Elsevier

Journal of Chromatography A

Volume 1594, 7 June 2019, Pages 54-64
Journal of Chromatography A

Development of a rapid ultra performance hydrophilic interaction liquid chromatography tandem mass spectrometry method for procyanidins with enhanced ionization efficiency

https://doi.org/10.1016/j.chroma.2019.02.007Get rights and content

Highlights

  • HILIC UPLC reduces analysis time over normal-phase or HILIC HPLC.

  • Post-column infusion with ammonium formate enhances procyanidin ionization.

  • Improved detection of larger procyanidins.

Abstract

Cocoa flavanols (catechins and procyanidins) can exist in various polymerization states and are commonly classified by their degree of polymerization (DP). There is increasing evidence that flavanols of distinct DP possess different biological activities, but separation and quantification of the higher DP procyanidins is challenging and has thus created the need for new methodologies that utilize advancements in columns and LC–MS/MS systems. An aqueous normal phase (hydrophilic interaction liquid chromatography, HILIC), UPLC method with post-column ESI adjuvant infusion was developed to reduce the total analysis time, increase peak separation, and increase detection specificity (compared to traditional fluorescence methods) by coupling with mass spectrometry detection. The total elution time was reduced from 70 to 90 min (typically used for normal phase and HILIC HPLC separation of procyanidins) down to 9 min by employing UPLC. Results indicate that by using a post-column 0.04 M ammonium formate infusion (5 μL/min), ionization of procyanidins was significantly enhanced. Lower limits of detection ranged from 3.19 × 10−2 to 4.56 pmol-on-column, and lower limits of quantification ranged from 2.79 × 10−2 to 1.17 × 102 pmol-on-column across compounds DP 1–9. This method builds upon the foundation set by existing analytical methods and employs new technologies to dramatically increase sample throughput and enhance detection limits and specificity, facilitating improved analysis for procyanidins.

Introduction

Cocoa is one of the most abundant sources of dietary flavanols, a subclass of flavonoids. Flavanols can exist as monomers ((±)-catechin (C), (–)-epicatechin (EC), etc.) or in various polymerization states (oligomers and polymers known as procyanidins, proanthocyanidins or condensed tannins) (Fig. 1). These compounds are commonly classified by their degree of polymerization (DP), and products such as cocoa contain high amounts of flavanols with DP 1–10 [[1], [2], [3]]. Cocoa flavanols have drawn significant interest due to their apparent health promoting effects, and there is increasing evidence that procyanidins with different DP possess structurally distinct biological activities [[4], [5], [6]]. Thus, qualitative and quantitative analysis of procyanidins by DP is important for understanding the biological activities of procyanidins.

Due to factors such as ease of analysis by and more widespread use of reverse-phase (RP) LC (which typically cannot resolve the large procyanidins adequately), wide commercial availability of standards and comparatively high bioavailability, much of the literature focuses on the food content and biological action of flavanol monomers, C and EC. However, the larger species (oligomeric and polymeric flavanols) are widely distributed in the diet, and recent evidence suggests that despite their limited bioavailability, these complex flavanols are effective at mediating the onset of obesity and insulin resistance as well as protecting against gut inflammation activity when compared to smaller flavanol fractions [[5], [6], [7]]. Although the relationship between DP and biological activity remains the subject of investigation, these complex flavanols are the subject of increasing investigation as potentially potent and novel complementary approaches to chronic disease prevention. Due to this interest, it is critical to develop analytical methods that can provide rapid, sensitive and specific detection and quantification of these individual procyanidins in order to more thoroughly understand their underlying mechanisms of action, plant distribution and consumption patterns.

Because of the structural complexity these high molecular weight procyanidins possess, their analysis is challenging [1]. There is generally an inverse relationship between flavanol DP and detector response (for various detection modalities), and thus limits of detection, limits of quantification, and analytical sensitivity [8]. Normal-phase (NP) or HILIC HPLC coupled to fluorescence detection (FLD) typically provide the best resolution of large flavanols, but efficiency and sensitivity remain the primary challenge of using these methods [1,[8], [9], [10], [11], [12]]. With run times typically ranging between 70–90 min, these methods are severely limited by low sample throughput and place high demand on analytical resources. Machonis et al. [10] was able to significantly optimize the robust, widely-used NP method proposed by Robbins et al. [9], reducing the elution time to 15 min as opposed to 86 min. Additionally, Hollands et al. [8] adapted the method proposed by Robbins et al. [9] but with a HILIC HPLC application to quantify large procyanidins extracted from apples. Although this method is able to quantify similar procyanidins, it has a lengthy 45 min run time. Recently, advances in UPLC column technology and the improvement of HILIC UPLC columns suggests that the speed of these methods may be optimized even further. However, despite reduced analysis time and increased throughput, sensitivity of high DP compounds remains low. This is due to the fact that NP methods typically rely on FLD [[9], [10], [11]]. While FLD is useful for procyanidins, use of FLD does not provide the specificity of definitive mass confirmation and fragmentation achieved by MS/MS. Therefore, coupling the NP or HILIC resolution power with the specificity of mass spectrometry (MS) is desired. However, NP LC is poorly compatible with the standard MS ionization source, electrospray ionization (ESI), which relies on the presence of high concentrations of protic solvents and other ionizable mobile phase reagents to facilitate charge transfer to analytes, while RP and HILIC LC are better suited for MS pairing. Though HILIC solvents are much better suited for ESI than NP, the comparatively low concentrations of water used for HILIC (vs. RP) suggest that ESI ionization efficiency could still be enhanced through post-column reagent infusion to achieve ionization performance similar to that of RP conditions, and therefore improving sensitivity and achieving adequate limits of detection and quantification [13,14]. Recently, the use of post-column ionization reagents has been proposed for improved ESI performance of flavanols [12].

To continue advancing our understanding of the role of procyanidins in human health, the quantification of these large procyanidins must be addressed. Long analysis times require extended instrument use and large quantities of solvents that could otherwise be used for other analyses. Additionally, long analysis times grouped with lengthy sample sets could lead to sample degradation. The objective of this study was therefore to develop an optimized methodology that rapidly and sensitively quantifies individual procyanidins based upon their DP to reduce instrument time, lower laboratory costs, and increase sample throughput. We sought to achieve this by pairing recent improvements in HILIC-UPLC with use of post-column ESI adjuvant infusion.

Section snippets

Reagents, standards, and samples

LC–MS grade acetonitrile (ACN), methanol (MeOH), and water were obtained from Thermo Fisher Scientific (Waltham, MA). Glacial acetic acid, methanol, and acetone were obtained from VWR (Radnor, PA). Ammonium formate and (−)-epigallocatechin gallate (EGCG) were obtained from Sigma-Aldrich (St. Louis, MO). Standards of (±)-catechin (C), (–)-epicatechin (EC), and procyanidin B2 (PCB2) were obtained from ChromaDex (Irvine, CA). Standards of procyanidin C1 (PCC1, DP3), cinnamtannin A2 (CinA2, DP4),

Results and discussion

In the present study, we developed a comparatively rapid HILIC-UPLC-MS/MS method to more efficiently and effectively quantify cocoa flavanols by their DP. We achieved this by employing a UPLC HILIC column to optimize peak resolution and then utilizing post-column ionization of ammonium formate to improve ESI-MS ionization. Although our method meets the definition of traditional NP through the utilization of mobile phases less polar than the stationary phase, it is in fact aqueous NP (HILIC) due

Conclusions

Growing interest in cocoa and the relationship between cocoa polyphenols and health has created a gap in the quantification of larger cocoa analytes. These large molecular weight compounds with varying DP are difficult to measure and thus, until recently, have been largely unexplored. Currently the only available methods to quantify these large DP procyanidins require large time commitments, large volumes of solvents, or are not compatible with MS, therefore being unable to identify any

Acknowledgements

Funding for this work was provided, in part, by 1) Waters Corporation, Milford, MA (purchase of authentic standards) and 2) the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture.

References (16)

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