Automated Powder Dosing in the Life Science Laboratory

Preparing analytical samples and standards for the life science laboratory, the pharmaceutical laboratory, and the analytical laboratory is typically a manual procedure that requires that solids be weighed into a volumetric flask and diluted to the mark. METTLER TOLEDO (Greifensee, Switzerland) critically reviewed the work flow and saw an opportunity to automate the sample preparation process while simultaneously reducing the impact of human variability and uncontrolled environmental factors. Specifically, it was predicted and verified that adding liquid by weight improves precision and reduces solvent consumption by 80%. More importantly, weight/weight sample preparation eliminates the variability of the human element.

Sample preparation

The traditional protocol for preparing an analytical sample or standard is to weigh out the specified amount of sample into a volumetric flask on an analytical balance and then dilute with solvent by filling to the mark. Typical volumetric flasks are 25 mL or larger; injection volumes in analytical instruments such as ultrahigh-performance liquid chromatographs (UHPLCs) are only 20 μL. Therefore, more than 99.9% of the prepared solution is disposed of without being used.

The reason for this excess is twofold: Analytical balances have a characteristic minimum net weight according to USP regulations. Smaller volumetric flasks are not practical because the smaller the flask, the greater the impact of an inaccurate reading of the meniscus. Indeed, when small volumetrics are used, their small size can affect the position of the meniscus by wall effects.

Weighing both sample and solvents instead of using volumetric flasks improves reproducibility and traceability and minimizes problems associated with volumetric glassware. The vials are small and disposable, which eliminates concern about possible cross-contamination. Hidden costs of washing and sample disposal are also reduced.

Measurement uncertainty

Figure 1  - Graph demonstrating that uncertainty decreases hyperbolically with net weight.

The relative measurement uncertainty of a balance is a hyperbolic function of the weight on the balance (see Figure 1). An upper limit on the relative measurement uncertainty means a lower limit of the weight on the balance. The latter is referred to as minimum sample weight or minimum weight. This minimum weight depends on environmental factors like air movements, stability of the supporting table, and the skills of the user. Therefore,it is recommended that the minimum weight be multiplied by a safety factor, typically two or three.

Adhering to the minimum weight for weighing the substance ensures that the required accuracy is achieved. Typical concentrations in the pharmaceutical industry require several milliliters of solvent or several grams. Quantifying these amounts on an analytical balance can be done with very high accuracy since this is several orders of magnitude higher than the minimum sample weight, and the uncertainty decreases hyperbolically with the net weight (see Figure 1).

Powder dosing
Substance savings

The minimum sample weight and the safety factor depend strongly on the user and environmental influences. Automated powder dosing with the Quantos powder dosing system (METTLER TOLEDO) within the closed draft shield can thus significantly reduce both the minimum sample weight and the safety factor.

For example, according to USP, the XP205 analytical balance (METTLER TOLEDO) has a minimum weight of typically 21 mg and a recommended safety factor of 2. The Quantos automated dosing balance has a minimum weight of typically 10 mg and a recommended safety factor of 1.5. This is a significant reduction in the cost of compliance: 65% of substance is saved while still remaining compliant with USP regulations.

Figure 2  - Graph illustrating the solvent consumption as a function of the desired concentration. The red curve applies when solvent is quantified volumetrically using flasks, the green curve when weighing the solvent.

Solvent savings

The red curves in Figures 2 and 3 illustrates how the minimum weight, the concentration, and the available flask determine the amount of solvent (Figure 2) and substance (Figure 3) needed. The green curves show the consumption of substance (Figure 3) and solvent (Figure 2) as a function of target concentrations.

The red curve in Figure 3 shows that only for four discrete concentrations the minimum net sample weight of 42 mg can be applied. In all other cases, significantly more substance is consumed because one is restricted to quantities of solvent for which glassware exists. In these cases, the amount of substance needs to be rounded up to the next flask size available.

The green curve shows that when the amount of liquid is weighed, for every target concentration only the minimum net weight of substance is required. When quantifying solvent by its weight and not by a volumetric flask, any amount of solvent to match the desired concentration can be used. No rounding up is required. Similarly, Figure 2 illustrates the solvent savings.

If a 1.5-mg/mL solution with a 1-g/mL density is prepared manually with a volumetric flask, 50 mL of solvent and 75 mg of substance are consumed. When the solution is prepared automatically and gravimetrically, 10 mL of solvent and 15 mg of substance are sufficient. Substance savings of 80% can be realized.

The use of volumetric flasks and manual powder weighing results in excessive use of solvent and substance.

Figure 3 - Graph illustrating the substance consumption as a function of the desired concentration. The red curve applies when solvent is quantified volumetrically using flasks, the green curve when weighing the solvent.

Reproducibility

Automated gravimetric powder and liquid dosing is very reproducible. To demonstrate this, nine solutions of an active pharmaceutical ingredient (API) were prepared individually, both automatically and manually. To measure the reproducibility, the solutions were analyzed by HPLC. To distinguish between the reproducibility of the sample preparation and the HPLC analysis, 10 repeat injections of the same solution were done.

Nine solutions with a target concentration of 0.603 mg/g were prepared. Ten milligrams of API were dispensed automatically into nine 20-mL brown glass vials. Automation allowed 10 mg to be accurately dispensed with an RSD of only 0.89%. Next, the solvent—a 80:20 acetonitrile:water mixture—was added gravimetrically based on the exact weight of the API dispensed. The RSD of the achieved concentration was 0.001%. The suspension was placed in an ultrasonic bath for 5 min until fully dissolved.

Next, a 2-μL sample was injected into the HPLC system. The peak areas for the nine individually automatically prepared samples varied with an RSD of 0.19%; the peak areas for the individually manually prepared samples varied with an RSD of 0.60%. When the same sample was injected 10 times, the peak areas varied with an RSD of 0.21%.

When a standard preparation is automated by weighing the substance and solvent, the variability in the prepared solutions is insignificant because it is lower than the variability of the analytical instrumentation itself. Automated, gravimetric sample and standard preparation is a paradigm shift that significantly reduces the cost of each assay and makes the process more reproducible, improving the analytical results.

Conclusion

Until now, the minimum weight of balances and volumetric flasks required that 99.9% of prepared samples and solvents were disposed of. Automated and gravimetric sample and standard preparation reduces the minimum weight of the balance and does not depend on volumetric flasks. Therefore, the cost of compliance is significantly reduced. Weight can be determined with very high accuracy, making the process more reproducible and improving the analytical results.

Jan Prochnow, Ph.D. is Head of Product Management and Business Development, Quantos Business Unit, METTLER TOLEDO, Im Langacher, 8606 Greifensee, Switzerland; tel.: +41 449442692; e-mail: jan.prochnow@mt.com.

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