научная статья по теме DETERMINATION OF TRACE NITRITES AND NITRATES IN HUMAN URINE AND PLASMA BY FIELD-AMPLIFIED SAMPLE STACKING OPEN-TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY IN A NANO-LATEX COATED CAPILLARY Химия

Текст научной статьи на тему «DETERMINATION OF TRACE NITRITES AND NITRATES IN HUMAN URINE AND PLASMA BY FIELD-AMPLIFIED SAMPLE STACKING OPEN-TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY IN A NANO-LATEX COATED CAPILLARY»

ОРИГИНАЛЬНЫЕ СТАТЬИ ^

УДК 543

DETERMINATION OF TRACE NITRITES AND NITRATES IN HUMAN URINE AND PLASMA BY FIELD-AMPLIFIED SAMPLE STACKING OPEN-TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY IN A NANO-LATEX COATED CAPILLARY

© 2015 Yanhao Zhang*, Liu Yang*, Xiangyu Tian**, Yaxiao Guo*, Wei Tang*, Ajuan Yu***, 2, Wenfen Zhang*, Baoguo Sun****, Shusheng Zhang*, 1

*College of Chemistry and Molecular Engineering, Zhengzhou University Kexue Rd 100, Zhengzhou 450001, P. R. China

1E-mail: zsszz@126.com **Basic Medical College of Zhengzhou University Zhengzhou 450001, P. R. China ***Key Laboratory of Chemical Biology and Organic Chemistry of Henan Province

450052, P. R. China 2E-mail: yuajuan@zzu.edu.cn ****Convance Laboratories Inc.

Wisconsin 53704-2523, USA Received 07.02.2014; in final form 19.12.2014

The associations of nitric oxide (NO) with many diseases are still being discovered, and sometimes, NO levels in the body fluids are too low to be detected. Thus, the determination methods for trace nitrites and nitrates in urine and plasma of healthy volunteers and rheumatoid arthritis (RA) patients are necessary. Using trimethylamine am-inated polychloromethyl styrene nano-latex coated capillary column (ccc-TMAPL) and field-amplified sample stacking (FASS) injection, the sensitive open-tubular capillary electrochromatography (OT-CEC) methods for determining trace nitrites and nitrates in urine and plasma of healthy volunteers and RA patients were set up. The limits of detection for both nitrite and nitrate were 1 ng/mL. The intra- and inter-day relative standard deviations (RSDs) of the methods were less than 1.9%, and recoveries were between 89 and 96%. Plasma nitrate (diluted 10 times) levels were 93—301 ng/mL for healthy volunteers and 222—421 ng/mL for RA patients, respectively. Meanwhile, plasma nitrite levels were 7—18 and 40—82 ng/mL, respectively. The urinary nitrite levels were significantly elevated in RA patients. The proposed sensitive FASS-OT-CEC methods for trace nitrites and nitrates hold the promise of practical applications in a wide array of biological analyses, clinical and pathophysiology studies of the diseases related with NO hyperproduction and metabolites.

Keywords: rheumatoid arthritis, plasma, urine, nitrite, nitrate, field-amplified sample stacking, open-tubular capillary electrochromatography, trimethylamine aminated polychloromethyl styrene nano-latex.

DOI: 10.7868/S0044450215070208

Rheumatoid arthritis is a chronic (long-term) disease. The previous works have provided evidence in RA for increased production of systemic nitric oxide [1]. As known, the endogenous NO has been identified as a mediator of many cellular functions such as vascular tone, signal transmission and phagocytosis [2]. It is synthesized in mammalian cells by a family of three NO synthases and rapidly oxidized to stable nitrite and nitrate in the blood. Clinically, nitrate and nitrite in plasma and urine increase during inflammation. For example, levels of nitrite are elevated significantly up to 36 ^M in patients with HIV infection [3]. Meanwhile, it is noteworthy that nitrite concentrations are of great difference on

(50 nM—26 ^M) for healthy persons resulted from different tested specimens and analytical methods [4]. For comprehensive investigations on relationship between NO (nitrate and nitrite) content and disease (for example, RA), it is necessary to develop a sensitive and practical method for determination of both nitrates and nitrites at ng/mL (sub ^M) levels in complicated body fluids.

To date, there are many different analytical methods [5] employed for the determination of nitrites and nitrates in body fluids, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass

DETERMINATION OF TRACE NITRITES AND NITRATES IN HUMAN URINE AND PLASMA

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spectrometry. As indicated in the works [5, 6], classical colorimetric and spectrophotometric methods lack sensitivity, as well as are laborious or suffer from matrix interferences. To remove the matrix interferences and obtain high detection sensitivity, HPLC with complicated sample pretreatment [7] and gas chromatogra-phy-mass spectrometry (GC-MS) with a derivatiza-tion reaction [8] have been carried out.

As an alterative method, capillary electrophoresis (CE) has been widely utilized in the biological samples [9—11] due to its many advantages (low sample consumption and little sample preparation, high separation performance and low running cost) over traditional GC and HPLC. As reported in the reviews [4, 10, 11] and work [6], various CE methods [12—17] have been developed for the determination of nitrites and nitrates by using direct UV detection at 214 nm. For achieving higher detection sensitivity, some capillary zone electrophoresis (CZE, a CE mode) with on-col-umn preconcentration techniques such as stacking and transient isotachophoresis have also been developed [18, 19]. For CZE mode with bare capillary, the major problems are associated with complicated background electrolyte (BGE), addition of electroosomtic flow (EOF) reverse additives, and unsteady EOF values (with pH changing). These problems may result in the relatively unsatisfactory repeatability and accuracy of CZE method for the determination of nitrates and nitrites.

To obtain relatively steady and reverse EOF values, the inner wall of the capillary column can be modified with the functionalized materials such as polyethyle-neimine [20] and quaternary ammonium nanopartic-ulates [21, 22], which form the new open-tubular capillary electrochromatogarphy mode for the determination of anions such as nitrites and nitrates.

In our previous work [23], we prepared a new trim-ethylamine aminated polychloromethyl styrene nano-latex and TMAPL-coated capillary column (Fig. 1), which has been successfully applied for trace bromate determination by FASS—OT-CEC method. Built on this success, we have in the present work crafted two major aims as follows: i) to validate the practicability of the ccc-TMAPL column; and ii) to develop a sensitive FASS-OT-CEC method for the determination of nitrites and nitrates in plasma and urine for healthy persons and RA patients. This method possesses practical applications in a wide array of biological, clinical and pathophysiology analyses.

EXPERIMENTAL

Reagents and apparatus. Unless specified otherwise, all chemicals used were of analytical grade and obtained from Beijing Chemical Factory (Beijing, China). Thiourea was used as neutral marker to measure the EOF. Water was purified using a Milli-Q water purification system (Millipore Corporation, Bedford, MA, USA). All of solutions were degassed prior to use and fil-

Si-O- + Nano latex

Fig. 1. Schematic representation of the coating of the ccc-TMAPL column.

tered through a 0.22 ^m membrane filter. Stock solutions (1 mg/mL) of sodium nitrate and sodium nitrite were prepared in deionised water. These solutions were preserved at 4°C in the darkness during the experiments. Working standard solutions were prepared by diluting the stock standard solutions with ultra-pure water. Each determination was performed in triplicate.

All experiments were performed on an Agilent CE system with a diode-array detector (Agilent Technologies, Waldbronn, Germany). The apparent pH was measured using a pH meter (Shanghai Weiye Factory, Shanghai, China). The dimension of the bare fused-sil-ica capillaries (Yongnian Optical Fiber Corporation, Hebei Province, China) was of inner diameter 50 ^m and length of 50 cm (41.5 cm to the detection window). The ccc-TMAPL (Fig. 1) is of 50 ^m i.d. and 50 cm length (41.5 cm to the detection window) coated with flushing TMAPL nano-latex through the bare fused silica capillary. The preparation procedures ofccc-TMAPL column were same as mentioned in our previous work [23] by introducing TMAPL nanoparticles into a treated bare fused silica capillary for three times, the nanoparticles were coated onto the inner wall surface of the capillary by dipole-dipole force, hydrogen bonding and electrostatic interactions. The ccc-TMAPL columns were characterized by infrared spectrum and EOF values. The absorption at 1400 and 1637 cm-1 was observed and associated to the C-N and benzene ring stretching vibration, respectively. Its EOF values are reversed and steady at ca. —16.8 x 10-5 cm2/(V s) with pH 7.30-8.03. These results indicate that TMAPL has been successfully coated onto the inner wall of the capillary.

Electrophoretic procedures. Before the electrophoret-ic procedure, the new ccc-TMAPL was flushed with water for 10 min. After each separation run, the ccc-TMAPL was flushed with water for 3 min and running buffer solution for another 3 min to re-equilibrate the ccc-TMAPL. All separation runs were undertaken at

10 mAU

(a)

(b)

(c)

(d) (e)

1 2

. .. ______________ .... ...1..

2

2

______ к

w - ■

0 12 3 4

Migration time, min

Fig. 2. Electrochromatogram of nitrite and nitrate standards and body fluids (all diluted 2 times). Conditions: column 50 ^m x 50 cm (effective length 41.5 cm to the detection window) ccc-TMAPL; BGE 20 mM Tris + 13.5 mM HQO4 (pH 7.87); separation voltage —20 kV; sample injection 50 mbar x 3 s; detection wavelength 214 nm; temperature 25°C. Chromatograms: (a) — nitrate (30 ^g/mL) and nitrite (30 ^g/mL) standards; (b) — patient urine 1; (c) — healthy urine 3; (d) — patient plasma 1; (e) — healthy plasma 3; 1 — nitrite, 2 — nitrate.

25°C. The separation voltage was —20 kV and injection pressure for water-plug was 50 mbar. Under the detection at 235 nm, the EOF (thiourea as marker) was determined when the buffer solution was 20 mM trisaminomathane (iris) and 12 mM HClO4 with pH 8.03.

Plasma and urine sample pretreatment. The blood samples collected (at 6.30 a.m.) from 10 healthy male volunteers and 10 male patients with RA (no dietary restriction) were drawn into centrifuge tubes. Within 5 min after samp

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