simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration...

Research Article

Simultaneous determination of nitriteand nitrate in human plasma by on-capillarypreconcentration with field-amplifiedsample stacking

A simple method for the determination of nitrite and nitrate in human plasma has been

developed using CZE with minimal sample preparation. Field-amplified sample stacking

(FASS) was used to achieve submicromolar detection by dilution of the plasma sample

with deionized water. In CZE, the separation of nitrite and nitrate was achieved within

10 min without adding EOF modifier. The optimal condition was achieved with 50 mM

phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydro-

dynamically for 40 s into a 60 cm� 75 mm id uncoated fused-silica capillary. The separation

voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The

linearity curves for nitrite and nitrate were obtained by the standard addition method. The

estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and

0.07 mM, respectively. The LODs for nitrite and nitrate in original plasma samples were

0.45 and 0.63 mM. The intra- and inter-day precisions for both analytes were o2.6% and

the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable

than nitrate in the plasma after the sample preparation. This proposed method was applied

to a number of human plasma samples and the measured nitrite and nitrate concentra-

tions in human plasma were consistent with the literature ranges.

Keywords:

CZE / Human plasma / Nitrate / Nitrite / Stacking DOI 10.1002/elps.201100285

1 Introduction

The endogenous gas nitric oxide (NO) plays an important

role in physiological and pathological processes in the

human body. It acts as a signal molecule in the cardiovas-

cular system [1]. Increased NO production has also been

related to many other diseases, such as shock and organ

dysfunction under pathological conditions [2, 3]. Therefore,

the amounts of NO have a critical influence on the human

health and disease, which makes it imperative to monitor

the levels of NO in clinical studies. However, direct

measurement of NO is difficult due to its short half-life. It

is known that NO can metabolize to two stable products,

namely nitrite and nitrate, which can be used as an indicator

or marker of NO generation in vivo [4].

Although the determination of nitrite and nitrate in

body fluids represents a challenge, many different analytical

techniques were employed to determine these two

compounds [5, 6]. The Griess reaction is the formerly used

method for the determination of nitrite and nitrate in

human blood. However, this colorimetric method lacks

sensitivity. Chemiluminescence is another more sensitive

technique, but it requires an expensive apparatus, which is

available in highly specialized laboratories only. Most HPLC

methods developed for biological samples require a

complicated sample preparation procedure to remove matrix

components [7]. The GC-MS methods developed for the

purpose require a derivatization reaction before analysis and

also a labeled internal standard [8].

Nowadays, CE has become one of the most attractive

techniques for biological samples due to its low sample

consumption and little sample preparation. Various CZE

methods have been employed for the determination of nitrite

and nitrate in biological fluids by using direct UV detection

at 214 nm [9–18]. In the meantime, many on-capillary

preconcentration techniques such as stacking and transient

isotachophoresis have also been developed to achieve the

satisfactory sensitivity [19–21]. Most of the CZE methods

Xu Wang1

Evi Masschelein2

Peter Hespel2

Erwin Adams1

Ann Van Schepdael1

1Laboratory for PharmaceuticalAnalysis, Faculty ofPharmaceutical Sciences, K.U.Leuven, Leuven, Belgium

2Research Centre for Exerciseand Health, Faculty ofKinesiology and RehabilitationSciences, K.U. Leuven, Leuven,Belgium

Received May 24, 2011Revised July 20, 2011Accepted August 29, 2011

Colour Online: See the article online to view Figs. 1 and 2 in colour.

Abbreviations: FASS, field-amplified sample stacking; NO,

nitric oxide

Correspondence: Professor Ann Van Schepdael, Laboratory forPharmaceutical Analysis, Faculty of Pharmaceutical Sciences,K.U. Leuven, O&N 2, PB-923, Herestraat 49, B-3000 Leuven,BelgiumE-mail: [email protected]: 132-16-323448

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2012, 33, 402–405402

dealt with a complicated BGE and EOF modifier, and few of

them focused on nitrite and nitrate in plasma with submi-

cromolar quantification. In [18], the authors describe a simple

and fast CZE method, but they state that good precision of

nitrite measurement could only be obtained in the plasma

of patients with increased nitrite levels. The reported levels of

basal nitrite in human plasma have ranged from ‘‘non-

detectable’’ to 26 mM and the reported concentrations of

nitrate in plasma ranged from 4 to 81 mM. The variations in

nitrite and nitrate levels reported in the literature could

possibly be explained by the problems arising from variable

diet, sample collection, preparation, contamination due to the

laboratory ubiquity of these ions and from lack of sensitivity

[1, 5]. Both high sensitivity and precautions taken during the

sample preparation procedure can improve the precision and

accuracy of the measurements.

The aim of the study was to develop a simple and

sensitive CZE method for the submicromolar determination

of nitrite and nitrate in human plasma with little sample

preparation for clinical routine analysis.

2 Materials and methods

2.1 Chemicals

All chemicals used were of analytical grade. Sodium nitrate

was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sodium nitrite and disodium hydrogen phosphate were

from Merck (Darmstadt, Germany). Sodium hydroxide was

from Fisher Scientific (Loughborough, UK). Ammonium

thiocyanate used as internal standard (IS) was from

AppliChem (Darmstadt, Germany).

All solutions were prepared in distilled water purified by

a Milli-Q Gradient system (Millipore, Molsheim, France).

The buffer solution was filtered through 0.2 mm regenerated

cellulose filter (Whatman, Dassel, Germany).

2.2 Instrument

All experiments were performed on an HP 3DCE system

(Agilent, Waldbronn, Germany). Separations were carried

out using an uncoated fused-silica capillary (60 cm� 75 mm

id; effective length 51.5 cm) at a voltage of �20 kV. The

temperature of the cassette was kept at 401C. Injection was

performed hydrodynamically for 40 s at 50 mbar. The UV

detection was set at 214 nm. The data were collected and

processed by Agilent ChemStation software (Hewlett-Pack-

ard, Waldbronn, Germany).

New capillaries (Composite Metal Service, Shipley, UK)

were conditioned at 451C by rinsing with 1 M NaOH

(10 min), 0.1 M NaOH (10 min) and water (10 min). At the

start of each day, the capillary was rinsed with 0.1 M NaOH

(10 min), water (10 min) and running buffer (5 min). Prior

to the injection of plasma samples, the capillary was flushed

between runs with 0.1 M NaOH (2 min), distilled water

(2 min) and running buffer (2 min). At the end of each day,

the capillary was rinsed with 1 M NaOH (10 min), 0.1 M

NaOH (10 min) and water (10 min).

The pH value of buffers was measured with a Metrohm

691 pH-meter (Metrohm, Herisau, Switzerland).

2.3 Sample preparation

The venous blood samples collected from 10 healthy male

volunteers (no dietary restriction) were drawn into lithium-

heparin tubes and plasma was separated by centrifugation

(3478� g for 10 min at 41C) within 3 min after sample

collection. Plasma was then isolated and immediately frozen

at �801C until later analysis. After thawing at room

temperature, 150 mL plasma sample was deproteinized by

spiking with 5 mL 1 M NaOH to pH 10, and then centrifuged

at 14 100� g for 10 min in a Minispin Plus microcentrifuge

(Eppendorf). Then, 100 mL supernatant was diluted nine

times with distilled water (100 mL supernatant with 800 mL

water) to achieve the sample stacking effect. The final

concentration of thiocyanate was always 10 mM both in the

calibration and sample solution. The study was approved by

the local Ethics Committee (K.U. Leuven) and the informed

consent was obtained from all subjects.

3 Results and discussion

3.1 Optimization of the CZE method

The selection of a simple BGE was important for routine

analysis. Thus, phosphate buffer was chosen as a separation

buffer. Increased resolution of nitrite and nitrate was

obtained when the buffer concentration increased from 25

to 75 mM with maximum at 50 mM. The effect of running

buffer pH on the resolution was also investigated by using

50 mM phosphate buffer at pH values ranging from 6.8 to

11. The resolution improved dramatically with the increas-

ing pH. However, the baseline became noisy at pH 11. The

effect of applied voltage on the separation was studied in the

range of 6–25 kV, showing that a high voltage not only

brought high separation speed but also improved resolution.

The use of a higher voltage was limited by a high current,

thus 20 kV was selected as a separation voltage. The

optimization of capillary temperature was studied in the

range of 15–401C, indicating that with increasing tempera-

ture, both the separation speed and resolution improved.

Thus, a capillary temperature of 401C was chosen as the

operation temperature. The effect of injection time was

investigated from 5 to 50 s at a pressure of 50 mbar. The

higher the injection time, the more sensitivity was obtained.

However, the nitrite peak was not resolved well from the

adjacent peak with an injection time of 50 s, thus an

injection time of 40 s was chosen for the sake of robustness.

The identity of this adjacent peak is unknown. The length of

capillary was raised from 50 to 70 cm, showing that the

Electrophoresis 2012, 33, 402–405 CE and CEC 403


resolution increased slightly, but the separation time was

long when the length of the capillary increased above 60 cm.

The initial length of the sample plug is about 11% of the

capillary length.

3.2 Injection condition

Biological samples containing high salts and protein content

only allow a small injection volume, making it difficult to

reach a satisfactory sensitivity. On-capillary preconcentration

with FASS can be simply achieved by diluting the sample

with deionized water. A long plug of sample, diluted in water

with low conductivity, can be injected hydrodynamically into

the capillary filled with separation buffer of high conductivity.

After the voltage is applied, the high electric field strength is

distributed over the sample plug due to its high resistivity,

while the rest of the capillary has relatively low electric field

strength. Therefore, the ions move fast in the sample zone

and slow down in the buffer zone. Stacking effect occurs at

the boundary between the sample zone and buffer zone

[22, 23]. The electropherogram of undiluted plasma sample is

shown in Fig. 1A. Direct injection of large volumes of

undiluted plasma sample could not be achieved because of

capillary clogging. Therefore, the direct injection of plasma

sample only allowed a small volume, which resulted in an

unsatisfactory detection limit. However, improved sensitivity

was achieved when the plasma sample was diluted nine times

with deionized water and a large volume was injected

(see Fig. 1B). This method provided a considerable signal

enhancement with acceptable analysis time.

3.3 Validation of the method

3.3.1 Figures of merit

The calibration curves were constructed by standard addition of

nitrite (0.27–11.1 mM) and nitrate (0.28–46.3 mM) to diluted

plasma samples containing 10 mM IS. The linearity regression

equations were y 5 0.1285x10.521 (r2 5 0.9991) for nitrite and

y 5 0.2028x11.1351 (r2 5 0.9999) for nitrate, respectively, where

x represents the concentration of nitrite or nitrate added to the

diluted plasma and y represents the relative peak area (the

corrected peak area of nitrite or nitrate divided by the corrected

peak area of IS peak). The corrected peak area is defined as the

peak area divided by the migration time. The LOD and LOQ

values of both analytes in diluted blank plasma were estimated

based on a signal-to-noise ratio of 3 and 10 respectively. The

LOD of nitrite and nitrate were estimated to be 0.05 and

0.07 mM, respectively. The LOQs for nitrite and nitrate were

estimated to be 0.17 and 0.24 mM, respectively. The sensitivity

obtained by this method was better than previous reported

values [9–16, 18–21]. The intra- and inter-day precisions were

determined on the diluted plasma with the standard solution

spiked samples. The intra-day RSD (n 5 6) of migration time is

1.0 and 1.6% for nitrite and nitrate, respectively. The intra-day

RSD (n 5 6) of relative peak areas is 0.9 and 1.1% for nitrite and

nitrate, respectively. The inter-day RSD (n 5 6) of migration

time is 0.8 and 2.6% for nitrite and nitrate, respectively. The

inter-day RSD (n 5 6) of relative peak areas is 2.0 and 1.9% for

nitrite and nitrate, respectively. The results indicated good intra-

day and inter-day precisions for both analytes.

3.3.2 Recovery

Known standard solutions of nitrite and nitrate were added

to the diluted plasma samples. Average results based on

three data points at each concentration were used. The

recoveries for different concentrations of nitrite and nitrate

were within the range of 92.3–113.3%, indicating good

accuracy of the method (see Table 1).

3.3.3 Stability study

The stability of nitrite and nitrate in plasma sample after

deproteinization was investigated at room temperature

(20–251C) for four days and in the refrigerator at 41C for six

days (see Fig. 2 for refrigerator data). It was found that nitrite

was stable in the deproteinized plasma for 3 days at room

temperature and 6 days at 41C. However, nitrate was only

stable in the deproteinized plasma for 3 days at both conditions.

The stability of nitrite and nitrate found in plasma is similar to

that found in saliva at 41C after deproteinization [16].

3.4 Applicability of the developed method

The proposed method was applied to determine the

concentrations of nitrite and nitrate in human plasma

Figure 1. Typical electropherograms of plasma samples contain-ing 3.9 mM nitrite, 35.9 mM nitrate and 10 mM IS (A) The undilutedplasma sample was injected hydrodynamically for 5 s at50 mbar. (B) The nine times diluted plasma sample was injectedhydrodynamically for 40 s at 50 mbar. Conditions: as discussedin Section 3.1.

Electrophoresis 2012, 33, 402–405404 X. Wang et al.


samples. Plasma samples of 10 healthy young male

volunteers were analyzed for nitrite and nitrate. The mean

basal nitrite concentration measured by this method was

3.6870.42 mM, which is in agreement with the literature

[4, 8, 11, 18, 24–26]. The mean basal nitrate concentration

was 40.0712.4 mM, which also agrees with previously

reported values [2, 9, 11, 18, 25, 27, 28].

4 Concluding remarks

The simultaneous determination of nitrite and nitrate in

human plasma by CZE with FASS has been successfully

performed. The results demonstrate that the proposed

method can be readily used for routine analysis in clinical

laboratories.

The authors acknowledge a Joint Excellence Scholarshipfrom China Scholarship Council and K.U. Leuven.

The authors have declared no conflict of interest.

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Table 1. Recoveries of nitrite and nitrate in human plasma

samples

Concentration (mM)Component

Added Found

Recovery (%)

Nitrite 0.936 0.935 99.8

1.87 1.77 94.6

4.68 4.33 92.3

Nitrate 2.48 2.78 113.3

4.96 4.89 98.6

12.7 13.2 103.6

25.47 25.51 100.2

Figure 2. The stability of nitrite and nitrate in spiked plasmasamples after deproteinization stored at 41C for up to 6 days.

Electrophoresis 2012, 33, 402–405 CE and CEC 405


simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration...

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