Validation inParticle Size Measurement

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By Steve Ward-Smith, PhD and Alan Rawle, PhD

Considerable information is available on the theory behind laser diffraction technology for particle sizing, as well as guidance on both sampling and dispersion techniques. Publications include the ISO 13320-1 document Particle size analysis — Laser diffraction methods — Part 1: General principles,1 and Particle Size Characterisation,2 the Special Publication 960-1 of the National Institute of Standards and Technology (NIST). However, information to guide validation of the chosen method is limited.

Validation is intended to determine the robustness and integrity of a particle sizing method by testing the parameters that could cause variation in the reported size. The United States Food and Drug Administration describes it as “establishing documentary evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specification and quality attributes.”

Development of a validated method should be undertaken using an instrument with validated software, which is regularly tested to confirm its performance. Validated software has life-cycle documentation detailing its development and maintenance and should be numerically validated using a peer package such as Microsoft® Excel. It should also be compliant to 21 CFR part 11 covering the use of electronic records and signatures.

The main variables to be considered are: sampling; sample preparation; instrument range; appropriateness of the technique; robustness of the analytical method; amount of light scattered by the sample; and the reproducibility and precision of the measurement.

Sampling
A sample should be representative of the bulk material. During transit, settling can occur — in powders, large particles tend to settle at the top of the sample container, whereas in suspensions, large particles will sediment. Material must be sampled in a way that removes the bias caused by these processes.

Using a spinning riffler gives the most reproducible and representative samples3 for powders when compared with alternative methods, such as scoop sampling, table sampling, cone and quartering, and chute riffling. Riffling works best for free-flowing particles but is time-consuming when large amounts of powder must be handled.

For slurries, re-suspending the sample overcomes sedimentation issues. This may require only simple stirring. However, certain stirrers, such as magnetic fleas, can cause large particles to be thrown to the outside of the container where they are not sampled.

Sample Prep
Having obtained a representative sample, correct presentation to the instrument is vital.4 The preparation method will depend on the interests of the user and should be investigated as part of method development rather than method validation. Where, for example, the primary particle size is important, correct sample dispersion will be important; if the natural agglomerated state is of interest, sample preparation should take this into account to avoid breaking up agglomerates. The dispersion medium — air or liquid — should not cause irreversible changes to the particle size through processes such as dissolution, milling or aggregation.

Measuring Range
The range of the particle sizing instrument should ideally cover the size range of the sample. This is not normally a problem for most pharmaceutical samples: modern laser diffraction systems can cover sizes from 20 nanometres to 2,000 micrometres in a single measurement. Older instrumentation, however, may need many lenses to cover the same dynamic range; in such cases, the lens covering the largest proportion of the size distribution should be used. Alternatively, the result from two lenses can be blended, but this is not recommended because the result depends on the mathematical efficacy of the blending routines.

Specificity
Specificity — whether or not the technique is appropriate to the material under analysis — should be addressed as part of method development and does not necessarily need revisiting for method validation. Different sizing techniques exhibit different sensitivities and will provide different results for the same sample. Selection of an appropriate technique depends on what is of interest. For instance, is detecting a small amount of over-sized material important, or must the technique differentiate between different particle types? Laser diffraction provides a good method for assessing small changes in size distributions, but differentiating between the different components in pharmaceutical dosage forms is more difficult.

Robustness
The robustness of an analytical method indicates its ability to remain unaffected by small variations in the test parameters and provides assurance of its reliability in routine use. Robustness should be considered ahead of repeatability, reproducibility and intermediate precision.

Measurement duration and stability are the two main variables. Others, including air pressure (dry measurements) and pump/stir rates (wet measurement), are normally considered as part of method development, but are briefly described here.

Measurement duration
To determine suitable measurement duration, a cycle of 10 measurements should be performed at two, five, seven, 10 and 15 seconds. Individual and mean readings for each time span can be over-plotted and any shift in particle size distribution noted. The appropriate duration period can be selected by looking at the relative standard deviation (RSD) of the median particle size. For particles with a median size of > 10 micrometres, the RSD should be < 3%; and for particles with a median size of < 10 micrometres, the RSD must be < 6%. This reflects the greater difficulty in dispersing smaller particles.

Table 1 shows an example of how the D(v, 0.5) reported for a lactose excipient varied with measurement duration. Here, a measurement duration of seven seconds was chosen. Although the RSD is low for the two-second measurement, the D(v, 0.5) is significantly smaller than for the other measurements, suggesting that the large particles were not correctly sampled.

Stability
Determining whether samples are stable over the analysis period, and are not subject to agglomeration, de-agglomeration or dissolution, requires monitoring of the particle size distribution at known points in time. At least five repeat measurements should be taken after one, three, five, seven and 10 minutes. The mean values and standard deviations for D(v, 0.1), D(v, 0.5) and D(v, 0.9) should be determined. The limits of acceptability for samples in different size ranges are defined in IS0 13320-1.

Air pressure and ultrasound
Other variables include the use of air pressure for dry dispersions, and ultrasound exposure when dispersing samples for wet measurements.

Air pressure titrations should be included in method development procedures. A suitable air pressure allows particle dispersion without milling. Most pharmaceuticals, for example, are friable and subject to grinding in a dry powder feeder if the air pressure is too high. Dispersion and milling often occur simultaneously, leading to a broadening of the particle size distribution. Measuring equal amounts of sample at different air pressures allows determination of the optimum pressure for maximum dispersion. Achieving near-identical results for both wet and dry dispersion of a sample is proof that full-dispersion has been achieved without attrition.1

Applying ultrasound to assist sample dispersion for wet measurements follows similar principles. Ideally, measurements should be made before, during and after ultrasound, to examine the effect on the robustness of the measurement. Ultrasound can increase the rate of particle-particle collisions and lead to agglomeration under incorrect dispersion conditions.

Pump and stir rates
Pump and stir rates should be examined during method development. The chosen conditions must allow suspension of all material without air entrainment. Figure 1 shows how the results obtained for a lactose sample varied according to the stirrer speed. The results reach a plateau at 2,000 rpm, at which point the material is correctly suspended and dispersed.

Obscuration
Obscuration is a measure of the amount of light scattered by a sample and is directly related to the concentration of material in the measurement zone. For most distributions the particle size should be independent of obscuration within a given concentration range. At extremely low concentrations, results with large RSDs may be obtained because of a high signal-to-noise ratio. At extremely high concentrations, the results may be smaller than expected due to multiple scattering. It is suggested that this be investigated by measuring the reported size at different obscurations, with the acceptable RSD being specified in the same way as for the measurement duration investigations.

Reproducibility
Reproducibility has been defined by some as an indicator of precision between laboratories.4 In our experience it is more likely to indicate the effectiveness of the sampling regime. It can also flag differences between instruments. A minimum of five samples from the same batch should be tested in accordance with the method under investigation in order to assess the reproducibility. From the average result for each repeat measurement, the RSD should be determined for the D(v, 0.5), D(v, 0.1) and D(v, 0.9). Acceptance limits are set out in IS0 13320-1.

Precision
The precision of the technique should be checked by a second analyst or against a second instrument (or both). This should, in essence, be a repeat of the reproducibility tests.

In Conclusion
The introduction of ISO 13320-1 and NIST 960-1 has provided information on theory, and guidance on dispersion and sampling, when using laser diffraction particle sizing systems. Here, we have outlined the important criteria that serve as a starting point for method validation. Considering these can lead to the specification of a robust, reproducible analysis procedure.

References:
(1) ISO 13320-1: 1999. Particle size analysis — Laser diffraction methods — Part 1: General principles.

(2) Jillavenkatesa, A., S.J. Dapkunas and L.-S.H. Lum. 2001. Particle Size Characterisation. Special Publication 960-1.

(3) Allen, T. 1999. Particle Size Measurement. 5th Edition. Volume 1, p38 Chapman & Hall, London.

(4) Bell, R., A. Dennis, B. Hendriksen, N. North and J. Sherwood. 1999. Position Paper on Particle Sizing: Sample Preparation, Method Validation and Data Presentation. Pharmaceutical Technology Europe. Volume 11(11): 36-42.

Steve Ward-Smith, PhD is technical product specialist — Diffraction Products at Malvern Instruments Ltd. (Worcestershire, U.K.). Alan Rawle, PhD is divisional manager — Applications Support at Malvern Instruments. www.malvern.co.uk