Advantages of Hybrid Surface Technology to Improve Chromatographic Analysis of Food, Beverages, and Dietary Supplements

Analyte adsorption due to metal-analyte interactions can cause issues such as peak tailing, carryover, analyte loss, or reduced response in liquid chromatography (LC) of food, beverages and dietary supplements. These issues can be effectively mitigated by using a hybrid surface technology that has been developed for LC instrument and columns. This article reviews three application areas, B-group vitamins, steviol glycosides, and dextran oligosaccharides, where the hybrid surface technology has been demonstrated to effectively mitigate those issues and helped to achieve accurate, sensitive, and reliable LC analyses of food, beverages and dietary supplements.

High-performance liquid chromatography (HPLC) is a reliable, reproducible analytical technique for identifying and quantifying specific target analytes in food, beverages, and dietary supplement matrices, important for food nutritional analysis, label claim testing and clinical diagnostics. Stainless-steel (SSL) has long been the preferred material for use in chromatographic systems and columns, due to its manufacturability and ability to withstand pressure. However, analyte molecules containing electron-rich moieties such as phosphate and carboxylate groups can interact with metal ions on stainless-steel surface, resulting in poor chromatographic peak shape, severe analyte losses and quantitative inaccuracies (1,2). In addition, it has also been reported that metals leaching from metal surfaces may be adsorbed onto column stationary phases and interact with metal-sensitive analytes (3,4).

Replacing SSL tubes and column frits with non-metal options like polyether ether ketone (PEEK) can significantly improve the signal to noise ratio (S/N), peak intensity and peak tailing for analytes prone to absorption on SSL surface. However, these non-metal materials have limited strength and cannot be used in high pressure applications. While alternative metals have been explored, they present several drawbacks (5). Alternatively, chromatographers have tried to address these performance issues by adding chelators like EDTA to the mobile phase or sample diluent (6,7), but this can negatively impact chromatographic selectivity and introduce other complications.

Application of a Novel Hybrid Surface Technology

In this article, we discuss the application of a hybrid organic/inorganic surface technology that forms a barrier between the sample and the metal surfaces of both the HPLC system and the chromatographic column. Formed by a vapor deposition of an ethylene-bridged siloxane polymer on metal substrates (8), this technology effectively addresses common chromatographic challenges such as analyte loss, carryover, and peak tailing due to metal-analyte interactions (9–11). It improves peak symmetry and areas, as well as reproducibility, thereby not only benefiting challenging analytes but also increasing confidence in analytical results (12). We demonstrate the benefits of this technology through the analysis of B-group vitamins, steviol glycosides, and dextran oligosaccharides typically found in food, beverages, and dietary supplements, showcasing its critical role in improving chromatographic performance.

Application 1: B-Group Vitamins

Recent innovations in dietary supplements have introduced new forms of vitamin B, such as flavin mononucleotide (FMN) and pyridoxal 5’-phosphate (PLP) (Figure 1), known for their enhanced absorption and efficacy. These “native” or coenzymated B vitamins contain phosphate and, therefore, often interact adversely with the SSL surfaces used in HPLC (13-14), leading to peak tailing, reduced peak height, and/or carryover, which compromise the accuracy and reliability of the HPLC analysis.

To address these challenges, a comparative study (15) for vitamin B vitamers as well as other B-vitamins was conducted using two HPLC systems, which differed in terms of the chemical inertness of the wettable surfaces. One system comprised an ACQUITY Premier System and an ACQUITY Premier BEH C18 (1.7 μm, 2.1 x 100 mm) Column (referred to as the HPS system), and the other comprised an ACQUITY UPLC H-Class PLUS System and an ACQUITY UPLC BEH C18 (1.7 μm, 2.1 x 100 mm) Column (referred to as the SSL system). These two HPLC systems were identical, with the exception that the HPS system incorporated the hybrid surface technology, while the SSL system had conventional SSL surfaces. Both systems were evaluated under identical conditions, in the analysis of B-group vitamins in commercially available energy drinks and dietary supplements following the AOAC Official Method 2015.14 (15) with minor modifications. In this comparative study, the HPS system demonstrated significantly higher peak intensities and larger peak areas for most of the 18 vitamins tested compared to the SSL system (16). We also evaluated the performance of the two systems through prolonged usage, as detailed in Figure 2. The HPS system maintained consistently higher peak intensities and areas, demonstrating robust performance even after extensive use. Although the SSL system’s performance gradually improved as the surfaces conditioned, it continued to underperform relative to the HPS system. Additionally, the SSL system continued to exhibit severe peak tailing for thiamine.

This comparative study has shown a reduction of peak tailing, higher responses and improved sensitivity, better calibration linearity, and reduced carryover when utilizing an HPLC and chromatographic column comprising hybrid surface technology to reduce unwanted interactions. These benefits were certainly more evident when the system was first used. After extensive use of the SSL system and column, the difference between the HPS and the SSL systems became less significant, but the improvement in chromatographic performance is still evident.

Application 2: Steviol Glycosides

Steviol glycosides (SG), from Stevia rebaudiana Bertoni (stevia), are widely used as non-caloric sweeteners in foods and beverages. More than 40 SG have been identified (17); the most abundant are rebaudioside A (Reb A) and stevioside (SV). The increasing demand for minor SGs, due to their higher sweetness intensity and less bitter aftertaste (18,19), has driven advances in their chromatographic analysis. This group of compounds shares the same steviol aglycone structure, but with different numbers and types of glycoside units (e.g., glucose, rhamnose, or xylose), thus making their analysis challenging.

The FAO/WHO JECFA Monograph 26 (2021) establishes the latest international standard for SG analysis, but this HPLC method achieves a resolution of only 1.0. Recognizing the need for improved separation efficiency, we optimized the method following the International Council for Harmonisation (ICH) Q14 guideline (20), without extending the analytical runtime. Commercial stevia extracts were used for the method development process, during which several columns were screened, and the elution conditions optimized (17). Figure 3 shows the structures of the SG used in the study.

These SG include the 13 compounds in the JECFA method for the major SG and two additional SG, Reb I and Iso Reb A.Using the final method, excellent resolution was obtained for SG on both C18 columns with the minimum resolutions of 1.51 for the critical pair (Reb A/SV) and 1.89 for the rest of the SG (Table 1). The columns with the hybrid surface technology (XSelect Premier HSS T3 Column 2.5 µm, 4.6 mm × 150 mm) provided improved resolution over the conventional SSL column with XSelect HSS T3 Column, 2.5 µm, 4.6 mm x 150 mm. Good analytical performance, in terms of linearity, sensitivity, accuracy, precision, and robustness has been demonstrated through this study (17), and are a significant improvement over the published JECFA method for SG.

Application 3: Dextran Oligosaccharides

β-galactooligosaccharides (GOS), which occur naturally in human milk and the milk of many animals, play a critical role as prebiotics and are commonly used as supplements in food products, including infant formula. AOAC Final Action Method 2021.01, a recently approved HILIC method, provides a reliable approach for GOS analysis. However, our research has identified potential challenges associated with analyte loss and carryover under this method’s conditions, particularly affecting oligosaccharides with a degree of polymerization (DP) of 6 or higher. These issues could potentially impact the accurate determination of oligosaccharide content in consumer products (21).

In this study, an ACQUITY UPLC H-Class PLUS System and an ACQUITY UPLC Glycan BEH Amide Column (1.7 μm 2.1 x 150 mm), the SSL system, was compared against an Arc Premier System with an XBridge Premier Glycan BEH Amide Column (2.5 m, 2.1 x 150 mm), the HPS system. Figure 4 shows a comparison of HILIC-FLR chromatograms obtained on the SSL and HPS systems for 2-AB labelled dextran (with an internal standard). Repeated injections demonstrated that when analysed on the HPS system, the dextran oligosaccharides (degree of polymerisation [DP] 6 and higher) have a more consistent response and greater peak areas compared to the SSL system (21). For a better comparison, the peak areas from the conventional SSL system have been normalized to those from the HPS system, as shown in Figure 5. The figure includes results from the first, 18th, and 21st injections to illustrate trends over time. The data shows that the oligosaccharides with a degree of polymerization (DP) of 6 or higher experienced significant analyte loss, and greater analyte loss with higher DP. As we have seen for the B-group vitamin analysis, while repeated injections on the SSL system slightly mitigated this loss, a substantial loss remained after 20 injections (30% for the DP 11 peak).

Additionally, carryover peaks from the previous injection were observed on the SSL system for dextran oligosaccharides with DP 6 and higher, as shown in Figure 6. The extent of carryover on the SSL system was also found to increase with the DP of the oligosaccharide. In contrast, no carryover was detected on the HPS system for any dextran oligosaccharide.

In summary, the absence of carryover and analyte loss on the HPS system for all dextran oligosaccharides demonstrates the effectiveness of hybrid surface technology in resolving these issues, which are clearly observed on the conventional SSL systems and columns.

Conclusion

The use of chromatographic systems incorporating the hybrid surface technology offers clear benefits for metal sensitive compounds in terms of analyte peak shape, height and area, and limit of quantitation (15). Additionally, it provides reduced carryover for various chromatographic modes, including reversed-phase and HILIC. This technology not only improves the analysis of B vitamins, steviol glycosides, and dextran oligosaccharides but shows promise for a wide range of food, beverage, and dietary supplement applications, including chlorate, perchlorate, and bromate in food of plant and animal origin (22), and aminoglycosides in foods (23). Additionally, use of this technology avoids the need for HPLC system passivation, simplifying instrument maintenance and improving long-term performance. Even after extensive use, HPS systems deliver consistent and reliable results compared to conventional SSL systems, highlighting the potential of this technology to transform analytical methodologies in various food testing applications.

Acknowledgement

The authors would like to acknowledge Robert Birdsall (Waters Corporation) for reviewing the manuscript.

Declaration of Competing Interest

The authors are employees of Waters Corporation (Milford, MA, USA), a manufacturer of chromatography systems and consumables.

Waters, ACQUITY, UPLC, BEH, Arc, XBridge, XSelect, MaxPeak, Atlantis, and Empower are trademarks of Waters Technologies Corporation

References

(1) Sakamaki, H.; Uchida, T.; Lim, L. W.; Takeuchi, T. Evaluation of column hardware on liquid chromatography–mass spectrometry of phosphorylated compounds. J. Chromatogr. A 2015, 1381, 125–131. DOI: 10.1016/j.chroma.2014.12.088

(2) Wakamatsu, A.; Morimoto, K.; Shimizu, M.; Kudoh, S. A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry. J. Sep. Sci. 2005, 28 (14), 1823–1830. DOI: 10.1002/jssc.200400027

(3) Siegel, D.; Permentier, H.; Bischoff, R. Controlling detrimental effects of metal cations in the quantification of energy metabolites via ultrahigh pressure-liquid chromatography–electrospray-tandem mass spectrometry by employing acetylacetone as a volatile eluent modifier. J. Chromatogr. A 2013, 1294, 87–97. DOI: 10.1016/j.chroma.2013.04.029

(4) Ma, L.; Carr, P. W. Loss of bonded phase in Reversed-Phase liquid chromatography in acidic eluents and practical ways to improve column stability. Anal. Chem. 2007, 79 (12), 4681–4686. DOI: 10.1021/ac0703303

(5) De Pra, M.; Greco, G.; Krajewski, M. P.; Martin, M. M.; George, E.; Bartsch, N.; Steiner, F. Effects of titanium contamination caused by iron-free high-performance liquid chromatography systems on peak shape and retention of drugs with chelating properties. J. Chromatogr. A 2020, 1611, 460619. DOI: 10.1016/j.chroma.2019.460619

(6) Heaton, J. C.; McCalley, D. V. Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation. J. Chromatogr. A 2016, 1427, 37–44. DOI: 10.1016/j.chroma.2015.10.056

(7) Myint, K. T.; Uehara, T.; Aoshima, K.; Oda, Y. Polar Anionic Metabolome Analysis by Nano-LC/MS with a Metal Chelating Agent. Anal. Chem. 2009, 81 (18), 7766–7772. DOI: 10.1021/ac901269h

(8) Lauber, M. A.; DeLano, M. H.; McCall, S. A.; Belanger, J. L.; Dourdeville, T. A.; Smith, K. M.; Rainville, P. D.; Shah, D. D.; Shiner,S. J.; Doneanu, C.; Donegan, M. Use of vapor deposition coated flow paths for improved chromatography of metal interacting analytes. U.S. Pat. App. Pub. No.: US 2019/0086371 A1, Sept 17, 2018. https://patents.google.com/patent/US20190086371A1/en.

(9) Lauber, M.; Walter, T. H.; Gilar, M.; DeLano, M.; Boissel, C.; Smith, K.; Birdsall, R.; Rainville, P.; Belanger, J.; Wyndham, K. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces (HPS); Application Note 720006930; Waters Corporation: Milford, MA, 2023. https://www.waters.com/webassets/cms/library/docs/720006930en.pdf.

(10) Birdsall R. E.; Kellet J.; Ippoliti S.; Ranbaduge N.; Shion H.; Yu Y. Q. Increasing Chromatographic Performance of Acidic Peptides in RPLC-MS-based Assays with ACQUITY Premier featuring MaxPeak HPS Technology; Application Note 720007003; Waters Corporation: Milford, MA, 2020. https://www.waters.com/nextgen/us/en/library/application-notes/2020/increasing-chromatographic-performance-of-acidic-peptides-in-rplc-ms-based-assays-with-acquity-premier-featuring-maxpeak-hps-technology.html.

(11) Boissel C.; Walter T. H. Improved Peak Shape and Wide Selectivity Range with ACQUITY Premier Columns; Application Note 720007014; Waters Corporation: Milford, MA, 2020. https://www.waters.com/nextgen/us/en/library/application-notes/2020/improved-peak-shape-and-wide-selectivity-range-with-acquity-premier-columns.html.

(12) DeLano, M.; Walter, T. H.; Lauber, M. A.; Gilar, M.; Jung, M. C.; Nguyen, J. M.; Boissel, C.; Patel, A. V.; Bates-Harrison, A.; Wyndham, K. D. Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93 (14), 5773–5781. DOI: 10.1021/acs.analchem.0c05203

(13) Wakamatsu, A.; Morimoto, K.; Shimizu, M.; Kudoh, S. A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry. J. Sep. Sci. 2005, 28 (14), 1823–1830. DOI: 10.1002/jssc.200400027.

(14) Asakawa, Y.; Tokida, N.; Ozawa, C.; Ishiba, M.; Tagaya, O.; Asakawa, N. Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry. J. Chromatogr. A 2008, 1198–1199, 80–86. DOI: 10.1016/j.chroma.2008.05.015

(15) McClure, S. Simultaneous Determination of Total Vitamins B1, B2, B3, and B6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS—A Multi-Laboratory Testing Study Final Action: AOAC Method 2015.14. J. AOAC Int 2020, 203 (4), 1060-1072. DOI: 10.1093/jaoacint/qsaa012 .

(16) Yang, J.; Wilson, I.; Rainville, P. Evaluation of hybrid surface technology for the analysis of the B-group vitamins by LC-ESI-MS/MS. J. Chromatogr. B 2022, 1204, 123336. DOI: 10.1016/j.jchromb.2022.123336

(17) Yang, J.; Rainville, P.; Harden, S. Improving chromatographic resolution of the JECFA method for the analysis of steviol glycosides; Application Note 720008236; Waters Corporation: Milford, MA, 2024. https://www.waters.com/nextgen/en/library/application-notes/2024/improving-chromatographic-resolution-of-the-jecfa-method-for-the-analysis-of-steviol-
glycosides.html.

(18) FAO and WHO. 2021. Compendium of Food Additive Specifications. Joint FAO/WHO Expert Committee on Food Additives (JECFA), 91st Meeting – Virtual meeting, 1–12 February 2021. FAO JECFA Monographs No. 26. Rome. DOI: 10.4060/cb4737en.

(19) Prakash, I.; Markosyan, A.; Bunders, C. Development of next generation Stevia Sweetener: Rebaudioside M. Foods 2014, 3 (1), 162–175. DOI: 10.3390/foods3010162

(20) ICH. Analytical Procedure Development Q14, (Final Version, 1 Nov 2023) https://database.ich.org/sites/default/files/ICH_Q14_Guideline_2023_1116.pdf (accessed Jan. 1, 2024).

(21) Yang, J.; Rainville, P.; Harden, S. Benefits of MaxPeakTM high performance surfaces in the determination of Β-Galactooligosaccharides in infant formula by HILIC; Application Note 720008260; Waters Corporation: Milford, MA, 2024. https://www.waters.com/nextgen/us/en/library/application-notes/2024/benefits-of-maxpeak-high-performance-surfaces-in-the-determination-of-galactooligosaccharides-in-infant-formula-by-hilic.html.

(22) Idialu, O.; Hancock, P.; Hird, S., Trivedi, E,; Ross, E. Determination of chlorate, perchlorate, and bromate in food commodities using LC-MS/MS with Atlantis™ Premier BEH C18 AX column; Application Note 720008070; Waters Corporation: Milford, MA, 2023. https://www.waters.com/nextgen/us/en/library/application-notes/2023/determination-of-chlorate-perchlorate-and-bromate-in-food-commodities-using-lc-ms-ms-with-atlantis-premier-beh-c18-ax-column.html

(23) Hird, S.; Rathmann, C.; Yang, J.; Woyzek, B. Development and validation of a confirmatory method for the determination of aminoglycosides in foods using LC-MS/MS with a zwitterionic HILIC column; Application Note 720008022; Waters Corporation: Milford, MA, 2023. https://www.waters.com/nextgen/us/en/library/application-notes/2023/development-and-validation-of-a-confirmatory-method-for-the-determination-of-aminoglycosides-in-foods-using-lc-ms-ms-with-a-zwitterionic-hilic-column.html

Dr. Jinchuan Yang is a Principal Scientist at Waters Corporation. Since he joined Waters in 2002, he has worked in method development for polymers, food nutrients, pesticides, and environmental pollutants contaminants using the cutting-edge technologies in liquid chromatography, supercritical fluid chromatography, mass spectrometry, and hyphenated techniques. His most recent research interests focus on the development of chromatographic methods to fill the emerging gaps for food manufacturers. He is an active participant in technical stakeholder groups at AOAC International and USP, serving as an expert panel member in AOAC SPIFAN and participating in review, recommendation, and muti-lab testing of AOAC methods.

Chantel Lee is a Principal Product Marketing Manager at Waters Corporation, driving marketing strategies for the HPLC product portfolio. With over a decade of experience in analytical chemistry, she specializes in chromatography and Empower software, supporting customers from pharmaceutical, food, and chemical industries. Since joining Waters in 2017, Chantel has progressed through roles in technical support, business development, and marketing. She holds an MBA from Boston University along with an MSc in Analytical Chemistry from the University of Alberta, Canada.

Dr. Stephanie N. Harden manages Waters small molecule HPLC product marketing and scientific teams. She has 25 years of experience in product and segment marketing and a proven track record leading cross-functional teams in B2B digital/omni-channel marketing, and in strategic program development and execution. She is a highly cited author with a PhD in Chemistry from the University of Bristol, UK.

Dr. Paul D. Rainville is a Scientific Advisor at Waters Corporation. He graduated from the University of Massachusetts with a BS in Biotechnology and received his doctorate from King’s College, London, UK. He has been with Waters for over twenty years. Prior to joining Waters, he worked as an analytical chemist and biochemist at various pharmaceutical companies. He has authored or contributed to over fifty peer-reviewed research articles. Dr. Rainville is the co-recipient of the 2011 Desty Award for Innovation in Separation Science and is a Fellow of The Royal Society of Chemistry.

E-mail: Jinchuan_Yang@waters.com

Website: www.waters.com