**2. Material and methods**

The chemicals and reagents such as the stock standard solution (100 mg/kg) for pesticide Baycarb, Carbaryl, Diazinon, Dursban, Metalaxyl, Propamocarb, Thiamethoxam and Thiobencarbwere purchased from AccuStandard® (New Haven, USA). The LC–MS grade organic solvents that include ACN and methanol were purchased from Merck (Germany). The formic acid was purchased from Fisher Scientific. The Millipore-filtered (deionized) water was obtained using Merck Millipore water purification system (Billerica, USA). While, the apparatus and equipments that include the 100 and 500 μL microsyringe were purchased from Agilent (Australia). The pH meter PB was purchased from Sartorius group (Germany). The HPLC autosampler vials were purchased from Agilent Technologies (USA). The Supelco HPLC column [Ascentis® Express C18 (5 cm x 2.1 mm, 2.7 μm)] was purchased from Sigma-Aldrich (USA). And theliquid chromatography–tandem mass spectrometry (LC–MS/MS) [triple quadrupole (G6490A) built in Electrosprays ESI (±) MS/MS Sensitivity and Jet stream Technology] instrument was purchased from Agilent (Singapore).

#### **2.1 Conditioning of the LC–MS/MS**

The following contributory parameters of the LC–MS/MS instrument were setup initially that include; analyte injection volume (5 μL), flow rate (0.1 mL/ min), column temperature (30°C), gas temperature (200°C), nebulizer gas (45 psi), gas flow (14 L/min), sheath gas temperature (400°C), capillary voltage


*Mobile Phase Selection by Optimization for the Determination of Multiple Pesticides... DOI: http://dx.doi.org/10.5772/intechopen.99029*

**Table 2.**

*The list of suggested and reported mobile phases used for the optimization.*

(3000 V), sheath gas flow (11 L/min), and delta(+) EMV (200 V). However, these factors contributed in determining optimum fragmentor voltage and the four-fragmentor product ions with their respective retention time (RT) and collision energy (CE) (**Table 2**). Moreover, the instrumental default settings were further used for the development of the best gradient program runs for the mobile phase-B elution time by adopting and modifying the methods used by Rajski *et al.* [35] and Vázquez *et al.* [39] for analysis of similar multi-pesticide compounds. This results in the best shortest elution time, which provided the best total ion chromatography (TIC) peaks resolution for the LC–MS/MS instrumentation (**Figure 2**). However, TIC resolution providedan optimum condition for the attainment of higher total chromatographic peak area (TCPA) [42] and mathematically expressed in Eq. (1) [43].

Therefore,

$$T \mathbf{CPA} = \sum \mathbf{CPA} \tag{1}$$

Where *TCPA*: The total chromatographic peak area; *CPA*: The chromatographic peak areas.

**Figure 2.** *The total ion chromatography (TIC) of the analyzed pesticide standards.*

Notably, the best setup of mobile phases were also selected using the initial settings of the instrument. Therefore, the TCPA obtained from LC–MS/MS analysis serves as an index used for estimating the number of target analytes that are present in the analyzed samples [31]. It is because of the close similarities range of the resulted peak areas due to the log*P* of targeted analytes. Moreover, the peak areas maybe correlated and categorically suitable for multiple pesticides analysis using the LC–MS/MS instrument [44].

#### **2.2 Sample treatment and methodology**

The stock standard solution of 100 μg/mL that is equivalent to 100 mg/kg (i.e. 100,000 μg/kg) or parts per million (ppm) [45] for each pesticide was diluted to 10, 1 and 0.1 mg/kg (100 μg/kg) with appropriate volumes of methanol. The appropriate volumes were calculated using the dilution formula as expressed in Eq. (2) [46], separately. Afterward, the prepared working standard solutions were preserved in a refrigerator at 4°C before carrying out the LC–MS/MS analysis.

$$\mathbf{C\_1C\_2} = V\_1V\_2 \tag{2}$$


*ATCPH, average total chromatographic peak height; ATCPA, average total chromatographic peak area; RT, retention time; AF, ammonium formate; FA, formic acid; STDEV, standard deviation; Ref, reference.*

#### **Table 3.**

*The ATCPH and ATCPA instrumental responses for the selection of mobile phase.*

*Mobile Phase Selection by Optimization for the Determination of Multiple Pesticides... DOI: http://dx.doi.org/10.5772/intechopen.99029*

Where *C*<sup>1</sup> : The concentration of the stock standard solution, *C*<sup>2</sup> : The concentration of the working standard solution; *V*<sup>1</sup> : The volume of the stock standard solution; *V*<sup>2</sup> : The volume of the working standard solution.

Meanwhile, the selection of the LC–MS/MS mobile phase was carried out by optimization technique using one factor or variable at a time (OFAT or OVAT) based on the documentation of Sherma [47]. However, the multivariate optimization technique was not favorable for the selection because responses for each of the mobile phase is required individually without interaction to estimate the actual effect of the mobile phase setup. Moreover, the two setups of mobile (organic and aqueous) phases are involved with interactive percentage flow of organic/aqueous changes to create an optimum condition of analytes detection.

Thus, comparative analysis was carried out on some assumed and selected mobile phases reportedly used for analysis of pesticides in various samples. Experimentally, the comparative analysis was carried out on the multipesticide mixture of 0.1 mg/kg multi-pesticide mixture of standard solutions. Consequently, the TIC of the instrumental runs for each of the mobile phases resulted in chromatographic peak heights (ATCPH), and areas (ATCPAs) as presented in **Table 3**. Then again, the addition of organic solvent into aqueous mobile phase could provide the optimum condition of log*P*, which contributes to the attainment of good condition for the multi-pesticide residues analysis in food samples using LC–MS/MS instrument as revealed [41]. For this reason, optimization was carried out by serial addition of ACN into the aqueous mobile phase (0.1% FA milli-Q-water). Thus, the mobile phase setup that provided the best separation of analytes and the highest TCPA was selected for further optimization by adding 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 7.5 and 10% ACN in mobile phase A. Moreover, the best pH solution was selected based on the results of the average TCPA responses of the LC–MS/MS instrument.

#### **3. Results and discussion**

The responses of the screened mobile phases were compared and recorded. The mobile phase setup [0.1% formic acid in Milli-Q-water (A) and 0.1% formic acid in ACN (B)] was the best based on the highest results obtained [ATCPAs ± standard deviation (STDEV) as well as ATCPH± STDEV)] in triplicates as tabulated and illustrated in **Table 3** and **Figure 3**, respectively. This result was also supported by other findings using the mobile phase for pesticides analysis [48, 49]. Meanwhile, further optimization result of mobile phase-A after addition of ACN (0–10%) revealed that the addition of 1% ACN into 0.1% FA Milli-Q-water at an average pH of 3.50 ± 0.07 STDEV (mobile phase A) coupled with 0.1% FA in ACN at pH 6.56 ± 0.04 STDEV (mobile phase-B) provided the highest ATCPA (**Table 4**). The results were supported by their respective pH readings as shown in **Table 4** and **Figure 4**, respectively. Moreover, the retention time (min) of the pesticide analytes were less than the results reported by some literatures such as thiamethoxam, 2.68 < 2.87 [50]; propamocarb, 1.36 < 1.47 [51]; carbaryl, 7.16 < 16.0 [52]; metalaxyl, 7.33 < 17.90 [53]; thiobencarb 10.34 < 10.76 [54], and dursban, 11.36 < 12.30 [55]. But the retention time (min) of baycarb (8.34) and diazinon (10.22) were more than 6.73 [56] and 7.09 [57] respectively. Fortunately, the optimized mobile phase contributes towards shortening the total run time (min) and improved the instrumental sensitivity of the LC–MS/ MS towards better analysis of multiple pesticides.

#### *Biodegradation Technology of Organic and Inorganic Pollutants*



*FA, formic acid; ApH, average pH reading; ATCPA, average total chromatographic peak area; STDEV, standard deviation.*

#### **Table 4.**

*The instrumental responses for the optimization of the selected mobile phase.*

*Mobile Phase Selection by Optimization for the Determination of Multiple Pesticides... DOI: http://dx.doi.org/10.5772/intechopen.99029*

#### **Figure 4.**

*Comparative illustration for the optimization of the selected aqueous mobile phase by ATCPA and ApH readings.*

## **4. Conclusion**

The selection and optimization of the best mobile phase setup was successfully carried out. Eventually, the optimized mobile phase setup [1% ACN and 0.1% FA in Milli-Q-water (mobile phase A) coupled with 0.1% FA in ACN (mobile phase-B)] improved the instrumental sensitivity on the targeted analytes. Thus, this justify the potential benefits of optimizing setup of the mobile phases prior to LC–MS/ MS instrumentation of multi-pesticide analytes. Also, the selected and optimized mobile phase setup could be used for the analysis of other contaminants with similar properties to the analyzed pesticide compounds.

#### **Acknowledgements**

The authors acknowledge the Postgraduate Research Project (IPPP) for supporting this research under the grant no. PG 174-2014B, University of Malaya Kuala Lumpur, Malaysia.

#### **Conflict of interest**

The authors of this research agreed with no conflicts of interest.
