CE Instruments have recently installed an Agilent 1100 HPLC system in one of our customer's laboratories. This system was put together by ourselves for this customer to perform the Lactulose:Mannitol test, otherwise known as the 'Leaky Gut' test - gut leakiness being a marker of certain gastrointestinal diseases. In this test, a patient must fast for 12 hours before consuming a drink containing mannitol and lactulose. They produce 3 urine samples at different time points and the concentration ratio of lactulose to mannitol is measured. The value of this ratio indicates whether the patient has abnormal intestinal leakage or not. This customer came to us, as we could not only provide the hardware, but create a turnkey analyser that is ready to use immediately after installation. Below is a brief summary on the entire process; from the planning stage to the delivery.
This method requires the separation and detection of sugars in a urine matrix. This poses several obstacles. First, the correct hardware is required. A refractive index detector (RID) must be employed to detect non-UV absorbing compounds such as sugars. RIDs require stable environments, so a column oven and stable pump are vital. Finally, for screening and monitoring purposes, as well as flexibility for further applications, a Diode Array Detector (DAD) was included too. This six module system (Degasser, Binary Pump, Autosampler, Oven, DAD, RID) was to be the hardware framework of the system.
The next step in delivering a turnkey system is to create a method that is robust and repeatable. Over a few weeks in our workshop, we built upon existing literature to formulate the ideal methodology for this analysis. After trials and tribulations, we prevailed with a repeatable method that could quantify low levels of both sugars. Below is a brief summary of our methodology, which may serve as an aid in the developing of similar methods in other laboratories.
Analytical Parameters
Flow rate: 1.0 ml/min
Oven Temperature: 40°C
RID Temperature: 40°C
Injection Volume: 100 ul
Mobile Phase: Acetonitrile: Water (3: 1)
1. Create 20 ml of a 2 mg/ml stock solution of lactulose and mannitol in blank, fasted urine using a 5-place balance and volumetric flask,
2. Dilute the stock to create 5 calibration levels (two Eppendorf vials per level) at the following concentrations – 500 ug/ml, 250 ug/ml, 125 ug/ml, 62.5 ug/ml, 31.3 ug/ml,
3. Centrifuge at 10,000 rpm for 10 minutes,
4. Take 1ml upper layer from each Eppendorf vial,
5. Add to a suitably sized vial with a 1:1 ratio of each amberlite resin e.g. 1 g of each resin for 2ml of standard or sample,
6. Shake sample using Vortexer/Whirlimixer for 30 seconds or by hand shaking for 60 seconds,
7. Remove the liquid phase with a syringe and needle, remove needle and inject 250 ul of this through a 0.2 um cellulose acetate syringe filter into a 2 ml HPLC vial intended for analysis,
8. In the HPLC vial, 250 ul of sample is further diluted with 750 ul of acetonitrile, vortex/shake to mix,
9. Label the vial and place into HPLC autosampler tray ready for analysis,
10. Perform a similar procedure for patient samples; treat the urine sample with a 1:1 ratio of Amberlite resins, filter through syringe filter, and dilute with acetonitrile as previously described.
11. Store stock solution and prepared samples in refrigerator when not in use.
Contact our team today for your hardware and application requirements at sales@ceinstruments.co.uk.
Authored by James Dobie, Applications Engineer at CE Instruments Ltd
Commentaires