Variation of patulin levels in citrus fruits from central cities of Punjab and Northern cities of Pakistan, and estimation of dietary intake

: The research aims to discover the natural occurrence of patulin (PAT) in selected citrus fruits from the central cities of Punjab and Pakistan's Northern cities. Total 2970 samples of twelve citrus fruits; kinnow, orange, grapefruits, bitter orange, mausami, red blood, pineapple, sweet orange, rough lime, sweet lime, kagzi lime and lemon were examined using liquid chromatography fitted with UV detector. The limit of detection (LOD) and quantification limit was 0.04 and 0.12 µg/kg, respectively. The results have shown that 56% of samples of citrus fruits from Punjab's central cities, Pakistan, were discovered to be infected from PAT. The elevated amounts of PAT ranging from 0.12 to 1150 µg/kg were found in citrus fruit samples from Multan cities. Furthermore, 31.7% of samples of citrus fruits from Northern cities of Pakistan were discovered to be infected with PAT, and the elevated amounts were found ranging from 0.12-320 µg/kg from Swat city. About 22.1% of samples of citrus fruits have levels of PAT greater than the suggested limits established by the European Union (EU). PAT's dietary intake levels ranged from 0.10-1.11 µg/kg bw/day from the central cities of Punjab, Pakistan, and 0.13-1.93 µg/kg bw/day were documented from Northern cities of Pakistan.


Highlights for Review
The daily intake levels of PAT were ranged from 0.10-1.11 µg/kg bw/day from central cities of Punjab, • Furthermore, the levels of dietary intake of 0.13-1.93 µg/kg bw/day were documented from Pakistan's Northern cities.

Introduction
Citrus fruits belonging to the Rutaceae family covering 130 genera are well-known worldwide due to their diversified potential in fruits, juices, confectionaries, and fresh fruits consumption. Citrus fruits can be cultivated in tropical, subtropical, and temperate environmental conditions, and these are produced almost in 137 countries. The essential citrus fruits are oranges, tangerines, limes, mandarins, grapefruits, lemons, citrons, and many hybrid varieties, with pleasant flavors, aroma, and attractive colors. Citrus apple fruits and 10 µg/kg in the apple-derived products along with baby foods, and for the young children and infants [21]. However, no regulations are established for PAT in fruits or juices in Pakistan [22].
In our previous study [ 22], considerable PAT amounts were found in juices, smoothies, and fruits from Pakistan. However, no comprehensive PAT surveillance in citrus fruits was conducted, considering their importance and production in Pakistan. Therefore, the present survey is designed to analyze PAT's existence in selected citrus fruits from the central cities of Punjab, and Northern cities of Pakistan, to evaluate the concentrations of PAT with EU permissible limits and estimate the daily intake of PAT in regional inhabitants. The findings will be useful for smallholders, consumers, and exporters to understand the toxic effect of this toxin.

Samples
Total 2970 samples of 12 citrus fruits (kinnow, orange, grapefruits, bitter orange, mausami, red blood, pineapple, sweet orange, rough lime, sweet lime, kagzi lime, and lemon) were collected from the major citrus crops producing cities of Pakistan (Toba Tek Singh, Sargodha, Multan, Jhang, Sawat, Peshawar, Mirpur) during September 2019 till December 2019. There was a random collection of samples, and each sample's size was maintained at 1kg. The samples were stored in plastic bags with proper labeling and kept at -20 °C in the freezer until further analysis.

Reagents and chemicals
Patulin standard with the concentration of 100 mg/mL, in acetonitrile was obtained from Sigma Aldrich: Saint-Louis, USA. Ethyl acetate, sodium carbonate, and acetic acid were obtained (Sigma-Aldrich, France). The solvents, including methanol and acetonitrile, were of HPLC grade, obtained from Fisher Chemicals (France). PAT's standard curve was made with a dilution of the pure amount of methanol ranging from 0.4, 1, 5, 10, 50, 100, 150, 200, 250, and 300 µg/L. The standard solutions were kept and stored in the sealed vials at -20 ºC, till further analysis. The other reagents and solvents were all analytical grade.

The cleanup procedure
Patulin extraction from the citrus fruit samples was achieved following the [22] method, with some modifications. About 10 g of solid citrus fruit sample was added in 10 mL water and homogenized. Then, a mixture of 20 mL of ethyl acetate was added and extracted using a vortex mixer for 3 min. After that, the mixture was centrifuged (5 min; at 25 ⁰C) at 4500 rpm. The upper organic layer was shifted in a centrifuge tube along with the addition of 10 mL of 1.5% (w/v) sodium carbonate with vigorous mixing. Again the addition of 5 mL of ethyl acetate with vigorous shaking for 5 min was done. The solution pH was maintained at 4 with the addition of a few drops of glacial acetic acid followed by the evaporation using a nitrogen stream at 60 ⁰C. A 5% solution of acetonitrile (5 mL) was then added to the residue and purified with a 0.22 mm syringe filter (Millipore), followed by the complete dryness using nitrogen stream. Finally, the residue was dissolved in 500 µL of methanol, and 20 µL was injected for HPLC analysis.

HPLC conditions
Patulin analysis of the citrus fruits was achieved on HPLC: Shimadzu LC-10A series, Kyoto, Japan, equipped with a UV detector (276 nm). A C18 column (4.6 X 250 mm, 5 µm) Discovery of Supelco, Bellefonte, USA, was used for evaluation. The mobile phase comprising the composition of 90% of acetonitrile, with a flow rate of 1 mL/min in the isocratic mode was employed.

Assessment of daily intake
The procedure for the estimation of dietary consumption in the citrus fruits was followed by our previously established method, i.e., Iqbal et al. [22]. The dietary consumption of patulin in the local consumers was calculated with a questionnaire based on food frequency, comprising 500 individuals. The recalling period of citrus fruits intake comprised of four weeks. Interviewees' memory was activated by collecting the information of citrus fruits intake by means of amount, size of fruits, or juices with glass or bowl measurements. Of 500 individuals, 450 individuals have returned the questionnaire with answers, 45 individuals have never responded, and five individual questionnaires were canceled due to the technical point of view. The body weight of the individual was 70 ±2. The daily intake was assessed utilizing the following procedure Dietary intake µg kg day = ⁄ ⁄ Daily intake of fruits juices ( g day ) × Patulin in fruits juices ( µg kg ) Average individual weight (kg)

Method Validation
The method was validated through the parameters including precision, linearity, reproducibility, limit of quantifications (LOQ), limit of detections (LOD), and repeatability and reproducibility of PAT. The LOD was obtained from the 3:1 signal-to-noise ratio, and LOQ was quantified by the 10:1 signal-to-noise ratio [23]. To assess the recovery analysis, three fortified PAT levels were spiked to uncontaminated oranges samples.

Statistical analysis
PAT data in selected citrus fruits were presented as mean ± standard deviation, and all the samples were analyzed in triplicate. The straight-line equation and coefficient of determination R 2 was calculated using the simple linear correlation/regression analysis. To determine the significant levels of PAT in citrus fruits from central cities of Punjab and Northern cities of Pakistan, the student T-test was applied using SPSS (IBM, PAW-27, USA).

Validation of Method and quality control
Method validation was achieved through the quality control parameters. The recovery rate was obtained by spiking blank samples with three patulin concentrations: 50, 100, and 200 µg/L. Mean values of the recovery varied from 85.5 -91.5%. The average relative standard deviation (RSD) variation ranged from 10.6 -14.5 %, as presented in Table 1. The recoveries range was within the requirement (70-110%) recommended by EC regulation 401/2006 [24]. PAT's standard curve ranging from 0.4 -300 µg/L has confirmed the linearity, with the coefficient of determination (R 2 ) value of 0.9947. The LOD and LOQ of PAT were 0.04 and 0.12 µg/kg, respectively. These values of LOD and LOQ are lower than the ones reported by [25], LOD and LOQ in sample matrix 0.5 μg/L and 2 μg/L, respectively. The accuracies of both these quantities mainly depend on the sensitivity of the instrument [26]. The chromatograms represented in Figure 1, showing the natural occurrence of PAT in orange (a), sweet orange (b), lime (c), and grapefruits (d). RSD = relative standard deviation, LOD = limit of detection, LOQ = limit of quantification. R 2 = coefficient of determination; LOQ = LOD x 3 Repeatability and reproducibility 2 are given as mean percent RSD (%).

Occurrence of PAT in the citrus fruit samples
The incidence levels of PAT in citrus fruit samples from central cities of Punjab, Pakistan, are shown in Table 2. The results have shown that 56%, i.e., 1101 out of 1967 samples of selected fruits samples, were positive with PAT. About 51.4%, 56%, 66%, and 50% samples of selected citrus fruits from Toba Tek Singh, Sargodha, Multan, and Jang cities were documented contaminated with PAT. The elevated amount of PAT 0.12-1150 µg/kg was found in sweet orange samples from Multan city.
The prevalence of PAT in selected citrus fruits from Northern cities of Pakistan is represented in Table 3. The results documented that 372 out of 1003, i.e., 37.1% of samples were found to be positive with PAT. The samples of 36.7%, 36.8%, and 37.8% were found positive from Mirpur, Peshawar, and Swat. The highest mean level of 163.3 ± 10.1 µg/kg was found in orange samples from the Swat City of Pakistan. About 657 out of 2970, i.e., 22.1% samples were found having a level of PAT greater than the EU permissible limit, and the highest 29.7% samples of citrus fruits were found levels higher than the recommended limit of EU. The PAT levels in citrus fruits from central cities of Punjab to Northern cities of Pakistan were found significant (p < 0.05).   The finding of current research on PAT levels is much higher than the previous study [27]. They documented that 74 out of 141 samples of orange fruits, juices, pulp, and orange jams were found contaminated with PAT and the elevated average amount was 8.7 ± 19.9 µg/kg in orange fruits. Only one sample was found to be contaminated with PAT with levels higher than EU limits. High levels of PAT in selected citrus fruits are observed because in Pakistan, Good Agriculture Practices are not followed, and during the growth of fruits, fungicides are not applied. During the ripening stage, wound on the skin of fruits would provide preharvest contamination of fruits, and in rotten or decayed areas, the levels of 113342 µg/kg were observed in apples [28]. In our previous study, Iqbal et al. [22] have observed 136 out of 237 (57.4%) samples of juices, smoothies, and fruits were observed contaminated with levels ranged up to 1100 µg/kg. The elevated mean level of 921.1 ± 22.4 µg/kg was found in red globe grapes, and 33.8% of samples were found to have levels higher than the EU recommended limit. Funes & Resnik [29] have studied that 21.6% of samples of solid, semisolid apple and pear products were found positive with PAT with average levels ranged from 17-221 µg/kg (mean levels 61.7 µg/kg). The elevated amount of PAT was observed in apple puree 123 µg/kg, and almost 50% of samples were observed to be positive with PAT.
Cho et al. [30], from South Korea, have analyzed 72 samples of fruits, including three apples, two orange, and four grapes samples, and reported the highest mean level of 30.9 µg/L in orange juice samples. Spadaro et al. [31] from Italy have shown that 47 out of 135 samples of different juices were contaminated with PAT with a mean level of 6.42 ± 4.48 µg/L, with the highest level of 55.4 µg/L. In another study from Greece, Moukas et al. [32] had reported the levels of PAT in orange juice samples ranging from 3.1-10.8 µg/kg. Zouaoui et al. [33] from Tunisia have observed 214 samples (including concentrated juice, apple juice, pear juice, mixed juice, compote samples, apple jams, and pear jams samples). They documented PAT levels in 50% of analyzed samples with concentrations ranging from 2 to 889 µg/L and an average amount of 89 µg/L. However, 22% of samples have a PAT concentration that exceeded the limit proposed by the EU. Murillo-Arbizu et al. [34] from the Spanish market have found that 66% of samples of apple juice were observed affected from PAT (LOD 0.7 µg/L) with a mean level of 19.4 µg/L and levels were ranging from 0.7-118.7 µg/L. They kept that 11% of the samples have PAT levels observed above the permissible limits of EU regulation. Similarly, very high levels of patulin are stated by Saxena et al. [35] from India, presenting branded juices including concentrate of apple, orange, guava, grape juice, etc. ranging 21-1839 µg/L, with an average level of 330 ± 141 µg/L. The maximum temperature of Northern cities of Pakistan remained 25 °C during summer and winter it drops below 0 °C, however, in central cities of Punjab, the weather during summers remains from 35 to 45 °C. During the winter season, it fluctuates from 7 to 15 °C. The climatic conditions would cause a high incidence of PAT in citrus fruits from the central cities of Punjab, Pakistan.

Daily intake assessment of patulin
The dietary intake of PAT from selected citrus fruits from central cities of Punjab, Pakistan, is presented in Table 5. The daily intake levels of PAT were ranged from 0.10-1.11 µg/ kg bw/day. PAT's highest daily intake was 1.11 µg/ kg bw/day in masambi fruits samples from Multan city. The levels of dietary intake of PAT from selected citrus fruits from Northern cities of Pakistan is presented in Table 6. The daily intake levels of PAT were ranged from 0.13-1.93 µg/kg bw/day. The elevated dietary intake level was 1.93 µg/kg bw/day in sweet orange from Sawt city. Rahimi & Jeiran [36] from Iran have analyzed different and estimated PAT's daily intake by fruit juice as 16.4, 45.9, and 74.6 ng/kg bw/day for Iranian adults, children, and babies, respectively, much lower compared the findings of current research. Saxena et al. [35], from India, have conducted a study to analyze apple juice's dietary intake among different consumer age groups and found the dietary intake ranging from 0.11-0.24 µg/kg bw/day, comparable to our research. In our previous study [22], low levels of daily intake of PAT were observed in fruits, juices, and smoothies 0.0049, and it was at least 0.0016, and at least 0.0014 µg/kg bw day, respectively. In China, Guo et al. [37] have estimated dietary intakes among adults, children, and babies of apple juice and found the levels of 28.1, 67.5, and 110 ng/kg bw/day, respectively. Piemontese et al. [38] from Italy have reported PAT's daily intake in different age groups and documented the levels ranging from 0.22 to3.41 ng/kg bw/day with a mean consumption of 21 g per day.  The body average weight = 70 ± 2. The body average weight = 70 ± 2.

Conclusions
In the current study, a considerable amount of PAT was observed in citrus fruit samples from central cities of Punjab and Pakistan's Northern cities. The results showed a significant difference in PAT levels from central cities of Punjab compared to Northern Cities of Pakistan. About 22.1% of citrus fruits from both locations have concentrations of PAT greater than the proposed limits established by the EU. The dietary intake of 1.11 and 1.93 µg/kg bw/day from masambi (Multan) and sweet orange (Swat) was determined. The high levels of PAT in citrus fruits could pose a significant health hazard for local consumers. Therefore, regular monitoring of fruits and their products would be desirable every sixth month, and recommendations should be shared with farmers, traders, exporters, and consumers.