Sensitive and accurate determination of 168 micropollutants including pharmaceuticals and pesticides in surface water and wastewater samples with direct injection using jet stream ESI LC-MS/MS

ABSTRACT This paper is to exhibit validation studies of developed analytical method for the simultaneous determination of 168 micropollutants including pharmaceuticals and several pesticides in surface water and wastewater samples with direct injection using jet stream ESI LC-MS/MS. Method development in the LC-MS/MS system was carried out to determine the best chromatographic column and MS conditions allowing the retention and the maximum number of analytes. The linearity of the analytical response across the studied range of 0.5–500 ng/L was evaluated as excellent, with correlation coefficients higher than 0.995 with a few exceptions such as bentazone (0.993), carbyl (0.994), etoxazole (0.994), fenthion (0.978), fludioxonil (0.985), metformin (0.844), metolachlor (0.992), naproxen (0.990) and sulfametoksazol (0.993). The LOD of 67% (113 analytes) of the analytes included important pesticides and pharmaceuticals was in the range of 0.5–2.0 ng/L. The recoveries of analytes in wastewater ranged mostly from 80% to 120% and RSD (%) values changed generally between 1 and 10. The recoveries of analytes in surface water were in the range of 80% and 100% and relevant RSD (%) values changed generally between 2 and 8. Repeatability studies were performed in the wastewater injected with three different concentrations of 25, 50 and 100 ng/L in seven measurements and in surface water with 100 ng/L in eight measurements. Reproducibility studies were performed by seven analyses on two different days. The matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and the standards prepared in the matrix, and the matrix effect was found in a range of 16–22%. In real samples, Propiconazole, Imidachloprid, Quinoxyfen, Carbendazim and Prochloraz are the five compounds detected in the highest amount in real samples. The results demonstrate that this LC-MS/MS method is suitable for the determination of pharmaceuticals and several pesticides in water samples.


Introduction
The protection of water resources and the recovery of all kinds of water have become inevitable in terms of human health and ecosystem with the climatic conditions that are getting harder day by day.Unfortunately, this resource, which is vital for life, is constantly polluted by organic pollutants from different chemical families in varying amounts as a result of increasing industrial activities and the consequences of human life.Considering the studies in the literature, pesticides, industrial pollutants, antibiotics, pharmaceuticals, personal care products and flame retardants are among the chemicals that cause water pollution [1][2][3].These chemicals took their place in the international regulations such as EU Water Framework Directive (2000/60/EC), Prevention and Control Regulation (PCR) and the Control of Pesticides Regulations (COPR/1986), and their amounts have been limited with the studies about continuous monitoring of waters [4,5].
Pesticides are one of the important groups of chemical compounds used not only in agriculture but also in many areas of life, whose application range is constantly expanding.For this reason, their presence and diversity in the aquatic environment is increasing day by day [6,7].The presence of pesticides in water is directly related to widespread pollution from runoff of agricultural fields.In addition, the presence of pesticides in urban wastewater has reached levels that need to be taken into account, with urban run-offs entering the sewer lines, gardening and landscaping works in urban areas.This situation has revealed that the pesticide amounts in urban wastewater may be in the same range as the most widely used agricultural pesticides in some studies [8][9][10][11].Thus, continuous monitoring of important micropollutants such as pesticides is necessary to assess their potential effects on the aquatic environment.
Another group of chemicals that represent a potentially significant environmental risk are pharmaceuticals.These substances are produced to perform a biological interaction in the human body, and they can be eliminated invariably from the body as a result of biological processes.This situation may cause unexpected results on the ecosystem by being released to the environment as a result of the processes in the wastewater treatment plant [12,13].In the studies conducted in different parts of the world, the results have been obtained that these substances like fluoxetine and acesulfame exist in surface water, groundwater, fresh and sea water bodies, tap water, and also at the inlets and outlets of wastewater treatment plants [14][15][16][17][18][19][20][21].For example, in the USA, Stackelberg et al. (2004) [14] investigated the persistence of pharmaceutical compounds and other organic wastewater contaminants in a conventional drinkingwater treatment plant in their study.In Italy, Castiglioni and co-workers (2005) [15] studied the pharmaceuticals in wastewaters of various sewage treatment plants.In Sweden, Bendz et al. (2005) [16] worked pharmaceutically active compounds in the environment through sewage treatment plants to constitute a health risk for humans and terrestrial and aquatic ecosystems or not.Bartelt-Hunt et al. (2009) [17] in their study exhibited the occurrence of illicit and therapeutic pharmaceuticals in wastewater effluent and surface waters in Nebraska.
In the light of this information, sensitive and rapid measurement of these substances in water and aquatic environment has become more important.With the 'Green Chemistry Approach', the reduction of solvent usage in the pre-treatment stages and solventless pre-treatment, if possible, allows more sensitive measurement [22].Today, wastewater contains most varied micropollutants, e.g.residues of household chemicals, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), organochlorinated pesticides (OCPs), dioxin/furans (PCDD/Fs), dioxin-like polychlorinated biphenyls (DL-PCBs), body care products and pharmaceuticals such as antibiotics and hormone-like substances, which are not eliminated in the wastewater treatment plants.Very low quantities of these pollutants are detectable in waters which may detrimentally affect aquatic organisms/life and drinking water production.Micropollutant analysis can be performed using gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis systems.In these systems, more resources (solvent, energy, etc.) and time are needed as extraction (solid-phase extraction (SPE), liquid/liquid extraction and so on) [23,24] and cleanup (silica, florosil, etc.), and then evaporation is performed in accordance with the accepted international methods (EPA 8141B, EPA 531.1, etc.).However, nowadays, direct injection with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the determination of micropollutants in water ensures that the pre-treatment stages are solvent-free.Accordingly, it can use precise measurements at the ng/L concentration level [25,26].
The purpose of this study was to validate our developed analytical method for the determination of 168 micropollutants including pharmaceuticals and pesticides in surface water and wastewater samples with large volume (100 µL) direct injection using jet stream electronspray ionisation (ESI) LC/MS/MS.This method provides for saving on time by enabling simultaneously measurement of many challenging analytes such as pharmaceuticals and pesticides, which are covered by national and international regulations such as the Water Framework Directive and surface waters, in critical matrices such as surface water and wastewater in the LC-MS/MS system.Also, it ensures significant gains in terms of both the protection of human/environment health and the reduction of cost by eliminating the use of high amounts of solvents/chemicals (green chemistry) that would be spent in pre-treatment stage for the measurement of the same analytes in gas chromatography systems.In addition, it is thought that it will contribute to obtaining acceptable proficiency test results in order for laboratories working in this context to be accredited.Finally, as far as we know, there are a limited number of analytical method studies in the literature that can simultaneously analyse important and critical micropollutants such as pharmaceuticals and pesticides in environmental water samples.

Preparation of stock and working solutions
Individual stock solutions of pharmaceuticals and pesticides from solid standards were prepared by dissolving 10 mg of each substance in 10 mL of acetonitrile at a concentration of 1000 mg/L.A mixed intermediate standard solution containing 168 compounds was prepared by diluting the stock standard solutions of pharmaceuticals and pesticides in acetonitrile at a concentration of 1 mg/L.Stock, intermediate standard solutions were stored at 4°C in amber flasks, and they are found stable for at least 6 months.Intermediate standard solutions were prepared by making gradual dilutions with acetonitrile in 10 ml flask.The working solutions (0.5, 1, 2, 5, 10, 25, 50, 100, 200 and 500 ng/L) to be used in the study were daily fresh prepared with distilled water.

Water samples
Surface water samples were gathered from the surfaces of Alibey lake (Istanbul), Omerli lake (Istanbul) and Sapanca lake (Sakarya) in November 2020.Wastewater samples were collected from the outlets of Istanbul Water and Sewerage Administration (ISKI) wastewater treatment plant (primary treatment) in Kadıköy, Istanbul, in December 2018.All samples were filled in 1000 mL glass bottles so that there were no bubbles in them and tightly closed.Samples were stored below 5°C until analysis was carried out.Samples were analysed within 48 hours of arriving at the laboratory.Some samples were reanalysed one week later to see if there were any deviations in the stability of the samples, and no significant difference was found in the analysis results.

Sample preparation procedure
All the water samples were filtered through a 0.45 µm filter.After filtration, 1 mL of sample was taken into a vial, and it was fortified with 20 µL of internal standard (4 µg/L Metolachlor D 6 ) prior to LC-MS/MS analysis.

LC-MS/MS analytical condition
The chromatographic analyses were performed using an HPLC system consisting of a binary pump

Validation study
Developed analytical method for the determination of micropollutants including pharmaceuticals and several pesticides in surface water and wastewater samples was validated according to the Guidelines for Standard Method Performance Requirements [27] and the Commission Decision EURACHEM Guideline [28] for a Laboratory Guide to Method Validation.The following parameters were evaluated in the validation procedure: selectivity, sensitivity (limit of detection (LOD) and limit of quantification (LOQ)), linearity, accuracy, precision (intraday and interday), trueness and matrix effect.

LC-MS/MS method development
Method development in the LC-MS/MS system was carried out to determine the best chromatographic column and MS conditions allowing the retention and the maximum number of analytes.LC and especially MS parameters were adjusted to get the best detection-quantification limits (highest sensitivity), good repeatability and the optimum separation of analytes of interest.Figure 1 shows the stages of the formation of the jet stream ESI LC-MS/MS method about 168 micropollutants, including pharmaceuticals and pesticides.After the method was formed in the software that allows the maximum number of analytes to be measured together, performance studies of the parameters such as sensitivity and robustness in two different LC-MS/MS systems with standard ESI and jet stream ESI were performed.The results showed that there have not been any problems in both the systems in terms of robustness, and detection limits of the jet stream ESI LC-MS/MS method in the study were 5 times lower in 9%, 7 times lower in 24% and 10 times lower in 67% of the analytes than standard ESI LC-MS/MS.Jet Stream ESI provides higher robustness and sensitivity than standard ESI because of heated sheath gas although it suffers significantly stronger signal suppressions.This situation regarding the detection limits was realised as expected, and the results were obtained in accordance with the literature [29].

Selectivity
The selectivity of the developed method was determined by duplicate analysis of 10 surface water and wastewater samples to check the stability of retention times and the ratio of precursor and product ion signals of each analyte.No peaks of interfering compounds were observed within the intervals of the retention time of the analytes in any of these samples.

Linearity
Linearity in the validation study was identified as the coefficient of determination (r 2 ) and from the slope of the calibration curve.In this study, linearity was evaluated from the calibration curves by triplicate analyses of the reference standard fortified with the analytes at 10 (0.5, 1, 2, 5, 10, 25, 50, 100, 200 and 500 ng/L) concentration levels in acetonitrile.The values of the correlation coefficient (r 2 ) from all calibration curves are illustrated in Table 2. Ferreira et al. (2016) [30] revealed that linear regression with correlation coefficient should be equal to or better than 0.99 (good linearity) in order to evaluate the linearity range in the measurement of an analyte.This is also supported by other similar studies in the literature [1,5,31,32].According to this approach, the r 2 values of the calibration curves of the analytes in the study are higher than 0.99 with a few exceptions such as Fenthion (0.978) and Metformin (0.844).The r 2 value of a significant part of the analytes is 0.999.The linearity of the analytical response across the studied range of 0.5-500 ng/L was evaluated as excellent, with correlation coefficients higher than 0.99 for most of the analytes.Also, Cotton and co-workers (2016) [1] in their study found correlation coefficients for some analytes in common with our study higher than 0.98, which was lower score than our study.

Sensitivity (LOD and LOQ)
The sensitivity of the developed method was specified with the determination of LOD and LOQ. Figure 2 demonstrates MRM chromatograms of surface water sample at the concentration level of 10 ng/L.The LOD and LOQ of each analyte were determined by analysis of 10 surface water samples.The LOD values were calculated as three times of the standard deviation (σ) divided by the slope (S) of the standard curve (LOD = 3σ/S).The LOQ was calculated by analysing 10 surface water samples spiked with concentration at LOD.Then, the LOQ value was added up to 10 times the corresponding standard deviation divided by the corresponding slope of the standard curve (LOQ = 10σ/S).Then, a preliminary experiment was conducted to check if all compounds were detected when spiked at their LOQ level.LOD and LOQ values of each analyte are presented in Table 2.The LOD of 64 analytes, mainly Acetamiprid, Azoxystrobin and Carbofuran was 0.5 ng/L.Detection level of 23 analytes such as Butralin, Diflubenzuron, Malaoxon and Simazine was 1.0 ng/L.Detection levels of 26 analytes including important pesticides and pharmaceuticals such as Acetaminophen, Atenolol, Fenthion and Methiocarb (Mercaptodimethur) were between 1.5 ng/L and 2.0 ng/L.The LOD of 67% (113 analytes) of the analytes included in the study was in the range of 0.5-2.0ng/L, and it is similar to other methods in the literature in which direct injection is made in LC-MS/MS [1,33].It even has a lower LOD than some studies [34,35].The results show that this method can be used to measure a large number of analytes including pharmaceuticals and pesticides simultaneously with very high sensitivity.Only nine analytes (Acetochlor, Aclonifen, Alachlor, Cyclanide, Cyromazine, DCOIT, Diafethriuron, Thidiazuron and Tolclophos methyl) have the highest LOD (6.0-20.0ng/L).

Accuracy, precision and trueness
The proposed method reliability was evaluated by accuracy, which is defined as the recovery test.The results of mean recovery and the relevant RSDs as percentage for each pharmaceutical and pesticide at spiked levels are illustrated in Table 2. Accuracy studies were carried out for the wastewater spiked at three different concentrations of 25, 50 and 100 ng/L in seven measurements and in surface water with 100 ng/L in eight measurements.The recoveries of analytes in wastewater ranged mostly from 80% to 120% and RSD (%) values were generally found below 20 with a few exceptions such as Fluquinconazole (recovery: 50%), Fenpropathrin (recovery: 69%), and Metformin (recovery: 135%) for 25 ng/L; Carbyl (recovery: 50%) at 100 ng/L.The RSD (%) values of seven analytes (Clarithromycin, Cyclanide, Diafethriuron, Diclofenac, Mepiquat, Thiophonate methyl and Thiobenuron methyl) were computed above 20%.This corresponds to 4% of the total amount of analyte, which is quite normal in a difficult matrix such as wastewater.The recoveries of analytes in surface water were often in the range of 80% and 100% and relevant RSD (%) values changed generally between 2 and 8.These results indicate that this method has substantially good quality for the accurate analysis of related pharmaceuticals and pesticides in wastewater and surface water.As can be seen, the accuracy results of the method were confirmed with validation guidelines for Standard Method Performance Requirements [27].Besides, Cotton and co-workers (2016) [1] for pesticides and drugs (87-90%) in water and Singer et al. (2010) [8] for pesticide (81-98%) in wastewater reported similar recoveries.The recovery of each analyte at each concentration level was calculated by using Eq. ( 1), The precision of the method was determined in two stages: repeatability (intraday) and intermediate precision (interday).The repeatability experiments were performed in the wastewater injected with three different concentrations of 25, 50 and 100 ng/L in seven measurements and in surface water with 100 ng/L in eight measurements.In wastewater, with a concentration of 25 ng/L, the recoveries ranged from 50% to 135% and the percentage of RSD values changed between 1 and 34.In wastewater with a concentration of 50 ng/L, the recoveries changed from 64% to 134% and the percentage of RSD values was found to be between 1 and 27.In wastewater with a concentration of 100 ng/L, the recoveries varied from 50% to 123% and the percentage of RSD values ranged between 1 and 28.In surface water with a concentration of 100 ng/L, the recoveries changed from 51% to 119% and the percentage of RSD values was found to be between 2 and 25.The intermediate precision was expressed by the RSD of the results of 14 analyses performed on two different days (n = 2), seven analyses/day, by the same analyst using the same instrument.In interday studies, the relative standard deviation (RSD) of pharmaceuticals and pesticides analysed by the present method was 2% to 27% and the recoveries ranged from 57% to 126% (Table 2) [27].In summary, it has been seen that this method has stable and sufficient applicability with its intraday and interday results.The performance of the developed jet stream ESI LC-MS/MS method was tested by evaluating with regard to trueness.From this perspective, trueness study defines how close the mean value (obtained from the method) of a series of studies is to the actual value [28].The trueness of some pesticides in this study was checked with PriorityPollutnT coded inter-laboratory test (P276-908, P276-718, P276-665) issued from ERA-A Waters Company.The results obtained from the proficiency test are shown in Table S1.The z-scores changed between −0.504 and 1.73 were satisfactory and comparable to those obtained by other laboratories participating in the round.Inter-laboratory test refers that the developed method is rather good and sufficient capability for the rapid and sensitive quantification of some pesticides in wastewater samples.

Matrix effects
The evaluation of matrix effect is important during validation of analytical methods using the ESI LC-MS/MS technique.The ionisation efficiency of the analytes in the ESI source may be affected by matrix interferences.The data about the rate of signal suppression or enhancement of related pharmaceuticals and pesticides spiked 100 ng/L to surface water and wastewater are shown in Figure 3(a).Ninety-two percent of analytes in wastewater and 97% of analytes in surface water were determined in the range of 80-120% recovery.Only Acetaminophen exceeded the 120% recovery rate in wastewater.Eleven analytes (such as Capecitabine, Diltiazem, DMSA, Fluquinconazole, Phenthoate) in wastewater and three analytes in surface water (like Butralin, Capecitabine, Fenbutatin Oxide) were detected below 80% recovery, and signal suppression due to the matrix effect occurred.In order to evaluate the degree of ion suppression or signal enhancement, the calibration curves were established with and without the matrix.Matrix-induced effects were assessed by comparing the slopes of these calibration curves using the following formula: Matrix effect (ME) = 1-(a matrix /a standard ) x100, where a matrix and a standard are the slopes of calibration straight lines for standard and matrix-matched calibration graphs [30].The matrix-matched calibration curves were constructed using wastewater samples spiked with pharmaceuticals and pesticides at concentration levels of 25 ng/L, 50 ng/L and 100 ng/L (Figure 3(b)).Matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and standards prepared in matrix and the matrix effect was found a range of 16-22%.These results showed that standard calibration, which was simpler and less time-consuming compared with matrix-matched calibration, could effectively be used for the quantitation of pharmaceuticals and pesticides in wastewater (Table 2).

Real samples
In Turkey and different parts of the world, the presence of pharmaceuticals and pesticides in the water has been controlled on the basis of the thresholds set out the relevant international and national regulations such as European Union's Drinking Water Directive (2020/2184), Prevention and Control (PPC) Regulations, the Food and Environmental Protection Act (FEPA/1985), the Control of Pesticides Regulations (COPR/1986), the United Kingdom (UK) Surface Waters (dangerous substances) Regulations (SI 1997/2560), and Turkey Surface Water Quality Regulation (environmental quality standard [annual average] in surface water [36][37][38].The proposed method was used in the analyses of 600 surface water and wastewater samples submitted to the laboratory for micropollutants by the region of Alibey lake (Istanbul), Omerli lake (Istanbul), Sapanca lake (Sakarya) and Istanbul Water and Sewerage Administration (ISKI) wastewater treatment plant in Kadıköy (Istanbul).Two transition ion pairs were  monitored for each of the analytes, and the ion ratios of the detected samples were compared well with those of standards.Table 3 presents important information about the study of real samples such as detected compounds, their minimum and maximum concentrations and the number of samples with positive results.Before the analysis of the relevant samples, retention times and precursor and product ions of analytes were also confirmed by the addition of known standards in the detected samples.In the real samples, there were no interfering compounds that would lead to false-positive or false-negative results in the chromatogram.As seen in Table 3, Acetochlor, Acetamiprid, Thiamethoxam, Carbendazim and Terbutryn are the five most detected compounds in the samples.Propiconazole (4155 ng/L), Imidacloprid (2979 ng/L), Quinoxyfen (822 ng/L), Carbendazim (773 ng/L) and Prochloraz (772 ng/ L) are the five compounds detected in the highest amount in the real samples.Chlorfenvinphos, Diazinon and Carbofuran were found in only one sample and at almost the lowest concentrations.Quantification of the samples was done by comparison with a seven-point calibration (0.5, 1, 2, 5, 10, 25 and 50 ng/L) in solvent reagent calibration.

Conclusions
A multi-class analysis procedure using jet stream ESI LC-MS/MS has been developed and validated for the simultaneous determination of 168 micropollutants including pharmaceuticals and several pesticides in surface water and wastewater samples.It has a very simple sample preparation method, such as filtering the water samples through a 0.45 µm filter prior to LC-MS/MS analysis, there is no need for any solvent extraction and clean-up steps.Method development in LC-MS/MS system was performed to determine the best chromatographic column and MS conditions allowing the retention and the maximum number of analytes.The validation of this method was performed according to Guidelines for Standard Method Performance Requirements [27] and the Commission Decision EURACHEM Guideline [28].The developed analytical method has been tested with parameters such as selectivity, sensitivity (LOD and LOQ), linearity, accuracy, precision (intraday and interday repeatability), accuracy and matrix effect of pharmaceuticals and various pesticides below the analytical level recommended in the relevant regulation and/or legislation.The linearity of the analytical response across the studied range of 0.5-500 ng/L was assessed to be excellent, with correlation coefficients higher than 0.99 with a few exceptions such as Fenthion (0.978) and Metformin (0.844).The LOD of 67% (113 analytes) of the analytes included important pesticides and pharmaceuticals such as Acetamiprid, Acetaminophen, Azoxystrobin, Butralin, Diflubenzuron and Fenthion was in the range of 0.5-2.0ng/L.The recoveries of analytes in wastewater ranged mostly from 80% to 120%, and RSD (%) values changed generally between 1 and 10.The recoveries of analytes in surface water were in the range of 80% and 100% and relevant RSD (%) values changed generally between 2 and 8.In the intraday, the recoveries ranged from 90% to 115% in general, and the percentage of RSD values changed between 1 and 18 in the wastewater injected with three different concentrations of 25, 50 and 100 ng/L at seven measurements and in surface water with 100 ng/ L in eight measurements.In interday studies, the relative standard deviation (RSD) of pharmaceuticals and pesticides analysed by the present method was 2% to 27% and the recoveries ranged from 57% to 126%.Based on the obtained results, jet stream ESI LC-MS/MS method indicated the suitability for sensitive quantification of 168 micropollutants including pharmaceuticals and several pesticides in surface water and wastewater samples for environmental safety applications.The validated method was applied on 600 surface water and wastewater samples.Acetochlor, Acetamiprid, Thiamethoxam, Carbendazim and Terbutryn are the five most detected compounds in the samples.Propiconazole, Imidacloprid, Quinoxyfen, Carbendazim and Prochloraz are the five compounds detected in the highest amount in the real samples.Chlorfenvinphos, Diazinon and Carbofuran were found in only one sample and at almost the lowest concentrations.This short analysis procedure can be suitable for a large number of water samples for routine analysis and rapid detection.

Figure 1 .Table 2 . 19 *
Figure 1.Steps for the formation of the jet stream ESI LC-MS/MS method for micropollutants including pharmaceuticals and pesticides.

Figure 2 .
Figure 2. MRM chromatograms of the surface water sample spiked with 10 ng/L of micropollutants including pharmaceuticals and pesticides.

Figure 3 .
Figure 3. Matrix effect: a) recovery of analytes in wastewater and surface water samples, b) RSD results of analytes in different concentrations of wastewater.

Table
. LC-MS/MS working conditions for all compounds.

Table 1 .
(Continued).Aldrich (St. Louis Missouri, USA) (43 compounds such as Azoxystrobin, Etoxazole, Fenthion and Simazine) and Chem Service (West Chester, Pennsylvania, USA) (26 compounds such as Carbayl, Lenacil and Quinoxyfen).Metolachlor D 6 used as internal standard was provided from Dr. Ehrenstrofer (Augsburg, Germany).LC-MS/MS grade methanol, acetonitrile (ACN), (Lichrosolve purity ≥99.9), formic acid, acetic acid (Emprove, 100%), and ammonium formate were purchased from Merck (Darmstadt, * Internal standard; DCOIT: Dichlorooctylisothiazolinone; DMSA: Dimercaptosuccinic acid; ETU: Ethylenethiourea and Terbutryn), Sigma- Waldbronn, Germany) was tested.The method used gradient mobile phases containing 0.1% formic acid in water (mobile phase A) and 5 mM ammonium formate in methanol (mobile phase B).The column temperature was maintained at 30°C with a flow rate of 0.6 mL/min.The gradient profile was scheduled as follows: 0 min, 80% A; 0-0.2 min, 80% A; 0.2-2 min 30% A; 2-6 5% A; hold at %5 A 2 min; return to %80 A for 4 min (total run time 12 min).The injection volume was 100 µL.The mass spectrometric system was coupled to the Jet Stream Electrospray Ionisation (ESI) source followed by an Agilent 6490 LC-MS.The MS/MS detector conditions were as follows: Gas flow: 11 L/min, nebuliser gas pressure: 30 psi, sheath gas temperature: 400°C, sheath gas flow: 123 L/min and scan type: dynamic MRM.The capillary voltage was set at 3000 V. MS data were acquired in the positive ion ESI mode using MS/MS scan events.Two transitions were monitored for each analyte.The selected molecular ion and optimised collision voltages of product ions used for quantification, confirmation and ion ratio are summarised in Table1.Agilent 6460 LC-MS/MS software Mass Hunter version B.05.01 was employed for data acquisition and processing.

Table 3 .
Overall results of the study on real samples.