Methodological investigation
Using rhamnose as an internal quality control monosaccharide. The glycans assembled in human cells consist of 10 monosaccharides, i.e. N-acetyl galactosamine, galactose, N-acetyl glucosamine, glucose, mannose, fucose, sialic acid, xylose, glucuronic acid, and iduronic acid. After hydrolysis, 8 out of 10 monosaccharides, including galactosamine (GalN), galactose (Gal), glucosamine (GlcN), glucose (Glc), mannose (Man), fucose (Fuc), xylose (Xyl), glucuronic acid (GlcA) could be detected by the PMP-derivatization and HPLC separation and detection method. Human fasting blood has no detectable rhamnose (Rha). Thus, we have added the Rha to each serum sample and during the assay optimizing process. Figure 1 showed the baseline separation of the 9 monosaccharide standards using the optimized HPLC method.
Linear relationship investigation
Each monosaccharide standard solution of 1mol/L was derivatized, diluted 2, 4, 6, 8 and 10 times respectively, and then analyzed by HPLC. The concentration of monosaccharide (mol/L) was taken as the abscissa and the corresponding peak area was taken as the ordinate. Coordinates, calculate the linear regression equation, the square of the linear correlation coefficient R2 is greater than 0.990 as a good linear relationship.. Mannose, glucosamine, galactosamine, glucuronic acid, glucose, galactose, xylose and fucose have a good linear relationship.
Standard concentration curve of the eight monosaccharides
Regression equation and Correlation coefficient R2
Man: y = 84680x + 448.84,0.9994;GlcN:y = 72933x + 348.8,0.9991;GalN:y = 67796x - 75.111,0.9993;Glc:y = 90330x + 525.00,0.9990;GlcA:y = 52893x +6.7293,0.9995;Gal:y = 82851x + 528.91,0.9992;Fuc:y = 98532x - 125.05,0.9996; Xyl: y = 12054x - 0.0683,0.9993.
Precision experiment
Precision of the method for monosaccharide standards. After the 9 monosaccharides standard mixture have gone through the optimized hydrolysis and derivatization procedure, the analysis was repeated 5 times with expected procession of a typical HPLC method. The relative standard deviation (RSD %) of both elution times and peak area were summarized in Figure 2.
The RSD for each of the monosaccharide standard analyzed 5 times by HPLC
Retention time/min and RSD%
Man:15.37±0.055,0.36;GlcN:17.15±0.048,0.28;GalN:22.08±0.060,0.27;Glc:29.53±0.067,0.23;
GlcA:24.27±0.059,0.24;Gal:31.25±0.072,0.23;Fuc:36.83±0.079,0.22;Xyl:33.33±0.074,0.22.
Peak area and RSD%
Man:12026.16±155.40,1.29;GlcN:10230.9±89.90,0.88;GalN:8157.62±18.91,0.23;Glc:9709.5±52.74,0.54;GlcA:5813.18±62.27,1.07;Gal:12099.24±138.69,1.14;Fuc:12251.66±35.80,0.29;Xyl:13274.46±64.80,0.48.
Repetitive experiment
Repeatability of the method for a serum sample. After the serum sample has gone through the optimized hydrolysis and derivatization procedure, the analysis was repeated 6 times with expected procession of a typical HPLC method. The relative standard deviation (RSD %) of both elution times and peak area were summarized in Figure 3.
The RSD for a serum sample analyzed 5 times by HPLC
Retention time/min and RSD%
Man:15.38±0.013,0.09;GlcN:17.25±0.006,0.04;GalN:22.22±0.012,0.05;Glc:29.54±0.022,0.07;GlcA:24.32±0.017,0.07;Gal:31.28±0.023,0.07;Fuc:36.84±0.021,0.05;Xyl:33.33±0.021,0.06.
Peak area and RSD%
Man:7458.3±132.23,1.77;GlcN:7409.06±66.13,0.89;GalN:4972.23±12.27,0.25;Glc:6639.26±163.54,2.46;GlcA:1703.36±49.13,2.88;Gal:7245.1±112.71,1.56;Fuc:6196.61±59.48,0.96; Xyl:5672.16±52.42,0.92.
Stability experiment
Stability of a hydrolyzed and derivatized serum sample before HPLC analysis. After the serum sample has gone through the optimized hydrolysis and derivatization procedure, the HPLC analysis was conducted immediately (0 h), 2 h, 4 h, 8 h, 16 h, 24 h, respectively. The relative standard deviation (RSD %) of both elution times and peak area were summarized in Figure 4. The hydrolyzed and derivatized serum sample showed excellent stability. Based on above experiment, all the serum samples after derivatization were analyzed in the same day.
The RSD for a serum sample analyzed at 0, 2, 4, 6, 8, 16, and 24 hours after derivatization by HPLC
Man:15.39±0.02,0.15;GlcN:17.23±0.03,0.21;GalN:22.18±0.05,0.25;Glc:29.49±0.03,0.10;GlcA:24.37±0.06,0.25;Gal:31.18±0.0,0.19;Fuc:36.75±0.06,0.17; Xyl:33.22±0.06,0.18.
Peak area and RSD%
Man:2128.55±2.77,0.13;GlcN:2733.2±5.42,0.2;GalN:603.3±1.16,0.19;Glc:2214.65±4.28,0.19;GlcA:151.91±0.93,0.62;Gal:1923.61±5.40,0.28;Fuc:808.35±1.25,0.16; Xyl:530.86±0.56,0.11.
Loss rate experiment
Since the properties of the eight monosaccharides in human serum are different, the effects of hydrolysis of trifluoroacetic acid will also be different. In order to explore the degree of influence of the hydrolyzed serum with trifluoroacetic acid on the eight monosaccharides, we conducted a hydrolysis method (Table 5). Exploratory test of the loss rate caused. We took 100 ml of serum from each of 10 healthy controls and 10 endometrial cancer patients and made a serum mixture. We then added monosaccharide standard into the sera to test the loss rate of monosaccharide standard using the optimized experimental procedure by HPLC analysis.
The loss rate of monosaccharide standards when added to the mixture of serum samples
Peak areas of Serum plus Monosaccharide standards /Serum alone /Monosaccharide standards alone
Man:14123.4/9794.7/4599.3;GlcN:18419.6/14381.2/4079.0;GalN:4117.75/780.3/3343.2;Glc:15247.1/11561.5/4830.7;GlcA:1039.55/71.85/2049.3;Gal:12745.1/8490.6/4527.4;Fuc:5468.45/1252.9/4854.5; Xyl:3627.4/80.85/5135.1.
The loss rate of monosaccharide standards/%
Man:5.882;GlcN:0.995;GalN:0.1708;Glc:23.70;GlcA:52.78;Gal:6.028;Fuc:13.16; Xyl:30.94.
Typical monosaccharide composition analysis of a serum sample
Using the optimized method, Figure 5 showed that baseline separation of 8 monosaccharides were achieved from a serum sample of a cancer patient in that 3 x 10 μL sample were independently hydrolyzed, PMP-labeled, and analyzed (Figure 5A-C) in comparison to a mixture of 8 monosaccharide standards (Figure 5D). Almost identical retention time and peak area for each monosaccharide were observed for three independent analysis of the same serum patient (Figure 5A-C), which showed that this method was dependable in performing monosaccharide quantification and monosaccharide compositional analyses of serum samples.