Estimation of boswellic acids in herbal formulations containing Boswellia serrata extract and comprehensive characterization of secondary metabolites using UPLC-Q-Tof-MSe.

Boswellia serrata is a widely used herb in Indian systems of medicine and is well known for its potential medicinal properties. A chromatographic method was developed for the analysis and quantification of six boswellic acid marker compounds, i.e., keto boswellic acid (1), 3-O-Acetyl 11-keto β-boswellic acid (2), ɑ-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic acid (5) and 3-O-Acetyl-β-boswellic acid (6) in commercial herbal products containing B. serrata as an ingredient. Combining UPLC with Q-Tof-MS/MS makes the better identification of secondary metabolites and adulterants in the herbal formulations containing B. serrata in rapid time using fragmentation approach than the traditional approaches. In this study quantification of boswellic acids with UPLC-PDA method was performed as per the pharmacopeia guidelines. Furthermore, minor phytochemical constituents were identified and characterized with the help of LC-Q-Tof-MS/MS fragmentation data and various isoforms of boswellic acids and tirucallic acids in B. serrata oleo-gum-resin extract were identified.

Introduction

The genus Boswellia (Fam: Burseraceae) grows on calcareous soil at high altitudes, slopes and ridges of hills. Oleo-gum-resin is collected from wild trees by making incisions in bark of the trunks and the gum resin exudes from incision point and solidifies into irregular mass as a result of exposure to open air [1]. Gum resin of Boswellia serrata, commonly known as Indian frankincense, has been used in Asian and African folk medicine for treatment of arthritis [2]. Terpenoids are by far the most abundant metabolites of this gum resin and boswellic acids in particular have been identified as active principles [[2], [3], [4]] whose chemical structures are given in Fig. 1. B. serrata gum resin has been reported potential therapeutics for a wide range of ailments including hepatoprotective, anti-inflammatory [5], anticancer [6], beneficial in liver fibrosis [7] and antibacterial activity [8]. Toxicological studies performed on animals with boswellia extract indicate that their inclusions in herbal formulation are safe [9].

Fig. 1

Chemical structures of standard boswellic acid (1–6) and putatively identified compounds (7–16).

This resin gum is being used widely in Ayurvedic herbal formulations which were prepared in accordance with the authorized methodology provided in Ayurvedic formulary of India [10]. The increased demand for frankincense has put pressure on supply of natural resources which ultimately results in use of substandard materials or substitution and adulteration. The content of six boswellic acids [keto boswellic acid (1), 3-O-Acetyl 11-keto β-boswellic acid (2), α-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic acid (5) and 3-O-Acetyl-β-boswellic acid (6)] had been used as the standard index to appraise the quality of boswellia gum resin and its products [4]. To control the adulteration and maintain quality and efficacy of product, analytical tools play a major role. Consequently, several analytical [[11], [12], [13], [14], [15]] and bio-analytical methods [[16], [17], [18]] reported in the literature include high performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC) for quantification of the above-mentioned markers. Keeping in view of the growing interest on herbal products, continuous studies were undertaken in our laboratory pertaining to their standardization and demonstrated the potential utility of the methods emphasizing the need to effectively and quantitatively identify the active ingredients [19,20]. In the present work, we developed a new, simple, rapid and specific reversed-phase UPLC-PDA method for the quantification of six markers (1–6) from crude resin and this method was successfully applied to determine these compounds in fourteen different marketed herbal formulations. Furthermore, optimized method was coupled to Q-Tof-MS/MS system for metabolite profiling and to characterize minor/unknown constituents from crude resin extract. Our study led to the identification of 16 tri terpenoids from crude extracts through detailed mass fragmentation study. To the best of our knowledge, metabolite profiling and identification of constituents from crude extracts using UPLC-Q-Tof-MS/MS is being reported for the first time.

Materials and methods

B. serrata resin gum material and samples

The B. serrata resin gum exudates were collected from Devgiri and Karhal region of Uttar Pradesh, India in spring season 2016. Four resin extract (methanolic) samples (S1–S4) and fourteen commercially available herbal drug formulations of capsules (#BS C1–C6), tablets (#BS T1-T3), powders (#BS P1–P2), gel formulations (#BS G1-G2) and tea fusion material (#BS F1) claimed to contain B. serrata were purchased from local suppliers and online market. The specimens of all samples were deposited at Natural Products Laboratory, CSIR-IICT, Hyderabad, India. Out of 14 marketed samples, eight are single herb formulations containing B. serrata gum resin as active constituent and remaining six formulations were polyherbal in nature, and B. serrata gum resin was one of the constituent along with other plant extracts in different concentrations.

Chemicals and standards

The reference standards keto boswellic acid (1), 3-O-Acetyl 11-keto β-boswellic acid (2), α-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic acid (5) and 3-O-Acetyl-β-boswellic acid (6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of reference standards was >98% (Confirmed with HPLC and MS). Acetonitrile and formic acid were obtained from BioSolv (ULC/MS Grade) (Dieuze, France). Ammonium hydroxide solution (25%), methanol and Milli Q water were procured from Merck KGaALi Chrosolv LC-MS grade (Darmstadt, Germany).

Preparation of standard stock and working solutions

Accurately weighed 2 mg of individual reference standards were dissolved in methanol to obtain concentration of 2.0 mg/mL. Mixture of six reference standards was prepared by the addition process to appropriate concentrations for construction of calibration curve by UPLC-PDA.

Preparation of resin material and commercial samples

Accurately weighed 1g of the sample was transferred into conical flask with 10 mL of MeOH and sonicated for about 45 min. Supernatant liquid was collected from samples after centrifugation process. Extraction process was repeated for three times to ensure effective extraction without any loss of analytes. Finally, supernatant liquid was diluted up to 50 mL with MeOH. Samples were taken about 20 mg/mL concentration, filtered through 0.20 μm PVDF membrane filters and used as stock solution throughout validation process. In addition, the process was also extended to UPLC-Q-Tof-MS/MS to characterize secondary metabolites in the methanolic gum extract which could be useful for species differentiation.

Solid dosage forms (capsules, tablets and powder) were pulverized with mortar and pestle in individual to the appropriate amount weighed and extracted. For the semi-solid dosage form (Gel), adequate amount was weighed and subjected for extraction process. Same extraction process was followed for commercially available formulations.

Chromatographic and mass spectrometric instrumental conditions

Sample analysis was performed with Waters acquity UPLC H-Class system (Waters Corp., Milford, U.S.A) which includes quaternary solvent manager (QSM), sampler manager-FTN with ANSI 2X48 well plate, thermostatic controlled column compartment with PDA eλ detector. UPLC column was acquity BEH C18 (100 mm × 2.1 mm, 1.9 μm) (Waters, Ireland). The column and auto sampler temperatures were maintained at 45 °C and 10 °C, respectively. The flow rate was 0.4 mL/min and mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% ammonium hydroxide (B). The gradient program was as follows: 0 min, 70 %B; 3.50 min, 75 %B; 7.00 min, 80 %B; 14.00 min, 95 %B. Column was equilibrated with initial mobile phase composition for 3.00 min before injection of each sample. Needle wash solution (strong wash, acetonitrile/water: 80/20, v/v) and seal wash solution (weak wash, acetonitrile /water: 10/90, v/v) were used. Injection volume was set at 3 μL throughout validation process. Mass spectrometric analysis was performed with Waters Xevo G2-XS Q-Tof-MS/MS (Waters, Manchester, U.K) with ESI source with Z-Spray technology using following parameters: capillary voltage, 3.0 kV; sample cone, 30 V; source temperature, 120 °C; desolvation temperature, 350 °C; cone gas flow rate, 50 L/h; desolvation gas (N2) flow rate, 1000 L/h; argon as CID gas for MS/MS experiments. To ensure accuracy and reproducibility, all analyses were performed using lock spray. Leu-enkephalin (YGGFL) (5 ng/mL) was used as lock mass generating reference ion in negative mode at m/z 554.2615 and was introduced by a lock spray at 10 μL/min for accurate mass acquisition. The data were processed with MassLynx v4.1 SCN937 workstation.

Method validation

Developed UPLC-PDA method was validated in terms of LOD, LOQ, system suitability, linearity, precision and accuracy as per ICH Q2 R1 Guidelines [21].

LOD and LOQ of individual analytes were determined by signal-to-noise ratio at lower concentration which was equal to 3:1 and 10:1, respectively. Linearity of the developed method was determined by plotting calibration curves with different concentrations of analytes versus peak area. From calibration data regression equation and correlation coefficients were calculated via regression analysis.

The system suitability was observed by repetition of standard analytes mixture with specific concentration in triplicate with developed method parameters. Precision of method was evaluated in terms of intra and inter-day precision by analyzing #BS T1 & #BS C1 samples for a week by following the same extraction procedure in triplicate manner. Further the samples were subjected to accuracy study. Accuracy was determined by analyzing samples to which reference analytes were added at three different concentrations followed by extraction and analysis.

Results and discussion

Ultra-performance liquid chromatography-diode array detection (UPLC-PDA) coupled to an electro spray ionization time of flight mass spectrometer (ESI-MS) has emerged as a new technique for identification and elucidation of metabolites in complex extracts. Therefore, in continuation of previous studies on fragmentation patterns on natural products [22,23], we extended UPLC/PDA/ESI-Q-Tof-MS analysis of crude resin extract of B. serrata for detailed characterization of secondary metabolites. Acquired total ion chromatograms (TIC) were analyzed in ESI negative ionization mode and the results are presented in Table 1, Figs. S1–S16 and Fig. 2. The metabolites assignments were made based on their MS spectral data (accurate mass and fragmentation pattern), retention times, comparison to the standard compounds (In-house library) and search in public online databases (DNP, Reaxys and SciFinder), respectively. Our studies led to the identification of fourteen compounds from methanolic extract of B. serrata resin. Majority of compounds in B. serrata are triterpeniods belonging to boswellic acids and tirucallic acid type metabolites. This can be easily distinguishable through mass fragmentation mechanism using CID-MS/MS. Based on developed analytical method, we could identify seven (9–12 & 14–16) isoforms of tirucallic acids along with six well characterized boswellic acids shown in Table 1 and Fig. S17.

Table 1

LC-Q-Tof-MSe data for Boswellia serrata gum resin extract (methanolic).

S No	Retention time (min)	HRMS		Error (Δppm)	Fragment ions	Fragments formula [M − H]-	λmax (nm)	Name of the compound (Putatively identified)
		m/z	Formula [M − H]-
1	3.20	469.3316	C30H45O4	−0.4	407.3308	C29H43O	250.42	Keto boswellic acid
					391.3000	C28H39O
					353.2880	C25H37O
					339.2719	C24H35O
					271.2066	C19H27O
					231.1762	C16H23O
2	6.13	511.3420	C32H47O5	−0.6	485.3627	C31H49O4	228.42	3-O-Acetyl 11-keto β-boswellic acid
					467.3571	C31H47O3
					439.3624	C30H47O2
3	8.41	455.3521	C30H47O3	−0.9	437.3412	C30H45O2	225.42	ɑ-Boswellic acid
					409.3462	C29H45O
					361.2930	C27H37
					233.1931	C16H25O
4	8.91	455.3527	C30H47O3	0.4	409.3466	C29H45O	225.42	β-Boswellic acid
					367.3059	C26H39O
					361.2906	C27H37
					283.2123	C20H27O
					233.1919	C16H25O
5	12.30	497.3637	C32H49O4	1.2	423.3326	C29H43O2	226.42	3-O-Acetyl-ɑ-boswellic acid
6	12.79	497.3633	C32H49O4	0.4	439.3222	C29H43O3	226.42	3-O-Acetyl-β-boswellic acid
					423.3318	C29H43O2
					285.1173	C17H17O4
					233.1944	C16H25O
7	3.42	513.3595	C32H49O5	2.9	487.3420	C30H47O5	190.42, 226.42	Unknown
					443.3571	C29H47O3
					415.3665	C28H47O2
					371.2997	C25H39O2
					249.1881	C16H25O2
					235.1749	C15H23O2
					209.1549	C13H21O2
8	4.60	471.3480	C30H47O4	1.3	409.3516	C29H45O	225.42	11-hydroxy-boswellic acid (new)
					393.3176	C28H41O
					377.2855	C27H37O
					253.1658	C18H21O
					233.1917	C16H25O
9	5.93	455.3523	C30H47O3	−0.4	425.3062	C28H41O3	191.42, 226.42	3-hydroxytirucallic acid isomer
					373.2748	C24H37O3
					339.2690	C24H35O
10	6.49	455.3523	C30H47O3	−0.4	373.2744	C24H37O3	224.42	3-hydroxytirucallic acid isomer
					355.2648	C24H35O2
					341.2894	C24H37O
					339.2757	C24H35O
					269.1953	C19H25O
11	6.95	453.3361	C30H45O3	−1.8	371.2587	C24H35O3	190.42, 222.42	3-oxo-tirucallic acid
					353.2510	C24H33O2
					339.2696	C24H35O
					309.2245	C22H29O
					269.1947	C19H25O
					255.1791	C18H23O
12	7.27	455.3531	C30H47O3	1.5	373.2746	C24H37O3	224.42	3-hydroxytirucallic acid isomer
					355.2758	C24H35O2
					341.2864	C24H37O
					281.2542	C18H33O2
13	8.67	485.3637	C31H49O4	1.2	441.3382	C29H45O3	225.42	Unknown
					397.3553	C28H45O
					381.3212	C27H41O
					365.2928	C26H37O
					337.3018	C25H37
					229.1437	C12H21O4
14	9.21	497.3635	C32H49O4	1.1	437.3427	C30H45O2	225.42	3-O-acetoxy tirucallic acid isomer
					415.2854	C26H39O4
					397.2770	C26H37O3
					355.2649	C24H35O2
15	9.80	497.3636	C32H49O4	1.0	437.3431	C30H45O2	225.42	3-O-acetoxy tirucallic acid isomer
					415.2856	C26H39O4
					397.2798	C26H37O3
					355.2671	C24H35O2
16	11.45	455.3527	C30H47O3	0.4	437.3432	C30H45O2	226.42	3-hydroxytirucallic acid isomer
					409.3477	C29H45O
					377.3214	C28H41
					361.2947	C27H37
					237.1601	C18H21
					175.1109	C12H15O

Fig. 2

LC-MS (TIC) chromatogram of gum resin extract (methanolic) of B. serrata.

Chromatographic method development

Two isoforms of tirucallic acids in B. serrata extract could be identified applying our TIC methodology. It is important to mention here that tirucallic acids present in boswellia have been reported to inhibit 5-lipoxygenase [24] and cyclooxygenase [25] enzymes, which may significantly contribute to the overall anti-inflammatory activities. Therefore, provision is warranted while preparing recommendation for standardization of B. serrata formulations.

In the present study, chromatographic separation was performed by using several reversed phase columns such as Acquity BEH C18 (100 mm × 2.1 mm, 1.7 μm), X Select Phenyl-Hexyl (100 mm × 2.1 mm, 1.9 μm) and Hypersil gold C18 (150 mm × 2.1 mm, 1.9 μm) to achieve a short run time, symmetric peak shapes and better chromatographic efficiency. This was investigated by appropriate changes in mobile phase composition (aqueous and organic phase) and flow rate. The standard boswellic acids were mid polar in nature and therefore suitable combination of aqueous and organic solvents was optimized with 0.1% formic acid and 0.1% NH4OH in both aqueous and organic phase. Mobile phase was operated at a flow rate of 0.4 mL/min. For better resolution between the polar and non-polar metabolites, gradient program was applied where gradual change in aqueous and organic solvents composition led to enhanced peak purity and resolution.

Chromatographic method validation

Initially, six reference analytes of boswellic acids (BA), namely, keto boswellic acid (1), 3-O-Acetyl-11-keto β-boswellic acid (2), ɑ-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic acid (5) and 3-O-Acetyl-β-boswellic acid (6), were quantified in crude resin using UPLC-PDA method and validated in terms of its linearity, LOD, LOQ, precision and recovery. Validation results are presented in Table 2, Table 3 and Table 1S. Linearity of six standard solutions at different concentrations (1, 5, 10, 50, 100 and 500 μg/mL) were analyzed. LOD and LOQ for standard analytes are given in Table 2. Precision data (Intra and inter day) in terms of % RSD and % recovery of analytes are shown in Table 3, Table 4, respectively. All the reference standards were found to be stable at room temperature for 72 h. Subsequently, applicability of method for quantification of marker compounds was demonstrated with fourteen commercial samples containing single or polyherbal formulations belonging to different classes viz tablets (#BS T1-T3), powder (#BS P1–P2), capsules (#BS C1–C6), semi-solid dosage [Gel (#BS G1-G2)] and tea fusion material (#BS F1), respectively. Selected formulations consisted of B. serrata as an important ingredient. PDA chromatogram of methanolic gum extract and commercial herbal formulations were recorded in the rage of 200–400 nm and quantitative analysis was performed at 210 nm. Reference standards (1–6) present in resin extracts and commercial formulations were identified by comparing retention time, peak purity and UV spectra of compounds in standard mixture. UPLC-PDA chromatograms at 210 nm of resin extract and commercial herbal formulations are shown in Fig. 3 and quantitative data for compounds (1–6) from different formulations are presented in Table 5.

Table 2

Linearity, LOD and LOQ data for six boswellic acid standards.

S No	Analyte	Retention time (min)	Linearity (μg/mL)	Regression equation	r2	LOD (μg/mL)	LOQ (μg/mL)	%RSD
1	Keto boswellic acid	2.95	1–500	y=18138x – 17092	0.999	0.10	0.50	0.44
2	3-O-Acetyl 11-keto β-boswellic acid	5.66	1–500	y=20645x – 49509	0.999	0.10	0.50	1.26
3	ɑ-Boswellic acid	7.89	1–500	y=6705x – 14231	0.999	0.50	1.00	3.72
4	β-Boswellic acid	8.35	1–500	y=3251x – 6391	0.999	0.50	1.00	2.61
5	3-O-Acetyl-ɑ-boswellic acid	11.72	1–500	y=4942x – 13251	0.999	0.40	1.00	3.42
6	3-O-Acetyl-β-boswellic acid	12.22	1–500	y=9085x – 23136	0.999	0.40	1.00	1.28

Table 3

Precision data (n=3) for analytes subjected to quantification (Mean conc. (μg/mL) ± %RSD).

Analyte	Nominal conc. (μg/mL)	Intra-day	Inter-day
Keto boswellic acid	1	0.91 ± 2.67	0.90 ± 3.72
	50	51.22 ± 0.91	50.08 ± 2.70
	500	495.84 ± 2.37	487.14 ± 0.65
3-O-Acetyl 11-keto β-boswellic acid	1	0.89 ± 4.41	0.92 ± 3.79
	50	49.82 ± 1.26	50.24 ± 1.33
	500	492.71 ± 0.97	495.82 ± 3.13
ɑ-Boswellic acid	1	0.94 ± 3.72	0.90 ± 3.55
	50	46.43 ± 1.15	45.09 ± 2.41
	500	492.36 ± 1.84	482.91 ± 2.55
β-Boswellic acid	1	0.91 ± 4.42	0.95 ± 4.61
	50	46.22 ± 1.19	47.26 ± 2.80
	500	488.61 ± 0.74	489.52 ± 1.56
3-O-Acetyl-ɑ-boswellic acid	1	0.90 ± 5.10	0.91 ± 3.54
	50	48.52 ± 2.65	45.66 ± 1.57
	500	491.05 ± 0.95	493.19 ± 3.36
3-O-Acetyl-β-boswellic acid	1	0.91 ± 5.04	0.92 ± 4.29
	50	46.25 ± 4.65	51.82 ± 3.23
	500	493.19 ± 1.49	483.07 ± 0.81

Table 4

% Recovery data (n=3) for single herbal formulations (Mean ± %RSD).

Sample code	KBA	AKBA	ɑ-BA	β-BA	3-O-ɑ-ABA	3-O-β-ABA
S1	94.23 ± 3.12	93.15 ± 0.87	93.85 ± 1.87	95.20 ± 0.81	94.58 ± 0.97	98.52 ± 2.70
S2	98.52 ± 2.17	102.11 ± 1.75	96.45 ± 1.41	98.85 ± 1.58	102.82 ± 1.71	98.74 ± 0.85
S3	99.81 ± 0.52	94.61 ± 2.85	96.58 ± 3.40	99.20 ± 1.85	101.63 ± 0.91	100.02 ± 0.75
S4	102.06 ± 2.15	98.62 ± 2.32	96.52 ± 0.75	102.45 ± 0.19	98.17 ± 2.03	102.11 ± 1.76
#BS C1	96.53 ± 3.60	101.21 ± 1.82	94.87 ± 3.64	95.94 ± 3.74	94.75 ± 2.61	99.23 ± 1.06
#BS C2	93.52 ± 1.44	94.91 ± 2.99	97.52 ± 1.57	102.49 ± 0.36	98.63 ± 0.62	94.50 ± 4.01
#BS C3	98.56 ± 3.01	99.86 ± 3.21	93.85 ± 0.82	93.11 ± 0.82	103.15 ± 3.78	95.00 ± 3.60
#BS C5	94.66 ± 0.92	96.61 ± 2.59	98.30 ± 0.93	100.34 ± 3.67	93.12 ± 3.92	98.62 ± 1.45
#BS T1	94.15 ± 1.61	95.73 ± 1.31	103.66 ± 2.41	95.83 ± 2.81	96.54 ± 2.73	101.85 ± 2.66
#BS T2	101.28 ± 1.79	99.57 ± 2.52	94.52 ± 1.20	98.60 ± 1.68	95.68 ± 0.49	102.16 ± 0.42
#BS P1	95.49 ± 2.56	98.28 ± 2.74	99.83 ± 1.88	95.27 ± 2.77	95.10 ± 1.82	98.66 ± 1.20
#BS P2	96.45 ± 1.09	102.63 ± 3.11	101.05 ± 0.63	101.83 ± 0.46	92.91 ± 3.75	93.19 ± 2.84

S1–S4: Methanolic extracts, #BS C1–C5: Capsule formulations, #BS T1-T2: Tablet formulations, #BS P1–P2: Powder formulations.

Fig. 3

UPLC-PDA chromatograms of herbal formulations samples containing B. serrata as active component or one of the active component in comparison with boswellic acids standard mixture (1–6).

Table 5

Content % of boswellic acids (BA) (1–6) in various herbal formulations; Keto boswellic acid (1), 3-O-Acetyl 11-keto β-boswellic acid (2), ɑ-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic acid (5) and 3-O-Acetyl-β-boswellic acid (6).

Sample code	Label claim (boswellia gum resin/extract)	Poly/Single	1	2	3	4	5	6	Total BA
S1	–	–	0.94 ± 0.02	0.26 ± 0.01	0.28 ± 0.01	1.22 ± 0.05	0.61 ± 0.02	0.25 ± 0.01	3.10
S2	–	–	2.81 ± 0.01	0.77 ± 0.01	0.82 ± 0.01	3.10 ± 0.07	0.38 ± 0.01	0.61 ± 0.02	8.49
S3	–	–	0.82 ± 0.01	0.24 ± 0.01	0.27 ± 0.01	1.36 ± 0.02	0.12 ± 0.01	0.20 ± 0.01	3.02
S4	–	–	1.43 ± 0.02	0.47 ± 0.01	0.58 ± 0.01	2.64 ± 0.03	0.27 ± 0.02	0.42 ± 0.01	5.82
#BS C1	250 mg (Capsule)	Single	1.02 ± 0.02	3.21 ± 0.05	1.06 ± 0.03	4.25 ± 0.19	0.51 ± 0.01	0.73 ± 0.01	10.80
#BS C2	500 mg (Capsule)	Single	1.44 ± 0.01	1.69 ± 0.01	1.48 ± 0.03	7.16 ± 0.05	0.87 ± 0.03	0.84 ± 0.01	13.50
#BS C3	500 mg (Capsule)	Single	1.08 ± 0.01	2.78 ± 0.23	1.51 ± 0.05	7.59 ± 0.01	1.24 ± 0.02	1.86 ± 0.01	16.08
#BS C4	300 mg (Capsule)	Poly	0.66 ± 0.02	0.55 ± 0.01	0.52 ± 0.01	2.55 ± 0.08	0.14 ± 0.01	0.13 ± 0.01	4.58
#BS C5	350 mg (Capsule)	Single	1.07 ± 0.01	2.33 ± 0.03	1.27 ± 0.02	5.51 ± 0.08	0.84 ± 0.02	0.97 ± 0.01	12.01
#BS C6	250 mg (Capsule)	Poly	1.49 ± 0.01	0.81 ± 0.03	1.14 ± 0.09	5.06 ± 0.02	0.17 ± 0.04	0.22 ± 0.01	8.91
#BS T1	75 mg (Tablet)	Single	0.15 ± 0.01	1.27 ± 0.01	0.11 ± 0.02	0.54 ± 0.02	0.12 ± 0.01	0.28 ± 0.01	2.45
#BS T2	125 mg (Tablet)	Single	0.37 ± 0.01	0.58 ± 0.01	0.33 ± 0.01	1.63 ± 0.03	0.21 ± 0.01	0.19 ± 0.01	3.32
#BS T3	240 mg (Tablet)	Poly	0.30 ± 0.01	0.24 ± 0.01	0.22 ± 0.01	1.22 ± 0.01	0.10 ± 0.01	0.12 ± 0.01	2.19
#BS P1	-(Powder)	Single	0.39 ± 0.01	0.41 ± 0.01	0.23 ± 0.01	1.66 ± 0.01	0.15 ± 0.01	0.24 ± 0.01	3.09
#BS P2	-(Powder)	Single	0.62 ± 0.01	0.60 ± 0.01	0.51 ± 0.02	2.52 ± 0.02	0.18 ± 0.01	0.15 ± 0.01	4.59
#BS G1	7.5 mg (Gel)	Poly	0.14 ± 0.01	ND	ND	ND	ND	ND	0.14
#BS G2	100 mg (Gel)	Poly	ND	ND	ND	ND	ND	ND	ND
#BS F1	Tea Fusion	Poly	0.20 ± 0.01	0.23 ± 0.01	0.17 ± 0.01	0.83 ± 0.01	0.13 ± 0.01	0.16 ± 0.01	1.72

ND: Not Detected; S1–S4: Methanolic extracts, #BS C1–C6: Capsule formulations, #BS T1-T3: Tablet formulations, #BS P1–P2: Powder formulations,. #BS G1-G2: Gel formulations and #BS F1: Tea fusion material.

As per the Indian and European pharmacopeias [2,4], the herbal preparations containing B. serrata must have minimum of 1.0% 11-keto-β-boswellic acid (1) and 1.0% acetyl-11-keto-β-boswellic acid (2) (w/w, dried drug) as active principles. From our study, it was found that authentic gum resin extract showed all reference standards in required quantity. However, analysis of commercial formulations indicated presence of markers (1–6) in the range of 0.14%–16.08% of dried material. Content of total boswellic acids (1–6) was found to be maximum of 16.08% in #BS C3 sample. It is important to mention that essential markers, KBA (1) and AKBA (2), were found to be in minimum of 0.14% and 0.23% and maximum of 1.49% & 3.21%, respectively. Commercial sample (#BS G1) showed only one reference standard, KBA (1).

Overall, our analysis of commercial samples containing Boswellia serrata showed that only four samples (#BS C1–C3, C5) could meet pharmacopeia guidelines (Fig. 1 containing compounds 1 and 2 with specified limits), hence warranting proper standardization of herbal formulations according to pharmacopeia guidelines for optimum presence of bioactive principles.

Q-Tof-MS/MS fragmentation study

Boswellic acids follow retro Diels-Alder reaction (RDA) at C-ring either on/or before loss of small molecules. Another class of triterpeniods called tirucallic acid and its derivatives following McLafferty rearrangement (MFR) at C-21 acid followed by breakage of bond between C-20 and C-17 as shown in Fig. 4. Based on these key fragments, compounds were identified and depicted in Table 1. Key fragmentation pattern and mechanism are shown in Fig. 4, Fig. 5, Fig. 6 and Figs. S1–S16. Although CID-MS/MS cannot discriminate between stereoisomers, CID-MS/MS coupling with UPLC resolves stereoisomer’s separation and alleged identification. This hyphenation helped us in separating 3-hydroxy tirucallic acid isomers (9, 10, 12 & 16) with the same key fragmentation (m/z 373 & m/z 83) formed through McLafferty rearrangement (MFR) at C-21. The compounds, ɑ-Boswellic acid (3) and β-Boswellic acid (4), were clearly separated in TIC with key fragment ion (m/z 237 and m/z 218) through RDA reaction and also matched with standard compounds along with other isoforms of boswellic acids shown in Table 1.

Fig. 4

Proposed common fragmentation of tirucallic acid type molecules.

Fig. 5

Proposed common fragmentation of boswellic acid (peak 1) type molecules.

Fig. 6

Proposed common fragmentation of boswellic acid (peak 8, new) type molecules.

In detail, peaks at retention time (Rt) 5.93, 6.49, 7.27 and 11.45 min with end absorption m/z 455 [M − H]- in ESI negative mode and m/z 437.3432 [M-H-H2O]- along with m/z 373 and m/z 83 formed through McLafferty rearrangement (MFR) were identified as 3-hydroxy-8,9,24,25-tetradehydrotirucallic acid [C30H48O3] isomers (9, 10, 12 and 16). On the other hand, peaks at Rt 9.21 and 9.80 min with end absorption m/z 497.3635 [M − H]- and 437 [M-AcOH-H]- along with m/z 416 and m/z 83 by McLafferty rearrangement (MFR) in ESI negative mode were identified as 3-O-acetoxy tirucallic acid isomer(14 and 15). Peak at Rt 6.95 min with end adsorption m/z 453.3361[M − H]- and m/z 425[M-H-CO]- along with m/z 310 and m/z 83 formed though MFR was characterized as 3-oxo-tirucallic acid (11). Similarly, boswellic acids (1–6) were also identified at Rt 3.20 (1), 6.13 (2), 8.41 (3), 8.91 (4), 12.30 (5) and 12.79 (6) min based on their fragmentation patterns along with RDA and also clearly matched their reference standards. In addition, peak at Rt 4.60 with molecular ion 471[M − H]- and 409 [M-H-CH2O3]- along with fragment ion at m/z 233 formed through RDA as shown in Fig. 6, clearly supports the position of hydroxyl at C-11 and was identified as 11-hydroxy boswellic acid (8) as a new compound.

Conclusion

A simple and efficient UPLC-PDA method was developed for quantification of six boswellic acids in B. serrata gum resin extract and various commercial herbal formulations containing B. serrata as one of the ingredients. The method was validated in terms of LOD, LOQ, precision and recovery. From quantitative data, it was observed that there was a wide variation in content of boswellic acids from the commercial formulations. The LC-Q-Tof-MS/MS technique was applied for the characterization of metabolites including boswellic acids and tirucallic acids in the B. serrata extract. The MS/MS fragmentation of boswellic acids and tirucallic acids would be a valuable tool for quality control assessment of commercially available formulations containing B. serrata.