Controlling Analytical Method Variability with Key Approaches to Sample Handling and Preparation

Sample handling and preparation can have a substantial effect on an analytical method’s variability and data quality.  It is important to consider and address the risks associated with sample preparation as part of the method development process.  This requires carefully designed and executed experiments and clear documentation in the method to give the analyst an effective analytical control strategy. Through proper technique, the choice of reproducible consumables and method lifecycle approaches, including an Analytical Target Profile (ATP), risk assessment (RA) and analytical control strategy (ACS), an analyst can effectively mitigate risk and improve the robustness of their analytical methods.

Sample preparation is an integral part of the analytical method and is often the most time-consuming step.  Method robustness and transfer problems frequently result from issues with sample preparation.¹ Even a thoughtfully designed LC analytical method may yield poor results due to some common errors that occur well in advance of the chromatographic separation. Through proper technique, the choice of certified clean, reproducible consumables and method lifecycle approaches such as creating an Analytical Target Profile, conducting an Analytical Quality by Design (AQbD) risk assessment, and implementing an appropriate Analytical Control Strategy, an analyst can mitigate the risk of method failure.

Here are some considerations to help control method variability by reducing errors in sample handling and preparation:

1. Create an Analytical Target Profile (ATP)

The ATP identifies critical method requirements and establishes the acceptance criteria for evaluating steps in sample handling and preparation.  The ATP should include the method’s allowable accuracy, precision, specificity and sensitivity requirements as these will influence acceptable sample preparation.  For more information on establishing a method’s analytical target profile, see “Defining the Analytical Target Profile”, Pharmaceutical Engineering,²

2. Conduct an AQbD Sample Handling Risk Assessment

Perform a sample handling Risk Assessment by evaluating each of the steps associated with the sample prior to analysis (e.g. by HPLC).  Consider how each step might impact the analytical results:

1. Ensure the sample or specimen is representative of the lot to be examined. This is often accomplished using statistical approaches.
2. Maintain sample integrity by properly labelling the sample and protecting it from environmental factors such as light, moisture, air, temperature and microbial contamination.
3. The homogeneity of the lot may also be a concern, so it is important to consider the content uniformity of tablets, or the effect of settling on a suspension.
4. Once the risk factors are identified, a critical step in controlling method risk is to specify low risk consumables. For instance, choosing the right vials can minimize mechanical effects (e.g. needle jams), contaminant peaks and adsorptive losses of the analyte. Refer to Control Method-Related Data Quality Risks During Chromatographic Analyses with Fit-for-Purpose, High Quality Glass Vials³  for more details.  In addition, innovative technologies have been designed to increase analyte recovery and reproducibility by minimizing analyte surface interactions that can lead to sample loss. See Achieving Maximum Protein and Peptide Recovery, Sensitivity and Reproducibility using QuanRecovery Vials and Plates⁴ for more information.
5. Choosing pipette tips that are appropriate for the chosen diluent and filters that minimize adsorptive losses can also reduce risks.
6. Lastly, factors deemed important to the success of the method, including consumables, should be clearly documented in the method as part of the Analytical Control Strategy (ACS) with the expectation that they will not be changed unless there is clear justification for alternatives.

3. Address Critical Risk Factors with Proper Technique

Once the sample enters the lab, there are a number of potential challenges getting the sample ready for chromatographic or spectroscopic analysis.  These include sample uniformity, choice of diluent and extraction technique.  For tablets, if ground or weighed, the resulting particles should be reasonably uniform.  Similarly, a suspension must be well mixed before it is sampled to ensure the solution is representative. Extraction of the analyte from the matrix is probably one of the most critical steps as it is affected by analyte solubility in the diluent, and the type of mixing and length of time. The diluent should be chosen based on solubility experiments for the analyte.

For a method to perform consistently, mixing should be well characterized (type of mixing, duration, speed, etc.). Preparation of the solution for analysis often involves weighing, pipetting or dilution steps, typically using volumetric flasks.  This points to the importance of good training and laboratory proficiency demonstrations for laboratory scientists.  Additionally, it may be necessary to evaluate various sample weights and volumes to assure the reportable results will meet the accuracy and precision criteria stated in the ATP.  The results from these experiments should be documented in the Method Development Report.  The outcomes, in terms of acceptable experimental parameters for the routine application of the method, should be documented in the method as elements of the ACS, with expectation that they will not be changed without appropriate justification.

4. Post-Extraction Considerations

An aspect sometimes overlooked is the filtering process for analytical solutions.  Filtering is necessary to remove particles, but it can have unintended effects such as adsorptive losses of the analyte. In addition to choosing the proper filtration products, this effect can be overcome by discarding the first few milliliters of filtrate, a volume that should be determined empirically.  Finally, it is important to evaluate stability for analytical solutions.  Various factors, such as light, temperature and time may result in changes to the solutions such as the loss of analyte or an increase in degradants.  Solution stability should be investigated as part of the method development process and documented in the method as part of the ACS.

5. Sample Preparation and Method Robustness

Even with a well-developed method, we can expect to see variations from day to day, analyst to analyst and lab to lab.  Experiments conducted on the same sample on multiple days by the same analyst, and by different analysts, can be used to evaluate expected variability.  If the variability is high relative to the requirements established in the ATP, it may be necessary to investigate further to understand the source. Perhaps the greatest challenge is with method transfer to another laboratory.  If initial transfer experiments are not successful, it may be necessary to carefully review the existing ACS, and potentially return to the AQbD RA with particular emphasis on those elements that may be different in the receiving laboratory.

6. Create and Implement an Analytical Control Strategy

When the sources of variability associated with the various risk factors have been identified and experimental controls on procedures, consumables, and reagents have been established, these constitute the Analytical Control Strategy (ACS).  The ACS should be well documented in the method to assure consistent application, including controls for reagents and equipment.  For further guidance on Analytical Control Strategies, see Analytical Control Strategy Elisabeth Kovacs, et al.  USP PF 42(5) Stimuli to the Revision Process⁵.

Sample handling and preparation can have a substantial effect on an analytical method’s variability and data quality.  It is important to consider and address the risks associated with sample preparation as part of the method development process.  This requires carefully designed and executed experiments and clear documentation in the method to give the analyst an effective analytical control strategy. Through proper technique, the choice of reproducible consumables and method lifecycle approaches, including an Analytical Target Profile, risk assessment and analytical control strategy, an analyst can effectively mitigate risk and improve the robustness of their analytical methods.

References:

1. Sample Preparation of Pharmaceutical Dosage Forms:  Challenges and Strategies for Sample Preparation and Extraction, Beverly Nickerson, p vii, AAPS Press, 2011.
2. Defining the Analytical Target Profile, Pharmaceutical Engineering, Kimber Barnett, Stephen Chestnut, Neil Clayton, Michael Cohen, Janice Ensing, Timothy Graul, Melissa Hanna-Brown, Brent Harrington, James Morgado. April 2018. Or Using the Analytical Target Profile to Drive the Analytical Method Lifecycle. Anal. Chem. 2019, 91, 4, 2577-2585. Patrick Jackson, Phil Borman, Marion Chatfield et al. January 2019.
3. Control Method-Related Data Quality Risks During Chromatographic Analyses with Fit-for-Purpose, High Quality Glass Vials, Tran Pham and Markus Wanninger. January 2020. Waters Corporation white paper.
4. Achieving Maximum Protein and Peptide Recovery, Sensitivity and Reproducibility using QuanRecovery Vials and Plates, Moon Chul Jung. May 2019. Waters Corporation white paper.
5. Analytical Control Strategy, Elisabeth Kovacs, et al.  USP PF 42(5) Stimuli to the Revision Process.