Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

The overall goal of this miniaturized solid phase extraction combined with LC-MS/MS assay is to accurately quantify free 3-nitrotyrosine in human urine for clinical applications. Free 3-nitrotyrosine help in yields and a biomarker for oxidative stress. However, it will remain a special challenge to accurately quantify 3-nitrotyrosine in biological emergencies for clinical applications due to insufficient sensitivity, selectivity, and cumbersome sample preparations.

This technique incorporating an effective solid phase extraction in a highly sensitive LC-MS/MS interaction allows rapid, specific, and accurate determination of low levels of endogenous 3-nitrotyrosine in human urine without the need of constitution and two-dimensional LC.Though this method can provide insight into oxidative stress investigation, it can also be applied to other pathological conditions such as complex inflammatory and neurodegenerative disorders. The implications of this technique could be extended toward monitoring the effectiveness of antioxidant treatment because the 3-nitrotyrosine level can be reduced by antioxidants. To begin this procedure, vortex previously prepared and thawed urine samples, standards, and QC samples.

After vortexing, add 250 microliters of each sample and standard to 32 wells of a clean two milliliter 96-well collection plate. Add 50 microliters of previously prepared internal standard working solution to each well except the double blank sample well. Then add 50 microliters of LC-MS grade water with 0.01%of formic acid to the double blank sample well.

Following this, add 250 microliters of LC-MS grade water with 0.1%of formic acid to the wells. Then mix the solutions three times with an eight-channel pipette and cover the plate until SPE loading. Next, place a mixed mode cation exchange 96-well extraction microplate and a collection reservoir on a positive pressure processor.

The solid phase extraction utilizing a mixed mode cation exchange microplate enables simultaneous sample cleanup and enrichment of 3-nitrotyrosine in a single extraction. Condition the extraction plate by flowing 200 microliters of LC-MS grade methanol through the sorbent. Turn the pressure adjusting knob on the positive pressure processor to set a low positive pressure in order to allow the mixture to flow through the sorbent slowly, adjusting the pressure if needed.

Equilibrate by flowing 200 microliters of LC-MS grade water with 2%formic acid through the sorbent. Using an eight-channel pipette, carefully load the entire volume of each of the pre-mixed samples onto the preconditioned extraction plate. Set low positive pressure on the positive pressure processor to allow the mixture to flow through the sorbent slowly, adjusting the pressure as needed.

Following this, wash the wells by flowing 200 microliters of LC-MS grade water with 2%formic acid through the sorbent. After washing with 200 microliters of methanol and water, set the positive pressure processor to high pressure in order to dry the wells. Once the wells are dry, replace the reservoir with a clean two milliliter 96-well collection plate.

Now, apply 50 microliters of a 25 millimolar ammonium acetate solution to slowly elute the retained analyte and internal standard from the extraction plate. Then add 50 microliters of LC-MS grade water with 5%formic acid to neutralize the eluent. The application of a mild ammonium acetate solution for elution of 3-nitrotyrosine provides substantial enhanced selectivity and sensitivity.

Mix the samples with an eight-channel pipette three times in preparation for liquid chromatography tandem mass spectrometry analysis. After setting up the LC-MS/MS instrument, equilibrate the 3-nitrotyrosine LC-MS/MS method for 10 minutes with the LC gradient elution. By equilibrating the method, the LC gradient will be equilibrated to the initial gradient elution.

The auto sampler temperature will be set to four degrees Celsius and the oven temperature will be set to 30 degrees Celsius. Create a batch list that includes the urine samples, standards, and QC samples. Then start the batch by injecting the prepared samples.

After completing all the injections, establish a standard curve with a range of 10 to 2, 500 picograms per milliliter for 3-nitrotyrosine quantitation by linear regression of the peak area ratio of 3-nitrotyrosine and internal standard versus the nominal 3-nitrotyrosine concentration. Finally, quantify all the other samples using the established standard curve and determine if the QC samples fall within the established range. LC data of human urine samples show that 3-nitrotyrosine is chromatographically separated from other structurally similar tyrosine analogs under the optimized condition which eliminates co-eluting interferences due to these vastly excessive compounds and consequently enhances the degree of assay selectivity.

A 3-nitrotyrosine signal is not observed in the multiple reaction monitoring chromatogram of the double blank sample, indicating that 3-nitrotyrosine is not formed during the entire process. Representative multiple reaction monitoring chromatograms of 3-nitrotyrosine and internal standard for a healthy individual are shown here. No interfering signals were observed at the retention times of 3-nitrotyrosine and internal standard.

A representative standard curve is shown here. The limit of detection defined as the lowest concentration with a signal to noise ratio greater than three was determined to be two picograms per milliliter. The lower limit of quantification was determined to be 10 picograms per milliliter and is defined as the lowest concentration within 20%of imprecision and accuracy with a signal to noise ratio of greater than 10.

The fine tuned integration assay delivers significant improved specificity, sensitivity, and throughput with reduced matrix effect, sorbent usage, and waste disposal. With this simple and rapid assay, urine sample can be processed within 24 hours. Take into account the non-invasive and inexpensive urine sampling.

This remarkably improved method is well suited to evaluate the role of 3-nitrotyrosine as an oxidative stress biomarker in preclinical and clinical studies.