Validating laser diffraction particle sizing methods to maintain integrity

When undertaking particle size measurements it is important to validate the analysis method to ascertain both its robustness and integrity. Steve Ward-Smith and Alan Rawle review the steps that should be taken when validating laser diffraction methods for particle size analysis.

Users of laser diffraction instruments for particle characterisation applications have a wealth of information on the theory behind the technology as well as guidance on both dispersion and sampling.

These include ISO 13320-1 (Particle size analysis ­ laser diffraction methods) and NIST 960-1 (Practice guide to particle size characterisation). However, the amount of information available on how to validate the method of analysis once it has been developed is more limited.

The purpose of validation is to ascertain the robustness and integrity of a particle sizing method by testing all possible parameters that could cause variation in the reported size.

Validation is described by the US Food and Drug Administration (FDA) 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“.

The development of a validated method should be carried out using an instrument that has validated software and is regularly tested to confirm its performance. Validated software has lifecycle documentation detailing its development and maintenance and should be numerically validated using a peer package such as Microsoft Excel.

In the current regulatory environment, itis also essential that the software is compliant to 21 CFR part 11, the FDA's rule regarding the use of electronic records and signatures.

When validating a laser diffraction method for particle characterisation, the following main variables must be considered: sampling; sample preparation; instrument range; appropriateness of the technique; robustness of the analytical method; the amount of light scattered by the sample; and the reproducibility and precision of the measurement.

Sampling

Whether a sample is representative of the bulk material depends on the sampling technique. During transit of the sample, settling can occur ­ in powders large particle tend to settle at the top of the sample container, 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.

Research has shown that use of a spinning riffler is the most reproducible method of obtaining a representative sample for powder samples when compared with other methods such as scoop sampling, table sampling, cone and quartering and chute riffling. Riffling works best for free-flowing particles but can take a great deal of time if a largeamount of powder is to be handled.

For slurries it is important to overcome sedimentation by re-suspending the sample. This can often be achieved by simple stirring. However, use of certain stirrers, such as magnetic fleas, can lead to the large particles being thrown to the outside of the container (in a similar way to hydrocyclone separation) where they are not sampled.

Once a representative sample is obtained it must be correctly presented to the instrument. The preparation method will depend on the interests of the user and should be investigated as part of the method development rather than the method validation process.

Where, for example, the primary particle size is important, correct dispersion of the sample will be important; if the natural agglomerated state is of interest, sample preparation should take this into account in order to avoid the break-up of agglomerated particles. In either case, the dispersion medium ­ whether air or a liquid ­ should not cause irreversible changes to the particle size through processes such as dissolution, milling or aggregation.

The range of the particle sizing instrumentshould ideally cover the size range of the sample. This normally does not present a problem formost pharmaceutical samples as modern laser diffraction instrumentation can cover a sizerange from 20 nm and 2000 µm in a single measurement.

Older instrumentation, however, may have to use many lenses in order to cover the same dynamic range. Here the lens that covers the largest proportion of the particle size distribution should be used. Alternatively the result from two lenses can be blended together, although this is not recommended since the result depends on the mathematical efficacy of the blending routines.

Specificity and robustness

Specificity, in terms of whether or not the technique is appropriate to the material under analysis, should be addressed as part of method development and does not necessarily have to be revisited for method validation.

It is, of course, true that different sizing techniques display different sensitivities and will therefore provide different results for the same sample. Selection of an appropriate technique depends on what is of interest ­ for instance is the detection of a small amount of over-sized material required or does the technique need to differentiate between different particle types?

Laser diffraction provides a good method for assessing small changes in the size distribution in this regard. However, it is difficult to differentiate between the different components within pharmaceutical dosage forms using the technique.

The robustness of an analytical method is an indication of its ability to remain unaffected by small variations in the test parameters and so provides assurance of its reliability during routine use. The method robustness should be consideredbefore repeatability, reproducibility andintermediate precision are assessed.

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

To determine suitable measurement duration, a cycle of ten measurements should be performed for 2, 5, 7, 10 and 15 seconds. The 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 µm, the RSD should be < 3 per cent; and for particles with a median size of < 10 µm,the RSD must be < 6 per cent. This reflects thefact that smaller particles are more difficult to disperse.

Table 1 shows an example of how the D(v, 0.5) reported for a lactose excipient varied with measurement duration. In this case a measurement duration of 7s was chosen. Although the RSD is low for the 2s measurement, the D(v, 0.5) is significantly smaller than for the other measurements,suggesting that the large particles were not correctly sampled.

Determining whether the samples are stable over the period of analysis 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 1, 3, 5, 7 and 10 minutes. The mean values and standard deviations for D(v, 0.1), D(v, 0.5) and D(v, 0.9) should then be determined. The limits of acceptability for samples in different size ranges are defined in IS0 13320.

Typical measurement stability results for a lactose sample are shown in Table 2. In this case reasonable measurements were obtained at each time point, showing the sample to be stable. A measurement time of one minute was therefore chosen inthis case.

Air pressure and ultrasound

Further variables to be considered include the use of air pressure for dry dispersions, and the amount of ultrasound used in dispersing samples for wet measurements.

Air pressure titrations should be performed as part of the method development process. A suitable air pressure is one at which particle dispersion is achieved without the occurrence of milling.

Most pharmaceuticals, for example, are friable and will be 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 with no milling. Achieving near-identical results for both wet and dry dispersion of a sample is proof that full-dispersion has been achieved without attrition.

Applying ultrasound to assist sample dispersion for wet measurements follows similar principles. Ideally measurements should be taken before, during and after the application of ultrasound, to examine the effect of sonication on the robustness of the measurement. While effective in separating particles, ultrasound can also increase the rate of particle-particle collisions, which may lead to agglomeration if the dispersion conditions are not correct.

Pump and stir rates should be examined at the method development stage, and the chosen conditions must allow suspension of all thematerial without air entrainment. Fig. 1 showshow the results obtained for a lactose samplevaried according to the stirrer speed. The results reach a plateau at 2000 rpm and it is at thispoint that the material is correctly suspendedand dispersed.

Linearity/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 particle size 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 has been defined by some authors as an indicator of precision between laboratories. In our experience, however, it is far more likely to indicate the effectiveness of the sampling regime. It can also flag differences between different instruments.

A minimum of five samples should be taken from the same batch and 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). Again the acceptance limits are set out in IS013320.

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 both ISO 13320 and NIST 960-1 has provided users of laser diffraction particle sizing systems with considerable information on theory and guidance on both dispersion and sampling. However, the process by which a method can be validated is not clear from these documents.

Here we have outlined the important criteria which should be considered as a starting point for method validation. Due consideration of each of these can lead to the specification of a robust, reproducible analysis procedure. This will aid in the detection of batch-to-batch sample differences which can otherwise be hidden by poor experimental technique.

Dr Steve Ward-Smith is Applications Engineer with Malvern Instruments Limited. Dr Alan Rawle is Divisional Manager ­ Applications Support with the same company, tel: +44 (0) 1684 892456, fax: +44 (0) 1684 892789. The email address for sales enquiries is Alison.vines@malvern.co.uk.

More information, plus a copy of the paper Validation of wet and dry laser diffraction particle characterisation methods which was presented at the Fourth World Congress on Particle Technology in Sidney, Australia, in 2002, is available at www.malvern.co.uk

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