1. INTRODUCTION:

Saliva is a special body fluid that is constantly in contact with the oral mucosa and teeth. It is secreted by salivary glands, and gingival crevicular fluid. Saliva plays an important role in self-cleaning, antimicrobial, lubrication, pronunciation, digestion, and olfactory functions.^1-4

Current studies on saliva are based on molecular diagnostics, which detect and quantify genetic materials. Technology related to biomarkers as predictors of systemic diseases is being developed.⁵ Diagnostics using saliva are particularly non-invasive, economical, and comfortable compared to blood tests. Therefore, saliva has potential application in diagnostic methods. Saliva contains diverse anionic compounds.

Several studies have reported physiological and biological states based on biomarkers such as inorganic ions and organic compounds.^6-9

Anions in saliva are mostly fluoride, chloride, nitrate, sulfate, phosphate, and iodide. These compounds can be evaluated in relation to the saliva buffer capacity for the purpose of dental caries risk assessment.

Fluoride, which is related to hard tissue demineralization, increases the resistance of dental structures, promotes remineralization, and inhibits microorganism enzymatic activities.^10,11 Fluoride also prevents caries. Chloride is an important diagnostic marker for cystic fibrosis. It is known that increased concentration of chloride in the sweat indicates cystic fibrosis.¹² It has also been reported that patients with autoimmune Sjogren syndrome exhibit increased Na+ and Cl- concentrations.^13,14 Nitrate and nitric oxide metabolites from body fluids can be used to estimate the magnitude of nitrosative and oxidative stress.¹⁵ Sulfate and phosphate in saliva are related to saliva buffer capacity. Hence, sulfate and phosphate can positively affect pH regulation and prevent caries. Phosphate, especially, is known for its role in cellular homeostasis. Phosphate levels in the blood and the saliva are proven biomarkers that indicate the initial stages of obesity.^16,17Iodine is an important substance of thyroid hormones, Deficiency is usually estimated by measuring iodine concentration in urine because about 90% of iodine is excreted in the urine.¹⁸

These anions can be analyzed using methods such as capillary electrophoresis,¹⁹ capillary isotachophoresis²⁰ and spectrophotometry.²¹ In this study, HPLC ion chromatography (IC) was used to develop a method of simultaneous analysis of fluoride, chloride, nitrate, sulfate, phosphate, and iodide in saliva. Real saliva samples were analyzed for their inorganic anion contents to validate the method and to provide reference material for future salivary anion analysis.

2. MATERIALS AND METHODS:

2.1. Reagents and materials:

Solvents and inorganic anion such as fluoride, chloride, nitrate, sulphate, phosphate and iodide were used for all experiments in this study(Sigma Aldrich St. Louis, USA). Mobile phase grade sodium hydroxide solution (NaOH) was purchased from Fisher Scientific (Fairlawn, NJ, USA). Water used to prepare solutions was purified using an Automatic Aquarius AW-1001 purification system (Top Trading, Seoul, South Korea).

2.2. Saliva sample preparation:

The subjects of this study were 20 healthy Korean adults without dental caries, periodontal diseases, metabolic diseases and who were not taking medication. All subjects were prohibited from drinking, eating, brushing their teeth, and rinsing with water for 1 hour before saliva collection. At 12 am, non-polar salivawere collected in 1.5 mL tubes. All biofluidsas saliva were centrifuged at 2,000 rpm for 12 minutes, diluted 20 times with distilled water.

2.3. HPLC:

A Dionex ion chromatography system GP50 gradient pump, ion column, and ED 50 conductivity detection was used. Standard mixtures of inorganic anions in the following concentrations were used for calibration by an external method. Each compound (0.25-2.5 mM). HPLC (High performance liquid chromatography) separation was carried out with DionexIonPac AG 11-HC column (50mm×4mm). The temperature in the column was 35°C and the flow rate of the NaOH solution was kept constant at 1,000 μL/min and isocratic elution with 120 mmol/L.

2.4. Method validation:

Standard stock solutions (10 mM) were prepared in diluted water. Calibration curves were obtained by plotting the compound peak area ratios using standard stock solutions diluted at five standard concentrations. Concentration of the external standard method. To assess the recovery test analytical method, solution mixtures were injected four times from blank human saliva.

3. RESULTS AND DISCUSSION:

The analyticalvalidation method for the inorganic anion analysis in this study is shown in Table 1 and Fig 1. Calibration curve was performed using standard solutions containing various concentrations of analyte range (0.25-2.5mM).

Figure 1. Linearity of each anion

Table 1. Linear range, linear equation, correlation coefficient (r²), LOD, and LOQ for inorganic anions

Compound	Linear range (mM)	Linear equation	r²	LOD (µM)	LOQ (µM)
Fluoride	0.25-2.5	y = 3.6128x + 0.027	0.9902	0.78	2.36
Chloride	0.25-2.5	y = 3.8785x - 0.0202	0.9987	0.83	2.50
Nitrate	0.25-2.5	y = 3.5283x + 0.036	0.9949	0.82	2.46
Sulphate	0.25-2.5	y = 1.2756x - 0.117	0.9950	0.88	2.64
Phosphate	0.25-2.5	y = 2.9294x - 0.0667	0.9987	0.93	2.75
Iodide	0.25-2.5	y = 3.0753x + 0.1585	0.9989	1.16	3.49

^*y=Sx+ σ

^aLOD: 3.3 σ/S;

^bLOQ: 10 σ/S (σ, the standard deviation of the y-intercept of the regression; S, the slope of the calibration curve).

The accuracy and precision and recovery of the analysis was evaluated by performing intra- and inter-day recovery assays. Human saliva containing known concentrations of each anions (0.50 and 1.00 mM) were extracted and the mean and SD of the precision and recovery was determined Table 2. This was performed four times per day for 4 days. The Relative Standard deviation (RSD) of intra- and inter-day was 3.37 - 11.48% respectively. The recovery ranges were 95.63 – 107.01% respectively.

Table 2. Recoveries (%) of inorganic anions in human saliva

Analyte	Added (mM)	Day 1		Day 2		Inter-day precision
Analyte	Added (mM)	Recovery (%) Mean±SD	RSD	Recovery (%) Mean±SD	RSD	Recovery(%)	RSD (%)
Fluoride	0.5	100.12±6.97	6.96	102.53±8.41	8.20	100.52	5.91
	1.0	104.18±2.97	2.85	102.81±7.29	7.09	103.44	4.46
Chloride	0.5	104.51±8.30	7.94	93.74±13.67	14.58	99.77	11.48
	1.0	105.38±6.51	6.17	101.17±6.75	6.67	103.98	5.72
Nitrate	0.5	93.19±9.90	10.62	93.73±11.14	11.89	95.63	11.03
	1.0	100.69±7.39	7.34	101.88±6.17	6.06	100.78	6.05
Sulphate	0.5	98.74±1.33	1.35	98.74±1.33	1.35	100.16	3.58
	1.0	109.56±9.75	8.90	105.40±4.96	4.71	107.01	6.62
Phosphate	0.5	104.61±2.66	2.55	104.47±2.82	2.70	105.90	3.37
	1.0	102.94±7.03	6.82	101.79±2.35	2.31	101.97	4.23
Iodide	0.5	113.99±16.50	14.47	107.05±3.32	3.10	107.77	7.49
	1.0	101.01±4.60	4.56	106.10±6.08	5.73	101.65	4.88

Table 2. Continue

Analyte	Added (mM)	Day 3		Day 4		Inter-day precision
Analyte	Added (mM)	Recovery (%) Mean±SD	RSD	Recovery (%) Mean±SD	RSD	Recovery(%)	RSD (%)
Fluoride	0.5	98.04±2.70	2.75	101.38±5.80	5.72	100.52	5.91
	1.0	103.60±3.36	3.24	103.17±4.79	4.65	103.44	4.46
Chloride	0.5	95.18±16.11	16.93	105.66±6.82	6.46	99.77	11.48
	1.0	103.91±5.27	5.08	105.45±5.22	4.95	103.98	5.72
Nitrate	0.5	98.92±10.26	10.37	96.69±10.88	11.25	95.63	11.03
	1.0	99.29±3.20	3.23	101.24±7.65	7.56	100.78	6.05
Sulphate	0.5	101.56±3.18	3.13	101.61±8.63	8.50	100.16	3.58
	1.0	102.41±5.68	5.55	110.67±8.09	7.31	107.01	6.62
Phosphate	0.5	105.98±3.31	3.13	108.54±5.52	5.09	105.90	3.37
	1.0	104.43±5.17	4.95	98.72±2.79	2.83	101.97	4.23
Iodide	0.5	101.84±6.11	6.00	108.19±6.90	6.38	107.77	7.49
	1.0	100.20±3.99	3.98	99.30±5.23	5.26	101.65	4.88

RSD, relative standard deviation.

Table 3. Inorganic anion concentrations in human saliva samples (mmol±SD)

Subjects	Fluoride	Chloride	Nitrate	Sulphate	Phosphate	Iodide
1	0.05±0.24	17.92±3.01	0.11±0.07	0.52±0.32	3.60±1.24	N.D.
2	0.07±0.19	12.44±2.98	N.D.	0.53±0.29	1.54±0.89	N.D.
3	N.D.	11.85±4.12	N.D.	N.D.	3.27±1.93	N.D.
4	0.04±0.02	9.56±2.32	N.D.	N.D.	1.60±0.92	N.D.
5	0.08±0.12	18.51±4.92	N.D.	0.94±0.35	5.58±2.01	N.D.
6	0.14±0.11	12.36±2.31	N.D.	2.61±0.03	2.19±1.90	N.D.
7	0.08±0.02	10.97±3.92	N.D.	N.D.	3.69±2.12	N.D.
8	0.03±0.00	6.36±1.09	N.D.	N.D.	2.32±0.95	N.D.
9	0.02±0.01	14.91±3.18	N.D.	1.03±0.16	2.76±0.98	N.D.
10	0.04±0.05	8.62±2.12	N.D.	0.21±0.06	3.10±0.45	N.D.
11	0.15±0.08	12.48±2.61	N.D.	1.37±0.31	3.82±0.87	1.57±0.23
12	0.10±0.09	17.46±4.90	0.26±0.08	0.82±0.52	5.72±2.12	0.47±0.04
13	0.05±0.04	5.49±2.10	N.D.	0.56±0.31	1.88±0.69	N.D.
14	0.08±0.05	10.54±3.01	N.D.	0.54±0.41	2.76±1.39	N.D.
15	0.04±0.03	13.31±1.98	N.D.	0.76±0.57	4.73±0.89	N.D.
16	N.D.	11.23±3.02	N.D.	0.37±0.32	4.76±0.92	N.D.
17	N.D.	14.89±5.17	N.D.	0.35±0.26	4.02±0.87	N.D.
18	N.D.	16.99±3.65	N.D.	0.35±0.26	9.38±2.31	N.D.
19	0.19±0.14	26.89±4.12	N.D.	1.43±0.89	9.90±2.64	1.25±0.34
20	1.51±0.32	12.41±3.57	0.81±0.71	74.2±5.98	3.56±0.94	N.D.

N.D, Not detected.

The results of anion analysis of human saliva samples are shown in Table 3 and fig 2. Analysis of 20 different saliva samples showed that inorganic anion concentrations were different in each participant (fluoride 0.00-1.51 mM, chloride 5.49-26.89 mM, nitrate 0.00-0.81 mM, sulfate 0.00-74.20 mM, phosphate 1.54-9.90 mM and iodide 0.00-1.57 mM).

Figure 2. Concentration of inorganic anions in the all human saliva

Clinical tests using saliva samples not only are useful for dental caries risk assessment but can also confirm hormone, antibody, microorganism, systemic, and pathological changes using proteins and ions.^{22, 23} Although changes in ion concentrations are observed due to several variables,² components of saliva samples are relatively stable and non-deteriorating. In addition, the similarity to plasma concentrations allows for effective testing.

In this study, a method to analyze fluoride, chloride, nitrate, sulfate, phosphate, and iodide levels using HPLC was developed. Real saliva samples were analyzed in order to establish a basic database of biomarkers that can diagnose oral and systemic diseases.

Capillary electrophoresis, capillary isotachophoresis, and spectrophotometry are simultaneous analysis methods similar to HPLC. Moreover, ion selective electrodes, atomic absorption,²⁴ and traditional spectrophotometric methods can be used for saliva analysis.^25,26 These analytical methods indicate that the saliva density is similar to the densities of other bodily fluids. In this study, a wide range of sample densities was measured using a relatively simple technique. Furthermore, HPLC conductivity detection, which is highly selective, was used for the analysis.

The results showed a relatively good linearity, LOD, and LOQ. The LOQ range was 2.36-3.49 µM, indicating that quantification is possible even at low concentrations. Although the recovery rate differed at 0.5 mM, the recovery test showed a normal recovery rate at 1.0 mM, which verified this analytical method.

Although deviations were observed, analysis on 20 subjects showed a good overall selectivity for saliva components. Ion concentrations and appearances in saliva were especially different depending on the salivary gland.²⁷ Stimulus is known to change the concentration of anions in saliva. Food and other stimuli increase chloride concentrations in saliva while decreasing phosphate concentrations.²⁸ In this study, there were no arbitrations on the subjects since it focused on the development of an analytical method. Using the method of analysis developed in this study, further studies should be performed to investigate changes in salivary anion concentrations due to systemic diseases.

Since other high and low ions concentration such as Na⁺, K⁺, Mg²⁺, Ca²⁺ and Cl^-exist in whole saliva,²⁹ future studies should take into account both cations and anions before analyzing the components of saliva in each subject.

This study focused on the development of an analytical method that can test for different anions simultaneously. Actual saliva samples were analyzed to confirm its accuracy. This analytical method could be used for the investigation of different biomarkers in saliva and the results of this study could be used as reference for quantification of saliva.

4. CONCLUSION:

The linearity of the sample’s linear range was 0.9902-0.9989, LOD was 0.78-1.16µM, and LOQ was 2.36-3.49 µM. The recovery test showed a recovery rate of 95.63-107.77% and RSD of 3.37-11.48%, which verified the analytical method. Although deviations were observed in different participants, analysis of real saliva samples showed relatively good selectivity. This study, which analyzed anions in saliva, could be used as reference for saliva biomarker studies in the future.

In this study, an analytical method to simultaneously quantify fluoride, chloride, nitrate, sulfate, phosphate and iodide amount in saliva was developed using HPLC ion chromatography (IC). Furthermore, inorganic anions in real saliva samples were analyzed for method validation.

5. ACKNOWLEDGMENT:

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (No. 2014R1A1A3051084 and 2017R1A2B4012865).

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Received on 12.12.2017 Modified on 16.01.2018

Research J. Pharm. and Tech 2018; 11(5):1820-1824.

DOI: 10.5958/0974-360X.2018.00338.4