Navigating Hurdles in Achieving HPLC Reproducibility

How to Guide 1 Navigating Hurdles in Achieving HPLC Reproducibility: 10 Common Challenges and Practical Solutions Imran Janmohamed, PhD Laboratories utilize analytical equipment to generate valuable data, enabling informed decisions to be made on various products or processes. Analysts are typically tasked with performing a range of testing procedures, from simple to complex, and are required to generate reproducible, accurate, consistent and reliable results, reflecting their overall performance. High-performance liquid chromatography (HPLC) is a common analytical technique and plays a pivotal role in separating, identifying and quantifying complex samples. Consequently, it has found applications in diverse fields including academic research, biotechnology, the chemical industry, environmental sciences and pharmaceutical, food and beverage testing. However, operational challenges in HPLC are common, including issues like poor reproducibility, poor peak shape, instrument sensitivity and detector drift,1 which can be linked to various factors. This guide provides practical advice for those aiming to address these types of issues and enhance workflow standards within their laboratories. Understanding key quality indicators in laboratory analyses: Definitions of the terminology Accuracy, precision, repeatability and reproducibility are terms used in relation to key indicators of the quality of experiments and analyses in a laboratory setting. However, these terms are often used inconsistently or even in contradictory ways across different scientific disciplines and institutions. Therefore, we will briefly define them to ensure a consistent understanding of the challenges associated with HPLC reproducibility. Accuracy is defined as the closeness of a measured value to the true or accepted value of a quantity, and it assesses how well a measurement reflects the actual or expected value.2 It is evaluated by comparing the measurement to a known standard or a reference material, assessing the bias or systematic error and is usually expressed as % recovery. For example, if the true concentration of paracetamol in a sample is 10 μg/mL (validated externally), and the measured value from your HPLC analysis is 10.1 μg/mL, the instrument accuracy is high. NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 2 How to Guide Precision expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.2 It is usually expressed as the variance, coefficient of variation or standard deviation. Precision is independent of accuracy (Figure 1A). For example, if the measured concentration of paracetamol from five replicate HPLC analyses is 10.1, 10.2, 9.8, 10.3, 10.1 μg/mL, this indicates high instrument precision, regardless of the true concentration. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility:2 • Repeatability is defined is the closeness of agreement between independent measurements obtained with the same method, under the same conditions, by the same operator using the same equipment and expresses the precision under the same operating conditions over a short interval of time. • Intermediate precision is the closeness of agreement between independent measurements obtained with the same method within laboratories but performed, for example, on different days, by different analysts and on different equipment. This can sometimes be referred to as “within-lab reproducibility”. • Reproducibility is the closeness of agreement between independent measurements obtained with the same method between different laboratories (e.g. in collaborative studies, usually on different days with different analysts and equipment (Figure 1B)). Figure 1: Representation of the differences between key terminology in lab quality indication. Figure 1A: The difference between accuracy and precision. Figure 1B: Reproducibility between laboratories A and B. Results from laboratory A shows low precision with high variation in results, while B shows high precision with low variation in the results. However, both results show good reproducibility within the measurement uncertainty. Credit: Imran Janmohamed In the context of the above definitions, let’s consider some of the sources of errors in HPLC precision (including reproducibility) and potential solutions to enhance the results of HPLC analyses. HIGH PRECISION, LOW ACCURACY HIGH PRECISION, HIGH ACCURACY LAB B LAB A 1A 1B NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 3 How to Guide 1. Equipment and method settings: Inconsistent performance of instruments and changes to method or instrument parameters, knowingly or unknowingly, can produce inconsistent analytical information, such as variations in retention times. Some solutions could therefore be to: • Establish detailed standard operating procedures (SOPs) and technical documents on the use of the instrument. This would help to maintain consistency among multiple users and uniformity in setup, usage and shutdown procedures. • Maintain the instruments regularly, including priming, flushing and purging of the system, to ensure that they are in a good working condition. • Understand, optimize and tailor method parameters for each instrument to ensure consistent analysis, e.g. allocate sufficient time for gradient equilibration to reach the column, as each system may have a different dwell volume due to the installation of different tube sizes and lengths. 2. Quality control: Quality control (QC) involves time-consuming but essential testing, calibration and verification procedures to monitor the performance of HPLC. The lack of routine quality checks can lead to the production of unreliable and error-prone data. The following solutions should be considered: • Implement a comprehensive QC program, including running appropriate system suitability checks and system blanks, to ensure that there is traceability of potential sources of errors. The type of blank could include solvent blanks, instrument blanks, trip blanks, vial blanks, sample blanks etc. • Periodically validate the entire HPLC system with reference standards and review the data to ensure consistency between different timelines. • Calibrate and validate different parts of the instrument, such as the autosampler, the pumps and the detectors, which enables targeted evaluation and can be used to define the potential sources of error. This is usually included in manufacturer or third-party preventative maintenance service plans. 3. Sample preparation: Sample preparation is often time-consuming and error prone in HPLC analysis. Inconsistent and inaccurate sample preparation can lead to poor chromatographic results and increased analysis time, generating variability and inaccuracies in analyses. The following solutions should be considered: • Develop standardized sample preparation methodologies with clear and comprehensive protocols specifying the type and amount of reagents, solvents and other materials used. Include specific instructions for the handling of different sample types, ensuring uniformity and homogeneity. • Implement rigorous sample handling protocols, including detailed procedures, to ensure consistency in sample weighing, dilution and mixing. • Implement automated sample preparation techniques to reduce human error, enhance reproducibility and improve consistency in tasks such as extraction e.g. solid-phase extraction (SPE), automated liquid–liquid extraction (LLE), QuEChERS (quick, easy, cheap, effective, rugged and safe).3 • Use calibrated equipment for sample preparation and transfer, including measuring equipment, to ensure the intended volume is dispensed accurately, especially when dealing with low-volume injections. NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 4 How to Guide 4. Mobile phase compatibility and preparation: The selection of a suitable mobile phase is crucial for achieving optimal separation, peak shape and retention times. Incompatibility between solvents used in samples and mobile phases can significantly impact the instrument’s performance. Consequently, the following should be taken into account: • Ensure compatibility of solvents used throughout the process from sample preparation to HPLC analysis by reviewing factors such as solvent boiling points, polarity index, ultraviolet (UV) cutoffs and solvent miscibility.4 • Use and prepare mobile phases using high-quality solvents and additives to avoid contamination and interferences. • Ensure precise process measurement during solvent preparation. For example, individually add accurately measured solvents such as methanol and water to a Duran bottle, rather than combining them directly, to achieve an accurate volumetric ratio (e.g. 40:60 methanol: water). 5. Column selection and care: The selection of a suitable column for complex analysis can be challenging and could affect factors such as resolution and efficiency. The following are potential solutions to ensure columns have high reproducibly: • Select and utilize columns from recognized manufacturers to ensure good availability and consistent performance, but also have a good understanding of the type of stationary phase, column dimensions and particle size used. • Consider the use of new column technology when selecting columns as they can offer improved performance and longevity, such as superficially porous particles (SPP) and monolithic columns. You should consult with column manufacturers or experts in the field to ensure applicability.5 • Ensure the columns used are maintained within their pH, temperature and pressure considerations during the analysis as well as pre- and post-analysis. • Ensure columns are handled with proper care and are maintained. This includes observing appropriate handling techniques, solvent flushing and conditioning to prevent damage to the stationary phase, thereby extending the column’s overall performance and life. 6. Temperature control: Temperature fluctuations can significantly impact the volume, flow rate, solubility and viscosity of solvents, consequently affecting injections throughout the analytical process. Such variations can have an influence on chromatographic separation and resolution. Consideration should be given to the following: • Use precise temperature control measures for key components of the HPLC system, including the autosampler, column oven and detector to guarantee a consistent volume and flow throughout the chromatographic process. • Maintain consistent temperature conditions during sampling, sample preparation and analysis to ensure reliable and reproducible results e.g. ensuring laboratory temperatures are consistent during sample preparation can help to minimize the loss of solvent and sample. NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 5 How to Guide 7. Injection technique: Variability in injection volumes can have an impact on peak areas, retention times and shapes. Additionally, knowledge of concentrations in the samples can be essential in the selection of appropriate injection volumes. The following can be considered: • Perform systematic injection volume studies to determine the optimal injection volume based on sample and column requirements. Consider factors such as sample concentration, solvent type and composition and column type and dimensions. • Understand and utilize automated injection systems with optimized wash settings to ensure consistent injection volume, minimize carry over and implement a suitable cleaning regime. 8. Data analysis and integration: Data analysis is critical for any analytical system, and variation in integrations can lead to inaccurate quantification, impacting the reliability of HPLC results. The following should be considered to reduce errors: • Select a reliable and fit-for-purpose data analysis software that is capable of accurately identifying, integrating and quantifying peaks for your application. • Regularly review, update and validate integration parameters using standard solutions as part of any regular QC checks. • Understand and evaluate spectra from photo diode array/diode array detector (PDA/DAD) detectors, reviewing factors such as lambda max and peak purity analysis, along with creating suitable UV libraries to allow spectral data to be evaluated faster. 9. Method development, validation and verification: Method development for complex samples, followed by method validation or verification, can be a lengthy and iterative process. However, it is crucial to ensure that the method parameters utilized continue to produce consistent analytical values and minimize variability. As part of validation, tests to evaluate parameters such as precision, repeatability and accuracy are performed and reported. The following are solutions to increase precision from method development and validation processes: • Use method development software tools as guides to develop your HPLC solvent gradient profiles, identify potentially suitable column types, appropriate solvent pHs and temperatures conditions. These tools utilize virtual physical-chemical retention models or empirically intelligent simulation of real HPLC data to provide information.7 • Leverage other tools, such as design of experiments (DoE) or quality by design (QbD), to aid method development and to identify the optimal separation conditions to allow for systematic exploration of variables and condition.8, 9 • Develop methods using innovative technological processes, such as ultra-high performance liquid chromatography (UHPLC) columns and gradient elution techniques, which can enhance efficiency and resolution, but exercise care and consideration regarding the types of samples, solvents and buffers that are used.10 • Ensure the methods developed are fit-for-purpose for the project stage and are validated at a suitable level with consideration on robustness and specificity. For example, in HPLC, variations of mobile phase pH and composition can have significant influence on the results. • Verify sample collection and sample preparation methods to ensure their reliability and reproducibility, including recovery studies to assess the efficiency of the sample preparation process. NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 6 How to Guide 10. Communication, documentation and training: Lack of communication among team members, insufficient record-keeping and documentation and untrained staff can lead to errors on the instrument, in the methods and data analysis. Reproducibly errors due to this can be resolved by the following solutions: • Encourage open communication to address instrument challenges promptly and encourage collaborations between analysts. • Maintain detailed records of instrument settings, mobile phase composition and sample preparation. • Create a logbook for instrument usage and maintenance including column records to ensure knowledge of the use of columns is available and shared. • Encourage ongoing education in chromatography techniques with regular training on targeted hardware and software, which increases the understanding of HPLC principles, data analysis and UV interpretations. By addressing these challenges and implementing the suggested solutions, laboratories can significantly enhance the reproducibility of HPLC analyses, leading to more reliable and accurate results. Regular monitoring, documentation and training are key elements in maintaining a high level of reproducibility in HPLC experiments. In summary, the reproducibility of HPLC results is not only crucial for scientific accuracy but also for meeting regulatory standards and ensuring the quality, safety and compliance of products and processes in numerous industries. References 1. Kromidas S. The HPLC Expert: Possibilities and Limitations of Modern High Performance Liquid Chromatography. 1st ed. John Wiley & Sons; 2016. doi:10.1002/9783527677610 2. European Medicines Agency. ICH topic Q 2 (R1) validation of analytical procedures: text and methodology. https://www. ema.europa.eu/en/documents/scientific-guideline/ich-guideline-q2r1-validation-analytical-procedures-text-and-methodology- step-5-first-version_en.pdf. Published June 1995. Accessed March 7, 2024. 3. QuEChERS: Home. www.quechers.eu. https://www.quechers.eu/. Accessed March 13, 2024. 4. Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. John Wiley & Sons; 2009:879-886. doi:10.1002/9780470508183 5. Hayes R, Ahmed A, Edge T, Zhang H. Core–shell particles: Preparation, fundamentals and applications in high performance liquid chromatography. J Chromatogr. 2014;1357:36-52. doi:10.1016/j.chroma.2014.05.010 6. Wang L, Zheng J, Gong X, Hartman R, Antonucci V. Efficient HPLC method development using structure based database search, physicochemical prediction and chromatographic simulation. J Pharm Biomed. 2015;104:49-54. doi:10.1016/j. jpba.2014.10.032 7. Pramod K, Tahir MA, Charoo NA, Ansari SH, Ali J. Pharmaceutical product development: A quality by design approach. Int J Pharm Investig. 2016;6(3):129. doi:10.4103/2230-973X.187350 8. Gurumukhi V, Bari S. Quantification and validation of stability-indicating RP-HPLC method for Efavirenz in bulk and tablet dosage form using quality by design (QbD): A shifting paradigm, J Chromatogr Sci, 2022:60 (2):143. doi:10.1093/chromsci/ bmab061 9. Guillarme D, Ruta J, Rudaz S, Veuthey JL. New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches. Anal Bioanal Chem. 2010;397(3):1069-1082. doi:10.1007/s00216-009-3305-8 NAVIGATING HURDLES IN ACHIEVING HPLC REPRODUCIBILITY: 10 COMMON CHALLENGES AND PRACTICAL SOLUTIONS 7 How to Guide About the author: Imran Janmohamed is a consultant, project leader, trainer and research scientist with over 17 years of experience specializing in R&D chemistry. His expertise encompasses a wide range of analytical techniques, including gas chromatography (GC), gas chromatography- mass spectrometry (GC-MS), ultra high pressure liquid chromatography (UHPLC/HPLC), liquid chromatography- mass spectrometry (LC-MS), Fourier transform infrared (FTIR) and ultraviolet-visible (UV-Vis) spectroscopies, across various sectors such as academia, food, medical devices and pharmaceuticals. Imran collaborates with clients to develop methodologies addressing analytical challenges as a director of Analyzd Ltd. He also served as the analytical lead in R&D product development, ensuring compliance with current good manufacturing practice (cGMP) guidelines while overseeing laboratory operations and equipment maintenance. Imran’s consultancy work includes delivering workshops for the Royal Society of Chemistry’s Pan Africa Chemistry Network (PACN) training program. With a strong academic background, Imran holds a PhD in analytical chemistry and a BEng Hons in chemical with biochemical engineering.