VALIDATION OF WET AND DRY LASER DIFFRACTION PARTICLE CHARACTERISATION METHODS.

Transcription

1 VALIDATION OF WET AND DRY LASER DIFFRACTION PARTICLE CHARACTERISATION METHODS. Ward-Smith, R.S., Gummery, N. and Rawle, A.F. Malvern Instruments Ltd, Enigma Business Park, Grovewood Road, Malvern, Worcs, U.K., WR14 1XZ. Tel: Fax: INTRODUCTION With the introduction of both ISO (Particle size analysis Laser Diffraction methods) [1] and NIST (Practice Guide Particle size Characterisation) [2] the user of a laser diffraction instrument has been supplied with a wealth of useful information on theory and guidance on both dispersion and sampling. The amount of information available on how to validate the method of analysis once it has been developed is a lot more limited. The Pharmaceutical Analytical Sciences Group (PASG) [3] have attempted to lay down some guidelines, but these did not provide sufficient detail regarding the actual experiments that need to be performed. This paper sets out to define the important areas of method development and provide both guidelines and example data to suggest what experiments might be done in order to validate a method. However just because these experiments have been performed, the authors cannot guarantee that an auditing body will always approve and rubberstamp the method. Rather the author s are suggesting a minimal amount of work that we d expect to be performed. Any more work will only strengthen the case of the candidate method. Lerke and Adams [4] have recently published their ideas on the subject in a useful paper. Their paper covers both method development and method validation, whereas this paper is mainly concerned with the latter. The purpose of creating a validated method is to ascertain the robustness and integrity of a particle size method by testing all possible parameters that could cause variation in the reported size. Validation is defined by the 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. METHOD VALIDATION It goes without saying that development of such methods should be performed on an instrument, which has validated software and is regularly tested to confirm its performance (both in the form of an IQ/OQ (at least yearly) and a secondary standard). Validated software is software, which has both lifecycle documentation and the ability to have its numeracy tested with the use of a peer package such as Excel. It is essential in the current climate that the software is also CFR21 Rule 11 compliant. This paper will now consider each of the main variables in turn.

2 SAMPLING The first step any analyst will take is the extraction of a representative sample from the bulk material. If the sample is not representative the result can be taken to be atypical and the experiment is a pointless exercise. The question that every analyst should ask themselves before they sub-sample is Is the small bag of powder I have representative of the production process? Is the use of devices such as sample thieves (to extract sample from the bulk) a regular part of the production process. It can be seen from table 1 that the use of a spinning riffler produces sub-samples of powder, which vary less than other more commonly used sampling techniques. Riffling works best for free flowing particles, but can take a great deal of time if a large amount of powder is to be dealt with. If the sample is a suspension then this brings its own set of problems. Whereas with a powder, settling in transit leads to the large particles at the top of the heap and the fine at the bottom, a suspension will show the reverse. Large particles must be adequately re-suspended without bias. Use of certain stirrers (especially magnetic fleas) can lead to the large particles being thrown to the outside of the container where they are not sampled. Method Relative Standard Deviation (%) Cone & Quartering 6.81 Scoop Sampling 5.14 Table Sampling 2.09 Chute Riffling 1.01 Spin Riffling Table 1: Sources of sampling error (Reproduced from Allen [5] ). SAMPLE PREPARATION Bell et al [3] defined sample preparation as the pre-treatment and the presentation of the sample to the measuring technique in a meaningful manner. This will depend on the interests of the user, sometimes it is the primary particle size that is important, and the sample should be dispersed to this, sometimes it is the natural agglomerated state that is of interest. In either case, the dispersion medium, air (for dry dispersion) or a liquid, should not cause irreversible change to the particle (dissolution, milling or aggregation). The whole sample preparation procedure should be carried out as part of method development rather than method validation. RANGE The range of the instrument should ideally cover the size range of the sample. For modern laser diffraction instrumentation with size ranges between 20nm and 2000µm this normally presents no problem for most pharmaceutical samples. Older instrumentation often has many lenses to cover the same range. In this case the lens which covers the largest proportion of the particle size distribution should be used or the result from two lenses could be blended together (though this is not recommended as the result depends on the mathematical efficacy of the blending routines).

3 SPECIFICITY Is the technique appropriate? There is no strict requirement to perform this exercise in particle size analysis as it is assumed that the operator is using the most suitable particle size technique for the material being analysed. The appropriate technique should have already been established in the method development. Of course different methods of particle size analysis will lead to different answers for the same sample. Also, due to the nature of the laser diffraction technique, it is difficult to develop a particle size analysis method that will distinguish the scattering from different components within a product dosage form. In the case of a suspension, it may be necessary to prepare a placebo mix and analyse this separately. ROBUSTNESS The robustness of an analytical method is an indication of its ability to remain unaffected by small variations in the test parameters and so provide assurance of its reliability during routine usage. The method robustness should be considered before repeatability, reproducibility and intermediate precision exercises are undertaken. The variations that are normally considered are that of measurement duration and measurement stability. Variables such as air pressure (dry measurement) and pump / stir rates (wet measurement), normally considered as part of method development, are also important and will be briefly touched on here. a) Measurement Duration The purpose of this exercise is to determine a suitable measurement duration, which will then be used for the repeatability, reproducibility and intermediate precision exercises. A sample should be prepared in accordance with the method under investigation. A cycle of ten measurements should be performed for durations of 2, 5, 7, 10 and 15 seconds. The individual and mean readings for each duration should be recorded. The results can be over-plotted and any shift in particle size distribution can be noted. The appropriate duration period can be selected by looking at the relative standard deviation of the median particle size. For particles with a median size (D(v,0.5)) of >10 micron the Relative Standard Deviation (RSD) should be <3% and for particles with a median size <10 micron the RSD must be <6%. This reflects the fact that smaller particles are more difficult to disperse. Table 2 shows how the D(v,0.5) measured for a lactose sample varies with measurement duration.

4 Measurement Measurement Duration Number 2 sec 5 sec 7 sec 10 sec 15 sec Mean / Microns %RSD Table 2: Variation of lactose D(v,0.5) with measurement duration. The RSD can be seen to increase over time, so an interim point where the RSD was low (5 seconds) was chosen for measurement. From looking at the data overall it can be deduced that 2 seconds is inadequate as it appears that it is not giving the larger particles sufficient time to be sampled, so the D(v,0.5) is significantly lower. b) Repeatability / Sample Measurement Stability In order to determine whether samples are stable over the period of analysis and not subject to agglomeration, de-agglomeration or dissolution, it is necessary to monitor the particle size distribution at known time points. A sample should be prepared in accordance with the method under investigation. It is recommended that at least five measurements at the previously established duration are taken. Measurements could be taken after 1, 3, 5, 7 and 10 minutes. At least five repeat measurements should be obtained at each time point. The mean and relative standard deviation for the D(v,0.1), D(v,0.5) and D(v,0.9) values should then be determined. For material that has a D(v,0.5) greater than 10 microns an acceptable time point is defined when the following conditions are met: D(v,0.5) RSD <3%, D(v,0.1) and D(v,0.9) RSD <5%. For material that has a D(v,0.5) less than 10 microns an acceptable time point is defined when the following conditions are met: D(v,0.5) RSD <6%, D(v,0.1) and D(v,0.9) RSD <10%. This reflects the fact that smaller particles are more difficult to disperse. These are the limits of acceptability as laid down in ISO13320 [1] and for many samples much lower RSDs should be easily achieved. Typical measurement stability results obtained for a lactose sample are show in table 3 and figure 1.

5 Measurement Stabilisation Time / Minutes D(v,0.1) / Microns %RSD D(v,0.5) / Microns %RSD D(v,0.9) / Microns %RSD Table 3: Variation of lactose size over time (mean of 5 repeat measurements are shown). Figure 1: Sample stability measurement summary. From the above results it is clear that the lactose sample is stable whilst in suspension. It was decided from these results that measurements should be taken after 1 minute in order to give the sample time to equilibrate. c) Air Pressure (Dry measurement) / Ultrasound (wet measurement) A pressure titration should have previously been performed as part of method development (see ISO section [1] ). A suitable pressure is one at which dispersion of the particles is achieved but milling of the particles is not occurring. Most pharmaceuticals are friable and will be ground in any dry powder feeder if the pressure is too high. Often both dispersion and milling occur simultaneously (which leads to a broadening of the distribution) [6]. By measuring the same amount of sample (ideally sampled to single shot size by a spinning riffler) at different pressures, the pressure at which maximum dispersion is achieved without milling can be ascertained. The best way of proving that no attrition is occurring is to achieve near identical results for both wet and dry dispersion [1].

6 Figure 2 shows a pressure titration on a typical material. A pressure of 1 bar is not enough to disperse the material. As long as the pressure is greater than 2 bar good results are obtained. Figure 2: Pressure titration data obtained for a typical lactose sample. The use of ultrasound in wet measurements is very similar. ISO recommends that ultrasound can be used to assist dispersion [1]. Too much ultrasound can fracture friable pharmaceutical particles, although this is extremely unusual. As part of method development the affect of varying the duration and power of ultrasound on the particle size distribution should be examined. Ideally measurements should be taken before, during and after ultrasound to examine what effect sonication has on the robustness of the measurement. As well as separating particles, ultrasound can also increase the rate of particle particle collision and can actually cause agglomeration d) Pump and Stir Rates The pump and stir speeds used during a measurement should be examined as part of method development. The chosen conditions should be capable of suspending all the material without causing air entrainment (a particular problem if surfactants are being used). Figure 3 shows how the result obtained for a lactose sample varies according to the stirrer settings. As can be seen, the result reaches a plateau above 2000rpm. It is at this point that all of the material is correctly suspended and dispersed. Sample sedimentation causes the results to be smaller than expected at stirrer rates below 2000rpm.

7 Figure 3: Effect of varying the stirrer rate on the result obtained for a typical lactose sample. e) Confirmation of refractive index choice As part of the method development work, the choice of refractive index should be examined. Index matching fluids can be used to provide experimental evidence of the real refractive index. The Fraunhofer approximation should not be used if there are particles smaller than 40λ (25 microns at the He-Ne laser wavelength of µm) present in the distribution [1] as it may erroneously report the presence of fine material. LINEARITY / OBSCURATION Obscuration is a measure of the amount of light scattered by the sample and is defined here as (1-Transmission)*100. For most particle size distributions within a given concentration range the particle size should be independent of concentration. At extremely low concentration results with large RSDs may be obtained (owing to high signal to noise ratios) and at extremely high concentrations, the result may be smaller than expected due to multiple scattering. It is suggested that obscurations of 5, 10, 15, 20 and 25% are investigated in the same way that the measurement duration test was performed, with the acceptable RSDs being similarly specified. REPRODUCIBILITY Reproducibility is defined by Bell et al [3] as an indicator of precision between laboratories. It can indeed show this, but in the authors experience is far more likely to show how effective the sampling regime in use is i.e. how homogenous or otherwise the sample is. It can also be used to flag differences between different instruments (be it of the same or different models). A number of samples (at least five) should be taken from the same batch and tested in accordance with the method

8 under investigation. For each sample at least five repeat measurements should be taken and the individual and average results should be obtained. From the average result for each of the five repeat measurements, the RSD should be determined for the D(v,0.1), D(v,0.5) and D(v,0.9) for all the samples taken. For material with a D(v,0.5) greater then 10 micron, good reproducibility is shown if the D(v,0.5) RSD <3%,D(v,0.1) and D(v,0.9) RSD<5%. For material that has a D(v,0.5) less than 10 micron, good reproducibility is shown if the D(v,0.5) RSD<6%, D(v,0.1) and D(v,0.9) RSD<10%. Again these are the limits set out within ISO [1]. Sample Number D(v,0.1) / Microns D(v,0.5) / Microns D(v,0.9) / Microns Mean %RSD Table 4: Variation in the results obtained for seven separate scooped-sampled lactose samples. Scoop sampling is being used here, and the RSDs are in line with those reported in table 1. INTERMEDIATE PRECISION The checking of the precision of the technique should be done by the use of a second analyst or a second instrument (or both). This should in essence be a repeat of the reproducibility test. The RSD of the D(v,0.5) on the second test should again be <3%. Both sets of results should then be combined to give a pooled mean and a pooled RSD (which should be <3%). Sample Number D(v,0.1) / Microns D(v,0.5) / Microns D(v,0.9) / Microns Mean %RSD Table 5: Results obtained for a second analyst measuring the same lactose sample.

9 Parameter Pooled Mean Value Mean Standard Deviation 0.68 %RSD 2.98 Table 6: Pooled mean values. The data here has a RSD<3% and shows acceptable intermediate precision. Validation terms such as detection limit and quantification limit do not apply to laser diffraction methods and are therefore outside the scope of this paper. The detection limit or instrument resolution will depend on the instrument in question. CONCLUSION This paper has attempted to lay down guidelines on how to validate a particle sizing method. Guidelines on achievable standard deviations have also been supplied. REFERENCES [1] ISO Particle Size Analysis Laser Diffraction Methods Part 1: General Principles (1999) [2] Jillavenkatesa, A, Dapkunas, S. J. and Lum, L. S., (2001) Particle Size Characterization Practice Guide, N.I.S.T [3] Bell, R., Dennis, A., Hendriksen, B., North, N. and Sherwood, J., (1999) Position paper on Particle Sizing: Sample Preparation, Method Validation and Data Presentation, Pharmaceutical Technology Europe, November 1999 [4] Lerke, S.A. and Adams, S.A, (2002) Development and Validation of a Particle Size Distribution Method for Analysis of Drug Substance, American Pharmaceutical Review, Fall 2002 [5] Allen, T. Particle Size Measurement, (1999) 5 th edition, Volume 1,p 38, Chapman and Hall, London [6] Rawle, A. F., (2000) "Attrition, dispersion and sampling effects in dry and wet particle size analysis using laser diffraction" Paper th International Congress of Chemical and Process Engineering "CHISA'2000", August, Praha, Czech Republic