4.1 Background - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/94141/10/10_chapter 4.pdf · Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite Ph. D Thesis:

Chapter 4: Liquid Chromatographic Determination of Nitrate and Nitrite

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 78

4.1 Background

Recently developed fluorometric HPLC method involves the precolumn

derivatization of nitrite with 2, 3-diaminonapthalene and enzymatic conversion of nitrite

to nitrate [1]. Measurement of nitric oxide itself is complicated by its short half life and

would, therefore, require on-line measurement which is suitable for routine use for

analysis of biological samples. After an exhaustive investigation, it appears that HPLC,

which is a very sensitive, rapid and accurate method with low detection limits for nitrite

and nitrate, carries none of these disadvantages and nitrite and nitrate can be measured

directly. The chromatographic system developed in this study, after investigating

alternatives, readily resolved NO2- and NO3

-, with peaks being separated within a minute

[2]. There are some analytical methods that has been reported for simultaneous

estimation of nitrite and nitrate cited in references [3-15].

Lulla (1984) proposed a stability-indicating analytical method for the

simultaneous determination of nitrate, and if present, its reductive degradation product,

nitrite, in toothpastes. Nitrate and nitrite were extracted from the toothpaste using

distilled water and separated from other water-soluble excipients by two RP-8 columns

(250 mm L x 4 mm i.d.) using a mobile phase containing 0.2 % (w/v) sodium acetate and

2.5 % (v/v) glacial acetic acid in distilled water. A UV detector set at 313 nm was used

for quantitation. The method was applied to commercial toothpastes containing 5 %

potassium nitrate and yielded an average recovery of 100.1 % with a relative standard

deviation of 1.43 %. Average recovery of nitrate and nitrite from spiked samples was

100.6 % and 96.4 %, respectively. The minimum detectable concentration for nitrite was

50 micrograms/g of toothpaste [3]. Marcelo et al. (1996) developed a high-performance

liquid chromatographic method for the determination of nitrite and nitrate anions derived

from nitric oxide in biological fluids. After separation on a strong anion-exchange

column (Spherisorb SAX, 250 x 4.6 mm I.D., 5 µm), two on-line post-column reactions

occur. The first involves nitrate reduction to nitrite on a copper-plated cadmium-filled

column. In the second, the diazotization-coupling reaction between nitrite and the Griess

reagent (0.05 % naphtylethylendiamine dihydrochloride plus 0.5 % sulphanilamide in 5

% phosphoric acid) takes place, and the absorbance of the chromophore is read at 540

nm. Before injection into the chromatographic system, the samples were diluted and



submitted to suitable clean-up procedures (urine and cell culture supernatant samples are

passed through C-s cartridges and serum samples were subjected to deproteinization by

ultrafiltration through membranes with a molecular mass cut-off of 3000). The method

has a sensitivity of 30 p mol for both anions, as little as 0.05-0.1 ml sample volume is

required and linearity is observed up to 60 n mol for each anion tested [4]. Rizzo et al.

(1998) proposed a specific and simple method for the direct simultaneous detection of

extracellular nitrite (NO2-) and nitrate (NO3-) using high-performance liquid

chromatography separation with UV and electrochemical detection in series. These stable

end products of nitric oxide (NO) were determined in dialysis perfusate obtained through

in vivo brain microdialysis during and after experimental photo induced cerebral

ischemia in rats. The chromatographic conditions were optimized with a reversed-phase

column (250 x 46 mm) using 10 mM n-octylamine pH 6.0 as a mobile phase. Absorbance

was measured at 220 nm for NO3- detection; electrochemical detection was performed at

+0.7 V for NO2- evaluation. This assay system holds the advantages of in vivo

consecutive measurements, high precision, good reproducibility, technical simplicity, fast

response (about 7 min) and wide availability [5]. A simple and rapid fluorometric HPLC

method was developed by Hui Lia (2000) for determination of nitrite through its

derivatization with 2, 3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell

culture medium, or biological samples, readily reacted with DAN under acidic conditions

to yield the highly fluorescent 2, 3-naphthotriazole (NAT). For analysis of nitrate, it was

converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with

DAN to form NAT. NAT was separated on a 5-µm reversed-phase C8 column (150 x 4.6

mm I.D.) guarded by a 40 µm reversed-phase C18 column (50 x 4.6 mm I.D.), and eluted

with 15 mM sodium phosphate buffer (pH 7.5) containing 50 % methanol (flow-rate, 1.3

ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm.

Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear

with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture

media, plasma and urine. The detection limit for nitrite and nitrate was 10 p mol/ ml [6].

Erk (2001) reported ratio spectra derivative spectrophotometry and high-performance

liquid chromatography (HPLC) method for determination of metronidazole and

miconazole nitrate in ovules. The first method depends on ratio spectra first derivative



spectrophotometry by utilizing the linear relationship between substances concentration

and first derivative peak amplitude. The ratio first derivative amplitudes at 242.6

(1DD242.6), 274.2 (1DD274.2), 261.8 (1DD261.8), 273.5 (1DD273.5) and 281.5 (1DD281.5) nm

were selected for the assay of metronidazole and miconazole nitrate, respectively. The

second method is based on high-performance liquid chromatography on a reversed-phase

column using a mobile phase of methanol-water-phosphoric acid (30:70:0.20 v:v) (pH

2.8) with programmable detection at 220.0 nm. The minimum concentration detectable

by HPLC was 0.9 mg/ml for metronidazole and 0.3 mg/ml for miconazole nitrate and by

ratio derivative spectrophotometry 4.0 mg/ml for metronidazole and 0.5 mg/ml for

miconazole nitrate [7]. Krzek (2003) developed a HPLC method for determination of

propane-1,2,3-triyl trinitrate and impurities: (2RS)-3-hydroxypropane-1,2-diyl dinitrate

and 2-hydroxypropane-1,3-diyl dinitrate in ointment. For individual constituents R.S.D.

was ranged from 0.7 to 9.9 %, while recovery was 100.1 % for propane-1,2,3-triyl

trinitrate and 95.1-/99.0 % for impurities. It has been found that propane-1,2,3-triyl

trinitrate used in medicine in the form of ointment contains such impurities which can be

identified and quantified at relatively low concentrations of 70 ng/ml [8]. Shin-Shou

(2003) developed a simple, rapid, precise and sensitive high performance liquid

chromatography (HPLC) method using an UV detector was developed for the

determination of nitrate and nitrite amounts in vegetables. The optimal conditions were

found and applied using 0.01 M octylammonium orthophosphate of aqueous 30 % (v/v)

methanol of pH 7.0 for the mobile phase at flow rate of 0.8 ml/min. The total time for

one sample analysis was within 10 min. Recoveries of nitrate and nitrite was between

96.6 to 105.7 %. The calibration curves of nitrate and nitrite were extremely linear, where

both correlation coefficients were greater than 0.9990 in the range of 0.1~100.0 µg/ml.

For application, nitrate and nitrite amounts in 12 marketed vegetables were determined

by this HPLC method. The results showed nitrate and nitrite contents varied in a range of

225-4, 410 mg/kg and 5-200 mg/kg, respectively [9]. Yuegang (2006) proposed a

simple, fast, sensitive and accurate reversed-phase ion-pair HPLC method for

simultaneous determination of nitrite and nitrate in atmospheric liquids and lake waters

has been developed. Separations were accomplished in less than 10 min using a reversed-

phase C18 column (150 mm x 2.00 mm I.D., 5 µm particle size) with a mobile phase



containing 83 %, 3.0 mM ion-interaction reagent tetrabutylammonium hydroxide (TBA-

OH) and 2.0 mM sodium phosphate buffer at pH 3.9 and 17 % acetonitrile (flow rate, 0.4

ml/min). UV light absorption responses at 205 nm were linear over a wide concentration

range from 100 g/ml to the detection limits of 10 µg/l for nitrite and 5 µg/l nitrate.

Quantitation was carried out by the peak area method. The relative standard deviation for

the analysis of nitrite and nitrate was less than 3.0 %. This method was applied for the

simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water

samples collected in southeast Massachusetts. Nitrate was found being present at 4.79-

5.99 µg/ml in dew, 1.20-2.63 µg/ml in rain, 0.32-0.60 µg/ml in snow and 0.12-0.23 µg/ml

in lake water [10].

Angel (2008) has been developed and validated a reversed-phase high

performance liquid chromatographic (RP-HPLC) method for determination of econazole

nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-

chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1,H-imidazole-1-ethanol) in cream

formulations. Separation was achieved on a column Bondclone C18 (300 mm x 3.9 mm

I.D., 10 µm particle size) using a gradient method with mobile phase composed of

methanol and water. The flow rate was 1.4 ml min−1, temperature of the column was 25

C and the detection was made at 220 nm. Miconazole nitrate was used as an internal

standard. The total run time was less than 15 min. The analytical curves presented

coefficient of correlation upper to 0.99 and detection and quantitation limits were

calculated for all molecules. Excellent accuracy and precision were obtained for

econazole nitrate. Recoveries varied from 97.9 to 102.3 % and intra and inter-day

precisions, calculated as relative standard deviation (R.S.D.), were lower than 2.2 %.

Specificity, robustness and assay for econazole nitrate were also determined [11]. A rapid

and cost-effective RP-HPLC method with diode array detector was optimized and

validated by Ferreira (2008) for quantification of nitrites and nitrates in ham. The

chromatographic separation was achieved using a HyPurity C18, 5 µm chromatographic

column and gradient elution with 0.01 M n-octylamine and 5 mM tetrabutylammonium

hydrogenosulphate to pH 6.5. The determinations were performed in the linear range of

0.0125-10.0 mg/l for nitrite and 0.0300-12.5 mg/l for nitrate. The detection limits for

nitrite and nitrate were 0.019 and 0.050 mg/kg, respectively. The reliability of the method



in terms of precision and accuracy was evaluated. Coefficients of variation lower than

2.89 and 5.47 % were obtained for nitrite and nitrate, respectively. Recoveries of residual

nitrite and nitrate ranged between 93.6 and 104.3 %, respectively. Analysis of cooked and

dried ham samples was performed, and the results obtained were in agreement with

reference procedures [12]. Kodamatani (2009) developed a simple, sensitive and selective

method for the simultaneous determination of nitrite and nitrate in water samples. The

method was based on ion-exchange separation, online photochemical reaction, and

luminol chemiluminescence detection. The separation of nitrite and nitrate was achieved

using an anion-exchange column with a 20 mM borate buffer (pH 10.0). After the

separation, these ions were converted to peroxynitrite by online UV irradiation using a

low-pressure mercury lamp and then mixed with a luminol solution prepared with

carbonate buffer (pH 10.0). The calibration graphs of the nitrite and nitrate were linear in

the range from 2.0x10-9 to 2.5x 10-6 and 2.0x10-8 to 2.5x10-5 M, respectively. Since the

sensitivity of nitrite was about 10 times higher than that of nitrate, the simultaneous

determination of nitrite and nitrate in the water samples could be efficiently achieved.

This method was successfully applied to various water samples, river water, pond water,

rain water, commercial mineral water, and tap water with only filtration and dilution steps

[13]. Yongtao Li et al. (2011) developed a new method for the analysis of nitrate and

nitrite in a variety of water matrices by using reversed-phase liquid

chromatography/electrospray ionization/mass spectrometry in the negative ion mode. For

this direct analysis method, nitrate and nitrite anions were well separated under the

optimized LC conditions, detected by monitoring m/z 62 and m/z 46 ions, and quantitated

by using an isotope dilution technique that utilized the isotopically labeled analogs. The

method detection limits, based on seven reagent water replicates fortified at 0.01 mg/L

nitrate and 0.1 mg/L nitrite, were 0.001 mg/L for nitrate and 0.012-0.014 mg/L for nitrite.

For the analysis of nitrate and nitrite in drinking water, surface water, and groundwater

samples, the obtained results were generally consistent with those obtained from the

reference methods. The mean recoveries from the replicate matrix spikes were 92-123 %

for nitrate with an RSD of 0.6-7.7 % and 105-113 % for nitrite with an RSD of 0.3-1.8 %

[14]. Anguo Wu (2013) developed a simple, cost-effective and accurate HPLC method

for the determination of nitrite and nitrate. On the basis of the reaction that nitrite is



oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the

determination of nitrite and nitrate was achieved by the following strategy: each sample

was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and

the second injection was to measure total nitrate including initial nitrate and the nitrate

from the conversion of nitrite with the addition of acid potassium permanganate in the

sample. The amount of nitrite can be calculated as difference between injections 2 and 1.

The HPLC separation was performed on a reversed phase C18 column for 15 min. The

mobile phase consisted of methanol-water (2:98 by volume); the water in the mobile

phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen

phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength

was set at 210 nm. Additionally, systemic investigation of the effects of the concentration

of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the

amount of acidic potassium permanganate added to the sample on the separation efficacy

was carried out. The results showed that the limits of detection (LOD) and the limit of

Quantitation (LOQ) were 0.075 and 0.25 µM for nitrate (containing the oxidized nitrite),

respectively. The linear range was 1-800 µM [15].

In this work, a new reversed-phase HPLC method has been developed

for the analysis of nitrite and nitrate in various samples. The method uses RP-HPLC

based on isocratic mode analogs to nitrite and nitrate. The nitrate and nitrite anions were

well separated by the selected HPLC column under a moderate pH condition.

Derivatization was not required for this method. The study was focused on the

selection and optimization of reversed-phase HPLC conditions, the demonstration of

method performance (sensitivity, accuracy and precision) and the investigation of

interferences from common inorganic anions.



4.2 Material and Methods

4.2.1 Equipments

Table 4.1: Instrumentation used in RP-HPLC Method Development

Sr. No. Name of Instrument Make Model

1 HPLC System Shimadzu, Japan Software–LC Solution Pump-LC 2010 CHT Detector- UV

2 Analytical Balance Shimadzu, Japan AUY 120

3 Digital pH Meter Electronic Corporation Ltd., India

pH 5651

4 Ultrasonicator Servewell Instrument, India RC System MU-1700

5 Degasser Tarsons, India Rockyvac-300

6 Milli-Q water purification system

Bedford, MA, USA

Millipore

4.2.2 Reagents and Materials

A. Standards 1. Potassium nitrate (Merck, India)

2. Sodium nitrite (Merck, India)

3. Sodium chloride, sodium sulfate, potassium phosphate (monobasic), and

sodium carbonate (Fisher Scientific, India)

4. Milli-Q water with 18.2 MΩ-cm resistances (Millipore water system)

B. Real Samples

1. Water samples- Tap, Ground and Surface water from local wells and river

2. Vegetable samples- Spinach, Coriander leaves, Beet, Radish leaves, Sorrel,

Cabbage, Tomato and Mint from local market

3. Soil sample- From local farm

4. Pharmaceutical preparation- Isosobidinitrate tablets from Nichoals Piramal

C. Chemicals and Reagents

1. Methanol (HPLC grade), Merck (India)



2. Potassium dihydrogen Phosphate AR Grade, Qualigens (India)

3. Orthophosphoric acid AR Grade, Qualigens (India)

4.3 Results and Discussion

4.3.1 Selection of Common Solvent (Diluent)

HPLC grade water was selected as common solvent for preparation of stock

solution and developing spectral characteristics of nitrate and nitrite, further dilutions from

stock solutions were made in mobile phase of aqueous HPLC grade methanol.

4.3.2 Determination of λmax of Nitrate and Nitrite

Preparation of Standard Stock solution: Weighed accurately 0.01500 gm sodium

nitrite and 0.016 gm potassium nitrate working standard in a 10.0 ml volumetric flask and

dissolved by sonication in sufficient mobile phase then make up the volume by mobile

phase. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase i.e. methanol.

The aliquot portion of standard stock solutions of nitrate and nitrite were diluted

appropriately with aqueous methanol to obtain concentration 10 ppm of each analyte. The

solutions were scanned in the range of 200-400 nm. The absorbance spectrum of nitrate

and nitrite is shown in Figure 4.1 & Figure 4.2, respectively whereas the overlain

spectra of both the ions in mixture are shown in Figure 4.3.

It is observed as the wavelengths of maximum absorption, λmax, for nitrate and

nitrite were 230 and 233 nm, respectively. From the overlain spectra, the wavelength

selected for simultaneous estimation of nitrate and nitrite was 222 nm as an isoabsorptive

point for nitrate and nitrite ions.

Figure 4.1; Absorbance Spectrum of Nitrate on UV Detector



Figure 4.2: Absorbance Spectrum of Nitrite on UV Detector

Figure 4.3: Overlain Spectra of Nitrate and Nitrite

4.3.3 Optimization of Chromatographic Conditions

The mobile phase was allowed to equilibrate with stationary phase until steady

baseline was obtained. The standard solution containing mixture of nitrate and nitrite was

run and different individual solvents as well as combinations of solvents have been tried to

get a good separation and stable peak. Each time mobile phase was filtered through 0.45

µm filter membrane. Based on sample solubility and stability, various mobile phase

compositions were evaluated to achieve acceptable separation using selected

chromatographic conditions.



The various conditions of mobile phases, flow rate and pH buffer tried are

represented in Table 4.2 and chromatograms obtained in Trial 1, 2 & 3 are shown in

Figure 4.4, 4.5 & 4.6, respectively..

Table 4.2: Trials for Variable Chromatographic Parameters

Chromatographic

Parameters

Trial 1 Trial 2 Trial 3

Column C13 C13 C13

Wavelength 222 nm 222 nm 222 nm

Flow rate 0.4 ml/min 0.4 ml/min 0.7 ml/min

Injection volume 20.0 µl 20.0 µl 20.0 µl

Column oven Temperature

25 0C 25 0C 25 0C

Run Time less than 10 minutes less than 10 minutes

less than 10 minutes

Mobile Phase Methanol:Water

( 30:70)

Methanol:Water

( 20:80)

Methanol:Buffer

( 20:80)

pH buffer

not used not used Potassium dihydrogen phosphate ( pH=3.0)

Figure 4.4: Chromatogram of Nitrate and Nitrite for Trial 1




.


Chromatogram of Trial 1 shows very poor resolution, Trial 2 also shows less

resolution between nitrate and nitrite; hence those method parameters were not suitable.

The chromatographic conditions of Trial 3 were established by trial and error and were

kept constant throughout the method because proper peak shape, resolution and system

suitability was observed within limits.

4.3.4 Optimization of the Mobile phase, pH and Flow Rate

The various aqueous methanol concentrations (10, 20, 25 and 30 %, v/v) and

different pH values (2.0, 2.5, 3.0 and 3.5) of mobile phase solutions at various flow rates

(0.4, 0.7 and 1.0 ml/min) were tested on running HPLC chromatograms. As shown in

Figure 4.7 the 20 % methanol gives the proper resolution. On 10 % methanol the

resolution is achieved but the retention time (RT) is long and 25 and 30 % methanol

concentrations in mobile phase gave less resolution. Figure 4.8 show that 3.7 is the



optimum pH for the best resolution in RT. This pH is intermediate between the pKa

values of both analytes. In the case of flow rate, Figure 4.9 shows that the 0.7 ml/min is

the preferred flow rate. On 0.4 ml/min flow rate the RT is too long and at 1.0 ml/min

flow rate, the resolution is not achieved. So the 20 % methanol at pH 3.0 and 0.7 ml/min

flow rate is considered as set of optimized chromatographic conditions. Figure 4.10

represents the resolved peak of nitrite and nitrate at optimized chromatographic

conditions.

Figure 4.7: Effect of Variation of Methanol Concentration in Mobile Phase

Figure 4.8: Effect of Variations of pH of Mobile Phase

0

2

4

6

8

10

0 10 20 30 40

Ret

enti

on

Tim

e

Methanol Concentration in %

NitriteNitrate

012345678

1.5 2 2.5 3 3.5 4

Ret

enti

on

Tim

e

pH of mobile phase

NitriteNitrate



Figure 4.9: Effect of Variation of Flow Rate of Mobile Phase

Figure 4.10: Separation of Nitrite and Nitrate in selected mobile phase showing retention time for Nitrite (1ppm) and Nitrate (10ppm)

4.3.5 Preparation of Calibration Curve

i) Preparation of Nitrite standard stock solution (1000 ppm) : An accurately weighed

quantity of sodium nitrite 0.015 gm was transferred to the 10.0 ml volumetric flask

and dissolved in 20 % methanol by sonication. The volume was made up to the mark

with 20 % methanol.

ii) Preparation of Nitrate standard stock solution (1000 ppm) : An accurately weighed

quantity of potassium nitrate 0.016 gm was transferred to the 10.0 ml volumetric

flask and dissolved in 20 % methanol by sonication. The volume was made up to the

mark with 20 % methanol.

0

2

4

6

8

10

0 0.2 0.4 0.6 0.8 1 1.2

Ret

enti

on

Tim

e

Flow rate of mobile phase (ml/min)

Nitrite

Nitrate



The aliquot portions of standard stock solutions of nitrite and nitrate were diluted

appropriately with 20 % methanol to obtain the series of concentrations from 1 to

100 ppm of nitrate and 0.1 to 10 ppm of nitrite.

ii) Procedure: The mobile phase was allowed to equilibrate with the stationary phase

until steady baseline was obtained. The series of concentrations from 1-100 ppm of

nitrate and 0.1-10 ppm of nitrite solutions were injected and peak area was recorded.

The standard calibration curve for nitrite and nitrate are represented in Figure 4.11

& 4.12, respectively.

Figure 4.11: Calibration Curve for Nitrite on HPLC Instrument

Figure 4.12: Calibration Curve for Nitrate on HPLC Instrument.

y = 1703.x + 69.43R² = 0.999

02000400060008000

1000012000140001600018000

0 2 4 6 8 10 12

Pea

k A

rea

NO2- concentration, ppm

y = 376.6x - 71.17R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

0 50 100 150

Pea

k A

rea

NO3- concentration, ppm



4.3.6 System Suitability Test

System suitability is used to verify, whether the resolution and reproducibility of

the chromatographic system are adequate for analysis to be carried out. It is performed to

ensure that the system is operating properly and ready to deliver results with acceptable

accuracy and precision. The tests were performed by collecting data from five replicate

injections of standard solutions. The Filtered mobile phase was allowed to equilibrate with

the stationary phase until steady baseline was obtained. A 20 µl standard nitrate and nitrite

solutions were injected which were made in five replicates and the system suitability

parameters were recorded as shown in Table 4.3 & 4.4 for nitrate & nitrite, respectively.

System suitability parameters criteria:

a. % R.S.D. of the area of analyte peakf in standard chromatogram should not be more

than 2 %.

b. Theoretical plates of analyte peaks in standard chromatogram should not be less than

2000.

c. Tailing factor (Asymmetry) of analyte peaks in standard chromatogram should be less

than 2.0.

d. Retention time (Rt) of analyte peaks in standard chromatogram should not be more than

1 %.

Table 4.3: System Suitability Test Results for Nitrate

Sr. No. Area

Reproducibility

Retention

Time

Tailing Factor (Asymmetry)

Theoretical

plates

1 3693.463 6.645 2.366 94054.844

2 3691.292 6.614 2.452 94136.76

3 3693.721 6.628 2.379 94095.27

4 3694.284 6.638 2.408 94183.18

5 3692.583 6.647 2.386 94068.25

Mean 3693.0686 6.6344 2.3982 94107.6608

SD 1.167 0.014 0.034 52.551

% RSD 0.0316 0.2052 1.4055 0.0558

Limit NMT 2.0 % NMT NMT 2 % NLT 2000



Table 4.4: System Suitability Test Results for Nitrite

Sr. No. Area

Reproducibility

Retention

Time

Tailing Factor (Asymmetry)

Theoretical

plates

1 1776.86 5.189 1.36 129055

2 1775.79 5.213 1.372 129067

3 1778.63 5.227 1.415 129112

4 1784.91 5.311 1.365 129236

5 1778.41 5.262 1.378 129125.4

Mean 1778.92 5.2404 1.378 73.785

SD 3.545 0.047 0.022 0.0571

% RSD 0.1993 0.9063 1.5808 0.0571

Limit NMT 2.0 % NMT 1% NMT 2 % NLT 2000

4.3.7 Application of Proposed Method on Real Samples

4.3.7.1 Preparation of Mobile Phase and Standard Solutions

The optimal conditions of the mobile phase (20 % aqueous methanol, pH 3.0 and

flow rate 0.7 ml/min) were used in the experiment. Other chromatographic conditions

were those used in trial chromatogram run. Standard solution diluted to a series of

concentrations containing 0.1, 5, 10, 50, 100 ppm of nitrate and nitrite were prepared and

stored at 4 oC for use. The solutions were freshly prepared every seven days. The

standard curve and calculated correlation coefficients represents linearity within the

tested range of concentrations.

4.3.7.2 Sample Preparation for Analysis

All vegetable samples including coriander leaves, radish leaves, spinach leaves

and cabbage were bought from the local morning market. The vegetable samples were

carefully rinsed with tap water and deionized water and were divided into smaller pieces.

They were dried at 105 °C for 24 h to constant weight. The dried samples were

pulverized and sieved to 60 mesh. The resultant powders were stored in desiccators until

analysis. Dry and powdered samples of vegetables weighing to 0.1 g were added to a

beaker containing approximately 15 ml water. The sample solutions were stirred and

heated in boiling water bath for 8 h and transferred to 25 ml volumetric flasks. The



sample solutions were shaken for 30 min in supersonic wave, and diluted to a final

volume of 25 ml with deionized water. All samples were filtered through membrane filter

before use.

4.3.7.3 HPLC Analysis

The mobile phase is filtered through 0.45 µm membrane filter and sonicated for

degassing in an ultrasonic bath and then allowed to pass through the HPLC column until a

stable baseline signal was observed. The flow rate was 0.7 ml/min and the detecting UV

wavelength was 222 nm. Equal volumes (20.0 µL) of standard and sample solutions were

injected separately after equilibrium of mobile phase with stationary phase. The

chromatograms were recorded at optimized conditions and the response i.e. peak area of

major peaks was measured. The content of nitrate and nitrite were calculated by

comparing a sample peak with that of standard.

The injections of the standard solutions gave reproducible retention times and

peak areas with relative standard deviation (RSD) below 2.0. The peaks of the sample

were identified by comparison to the respective peaks of the standards. The amounts of

nitrate and nitrite in the test solution were calculated from the peak areas by using linear

regression equations of nitrate and nitrite standard curves. If the curve of the peak areas

was larger than that of the maximum amount from the standard curve, the test solution

was diluted to suitable concentrations.

4.3.7.4 Determination of Nitrate and Nitrite Contents in Vegetables and Water

The results for nitrate and nitrite analysis of the eight selected vegetables from

local market showed that the nitrite and nitrate contents varied significantly in the range

of 0-9.8 ppm and 57-1540 ppm, respectively (Table 4.5). The wide ranges and large

standard variations in nitrate and nitrite levels for the same vegetables purchased from

different places and periods due to the fact that nitrate levels in vegetable plants are

highly sensitive to inherent and environmental variables such as species, maturity,

fertilizer application and storage temperature. The nitrate amounts in vegetables have

reached to hazardous levels.



Table 4.5: Nitrite and Nitrate Content of Vegetables and Water Samples (n= 3)

Sample Nitrite content

ppm±SD Nitrate content

ppm±SD Coriander leaves 5.22±0.08 114.4±0.51

Radish leaves 7.11±0.07 220.7±0.58

Beet 9.83±0.15 1200.7±1.53

Spinach 9.40±0.10 1539.7±0.58

Sorrel 2.13±0.06 135.3±1.53

Cabbage 3.77±0.42 59.6±1.44

Tomato 3.90±0.10 57.8±0.76

Mint 6.0±0.10 164±1.0

Tap Water 0.21±0.01 2.97±0.15

Ground water 0.4±0.02 5.0±0.17

4.4 Validation of Developed RP-HPLC Method Parameters

4.4.1 Accuracy

The recoveries of nitrite and nitrate in the study are shown in Table 4.6 and 4.7.

The recoveries of nitrite for five concentrations (0.1, 0.5, 1, 5 and 10 ppm) into vegetable

samples were in the range of 96.4 to 100.3 % and nitrate for five concentrations (1, 5, 10,

50 and 100 ppm) into vegetable samples were in the range of 98.3 to 100.3 %. The

average of recovery is determined and recovery precision values were characterized by

the relative standard deviation (RSD %). The average recoveries of nitrite and nitrate

were 98.73% and 99.03 %, indicating that the method is quite accurate.

Table 4.6: Recovery Results of Nitrite Analysis for HPLC Method (n=3)

Spiked level

ppm % Average

Recovery SD

% RSD

0.1 96.47 0.9 0.8 0.5 99.43 1.0 1.0

1 97.11 0.7 0.7

5 100.26 0.4 0.4

10 100.37 0.9 0.9

Average 98.73 0.8



Table 4.7: Recovery Results of Nitrate Analysis for HPLC Method (n=3)

Spiked level

ppm % Average

Recovery

SD

% RSD

1 98.30 1.2 1.2

5 99.11 1.1 1.1

10 100.30 0.6 0.6

50 98.90 0.8 0.8

100 98.54 0.9 0.9

Average 99.03 0.9

4.4.2 Precision

Reproducibility tests, Intra-day (running 3 times on the same day), and inter-day

tests (running 3 times within successive 7 days with at least 24 h as intervals) were

conducted. The reproducibility precision values were characterized by the relative

standard deviation (RSD %). The intra-day and inter-day precision showed that the

results were within acceptable limit i.e. % R.S.D. below 2.0 indicating that the method is

reproducible. Intra-day and Inter-day Precision are represented in Table 4.8 & 4.9.

Table 4.8: Intra-day and Inter-day Precision Data of Nitrite for HPLC Method

Conc. of Nitrite ppm

% RSD

Intra-day Inter-day

0.1 1.2 1.5

0.5 0.9 1.2

1 0.6 0.2

5 0.4 0.4

10 1.3 1.2



Table 4.9: Intra-day and Inter-day Precision Data of Nitrate for HPLC Method

Conc. of Nitrate ppm

% RSD

Intra-day Inter-day 1 1.1 1.4

5 0.3 0.8

10 0.8 0.9

50 0.2 0.3

100 0.1 0.2

4.4.3 Specificity

In order to remove potentially interfering substances, the sample can be further

cleaned on an SPE anion exchange cartridge. The filtrate can then be used directly for

HPLC analysis. Fluoride, chloride, sulphate and sulfite are optically transparent, do not

interfere the UV absorbance measurement and thus make the simultaneous determination

of nitrite and nitrate possible [ 35]. Thus the same chromatogram for real sample analysis

was obtained as shown in Figure 4.10.

4.4.4 Limit of Detection(LOD) and Limit of Quantitation(LOQ)

Nitrite and nitrate sample solution was subjected to Limit of Detection(LOD) and

Limit of Quantitation(LOQ) studies,results are given in Table 4.10.

Table 4.10: LOD and LOQ of Nitrite and Nitrate for HPLC Method

Sample LOD (ppm) LOQ (ppm)

Nitrite 0.278 0.844

Nitrate 2.592 7.853

4.4.5 Linearity and Range

To show linerity and range of sample solution, the working range of analyte was

set between 0.1 to 10 ppm for nitrite and 1 to 100 ppm for nitrate. Figure 4.11 & 4.12

provided the standard curves of nitrite and nitrate. Linearity was obtained over the tested

concentration range of 0.1, 0.5, 1, 5 and 10 and 1, 5, 10, 50 and 100 ppm of nitrite and

nitrate, respectively. The linear regression equations of nitrite and nitrate standard curves

were calculated as y = 1703x + 69.43 (R2 = 0.9990) and y = 376.6x + 71.17 (R2 = 0.9990)



respectively. y is the value of peak area and x is the value of various concentrations of

standard solutions The correlation coefficients were both greater than 0.999, which

showed a very good linearity within the range receptive to nitrite and nitrate.

4.4.6 Robustness

The real sample of nitrite was analyzed using proposed method after a deliberate

change in detection wavelength for estimation by ±2 nm.

Table 4.11: Robustness Study Data of Nitrite and Nitrate for HPLC Method

Sr. No. Change in

wavelength

(±2 nm)

% Estimation

Nitrite Nitrate

1 220 98.66 98.91

2 222 98.75 99.01

3 224 99.09 98.65

Mean 98.83 98.86

±SD 0.2268 0.1858

% RSD 0.2295 0.188

The results indicates that the selected wavelength was unaffected by small variation in

the selected method parameters.

4.4.7 Ruggedness

Ruggedness study was carried out using only one parameter i.e. different analyst.

The result showed that the % RSD was in the range of 0.02-0.06 % and the result were

shown in Table 4.12. This study was signifies the ruggedness of the method under

varying conditions of its performance.

Table 4.12 Ruggedness Study Data of Nitrite and Nitrate for HPLC Method

Matrices Concentration

(ppm) Amount Found (%)

Analyst I Analyst II

Nitrite 1 99.86 97.75

Nitrate 10 96.51 96.82



4.5 Conclusion

1. The phosphate buffer of pH 3.7 and 20 % methanol at flow rate of 0.7 ml/min as

mobile phase is optimum for nitrite and nitrate analysis using HPLC method.

2. The wavelength of 222 nm is useful for simultaneous estimation of nitrate and nitrite

on UV detector.

3. Recoveries of nitrite and nitrate were better than 97 %.

4. A statistical analysis based on % R.S.D. indicates that the method is sufficiently

accurate as the values are less than 2 %.

5. The developed method is rapid, precise and sensitive and successfully applied for

determining nitrite and nitrate amounts in vegetable and water samples.



References:

1. H. Li, C. J. Meininger and G. Wu, Rapid determination of nitrite by reversed-phase

high-performance liquid chromatography with fluorescence detection, J.

Chromatography B, 746 (2000) 199-207.

2. S. A. Everett, M. F. Dennis, G. M. Tozer, V. E. Prise, P. Wardman and M. R. L.

Statford, Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-

performance ion chromatography, J. Chromatography A, 706 (1995) 437-442.

3. H. Lulla, S. S. Chen and F. J. Sena, Simultaneous determination of nitrate and nitrite

in toothpastes by high-performance liquid chromatography, J. Pharm. Sci., 73 (7)

(1984) 1004-6.

4. M. N. Muscarfi and G.de Nucci, Simultaneous determination of nitrite and nitrate

anions in plasma, urine and cell culture supernatants by high-performance liquid

chromatography with post-column reactions, J. Chromatog. B, 686 (1996) 157-164.

5. V. Rizzo, L. Montalbetti, A. L. Rozza, W. Bolzani, C. Porta, G. Balduzzi, E. Scoglio

and R. Moratti, Nitrite/nitrate balance during photoinduced cerebral ischemia in the

rat determined by high-performance liquid chromatography with UV and

electrochemical detection, Journal of Chromatography A, 798 (1998) 103-108.

6. Hui Lia, C. J. Meiningerb and G. Wua, Rapid determination of nitrite by reversed-

phase high-performance liquid chromatography with fluorescence detection, Journal

of Chromatography B, 746 (2000) 199-207.

7. N. Erk and M. Levent Altun, Spectrophotometric resolution of metronidazole and

miconazole nitrate in ovules using ratio spectra derivative spectrophotometry and RP-

LC, Journal of Pharmaceutical and Biomedical Analysis, 25 (2001) 115-122.

8. J. Krzek, M. Moniczewska, G. Zabierowska-Slusarczyk, The HPLC determination of

propane-1,2,3-triyl trinitrite and impurities: (2RS)-3-hydroxypropane-1,2-diyl

dinitrate and 2-hydroxypropane-1,3-diyl dinitrate in ointment, Journal of

Pharmaceutical and Biomedical Analysis, 33 (2003) 403-409.

9. Shin-Shou Chou, Jen-Chien Chung and Deng-Fwu Hwang, A High Performance

Liquid Chromatography Method for Determining Nitrate and Nitrite Levels in

Vegetables, Journal of Food and Drug Analysis, 11 (3) (2003) 233-238.

10. Y. Zuo, Chengjun Wang and Thuan Van, Simultaneous determination of nitrite and



nitrate in dew, rain, snow and lake water samples by ion-pair high-performance liquid

chromatography, Talanta, 70 (2006) 281-285.

11. Angel A. Gaona-Galdos, Pedro López García, María S. Aurora-Prado, Maria Inês

Rocha Miritello Santoro and Érika Rosa Maria Kedor-Hackmann, Simultaneous

determination of econazole nitrate, main impurities and preservatives in cream

formulation by high performance liquid chromatography, Talanta, 77 (2008) 673-678.

12. I. Ferreira and S. Silva, Quantification of residual nitrite and nitrate in ham by

reverse-phase high performance liquid chromatography/diode array detector, Talanta,

74 (2008) 1598-1602.

13. H. Kodamatani, S. Yamazaki, Keiitsu Saito, Takashi Tomiyasu and Yu Komatsu,

Selective determination method for measurement of nitrite and nitrate in water

samples using HPLC with post-column photochemical reaction and

chemiluminescence detection, J. Chromatography A, 1216 (2009) 3163-3167

14. Y. Li, J. S. Whitaker and Christina L. McCarty, Reversed-phase liquid

chromatography/electrospray ionization/ mass spectrometry with isotope

dilution for the analysis of nitrate and nitrite in water, J. Chromatography A,

1218 (2011) 476-483.

15. A. Wu, T. Duan, D. Tang, Y. Xu, L. Feng, Z. Zhu, R. Wang and Q. Zhu,

Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by

HPLC Coupled to Nitrite Oxidation, Chromatographia, 76 (2013) 1649-1655.

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