A HPAE-PAD Chromatographic Assay for Carbohydrates in Urine as a Measure of Intestinal Permeability

Transcription

1 A HPAE-PAD Chromatographic Assay for Carbohydrates in Urine as a Measure of Intestinal Permeability Deanna Hurum and Jeffrey Rohrer, Thermo Fisher Scientific, Sunnyvale, CA, USA

2 Overview Purpose: A high-performance anion-exchange with pulsed amperometric detection (HPAE-PAD) chromatographic assay was developed to quantify carbohydrates in urine. Methods: Using a Thermo Scientific Dionex ICS Ion Chromatography System with PAD, an assay is developed to separate five common intestinal permeability probes in urine. Results: The method developed determines the five most commonly used probes of intestinal permeability with an analysis time of 16 min (23 min between injections). Simple dilution of urine samples is the only sample preparation required. Introduction A wide range of diseases and pharmaceutical therapies can cause damage to the small intestinal mucosa leading to changes in intestinal permeability. Examples include celiac disease, rheumatoid arthritis, HIV, food allergies and intolerance, nonsteroidal antiinflammatory drug use, and stress. These changes have additional consequences, including poor absorption of nutrients, further complicating disease treatment. Measuring the absorption of metabolically inert carbohydrates, such as D-mannitol and lactulose, is a noninvasive method for evaluating intestinal damage. Additional carbohydrates commonly used for this evaluation include L-rhamnose, 3-O-methyl-D-glucose, and D-xylose. The amounts of these carbohydrates in urine after ingestion of a mixed sugar solution are indicative of four types of carbohydrate absorption: paracellular diffusion between cells in the intestinal lining (lactulose), passive transcellular diffusion (D-mannitol or L-rhamnose), active transcellular transport (D-xylose) and ATP-dependent active transcellular transport (3-O-methyl-D-glucose). 1 Although the individual recoveries of ingested carbohydrates in the urine are indicators of intestinal health, frequently, the ratio of either D-mannitol or L-rhamnose to lactulose is used as a more reliable indicator of intestinal permeability. Methods Sample Preparation Aqueous Saline-Based Samples: Dilute samples by a factor of 600 in DI water prior to analysis. Liquid Chromatography Dionex ICS-3000 or ICS-5000 including: DP Dual Pump DC Detector/Chromatography Compartment AS Autosampler ED Electrochemical Detector ED Cell Gold on PTFE Disposable Electrode ph, Ag/AgCl Reference Electrode 2 A HPAE-PAD Chromatographic Assay for Carbohydrates in Urine as a Measure of Intestinal Permeability

3 Columns: Thermo Scientific Dionex CarboPac PA20, mm (P/N ) or with manually prepared eluent: Dionex CarboPac PA20, mm (P/N ) and Dionex CarboPac PA20, 3 30 mm (P/N ) Eluent Gradient: 10 mm KOH from -7 to 1 min, mm KOH from 1 9 min, mm KOH from 9 16 min Eluent Source: Thermo Scientific Dionex EGC III KOH Potassium Hydroxide Eluent Generator Cartridge with the Thermo Scientific Dionex CR-ATC Continuously Regenerated Anion Trap column or 100 mm NaOH manually prepared 2 Flow Rate: Inj. Volume: Temperature: Detection: Background: Noise: System Backpressure: 0.5 ml/min 10 ml (full loop) 30 C (column and detector compartments) Pulsed amperometric, Au on PTFE Disposable Electrode, 2 mil gasket, standard quad. Waveform 3 ~45 nc (using the carbohydrate 4-potential waveform and eluent generation) ~ 30 pc ~ 2400 psi Chromatographic Data Analysis The Thermo Scientific Dionex Chromeleon 7.1 Chromatography Data System (CDS) software was used for chromatographic data collection and processing. Eluent Generation Eluents were generated using a Dionex EGC III KOH (Figure 1). This module automatically generates purified hydroxide eluents by electrolyzing water. The Chromeleon CDS software will track the amount of KOH used and calculate the remaining lifetime as well as control the module. FIGURE 1. Eluent Generation Using Reagent-Free Ion Chromatography (RFIC ) Technology: Degassed water is delivered to the eluent generator by the pump. Current is applied to the cathode, generating potassium hydroxide. The eluent is then degassed and any anionic contaminants are trapped in the Dionex CR-ATC column, providing purified potassium hydroxide eluent. [KOH] α Current Flow Rate [+] Pt. Anode (H 2 O 2 H + +1/2 O 2 + 2e - ) KOH Generation Chamber K + Electrolyte Reservoir K + Cation-Exchange Connector H 2 O Pump KOH Degas Unit [-] Pt. Cathode (2 H 2 O +2e - 2 OH - + H 2 ) CR- ATC KOH Thermo Scientific Poster Note PN70137_e 06/12S 3

4 Results Although many similar methods have been previously published using the Dionex CarboPac PA1, PA100, and MA1 columns, 4 6 here the Dionex CarboPac PA20 column was used for the separation of endogenous carbohydrates and intestinal permeability probes that may be present in urine (Figure 2). FIGURE 2. Separation of ten carbohydrates of interest using a gradient elution from the Dionex CarboPac PA20 column 110 nc Column: Dionex CarboPac PA mm Eluent: 10 mm KOH from -7 to 1 min, mm KOH from 1 9 min, mm KOH from 9 16 min (using eluent generation) Flow Rate: 0.5 ml/min Inj. Volume: 10 µl Temperature: 30 C Detection: PAD, Au on PTFE (Disposable) Samples: Mixed carbohydrate standard in DI water, 1.67 μg/ml each with 0.32 µg/ml ribose Peaks: 6 1. Mannitol 2. 3-O-methylglucose 3. Rhamnose 4. Galactose 5. Glucose 6. Xylose 7. Sucrose 8. Ribose 9. Lactose 10. Lactulose Minutes Method precision for each of the carbohydrates studied, as relative standard deviation of peak area, was <1.5. Retention times are stable to within an RSD of 0.27 as a result of the consistency of eluent concentration delivered from the eluent generator (Table 1). TABLE 1. Linearity ( µg/ml for each carbohydrate), precision, LOQs (as 10 times S/N), and recoveries from synthetic urine for nine chosen carbohydrates when samples are analyzed on the Dionex CarboPac PA20 column Analyte Retention Time (min) Retention Time Peak Area (nc min) Peak Area Coeff. of Determin. (r 2 ) Recovery from Urine Matrix LOQ (µg/ml) Mannitol Omethylglucose 4.56 < Rhamnose Galactose Glucose 7.16 < Xylose Sucrose Lactose Lactulose A HPAE-PAD Chromatographic Assay for Carbohydrates in Urine as a Measure of Intestinal Permeability

5 The five metabolically inert carbohydrates are resolved from each other and response is linear within the range studied, with coefficients of determination >0.999 (Figure 3). FIGURE 3. Calibration curves for the five carbohydrates most frequently used to evaluate intestinal permeability 10.0 Xylose Mannitol Rhamnose Peak Area (nc min) Lactulose 3-O-methylglucose Concentration (µg/ml) Injections of synthetic urine spiked with standards and standards in DI water were nearly identical with little evidence of matrix interference as determined by retention time stability and equivalent peak areas when comparing the two matrices (Figure 4). FIGURE 4. Comparison of standards in DI water and synthetic urine spiked with 1.67 mg/ml of each of the five commonly used carbohydrates for intestinal permeability evaluation Column: Dionex CarboPac PA mm Eluent: 10 mm KOH from -7 to 1 min, mm KOH from 1 9 min, mm KOH from 9 16 min (using eluent generation) Flow Rate: 0.5 ml/min Inj. Volume: 10 µl Temperature: 30 C Detection: PAD, Au on PTFE (Disposable) 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 nc B) 3 4 Peaks: 1. Mannitol 2. 3-O-methylglucose 3. Rhamnose 4. Xylose 5. Lactulose 2 5 A) Minutes Thermo Scientific Poster Note PN70137_e 06/12S 5

6 Carbohydrate recoveries (measured by spiking samples prior to sample preparation) ranged from % across three days of sample analysis, indicating method accuracy (Table 2). TABLE 2. Method precision and accuracy evaluations for triplicate sample preparations across three days: accuracy and both intraday (n = 3) and between-day (n = 9) precisions, as measured by recoveries, are reported. Sample Carbohydrate Spiked Synthetic Urine Low-level Lactulose Spike Analyte Amount Spiked (µg/ml) Amount Determined (µg/ml) Recovery (%) Intraday Between- Day Xylose ± ± Lactulose ± ± Mannitol ± ± Omethylglucose ± ± Rhamnose ± ± Mannitol Omethylglucose Rhamnose Xylose Lactulose Conclusion The Dionex CarboPac PA 20 column separates commonly used intestinal permeability probes and additional endogenous carbohydrates within 16 min (total method time of 23 min) with excellent precision and recoveries. This method utilizes the RFIC system capabilities, requiring only a source of degassed DI water for eluent generation. This assay allows the sensitive detection of multiple indicative carbohydrates without complex sample preparation, time-consuming derivatization, or matrix elimination steps. 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. Cardiopulmonary Bypass Impairs Small Intestinal Transport and Increases Gut Permeability. Ann. Thorac. Surg. 1993, 55, Dionex (now part of Thermo Scientific) Technical Note 71: Eluent Preparation for High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. [Online] HPAE-PAD-16Sept2009-LPN pdf (accessed May 21, 2012). 3. 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] (accessed May 21, 2012). 4. Kynaston, J.A.; Fleming, S.C., Laker, M.F.; Pearson, A.D.J. Simultaneous Quantification of Mannitol, 3-O-Methyl Glucose, and Lactulose in Urine by HPLC with Pulsed Electrochemical Detection, for Use in Studies of Intestinal Permeability. Clin. Chem. 1993, 39, Sorensen, S.H.; Proud, J.F., Adam, A.; Rutgers, H.C.; Batt, R.M. A Novel HPLC Method for the Simultaneous Quantification of Monosaccharides and Disaccharides Used in Tests of Intestinal Function and Permeability. Clin. Chim. Acta, 1993, 221, Bao, Y.; Silva, T.M.J.; Guerrant, R.L.; Lima, A. A. M.; Fox, J.W. Direct Analysis of Mannitol, Lactulose and Glucose in Urine Samples by High-Performance Anion- Exchange Chromatography with Pulse Amperometric Detection. Clinical Evaluation of Intestinal Permeability in Human Immunodeficiency Virus Infection. J. Chromatogr. B, 1996, 685, A HPAE-PAD Chromatographic Assay for Carbohydrates in Urine as a Measure of Intestinal Permeability

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