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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 in inflammatory conditions³, neurodegenerative disorders^4,5, cardiovascular disease⁶ and diabetes⁷ as well as conditions related to oxidative stress. However, these observations are based on results from methodologies lacking in sensitivity and/or selectivity^8,9,10,11. The enormous 3-NT concentration ranges for the biological samples previously reported in the literature reveal that serious analytical problems are associated with these assays and technical improvement is needed to accurately quantify the levels of 3-NT and verify its role in the pathology of these conditions.

The quantitation of free 3-NT in biological matrices presents a special challenge to man and instrument^8,9,10,11. First, the trace level of endogenous 3-NT demands an ultra-sensitive detection; second, the existence of numerous structurally similar analogues, especially tyrosine, which is present in vast excess, requires a high degree of selectivity; third, the artefactual formation of 3-NT by tyrosine nitration with ubiquitous nitrate and nitrite requires special consideration during sample preparation to avoid false overestimation of 3-NT.

Among a wide variety of methodologies employed to measure 3-NT, MS/MS has been considered the gold standard method due to its superior sensitivity and selectivity^11,12,13,14. Gas chromatography (GC) coupled MS/MS offers the best sensitivity, however, the indispensable sample derivatization steps are too tedious and time-consuming to be efficient for clinical utility^15,16. LC-MS/MS does not require complex sample derivatization, making it the more promising option. Nonetheless, there are several obstacles to overcome such as the sensitivity of LC-MS/MS methods reported in the literature needs to improve for the measurement of low abundant 3-NT^7,17,18 and the relatively long turnaround time must be shortened for high-throughput applications^12,13,17,19.

Additionally, when considering clinical applications, the biological matrix used plays a significant role. It should be easy and inexpensive to obtain and non-invasive if possible^20,21,22. Plasma, the traditionally used sample in the literature, is not a clinically desirable matrix, so a methodology utilizing urine which is non-invasive and cost-effective, was sought.

Several attempts to develop reliable and specific LC-MS/MS methodologies have been made using urine^9,10,11. However, they have all fallen short of being either selective, reliable or efficient enough for clinical use. The effectiveness of the predominant SPE using traditional reversed-phase cartridge (C18 type) as sample cleanup for the 3-NT analysis has been questioned and a sequential SPE of strong cation exchange (SCX) and reversed phase C18-OH has been proposed^6,7,19. One recently developed LC-MS/MS method utilized a multi-step purification process of manual C18 SPE, preparative high pressure liquid chromatography (HPLC), and online SPE for analysis of 3-NT²³. Although this method was sensitive enough for clinical purposes, with an LLOQ of 0.041 nM, the cleanup process was intensive and tedious and required 3 mL of urine, limiting its feasibility for high-throughput. A molecularly imprinted polymer was employed as the SPE sorbent to improve the efficiency of the cleanup process¹⁴, but the resulting LLOQ (0.7 µg/mL) was not low enough for clinical specimens. Another method required two-dimensional (2D) LC-MS/MS and immunoaffinity chromatography for sample cleanup in order to achieve a limit of detection (LOD) of 0.022 nM²⁴. While all these methods have made advancements in the assessment of 3-NT, none have achieved the sensitivity, reliability, and efficiency necessary for clinical applications.

In order to investigate the pathology of free 3-NT and its role as a biomarker of oxidative stress in clinical settings, we have developed a methodology that is simple, efficient, accurate and precise, enabling for high-throughput clinical applications²⁵. A miniaturized mixed-mode cation exchange (MCX) 96-well extraction microplate was implemented to achieve simple and effective sample cleanup and enrichment of 3-NT in a single extraction bypassing the drawbacks seen in the existing methods that require derivatization, evaporation and 2D-LC. Liquid chromatography with 0.01% HCOOH as an additive in mobile phase offered an enhanced signal response with a rapid cycle time. Selectivity was further improved through application of a mild NH₄OAc elution solution for selective elution of 3-NT, and use of MRM transition for both 3-NT and the internal standard (IS). The matrix effect was compensated for by using a reduced amount of a preferred ¹³C-labeled isotopic IS for quantification. With the advent of this methodology, researchers and clinicians will be able to verify the role of 3-NT in clinical conditions and further explore the impact of oxidative stress.

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 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. Thaw and vortex 5 mL transport urine A tube and centrifuge in a centrifuge (e.g. Sorvall) (2297 x g, 10 min). Aliquot 1 mL of urine twice from the 5 mL transport tube A. Determine Cr by a urinary creatinine method26. 2. Preparation of Standard, IS and Quality Control (QC) Samples Prepare a stock solution of 3-NT at 100 µg/mL in water with 0.01% HCOOH mobile phase A (MA) and store at -20 °C. Make a 3-NT working standard solution at concentration of 100 ng/mL by diluting the 100 µg/mL 3-NT stock solution with MA. Generate standards ranging from 5 to 2500 pg/mL by dilution of the 100 ng/mL working standard with 0.15 M HCl acidified blank urine free of 3-NT along with blank and double blank samples. Prepare a stock solution of IS 13C9-3-NT at 100 µg/mL in water with MA and store at -20 °C. Make an IS working solution at 500 pg/mL by dilution of the 100 µg/mL 13C9-3-NT IS stock solution with MA. Establish five levels of QC samples covering the LLOQ, low, medium and high levels (i.e., 10, 25, 100, 500 and 1,250 pg/mL), by dilution of 3-NT working standard in the acidified blank urine. 3. Solid Phase Extraction Procedure Thaw and vortex urine samples, standards and QC samples. Add urine samples, standards and QC samples (250 µL each) to 32 wells of a clean 2 mL 96-well collection plate. Introduce the 500 pg/mL IS working solution (50 µL) to each well except double blank sample well. Add 0.01% HCOOH (50 µL) to the double blank sample well. Add LC-MS/MS water with 0.1% HCOOH (250 µL). Mix the above mixture with an 8-channel pipette three times. Cover the plate until SPE loading. Place an MCX 96-well extraction plate and a collection reservoir on a positive pressure processor. Condition the extraction plate with flowing MeOH (200 µL) through the sorbent. Equilibrate by flowing water with 2% HCOOH (200 µL) through the sorbent. Load the entire volume of each of the pre-mixed samples onto the pre-conditioned extraction plate carefully with an 8-channel pipette. Set low positive pressure (e.g., 3 psi) on the positive pressure processor to allow the mixture to flow through the sorbent slowly, adjust the pressure if needed. Wash the wells by flowing water with 2% HCOOH (200 µL) through the sorbent. Wash the wells by flowing methanol (200 µL) through the sorbent. Wash the wells by flowing water (200 µL) through the sorbent. Dry the wells completely with high positive pressure setting (e.g., 40 psi) on the positive pressure processor. Replace the reservoir with a clean 2 mL 96-well collection plate. Apply 25 mM NH4OAc at pH 9 (50 µL) to elute the retained analyte and IS from the extraction plate. Pipette LC-MS water with 5% HCOOH (50 µL) to neutralize the eluate. Mix with the 8-channel pipette three times and submit to LC-MS/MS station for analysis. 4. LC-MS/MS Analysis Accurately measure 2,000 mL of LC-MS water with a graduated cylinder and transfer it into a 2 L bottle. Pipette 200 µL pure HCOOH into the above bottle containing LC-MS water. Mix thoroughly and label as mobile phase A (MA). Include initials, preparation date and expiration date. Take a 2 L bottle of LC-MS methanol and label as mobile phase B (MB) with starting date and expiration date. Set temperature of auto-sampler to 4 °C. Place the collection plate with prepared samples into the autosampler. Place a PFP column (150 mm x 2.1 mm i.d., 3 µm) and guard column in the oven. Set the oven temperature to 30 °C. Equilibrate 10 min using the acquisition method with the LC gradient elution as shown in Table 1. Time (min) Module Events Parameter 0 Pumps Pump B Conc. 5 0.5 Pumps Pump B Conc. 20 1 Pumps Pump B Conc. 50 3 Pumps Pump B Conc. 80 4 Pumps Pump B Conc. 90 4.01 Pumps Pump B Conc. 95 5.5 Pumps Pump B Conc. 95 5.6 Pumps Pump B Conc. 5 7 Controller Stop Table 1: Liquid Chromatography Gradient Elution Conditions Create a batch list including standards, QC and urine samples. Start the batch by injection of the prepared samples (12 µL). 5. Peak Identification, Integration and Data Process Control data acquisition and processing using the software. Identify and integrate 3-NT and IS peaks for all the samples. Establish a standard curve with the range of 10-2,500 pg/mL for 3-NT quantitation by linear regression of the peak area ratio of 3-NT and IS versus the nominal 3-NT concentration with a weighting factor of 1/x. Quantify all the samples using the standard curve. Determine if the QC samples fall in the established range. Convert the detected concentrations of urine samples to final results in the unit of nM or nmol/mmol Cr.

Representative 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

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
2mL 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 32mm screw neck vial	Waters	600000751CV
LCGC certified 12 x 32mm 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