Instrument Based Testing: A More Modern and Robust Approach to Titration

Abstract

Manual titration is a well-established, and widely applicable technique for quantitation of analytes that has been in use for hundreds of years. While this technique can be used for analysis of a variety of materials and is readily accessible, it is not without limitations – primarily subjectivity in endpoint selection and manual recording of data. Migration to the instrument-based (auto titrator) methods address these shortcomings, allowing for more precise and repeatable methods, and less subjectivity in the measurements.

Analytical chemistry uses a variety of techniques to identify and quantify analytes of interest, including both desired and undesired compounds (impurities). This field utilizes both classical wet chemistry (non-instrument-based) and more modern instrumental methods.¹ Manual titration is a wet chemistry technique that has been in use since the 1800s, with the first text on titrimetric methods dating back to 1855.² Despite its age, it remains a powerful analytical technique that balances repeatability, accuracy, and precision. In addition, manual titration reactions are specific and cost-effective as the requisite reagents are typically easy to source and inexpensive. Very little is required in the way of costly equipment to run manual titration, making it nearly universally accessible. That said, it is not without its limitations and areas for improvement, especially in the modern pharmaceutical chemistry space. The two primary shortcomings inherent to manual titration can be summarized as subjectivity and manual data entry, both of which can be easily addressed with the use of auto-titrator equipment and methods.

Titrimetric analysis can be categorized based on the chemical reaction taking place and includes acid/base (the most common), oxidation-reduction (redox), complexometric,c, and others. Titrations can also be considered direct or residual depending on how the analyte concentration value is calculated. For direct titrations, the analyte is reacted with the titrant, and the volume of titrant required is used to directly calculate an assay value. With residual titrations, a precisely measured quantity of titrant is added (in excess concerning the analyte of interest), the unreacted/ excess titrant is then titrated with a secondary titrant, and the assay valueis  calculated (indirectly) based on the excess. In addition to categorizing titrations in terms of the reaction taking place, and whether they are direct or residual, titrations can be classified based on how the endpoint is determined and the data captured. From this perspective, titrations can be either manual (visual) or potentiometric (electrochemical/automatic).

The first shortcoming seen with manual titrations is the subjectivity in identifying the endpoint. In all manual titrations, there is an evaluation of the endpoint based on visual observation of the flask and (typically) a color change brought on by the change in environment (e.g., pH, oxidation state) experienced by the indicator used. This observation of the endpoint is the first place subjectivity comes into play. The final calculated result relies entirely on the analyst’s identification of this color change, and the volume of titrant required to cause this change. In some cases, the difference in color before and after the endpoint is reached can be subtle and may require extensive training and experience to accurately identify. In other cases, the timing of the addition of indicator is crucial (e.g. starch indicator for iodometric titration), and when performed incorrectly can make accurate endpoint assessment extremely difficult, leading to inaccurate results. Adding to and compounding the subjectivity of the endpoint identification for manual titrations is the reading of the volume of titrant used. Titrants in manual titration are most often dispensed from a burette that must be manually read from a lined scale similar to a ruler or graduated cylinder, introducing additional subjectivity and opportunity for inaccuracy, including parallax error. The subjectivity around the reading of the burette is compounded for manual titrations, as it must occur at least twice per analysis (starting and ending volumes). Burettes also suffer from an inherent lack of precision, as their certified accuracy is limited – a typical borosilicate laboratory burette has a resolution of 0.10 mL and a tolerance of 0.05 mL. This lies in stark contrast to dispensers/burettes on auto titrators, which are often accurate to as low as 0.0001 mL.

The second major shortcomingofh manual titration is the manual recording/entry of data. With manual titrations, the analyst must record the endpoint into a paper or electronic notebook directly, following internal procedures that are typically aligned with the requirements of a regulatory agency such as the Medicines and Healthcare Products and Regulatory Agency (MHRA) in the UK, which states, “Where manual transcriptions occur, these should be verified by a second person or validated system.”³ In keeping aligned with guidance from the MHRA, et al., the pharmaceutical industry has been steadily pushing towards real-time electronic capture of original raw data, as auditstraild, electronic recording of data eliminates questions on accuracy in the recording of data. Non-instrument-based methodologies such as manual titration lack an avenue by which this data can be automatically captured and recorded. The second-person verification requirement for manually recorded data adds significant resource constraints and increases costs, particularly for contract testing laboratories.

Almost all modern analytical techniques such as chromatography are run through 21CFR part 11 compliant systems, complete with robust audit trails. Automatic (potentiometric) titrator systems make use of similar systems that automatically capture and store audit-trailed data (e.g. titrant volume and inflection/endpoint), eliminating concerns around manual recording, as well as drastically increasing the precision of endpoint volume captured. In addition, all parameters, etc., are captured within the system in an audit-trailed fashion, addressing questions on adherence to the prescribed test method, and ensuring consistent results, helping to improve comparison of historical data. Auto titrator instruments also allow for higher throughput with the use of autosamplers, and efficiency gains by carefully writing methods wo quickly add the majority of the required titrant before slowing additions down so as not to overshoot the equivalence point. The use of saved methods on auto titrator instruments also carries the advantage of reducing variability that is seen with technique-driven methods such as manual titration.

With foresight, titrations can be developed and verified or validated initially utilizing an auto titrator. This is a good approach to take as it preemptively addresses the previously mentioned concerns. Each compendium has specific requirements and allowances about the utilization of alternate methods of analysis. These requirements must be considered if the user’s end goal is to claim compliance with a compendial monograph. The United States Pharmacopeia (USP) specifically addresses the use of potentiometric titration in section 6.20 of its General Notices (USPNF 2023 Issue 3), stating that “Automated and manual procedures employing the same basic chemistry are considered equivalent provided the automated system is properly qualified as being suitable to execute the compendial manual method and the analytical procedure is verified under the new equipment conditions.” The Japanese Pharmacopeia (JP), does not specifically address potentiometric end point determination, however, has a stipulation in section 14 of its general notices section allowing for use of “alternative methods which give better accuracy and precision”. This provision gives the testing laboratory the freedom to perform the initial method verification using an instrument-based method. The European Pharmacopeia (Ph. Eur.), however, does not contain specific language allowing for this, necessitating a full method validation when the use of a potentiometric method is preferred. Both are viable options, however, the full method validation required for the Ph. Eur. carries an increased scope of work, adding to both the time and cost required to onboard a method.

As a case study, we can examine the potassium chloride assay from the USP and JP. These are both written as manual titrations with silver nitratetitrantst but utilizing different solvent systems and indicators. The decision was made to onboard these methods initially as potentiometric titrationtoto leverage their inherent advantages over the prescribed manual methods. As the USP and JP allow for potentiometric endpoint evaluation, a standard verification scope of work was undertaken – this included evaluations of specificity, repeatability, and linearity. This is significantly abbreviated in comparison to the scope of work required for method validation, allowing for faster completion with lower costs. For both compendia, the method performed well, giving relative standard deviations of less than 0.05%, and correlation coefficient of >0.9999, and were considered verified, and compliant to their respective compendium.

While it is ideal to onboard a method using an auto titrator from the start, in many cases, laboratories utilize legacy titration methods that were developed and verified manually. Even in these cases, there is a path forward to converting testing methods to an auto titrator. For an established and well-performing compendial monograph method, the conversion from manual to automatic titration can be done by demonstrating the equivalency of the techniques. A typical scope of work for this would include assessments of precision, intermediate precision, and linearity performed using both manual and potentiometric procedures. As discussed above, the Ph. Eur. requires a formal method validation and therefore more extensive scope of work for this exercise.

As a case study, we can examine the Ph. Eur. assay of sodium citrate. This is a direct manual titration method using perchloric acid in an acetic acid titrant with a naphtholbenzein indicator. The lab was experiencing a higher-than-expected rate for out-of-specification results, many of which were attributed to endpoint recognition. This, coupled with the fact that both the USP and JP methods were effectively identical (with only minor differences such as sample and solvent amounts) performed potentiometrically drove the decision to migrate the EP assay to an auto titrator method. Because the auto titrator assay was already in place and well-performing (for USP and JP), development work was abbreviated, consisting only of a small number of feasibility runs to confirm instrument parameters. The validation study was set up as a means of demonstrating that the new (potentiometric) method performed as well as, or better than, the prescribed manual titration method.

Both methods were run through the following parameters, and assessed against one another: precision, intermediate precision, linearity, accuracy, range, and robustness (solvent volume). The results of this study indicated that the potentiometric method performance was equal to the original method, and could be considered equivalent, and use of it moving forward justified. While the potentiometric method did not necessarily outperform the manual method in the context of the validation study, it still carries several advantages, including lack of subjectivity (endpoint recognition, reading of the burette), and traceability (automatically captured data). In addition, it carries the potential for automation when run on an auto titrator instrument with an autosampler, increasing throughput, as well as allowing for lower-risk testing with less experienced staff, both of which will result in cost savings.

While utilizing automatic titrators offers numerous advantages, as with any technology there are some drawbacks, although they are relatively minor in this case. As with all instruments, the primary obstacles are around startup and maintenance in terms of both time and cost. Transitioning to automatic titrations will require some startup costs to bring instruments in-house and have them qualified for use, particularly in a GMP environment. In addition, routine scheduled maintenance is typically required, which adds to overall costs andbringsg with it some transient instrument downtime. The advantages offered by migrating titration work to instruments are numeros, and include both the potential for increased throughput, and decreased variability in results. These advantages far outweigh the added costs, especially in today’s environment, which has seen increased focus on data integrity. The future of titrations lies in automated instrumentation and the expected cost savings gained by moving away from older, manual techniques will pay dividends in short order. Changing technology and attaining FDA 21CRF part 11 compliance greatly reduces the potential for human error and will yield more repeatable, reliable, and traceable data.

References

1. Skoog, Douglas A.; Holler, F. James; Crouch, Stanley R. Principles of Instrumental Analysis 2007
2. Mohr, Friedrich; Textbook of Analytical Chemistry Titration Methods 1855
3. Medicines & Healthcare products Regulatory Agency ‘GXP’ Data Integrity Guidance and Definitions March 2018

Author Details

Matt O’Connell, Senior Business Manager, PPD clinical research business - Thermo Fisher Scientific

Matt O’Connell received a B.S. in biochemistry from Benedictine University and hismaster'ss in organic chemistry from the Illinois Institute of Technology. He has been working in the pharmaceutical industry for 25 years, covering many facetofin his professional career ranging from drug discovery to highly potent API manufacturing. Matt is currently a Senior Business Manager with the PPD clinical research business of Thermo Fisher Scientific and supports the physical and compendial testing team.

Publication Details

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