There has been much discussion surrounding the benefits of gravimetric sample preparation during the last 12 months <1 – 5>. It has been recognised by industry organisations such as the United States Pharmacopeia (USP) who proposed an update to sub-chapter <1251> “Weighing on an Analytical Balance” in Pharmacopeial Forum (PF) Sept/Oct 2012. This proposal includes a detailed description of the steps involved in gravimetric dosing for sample and standard preparation.

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Tab. 1 Manual sample preparation – reproducibility and precision

Pfizer’s Analytical Research and Development Group (AR&D) in Groton, USA have embraced this new approach to sample preparation using a pioneering fully automated gravimetric sample preparation workstation. Detailed studies have been carried out which compare the differences between preparing samples and standards using manual volumetric processes and the new automated gravimetric methods.

This article presents data generated by two specific experiments performed by the AR&D group in Groton. ­
The solid sample used in both experiments is the non-proprietary material caffeine. The first experiment focuses on the reproducibility and precision of sample

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Tab. 2 Automated gravimetric sample preparation – reproducibility and precision

preparation. The second experiment is a linearity study. In both experiments sample preparation is performed manually with volumetric flasks and compared with a new fully automated method, where samples are prepared on a gravimetric sample preparation workstation, the Quantos QX1 from Mettler Toledo. The QX1 workstation incorporates a 6-place microbalance for weighing of the solids and solvents with automated interchange of up to 10 ­solid dosing heads and 5 solvent dosing heads.  

Reproducibility and precision

The first experiment is designed to investigate the reproducibility and precision of manual sample

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Tab. 3 “Unknown” samples for manual linearity study

preparation compared to the new automated gravimetric method.

Six replicate solutions were prepared by weighing 20 mg of caffeine and making it up to 50 ml in a volumetric flask using a 30:70 methanol/water mixture. This procedure took a total of 50 minutes. Dissolving/mixing the samples on a shaker took 15 minutes of this time, ­so the manual labour time is 35 minutes full-time equivalent (FTE) – 5 minutes per sample for each weighing and dilution plus 5 minutes for the diluent ­prep. Table 1 ­shows the data. The key metric is that the relative standard deviation of these samples is 1.67 % (Tab.1).

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Tab. 3 “Unknown” samples for manual linearity study

Table 2 shows the equivalent data from the automated gravimetric method. Six replicate solutions were prepared by weighing 5mg of caffeine and adding 12.5 g of the same diluent (30:70 methanol/water) directly into a vial. This procedure took a total of 30 minutes, consisting of ­­10 minutes preparation time (such as filling and installing powder and liquid dosing heads and setting up the sequence) with an additional 20 minutes running time.The RSD of these samples is 0.49 % (Tab. 2).

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This experiment highlights three advantages of automated gravimetric sample preparation. 75 % less substance and solvent are used to prepare the sample solutions; more than 70 % of labour time is saved in the sample preparation steps and most importantly the precision is improved by a factor of more than three.

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Fig. 1 Linearity plot of automated sample preparation data

Linearity testing study

The second experiment is a linearity study. To generate the manual data, five different concentrations from 0.2 ­
to 0.6 mg/ml were prepared in 100ml volumetric flasks, which took 60 minutes. Linear regression analysis resulted in a correlation coefficient of 0.99473.

Five unknown samples were prepared with concentrations that varied between 0.3 to 0.5 mg/mL. Table 3 indicates the actual amount of caffeine weighed into the solution (Wv) versus the amount determined from comparison with the linearity plot (Wa). The agreement between these 2 values varies between 97 and 100 % (final column) (Tab. 3).

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Tab. 4 “Unknown” samples for automated gravimetric linearity study

An equivalent experiment was carried out to generate the automated gravimetric data. This time the solutions were prepared in 10g solvent rather than 100ml solvent. It took 40 minutes to prepare the solutions (plus 5 mins to set up the sequence). In this experiment, the linear regression analysis resulted in a near-perfect correlation coefficient of 0.99998 (see Figure 1).

As in the manual experiment, five unknowns were prepared with concentrations that varied between 0.3 to 0.5 mg/g with the mass of each solution recorded. ­(Tab. 4). The final column in the “unknowns” Table 4 indicates that the actual versus determined caffeine weights for each unknown are in 100 % agreement in each case. ­

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The data generated during the linearity study reinforces the superior quality of the automated gravimetric approach: 90 % less substance and solvent are used; 25 % of time is saved; the correlation coefficient is improved and the unknown samples are accurately identified.

Practical advantages of automated gravimetric sample preparation

The accuracy of the samples prepared is significantly improved, which will have a knock-on effect in the
q­uality of the analytical results generated. The reduced labour time and amount of substance and solvent saved has the potential to have a dramatic impact on laboratory efficiency and running costs.

Literature:
[1] „One-click sample preparation“, Joanne Ratcliff, World Pharmaceutical Frontiers, März/April 2012, S. 77.
[2] „Innovations in Gravimetric Preparation of Analytical Solutions: Regulatory and Compendial Perspectives“, Gregory Martin, American Laboratory, Juni/Juli 2012
[3] „The benefits of automated gravimetric sample preparation“, Joanne Ratcliff & Jan Prochnow, q&more, März 2012.
[4] „Reducing Variability and Out-of-Specification Results by Implementing High Quality Gravimetric Sample Preparation (GSP)“, Charles Ray, Klaus Fritsch, Joanne Ratcliff, ISPE, Februar/März 2012.
[5] On-demand-Webinare auf www.mt.com/Q-webinars: „Recent USP changes: Regulatory and quality aspects of sample preparation“; „What’s the matter with Sample Prep? Novel approaches and solutions“; „Preventing costly out-of-specification investigations“.