Simultaneous Quantification of κ-Carrageenan Oligosaccharides of DP 3, 5 and 7 by LC-MS/MS: Application to an in vitro Absorption Study

YANG Yali ZHANG Pengpeng LIU Guilin YANG Shuang 2) 3) 4) WANG Yuanhong 2) 3) 4) JIANG Tingfu 2) 3) 4) LVZhihua 2) 3) 4) * and YU Mingming 2) 3) 4) *

Simultaneous Quantification of κ-Carrageenan Oligosaccharides of DP 3, 5 and 7 by LC-MS/MS: Application to anAbsorption Study

YANG Yali1), ZHANG Pengpeng1), LIU Guilin1), YANG Shuang1), 2),3), 4), WANG Yuanhong1), 2),3), 4), JIANG Tingfu1), 2),3), 4), LVZhihua1), 2),3), 4), *, and YU Mingming1), 2),3), 4), *

1)School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China 2) Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China 3) Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, Qingdao 266003, China 4) Key Laboratory of Marine Drugs, Ministry of Education of China, Qingdao 266003, China

Carrageenans are widely utilized in many commercial applications such as the food and pharmaceutical industry, due to their excellent functional properties. In this study, a sensitive LC-MS/MS method was developed to determine κ-3, κ-5, and κ-7 carrageenan oligosaccharides simultaneously. Optimum MRM transitions for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were (645.079→565.111, [M-H]−), (515.137→474.946, [M-2H]2−), and (471.484→445, [M-3H]3−), respectively. Chromatographic separation was performed on anAmide column coupled with a guard column operated at 60℃ under stepwise gradient elution. The linearity of the LC-MS/MS method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides, evaluated over the concentration range of 0.10-20.0μmolL−1, was excellent. The precisions of the method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were from 0.91% to 9.66%, and the inter-day precisions were from 0.92% to 10.5%. Validation of theLC-MS/MS method indicated that the method was precise and in line with the CFDA guidance. This method has been successfully applied to anabsorption study.

κ-carrageenan oligosaccharide; Caco-2 cell; LC-MS/MS; degree of polymerization

1 Introduction

Carrageenans are highly sulfated polysaccharides extracted from the cell walls of marine red seaweeds (Campo, 2009). Carrageenans are formed by alternate units of D-galactose and 3, 6-anhydro-galactose joined by β-1,4 and α-1,3-glycosidic linkage (Shukla, 2016). Carrageenans are widely utilized in many com- mercial applications such as the food and pharmaceutical industry (Li, 2014; Prajapati, 2014), due to their excellent functional properties. Carrageenans are divided into six basic forms: Iota (ι)-, Kappa (κ)-, Lamb- da (λ)-, Mu (μ)-, Nu (ν)- and Theta (θ)-carrageenan (Campo., 2009). Kappa-Carrageenan (κ-carrageenan) is predominately obtained from the tropical seaweed, and it consists of D-galactose having sulfate at C4position linked to anhydro-galactose (Fig.1) (Vera, 2011).

Fig.1 The chemical structure of κ-carrageenan.

The high viscosity properties limit the application of carrageenan, while their oligomers are widely used. Carrageenan oligosaccharides are the degradation products of κ-carrageenan. Many studies have demonstrated that the carrageenan oligosaccharides have a variety of pharmacological activities including antioxidant, anticoagulant anti-inflammatory, and antiviral activities (Yuan, 2005, 2006; Nardi, 2007; Abad., 2013). These pharmacological activities are closely related to the number of sulfate groups and the degree of polymerization (DP) (Sun, 2014). Many studies showed that the degree of polymerization and molecule weight impact the absorption and bioavailability of compounds (Veber, 2002; Kruger, 2003; Tsai, 2012). However, there are few studies about the relationship between the DP and absorption of κ-carra-oligo- saccharides.

A rapid and stable analytical method for the determination of κ-carrageenan oligosaccharides of different DP is very important to study their absorption processes. However, few reports are demonstrating the quantification of κ-carrageenan oligosaccharides. In this study, we report an analytical method that is able to determine κ- carrageenan oligosaccharides of DP with 3, 5 and 7, simultaneously in HBSS (Hank’s Balanced Salt Solution) using an LC-MS/MS system. The application of this method to support the study of the absorption and transport mechanism of κ-carrageenan oligosaccharides in Caco-2 model.

2 Materials and Methods

2.1 Materials

Κ-carrageenan oligosaccharides (DP of 3, 5, and 7) were purchased from Bozhi Huili Biological Technology Co., Ltd. (Qingdao, China); the human cell line Caco-2 (passages40-50) was purchased from American Type Culture Collection (Virginia, USA); MTT, Propranolol, Atenolol, penicillin, and Streptomycin were purchased from Sigma (St. Louis, MO, USA). Ammonia, Acetonitrile, and Ammonium acetate (LC/MS grade) were obtained from Corning Inc. (New York, USA).

2.2 LC-MS/MS Instruments and Conditions

Chromatographic separation was performed on aWaters X-Bridge Amide (3.5μm, 2.1mm × 150mm, Waters, Milford, MA, USA) coupled with guard column operated at 60℃. The mobile phases consisted of solvent, NH4OAc solution in water (A) and acetonitrile (B). Stepwise gradient elution was used with a flow rate of 0.20 mL/min as follows: 95% B for 0-2.0min, 95%-50% B for 2.0-7.0 min, 50% B for 7.0-9.0min, 50%-95% B for 9.0-9.1 min, and 95% B for 9.1-17.0min.

The κ-carrageenan oligosaccharides of DP with 3, 5, and 7 were simultaneously determined by using UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, USA) with the TSQ QUANTIVA system (Thermo Scientific, San Jose, USA) in negative ion mode (multiple reaction monitoring (MRM) scan). The MRM transitions and parameters for κ-carrageenan oligosaccharides were as follows: Ion Spray voltage of 2500V, the temperature of 300 ℃. Transitions were m/z 645.079→565.111, m/z 515.137 →474.946, and m/z 471.484→445 for κ-3, κ-5, and κ-7 carrageenan oligosaccharides, respectively.

2.3 Assay Validation

The assay method was validated to determine selective ity, the lower limit of quantitation (LLOQ), linearity, accuracy, precision, and stability. Assessment of precision and accuracy of the assay was performed by linear regression of each analyte using a calibration curve over the concentrations of 0.10, 0.20, 0.50, 1.00, 2.00, 5.00, 10.0, and 20.0μmolL−1. According to the concentrations in samples and standard curves, 0.20, 2.00, and 15.0μmolL−1were selected as quality control (QC). QC samples were prepared as three sets of six replicate samples using concentrations of 0.20, 2.00, and 15.0μmolL−1to assess the intra- and inter-day precision and accuracy of the assay. Stabilities of κ-carrageenan oligosaccharides in HBSS were evaluated under −20℃ storage conditions for 1 month.

2.4 Assessment of κ-Carrageenan Oligosaccharides Transport with Caco-2 Model

Caco-2 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 20% FBS and 1% penicillin-streptomycin mixture (Huang, 2006). The medium was replaced every 2d after incubation. Once the cells reached 80% confluence, cells were passaged approximately using trypsin-EDTA at a split ratio of 1:4. Cells were trypsinized and seeded at 5×104cells per insert onto Transwells 12-well for transport studies (Monente, 2015). The quality of the mono- layers grown on the polyester membrane was assessed by measuring the transepithelial electrical resistance (TEER) using a Millicell-ERS Epithelial Tissue Voltohmmeter. Propranolol and atenolol were used as the markers of passive diffusion (Madgula, 2008). Transport studies were performed at 17-21d post-seeding (Farrell, 2012). 0.2/0.6mL HBSS containing κ-3, κ-5, and κ-7 carrageenan oligosaccharides (150, 250, and 350μmolL−1) was added to the AP/BL side, and 0.6/0.2mL of HBSS was added to the BL/AP side. The monolayers were incubated at 37℃. Samples were collected at 30, 60, 90, 120, 150, and 180min.

3 Results and Discussion

3.1 Development of LC-MS/MS Conditions

There are few reports about the quantification of κ-car- rageenan oligosaccharides. Determination of the optimal MRM transitions for κ-carrageenan oligosaccharides was performed using full scan mode. Optimum MRM transitions for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were (645.079→565.111, [M-H]−), (515.137→474.946, [M-2H]2−, and (471.484→445, [M-3H]3−), respectively (Fig.2).

The chromatographic conditions were assessed to achi- eve sensitivity, good resolution, and peak shapes for κ- carrageenan oligosaccharides. Several types of HPLC columns were evaluated including C18 and Amide column. As a result, Waters X-Bridge Amide (3.5μm, 2.1 mm×150mm) coupled with guard column under stepwise gradient elution produced higher sensitivity and good peak shape (Fig.3).

Fig.2 Full production scan of κ-3 (A), κ-5 (B), and κ-7 (C) carrageenan oligosaccharides.

Fig.3 Representative MRM chromatograms (A) blank HBSS sample; (B) HBSS spiked with LLOQ of carrageenan oligosaccharides (0.10μmolL−1); (C) selected caco-2 cell sample from the in vitro study.

3.2 Method Validation

Results from blank samples demonstrated the absence of endogenous interference at retention time for κ-carrageenan oligosaccharides. The results confirmed the selectivity of the method toward κ-carrageenan oligosaccharides in HBSS. The linearity of the LC-MS/MS method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides, evaluated over the concentration range of 0.10-20.0μmolL−1, was excellent. The linear regression equation was=114900−2561.06 (²=0.9998) for κ-3, and=237546−2798.37 (²=0.9999) for κ-5, and=31618.6−641.531 (²=1.00) for κ-7 carrageenan oligosaccharides. The precisions of the method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were from 0.91% to 9.66% (Table 1), and the inter-day precisions were from 0.92% to 10.5%. κ-3, κ-5, and κ-7 carrageenan oligosaccharides in HBSS were found to be stable for 1 month according to the stability test. Validation of theLC-MS/MS method indicated that the method was precise and in line with the CFDA guidance.

3.3 Caco-2 Permeability Studies

Before the transport experiments, the TEER values were measured >400Ωcm2indicating that the caco-2 cell monolayers were integral. The results of permeability studies for κ-carrageenan oligosaccharides were shown in Table 2 and Fig.4. The cumulative amount transported was concentration and time-dependent (Fig.4). The κ-car- rageenan oligosaccharides (150μmolL−1, 250μmolL−1, and 350μmolL−1) exhibited a bi-directional transport, and the apparent permeability coefficients (app) for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were shown in Table 2. The efflux ratios were increased from κ-3 to κ-7.appvalues of κ-carrageenan oligosaccharides were from 1×10−6cms−1to 1×10−5cms−1, which lower than that of propranolol (a high permeability drug) and higher than that of atenolol (a low permeability drug) (Table 3). These results indicated a passive diffusion of κ-carrageenan oligosaccharides in both directions, and the permeability of κ-carrageenan oligosaccharides was decreased as the degree of polymerization increased.

Table 1 Intra- and inter-day precision and accuracy data for the quantification of κ-carrageenan oligosaccharides

Note: RSD = Relative standard deviation.

Table 2 Stability of κ-carrageenan oligosaccharides in HBSS

4 Conclusions

This study describes a sensitive and validated LC-MS/ MS method for the quantification of κ-3, κ-5, and κ-7 car- rageenan oligosaccharides simultaneously. This method offers the advantages of higher sensitivity and shorter run time. Moreover, this method has been successfully applied to anabsorption study. These results of per- meability studies indicated that the permeability of κ- carrageenan oligosaccharides was decreased as the degree of polymerization increased.

Acknowledgements

This work was supported by theShandong Provincial Natural Science Foundation, China (No. ZR2019BC025), Open Research Fund Program of Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology (Ocean University of China), and the Fundamental Resear- ch Funds for the Central Universities (Nos. 201912008,201964019).

Fig.4 Cumulative amount transported of κ-carrageenan oligosaccharides components across Caco-2 monolayers during a 180-min incubation: (a1), κ-3 AP-BL; (a2), κ-3 BL-AP; (b1), κ-5 AP-BL; (b2), κ-5 BL-AP; (c1), κ-7 AP-BL; (c2), κ-7 BL- AP.

Table 3 Apparent permeability coefficients (Papp) and the efflux ratio of κ-carrageenan oligosaccharides across Caco-2 cell monolayers

Notes: AP-BL, apical-basolateral;BL-AP, basolateral-apical.

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E-mail: lvzhihua@ouc.edu.cn

E-mail:yumingming@ouc.edu.cn

December 31, 2019;

April 13, 2020;

April 28, 2020

(Edited by Ji Dechun)