Determination of glucosamine and galactosamine in food by liquid chromatography with pre-column derivatization

ABSTRACT

A pre-column derivatization liquid chromatographic method was developed for the simultaneous determination of both glucosamine and galactosamine in selected food samples. The derivatization of glucosamine and galactosamine with 9-fluorenylmethoxycarbonyl chloride was accomplished under alkaline conditions and UV irradiation. When the detection wavelength was 265 nm and acetonitrile-0.1% phosphoric acid was the mobile phase at a flow rate of 1.0 mL/min, the linear range of ultraviolet intensity of glucosamine and galactosamine was 10–100 μg/mL,correlation coefficient of glucosamine was 0.9994 and correlation coefficient of galactosamine was 0.9993. The standard recovery of three kinds of food was in the range of 94.09–114.5%, the relative standard deviation (RSD) was 0.38–5.29%, the detection limit was 1.0 mg/kg, and the quantitative limit was 4.0 mg/kg. The results of this research indicate that this method is suitable for the simultaneous determination of glucosamine and galactosamine in food.

KEYWORDS:

• High-performance liquid chromatography (HPLC)
• glucosamine (GlcN)
• galactosamine (GalN)
• 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl)

Highlights

• Simultaneous detection of HPLC for GlcN and GalN in food were built.

• Galactosamine was derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl).

• The established method was applied to the detection of actual samples.

• It was subjected to quantitative and qualitative analytical GlcN and GalN in food.

1. Introduction

Glucosamine (GlcN) and galactosamine (GalN) are C-4 differential isomers formed by replacing the hydroxyl group at the C2 position of glucose with an amino group. GlcN and GalN show multiple biological activities. Glucosamine is a natural amino monosaccharide that is an intermediate in the synthesis of proteoglycan macromolecules. Mucopolysaccharides, glycoproteins, and proteoglycans can be synthesized from glucosamine. Glucosamine is an intermediate in the synthesis of articular cartilage and synovial fluid molecules promote the synthesis of cartilage and inhibit the breakdown of articular cartilage. Glucosamine also has an anti-inflammatory effect and stimulates chondrocyte growth (Ilic et al., 2008; Jin et al., 2011; Kosman et al., 2017; Zhou et al., 2004). Chinese drug standard WS1-(X-090)-2005Z is used to determine the content of glucosamine hydrochloride capsules by UV spectrophotometry after the Elson-Morgan reaction. Following the standard GB/T20365-2006, when the detection wavelength was 195 nm, the baseline noise was higher and the detection sensitivity of the instrument was reduced (Hairui et al., 2016; Zhang et al., 2006). With the widespread use of glucosamine in health products, its detection methods have become a contemporary research hotspot in the food industry. Galactosamine is a hepatocyte uracil phosphate nucleoside disruptor. Uridine phosphate was depleted after galactosamine enters the body, resulting in damage to a series of organelles as well as protein synthesis disruption, which results in liver cell damage (Gong et al., 2007; Liang-ming et al., 2006; Nakama et al., 2001; Sinha et al., 2007; Tunc & Sehnaz, 2008;Woo-Cheol et al., 2009). Because of the cytotoxic effect of galactosamine, monitoring its level in food is important to protect the health of consumers. In view of the difference in biological activity and functional properties of glucosamine and galactosamine, simultaneous determination of glucosamine and In view of the difference in biological activity and functional properties of glucosamine and galactosamine in the same substance is of great significance for human health.

At present, there are many reports on the detection methods of glucosamine at home and abroad, but there are few methods to detect glucosamine and galactosamine simultaneously. The main methods for determining glucosamine and galactosamine were colorimetric (Thomas A. & Samuel P., 1964) and ion chromatographic methods (Maurizio et al., 2001). However, these methods are less stable, less accurate, and less sensitive, especially for the determination of trace substances.

With advancements in technology, the use of analytical tests is becoming more widespread. Currently, the most commonly used method for sugar detection is HPLC (Rajarshi & Paul, 2019). More accurate qualitative and quantitative analysis can be obtained by selecting the appropriate derivatization reagent, derivatization mode, mobile phase, detector, etc. Due to its sensitivity and high efficiency, pre-column derivatization liquid chromatography is gradually becoming a popular method for the analysis of glucosamine and galactosamine.9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) can react with primary and secondary amino sugars or amino acids as a derivative reagent. The principle of this study is that under alkaline conditions, glucosamine (Figure 1(a)) and galactosamine (Figure 1(b)) were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) (Figure 2). Glucosamine and galactosamine are C-4 differential isomers of each other and react following the same principle.

Figure 1. Glucosamine and Galactosamine structural formula. (a) Glucosamine (b) Galactosamine.

Figure 2. Reaction equation of 9-fluorenylmethoxycarbonyl chloride with glucosamine.

2. Materials and methods

2.1 Reagents and instruments

2.1.1 Experimental materials and reagents

Bird's nests (instant stewed bird's nest, triangle bird's nest, pure bird's nest, dried bird's nest) as solid substrate, milk as liquid substrate, and starch as blank substrate were purchased from a retail market.

Glucosamine hydrochloride, galactosamine hydrochloride, and 9-fluorenylmethyloxycarbonyl chloride were obtained from Shanghai Macklin Biochemical Co., Ltd. Acetic acid was ordered from Tokuyama Pharmaceutical Co., Ltd., Shanghai, China. Amino acids mixture standard solution was ordered from Fujifilm Wako Pure Chemical Co., Ltd., Japan. Boric acid was ordered from Shanghai Yunling Chemical Plant. Hydrochloric acid, phosphoric acid, and sodium hydroxide were obtained from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water was used in all experiments.

2.1.2 Experimental instruments

Derivatised compounds were detected by HPLC apparatus-2695 Series HPLC – (Waters Corporation, USA).

Reaction system was occured in Digital display constant-temperature six-hole water bath – (Yinzhou Qun'an Experimental Instrument Co., Ltd., Ningbo, China)

Solution pH need pH meter – (Instrument Co., Ltd. Shanghai, China) to measure in pretreatment and reaction system.

High-speed centrifuge instrument – (Xiangyi Laboratory Instrument Development Co., Ltd., Hunan, China) was used to extract supernatant.

Electronic analytical balance – (Huazhi Electronic Technology Co., Ltd., China) was used to weight materials and samples.

Vortex Mixer – (IKA Works Co., Ltd., Guangzhou China) was used to mix reactants and products.

Ultrapure water was prepared by ultrapure water system – (Hetai Instrument Co., Ltd., Shanghai, China).

2.1.3 Solution preparation

The glucosamine hydrochloride and galactosamine hydrochloride standard reference reserve solution was prepared by precisely weighing 0.4 g each of glucosamine hydrochloride and galactosamine hydrochloride standards, which were then dissolved in 0.1 M hydrochloric acid in a 100 mL brown volumetric flask. The flask was kept cold at 0–4 ^oC and away from light. When using the solution, it was diluted with 0.1 M hydrochloric acid to the required concentration.

The glucosamine hydrochloride standard reference solution was prepared by precisely weighing 0.4 g of glucosamine hydrochloride standard and then dissolving it in 0.1 M hydrochloric acid in a 100 mL brown volumetric flask. The flask was kept cold at 0–4 ^oC and away from light.

The galactosamine hydrochloride standard reference solution was prepared by precisely weighing 0.4 g of galactosamine hydrochloride standard and then dissolving it in 0.1 M hydrochloric acid in a 100 mL brown volumetric flask. The flask was kept cold at 0–4 ^oC and away from light.

The 9-fluorenylmethoxycarbonyl chloride standard reference solution was prepared by precisely weighing 0.05 g of 9-fluorenylmethoxycarbonyl chloride standard and then dissolving it in acetonitrile in a 10 mL brown volumetric flask. The flask was kept cold at 0–4 ^oC and away from light. It was used within 48 h of preparation.

The sodium hydroxide reserve solution was prepared by dissolving sodium hydroxide (0.1 g) in 250 mL ultrapure water in a volumetric flask. Then, a 0.01 M sodium hydroxide solution was prepared and stored until later use.

The borate buffer solution reserve solution was prepared by mixing boric acid (2.40 g) and sodium hydroxide (1.14 g) and then dissolving them in ultrapure water in a 100 mL volumetric flask. A pH 10 borate buffer solution was prepared.

The derivatization termination solution was prepared by mixing acetic acid and acetonitrile (volume ratio 2: 8) and storing the mixture away from light.

2.2 Liquid chromatography

HPLC was performed using a 5 µm 4.6 × 150 mm Eclipse XDB-C18 column (Agilent). The mobile phases were 0.1% phosphoric acid solution (A) and HPLC-grade acetonitrile (B). The flow rate was 1 mL/min, the injection volume was 20 µL, the detection wavelength was 265 nm, and the column temperature was 25 ℃. Gradient elution was performed using the time gradient shown in Table 1.

Table 1. Mobile phase time gradient.

Retention time/min	Acetonitrile (vol.%)	Phosphoric acid (vol.%)
0	20	80
5	25	75
20	50	50
25	70	30
30	20	80
35	20	80

2.3 Experimental methods

2.3.1 Sample preparation

First, after all samples were crushed and homogenized, 0.10 g of each sample was weighed and dissolved in 4.0 M hydrochloric acid in a 5 mL volumetric flask. The solution was shaken well by a water bath (90 ℃) for 3.0 h. After the solution was blown by N₂ gas, the residue was diluted in ultrapure water to 10 mL in a volumetric flask. The sample was centrifuged for 5 min at 10,000 r/min, and then the supernatant was removed and set aside.

2.3.2 Derivatization method

A 0.2 mL sample of glucosamine hydrochloride and galactosamine hydrochloride standard solution was precisely extracted. Then, 0.2 mL of borate buffer solution and 0.1 mL 9-fluorenylmethoxycarbonyl chloride solution were successively shaken on a vortex mixer. After mixing, the system was allowed to stand for 10 min. Then, 0.2 mL derivatization termination solution was added, and when the reaction was complete, it was filtered through a 0.45 μm microporous filter. The filtrate was collected in an HPLC injection vial (Zhu et al., 2005).

2.3.3 Sample pretreatment

A 0.1 g sample was precisely weighed and then added to 4.0 M hydrochloric acid (HCl) or 4.0 M trifluoroacetic acid (TFA) solution separately into 5 mL volumetric flasks. The solution was shaken on a vortex mixer. The solution was reacted in a constant-temperature water bath at temperature 90 ^oC for 3.0 h. After the solution was blown using N₂ gas, the residue was diluted in ultrapure water in a 10 mL volumetric flask. The solution was centrifuged for 5 min at 10,000 r/min, and then the supernatant was removed,and the derivatization reaction was monitored according to the derivatization method in section 2.3.2. Derivatised compounds were collected in an HPLC injection vial.

2.4 Standard curve establishment

The glucosamine hydrochloride and galactosamine hydrochloride standard reference reserve solution was diluted to a concentration of 100 µg/mL, and 0.1, 0.2, 0.5, 0.8, and 1.0 mL samples were extracted. The derivatization reaction was carried out according to the method in section 2.3.2. After filtering through a 0.45 μm microporous filter, followed by high-performance liquid chromatography and UV detection, the standard curve was obtained.

3. Results and discussion

3.1 Chromatography

3.1.1 Detection wavelength

UV spectrophotometry was used to obtain a wavelength scan spectrum from 200 to 400 nm, and spectral peaks appeared at 264.8 nm. As shown in Figure 3, the absorption wavelength of derivatised compounds via derivatization reaction of glucosamine hydrochloride and galactosamine hydrochloride were measured to be 265 nm.

Figure 3. UV spectrum of derivatised compounds.

3.1.2 Selection of the mobile phase

Different mobile phases were investigated. Acetonitrile was selected as the organic phase because it has a better separation effect for derivatised compounds than methanol(Figure 4(a and b)). In terms of the aqueous phase, the effects of dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer, phosphoric acid, and triethylamine on the separation were investigated. According to the separation of the target peak (Figure 4(c and d)), when the acetonitrile-0.1%phosphoric acid mixture was selected as the mobile phase, the elution effect was stronger than others.

Figure 4. (a) Chromatograms of derivatised compounds under methanol-triethylamine mobile phase. (b) Chromatograms of derivatised compounds under acetonitrile-triethylamine mobile phase. (c) Chromatograms of derivatised compounds under acetonitrile-dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer mobile phase. (d) Chromatograms of derivatised compounds under acetonitrile-0.1%phosphoric acid mobile phase.

3.1.3 Selection of the elution method

During the experiment, continuous elution and gradient elution methods were studied. During continuous elution, environmental factors were unstable. Therefore, gradient elution was selected to separate the target peaks. The elution ratio of the mobile phase was continuously adjusted according to the degree of elution, and the gradient elution rate was determined, as shown in Table 1.

3.2 Selection of derivatization conditions

3.2.1 Selection of derivatization pH

A 0.2 mL sample of the glucosamine hydrochloride and galactosamine hydrochloride standard solution was precisely extracted. Then, 0.2 mL of borate buffer solution at pH values of 9.0, 9.5, and 10 were added separately. The derivatization reaction (2.3.2) was shown in Figure 5. When the solution pH was belowed 9.0, the derivatization reaction could not occur. The effect of the derivatization reaction at pH 10 was better than at pH 9.5. If the solution pH was greater than 10, the Eclipse XDB-C18 column that was used in the HPLC would be destroyed.

Figure 5. Chromatograms of derivatised compounds under different pH.

3.2.2 Selection of the derivatization time

The derivatization reaction (2.3.2) was conducted under different reaction times of 5, 10, 15, 20, and 30 min, and the resulting chromatograms of derivatised compounds were shown in Figure 6. When the reaction time was 10, 15, or 20 min,the chromatogram peaks area of derivatised compounds were better than other reaction times. So, a reaction time of 10 min was the optimal condition.

Figure 6. Chromatograms of derivatised compounds under different reaction time.

3.2.3 Selection of derivatization temperature

During the derivatization reaction (2.3.2), temperatures of 15, 23(room temperature), 30, 35, and 40 ℃ were used. The resulting chromatograms of the derivatised compounds were shown in Figure 7. When the reaction temperature were 23 ℃and 30℃, the chromatogram peaks area of derivatised compounds were greater. To facilitate the test operation, a reaction temperature of room temperature was the optimal condition.

Figure 7. Chromatograms of derivatised compounds under different reaction temperature.

3.3 Determination of derivatization peaks

Derivatization of glucosamine and galactosamine produced two isomers, which displayed separate α and β double peaks in the HPLC chromatograms of derivatised compounds in Figure 8. Since the β peaks of derivatised compounds of glucosamine and galactosamine overlapped, due to the derivatization reaction (2.3.2). So the derivatization effect was qualitatively and quantitatively assessed using the α peak. It was shown that the derivatised compounds of galactosamine (GalN) peak was at 10.75 min, and the derivatised compounds of glucosamine (GlcN) peak appeared at 11.17 min.

Figure 8. Chromatograms of derivatised compounds of GlcN and GalN standards solutions.

3.4 Interference experiment of amino acids in food

Free amino acids in food can react with 9-fluorenylmethoxycarbonyl chloride (Elisabeth L et al., 2005; Herbert et al., 2000; Jámbor & Molnár-Perl, 2009; López-Cervantes et al., 2006). In the experiment, a standard amino acid solution was added to the sample solution, and the derivatization reaction of amino acids was monitored according to the derivatization method in Section 2.3.2.

According to the chromatograms (Figure 9), within the retention time range of 10–12 min, amino acids did not interfere with derivatised compounds of glucosamine and galactosamine.

Figure 9. Amino acid interference chromatograms.

3.5 Selection of sample pretreatment conditions

3.5.1 Selection of acid

Sample was added to 4.0 M hydrochloric acid (HCl) or 4.0 M trifluoroacetic acid (TFA) solution. After the solutions were prepared via Section 2.3.3, and derivatised compounds were prepared via Section 2.3.2. As shown in Figure 10 and Table 2, the derivatization effect using hydrochloric acid was better than trifluoroacetic acid.

Figure 10. Sample pretreatment effect under different acids.

Table 2. Sample pretreatment effects under different acids.

Acid	Name	Peak area
HCl	GalN	431414
	GlcN	487132
HCl	GalN	414171
	GlcN	479958
TFA	GalN	284519
	GlcN	560987
TFA	GalN	258555
	GlcN	523080

3.5.2 Selection of sample pretreatment temperature

Sample was added to 4.0 M hydrochloric acid solution. According Section 2.3.3,The solution was reacted in an adjustable-temperature water bath at 70°C, 80°C, 90°C and 100 ^oC for 3.0 h, and derivatised compounds were prepared via Section 2.3.2. As shown in Figure 11 and Table 3, 90 ^oC provided better results than other temperatures.

Figure 11. Sample pretreatment effect under different temperature.

Table 3. Sample pretreatment effect under different temperature.

Pretreatment temperature	Name	Peak area
70 ℃	GalN	254806
	GlcN	481159
80 ℃	GalN	482550
	GlcN	615871
90 ℃	GalN	523625
	GlcN	565990
100 ℃	GalN	322477
	GlcN	497429

3.6 Methodology validation

3.6.1 Linearity

According to the method in Section 2.3, the standard curve was established using standard solutions with concentrations of 10, 20, 50, 80, and 100 µg/mL for liquid-phase determination. Using the peak area of derivatised compounds as the ordinate, and the amount of standard glucosamine hydrochloride and galactosamine hydrochloride standards added as the abscissa, two standard curves were drawn. Regression was conducted to obtain the glucosamine hydrochloride linear equation Y = 5.06×10³X + 8.60×10³, R² = 0.9994, and the galactosamine hydrochloride linear equation was Y = 4.06×10³X+ 6.75×10³, R² = 0.9993. In the range of 10–100 μg/mL, a good linear relationship was obtained, as shown in Figure 12(a and b).

Figure 12. (a) Standard curve of glucosamine hydrochloride. (b) Standard curve of galactosamine hydrochloride.

3.6.2 Detection limit determination

According to the research method described in section 2.3, pretreament of samples and analytical detection of derivatised compounds were carried out. The detection limit was 1.0 mg/kg, which was calculated as three times the signal-to-noise ratio (S/N = 3), and the quantitative limit was 4.0 mg/kg, which was calculated as 10 times the signal-to-noise ratio (S/N = 10). These values indicate the high sensitivity of the method.

3.6.3 Recovery experiment and precision

According to the method described in section 2.3, a labelled recovery test was carried out for three kinds of food, including edible bird's nest, milk, and starch (chromatograms were shown in Figure 13). Three different concentrations of glucosamine hydrochloride and galactosamine hydrochloride standards solution (10, 20, and 100 μg/mL) were also added. The recovery and relative standard deviation (RSD) were calculated, and the results were shown in Table 4. As shown in Table 4, the sample recovery rate ranged from 94.09 to 114.15%, and the RSD ranged from 0.38 to 5.29% (n = 6). Equation (1) was used to calculate the sample recovery rate (P) from the concentrations, and Equation (2) was used to calculate the relative standard deviation. These metrics were met the requirements for an experimental analysis method and shown that this method has good precision and high accuracy. (1) $P={\displaystyle \frac{c_2-c_1}{c_3}\times 100\text{\%}}$(1) (2) $RSD={\displaystyle \frac{SD}{X}\times 100\text{\%}}$(2)

Figure 13. Chromatograms of samples with added standards.

Table 4. Recovery and precision of GlcN and GalN in different food substrates.

Sample	Spiked level/(mg/kg)		Found/(mg/kg)						Back-ground/mg/kg	Recovery rate/%	RSD/%
			1	2	3	4	5	6
Starch	10	GalN	10.863	10.867	10.897	10.831	10.942	10.948	0	108.91	0.43
		GlcN	9.785	9.741	9.865	9.74	9.844	9.905	0	98.13	0.70
	20	GalN	22.145	22.138	22.140	22.371	22.308	22.197	0	111.08	0.45
		GlcN	20.747	20.551	20.667	20.909	20.833	20.567	0	103.56	0.70
	100	GalN	113.478	114.232	114.318	113.823	114.697	114.362	0	114.15	0.38
		GlcN	113.184	114.255	114.310	113.414	114.762	114.483	0	114.07	0.55
Bird's Nest	10	GalN	10.290	10.758	11.813	10.752	10.811	11.572	0.357	106.42	5.22
		GlcN	10.704	10.023	11.383	9.889	10.295	10.659	0.344	101.48	5.20
	20	GalN	20.021	19.656	18.378	20.634	18.634	20.062	0.363	96.01	4.50
		GlcN	20.117	19.250	19.866	19.422	17.253	19.084	0.348	94.09	5.29
	100	GalN	99.941	105.780	104.479	112.230	105.129	106.870	0.357	105.38	3.76
		GlcN	97.103	100.544	96.103	109.482	101.219	99.096	0.345	100.25	4.75

3.6.4 Stability of derivatised compounds

The stability of the derivatised compounds was investigated. According to the experimental method in section 2.3, the derivatized sample solution was stored at 4 ℃ in the dark, and samples were injected every 1.0 h for determination. As shown in Table 5, after 10 h, the derivatised compounds remained stable, and the difference between the maximum concentration and the minimum concentration was less than 0.48%.

Table 5. Change in the front area within 10 h.

Time/ h		1	2	3	4	5	6	7	8	9	10	RSD/ %
Standard solution	GalN	10.032	10.002	9.991	10.101	10.105	9.986	10.083	10.036	10.087	10.036	0.45
	GlcN	10.029	9.997	10.002	10.088	10.022	10.021	10.029	10.036	10.006	10.027	0.25
Bird's Nest	GalN	0.271	0.270	0.272	0.270	0.271	0.271	0.271	0.272	0.272	0.271	0.27
	GlcN	0.281	0.278	0.280	0.279	0.279	0.280	0.279	0.279	0.282	0.281	0.48

3.7 Actual sample testing

This method can be applied to a variety of food substrates. In this experiment, the glucosamine and galactosamine contents of three kinds of commercially-available preserved foods (edible bird's nest, milk, and starch) were analysed according to the newly-established method (chromatogram was shown in Figure 14). The detection results were shown in Table 6.

Figure 14. Chromatograms of samples.

Table 6. Actual sample detection values.

Sample	Found /(μg/kg)		Sample	Found /(μg/kg)
Instant stewed bird's nest	GalN	273.192	Dried bird's nest	GalN	355.183
	GlcN	296.386		GlcN	348.168
Triangle bird's nest	GalN	224.223	Milk	GalN	0.223
	GlcN	233.506		GlcN	0.265
Pure bird's nest	GalN	275.07	Starch	GalN	undetected
	GlcN	283.773		GlcN	undetected

4. Conclusion

A method for the simultaneous detection of glucosamine and galactosamine in foods has rarely been reported. In this work, an HPLC analysis method was established in which glucosamine and galactosamine samples and 9-fluorenylmethoxycarbonyl chloride were used to generate compounds via UV absorption. The target compounds were separated by liquid chromatography. The results were shown that the linear range of the peak area of glucosamine standard was 10–100 µg/mL, as so as galactosamine standard. The recoveries of three kinds of food were approximately 94.09–114.15%, the RSD was 0.38–5.29%, and the detection limit was 1.0 mg/kg. This method was shown that the pretreatment conditions were optimized and the detection time was shortened. This method was suitable for simultaneously detecting glucosamine and galactosamine in a variety of foods and has high sensitivity and strong specificity.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analysed during this study are included in this published article.

Additional information

Funding

This work was supported by Climbing Project Fund of Changchun University: [Grant Number ZKP202121]; Jilin Province Education Department Fund: [Grant Number JJKH20210616KJ].