LAAN-A-LM-E045

No.C74Liquid Chromatography Mass Spectrometry

Quantitative Analysis of Carbohydrates by LC/MSA variety of separation and detection methods are available for the HPLC analysis of saccharides. Typical separation techniques for carbohydrate analysis include normal phase distribution and ligand exchange. Differential refractive index detection and post-column fluorescence derivatization are known to be effective for detection.In carbohydrate analysis using LC/MS, derivatization is often used to improve detection sensitivity. However, to c i rcumvent th is cumbersome derivatization operation for ESI, solvent is added after column using APCI to allow ionization of low-polarity compounds. This solvent addition promotes ionization, thereby allowing stable ion detection. Here we introduce examples of quantitative analysis of carbohydrates in foods using post-column solvent addition.

n Analysis of Carbohydrates Using LCMS-2020Fig. 1 shows mass chromatograms of carbohydrates. Methanol – chloroform (4:1) was used as the solvent that was added after the column. This solvent encourages the formation of the chloride ion adduct ([M+Cl]-) preferentially, allowing stable detection of the ion.Since post-column solvent addition means that substances eluting from the column (including mobile phase) are mixed with the solvent, a connector is used between the column and interface (in this case, the APCI), and a separate pump is used for delivery of the solvent.

0.0 2.5 5.0 7.5 10.0 12.5 min

0.40

0.30

0.20

0.10

0.000.600.50

0.40

0.30

0.20

0.10

0.001.201.000.800.600.400.200.00

2.00

1.00

0.00

2,657,009(×1,000,000)

1,381,057(×1,000,000)

615,159(×1,000,000)

434,132(×1,000,000)

376.90@1

214.80@1

198.80@1

184.80@12: Xylose

1: Ribose

5: Glucose

7: Maltose

3: Rhamnose

4: Fructose

6: Sucrose

Fig. 1 LC/MS (SIM) Analysis of Carbohydrates Using Post-Column Solvent Addition (100 mg/L each)(1: Ribose, 2: Xylose, 3: Rhamnose, 4: Fructose, 5: Glucose, 6: Sucrose, 7: Maltose)

SHIMADZU CORPORATION. International Marketing Division3. Kanda-Nishikicho 1-chome, Chiyoda-ku, Tokyo 101-8448, Japan Phone: 81(3)3219-5641 Fax. 81(3)3219-5710Cable Add.:SHIMADZU TOKYO

No.C74

Table 1 shows the quantitation results for each substance. The RSD (%) values shown in the table correspond to the RSD (%) for the smallest

concentrations used in generating the calibration curves.

n Calibration Curves

Table 1 Calibration Curves

MW m/z Range (mg/L) Correlation Coefficient (R) RSD (%)

Ribose 150.1 184.8 [M+Cl]- 0.5-100 0.9999 3.40

Xylose 150.1 184.8 [M+Cl]- 0.1-100 0.9996 5.13

Rhamnose 164.2 198.8 [M+Cl]- 0.5-100 0.9999 5.04

Fructose 180.2 214.8 [M+Cl]- 0.1-100 0.9999 2.81

Glucose 180.2 214.8 [M+Cl]- 0.5-100 0.9999 0.51

Sucrose 342.3 376.9 [M+Cl]- 1-100 0.9996 10.22

Maltose 342.3 376.9 [M+Cl]- 5-100 0.9996 4.92

n Analysis of Foods Using the LCMS-2020Fig. 2 and 3 show the results of food analysis. The samples were each diluted 10-fold using distilled water, and then ultrafiltrated (Microcon YM-3, MILLIPORE). The recovered sample was further diluted 1000-fold in distilled water, and 1 μL of each

sample was injected. The carbohydrate concentrations in the foods used for analysis were 360 mg/mL (glucose) and 41.7 mg/mL (maltose) in the mirin (sweet sake), and 8.6 mg/mL (fructose), 12.1 mg/mL (glucose), and 32.4 mg/mL (sucrose) in the soft drink.

3.00

2.00

1.00

0.00

0.020.0 2.5 5.0 7.5 10.0 12.5 min

0.040.060.080.100.120.140.160.180.200.220.240.260.28

0.320.30 Maltose

Glucose(×100,000)346,065

(×100,000)32,528

214.80@1

376.90@1

1.501.401.301.201.101.000.900.800.700.600.500.400.30

7.00

6.00

5.00

4.00

3.00

2.00

1.00

(×10,000)15,240

(×10,000)78,330

214.80@1

376.90@1

Fructose Glucose

Sucrose

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Fig. 2 Sweet Sake (1 μL injected) Fig. 3 Soft Drink (1 μL injected)

Table 2 Analytical Conditions

Column : Shodex Asahipak NH2P-50 MS : LCMS-2020(150 mmL. × 2.0 mmI.D., 5 μm) Probe Voltage : -3.5 kV (APCI-Negative mode)

Mobile Phase : acetonitrile/water (3:1) Probe Temperature : 400 ˚CFlow Rate : 0.2 mL/min Nebulizing Gas Flow : 4.0 L/minPost Column Solvent : methanol-chloroform (4:1) Drying Gas Flow : 5.0 L/minFlow Rate : 0.075 mL/min DL Temperature : 250 ˚CColumn Temperature : 30 ˚C Block Heater Temperature : 200 ˚CInjection Volume : 1 μL DL, Q-Array Voltages : default values

Event Time : 0.5 secSIM : m/z 184.8 (Ribose, Xylose)

m/z 198.8 (Rhamnose)m/z 214.8 (Fructose, Glucose)m/z 376.9 (Sucrose, Maltose)