8 µm. Since the SPOS technique can work at much lower concentrations than the laser diffraction method, two different PSL standards were used.

Experiment

The following procedure was used to prepare the API sample for the SPOS measurements:

• 0.05 g of API was weighed and transferred into a 250 mL beaker
• 3 drops of 0.1% Igepal CA 630 was pipetted onto the powder
• 150 mL of DI water was added to the beaker
• The powder was dispersed using an ultrasonic probe for 60 seconds

The following procedure was used to prepare the API sample for the laser diffraction measurements:

• 0.1 g of API was weighed and transferred into a 250 mL beaker
• 3 drops of 0.1% Igepal CA 630 was pipetted onto the powder
• 100 mL of DI water was added to the beaker
• The powder was dispersed using an ultrasonic probe for 60 seconds

The above sample preparations were slightly different because as mentioned above, the SPOS method can work at much lower concentrations when compared to the laser diffraction technique.

The background count was reduced to less than 200 particles/mL by flushing the Entegris AccuSizer A7000. The following measurement protocol is used:

• Flow rate: 60 mL/minute
• Sample volume: 100 µL
• Size threshold: 0.56 µm
• Sensor mode: summation
• Target concentration: 3500/mL
• Stirrer speed: 60%
• Equilibration volume: 2 mL
• Baseline offset*: 0

*A 0 baseline offset means that all counts from all channels were included in the result calculations.

Shown below is an overview of how the SPOS measurements are carried out:

1. The sample is continuously mixed by placing the beaker on a stir plate. This reduces any potential error caused by sub-sampling from the beaker into the analyzer.
2. The filtered DI water flows through the sensor until the background count of 200 particles/mL is obtained.
3. 100 µL of the sample was then pipetted into the 60 mL mixing bowl in the AD sampler.
4. The sample undergoes automated single-stage exponential dilution until the count rate falls below the target concentration of 3500 particles/mL.
5. Before the measurement begins, the 2 mL equilibration volume is allowed to pass through the sensor.
6. The sample is then measured for 60 seconds.
7. The system flushes until the background count is again obtained.

The following Mastersizer measurement protocol is used:

• Sensitivity: Enhanced
• Analysis model: Multiple narrow modes*
• Particle RI: 1.590, 0.01**
• Sample time: 12 seconds
• Dispersant RI: 1.33
• Pump/stir speed: 2500 rpm
• Ultrasound = off

* This model offers the highest resolution possible in order to resolve multiple peaks. It is seldom used for routine particle size analysis, but was selected to best detect the 50 μm PSL spikes.

** These RI values produced the lowest weighted residual values – the recommended method for choosing the RI of unknown samples (most APIs).

Shown below is an overview of how the Malvern Mastersizer laser diffraction measurements are carried out:

1. The sample is continuously mixed by placing the beaker on a stir plate. This reduces any potential error that may arise from sub-sampling from the beaker into the analyzer.
2. Clean DI water was allowed to recirculate through the system, while the optics was automatically aligned and the background was established to be less than 20 on the 20^th detector.
3. Sample was then pipetted into the HydroS sampler until the obscuration range falls between 5 and 15%.
4. The sample was then measured for 12 seconds.
5. The sampler was flushed two times until the background reduces to below 20 on the 20^th detector.

Results – Basic Particle Size Analysis

Figure 3 presents a graph showing four SPOS repeat results of the API suspension, and Table 1 shows a table summarizing the results.

Figure 3. Overlay of four SPOS results.

Table 1. SPOS result summary.

The AccuSizer software is capable of providing quantitative result calculations such as absolute volume and volume fraction, ppm/ppb. In this study, the tabular results were exported into Excel where calculations were made to establish the number of particles (droplets)/gram above the given sizes. Table 2 shows results, which reveal cumulative number of particles/gram greater than 0.63, 1.9, 5.4, and 10 μm for measurement R2.

Table 2. Cumulative number of particles/gram above given size.

Figure 4 shows a graph showing three laser diffraction repeat results of the API suspension, and Table 3 shows a table summarizing the results.

Figure 4. Overlay of three laser diffraction results.

Table 3. Laser diffraction result summary.

Discussion – Basic Particle Size Analysis

The AccuSizer SPOS and laser diffraction results are in good agreement, considering that these are two entirely different methods based on different principles. Compared to the Mastersizer laser diffraction analyzer, the AccuSizer can report a more narrow distribution. The span is a typical way to report the width of the particle size distribution, defined as

Span = (D90 – D10)/ D50 (Equation 1)

The span for the AccuSizer = (18.752 – 4.893)/9.997 = 1.386
The span for the Mastersizer = (23.214 – 4.592)/10.896 =1.709

Since laser diffraction is a lower resolution technique than SPOS, the 23% increase in the span for the former is not unusual. The AccuSizer results are produced by changing the individual pulses from particle and light interactions into a particle size based on a calibration curve. Therefore, each individual particle equally contributes to the final reported distribution, producing an essentially infinite resolution result.

The Mastersizer results are produced by averaging the ensemble light scattering from all of the particles over some time period. This averaged light scattering is subsequently converted to the reported distribution using a resolution-limited algorithm. Two properties of the resolution-limited laser diffraction results are (i) reduced sensitivity to tails of distributions outside of the main population and (ii) a broadening of the distribution (increased span) as observed in these results. The first effect is analyzed in the next section of this study.

Particle size results should be reproducible and repeatable. According to the USP test 429 – Light diffraction measurement of particle size – the predicted repeatability for three measurements should agree within a coefficient of variation (COV) of less than 15% at the D10 and D90, and less than 10% at the D50. The COV is defined as:

COV = (standard deviation/mean) x 100 (Equation 2)

The SPOS results were extremely repeatable, surpassing the requirements specified in USP test 429. The SPOS results reported COVs of 2.68% at the D10, 2.85% at the D50 and 1.93% at the D90, as shown in Figure 4. While no official USP test is available for the SPOS method, these results indicate that this is an appropriate method for particle size analysis of APIs. On the other hand, the laser diffraction results reported COVs of 8.652% at the D10, 3.823% at the D50 and 3.944% at the D90, as depicted in Table 3. These values are in accordance with the USP 429 guidelines.

Sensitivity to Tails

SPOS offers a number of benefits over laser diffraction such as greater sensitivity to tails and higher resolution results. Earlier studies have reported that the SPOS technique is about 600 times more sensitive to tails than the laser diffraction technique ^6,7. In this analysis, 50 μm polystyrene latex (PSL) standards were used to spike the API suspension in order to test for sensitivity to small concentrations of tails outside of the main distribution.

First 100 μL of the same API suspension used to create the results shown in Figures 2 and 3 were pipetted into the AccuSizer A7000AD, followed by pipetting a small amount of 50 μm PSL standard into the system. System sensitivity to the PSL spike was tested by introducing 100 μL and then 10 μL of the 50 μm PSL standard. The volume distribution result from the 10 μL spike of 50 μm PSL is shown in Figure 8. It is clear that the AccuSizer A7000AD had the required sensitivity to detect the 10 μL spike of 50 μm particles.

Shown in Figure 6 is the same result plotted as counts on the Y axis using the full 1024 size channel resolution as well as the defined region from 45 to 55 μm. The statistics for the defined region is shown in Figure 6. It was seen that the “counts” value of 33 is very close to the theoretical recovery value of 26. The data obtained from the counts versus size data could be used to define the presence of fines in the sample in a better way, because such fines can negatively affect properties like tablet compression or powder flow.

Figure 5. 10 μL spike of 50 μm PSL standard, volume Distribution.

Figure 6. 10 µL spike of 50 µm PSL standard, counts distribution and statistics for the 50 µm region.

Next, the Mastersizer laser diffraction system was used to perform a similar spiking study. To the API suspension, different amounts of a 50 µm PSL standard were added until the second peak was resolved by the laser diffraction instrument. The 50 µm peak was resolved after the addition of 250 µL of the standard into 100 mL of the API suspension (Figure 7), demonstrating an overlay of results from a 150, 175 µL and 250 µL spike of the 50 µm particles. While laser diffraction may resolve the second peak notice, it is still not an entirely separate population as predicted and as detected by the SPOS technique.

Figure 7. Spikes of 50 µm PSL standard, volume distribution.

Given that the laser diffraction technique fails to report the exact concentration, a direct, quantitative comparison of sensitivity to the spike of PSL particles was not performed in this study. However, when compared to the laser diffraction technique, a qualitative calculation indicated that the SPOS technique is about 700 times more sensitive to the presence of a second population. This compares well to other analyzes exploring the comparative sensitivity of both these methods^6,7. It must be remembered that the higher resolution “Multiple Narrow Modes” algorithm was used to measure the laser diffraction results, and not the “General Purpose” algorithm which most customers would use for standard analysis. As a result, the sensitivity could in fact be much lower for standard operation of the Mastersizer laser diffraction analyzer.

Conclusions

The SPOS technique is a high-resolution, high-0accuracy technique that can be used for measuring both particle size and concentration. The SPOS technique, compared to laser diffraction, reports a more accurate width of the particle size distribution with no false broadening. It is also very sensitive to tails separated from the main distribution, making it useful for detecting a few large particles that could occur in over dosage in tablets and content uniformity problems.

References

1. Hintz, R. Johnson, K. The effect of particle size distribution on dissolution rate and oral absorption, International Journal of Pharmaceutics Volume 51, Issue 1, 1 April 1989, Pages 9-17

2. Wang et al., Analysis of Diffusion-Controlled Dissolution from Polydisperse Collections of Drug Particles with an Assessed Mathematical Model, Journal of Pharmaceutical Sciences, 104:2998–3017, 2015

3. Zhang, Y., Johnson, K., Effect of drug particle size on content uniformity of low-dose solid dosage forms, International Journal of Pharmaceutics 154 (1997) 179-183

4. Yalkowsky, S.H., BoRon, S., Particle size and content uniformity, 1990, Pharm. Res. 7, 962-966.

5. Automated Microbial Identification and Quantitation: Technologies for the 2000s (book preview), section laser diffraction, edited von Wayne P. Olson and Laser Diffraction

6. D.F. Driscoll et al., Physicochemical assessments of parenteral lipid emulsions: light obscuration versus laser diffraction / International Journal of Pharmaceutics 219 (2001) 21–37

7. Nichols, K., et. al., Perturbation Detection Analysis: A Method for Comparing Instruments That Can Measure the Presence of Large Particles in CMP Slurry, report published by BOC Edwards, Chaska, MN

This information has been sourced, reviewed and adapted from materials provided by Entegris

For more information on this source, please visit Entegris