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AnalyticalMethods
PAPER
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A rapid analytica
aSophisticated Analytical Instrument Facili
[email protected] of Scientic and Innovative ReseacBiometry and Statistics Division, CSIR-CDRdBotany Division, CSIR-CDRI, Lucknow, IndeMicrobiology Division, CSIR-CDRI, Lucknow
† Electronic supplementary informa10.1039/c4ay00975d
Cite this: Anal. Methods, 2014, 6, 7349
Received 24th April 2014Accepted 28th June 2014
DOI: 10.1039/c4ay00975d
www.rsc.org/methods
This journal is © The Royal Society of C
l method for characterization andsimultaneous quantitative determination ofphytoconstituents in Piper betle landraces usingUPLC-ESI-MS/MS†
Renu Pandey,ab Preeti Chandra,ab Mukesh Srivastva,bc K. R. Arya,bd Praveen K. Shuklabe
and Brijesh Kumar*ab
A rapid ultra performance liquid chromatography electrospray ionisation tandem mass spectrometry
(UPLC-ESI-MS/MS) method was developed and validated for identification and characterization of
phytoconstituents with simultaneous quantitative determination of three major bioactive phenolics
namely allylpyrocatechol-3,4-diacetate, eugenyl acetate and eugenol from thirteen landraces of Piper
betle leaf extracts. Among the 29 phytoconstituents detected, 19 were identified and characterized
based on their fragmentation pattern obtained via MS/MS by means of collision-induced-dissociation
(CID) and by comparison of their retention time with authentic standards or by previously reported
literature. The proposed method was fully validated in terms of linearity, LOD, LOQ, precision, stability
and recovery. All calibration curves showed a good linear relationship (r2 $ 0.9981). The precision
evaluated by an intra- and inter-day study showed RSD # 1.0% and good accuracy with overall recovery
in the range from 96.14–98.46% (RSD # 1.6%) for all analytes. The quantitative results showed that there
were remarkable differences in the contents of the major bioactive phenolics in all the thirteen landraces
of P. betle. The developed UPLC-ESI-MS/MS method was found to be simple, precise, sensitive and
accurate. Hence, it can be reliably utilized as a quality control method for P. betle or derived herbal
formulations. In vitro antimicrobial activity of the crude extract of P. betle landraces was also evaluated,
and all extracts tested exhibited antifungal activity but none of the extracts were found to be active
against bacteria even at 500 mg mL�1 concentration.
1. Introduction
Piper betle Linn., commonly known as the betel vine, is a trop-ical plant belonging to the family piperaceae. It is a perennialdioecious, semi-woody climber extensively grown in India, SriLanka, Malaysia, Thailand, Taiwan and other Southeast Asiancountries. The betel plant is an attractive and aromatic creeperwith alternate, heart shaped, smooth, shining, and long stalkedleaves with a pointed apex. An ancient Indian system of medi-cine, Ayurveda, recognized the therapeutic uses of P. betlebesides its ethnomedicinal uses. Phenol rich leaves of P. betlehave been reported to exhibit antimicrobial,1,2 antioxidant,3,4
anti-inammatory,5,6 antiulcer/wound healing,7 antiplatelet,8
ty, CSIR-CDRI, Lucknow, India. E-mail:
rch, New Delhi, India
I, Lucknow, India
ia
, India
tion (ESI) available. See DOI:
hemistry 2014
antifertility,9 antileishmanial,10 antidermatophytic,11 anti-cancer,12,13 antimalarial,14 antidiabetic,15 anthelmintic,1 mos-quitolarvicidal,16 anticoagulant,17 antilarial18 andimmunomodulatory activities.19 It is estimated that in Indianearly one hundred landraces of P. betle are in cultivation.20
Biodiversity within a species is represented by landrace orvariety. Since P. betle is an important cultural and medicinalplant it is important to explore the available biodiversity for itstherapeutic potential. Thus, in order to select the most suitableones for medicinal uses, it is essential to screen the availablebiodiversity based on qualitative and quantitative analysis ofphytoconstituents in P. betle landraces. Hence it is required todevelop a quality control method for standardization of P. betleleaf extracts. Plant phenolics are important nutritional antiox-idants which could assist in overcoming chronic diseases suchas cardiovascular disease and cancer, the two leading causes ofdeath in the world.21 It is also reported that biological activitiesin P. betle landraces are mainly due to phenolics. In view of this,we have developed and validated a rapid and sensitiveultra performance liquid chromatography tandem massspectrometry (UPLC-ESI-MS/MS) method for identication and
Anal. Methods, 2014, 6, 7349–7360 | 7349
Analytical Methods Paper
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characterization of phytoconstituents with simultaneousquantitative determination of three major bioactive phenolicsnamely allylpyrocatechol-3,4-diacetate, eugenyl acetate andeugenol from thirteen landraces of P. betle leaf extracts for theirquality assessment. The principal phytoconstituents of P. betleleaves are propenylphenols, their derivatives and terpenes, withvarious other bioactive molecules such as alkaloids/amides,steroids and avonoids.22 Previously GC-MS and DART-MSmethods were reported for the analysis of chemical constituentsof betel leaf.23–27 GC-MS studies on P. betle leaf oil showed thepresence of safrole, allylpyrocatechol-3,4-diacetate, eugenol andeugenyl acetate as the major components and their quantitativevariation in different landraces due to the difference ingeographical conditions of growth.23–25 P. betle landraces werealso analyzed using the Direct Analysis in Real Time (DART)mass spectral technique which showed the presence of mainlyterpenes and phenols in betle leaves. Some variations wereobserved in the composition and percentage of constituents.26,27
Analytical methods based on HPLC-PDA and HPTLC havebeen reported for the determination of eugenol and eugenylacetate in the ower and leaf of P. betle.2,28 Eugenol is alsoquantied in plasma using tandem mass spectrometry coupledwith liquid chromatography.29 These methods contributedsignicantly but have the limitation of long analysis time. Onthe other hand, quantication by UPLC-ESI-MS/MS in multiplereaction monitoring (MRM) acquisition mode has drawn muchattention in the analysis of natural compounds due to its highspeed, improved sensitivity, compound specicity and betteraccuracy compared to HPLC analysis.
Ultra high performance liquid chromatography (UPLC) is arecent technique in liquid chromatography, which enablessignicant reductions in separation time and solventconsumption. UPLC columns packed with <2 mm particle sizeprovide faster, more efficient separation, narrow peak widthwith good symmetry for maximum peak height, thus allowingfor higher peak capacities. The UPLC-QTRAP-MS/MS systemcombines UPLC and hybrid linear ion trap triple quadrupoledetection effectively, thus providing efficient separation, highselectivity, high peak capacity and multiple ion detection basedon selective ion fragmentation. Due to the high sensitivity andhigh selectivity of MRM acquisition mode, optimization ofchromatographic separation is greatly simplied. Furthermore,precursor and product ion monitoring can be used to increasethe specicity of detection and identication of the knownmolecules. Recent success in the use of liquid chromatographycoupled with triple quadrupole mass spectrometry (LC-MS/MS)for characterizing and quantifying a wide variety of compoundsin complex samples suggests that LC-MS/MS might be auniversal technique in the determination of phytoconstituentsin complex plant extracts.
Therefore UPLC-ESI-MS/MS was selected for qualitative andquantitative analysis of phytoconstituents in P. betle extract. Inthe present work using UPLC-ESI-MS/MS we have quantied themajor bioactive compounds, namely, allylpyrocatechol-3,4-diacetate, eugenyl acetate and eugenol known to possess anti-cancer/antitumor activity.13,30,31 Investigation of the contents ofmajor bioactive phenolics in thirteen landraces of P. betle using
7350 | Anal. Methods, 2014, 6, 7349–7360
the UPLC-ESI-MS/MS method in MRM acquisition mode stillhas not been reported.
2. Experimental2.1 Chemicals and solvent
Analytical standards allylpyrocatechol-3,4-diacetate and eugenylacetate were purchased from Sigma-Aldrich (St. Louis, USA),eugenol and daidzein were purchased from Extrasynthese (Z.ILyon Nord, Genay Cedex, France). Acetonitrile (LC-MS grade)and formic acid (analytical grade) were purchased from Sigma-Aldrich and water was puried using a Milli-Q system (Milli-pore, Milford, MA, USA).
2.2 Extraction and sample preparation
All the thirteen landraces of P. betle, namely, Meetha Patta(West Bengal), Sanchi (West Bengal), Shirpurkata, Kapoori,Assam Pan, Nagpuri, Jalesar Green, Jagarnathi, Deshi, Mahoba,Saua, Jalesar White and Bangladeshi were collected from thelocal markets. Specimen vouchers of Meetha Patta, Jagarnathi,Jalesar Green, Deshi, Kapoori, Jalesar White, Saua, Banglade-shi and Mahoba (voucher no – KRA 24476, KRA 24477, KRA24478, KRA 24479, KRA 24480, KRA 24481, KRA 24482, KRA24483 and KRA 24484 respectively) have been deposited at theBotany Department of the Central Drug Research Institute(CDRI) Lucknow, India. Specimen vouchers of Shirpurkata,Sanchi (West Bengal) and Assam Pan (voucher no – IIHRBV9,IIHR BV 24 and IIHR BV 45 respectively) have been deposited atIndian Institute of Horticultural Research (IIHR) Bangalore,India. The dried leaves of thirteen landraces of P. betle werecrushed and suspended in ethanol and the suspension wasplaced in an ultrasonic bath for 30 min and then le for 24 h atroom temperature for extraction of secondary metabolites. Theprocess of extraction was repeated three times. The supernatantwas ltered (using Whatman lter paper) to eliminate the largemolecular impurities and evaporated to dryness under reducedpressure using a Buchi rotary evaporator at 40 �C. Samples forUPLC-ESI-MS/MS analysis were obtained by dissolving andltering (using Millex, syringe-driven lter, pore size 0.22 mm,Merk Millipore) the dried residues (about 1 mg accuratelyweighed) with 1 mL of acetonitrile which is further diluted foranalysis.
2.3 Preparation of standard solutions
Standard stock solutions of 1 mg mL�1 of selected analytes(allylpyrocatechol-3,4-diacetate (1), eugenyl acetate (2), andeugenol (3)) and internal standard daidzein were prepared inacetonitrile. Aliquot of each stock solution was mixed anddiluted with acetonitrile to make a standard mixture and it wasfurther diluted to provide a series of 9 different concentrationsin the range of 1, 10, 25, 50, 75, 100, 200, 500, 1000 ng mL�1
used for plotting calibration curves. The standard stock andworking solutions were all stored at �20 �C until use and vor-texed prior to injection.
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2.4 Instrumentation and analytical conditions
The UPLC-ESI-MS/MS analysis was performed on a WatersACQUITY UPLC™ system (Waters, Milford, MA, USA) interfacedwith a hybrid linear ion trap triple-quadrupole mass spec-trometer (API 4000 QTRAP™ MS/MS system from AB Sciex,Concord, ON, Canada) equipped with an electrospray (Turbo V)ion source. The Waters ACQUITY UPLC™ system was equippedwith a binary solvent manager, a sample manager, a columnoven and a photodiode array detector (PDA). AB Sciex Analystsoware version 1.5.1 was used to control the LC-MS/MS systemand for data acquisition and processing.
The chromatographic separation of phytoconstituents andanalytical standards was achieved on an ACQUITY UPLC BEHC18 column (50 mm � 2.1 mm, 1.7 mm) at a column tempera-ture of 40 �C. Analysis was completed with gradient elution of0.1% formic acid in water (A) and acetonitrile (B) as the mobilephase. The four min UPLC gradient system was as follows: 0–1.5min, 30–70% B; 1.5–3 min, 70–70% B; 3–3.5 min, 70–30% B;3.5–4 min, 30–30% B. Sharp and symmetrical peaks wereobtained at a ow rate of 0.3 mL min�1 with a sample injectionvolume of 1 mL. The UV spectra (PDA) were recorded between190 and 400 nm and 254 nm was chosen for determination.
The identication and characterization of phytoconstituentswas performed in positive ESI mode with Q1MS Scan andproduct ion scan (MS/MS) conducted at unit resolution for bothQ1 and Q3 and the collision gas set at “medium”. Q1 massspectra were recorded by scanning in the range of m/z 100–1000at a cycle time of 9 s with a step size of 0.1 Da. Nitrogen was usedas the nebulizer, heater, and curtain gas as well as the collisionactivation dissociation (CAD) gas. The source parameters were:ion spray voltage set at 5500 V, curtain gas, nebulizer gas (GS1)and heater gas (GS2) set at 20, 50 and 50 psi, respectively andsource temperature set at 550 �C. Compound dependentparameters, declustering potential (DP) and entrance potential(EP) were set at 80 and 12 V, respectively.
For quantitative analysis the detection of the analytes andinternal standard (IS) was performed in positive ESI mode usingmultiple reaction monitoring (MRM) acquisition mode at unitresolution. The ion spray voltage was set at 5500 V, curtain gas,nebulizer gas (GS1) and heater gas (GS2) were set at 20, 50 and50 psi, respectively and source temperature set at 550 �C.Quantitative parameters are listed in Table 1.
2.5 Antimicrobial activity
In vitro antimicrobial activity of ethanolic leaf extracts of P. betlelandraces, Nagpuri, Jalesar Green, Jagarnathi, Deshi, Mahoba,
Table 1 List of selected MRM parameters, declustering potential (DP), enfor each analyte and IS
S. no. RT (min) Analyte Q1 mass (D
1 0.89 Daidzein (IS) 2552 1.78 Allylpyrocatechol-3,4-diacetate 2353 1.86 Eugenol 1654 1.87 Eugenyl acetate 207
This journal is © The Royal Society of Chemistry 2014
Saua, Jalesar White and Bangladeshi, which showed highcontent of major bioactive phenolics in quantitative analysis,was tested against four bacteria; E. coli (Ec, ATCC 9637),P. aeruginosa (Psu, ATCC BAA-427), S. aureus (Sa, ATCC 25923),K. pneumoniae (Kpn, ATCC 27736) and 6 fungi; C. albicans (Ca,patient isolate), C. neoformans (Cn, patient isolate), S. schenckii(Ss, patient isolate), T. mentagrophytes (Tm, patient isolate),A. fumigatus (Af, patient isolate) and C. parapsilosis (Cp, ATCC-22019). The susceptibility testing was performed by a StandardBroth Microdilution method as per Clinical and laboratoryStandard Institute (CLSI) guidelines using RPMI 1640 Mediumbuffered with MOPS [3-(N-morpholino) propanesulphonic acid]for fungal cultures and Mueller Hinton Broth (Difco) forbacterial cultures in 96 well microtitre plates. The maximumconcentration tested was 500 mg mL�1 and the inoculum load ineach test well was in the range of 1–5 � 103 cells. The plateswere incubated for 24–48 h for yeast, 72–96 h for mycelial fungiat 35 �C and 24 h for bacteria at 37 �C and read visually as well asspectrophotometrically (SpectraMax) at 492 nm for determina-tion of minimal inhibitory concentrations (MIC).
3. Results and discussion3.1 Optimization of UPLC-MS/MS conditions
In order to achieve optimum separation in a short analysis time,the chromatographic conditions such as the column, mobilephase and gradient program were optimized in the preliminarytest. Two brands of analytical columns, the ACQUITY BEH C18column (50 mm � 2.1 mm, 1.7 mm) and the ACQUITY CSH C18column (100 mm � 2.1 mm, 1.7 mm), were compared. Theresults showed that the ACQUITY BEH C18 column (50 mm �2.1 mm, 1.7 mm) produced chromatograms with better resolu-tion within a shorter time. In this study, various combinationsof mobile phases (water–methanol, 0.1% formic acid in water–methanol, water–acetonitrile and 0.1% formic acid in water–acetonitrile) at different ow rates (0.1, 0.2, 0.25, 0.3, and 0.4),column temperatures (25, 30, 40 and 50 �C) and time wereoptimized for better chromatographic behaviour and appro-priate ionization. A good chromatographic separation wasachieved within four minutes using gradient elution with 0.1%formic acid in water and acetonitrile at 40 �C column temper-ature with a ow rate of 0.3 mL min�1. The detection wave-length was set at 254 nm because of the good response of mostconstituents at this UV wavelength. MS analyses were per-formed in both positive and negative ionization modes. ESIpositive ionization mode was preferred because it providedmore information. The most abundant pseudomolecular ion in
trance potential (EP), collision energy (CE) and cell exit potential (CXP)
a) Q3 mass (Da) DP (V) EP (V) CE (eV) CXP (V)
199 55 10 36 13193 80 12 11 7124 80 12 12 15165 70 12 13 12
Anal. Methods, 2014, 6, 7349–7360 | 7351
Tab
le2
The(M
+H)+
ions,theirMS/MSfrag
mentionsan
dUVab
sorptionmaxim
aforco
mpoundsidentifiedfrom
leaf
extractsofPiperbetlebytheUPLC
-ESI-M
S/MSexp
eriment
Peak
/com
pound
no.
Rt(m
in)
Precursor
ion
(M+H)
MS/MSfrag
men
tions(%
intensity)
Iden
tication
lmax
(nm)
CE
10.39
149
134(1),1
31(29),1
21(64),1
19(5),10
6(7),1
03(100
),93
(45),
91(49),7
7(41
),69
(1),65
(3),55
(14)
Unkn
own
209
19
21.09
223
205(3),1
95(10),1
91(5),18
1(10
0),1
63(5),15
4(50
),14
9(23
),14
0(3),1
35(3),12
1(5),1
03(2)
Nerolidol
230,
282
25
31.09
303
285(8),2
57(20),2
39(2),22
9(49
),20
1(24
),18
3(7),1
65(24),
153(68
),13
7(10
0),1
35(14),1
21(15),1
09(17),9
3(3),8
1(4),6
9(15
)Que
rcetin
230,
282
25
41.21
177
162(2),1
49(50),1
45(30),1
35(20),1
34(5),13
1(25
),11
7(10
0),
107(25
),10
5(15
),91
(47),8
9(10
),81
(2.8)
Chavicol
acetate
248,
277
28
51.25
229
211(1),2
01(4),18
3(2),1
69(12),1
59(5),13
7(59
),10
9(10
0),1
05(11),
99(3),95
(18),9
1(2),6
9(2),5
5(1)
Unkn
own
252,
256
28
61.39
257
229(1),1
97(1),18
3(2),1
79(4),16
9(28
),16
5(8),1
55(2),14
7(1),
137(57
),11
9(13
),10
9(10
0),9
5(13
),91
(3),81
(1),67
(1)
Unkn
own
228,
275
25
71.5
269
241(1),2
27(7),19
5(4),1
91(6),18
1(22
),16
3(68
),15
3(2),1
49(16),
135(98
),12
1(15
),10
7(10
0),9
7(1),9
3(5),8
1(1)
Unkn
own
244,
280
10
81.5
287
269(13
),23
1(5),2
01(5),19
5(5),1
67(8),16
3(10
0),1
49(13),
135(45
),12
1(15
),10
7(35
),10
5(10
)Unkn
own
244,
280
30
91.5
291
273(2),2
41(2),23
1(3),2
07(11),1
99(10),1
79(10),1
65(12),
161(67
),14
7(16
),13
9(10
0),1
23(25),1
05(2),95
(1),71
(1),57
(1)
Catechin
203,
232,
280
45
101.65
165
150(21
),13
7(66
),13
3(39
),12
4(97
),10
9(21
),10
5(10
0),9
5(5),9
1(5),7
9(5)
Eug
enol
238
1711
1.65
301
283(4.1),2
69(100
),25
5(16
.3),24
1(83
),22
7(10
),20
9(20
),19
7(10
),18
1(37
),16
7(4),1
63(15),1
07(4)
8-Hyd
roxy-5,7-
dimethoxy
avan
one
238,
256
29
121.73
151
133(10
),12
3(10
0),1
10(27),1
05(44),1
03(6),95
(6),91
(6),79
(5),77
(5)
Hyd
roxych
avicol
264,
271
3013
1.73
235
207(54
),20
5(17
),19
3(75
),18
1(10
),17
5(20
),15
7(12
),15
1(10
0),1
33(3),12
3(3),1
05(1)
Ally
lpyrocatechol-
3,4-diacetate
264,
271
20
141.73
255
237(7),2
27(7),21
3(45
),19
5(17
),18
1(2),1
67(26),1
63(41),1
53(19),
149(55
),13
9(11
),13
5(88
),12
1(58
),11
7(12
),10
7(10
0),9
3(61
)Unkn
own
264,
271
28
151.73
357
339(3),3
25(4.4),29
3(3),2
73(7),26
5(11
),23
3(7),2
05(100
),19
1(15
),17
7(35
),16
3(4),1
49(6),12
3(99
),10
5(26
),86
(2),69
(3)
Pluv
iatilol
264,
271
25
161.81
135
120(2),1
07(100
),10
5(45
),10
3(45
),93
(5),91
(75.5),7
9(30
),77
(76),
69(5),67
(3),57
(2),55
(2)
Chavicol
220,
273
30
171.81
207
189(97
),17
9(14
),17
4(48
),16
5(10
0),1
61(11),1
57(56),1
50(11),
137(81
),13
1(15
),13
3(47
),12
9(47
),12
4.1(10
),11
9(3),1
05(30),8
3(6)
Eug
enyl
acetate
220,
273
22
181.86
181
166(1)
163(9),1
53(22),1
40(69),1
37(1),13
5(18
),12
5(28
),12
1(50
),11
1(15
),10
9(8),1
07(100
),97
(1),95
(4),93
(38),9
1(65
),81
(4),79
(29),7
7(13
),67
(5),55
(4)
Unkn
own
258,
268,
277
28
191.93
127
109(40
),10
1(50
),99
(100
),86
(60),8
1(75
),79
(38),6
9(38
),67
(30),5
8(45
)Py
roga
llol
217,
327
2520
1.93
209
194(1),1
91(2),18
1(5),1
77(9),16
7(18
),16
3(25
),14
9(30
),13
7(25
),13
5(10
0),1
33(3),12
1(26
),12
3(18
),11
7(45
),10
7(33
),10
5(9),9
5(8),
93(7),87
(25.5),8
3(3),6
9(2)
Sinap
inalde
hyd
e21
7,32
735
211.93
355
263(18
),23
5(3.3),2
03(24.4),1
75(5),16
3(10
0),1
61(5),14
5(22
),13
5(15
),97
(8),85
(3),57
(5)
Chlorogenic
acid
217,
327
19
222.10
285
267(2),2
55(2),25
3(1),2
25(17),2
07(19),1
97(65),1
93(89),
183(7),1
79(25),1
65(100
),15
1(31
),13
7(42
),13
3(7),1
23(3),
107(10
),95
(7.1),87
(1),67
(1)
3-(2,4,5-Trimethoxy-
phen
yl)-2-acetoxy-l-
hyd
roxyprop
ane
253,
280
20
7352 | Anal. Methods, 2014, 6, 7349–7360 This journal is © The Royal Society of Chemistry 2014
Analytical Methods Paper
Publ
ishe
d on
31
July
201
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rsity
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Tab
le2
(Contd.)
Peak
/com
pound
no.
Rt(m
in)
Precursorion
(M+H)
MS/MSfrag
men
tions(%
intensity)
Iden
tication
lmax
(nm)
CE
232.13
193
175(13
),16
1(87
),15
7(10
),15
1(45
),13
7(1.3),1
33(19),1
23(100
),11
0(13
),10
5(22
),95
(1),91
(1),79
(1),55
(1)
Ally
lpyrocatechol
mon
oacetate
260
20
242.30
195
180(3),1
77(6),16
7(3),1
63(3),15
3(39
),14
9(2),1
45(2),13
5(10
0),
133(3),1
29(3),12
5(62
),12
3(45
),12
1(91
),11
7(15
),10
7(50
),10
5(40
),95
(2),93
(26),9
1(2),7
9(3)
Methoxyeu
genol
250
15
252.30
273
227(2),2
13(25),1
95(2),18
5(31
),18
1(10
),17
1(21
),16
7(55
),16
5(1),1
57(56),1
43(66),1
41(5),12
9(12
),10
5(10
0),
91(19),7
2(1),6
9(27
)
Unkn
own
250
15
262.30
305
273(32
),25
9(6),2
45(12),2
41(100
),23
1(5),2
27(47),2
13(74),
203(1),1
99(43),1
85(10),1
57(10),1
43(4),10
5(5),1
01(1),69
(8)
Unkn
own
250
27
272.34
373
355(1),3
27(4),27
7(1),2
63(23),2
56(9),22
9(9),2
05(4),17
3(10
0),
154(2),1
39(48),1
21(53),1
17(7),10
9(3),8
7(1),6
8(1)
Unkn
own
250
30
282.34
179
164(5),1
61(24),1
51(59),1
47(20),1
37(45),1
36.2(21),
133(76
),12
3(39
),12
1.1(32
),11
9(76
),10
5(3),1
03(29),
93(25),9
1(10
0),7
7(12
),67
(3)
Con
iferalde
hyd
e25
039
292.79
371
353(2),3
25(6),31
1(4),2
81(8),25
1(12
),23
3(5),2
23(3),
219(88
),20
5(16
),19
1(10
0),1
73(5),16
3(17
),14
5(4),
135(12
),12
3(8),8
5(2),7
3(4)
Kus
unok
inin
264,
271
30
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Q1MS scan mode was then selected as the precursor ion forproduct ion scan and the product ions were recorded at variousCID collision energies. The efficiency of selected parameters hasbeen experimentally evaluated according to the obtainedresults. Finally, the optimized UPLC-MS/MS method wasapplied for quantitation of phenolics using daidzein as aninternal standard.
3.2 Qualitative analysis
Propenyl phenols, their derivatives, terpenes, avonoids,steroids, alkaloids/amides and hydroxycinnamic acid deriva-tives were previously reported in P. betle. In this study a total of19 phytoconstituents including propenyl phenols and theirderivatives, avonoids, lignans, hydroxycinnamic acid deriva-tives, polyphenol and terpenes were tentatively identied andcharacterized. Among them three propenyl phenols (allylpyr-ocatechol-3,4-diacetate, eugenyl acetate and eugenol) wereunambiguously identied based on their retention time, MSand MS/MS spectra compared with the authentic standards.The tentative identication of the other constituents was basedon the interpretation of their fragmentation patterns obtainedvia MS/MS and by comparison with previously reported litera-ture. The (M + H)+ ions, their MS/MS fragment ions and UVabsorption maxima for compounds identied from leaf extractsof Piper betle are listed in Table 2. Fig. 1 represents UPLCchromatograms of mixed reference standard compounds andleaf extract of P. betle (Bangladeshi). Several peaks wereunidentied due to the lack of sufficient data and literaturesupport.
3.2.1 Identication of propenyl phenols and their deriva-tives. Peak 12 (m/z 151) and peak 16 (m/z 135) were tentativelyidentied as hydroxychavicol and chavicol, respectively on thebasis of the ESI-MS/MS fragmentation pattern (Scheme S1†).Base peak ions for chavicol and hydroxychavicol were observedat m/z 107 and 123, respectively due to the loss of ethylenefrom (M + H)+ ions and further elimination of neutral COgenerated fragment ions at m/z 79 and 95 for chavicol andhydroxychavicol, respectively. The presence of two vicinalhydroxyl groups in hydroxychavicol favoured the eliminationof water resulting in a fragment ion at m/z 133 which indicatespeak 12 as a hydroxy derivative of chavicol. A further loss ofethylene molecules from the fragment ion atm/z 133 generatesthe fragment ion at m/z 105. Hydroxychavicol and chavicolwere previously reported from P. betle.13,32,33 Peak 10 (m/z 165)was identied as eugenol by comparing the retention time andthe characteristic fragment ions with the correspondingauthentic standard. It was also observed that peak 24 (m/z195), tentatively identied as methoxy eugenol, generatedsimilar types of fragment ions. The ESI-MS/MS fragmentationpattern as shown in Scheme 1 reected both compounds to beanalogous. Fragmentation of (M + H)+ ions showed the loss ofmethanol (m/z 163 and 133) and ethylene (m/z 167 and 137)from eugenol andmethoxy eugenol, respectively. A further lossof ethylene from fragment ions m/z 133 and 163 yielded frag-ment ions at m/z 105 and 135 for eugenol and methoxyeugenol, respectively. Fragment ions observed at m/z 150 and
Anal. Methods, 2014, 6, 7349–7360 | 7353
Fig. 1 (A) UPLC chromatogram of Piper betle (Bangladeshi) leaf extract and (B) UPLC chromatogram of reference standards.
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180 were due to the loss of methyl radicals from eugenol andmethoxyeugenol, respectively. A fragment ion observed at m/z133 was due to the loss of formaldehyde from the fragment ionof methoxy eugenol at m/z 163 which proved peak 24 as amethoxy derivative of eugenol. The loss of formaldehyde is acharacteristic fragmentation of methyl aryl ether. These frag-mentation patterns were in agreement with the reportedliterature.34
Peaks 13 (m/z 235) and 17 (m/z 207) were identied as allyl-pyrocatechol-3,4-diacetate and eugenyl acetate, respectively bycomparing their retention time and the characteristic fragmentions with the corresponding authentic standards. Peaks 4 (m/z
Scheme 1 Proposed ESI-MS/MS fragmentation pathway for eugenol (1)
7354 | Anal. Methods, 2014, 6, 7349–7360
177) and 23 (m/z 193) were tentatively identied as chavicolacetate and allylpyrocatechol monoacetate, respectively on thebasis of the ESI-MS/MS fragmentation pattern as shown inScheme 2, which was found to be similar to the above propenylphenol esters matched with authentic standards. Fragment ionsobserved atm/z 135, 151, 165, 193 were due to the loss of neutralketene molecules from (M + H)+ ions of chavicol acetate (m/z177), allylpyrocatechol monoacetate (m/z 193), eugenyl acetate(m/z 207) and allylpyrocatechol-3,4-diacetate (m/z 235), respec-tively. A loss of acetic acid from the corresponding (M + H)+ ionsand fragment ions was observed atm/z 117, 133, 147 and 175 forchavicol acetate, allylpyrocatechol monoacetate, eugenyl acetate
and methoxy eugenol (2).
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Scheme 2 Proposed ESI-MS/MS fragmentation pathway for chavicol acetate (1), allylpyrocatechol monoacetate (2), eugenyl acetate (3) andallylpyrocatechol-3,4-diacetate (4).
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and allylpyrocatechol-3,4-diacetate, respectively. Thesecompounds were previously reported from P. betle.13,23,32,33
In the present study, peaks 20 (m/z 209) and 28 (m/z 179) weretentatively identied as sinapinaldehyde and coniferaldehyde,respectively on the basis of the ESI-MS/MS fragmentationpattern (Scheme S2†) which is in agreement with the reportedliterature.34 Similar fragmentation patterns were observed forboth compounds which indicate that these are analogous.Elimination of methanol, methyl radicals and water from (M +H)+ ions of coniferaldehyde and sinapinaldehyde producedfragment ions at m/z 147, 177, at m/z 164, 194 and at m/z 161,191, respectively. Fragment ions observed at 151 and 181 forconiferaldehyde and sinapinaldehyde, respectively were due tothe elimination of CO molecules from (M + H)+ ions.
3.2.2 Identication and characterization of other phyto-constituents. In the present study three avonoids, peaks 3 (m/z303), 9 (m/z 291) and 11 (m/z 301), were tentatively identied asquercetin, catechin and 8-hydroxy-5,7-dimethoxyavanone,respectively and characterized on the basis of the ESI-MS/MSfragmentation pattern (Scheme S3–S5†) which was previouslyreported in the literature.35,36 Fragments formed by retro-Diels–Alder (RDA) reactions are the most diagnostic fragments foravonoid identication.37 Fragment ions observed at m/z 153,139, 197 for quercetin, catechin and 8-hydroxy-5,7-dimethoxy-avanone, respectively, were due to retro-Diels–Alder reaction.Quercetin and catechin were previously reported from P. betle
This journal is © The Royal Society of Chemistry 2014
and 8-hydroxy-5,7-dimethoxyavanone was previously reportedfrom P. hispidum.32,38
Two lignans, peaks 15 (m/z 357) and 29 (m/z 371), weretentatively identied as pluviatilol and kusunokinin, respec-tively and characterized on the basis of the ESI-MS/MS frag-mentation pattern (Scheme S6 and S7†). Pluviatilol andkusunokinin were reported from P. brachystachyum, P. chabarespectively.32 Base peak ions for pluviatilol observed at m/z 205were due to the loss of C7H8O2 followed by the loss of neutral COmolecules and the peak at m/z 123 was due to the loss ofC13H14O4. The fragment ion observed at m/z 339 was due to theloss of water molecules. The fragment ion observed at m/z 219for kusunokinin was due to the loss of C9H12O2, a further loss ofneutral COmolecules generates a fragment ion atm/z 191 whichis a base peak ion. The fragment ion observed at m/z 353 wasdue to the loss of water molecules. The proposed fragmentationof lignans is in agreement with the reported literature.39
Peak 21 (m/z 355) was tentatively identied as chlorogenicacid and characterized on the basis of the ESI-MS/MS frag-mentation pattern (Scheme S8†). Previously chlorogenic acidreported from P. betle leaf showed activity against cancer.40 Apredominant fragment ion at m/z 163 was observed due to theloss of stable acidic part (C7H12O6), a further loss of neutral COmolecules generated a fragment ion at m/z 135. The presence oftwo vicinal hydroxyl groups favoured the elimination of waterobserved at m/z 145. Fragmentation of chlorogenic acid in
Anal. Methods, 2014, 6, 7349–7360 | 7355
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positive mode is in agreement with previously reportedliterature.35
Peak 19 (m/z 127) was tentatively identied as pyrogallol andcharacterized on the basis of the ESI-MS/MS fragmentationpattern (Scheme S9†). A base peak for pyrogallol observed atm/z99 was due to the loss of CO which is a characteristic frag-mentation of phenols. A further loss of water from the peak atm/z 99 gave a fragment ion atm/z 81. The presence of two vicinalhydroxyl groups on the benzene ring favoured the eliminationof water observed at m/z 109.
Peak 22 (m/z 285) was tentatively identied as 3-(2,4,5-tri-methoxyphenyl)-2-acetoxy-1-hydroxypropane and characterizedon the basis of the ESI-MS/MS fragmentation pattern (SchemeS10†). 3-(2,4,5-Trimethoxyphenyl)-2-acetoxy-1-hydroxypropanewas previously reported from P. clusii.32 A fragment ion at m/z225 was observed due to the loss of two formaldehyde mole-cules, a further loss of acetic acid produced an ion at m/z 165.Elimination of water from the (M + H)+ ion generated a frag-ment ion atm/z 267 which gave a fragment ion atm/z 207 due tothe loss of two formaldehyde molecules.
Peak 2 (m/z 223) was tentatively identied as nerolidol andcharacterized on the basis of the ESI-MS/MS fragmentationpattern (Scheme S11†). Nerolidol was previously reported fromPiper species (P. falconeri, P. guineense, P. marginatum andP. nigrum).32 The fragment ion observed at m/z 205 was due to
Fig. 2 (A) UPLC-MRM extracted ion chromatogram of standard solut(Bangladeshi) leaf extract, (1) daidzein (IS), (2) allylpyrocatechol-3,4-diac
7356 | Anal. Methods, 2014, 6, 7349–7360
the loss of water molecules and the fragment ion atm/z 181 wasdue to the loss of a propene moiety from the parent ion.
3.3 Validation procedure for quantitative analysis
The UPLC-MRM method was validated according to the guide-lines by international conference on harmonization (ICH,Q2R1) with respect to linearity, lower limit of detection (LOD),lower limit of quantication (LOQ), accuracy, precision,stability and recovery. Fig. 2 shows UPLC-MRM extracted ionchromatograms (XIC) of standard solution and Piper betle(Bangladeshi) leaf extract.
3.3.1 Calibration curves, limits of detection (LOD) andquantication (LOQ). A series of concentrations of standardsolution were prepared for the establishment of calibrationcurves. The linearity of calibration was performed by the ana-lytes-to-IS peak area ratios versus the nominal concentrationand the calibration curves were constructed with a weight (1/x2)factor by least-squares linear regression. The applied calibra-tion model for all curves was y ¼ ax + b, where y ¼ peak arearatio (analyte/IS), x ¼ concentration of the analyte, a ¼ slope ofthe curve and b¼ intercept. The LODs and LOQs weremeasuredwith the S/N of 3 and 10 respectively, as criteria. The results arelisted in Table 3. The LOD for each analyte varied from 0.13–0.48 ng mL�1 and LOQ from 0.41–1.47 ng mL�1. All the
ion and (B) UPLC-MRM extracted ion chromatogram of Piper betleetate, (3) eugenyl acetate, and (4) eugenol.
This journal is © The Royal Society of Chemistry 2014
Tab
le3
Linear
regressionequations,lin
ear
ranges,LO
Ds,LO
Qs,intra-day,inter-day
precisions,stab
ility
andreco
very
forthreean
alytes
Analytes
Regressioneq
uation
ar2
Linearrange
(ngmL�
1)
LODb(ngmL�
1)
LOQc(ngmL�
1)
PrecisionRSD
(%)
Stab
ilityRSD
(%)(n
¼6)
Recovery(n
¼3)
Intra-da
y(n
¼6)
Inter-da
y(n
¼6)
Mean(%
)RSD
(%)
Ally
lpyrocatechol-3,4-diacetate
Y¼
24.35x
+0.62
60.99
921–20
00.14
0.42
0.83
0.91
2.45
97.65
1.58
Eugenyl
acetate
Y¼
1.13
3x+0.22
10.99
8110
–500
0.48
1.47
0.75
0.93
2.52
96.14
1.14
Eug
enol
Y¼
0.17
4x�
0.00
70.99
9910
–500
0.13
0.41
1.0
1.04
2.31
98.46
0.66
ayis
thepe
akarea
andxis
theconcentrationof
stan
dard
solution
s.bLO
Drefers
tothelimitsof
detection,3
.3�
Syx/slop
e.cLO
Qrefers
tothelimitsof
quan
tity,1
0�
Syx/slop
e.
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calibration curves showed good linearity (r2 $ 0.9981) withinthe test ranges.
3.3.2 Precision, stability and recovery. The intra-day andinter-day variations, which were chosen to determine theprecision of the developed method, were investigated bydetermining three analytes with IS in six replicates during asingle day and by duplicating the experiments on threeconsecutive days. Variations of the peak area were taken as themeasures of precision and expressed as percentage relativestandard deviations (RSD). The overall intra-day and inter-dayprecisions were not more than 1.0%. Stability of sample solu-tions stored at room temperature was investigated by replicateinjections of the sample solution at 0, 2, 4, 8, 12 and 24 h. TheRSD values of stability of the three analytes were #2.5%. Arecovery test was applied to evaluate the accuracy of thismethod. Three different concentration levels (high, middle andlow) of the analytical standards were added into the samples intriplicate and average recoveries were determined by thefollowing equation:
Recovery (%) ¼ (observed amount � original amount)/
spiked amount � 100%.
The analytical method developed had good accuracy withoverall recovery in the range from 96.14–98.46% (RSD # 1.6%)for all analytes (Table 3).
3.3.3 Quantitative analysis of samples. The proposedUPLC-MRMmethod was subsequently applied to determine thecontents of three major bioactive phenolics in all the thirteenlandraces of P. betle. Quantitative analysis of these phenolicsshows that allylpyrocatechol-3,4-diacetate is below the detectionlevel in Meetha Patta, Shipurkata, Assam Pan, Mahoba andSaua; similarly eugenyl acetate is below the detection level inShirpurkata, Kapoori, Assam Pan and Mahoba, whereaseugenol is detected in all the landraces except Meetha Patta andKapoori. The contents of three phenolics in thirteen landracesare summarized in Table 4. The results showed that there wereremarkable differences in their contents in all the thirteenlandraces of P. betle. For example, the total contents of threephenolics in Jalesar Green (41.4%) weremuch higher than otherlandraces of P. betle, other notable landraces with high contentswere Bangladeshi (33.2%) > Deshi (31.3%) > Jalesar White(22.7%). With respect to the content of each analyte eugenol ismost abundant in all the landraces and highest in JalesharGreen reaching up to (23.1%). The study showed that theconcentration of phenolics varies signicantly with thegeographical origin of P. betle. Therefore the developed methodmight be quite suitable and reasonable for quality control ofP. betle or derived herbal formulations.
3.4 Evaluation of antimicrobial activity
All the ethanolic leaf extracts tested exhibited antifungal activitywhere Jalesar Green which shows higher contents of quantiedphenolics was best against Candida parapsilosis and Crypto-coccus neoforman with MIC values 6.25 and 31.2 mg mL�1
respectively (Table 5). Other notable landraces, Bangledeshi,
Anal. Methods, 2014, 6, 7349–7360 | 7357
Table 4 Contents (mg g�1) of three analytes in thirteen landraces of Piper betlea
P. betle Landraces
(Mean � SD) mg g�1
Allylpyrocatechol-3,4-diacetate Eugenyl acetate Eugenol Total (mg g�1) Total (%)
Meetha Patta* BDL 0.4 � 0.06 BDL 0.4 0.040Sanchi* 0.16 � 0.37 1.5 � 0.02 1.71 � 0.04 3.37 0.337Shirpurkata BDL BDL 0.06 � 0.11 0.06 0.006Kapoori 0.03 � 0.14 BDL BDL 0.03 0.003Assam Pan BDL BDL 4.72 � 0.06 4.72 0.472Nagpuri 4.25 � 0.22 56.4 � 0.25 113.20 � 0.10 173.85 17.385Jalesar Green 3.19 � 0.26 180.0 � 0.31 230.67 � 0.14 413.86 41.386Jagarnathi 0.6 � 0.02 34.8 � 0.18 66.80 � 0.21 102.2 10.220Deshi 2.2 � 0.04 133.33 � 0.24 177.33 � 0.22 312.86 31.286Mahoba BDL BDL 24.27 � 0.26 24.27 2.47Saua BDL 34.53 � 0.61 85.20 � 0.31 119.73 11.976Jalesar White 16.8 � 0.05 90.4 � 0.59 119.33 � 0.35 226.53 22.653Bangladeshi 20.4 � 0.16 121.3 � 0.06 190.67 � 0.05 332.37 33.237
a BDL – below detection level, *West Bengal.
Table 5 Minimum inhibitory concentration (MIC) in mg mL�1 of ethanolic leaf extracts of Piper betle landraces against bacteria and fungia
P. betle Landraces
Minimum inhibitory concentration (MIC) in mg mL�1 against
Bacteria Fungi
1 2 3 4 5 6 7 8 9 10
Nagpuri >500 >500 >500 >500 250 125 500 500 >500 125Jalesar Green >500 >500 >500 >500 62.5 31.2 250 250 500 6.25Jagarnathi >500 >500 >500 >500 62.5 62.5 250 250 500 125Deshi >500 >500 >500 >500 31.2 15.6 125 125 125 31.2Mahoba >500 >500 >500 >500 62.5 62.5 500 500 >500 125Saua >500 >500 >500 >500 125 125 250 500 >500 125Jalesar White >500 >500 >500 >500 31.2 15.6 125 125 250 31.2Bangladeshi >500 >500 >500 >500 62.5 31.2 125 125 500 62.5
StandardsGentamycin 12.5 3.12 6.25 6.25 ND ND ND ND ND NDNoroxacin 0.024 0.78 0.39 0.05 ND ND ND ND ND NDFluconazole ND ND ND ND 1 2 2 >32 >32 2Amphotericin B ND ND ND ND 0.02 0.125 0.3 0.25 0.5 0.016
a (1) E. coli (ATCC 9637), (2) Pseudomonas aeruginosa (ATCC BAA-427), (3) Staphylococcus aureus (ATCC 25923), (4) Klebsiella pneumoniae (ATCC27736), (5) Candida albicans, (6) Cryptococcus neoformans, (7) Sporothrix schenckii, (8) Trichophyton mentagrophytes, (9) Aspergillus fumigates, (10)Candida parapsilosis (ATCC-22019), ND – not detectable.
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Jalesar White and Deshi were best against Candida albicans,Candida parapsilosis and Cryptococcus neoforman with MICvalues ranging from 15.6–31.2 mg mL�1 (Table 5). However,none of the extracts were found to be active against bacteria ateven 500 mg mL�1 concentration.
3.5 Principal component analysis
Principal component analysis (PCA) was applied in this study toestablish the correlation and discrimination of P. betle land-races using soware STATISTICA 7.0. The UPLC-MS data(including Rt, mass and % area of peaks) for all the thirteenP. betle landraces were subjected to PCA using 12 variables (m/z223, 303, 177, 291, 165, 301, 151, 235, 357, 207, 135, 127, 209,355, 285, 193, 195, 179 and 371). The PC1 vs. PC2 plot clearly
7358 | Anal. Methods, 2014, 6, 7349–7360
brings out the relationship among all the thirteen landracesand classied them into four well-dened groups based onrelative contents of identied compounds (Fig. 3(A) is the PCAscore plot showing the discrimination of P. betle landraces and(B) is the PCA score plot showing loading of variables). Thedimensions of 12 peaks were reduced to three principalcomponents explaining about 72.6% variation. The 33.21%variation was explained by PC1, composed of peaks at Rt 1.73min (m/z 151, 235, 357), Rt 1.93 min (m/z 127, 209, 355) and Rt2.34 min (m/z 179). PC2 explains 23.2% of variations in P. betlelandraces. The similarity in Meetha Patta and Nagpuri land-races was due to the compound at Rt 1.5 min (m/z 291) as theircontribution was highest (20–23%). Landraces Shirpurkata andSaua were together due to compounds at Rt 1.09 min (m/z 223,303); similarly Deshi, Bangladeshi and Jalesar White were in the
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Fig. 3 (A) PCA score plot showing discrimination of Piper betle landraces and (B) PCA score plot showing loading of variables.
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same group due to similarity in the relative abundance ofcompounds at Rt (1.09 min, 1.5 min, 1.81 min and 2.34 min).The highest abundance (23–47%) of compounds was present atRt 1.09 min (m/z 223, 303). However, compounds at Rt 1.5 min(m/z 291), Rt 1.81 min (m/z 135, 207) and Rt 2.34 min (m/z 179)were present in equal amounts (6–7%). Sanchi, Kapoori, AssamPan, Jalesar Green, Mahoba and Jagnathi were very similar dueto high abundance of compounds at Rt 1.09 min (m/z 223, 303)which was 60% in Assam Pan and more than 70% in JalesarGreen, Mahoba and Jagnathi. The compound at Rt 1.5 min (m/z291) was present in 10–12% abundance in Assam Pan, JalesarGreen, and Jagnathi but completely absent in Sanchi, Kapooriand Mahoba landraces.
4. Conclusion
In this study, a UPLC-ESI-MS/MS method was developed andvalidated as a simple, fast, sensitive and reliable analyticaltechnique for identication and structural characterization ofphytoconstituents with simultaneous determination of majorbioactive phenolics, namely, allylpyrocatechol-3,4-diacetate,eugenyl acetate and eugenol from thirteen landraces of P. betleleaf extracts. In this experiment 19 compounds were identied,characterized and schematic fragmentation pathways for all theidentied compounds were proposed. This method was alsoapplied to investigate the contents of major bioactive phenolicsin thirteen landraces of P. betle and results showed remarkabledifferences in their contents. In vitro antimicrobial activity ofonly those landraces which showed high contents of bioactivephenolics in quantitative analysis was evaluated. Therefore thismethod provided an excellent quantitative tool for qualityassessments of P. betle or derived herbal formulations due to itshigh capacity, high sensitivity, high selectivity and shorteranalysis time.
Acknowledgements
A grateful acknowledgement is made to SAIF-CDRI, Lucknow,where all the mass spectral studies were carried out. BK is
This journal is © The Royal Society of Chemistry 2014
thankful to Dr K. P. Madhusudanan and Dr N. Kumar for theirhelpful discussions, RP to UGC, New Delhi, for nancialsupport. CDRI Communication No. 8740.
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