(T0930-09-59) Analysis of Semi- and Non-Volatile Leachables via an Enhanced HPLC-UV Method

Purpose: Drug products in all dosage forms and storage containers contain leachables to varying degrees and pose significant quality and safety risks.¹ Due to the wide variety of potential leachables from container closure systems (CCS) and formulations, the creation of a single method to analyze more than a handful of drug products at a time is nearly impossible.² Currently, many analytical labs have developed their own methods for the quantification of both extractables and leachables using one or more surrogate standards, primarily relying on LC/MS for semi- and non-volatile leachables.³ Although MS excels at identification, large variations in responses based on factors such as ionization and functional group pose a significant problem for the quantification of species (ex. From Jordi et al: across 217 species quantified via LC-MS, only 33% were accurate within ± 20%).^3,4 To account for these variations, uncertainty factors are typically applied in an attempt to overestimate the actual amount of impurity.⁵ For some analytes the Safety Concern Threshold (SCT) is high and does not pose a risk even if underestimated. However, underestimating the exposure for toxic, carcinogenic, or mutagenic species poses a risk of harm to patients.^3,4 Recently, interest in LC with UV detection has increased due to the reliability of Relative Response Factors (RRF; across various labs and instruments 7% RSD for like-species when used in extractable quantification).⁶ Jordi et al have shown that implementation of UV detection used concomitantly with MS show significant improvement in quantitation (85% of analyzed compounds were accurate to ± 20%).⁴ However, the multidetector approach presented requires 10 standards and has limitations due to UV-detection at 230 nm.⁴

Methods: To bridge this gap, we have developed a method with enhanced sensitivity using HPLC-UV. As a modifier, phosphoric acid is used to allow for low wavelength analysis. Additionally, by equipping a Ghost Buster™ column we reduce the baseline noise, increasing method sensitivity. With this method, we have determined the relative response factors for common – and not-so-common – E/L species (Figure 1).

Results: BHT (butylated hydroxytoluene) was selected as a surrogate standard due to its prominence as an extractable and leachable and being readily available at low costs. Other leachables were selected due to their presence in previous E/L studies performed at Frontage Laboratories. Increasing the sensitivity of this method further can be easily performed by varying the injection volume. In Figure 2, solutions at various concentrations are all compared with 10 µL injection volumes. With S/N values higher than 10, we could accurately quantify these species. However, as our library of leachables expands, the need for increasing the injection volume for analytes containing weaker chromophores may be necessary. Below, injections of citric acid solution at 50 µg/mL were completed by increasing the injection volume from 20 to 100 µL with minor obfuscation of peak shape and a slight shift in retention time (Figure 3). However, in each case, we see clear resolution between the adjacent diluent peaks and can account for slight changes in retention time for identification using Relative Retention Time (RRT).

Conclusion: By creating a library of known leachables with RRTs and RRFs we can use a single standard (BHT) to confidently quantify known leachable species. Using this HPLC-UV method we can quantify at low levels due to enhanced sensitivity and see species, such as citric acid, which normally cannot be seen without low wavelengths such as 200 nm. With these developments, we can confidently identify and quantify leachables to meet regulatory guidelines for E/L studies.

References: 1 United States Pharmacopeia, USP Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems, Retrieved from https://doi.org/10.31003/USPNF_M7127_03_01 (accessed 10 June, 2023), Official as of 01 December 2020.
2 Riley, Christopher M. Rosanske, Thomas W. Reid, George L.. (2020). Specification of Drug Substances and Products - Development and Validation of Analytical Methods (2nd Edition). Elsevier. Retrieved from
https://app.knovel.com/hotlink/toc/id:kpSDSPDVA1/specification-drug-substances/specification-drug-substances
3 Singh, G.; Lu, D.; Liu, C. and Hower, D. Trends in Analytical Chemistry 2021, 114, 116286.
4 Jordi, M.A.; Rowland, K.; Liu, W.; Cao, X.; Zong, J.; Ren, Y.; Liang, Z.; Zhou, X.; Louis, M. and Lerner, K. Journal of Pharmaceutical and Biometical Analysis 2020 186, 113334.
5 Norwood, D.L.; Nagao, L.M. and Stults, C.L.M. Perspectives on the PQRI extractables and leachables “safety thresholds and best practices” recommendations for inhalation drug products. J. Pharm. Sci. Technol. 2013, 67, 413-429.
6 Jenke, D.; Liu, N. Chromatographic considerations in the standardization of liquid chromatographic methods used for extractables screening, J. Liq. Chromatogr. Relat. Technol. 2016, 39, 613-619.

Figure 1. Representative Chromatogram of BHT, our surrogate standard with a selection of other leachables of interest.

Figure 2. Citric acid, typically not visible using traditional UV techniques, can be seen at 50 µg/mL while bis(2-hydroxyethyl)-terephthalate and 2-hydroxy-2-methyl-propiophenone are both quantifiable at 2 µg/mL.

Figure 3. Injection volume variations for citric acid at 50 µg/mL.