A Simple Analysis Method for 4-Deoxy-l- erythro -5-hexoseulose Uronic Acid by HPLC-ELSD with Column for Anion Analysis

Abstract

4-Deoxy-l-erythro-5-hexoseulose uronic acid (DEH) is a rare deoxy sugar produced from alginate by the action of an exotype alginate lyase. A simple and rapid method for analyzing DEH using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed in this study. For chromatography, an isocratic elution of ammonium formate buffer including formic acid and a column for anion chromatography were used. In the developed method, DEH was detected at a retention time of 3.038 minutes and limits of detection (signal-noise ratio = 3) and quantification (signal-noise ratio = 10) were 37.5 and 124.9 µg/mL as a sodium DEH, respectively. In addition, separation and detection of alginate unsaturated oligosaccharides were also tested using the method. Within an analysis time of 10 minutes, it was possible to separate and detect unsaturated disaccharide, unsaturated trisaccharide, and unsaturated tetrasaccharide prepared using poly(β-d-mannuronate) lyase and sodium alginate of high mannuronate type. The HPLC-ELSD method established in this study will be applicable for quantitative analysis of DEH and measurement of exotype alginate lyase activity.

4-Deoxy-l-erythro-5-hexoseulose uronic acid (DEH) is a monosaccharide produced by the action of an exotype alginate lyase from alginate of intercellular mucopolysaccharide in brown algae ¹ (Figure 1). In the Carbohydrate-Active EnZymes database, alginate lyases are classified as one of 28 different polysaccharide lyase families. The exotype alginate lyase is defined in the PL-14, PL-15, and PL-17 families of enzymes, but the findings concerning its acquisition from microorganisms are extremely limited. From such a scientific background, DEH is regarded as a rare sugar of unknown physiological function, and studies on its production in the field of bioengineering and marine biochemistry are attracting attention. ^2-5

Figure 1

Structure of 4-deoxy-l-erythro-5-hexoseulose uronic acid.

Among the enzymatic degradation products of alginate, the unsaturated oligosaccharides (AUO) produced by the endotype alginate lyase has 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid residue with a double bond at the nonreducing end. ⁶ Therefore, AUO can be detected by direct optical measurement with absorbance at 235 nm. In fact, there are some reports on the analysis of AUO using liquid chromatography with UV detection. ^7,8 On the other hand, detection of the monosaccharide DEH by UV detection is difficult because there is no absorption at 235 nm due to the desaturation transition to the ketone group. ^1,9-11 For detection of DEH in the enzymatic degradation products, TLC with a coloring reagent (eg., thiobarbituric acid) is often used. ^9,10,12 However, since each of DEH standard and exotype alginate lyase is not commercially available, qualitative and quantitative analyses of DEH using TLC cannot be performed.

The evaporation light scattering detector (ELSD) has semi-universal detection characteristics, and in principle it is possible to directly detect nonvolatile molecular species. Since the detection sensitivity is almost constant irrespective of the structure of the compound, ELSD is suitable for analysis of substances with poor light-absorbing properties such as carbohydrates. ^13,14 Furthermore, since high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) uses the same eluent as liquid chromatography-mass spectrometry (LC/MS), there is also an advantage that the compounds detected on the ELSD chromatogram can be analyzed in-line using a mass spectrometer. In preceding studies, ^15,16 we reported the isolation of Falsirhodobacter sp. strain alg1 as a novel alginate degrading bacterium. A novel exotype alginate lyase (AlyFRB) possessed by the strain alg1 is an enzyme capable of depolymerizing alginate and producing only DEH without intermediate products. ^15,16 In this study, we prepared DEH from sodium alginate using AlyFRB, and developed a method for analyzing DEH using HPLC-ELSD.

Alginate and its related molecules including DEH and AUO have a carboxyl group in the uronic acid unit, and so they have a negative electric charge. Therefore, it is considered that an analytical column for anion chromatography is effective for holding DEH. In this study, we attempted detection of DEH using a column for anion analysis with quaternary ammonium as the functional group and ammonium formate buffer as the mobile phase. Figure 2 shows a typical chromatogram of DEH obtained by HPLC-ELSD analysis. In the chromatography using a 40 mM ammonium formate buffer containing 0.1% formic acid as the mobile phase, a peak of DEH was detected on the ELSD chromatogram at a retention time of 3.038 minutes (Figure 2). The larger peak detected at 2.273 minutes indicates that of the component eluted in the void volume (Figure 2). This larger peak also shows sodium ions dissociated from the sodium salt of alginate molecules, including DEH and AUO. The relative standard deviations of the retention time (t _R-RSD), area values (Area-RSD), and the value of the capacity factor (k′) are shown in Table 1. Each value of the RSD suggests that the analysis conditions of DEH established in this study have high reproducibility. The response of ELSD shows an exponential response to the amount of injected substance. Therefore, a calibration curve was prepared from a logarithmic plot of the concentration of sodium DEH and the area value of DEH detected on the ELSD chromatogram. As shown in Table 2, the correlation factor of the calibration curve was higher than 0.996. This result suggests that the developed conditions have good linearity within the range of concentrations tested. The limit of detection (signal-noise ratio = 3) and limit of quantitation (signal-noise ratio = 10) for sodium DEH were 37.5 and 124.9 µg/mL, respectively (Table 2).

Figure 2

HPLC-ELSD chromatogram of DEH. Ten microliters of sodium DEH solution (1 mg/mL) was injected into the HPLC-ELSD system. DEH: 4-deoxy-l-erythro-5-hexoseulose uronic acid; HPLC-ELSD: high-performance liquid chromatography with evaporative light scattering detection.

Table 1

The Evaluation of Precision and Specificity of the Developed HPLC-ELSD Condition for DEH Analysis.

t R (min)	t R-RSD (%)	Area	Area-RSD (%)	K ′ a
3.038	0.056	1048	1.118	0.337

HPLC-ELSD, high-performance liquid chromatography with evaporative light scattering detection; DEH, 4-deoxy-l-erythro-hexoseulose uronic acid.

The analytical data of t _R, t _R-RSD, Area, and Area-RSD in the table were calculated from lthe ELSD chromatograms obtained by a 10 µL injection (n = 5) of sodium DEH (1.0 mg/mL).

^aCapacity factor.

Table 2

Limits of Detection and Quantification for DEH in the HPLC-ELSD Analysis.

Regression equation a	Correlation factor	Linearity range (µg/mL)	LOD(µg/mL) b	LOQ(µg/mL) c
y = 1.8839x-2.7696	0.9969	125-1000	37.5	124.9

HPLC-ELSD, high-performance liquid chromatography with evaporative light scattering detection; DEH, 4-deoxy-l-erythro-5-hexoseulose uronic acid; LOD, limit of detection; LOQ, limit of quantification.

^a y is the logarithmic value of the peak area; x is the logarithmic value of the concentration injected in the HPLC-ELSD.

^bLimit of detection (signal-noise ratio = 3).

^cLimit of quantification (signal-noise ratio = 10).

We examined the separation and detection of AUO using the developed condition for DEH analysis. The endotype alginate lyase used in this study produces unsaturated oligosaccharides ranging from 2 to 4 residues with 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid residue at the nonreducing end and β-d-mannuronate at the reducing end. ⁶ As shown in Figure 3, three characteristic peaks were detected from AUO prepared from the endotype alginate lyase and sodium alginate of high mannuronate type. As a result of in-line electrospray ionization-mass spectrometry (ESI/MS) in negative mode to identify each oligosaccharide, an m/z 371 ion corresponding to a deprotonated molecule [M-H]^- of an unsaturated disaccharide was detected at peak a. Peak b had an m/z 527 ion and peak c had an m/z 703 ion as a signal each with a relative intensity of 100%. Each value of m/z corresponds to a mass number of a deprotonated molecule [M-H]⁻ of an unsaturated trisaccharide or an unsaturated tetrasaccharide. Therefore, it was found that peak a is an unsaturated disaccharide, peak b is an unsaturated trisaccharide, and peak c is an unsaturated tetrasaccharide. To validate the precision of analysis, the t _R, peak area, k′, Rs, and α for each AUO were calculated, respectively (Table 3). The t _R-RSD values were each less than 0.659%, and the Area-RSD values were each less than 3.200%. The Rs value is a parameter indicating separation of adjacent peaks. An Rs value of 1.5 or higher means complete separation of adjacent peaks. The values of Rs obtained in this study were 2.970, 2.622, and 2.913 (Table 3). Therefore, it was found that the analysis conditions of DEH developed can also be used for separation and detection from unsaturated disaccharide to tetrasaccharide.

Figure 3

HPLC-ELSD chromatogram of AUO. Ten microliters of sodium AUO solution (10 mg/mL) was injected into the HPLC-ELSD system. a, an unsaturated disaccharide; b, an unsaturated trisaccharide; c, an unsaturated tetrasaccharide. AUO: unsaturated oligosaccharides; HPLC-ELSD: high-performance liquid chromatography with evaporative light scattering detection.

Table 3

The Evaluation of Precision and Specificity for AUO Analysis.

AUO	t R (min)	t R-RSD (%)	Area	Area-RSD (%)	K ′a	Rs b	α c
Disaccharide	3.929	0.184	153	0.992	0.729	2.970	2.163
Trisaccharide	5.240	0.325	2305	2.120	1.305	2.622	1.790
Tetrasaccharide	7.857	0.658	1525	3.199	2.457	2.913	1.883

AUO, unsaturated oligosaccharides.

The analytical data of t _R, t _R-RSD, Area, and Area-RSD in the table were calculated from the evaporative light scattering detection chromatograms obtained by a 10 µL injection (n = 5) of sodium AUO (10 mg/mL).

^aCapacity factor.

^bResolution factor.

^cSeparation factor.

In other recent studies, there are some reports using the LC/MS ² or the gas chromatography-mass spectrometry (GC/MS) ^3,17 for detection of DEH. In the report using LC/MS, DEH was detected at a retention time of about 9 minutes using an ODS column and a gradient elution of two kinds of mobile phases. ² In the case of the GC/MS, the methoxyaminated DEH was detected at a retention time of about 30 minutes using a fused silica capillary column. ^3,17 Our HPLC-ELSD method can be regarded as a more convenient method because it can directly detect DEH at a retention time of about 3 minutes without derivatization of samples or a complicated elution program. It is also possible to separate and detect from DEH to alginate unsaturated tetrasaccharide in an analysis time of less than 10 minutes (Figure 4). Therefore, it can be used for measurements such as search for exotype alginate lyase and quantitative analysis of DEH in the enzymatic degradation products. In conclusion, the HPLC-ELSD method established in this study suggests this use of DEH analysis as a simple and rapid method alternative to the TLC method.

Figure 4

HPLC-ELSD chromatogram of mixed solution of DEH and AUO. Equal volumes of DEH solution (1 mg/mL) and AUO solution (10 mg/mL) were mixed. Ten microliters of mixed solution was injected into the HPLC-ELSD system. (a) an unsaturated disaccharide; (b) an unsaturated trisaccharide; (c) an unsaturated tetrasaccharide. AUO: unsaturated oligosaccharides; DEH: 4-deoxy-l-erythro-5-hexoseulose uronic acid; HPLC-ELSD: high-performance liquid chromatography with evaporative light scattering detection.

Experimental

Materials

Poly(β-d-mannuronate) lyase from Flavobacterium multivorum (EC 4.2.2.3) was purchased from Sigma-Aldrich. Sodium alginate of high mannuronate type (IL-6M, the ratio of mannuronate to gluronate: about 2.56) was kindly donated by Kimica. Anmonium formate, formic acid, and ultrapure water for LC/MS were purchased from FUJIFILM Wako Pure Chemical. All other reagents used in this study were of analytical grade.

Preparation of Sodium DEH

Sodium DEH was prepared according to the same method as the preceding report. ¹⁶ The solution of sodium alginate degradation prepared by AlyFRB was ultrafiltered using Vivaspin Turbo 15 (3000 nominal molecular weight limit, Sartorius). The filtrate was lyophilized using a freeze dryer FDU-2200 (Eyela), and the obtained lyophilized powder was used as sodium DEH. It was confirmed that the obtained DEH had an m/z 175 ion corresponding to the molecular mass of the deprotonated form by negative ion measurement of ESI/MS. ¹⁸ The sodium DEH was stored at −80°C until use.

Preparation of Sodium AUO

Sodium AUO were prepared using the method described in the preceding report. ¹⁸ Degradation of sodium alginate by poly(β-d-mannuronate) lyase was confirmed via the increase in absorbance at 235 nm. The enzyme-decomposed solution of alginate was ultrafiltered by Vivaspin Turbo 15. The filtrate was freeze-dried using the FDU-2200. The obtained lyophilized powder was used in HPLC-ELSD analysis as sodium AUO. Sodium AUO was stored at −30°C until use.

HPLC-ELSD Analysis

The separation and detection of DEH and AUO was carried out using a 1260 Series HPLC system consisting of a 1260 Infinity ELSD, a 1260 quaternary pump, a 1260 autosampler, and a 1260 thermostatted column compartment (Agilent Technologies). In this study, a Shodex I-524A (4.6 i.d. x 100 mm, Showa Denko) was used for the analytical column. Elution was performed at a flow rate of 0.5 mL/min with a 40 mM ammonium formate buffer including 0.1% formic acid (pH 3.5) as the mobile phase. The column oven was set at 40°C. Experimental conditions for ELSD were as follows: evaporator temperature, 30°C; nebulizer temperature, 30°C; the nitrogen gas flow rate, 1.6 L/min. The sodium DEH and sodium AUO were each dissolved in the mobile phase, and used as samples for analysis experiments.

Validation of the Developed Method

The precision test was demonstrated by replicative injections (n = 5) of both sodium DEH and sodium AUO solutions. Measurements of t _R, Area, k′, Rs, and α were used to assess the reproducibility of the developed method. The parameters were defined using these standard formulas:

k′ = (t _Rn − t ₀) / t ₀,

Rs = 2( t _Rn+1 − t _Rn ) / (W _{n +1} + W _n ),

α = k′ _{n +1} / k′ _n .

In these formulas, t ₀ is the retention time of the component in the void volume (2.273 minutes) and Wn is the width of the peak at baseline. The subscript n represents the order of elution of AUO. LOD and LOQ for DEH analysis under the present chromatographic conditions were determined at a signal-noise ratio of 3 and 10, respectively.d