Physico-Chemical Condition Optimization during Biosynthesis lead to development of Improved and Catalytically Efficient Gold Nano Particles

Biosynthesis of nanoparticles has gained great attention in making the process cost-effective and eco-friendly, but there are limited reports which describe the interdependency of physical parameters for tailoring the dimension and geometry of nanoparticles during biological synthesis. In the present study, gold nanoparticles (GNPs) of various shapes and sizes were obtained by modulating different physical parameters using Trichoderma viride filtrate. The particles were characterized on the basis of visual observation, dynamic light scattering, UV-visible spectroscopy, transmission electron microscopy, fourier transform infrared spectroscopy, and X ray diffraction. While the size varied from 2–500 nm, the shapes obtained were nanospheres, nanotriangles, nanopentagons, nanohexagons, and nanosheets. Changing the parameters such as pH, temperature, time, substrate, and culture filtrate concentration influenced the size and geometry of nanoparticles. Catalytic activity of the biosynthesized GNP was evaluated by UV-visible spectroscopy and confirmed by gas chromatography-mass spectrometric analysis for the conversion of 4-nitrophenol into 4-aminophenol which was strongly influenced by their structure and dimension. Common practices for biodegradation are traditional, expensive, require large amount of raw material, and time taking. Controlling shapes and sizes of nanoparticles could revolutionize the process of biodegradation that can remove all the hurdles in current scenario.


Results and Discussion
Biosynthesis and characterization of gold nanoparticles. Biosynthesis of gold nanoparticles was evident from the color change of the reaction mixture and confirmation was done by UV-visible spectroscopy (Fig. 1A). The reduction process was very quick and color change was evident 10 min after addition of HAuCl 4 16 .
Gold nanoparticles are known to exhibit a range of colors according to their shapes and sizes, due to surface plasmon resonance and accordingly can be characterized by obtaining the characteristic peak in 500-600 nm range 25 . The crystalline nature of gold nanoparticles was confirmed by X-ray diffraction (XRD) studies (Fig. 1B). The XRD pattern recorded from the solution cast film of T. viride reduced gold nanoparticles, showed a very intense Bragg reflection at 38.6, 44.38, 64.57 and 77.5 degree corresponding to (111), (200), (220) and (311) crystal lattice of face-centered cubic (FCC) gold. Fourier-transform infrared spectroscopy (FTIR) was carried out to identify the biological moieties involved in biosynthesis. The FTIR spectrum of the cell-free extract and the biosynthesized gold nanoparticles (Fig. 1C,D) exhibited intense and distinct absorption bands at 757.58 and 1215.2 cm −1 . The intense absorbance at 757.58 cm −1 corresponds to C-H "oop" of aromatics while 1215 cm −1 can be assigned to C-N stretch of aliphatic amines. The FTIR spectrum also showed bands at 668.96, 2855.06, 3020, and 3374.82 cm −1 with some other bands. The band at 668.962 cm −1 corresponds to C-Br stretch of alkyl halides and 2855.06 cm −1 demonstrates H-C= O: C-H stretch of aldehydes. The medium band at 3020 cm −1 demonstrates = C-H stretch of alkenes and band at 3374.82 cm −1 can be assigned to O-H stretch of alcohols and phenols. The results clearly indicated the role of several biological moieties, including proteins and organic compounds which might be the major components of secondary metabolites in biosynthesis of nanoparticles. Secondary metabolites of Trichoderma are rich in non-ribosomal peptides, peptaibols, siderophores, polyketides, terpenoids/steroids, pyrones and hydrolytic enzymes 25 . These organic moieties contain several phenolic groups and their derivatives which can take part in redox reaction generating quinones and donating electrons. These electrons can further reduce and stabilize the metallic ions into nanoparticles. Many reports of biosynthesis have demonstrated the role of organic compounds and proteins in reduction and stabilization of nanostructures 15,25 . Morphological observations of biosynthesized gold nanoparticles. A wide range of colors were observed at different conditions of pH, temperature, reaction time, salt, and extract concentrations (Fig. S1). Dynamic light scattering (DLS) size also indicated the reduction of gold ions and their conversion into nanoparticles of different sizes with good monodispersity (Figs S2 and S3). DLS provides the size of whole conjugate including size of individual nanoparticles and the ligand shell surrounding them, thus providing larger size than obtained during TEM measurements 15 . Effect of each parameter with respect to the other was studied in detail (Table 1) and a mechanisms/suitable conditions were proposed for obtaining monodispersed population of a single geometry.
Effect of concentration of cell free extract. Among 20 and Song et al. 26 have suggested the reduction in particle size with increase in cell free extract concentration. Contrary to these results, in this study by decreasing the concentration of reducing agent (cell free extracts), reduction in particle size was obtained. Higher concentration of reducing agents might increase the catalytic activity which leads to faster reaction, thus size of nanoparticles increased in presence of higher concentration of cell free extract. Higher concentration of cell free extract increased the concentration of reducing agent, thus increasing the reaction rate which resulted in rapid growth of nanoparticles 27 , so increase in size was observed as the concentration increased.
Effect of concentration of HAuCl 4 . Two concentrations of gold salt viz. 250 and 500 mg/L were taken and their effect on shapes and sizes at different temperatures viz. 30, 40 and 50 °C were observed with 10% cell free extract. At the lower concentration, particles formed were smaller as compared to the particles formed at 500 mg/L (Figs 2b, S4b and S11). Temperature also played a crucial role at both the concentrations. At 30 °C, 250 mg/L, (G10.250.7.72.30) mixed population of particle were synthesized having mean size of 34 nm, while at 500 mg/L (G10.500.7.72.30), particles were predominantly hexagons of 85.2 nm. At 40 °C, nanoparticles were triangular at both concentrations, while the mean size increased from 164 (G10.250.7.72.40) to 335 nm (G10.500.7.72.40) when concentration of gold salt was increased from 250-500 mg/L. At 50 °C, very large triangles of mean size 699 nm were formed at 500 mg/L (G10.500.7.72.50), while triangles of 273.6 nm were biosynthesized at 250 mg/L (G10.250.7.72.50). At higher concentration of gold salt, the cell free extract concentration becomes less, resulting in insufficient capping and stabilizing action of reducing agents 20 . This situation resulted in exposure of different facets on surface, which after random collisions and fusion of nuclei formed larger nanoparticles. As the temperature was increased, there was an increase in the rate of reaction resulting in the formation of small nuclei quickly 26 , which might further collide rapidly and randomly to form larger particles at higher concentrations and higher temperature at a given interval of time.
Effect of pH. Among the growth parameters, pH plays a substantial role on the oxidation state and reducing power of enzymes and secondary metabolites present in cell free extract 20 . A wide pH range was tested for biosynthesis of gold nanoparticles at 30 °C, time interval of 72 h and HAuCl 4 concentration of 250 mg/L (Figs 3 and S12). At pH 5.0 (G10.250.5.72.30) particles of varying shapes i.e large prisms, triangles, pentagons, hexagons and rods were synthesized, with size range of 5-200 nm. As the pH was increased, the shape of the particles changed to star/hexagonal at pH 5. , which were further converted into penta/hexagons of size 50-80 nm at pH 8.5(G10.250.5.72.30). When the pH was further increased to 9.0 (G10.250.9.72.30), particle size went down to 3-10 nm and shape changed to spherical and it changed to irregular at pH 9.5 and 10.0(G10.250.9.5.72.30 and G10.250. 10.72.30). It was observed that the particle size decreased with increase in pH. At lower pH, a mixed population was synthesized, similar to the observations of Armendariz et al. and Sneha et al. 18,28 who demonstrated the synthesis of tetrahedral, hexagonal, decahedral and rod-shaped nanoparticles at pH 2.0-6.0. Each methodology involves two steps for gold nanoparticles synthesis: (I) nucleation and (II) crystal growth 27,29 . At lower pH, repulsion between negatively charged AuCl 4 − ions and carboxylic group of extract is reduced resulting in uncontrolled nucleation of seeds and formation of larger mixed shape particles 18 . Lower pH, with due course of time allows coagulation of smaller nuclei resulting in larger colloids 30 . After 72 h of reaction time, at pH 5, smaller nuclei aggregated, forming larger particles. At neutral range of pH (6.0-8.0), 70-80% of population obtained consisted of nanotriangles and prisms. This range of pH is predominated by AuCl 4 −30 and most of the active biological moieties, predominantly synthesized nanoprism in due course of time. As the pH was increased to 9.0, ≥ 90% of the population consisted of spheres with significant decrease in size to 3-10 nm. Earlier, role of higher pH in synthesis of small nanospheres has been discussed by many researchers 18,20,31,32 . At higher pH, Cl − ions present in AuCl 4 − get substituted by OH − present in cell free extract resulting in repulsion between the two negatively charged moieties of cell free extract and gold ions, thereby reducing the possibility of further growth of nuclei maintaining them as small spheres.
Effect of reaction time. Effect of time on morphology of gold nanoparticles biosynthesized was studied at 24, 48 and 72 h of time interval at three different pH 5.0, 7.0 and 9.0 (Figs 4 and S13, Table 1). After 24 h, at pH 5.0 (G10.250.5.24.30), 100% of the particles synthesized were small spheres (7-24 nm), indicating the initiation of nucleation, which was gradually followed by synthesis of mixed population of spheres, triangles and prisms of larger size (7-  Effect of temperature. To study the effect of temperature on morphology of gold nanoparticles, experiments were carried out at 20, 30, 40 and 50 °C at different gold ion concentrations (250 and 500 mg/L) and pH (5.0, 7.0 and 9.0) (Figs 5 and S5, S6, S14, S15, S16; Table 1).
Biosynthesis of gold nanoparticles at 50 °C yielded large prisms of 200-600 nm irrespective of the pH and gold ion concentrations. This temperature was neither low nor very high for growth of nuclei. It was an optimum temperature for generation of activation energy required for catalytic activity to synthesize larger prisms. At 40 °C, and at pH 7.0 (G10.250.7.72.40) and 5.0 (G10.250.5.72.40), there was formation of mixed population, while percentage of nanoprisms was more in 500 mg/L (G10.500.7.72.40 and G10.500.5.72.40) of HAuCl 4 concentration (Figs 5, S5 and S14). However, at pH 9.0 (G10.500.9.72.40), 82% population observed was pentagons and hexagons. A transition phase was created during nucleation and crystal growth at 40 °C and pH 9.0. This situation becomes favorable for formation of pentagons and hexagons (Fig. 6). Crystal growth without any hindrance could lead to formation of nanoprisms at lower pH, but at higher pH, due to decrease in crystal growth because of repulsion between the negatively charged groups, evolution in morphology was slow, resulting in penta/ hexagonal particle synthesis.
Similarly   Table 1 for detail).
When the reaction was carried out at 20 °C and at the pH 9.0 (G10.250.9.72.20), pH factor played the dominant role for the formation of small nanoparticles (10-60 nm). At pH 7.0 (G10.250.7.72.20), the geometry of the particles became more uniform. 70% of the total population consisted of nanosheets, not reported yet by any biological methods, while the rest comprised of pseudospheres of 10-50 nm. At pH 5.0 (G10.250.5.72.20), nanoparticles biosynthesized consisted of a mixed population of triangles, prisms, rods and penta/hexagons of various sizes at both the HAuCl 4 concentrations. The mechanism involved behind this phenomenon is yet to be understood.

Conditions for synthesis of multishaped gold nanoparticles and the proposed mechanism.
Suitable conditions were deciphered to synthesize the desired shape and size of nanoparticles by regulating combination of pH, reaction time, temperature, concentration of gold salts, and cell free extract (Table 1 and Fig. 7).
Spherical nanoparticles. G10.250.9.24.30, G10.250.9.48.30, G10.250.9.72.30 and G10.250.5.24.30 were the 100% homogeneous spherical GNPs (Fig. S7). For obtaining monodispersed population of nanospheres, it is necessary to stop the growth of nuclei. This can either be achieved at very high temperature 16 , or at high pH at 30 °C as reported in our study, where repulsion was higher between the negatively charged biological moieties and chloroaurate ions. Spherical nanoparticles can also be formed by inhibiting further crystal growth after development of nuclei by stopping reaction at an early stage (Fig. S7).  (Fig. S8). At pH 5.0 and 7.0, there was no hindrance in the growth of crystals at 40 °C, 72 h, leading towards formation of nanotriangles. After 48 h, nanotriangles were observed which were smaller in size. When the incubation was beyond 24 h, the nuclei increased in size (upto 400 nm) but shape remained the same. Mukherjee et al. 21 have also demonstrated the evolution of morphology from sphere to triangles in a time dependent manner. Time plays a major role in nanotriangle formation but the major constraint  Table 1 for detail). associated with this is the growth of the crystal along with formation of nanoprisms. Biosynthesis of smaller triangles and prisms below 50 nm still remains a mammoth task to be achieved.  Table 1 for detail). Penta and hexagon nanoparticles. Favourable situations for biosynthesis of penta and hexagons were obtained at G 10.500. 9.72.40 (80-85 nm) and at G10.500.7.72.30 (Fig. S8). In the first case, at higher pH, growth of crystal is retarded while the rate of reaction is high at high temperature, so after 72 h of reaction, spherical nuclei could have been converted into hexagons and pentagons instead of triangles, because of two opposing forces of temperature and pH were working in synergism. In the second case, at pH 7.0 and 30 °C, all conditions were favorable for the reaction where concentration of gold salt might play an important role. Insufficient amount of the cell free extract to cover all gold ions led to incomplete capping 23 of salt, leading to selective growth for the construction of penta and hexagonal nanoparticles. Star shaped hexagons were observed at pH 5.5 indicating the evolution of morphology from spherical seeds to triangles.
Nanosheets. Nanosheets were synthesized at pH 7.0, 250 mg/L concentration of gold salt, 20 °C after 72 h of reaction (G10.250.7.72.20) (Fig. S8). However, on keeping the conditions same, when temperature was changed to 30 and 40 °C, nanotriangles were formed. There might be fusion of small nuclei to form straight chains, which when clubbed together gave the appearance of a sheet. There might also be the effect of temperature on formation of nanosheets, the mechanism of which still needs to be explored.

Effect of different morphologies of GNP in catalytic conversion of 4-nitrophenol.
Biogenic nanoparticles have significantly higher catalytic activity due to presence of protein corona on their surface which acts as an effective host for the substrate 33 . Different shapes of particles obtained in our study viz: spheres, triangles, penta/hexagons and sheets (Fig. 6) were evaluated for their catalytic activity. Among the particles of different shapes, spherical gold nanoparticles (3-10 nm) acted as the best catalyst, demonstrated a sharp decrease in absorbance at 400 nm followed by penta/hexagonal, triangles and nanosheets (Fig. 8A). The decrease in nitrophenolate ion and disappearance of yellow colour indicated that 4-nitrophenol (4-NP) was converted to 4-aminophenol (4-AP) within 5 min, just after addition of spherical GNPs to the reaction mixture, while it took 30 min and 120 min for complete degradation of 4-NP in case of penta/hexagonal, and triangular nanoparticles respectively (Fig. 8B).
4-NP exhibits an absorption peak at 400 nm in alkaline condition because of formation of nitrophenolate ion 34 . In the presence of gold nanoparticles as biocatalyst, 4-NP was converted into 4-AP resulting in decrease in absorbance at 400 nm and increase in 300 nm due to formation of 4-AP 15 .
Since the concentration of NaBH 4 is much higher than 4-NP, the reduction rate can be assumed to be independent of NaBH 4 concentration. Therefore, the catalytic rate constant (K) in this case can be evaluated by studying the pseudo-first-order kinetics with respect to 4-NP concentration. The kinetics study demonstrated that the rate constant was maximum in spheres (3.94 × 10 −3 section −1 ), followed by penta/hexagonal particles (2.4 × 10 −3 section −1 ), triangles (2.3 × 10 −3 section −1 ), and nanosheets (0.79 × 10 −3 section −1 ). The higher catalytic activity of nanospheres may be attributed to their smaller size and large volume to surface area ratio in comparison to nanoparticles of other shapes, providing maximum number of reaction sites for catalysis. The smaller size results Further, when spherical gold nanoparticles of two different sizes were compared, it was found that particles of 3-10 nm (6.75 × 10 −3 section −1 ), had higher rate constant than particles of 7-24 nm (3.94 × 10 −3 section −1 ).

Comparison of different sizes of spherical GNP for catalytic activity by GC-MS/MS analysis.
The catalytic activtity of two spherical nanoparticles showing maximum rate constant (3-10 and 7-24 nm) were further evaluated by gas chromatography-mass spectrometric analysis (GC-MS). Mixture of 4-NP and 4-AP; obtained at retention time 7.13 and 7.34 respectively (Fig. 9). In blank transformation of 4-NP (RT at 7.13) to 4-AP (RT at 7.34) was also observed without addition of gold nanoparticles after 5 min, but it was not stable and we further observed the starting material (4-NP, RT-7.14) at 10 min along with the product (4-NP, ). This may be due the instability of the product in the absence of any catalyst (Fig. S9). While in treatment of both spherical GNP (3-10   spherical gold nanoparticles. This was clearly evident from the complete conversion of 4-NP into 4-AP, when we use spherical nanoparticles as catalyst which is proved by GC-MS analysis of the reaction mixture. In agreement with the earlier results, smaller nanoparticles (3-10 nm) acted as better catalyst depicting almost double the peak area of 4-AP than the larger spherical nanoparticles (7-24 nm) after 5 and 10 min of addition of GNPs to the reaction mixture (Fig. S9). Though spherical particles of both sizes were excellent catalyst, smaller nanoparticles (3-10 nm) emerged as a better catalytic agent for conversion of 4-NP into an eco-friendly product (4-AP) and it helps to degrade the toxic organic pollutants.
Particles below 10 nm have previously been known for their excellent catalytic properties 24,38 . Decrease in size below 10 nm results in different geometric and electronic properties that strongly affect the adsorption and activation of the reactants, resulting in creation of more catalytic active sites with low-coordinated atoms that are usually located in the defects such as terraces, edges, kinks, and vacancies 36 .
The ability of heterogenous gold nanocatalysts to recycle themselves 39 for further degradation of organic pollutants and enhanced selectivity and reactivity of biological nanocrystals than their conventional counterparts makes them a very promising candidate for application in biotransformation. Our work provides a detailed study of structure and dimension controlled biosynthesized GNP for the degradation of 4-nitrophenol into 4-aminophenol (Fig. 10).

Conclusion
The cost-effective, simple and green protocols were developed to synthesize biogenic gold nanoparticles of different shapes and sizes with good yield and monodispersity. The peculiarity of our study is synthesis of gold nanoparticles of size 3-10 nm, having highest catalytic activity among the other different shapes and sizes of the particles by modulating the physical parameters. A complete control on physical and biological parameters (pH, time, temperature, concentration of salts and cell free extract) to obtained various shapes and sizes (spherical, penta/hexagons, triangle) were found during biosynthesis of GNPs. Deciphering of the unique balance of physical and biological factors networking to find highly reactive nanocatalyst, that bears a great potential in heterogenous catalysis may revolutionise the organic transformation for bioremediation.

Materials and Methods
Materials. The Gold (III) chloride (ACS reagent) was purchased from Sigma Aldrich, USA and used as received. All other reagents used were of analytical grade. All other reagents 4-Nitrophenol, 4-Aminophenol and NaBH 4 were also from Sigma Aldrich, USA used were of analytical grade.
Isolation of fungal isolate. T. viride (MTCC 5661) was isolated from CSIR-NBRI garden campus, and deposited in Microbial Type Culture Collection (MTCC), Chandigarh, India 16 . The culture was grown and maintained on Potato dextrose agar (PDA) medium at 28 °C for 4 days and maintained at 4 °C in refrigerator. Fungal cell free extract preparation and synthesis of gold nanoparticles. To prepare cell free extract, two bids of 6 mm were cut from the seven-day old culture of T. viride maintained on PDA and inoculated in 100 ml of potato dextrose broth (PDB). It was allowed to grow for4 days at 28 °C at 80 rpm. After 4 days, the biomass obtained was filtered through Whatman filter paper no. 1 and washed with autoclaved MQ water thrice. The biomass thus obtained was re-suspended in 100 ml of sterile deionised water for 3 days in similar conditions. The cell free extract was obtained by filtering out the biomass after 3 days.

Morphological observations by varying biological and physical parameters.
To evaluate the effect of various biological and physical parameters, the reaction was carried out under different reaction conditions. Fungal cell free extract and gold salt preparation. Three different fungal cell free extract concentration (10, 50 and 100%) were used in this study. The cell free extract obtained without any dilution served as 100% concentrated cell free extract. For preparing 10 and 50% of concentration, 10 ml and 50 ml of 100% concentrated cell free extract were added to 90 ml and 50 ml of MQ in sterilized conditions. Two different concentration of HAuCl 4 (250 and 500 mg/L) was prepared in sterilized MQ water for assessing the synthesis of gold nanoparticles. Characterization of gold nanoparticles. Preliminary characterization of GNPs synthesized was done by visual observation for change in colour of cell free extract, and further formation of GNPs was confirmed by UV-visible spectroscopy (Thermo spectrascan UV 2700) for appearance of characteristic surface plasmon resonance band of gold nanoparticles 40 . To study the hydrodynamic size and poly dispersity index (PDI) of the nanoparticles biosynthesized, particle size analyzer was used (Malvern, nanoseries zeta sizer, UK) 41 . Particle size and morphology were investigated using transmission electron microscopy (Technai G2 spirit, FEI, Netherland) with Gatan Orius camera. Samples were prepared by filtering them through 0.45 μ syringe millipore filters and sonicated for 2 min. After sonication, a drop of solution was placed immediately on formvar-coated copper grid and left overnight for drying. Bright field TEM studies were carried out at 80 KV. Size of ten different nanoparticles were measured randomly in one field during TEM studies and average size was calculated (n = 3). The crystalline phase was detected using X-ray diffraction (XRD) analysis. For fourier transform infra-red spectroscopy (FTIR) measurements, the nanoparticles were freeze dried and diluted with potassium bromide in the ratio of 1:100. The FTIR spectra of samples were recorded on a FTIR instrument (Agilent Cary 630, USA). All measurements were carried out in the range of 400-4,000 cm −1 .
Effect of different morphologies of GNP in catalytic degradation of 4-nitrophenol. Effect of different shapes and sizes of biosynthesized GNPs on their catalytic activity to reduce 4NP into 4AP was assessed by the method of Gangula et al. 42 , with some minor modifications. In a 3 ml of quartz cuvette, 1.7 mL of water, 0.3 mL of 2 mM solution of 4-nitrophenol and 1 ml of 0.03 M of freshly prepared NaBH 4 solution were added 43,44 . To this reaction mixture, 50 μ L of gold nanoparticles of different morphologies viz. spherical, triangular, hexagonal and sheet shaped were added. The reaction temperature was kept constant at room temperature (25 °C) to avoid the thermal effect on the process of catalysis. The reaction mixture was stirred well with microstirrer and quickly scanned between 200-600 nm in UV-vis spectroscopy (Thermo spectrascan UV 2700). Further, to assess the effect of size, spherical gold nanoparticles of two different size range (3-10 and 7-24 nm) were used as biocatalyst. The 4-nitrophenol shows an absorbance peak at 400 nm in presence of NaBH 4 due to formation of nitrophenolate ions, so the progress of the reaction was monitored by tracking the decrease in the absorption spectra of 4-nitrophenolate ion at 400 nm. Kinetics of the reaction was monitored for 15 min which showed a decrease in absorbance at 400 nm and increase in absorbance at 290 nm at an interval of 1 nm.
Gas chromatography-Mass spectrometric (GC) analysis for catalytic conversion of p-nitrophenol into p-aminophenol. Catalytic degradation of 4-nitrophenol into 4-aminophenol by gold nanoparticles (Spherical gold nanoparticles showing highest reaction kinetics; 3-10 nm and 7-20 nm) were confirmed by gas chromatography mass-spectrometric analysis (Trace GC Ultra TSQ Quantum XLS, Thermo Scientific). Reaction mixtures (4-NP + GNP + NABH 4 ) were harvested at time intervals of 0, 5, 10, 20, 30 min. then the reaction was stopped by using 2 M HCl and the resultant mixture was, lyophilized (Thermo Scientific Heto Freeze Dryer 1.0-110) and subjected to GC-MS/MS analysis. The instrument was equipped with Tri-Plus auto-sampler to perform injector port silylation at the injector port of GC. The instrument was equipped with DB-5 MS capillary column, and ionization voltage was 70 eV. An aliquot of 1.5 μ l of sample was injected and analysed using the following condition. The oven temperature programming was set initially at 100 °C for 1 min, then increased to 160 °C at the rate of 20 °C/min after that increased to 175 °Cwith a rate of 2 °C/min, and then the final temperature was set at up to with a 290 °C with a rate of 60 °C/min. The analysis was done in the full scan mode of GC-MS and the confirmation of peaks done by using Selected Reaction Mode (SRM) at a transitions of 253→ 238 for 4-AP and 211→ 196 for 4-NP with a collision energy of 15. The calibration curve was constructed for 4-AP and 4-NP in the concentration of range of 0.01-10 μ g/mL and found to be linear with a regression coefficient of 0.994 and 0.998 respectively (Fig. S17).