Abstract

The particle size distributions of powders have significant and potential effects on the quality of the final product. In case of biopharmaceutical industry the drug product properties like product appearance, solubility, processability, bioavailability, stability, and content uniformity are influenced to a considerable extent by the particle size of the drug substances. Particle size for Active Pharmaceutical Ingredient is a major parameter that can strongly affect the Active Pharmaceutical Ingredient processability, blending, flowability, compressibility, segregation propensity etc, in such instances the particle size distribution of the powders should be controlled using appropriate validated analytical methods.  Laser diffraction is one of the most widely used techniques for measuring the size of a wide range of particles from very fine to very coarse. The method is popular because it is quick and easy to use, flexible, and it can be adapted to measure samples presented in various physical forms. Laser diffraction provides several advantages including a wide dynamic range of the instrument, both dry powders and liquid suspensions can be measured and the technique is rapid and reproducible. As a case study it is necessary to have a systematic and standardized approach to the development and validation of a laser diffraction particle size method. To monitor production and particle stability, efficient and rapid methods for particle sizing are needed. Modern drug formulations are may also be particulate formulations where the particle size will be below 100 µm, reliable and reproducible methods are needed for the quality control of these drug products.

Keywords: Biopharmaceutical industry, Laser diffraction, Dry powders, liquid suspensions, Development, Validation.

Introduction

1. Laser Diffraction

Laser diffraction is on based particle size analysis relies on the fact that particles passing through a laser beam will scatter light at an angle that is directly related to their size. The particle size decreases, the observed scattering angle increases logarithmically and the scattering intensity dependent on its particle size, diminishing with particle volume.  Large particles therefore scatter light at narrow angles with high intensity whereas small particles scatter at wider angles but with low intensity as shown below in the figure-2.

Fig. 1: Light scattering pattern observed for a small particle.         Fig. 2: Light scattering patterns observed for a large particle

A typical setup for a laser diffraction instrument a representative sample, dispersed at an adequate concentration is passed through the laser beam in a measuring zone by a transporting fluid (air pressure or liquid), this measuring zone should be within the working distance of the lens used.

The light scattered by the particles at various angles is measured by a multi-element detector, and numerical values relating to the scattering pattern are then recorded for subsequent analysis. These numerical scattering values are then transformed, using an appropriate optical model and mathematical procedure to yield the proportion of total volume to a discrete number of size classes forming a volumetric particle size distribution. e.g., d₅₀ describe a particle diameter corresponding to 50% of the cumulative undersize distribution.

"In this article I made an attempt to explain some basic theory of light diffraction that can be understood by the users along with the guidelines which can be used during the initial stage of product development".

2. Apparatus and Equipment

The Particle size analysis was conducted using a long bed laser diffraction particle size analyzer equipped with wet and dry sample feeders. The Particle Size range for analysis using the instrument range of minimum 0.02 µm to maximum 2000 or to 3500 µm depending up on the requirement.

3. Method Development

Method development involves the selection of proper instrument and operating conditions to reduce method variability. A developmental study was conducted to assure that the analytical methodology provides the appropriate information by determining the critical product parameters the method is measuring.

4. Wet method development for laser diffraction measurements

The particle size analysis using wet dispersion is by far the most widespread method for obtaining reproducible results using laser diffraction. Wet analysis provides a method of dispersion for samples across a wide particle size range. It is possible to obtain good results using wet dispersion; can be achieve if develops a robust method. While developing a method, users should considered some important parameters, when defining a Standard Operating Procedure (SOP) for measuring wet dispersions using laser diffraction. However, it is important that the following factors associated with the sample are considered if realistic measurements are to be made:

a)Representative sampling

b)Dispersant selection

c)Measurement setting

d)Effect of Ultrasound

a)Representative sampling

Sampling of the material is the important aspects of particle size analysis. One of the goals of the proper method development is to minimize the total error. In general the larger particle in a bag of powder originates on the top in transit (figure-3). It is important to consider how to handle the sample, before taking the measurement. If sampling is ignored the developer doesn’t know which portion of the total error comes from the sampling. This is particularly true if large particles >75 microns in size are present in the sample will be the biggest potential error in the measurement. Whereas suspensions show the reverse within large particles undergoing sedimentation. In each case the material must be sampled in such a way as to remove the bias caused by these processes.

Fig 3: Classification of particles in transit

Laser diffraction is volume based measurement technique and is therefore sensitive to small changes in the amount of large material in the sample. This is because coarse particles occupy a large volume compared to smaller volume. One 100 micron particle has the same volume as one million 1 micron particles and will therefore give the same scattering response.

a)One million of 1 µ particle b)One 100 µ particle

The spinning riffler is the most reproducible method for obtaining a representative sample for powder samples when compared with other methods as describe in the (Table-1). Riffling of particle works best for free-flowing particles, can take a great deal of time when we have a large amount of powder to be handled.

Method	Estimated maximum error
Cone and Quartering	22.7%
Scoop Sampling	17.1%
Table Sampling	7.0%
Chute Riffling	3.4%
Spinning Riffing	0.42%
Table-1: Typical error’s associated with different sampling techniques

Spinning rifflers

If the distribution of particles within a sample is particularly broad, representative sampling can be difficult. If problems continue, using a spinning riffler may help. This is a vibrating hopper which vibrates the sample down a chute. The act of vibrating the sample causes the larger particles to separate out and travel down the chute first. At the end of the chute a set of rotating pans collects the sample evenly. When the entire sample has passed down the chute each collecting pan will contain a representative sample.

b) Dispersant selection

Factors such as sample quantity, safety requirement, flow properties and particle morphology must consider when selecting a dispersion technique. Selection of an appropriate dispersant for wet laser diffraction measurements, it is necessary to use a dispersion medium in which the particles are not soluble. Sometimes this is not possible so as a last resort, a saturated solution of the compound in the dispersion medium can be prepared and filtered through a suitable filter immediately prior to use.

Dispersant selection is one of the most important activities determining the success of a particle size measurement. It is important that the chosen dispersant:

§ Solubility: Does not dissolve the material under test during the measurement. To check the suitability of the dispersion medium the particles are observed microscopically and by laser diffraction over the course of the experiment to ensure they do not dissolve or agglomerate and dispersion stability is achieved. In addition, the solubility in the dispersion medium is evaluated using HPLC. Solubility must be 5g powder in 1 kg liquid.

§ Polarity: The polarity of the liquid medium should not cause the material to preferentially adhere to glass surfaces - check this by placing some of the sample material in a small beaker and checking that it remains in suspension.

§ The dispersant used should be transparent to the laser beam, ensuring the light scattered measurements can be made.

§ Dispersant should have the different refractive index to the particles being measured; it is the refractive index differences which defines the intensity of scattering observed.

§ Can wet the particles being measured, ensuring agglomerate dispersion.

§ The sample should not form solvates or hydrates in a particular dispersion medium must be known so those dispersion medium can be avoided.

§ Evaluate stability of the sample in dispersant over the course of the experiment, such that re-agglomeration does not occur.

§ Dispersant should not too viscous as otherwise bubble formation may occur during measurements.

§ Vary sample sonication time until particle size does not change with increase in sonication time and particles remain intact based on optical microscopy.

§ Dispersant should not react with sample being measured does not swell or shrink particles by more than 5% in diameter and compatible with materials in the analyzer.

§ Should not form sedimentation and breakups the sample being measured in the dispersion medium.

§ Dispersant not be hazardous to heath and meet safety requirement.

If there is significant material below 20µ in the sample then the major factor governing repeatability will be the dispersion of the sample. Lesser the sample size, dispersion will be the important factor to look in to consideration.

It may also be necessary to add surfactants and other stabilizers to prevent agglomeration of fine particles. Many materials, especially pharmaceuticals actives, are soluble in water. For such materials it will be necessary to use a less polar dispersant (table 2). The choice of dispersant will be depend on the polarity of the material under test. The cost of disposal of some nonaqueous dispersants is also an important consideration.

Table- 2: Dispersants for laser diffraction measurements

Surfactants

Surfactants (Surface active agents) are often required to wet the powder for proper dispersion. In some cases it may be necessary to use a surfactant to cause wetting to occur. Surfactants works by lowering the surface tension of the dispersant. This reduces the contact angle between the dispersant and the particle surface. The choice of surfactant depends on the particle surface chemistry and nature of the dispersant. The frequent use of surfactants including Micro 90 solution (also good for cleaning the instrument). Non-ionic surfactants (e.g.  Tween 20/80, Span 20/80, Triton X-100, Igepal, CA630, Aerosol OT and lecithin are generally considered safe for use within pharmaceuticals or food application. In some applications anionic surfactants (e.g. SDS-sodium dodecylsulfate and sodium bis 2- ethyl hexylsuccinate or cationic surfactants (e.g. CTAB- Cetyl triethyl Ammonium bromide, Hyamine 2389) can use not only improve wetting but also stabilize the particles surface, thus providing a barrier to agglomeration.

In all cases the concentration of surfactant should be carefully controlled. Surfactants are active at very low concentrations (>0.001 g/l). Generally 1-2 drops of a diluted surfactant solution (10% concentration) is sufficient to improve particle wetting. Using higher concentrations can lead to bubble formation within the dispersion unit, causing the reporting of large particles that are not present in the sample.

Stabilizers

It sometimes helps to add a stabilizer (or admixture) to the sample, admixtures can increase or alters the particle charge, it is important to ensure that the re-agglomeration is prevented. Within aqueous dispersants controlling the conductivity of the suspension can ensure stability. This can simply involve using deionised water as a dispersant rather than tap water. It is possible to use charge-stabilizing agents such as sodium hexametaphosphate (Calgon), Sodium Pyrophosphate, Ammonium Citrate, Calcium chloride, Trisodium Phosphate, Sodium Oxalate  and pH adjustment can also important. Each of these increases the stability of a suspension by increasing the zeta-potential (this is related to the particle charge). However, just as with surfactants, adding too much of these stabilizing agents can cause dispersion problems. Typically the concentration of these species should not be higher than 1w/v% otherwise the conductivity of the solution will be too high, causing agglomeration to occur. As many of these are solid materials that are dissolved into the dispersant, the solution should be filtered after preparation to remove impurities.

Optimizing Dispersant Selection

It is possible to choose the correct dispersion conditions without the need for carrying out particle size measurements. The sample under test can be dispersed in a series of vials containing different dispersants. If the particles float on the top of the dispersant this suggests that wetting is poor – this can be improved by either adding surfactants or by moving to a dispersant with a lower surface tension. If the particles are wetted by the dispersant then the user should sonicate the sample and then observe the settling behavior.  

A simple wetting with admixture with i.e. sodium hexameta phosphate leads to good dispersion test. Addition of more sodium hexameta phosphate admixture will lead to dissolve or deagglomeration.

While optimizing the dispersion selection, the user must need to consider and understand that most of errors during particle size measurement arise through poor sampling and dispersion and not through instrument inadequacies.

Determine the Refractive Index (RI)

It is important to enter the appropriate refractive index (RI) value for the sample and for dispersion medium. Methods to obtain the sample RI include literature and internet searches, use of a suitable refractometer to determine the refractive index values for the sample and dispersant. It is better to determine the refractive index early in the method development process in order to understand the general shape of the distribution before conducting others parameters for method development and method validation.

Small difference in the assumed complex refractive index may cause significant differences in the resulting particle size distributions.

c) Measurement Settings

During method development it is important to consider how the amount of sample and the dispersion unit setting that affect the measurement of particle size.

Pump and stirrer setting

The pump and stirrer speeds for the liquid sampler may also have an effect on results and need to be investigated during the method development process. The goal is to keep the system well mixed so that all of the particles are analyzed while keeping the agitation below the level that creates bubbles or entrains air. Large, heavy particles require higher pump and stir speeds. Optimization of the pump / stirrer rate can be simply be achieved by determining how the reported particle size changes as the pump speed is increased.  

Note that users should not default to using the fastest possible pump rate as this may lead to bubble formation in some dispersants. High pump speeds can also cause particle break-up, especially for emulsion samples. To avoid bubble formation more gentle agitation may be better for samples containing surfactants.

Sample Concentration

To get good reproducible results the obscuration range what we are measuring should get good signal to noise ration. The sample concentration used for wet laser diffraction measurements must be set so as to allow reproducible scattering data to be obtained without observing multiple scattering .For fine particle measurements (<10 microns in size) this will require users to measure at not more than 10% obscuration. For coarser materials measurement obscurations of up to 20% can be used in order to maximize the single to- nose ratio. The concentration at which multiple scattering occurs can be assessed by measuring the particle size of a stable dispersion as a function of the measurement obscuration. At low obscurations the reported particle size will be constant – this is where the correct result is obtained. The reported size will shift towards the finer particle sizes at higher obscurations as multiple scattering starts to occur.

The good obscuration range for determination of particle size measurement using wet method is between 5 – 10%. (Figure-4)  

Fig 4:Particle Size as a function of sample concentration

d) Effect of Ultrasound

Ultrasound can be used to add energy into the system in order to disperse agglomerates into individual particles. Very difficult/agglomerate sample particles require the use of high external effect of ultrasound. It suggests that users must understand how the input of dispersion energy affects the particle size. For wet measurements this involves determining how the dispersion is affected by the application of ultrasound. This is typically accomplished by exposing the sample to ultrasound for varying lengths of time. As an example the wet method developer should follow/observe the optimization conditions while applying ultrasound for the measurement of solid particles as follows:

§Initially wetting of sample is needed for solid particle measurement. (as described in  sub-section: Imperfect wetting)

§Analyze the solid sample prior to applying ultrasound.

§Turn on the ultrasound in the sampler unit for 5 second to understand the dispersion of the sample.

§ Turn off the ultrasound and measure the sample to ensure the dispersion is stable.

§Turn on the ultrasound in the sampler unit for 5 more second and measure the sample.

§Repeat this procedure until results begin to stabilize or an indication of particle breakage is observed.

Note: The 5 second interval of ultrasound may vary depending on the sample sensitivity to additional energy input.

Solid particle measurement for Wet Method Development

§Obtain the representative sampling especially for solid particle measurement.

(Refer section: representative sampling)

§Initial wetting of sample in the dispersion unit the particle size may slowly decrease this is due to the dispersion of loosely bound agglomerates under of action of pump and stirrer.

§If the sample particles float on the dispersant surface add surfactants or stabilizers/add mixtures to obtain a uniform dispersion of sample.

Note: At this point of time if the user observed the rapid decrease in obscuration, it is suggested that the material under test is rapidly dissolving in the proposed / chosen dispersant; this is associated with an increase in the measured particle size, as the fines present in the sample will be dissolved more rapidly. In this case a different dispersant should be used.

§The initial stage of dispersion, ultrasound is applied if rapid dispersion is normally observed at this stage as strongly bound agglomerates are dispersed. As the sonication time is increased the particle size should reach a plateau where the particle size becomes stable which represents the fully dispersed state.

Note: If the particle size continues to reduce over time this may suggest that particle breakup occurs during sonication. Sonication may also cause agglomeration to occur which suggest that the dispersion is unstable.

§The particle size should be monitored with the ultrasound off once the full dispersion is achieved. If the particles size remains stable then the dispersion conditions are optimized.

§The user must understand the need of extent of sonication, pump/ stirrer rate, use surfactants/stabilizers to adjust the dispersion conditions as described below in the flow chart. (Figure-5).

Fig 5.Flow chart diagram for Solid particle measurement for Wet Method Development

Examples for Wet Method Development

The below following example show how the wet dispersion of material can be optimized following the above guidelines.

Prednisolone Acetate

During the initial stage of method development, representative sample been obtain and select the dispersant medium as de-ionized water based up on the solubility of the particles. De-ionized water having different refractive index selected when compared with refractive index of the above sample. Polarity of the sample been observed and it was not adhere to glass surfaces of the beaker and it remains in suspension during solid sample preparation for particle size measurement. Added 1-2 drops of 10% Tween 80 used as diluted surfactant in order to improve the particle wetting. Particle size measured during dispersion under the action of the pump and stirrer within the dispersion unit. As seen in the (figure-6) the particle size of Dv90 reduces during this process, as loose the agglomerates are dispersed.

Secondly ultrasound within the dispersion unit is switched on, causing further rapid dispersion. The ultrasound continues until a stable particle size is obtained. The fact that the particle size stabilized shows that full dispersion is achieved based up on the fact that the particles within the dispersion medium is observed without particle break-up, the size of the particle gradually reduce if break-up of the particle occurred.

Thirdly the ultrasound was switched off in order to assess the dispersion was stable and particle size remain constant showing the dispersion is stable.

Fig 6: Particle size distribution statistics obtained during the dispersion of Prednisolone acetate.

Reproducibility Check

Once all the important aspects of the method development been established it is time to check for the reproducibility. Both ISO 13320 and USP <429> establish reproducibility goals at the Dv10, Dv50, and Dv90 based on the coefficient of variation (COV) , standard deviation (SD) and average on five different measurements.

Standard	COV
	Dv 50/ microns	Dv 10 and Dv 90/microns
ISO 13320-1 (Section 6.4)	< 3%	< 5%
USP <429> and EP 2.9.31	< 10%	< 15%

Table- 3   Reproducibility acceptance criteria

Note: The above limits can be doubled if the Dv 50 <10µm.

The results and Co-efficient of variation for prednisolone acetate (table-4) measured on six different sample of prednisolone acetate based up on the above dispersant and measurement settings on advance laser diffraction particle size analyzer.

Measurement	Sample	Dv 50/micron	Dv 10/micron	Dv 90/micron
1	Prednisolone Acetate-1	7.940	3.156	16.222
2	Prednisolone Acetate-2	8.046	3.266	16.828
3	Prednisolone Acetate-3	7.996	3.207	16.286
4	Prednisolone Acetate-4	7.874	3.148	16.323
5	Prednisolone Acetate-5	8.022	3.106	16.456
6	Prednisolone Acetate-6	8.091	3.228	16.665
Mean		7.990	3.19	16.46
Standard deviation (SD)		0.08	0.06	0.24
Coefficient of Variation (COV)		0.97%	1.85%	1.45%

Table-4: Six different readings of prednisolone acetate with wet dispersion method

Conclusion

For development of a wet method requires user to explore the systematic and comprehensive approach and understand the way to obtain the representative sample. Selecting the choice of dispersant and surfactants may involve trial and error for unknown samples. Optimizing the other measurement such as pump and stirrer settings, sample concentration and application of ultrasound following the procedures as described and assurance should be provided that the results generated are robust and reproducible and control the quality of the product can be achieved by validating the method ensuring good batch to batch reproducibility.

References

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About Authors:

Abdul Kaleem