HPLC–QTOF–MS/MS-based rapid screening of phenolics and triterpenic acids in leaf extracts of Ocimum species and their interspecies variation

ABSTRACT Species of genus Ocimum are traditionally used for their medicinal and flavoring properties. These are rich sources of essential oils and found as an ingredient in many Ayurvedic preparations and food products. Phenolics and triterpenic acids are the medicinally active compounds mainly concentrated in the leaves of Ocimum species. This study aimed to develop an efficient and reliable analytical method for the rapid screening and characterization of phenolics and triterpenic acids in the leaf extracts of 6 Ocimum species using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC–ESI–QTOF–MS/MS). A total of 50 compounds were identified and characterized on the basis of their accurate MS and MS/MS information, out of which 23 compounds were confirmed by authentic standards. Identified compounds include 28 flavonoids, 4 propenyl phenol derivatives, 2 triterpenic acids, 11 phenolic acids, and 5 phenolic acid esters. The developed method was applied to study the interspecies variation of identified compounds. Significant variation in the distribution of identified phenolics and triterpenic acids was observed among studied Ocimum species. Hence, the established method provides an effective and reliable tool for screening and characterization of phytoconstituents in Ocimum species. GRAPHICAL ABSTRACT

Introduction plant metabolites than above-mentioned methods due to their high sensitivity, selectivity, specificity, and shorter analysis time. [46] So far, there are few LC-MS/MS methods reported using triple quadrupole and ion trap mass spectrometers for the phytochemical analysis of Ocimum species but these are mainly focused on the screening of flavonoids. [13,47] Previously, we have done quantification of targeted 16 marker compounds in the leaf extracts of 6 Ocimum species and marked herbal formulations of O. sanctum using UHPLC-QqQ LIT -MS/MS. [48,49] As the reported LC-MS/MS methods have limited data and also these are based on the application of low-resolution mass analyzers, e.g., triple quadrupole/hybrid linear ion trap triple quadrupole and ion trap mass spectrometers that ensure excellent selectivity and sensitivity for quantitative analyses/ targeted analyses but give nominal mass and so may not be effective in untargeted metabolite profiling/screening.
On the other hand, high-resolution mass analyzer time-offlight (TOF) has better accuracy, it provides accurate precursor and product ions information and molecular formula with mass error < 5 ppm, which greatly enhances the metabolite characterization reliability, especially when standard compounds are not available. [50] Therefore, there is a need to develop a versatile high-resolution LC-TOF-MS/MS method for screening and characterization of multiclass phytoconstituents of Ocimum species.
Hence, the goal of the present work is to develop an efficient and specific HPLC-ESI-QTOF-MS/MS method for rapid screening and characterization of triterpenic acids and phenolics including flavonoids, propenyl phenol derivatives, phenolic acids, and their esters in leaf extracts of 6 Ocimum species, viz. O. americanum, O. basilicum, O. gratissimum, O. killimandscharicum, O. sanctum "green," and O. sanctum "purple." The developed method is also applied to study the interspecies variation of identified compounds.

Extraction and sample preparation
The leaves of selected Ocimum species were air dried at room temperature and ground into powder with 40 mesh. Aqueous methanol (80%) was selected for extraction process due to its high efficiency in extracting phenolics and triterpenic acids from plant samples. [51,52] The dried powder (20 g) of the leaves of each species was extracted with 200 mL of 80% aqueous methanol in an ultrasonic water bath (Fisherbrand FB15067, Fisher Scientific, UK) for 30 min and left for 24 h at room temperature (22-24°C). Three replicates of the extraction process were carried out on each sample. The extract was filtered through Whatman filter paper and concentrated under reduced pressure (20-50 kPa) using a rotary evaporator (Buchi Rotavapor-R2, Flawil, Switzerland) at 40°C. Dried residues (1 mg) were weighed accurately, dissolved in 1 mL of methanol, and sonicated using an ultrasonicator (Bandelin SONOREX, Berlin, Germany). The solutions were filtered through 0.22 µm syringe filter (Millex-GV, PVDF, Merck Millipore, Darmstadt, Germany). The filtrates were diluted with acetonitrile to final working concentration for analysis. A mixed standard stock solution (1 mg/mL) of 23 reference compounds was prepared in acetonitrile. The working standard solution was prepared by diluting the mixed standard solution with acetonitrile. The standard stock, working standard, and sample solutions were stored at À 20°C until use and vortexed before injection.

Mass spectrometry
The MS analyses were performed on a QTOF-MS/MS instrument of Agilent 6520 series, connected with an Agilent 1200 HPLC (Agilent technologies, USA) through a dual ESI interface. Nitrogen was used as drying and collision gas in the ESI source. The ion source parameters were as follow: drying gas flow rate, 12 L/min; heated capillary temperature, 350°C; nebulizer pressure, 45 psi; VCap, fragmentor, skimmer and octopole RF peak voltages set at 3500, 150, 65, and 75 V, respectively. The detection was carried out in positive and negative electrospray ionization mode, and spectra were recorded by MS scanning in the range of m/z 50-1000. The MS/MS analyses were carried out by targeted fragmentation and collision energy was set at 8-40 eV. Mass Hunter software version B.04.00 build 4.0.479.0 (Agilent Technology) was used to control LC-MS/MS system, data acquisition, and processing including the prediction of chemical formula and exact mass calculation.

Optimization of Conditions for HPLC-ESI-QTOF-MS/MS analysis
The HPLC conditions were optimized to obtain maximal resolution and signal within a minimal run time. Various chromatographic conditions such as mobile phase composition, flow rate, injection volume, column temperature, and gradient program were studied and optimized for the separation of phenolics and triterpenic acids. Different mobile phase compositions (methanol-water, methanol-0.1% (v/v) formic acid aqueous solution, acetonitrile-water, and acetonitrile-0.1% (v/v) formic acid aqueous solution) were tested in the gradient program at 0.4 mL/min flow rate. A mobile phase composed of 0.1% (v/v) formic acid aqueous solution (A) and acetonitrile (B) at 0.4 mL/min flow rate and 25°C column temperature was found optimal for resolution of the maximum number of peaks in leaf extracts of Ocimum species within 65 min. In the MS analysis, both positive and negative ESI modes were tested, and the result showed that all the constituents exhibited good sensitivity in positive mode except phenolic acids and their esters, which showed high sensitivity in negative mode. Therefore, flavonoids, propenyl phenol derivatives, and triterpenic acids were analyzed in positive mode while phenolic acids and their esters were analyzed in negative mode.

HPLC-ESI-QTOF-MS/MS analysis
Metabolic profiling of leaf extracts of Ocimum species was resulted in the identification of 50 compounds including 28 flavonoids, 4 propenyl phenol derivatives, 2 triterpenic acids, 11 phenolic acids, and 5 phenolic acid esters. The structural identification of each compound was carried out on the basis of their accurate mass, molecular formula, and MS/MS fragmentation by HPLC-ESI-QTOF-MS/MS. Twenty-three compounds were unambiguously identified and characterized by comparing their retention time and fragmentation pattern with authentic standards. Other compounds were tentatively identified by comparing their MS and MS/MS data with available literature. All the identified compounds along with retention time, molecular formulas, m/z calculated and observed, error (Δ ppm), MS/MS data, and their comparative profile for 6 Ocimum species are summarized in Table 1a and b. The HPLC-ESI-QTOF-MS/MS analysis of flavonoids, propenyl phenol derivatives, and triterpenic acids was carried out in positive ionization mode, whereas phenolic acids and their esters were analyzed in negative ionization due to higher sensitivity. The base peak chromatograms (BPCs) of the leaf extracts of 6 Ocimum species in positive and negative ionization modes are presented in Figure 1a and b, respectively.

Identification of triterpenic acids
Two isomeric triterpenic acids, peaks 33 and 34, were identified and characterized as oleanolic and ursolic acid, respectively. These isomeric acids showed similar [MþH] þ at m/z 457.3675 and fragment ions in MS/MS scan but eluted at different retention time, 55.2 and 57.9 min. The identity of ursolic acid (peak 34) at t R 57.9 min was confirmed by authentic standard. The protonated molecular ions of oleanolic (peak 33) and ursolic acid (peak 34) generated base peak ion at m/z 439.3551 due to loss of H 2 O. They also produced fragment ions at m/z 411.3604 [MþH-COOH] þ and 393.3489 [MþH-COOH-H 2 O] as reported earlier. [58] The structures, fragmentation, and MS/MS spectra of triterpenic acids are shown in Figure 6.

Interspecies variation of phenolics and triterpenic acids in Ocimum species
The LC-MS/MS profile of identified 50 compounds was used to study the interspecies variation of phenolics and triterpenic acids in leaf extracts of 6 Ocimum species. Considering the area of extracted ion chromatogram (EIC) of each compound, it was observed that flavonoids were the most abundant class of compounds in O. sanctum purple, O. sanctum green, and O. basilicum, whereas in other species phenolic acid and their esters were the abundant class of compounds. It was also observed that maximum abundance of flavonoids was found in O. sanctum purple, propenyl phenol derivatives in O. americanum, triterpenic acids in O. killimandscharicum, and phenolic acids and their esters in O. gratissimum. Significant variation in the abundance of identified compounds was observed among 6 Ocimum species, which is graphically represented in Figure 7. These observations were based on the calculation of averaged relative percent peak area obtained from extracted ion chromatograms (EICs) of all the 50 compounds in triplicate.

Conclusion
In summary, the present study described first systematic comparative screening of phenolics and triterpenic acids in leaf extracts of 6 Ocimum species using HPLC-ESI-QTOF-MS/ MS. A total of 50 compounds including 28 flavonoids, 4 propenyl phenol derivatives, 2 triterpenic acids, 11 phenolic acids, and 5 phenolic acid esters were identified from leaf extracts of 6 Ocimum species. They were characterized by their retention behavior, exact mass measurement, molecular formula, MS/ MS spectral patterns, and authentic standards. The interspecies variation of identified phenolics and triterpenic acids was studied using LC-MS/MS profile. The results indicated that O. sanctum purple is the richest source of flavonoids and O. killimandscharicum is of triterpenic acids, while O. americanum is the richest source of propenyl phenol derivatives and O. gratissimum is of phenolic acids. This information might be helpful for better swapping of Ocimum species. Comparative metabolite profiling could be helpful for consumers in selecting best Ocimum species with a better expression of active compounds for their commercial use. Therefore, the developed analytical method is rapid and accurate for the screening and structural characterization of phenolics and triterpenic acids in Ocimum species.