UHPLC ANALYSIS OF POLYCYCLIC AROMATIC HYDROCARBONS (PAH) COMPOUNDS FROM THE SOIL BY QuEChERS AOAC METHOD FROM MANESAR INDUSTRIAL AREA, HARYANA, INDIA

in Manesar located 60 km away from Delhi, capital of India 28.3542° N, 76.9400° E in different seasons. The sampling was done in the industrial areas, agricultural fields, and residential plots all within 10 km radius of the industrial area. In the present study, the QuEChERS method was used for sample preparation, which has shown good recoveries around 80 to 120% The extraction time was only 10 minutes, followed by the UHPLC technique of run time of just 16 minutes. Average PAH concentration in Manesar soil was found highest in the winter session of industrial areas. ∑16 PAH was 141 ppb in the industrial area in winter, 64 ppb in agricultural fields, and 54.7 ppb in residential plots. The lowest PAH levels were observed in the monsoon season. These values are very less if compared to Brazil, UK, Germany, and Korea.


INTRODUCTION
Polycyclic Aromatic Hydrocarbons (PAH) are known carcinogenic and mutagenic compounds which are formed by burning organic matter with less oxygen presence and heavy vehicle emissions (Sorensen, 1994;Nam et al., 2003). Such compounds need to be monitored and checked from time to time in industrial areas. Apart from mutagenicity, they are responsible for many other lungs and respiratory diseases (Grimmer et al., 1983;Yang et al., 1991;Rost and Loibner, 2002). In soil, these compounds are retained in humus and organic matter for a very long time as they are hydrophobic (Krauss et al., 2000). Hence soil is the best place to trace out and check for PAH contamination (Ockenden et al., 2003). Few studies have already been done in India, like in Delhi (Kannan and Kapoor, 2004), Mumbai (Sahu et al., 2001), and Ahmedabad (Raiyani and Shah, 1993). PAHs are one of the most dangerous pollutants found in soil as per IARC. Higher ring PAHs are more carcinogenic Yang et al., 1991;Massei and Ollivon, 2004). The study of PAH becomes very essential in areas where the industries are very close to residential apartments and agricultural fields. Manesar located 60 km away from Delhi, the capital of India 28.3542° N, 76.9400° E is one of the fastest-growing industrial areas and townships. As per Human Rights Documentation Indian Social Institute, (India) Manesar has fertile lands which are used for farming. It's becoming more important to check the PAH levels in Manesar as it's a hub of farming lands, residential, industries, and all within 10 km area. The PAH-contaminated soil can be harmful to farming as PAH can percolate from soil to vegetables (Wennrich et al., 2002). Many studies have shown that if vegetables are grown in PAHcontaminated soil, it may lead to the uptake of PAH in them (Wennrich et al., 2002). High concentrations of PAH compounds were found in vegetables like a tomato at 0.2 µg/kg, spinach at 6.6µg/kg, and cabbage 20.4µg/kg (M. Kluska.,2003) So, the study of soil is very crucial to trace out PAH. Currently available and reported PAH analysis methods are long and time taking and use a classical sample preparation and analysis approach. Classical soxhlet extraction technique takes 8 to 12 hours of extraction of PAH from soil and uses a huge amount of extraction solvents which is then analyzed by HPLC technique whose run time was around 30 to 40 minutes for analysis of 16 PAH. This study has identified the use Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) AOAC method which is very quick and rugged. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) is an acetonitrile-based extraction technique followed by dispersive solid-phase extraction (SPE) (Anastassiades et al., 2003). Followed by this extraction technique a new UHPLC method was also developed and validated as per ICH guidelines which separate 16 PAHs in just 16 minutes.

Regional site description
Manesar located 60 km away from Delhi, the capital of India 28.3542° N, 76.9400° E has become the hub of industrialization and real estate. The faster-growing industrialization needs the huge movement of heavy vehicles run on diesel. NH8 is the highway that connects Manesar to Gurugram and Delhi. Apart from the main highway NH8 various other roads pass through agricultural fields. One such road is Pataudi road which is the easiest and most popular road considered as an alternative to NH8. The frequency of heavy and small vehicles increased drastically throughout the last 5 years due to the sudden outburst of industrialization and real estate construction in Manesar. This road is surrounded by farmlands. PAH study is very essential to check the levels as heavy vehicle exhausts are a potential source of PAHs. Manesar itself has many factories which release smoke from chimneys. Chimneys are potential sources for PAH releases. During winter seasons, many organic materials are burnt like wood and coals as a source of fuel, which is again a potential source of PAH. Hence, PAH study has become essential in Manesar and surrounding areas. The climate of Manesar is dry and hot with a temperature range from 32°C to 48°C. In winter, the temperature ranges from 4°C to 30°C.

Sample collections
For sample collection, the entire Manesar area was categorized into three parts industrial area, an agricultural area, and a residential area. For analysis, 5 cm topsoil was taken through an auger and sieved through 20-mesh sieve, and stored in airtight plastic tubes in a refrigerator at 4°C. Total of 240 samples were collected, 80 samples from each location.

Extraction and analysis of PAH
QuEChERS AOAC technique was used for the extraction of PAH from the soil. In a 50 ml tube, 5gm soil samples were taken, followed by 5ml water and sonicated An Ultra-High-Performance Liquid Chromatography (UHPLC) method was developed and validated as per ICH guidelines for the analysis of 16 PAH compounds as per USEPA and various soil samples were analyzed in Manesar located 60 km away from Delhi, capital of India 28.3542° N, 76.9400° E in different seasons. The sampling was done in the industrial areas, agricultural fields, and residential plots all within 10 km radius of the industrial area. In the present study, the QuEChERS method was used for sample preparation, which has shown good recoveries around 80 to 120% The extraction time was only 10 minutes, followed by the UHPLC technique of run time of just 16 minutes. Average PAH concentration in Manesar soil was found highest in the winter session of industrial areas. ∑16 PAH was 141 ppb in the industrial area in winter, 64 ppb in agricultural fields, and 54.7 ppb in residential plots. The lowest PAH levels were observed in the monsoon season. These values are very less if compared to Brazil, UK, Germany, and Korea.
for 2 minutes, then 10ml acetonitrile was added and again sonicated for 2 minutes. Agilent QuEChERS AOAC pouch (PN-5982-0755) was added to the mix and vortexed for 15 minutes and the contents were centrifuged for 5 minutes at 10000 RPM. The volume of about 5ml of the supernatant was transferred to the dispersive SPE 15ml tube and vortexed for 2 minutes.

Figure 1 Sampling points at Manesar
The supernatant was filtered through 0.2µm syringe filter and injected into UHPLC (Ultra-High-Performance Liquid Chromatography). The analysis of PAH was done by Agilent 1290 Binary UHPLC with Agilent PAH column (2.1mm x 100mm x 1.8µm). Milli Q water was used in mobile phase channel A and HPLC grade acetonitrile was used in mobile phase channel B, flow was optimized at 0.35ml/min with a stop time of 18 minutes. The gradient elution program was given as 55% mobile phase B at the start, ramp till 95% mobile phase B at 9.5 minutes, 95% mobile phase B at hold till 18 minutes, 1 minute post time for column equilibration at an initial gradient, the column was kept at room temperature. For trace level detection DAD (Diode array detector) and FLD (Fluorescence detector), both were used with UHPLC. DAD at 230nm was only used for detection of Acenapthalene, for others, FLD was used with a fixed excitation at 260nm and multiple emission was used at 350nm, 420nm, 440nm, and 500nm.
The new UHPLC method was validated as per ICH guidelines and calibration was done for different levels. To check the robustness of the UHPLC method 2% deviation was introduced to the flow rate and injection volume and 5% deviation in the column temperature and found that the retention time (RT) deviation was within 3% limit. For repeatability, 10 replicate injections were done, and % RSD (Relative Standard Deviation) for area and RT were calculated. To validate the sample prep spiking studies were performed at low, mid, and higher concentration levels, and recoveries were found between 80% to 120%. The chromatogram for UHPLC is shown in Figures 2& 3.

PAH extraction from soil
Manesar was divided into industrial, agricultural, and residential areas, and samples were collected in summer (S), monsoon (M), and winter (W), average concentrations are reported in table 2 and figure 6. The average level of PAH in the industrial area was an all-time high as compared to agricultural lands and residential areas. This was well justified as the agricultural lands were close to the PAH emission zones. The residential areas were located 10 km far and close to only NH8 which is the highway so the only potential PAH source is vehicle emission.

Seasonal variations
Average concentrations of PAH were plotted in the graph in which the Y-axis is the ppb concentration of PAH. It was found that in winter the PAH levels were higher relatively. In winter it was noticed that the burning of organic wastes was more in village areas near agricultural fields which contributes to higher PAH levels. The entire amount of average PAH in the industrial area is 89.3 ppb in the summer and 141 ppb in the winter. This value is very low if compared to other case studies (Wennrich et al., 2002). The average PAH concentrations are represented for different countries, Table 3.

PAH presence profile assessment
Till date, the PAH concentration and exposure limit are not directly linked to any disease, but few are categorized as most carcinogenic like benzo(a)pyrene, and classified as (2A) and (2B) human carcinogens by the International Agency for Research on Cancer (IARC, 1987). BaP has 5 rings (C20H12) which have shown potential mutagenic character for human cells in cultures environment (Osborn and Crosby, 1987), and animal assay (Cerna et al., 2000). As per the toxic equivalent factor, BaP is one (1) which is the highest of all. PAH concentration below 1ppm shows the soil of Manesar is non-carcinogenic presently.

CONCLUSION
In the location of Manesar, 16 PAH were observed in the soil. The total PAH concentrations were found to be 141µg/Kg, 64 µg/Kg, 54.7 µg/Kg in industrial areas, agricultural areas, and residential areas in the winter season respectively. Out of summer, monsoon, and winter, the winter season has shown the highest amount of PAH concentration. In the monsoon season the concentrations were very low, 61 µg/Kg, 16.3 µg/Kg, 17.7 µg/Kg respectively in industrial areas, agricultural areas, and residential areas. Anyhow, in winter the concentration of PAH for industrial areas is also very low concerning other countries' industrial areas, the reason could be the establishment of Manesar is new around 10 years old. But such monitoring is very important from time to time. The new UHPLC method was developed and saved a lot of time and solvent, and good separation was achieved. The sample preparation technique also saves a lot of time. The most carcinogenic PAH is BaP which is also reported very low and insignificant in Manesar soil at present as per table 3.

S E A S O N A L P A H V A R I A T I O N S I N M A N E S A R I N D U S T R I A L A R E A ( P P B )
Industrial area Summer Industrial area Monsoon Industrial area Winter