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Conclusion Both proposed methods accurately determine carbohydrate probes in urine. The first method takes advantage of eluent generation (requiring only DI water for system performance) to determine the intestinal permeability probes in 16 min. The second method strongly retains mannitol, eliminating potential interferences, and provides a convenient isocratic method with a 40 min run time. References 1.Ohri, S.K.; Bjarnason, I.; Pathi, V.; Somasundaram, S.; Bowles, C.T.; Keogh, B.E.; Khaghani, A.; Menzies, I.; Yacoub, M.H.; Taylor, K.M. Ann. Thorac. Surg. 1993, 55, 1080–1086. 2.Kynaston, J.A.; Fleming, S.C., Laker, M.F.; Pearson, A.D.J. Clin. Chem. 1993, 39, 453–456. 3.Sorensen, S.H.; Proud, J.F., Adam, A.; Rutgers, H.C.; Batt, R.M. Clin. Chim. Acta, 1993, 221, 115–125. 4.Bao, Y.; Silva, T.M.J.; Guerrant, R.L.; Lima, A. A. M.; Fox, J.W. J. Chromatogr. B, 1996, 685, 105–112. 5.Dionex (now part of Thermo Scientific) Application Update 141: Improved Long-Term Stability of N-Acetylneuraminic Acid and N- Glycolylneuraminic Acid Peak Area Responses Using Waveform A, a Quadruple Potential Waveform. [Online] www.dionex.com/en- us/webdocs/6511-AU141_LPN1225.pdf (accessed May 21, 2012). 6.Dionex (now part of Thermo Scientific) Technical Note 71: Eluent Preparation for High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. [Online] www.dionex.com/en-us/webdocs/58087-TN71-Eluent-Prep-HPAE- PAD-16Sept2009-LPN1932-02.pdf (accessed May 21, 2012). Overview Purpose: Two high-performance anion-exchange with pulsed amperometric detection (HPAE-PAD) chromatographic methods were developed and compared to quantify carbohydrates in urine. Methods: Using a Thermo Scientific Dionex Ion Chromatography System, carbohydrates are determined in urine. Results: The first method developed determines the five most common carbohydrate probes with an analysis time of 16 min (23 min between injections). The only sample preparation needed is simple dilution of urine samples. The second method provides an isocratic chromatographic assay that determines four probes with reduced sample dilution and a wider linear range. Introduction Measuring the absorption of metabolically inert carbohydrates, such as D-mannitol and lactulose, is commonly used for researching intestinal permeability. Additional carbohydrates commonly determined include L-rhamnose, 3-O-methyl-D-glucose (3-OMG), and D-xylose. 1 Frequently the ratio of either D-mannitol or L-rhamnose to lactulose is monitored by HPAE-PAD. 2–4 Methods Sample Preparation Dilute samples by a factor of 600 with DI water for Method 1 and 200-fold with DI water for Method 2. Liquid Chromatography—Method 1 Dionex ICS-3000 or ICS-5000 ion chromatography system including: DP Dual Pump DC Detector/Chromatography Compartment EG Eluent Generator AS Autosampler ED Electrochemical Detector Electrochemical Cell Gold on PTFE Disposable Electrode (working) pH, Ag/AgCl Reference Electrode Columns: Thermo Scientific Dionex CarboPac PA20, 3 × 150 mm Eluent Source: Thermo Scientific Dionex EGC III KOH with Dionex CR-ATC Continuously- Regenerated Anion Trap Column Gradient: 10 mM KOH from -7 to 1 min, 10–30 mM KOH from 1–9 min, 30–35 mM KOH from 9–16 min Flow Rate: 0.5 mL/min Inj. Volume: 10 μL, (full loop) Temperature: 30 °C (column and detector compartments) Detection: PAD, Gold on PTFE Disposable Electrode, 2 mil gasket, standard quad. waveform 5 Background: ~45 nC Noise: ~ 30 pC System Backpressure: ~ 2400 psi Chromatographic Data Analysis The Thermo Scientific Dionex Chromeleon 7.1 Chromatography Data System (CDS) was used for chromatographic data collection and processing. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Liquid Chromatography—Method 2 Columns: Dionex CarboPac MA1, 4 × 250 mm and Dionex CarboPac MA1, 4 × 50 mm Eluent: 480 mM NaOH, manually prepared 6 Flow Rate: 0.4 mL/min Inj. Volume: 25 μL, (full loop) Temperature: 30 °C (column and detector compartments) Detection: PAD, Gold on PTFE Disposable Electrode, 2 mil gasket, standard quad. waveform. Background: ~37 nC Noise: ~ 50 pC System Backpressure: ~ 1345 psi Chromatographic Determination of Carbohydrates in Urine Deanna Hurum, Deepali Mohindra, and Jeffrey Rohrer, Thermo Fisher Scientific, Sunnyvale, CA, USA FIGURE 1. Separation of ten carbohydrates of interest using a gradient elution from the Dionex CarboPac ™ PA20 column. Column: Dionex CarboPac PA20, 3 × 150 mm Eluent Source: Dionex EGC III KOH with Dionex CR-ATC Gradient: 10 mM KOH from -7 to 1 min, 10–30 mM KOH from 1–9 min, 30–35 mM KOH from 9–16 min Flow Rate: 0.5 mL/min Inj. Volume: 10 µL Temperature: 30 °C Detection: PAD, Gold on PTFE Disposable Electrode Samples: Mixed carbohydrate standard in DI water, 1.67 μg/mL each with 0.32 µg/mL ribose Peaks: 1. Mannitol 2. 3-O-methylglucose 3. Rhamnose 4. Galactose 5. Glucose 6. Xylose 7. Sucrose 8. Ribose 9. Lactose 10. Lactulose 0 2 4 6 8 10 12 14 16 30 110 Minutes nC 1 2 3 4 5 6 7 8 9 10 FIGURE 2. Comparison of standards in DI water and synthetic urine spiked with 1.67 mg/mL of each of the five commonly determined carbohydrates in urine. Column: Dionex CarboPac PA20, 3 × 150 mm Eluent Source: Dionex EGC III KOH with Dionex CR-ATC Gradient: 10 mM KOH from -7 to 1 min, 10–30 mM KOH from 1–9 min, 30–35 mM KOH from 9–16 min Flow Rate: 0.5 mL/min Inj. Volume: 10 µL Temperature: 30 °C Detection: PAD, Gold on PTFE Disposable Electrode Sample Prep: 600 fold dilution Samples: A) Standards in DI water (black) B) spiked synthetic urine, (blue) 1.67 µg/mL each carbohydrate after dilution Peaks: 1. Mannitol 2. 3-O-methylglucose 3. Rhamnose 4. Xylose 5. Lactulose 0 2 4 6 8 10 12 14 16 30 120 Minutes nC 1 2 3 4 5 A) B) Columns: Dionex CarboPac MA1, 4 × 250 mm Dionex CarboPac MA1 guard, 4 × 50 mm Eluent: 480 mM NaOH Flow Rate: 0.4 mL/min Inj. Volume: 25 µL Temperature: 30 °C Detection: PAD, Gold on PTFE Disposable Electrode Sample Prep: 200 fold dilution Samples: A) spiked synthetic urine, 5.0 µg/mL each after dilution (Note, lactose was not included in spiked urine samples) B) synthetic urine C) carbohydrate standards in DI water, 5.0 µg/mL each Peaks: 1. 3-O-methylglucose 2. Mannitol 3. Xylose 4. Lactulose 5. Lactose 0 10 20 30 40 20 160 Minutes nC 20% signal offset applied 1 2 3 4 5 1 2 3 4 A) B) C) Analyte RT (min) Peak Area (nC*min) Peak Area Precision (RSD) Recovery Mannitol 21.48 5.426 0.90 101–103 3-OMG 14.90 0.7861 1.72 105–107 Xylose 27.30 2.746 1.43 99.5–101 Lactulose 29.44 2.068 0.68 90.6–98 Method Parameter Method 1 Method 2 Run time (min) 23 40 Elution type Gradient hydroxide Isocratic hydroxide Linearity (r 2 ) >0.999 >0.998 Linear range (μg/mL) 0.050–2.50 0.500–150 Precision (as Peak area RSD for lactulose) 1.47 0.68 Accuracy (as % recovery) 93.8–112 90.6–107 Mannitol RT (min) 1.62 21.48 Lactulose RT (min) 14.09 29.44 FIGURE 3. Separation of carbohydrates of interest using a gradient elution from the Dionex CarboPac MA1 column. Table 1. Precision and accuracy for Method 1. Precision values were determined at a concentration of 1.00 µg/mL for each carbohydrate. Table 2. Precision and accuracy for Method 2. Precision values were determined at a concentration of 1.00 µg/mL for each carbohydrate. Table 3. Comparison and summary of the two proposed methods. As shown in Figure 3, this method provides excellent retention of carbohydrate probes compared to the synthetic urine matrix. Mannitol is well resolved from the void. Accuracy and precision are similar to Method 1 (Table 2). The urine matrix does not effect the chromatography, as shown by the overlaid chromatograms in Figure 2. Multiple analyses of synthetic urine samples (n = 9) had recoveries ranging from 93.8–112%. PO70151_E 08/12S Analyte RT (min) Peak Area (nC*min) Peak Area Precision (RSD) Recovery Mannitol 1.62 1.918 1.16 95.9 3-OMG 4.56 0.7491 0.54 112 Rhamnose 4.79 1.449 1.14 103 Galactose 6.63 2.398 0.25 93.8 Glucose 7.16 2.169 0.54 113 Xylose 7.88 3.280 0.45 97.3 Sucrose 8.93 1.208 0.61 95.5 Lactose 13.76 1.907 0.99 99.6 Lactulose 14.09 1.525 1.47 101 Results Method 1 The five carbohydrate probes are determined easily using this method. Ribose is included in the chromatogram in Figure 1 as a potential internal standard. Determination precision and accuracy, as recovery, are shown in Table 1. Method 2 For Research Use Only. Not for use in diagnostic procedures.
