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

Summary

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Abstract

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH₄OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 µm) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

Introduction

The effects of oxidative stress on clinical presentation have been thrust into the forefront in recent years¹. One of the biomarkers being explored is 3-nitrotyrosine (3-NT), an end stable product formed when reactive nitrogen species (RNS) interact with tyrosine, a catecholamine neurotransmitter precursor. While 3-NT may have clinical value as a biomarker for RNS in vivo, the substantial changes of the properties and functions of tyrosine may adversely affect corresponding proteins and cellular functions^1,2. Emerging research has suggested that 3-NT may play an important role ....

Protocol

All studies involving human urine samples were conducted adherence to the procedure approved by Pharmasan/Neuroscience Institutional Review Board (IRB).

1. Urine Sample Collection and Creatinine (Cr) Determination

1. Collect 5 mL of the next morning urine samples after ca. 10 h overnight fasting in a 5 mL transport tube A containing 250 µL of 3 N HCl as preservative and store at -20 °C until use.
2. Thaw and vortex 5 mL transport urine A tube and centrifuge in a centrifuge (e.g. Sorvall) (2297 x g, 10 min).
3. Aliquot 1 mL of urine twice from the 5 mL transport tube A.
4. Determine Cr by a ur....

Results

Figure 1 illustrates that 3-NT is completely chromatographically separated from other structurally similar tyrosine analogues under the optimized LC condition, which eliminates the co-eluting interferences due to these vastly excessive compounds and consequently enhances the degree of assay selectivity. In addition, the gradient elution with 0.01% HCOOH as additive in MA and methanol at a flow rate of 0.45 mL/min allows rapid elution of 3-NT (i.e., 3.......

Discussion

Substantial variations in concentrations previously reported in the literature for the endogenous free 3-NT in human urine samples reveal methodological problems associated with available assays^8,9,10,11. Accurate determination of the low basal level of 3-NT in human urine remains a challenging task that requires special precautions for sample preparation and LC-MS/MS analysis. This protocol ou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Nitro-L-tyrosine	Sigma	N7389-5g
3-Nitro-L-tyrosine-13C9	Sigma	652296-5.0mg
Mass Spec Gold Urine	Golden West Biologicals	MSG 5000-1L
Oasis MCX 96-well µElution plate	Waters	186001830BA
2 mL 96 well collection plate	Phenomenex	AH0-7194
96 positive processor	Waters	186005521
LC-MS Ultra CHROMASOLV methanol	Sigma	14262-2L
LC-MS Ultra CHROMASOLV water	Sigma	14263-2L
Formic acid for mass spectrometry	Sigma	94318-50ML-F
Ammonium hydroxide solution	Sigma	338818-1L
Ultra PFP propyl columns	Restek	9179362
5500 Triple quad	AB Sciex	/	Contact manufacture for more detail
UFLC-XR	Shimadzu	/	Contact manufacture for more detail
Integra 400 Plus	Roche	/	Urinary Creatinine Jaffé Gen 2 method
LCMS certified 12 x 32 mm screw neck vial	Waters	600000751CV
LCGC certified 12 x 32 mm screw neck total recovery vial	Waters	186000384C
5 mL transport tube	Phenix	TT-3205
50 mL Centrifuge tube	Crystalgen	23-2263
15 mL Centrifuge tube	Crystalgen	23-2266
eLine electronic pipette	Sartorius	730391
Microfuge centrifuge	Beckman Coulter	A46474
OHAUS balance	Kennedy Scales, inc.	735
Vortex mixer	Bernstead Thermolyne	M16715