In this one step metabolomics procedure, biological samples are prepared for analysis of carbohydrates, organic and amino acids by gas chromatography mass spectrometry in a single procedure. The samples are first treated with urease enzyme to remove urea. The second step of the procedure is to dehydrate the samples in the presence of a detergent comprised of two volatile components, triathlon, ammonium, and tri fluoro acetate.
This is followed by derivitization of the components by trimethylation. And finally, analysis by gas chromatography, mass spectrometry tree, automated quantification of carbohydrates, organic and amino acids, isol, glycine, and amino sugars allows confirmation and follow-up of most genetic disorders found on neonatal screening and an additional 38%of disorders not currently screened for by tandem mass spectrometry. Hello, I'm Jim Shoemaker at the Metabolic Screening Lab at St.Louis University.
I'm here to demonstrate one step metabolomics, a method for analyzing biological fluids for the intermediary metabolites without separating acids, bases and neutral compounds. This makes it possible to follow up tandem mass spec neonatal screening with a single procedure that allows you to analyze for organic acids, amino acids, carbohydrates, carbohydrate derivatives, and ACell glycine all in one step. The upshot of it is that in the graph behind me, you can see that the diagnoses in red can be made using one step metabolomics, but would not have been picked up on current neonatal screening by tandem mass spectrometry.
So let's start the demonstration. To begin this procedure, haw the urine sample in a 37 degree Celsius water bath. If the original container is compromised, decamp a sample into a new container.
Take an aliquot of sample and store it at minus 20 degrees Celsius in a labeled conical centrifuge tube as a backup. Next measure and record the optical density of the sample by placing a couple of drops of urine in the handheld refractometer. After measuring the optical density, filter the sample through a 0.2 micron filter.
Measure out the volume of sample according to the optical density measured prior to filtration. For values between one and 1.009, use one milliliter of sample for values between 1.01 and 1.019. Use 0.5 milliliters of sample and for values between 1.02 and 1.05, combine 0.25 milliliters of sample with 0.25 milliliters of water.
Transfer the specified volume to a reactive vial already containing the isotope labeled internal standards specified in the table shown here. The standards include creatine, methylmalonic acid, lactate, pyruvate, glycine, sine phenyl alanine, hexa oil, glycine, orate, SEBA acid glucose, inositol, and tryptophan. Then add 20 microliters of a 7.5 units per microliter solution of urease.
Flush the sample and seal under carbon dioxide through an inert septum. Hold the sample at 37 degrees Celsius for 30 minutes while adding more carbon dioxide gas at 15 minute intervals to maintain the pressure at the end of the 30 minute incubation at 20 microliters more of the urease solution. Flush the vial with carbon dioxide and maintain the sample at 37 degrees Celsius for another 15 minutes.
Once the urease treatment is complete, proceed directly to dehydrate and derivate the sample immediately following the urease treatment. At 500 microliters of a 30 to 70 acetone to methanol solution. To the samples, replace the rubber septum with a Teflon coated septum.
Chill the sample at minus 20 degrees Celsius for 15 minutes. Remove solids by centrifugation at 1500 RPM for 10 minutes, and then decamp the supinate into a clean 2.0 CC reactive vial. Now add tri ammonium, tri fluoro acetate or T-E-A-T-F-A, which is the catalyst for trimethylation for one milliliter samples at 20 microliters, and for 0.5 milliliters or less samples at 40 microliters.
Top off with aceto nitrile and place under a nitrogen stream at 70 degrees Celsius until constant volume is achieved, indicating that only T-E-A-T-F-A remains. This takes about 15 minutes. Again, top off with Acetonitrile and place under a nitrogen stream at 70 degrees Celsius until a constant volume is achieved, repeating up to four times until a precipitate forms.
This takes about 10 minutes each time after dehydration, let cool for about two minutes. Top off with methylene chloride, being careful of boil over and dry. This takes about four minutes.
Top off with methylene chloride and dry. One more time. Next, derivatize the sample.
Add 150 microliters OFM, STFA for one milliliter samples and 200 microliters per 0.5 milliliter or less. Samples cap under a nitrogen atmosphere and incubate at 70 degrees Celsius for one hour. At end of the one hour incubation, work under a nitrogen atmosphere to transfer the samples to micro vials for analysis on a gas chromatograph mass spectrometer, the Agilent 5 9 7 5 GCMS is used here with a DB five capillary.
For the mass spec, set the source to 230 degrees Celsius, the quad to 150 degrees Celsius, and the scan mass range to 50 to 650 A MU at 2.46 scans per second. Solvent delay is at 3.5 minutes. The instrument temperatures are 200 degrees Celsius for the injector and 250 degrees Celsius.
For the interface, set the oven to 80 degrees Celsius for one minute, then ramp at four degrees Celsius per minute from 80 to 130 degrees Celsius, after which ramp at six degrees Celsius per minute between 130 to 200 degrees Celsius, and finally ramp at 12 degrees Celsius per minute from 200 to 285 degrees Celsius and hold for 10 minutes. Place the micro vials holding the deriv eye samples for automated injection by the GCMS and start the analysis. A representative spectrum of a urine sample analyzed by GCMS following Urease treatment is shown here with the peaks corresponding to the metabolites pyro glutamic acid, four hydroxy cyclo acetic acid, and hawkinson marked.
These metabolites are indicative of Hawkins Anuria, a rare genetic disorder. All the peaks in the spectrum can be assigned to known metabolites and quantified. The results of initial newborn screening tests can be nonspecific.
However, the elevated levels of Hawkins UR metabolites in the urine sample analyzed by GCMS as shown here, confirmed the diagnosis of this inborn error of metabolism disease. Using the same URIs method, inborn errors were diagnosed in 184 patients. Close to 40%of the diagnoses shown in red would not have been made by tandem mass spectrometry commonly used in neonatal screening.
So that's our demonstration of how to do one step metabolomics using the urease method to analyze biological fluids for carbohydrates, organic and amino acids, and acyl glycine, all in the same procedure. It's important to remember that when you're evaluating the effectiveness of the procedure, the urease digestion, the dehydration and the trimethyl simulation can all be assessed for their completeness. Just by checking for the presence of all of the internal standards.
The internal standards were deliberately chosen to demonstrate that, for example, dehydration went to completeness because if it didn't, the creatinine TRI TMS would not be present in the full amount. So that's our demonstration. If you're interested in the macros and libraries that we have developed to interpret this procedure using chem station, please email me at the address shown on your screen, and more information about our laboratory can be found at the URL on your screen.
Thanks a lot. This is Jim Shoemaker.
