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Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry
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Syed Ghulam Musharraf ⇑, Madiha Goher, Bibi Zareena
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H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
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Article history: Received 17 November 2014 Received in revised form 7 February 2015 Accepted 31 March 2015 Available online xxxx Keywords: Steroidal alkaloids Electrospray ionization mass spectrometry Buxus papillosa ESI-QQQ-MS/MS
a b s t r a c t Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486–8.08 ng/mL and 1.473–24.268 ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15 min run time. This paper describes the application of LC– QQQ-MS technique for steroidal alkaloids analysis in plant samples. Ó 2015 Published by Elsevier Inc.
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1. Introduction
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Buxus alkaloids represent a unique class of compounds possessing a triterpenoidal skeleton in which one or two nitrogen atoms are incorporated as side chains. About 100 species of genus Buxus are present in Africa, America, Asia and Europe [1]. The genus Buxus is among the richest source of steroidal alkaloids and more than 200 new steroidal alkaloids have already been reported [2]. Herbal formulations based Buxus species are widely used in Ayurvedic, Greco-Arabic and Chinese medicines in Asian countries due to their impressive biological activities. Extracts of genus Buxus have been used in the indigenous systems of medicine for the treatment of various disorders, such as malaria, rheumatism and skin infections. They also showed anticholinesterase [3], anti-HIV [4], immunosuppressive, and cytotoxicity activities [5,6]. Most of the biological activities of Buxus species are largely due to the steroidal alkaloids present therein. Buxus papillosa is widely distributed in Khyber Pakhtunkhwa (KPK) province of Pakistan. It is a well studied species of the genus Buxus with over 50 triterpenoidal and steroidal alkaloids. Many of the alkaloids are known for the various pharmacological activities, such as acetylcholinesterase and butyrylcholinesterase inhibitory activity [7]. Buxus alkaloids, also called alkamine, have nitrogen atoms, either at C-3 or C-20 or at both positions. On the basis of their basic
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⇑ Corresponding author. Tel.: +92 21 34824924 5, +92 21 4819010; fax: +92 21 34819018 9. E-mail address: [email protected] (S.G. Musharraf).
skeleton, Buxus alkaloids can be divided into the following groups: (1) derivatives of 9b,19-cyclo-4,4,14a-trimethyl-5a-pregnane: having a basic skeleton with a cyclopropane ring between C-9 and C19. (2) derivatives of 9(10–19)abeo,14a-trimethyl-5a-pregnane, resulting from the opening of the 9b-19-cyclopropane ring system. Numerous studies on quantitative and qualitative analysis of different steroidal alkaloids have been published. The identification of glycosides of solasodine steroidal alkaloid have been reported by using techniques such as thin layer chromatography (TLC) [8], capillary electrophoresis mass spectrometry (CE-MS) [9] and high-performance liquid chromatography (HPLC) [10]. An HPTLC method was developed for the determination of steroidal glycoalkaloids in Solanum xanthocarpum [11]. Similarly, method was developed for the simultaneous identification and analysis of Veratrum alkaloids [12]. Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum), have been identified by using optimized reversed-phase LC with electrospray ionization (ESI) and ion trap mass spectrometry (IT-MS) [13]. An HPLC–MS method for characterizing cevanine-type, veratramine-type, jervine-type and secosolanidine-type alkaloids in Fritillaria species has been developed [14,15]. Electrospray ionization multi-stage mass spectrometry was used to study the fragmentation of protoverine-type, germine type and zygadenin-type alkaloids, isolated from the Chinese herb, Veratrum nigrum L. [16]. However, only one report on the quantification of steroidal alkaloids through LC–ESI-TOF-MS method is found in literature used for quantification of steroidal alkaloids from Fritillaria species [17].
http://dx.doi.org/10.1016/j.steroids.2015.03.018 0039-128X/Ó 2015 Published by Elsevier Inc.
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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In continuation of our studies on the application of electrospray ionization mass spectrometry and high throughput dereplication strategy development [18–22], we describe here the development and validation of an LC–ESI-QQQ-MS/MS method for the quantification of steroidal alkaloids in B. papillosa with better limit of detection in short analysis time. This is the first report on the quantification of steroidal alkaloids in any plant using ESI-QQQ-MS.
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2. Experimental
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2.1. Chemicals and reagents
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Chemicals and solvents were of analytical reagent and HPLC grades, respectively, and purchased from Aldrich–Sigma (USA). Deionized water (Milli-Q), used throughout the study, was obtained from Millipore Milli Q Plus System (Bedford, USA). The steroidal alkaloid standards, cyclomicrobuxine (1), cyclomicrobuxinine (2), E-buxenone (3), N-benzoylbuxahyrcanine (4), buxidine (5), buxandrine (6), and N-Isobutyroylbuxahyrcanine (internal standards, I.S.) (Fig. 1) were obtained from the Molecular Bank facility of the Dr. Panjwani Center for Molecular Medicine and Drug Research (International Center for Chemical and Biological Sciences), University of Karachi. The isolation and characterization details of the analyzed steroidal alkaloids have been reported previously [23,24].
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2.2. Standard solution and calibration standards
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Stock standard solutions were prepared in methanol at a final concentration of 1 mg/mL. These solutions were stored at 4 °C. Working solutions, used for LC–QQQ-MS analysis, were obtained by diluting the stock solutions with acetonitrile (with concentrations between 0.1 lg/mL and 19 lg/mL). Stock solution (260 lg/ mL) of internal standard, I.S. (N-Isobutyroylbuxahyrcanine) was
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prepared in methanol and stored at 4 °C until use, and the final concentration of the internal standard was 0.5 lg/mL in the calibration standards range of 0.1–1 lg/mL and 12 lg/mL in the calibration range of 3–19 lg/mL, respectively.
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2.3. Sample preparation
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The plant B. papillosa (leaves, stems & roots) was collected from the Swat, Pakistan (Voucher # 9718). The plant material was identified by Mr. Mehboob ur Rehman, Plant taxonomist, the Department of Botany, Government Postgraduate Jahanzeb College, Swat. The dried leaves, roots and stems of B. papillosa were separately powdered to a homogeneous size, and sieved through a 400 lm mesh, followed by drying at 60 °C in the oven for 2 h. The dried leaves, roots and stem powder (500 mg) was pre-alkalized with 5 mL ammonia solution (33%) for 1 h, and immersed in 12 mL methanol overnight, then ultrasonicated for 1 h. After being filtered, the extract was concentrated to dryness in vacuum at 45 °C. 10 mL methanol was added in the residue and ultrasonicated for 5 min. Two working solutions for two different concentrations of internal standard were prepared by taking 100 lL of this reconstituted residue solution, and both were made up to 1 mL with acetonitrile containing a final concentration of 0.5 and 12 lg/mL internal standard respectively. The resultant solutions were centrifuged at 12,000 rpm for 10 min, the supernatants were filtered through Millipore filter (0.45 lm), and transferred to an autosampler vial for LC–QQQ-MS analysis.
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2.4. LC–QQQ-MS analysis
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Liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) in positive ionization mode, was used to detect the steroidal alkaloids. The LC analysis of the selected steroidal alkaloids was carried out using an
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O
H N
O
H
OH
H N H
H
Cyclomicrobuxine (1)
H
H
H
OH N H
H
H
E-Buxenone (3)
Cyclomicrobuxinine (2)
N
N H
O
H N H
H
H
Buxidine (5)
N H
O
N O
O
H O
N H HO
Buxandrine (6)
H
OH N H
H
OH
HO
N-Benzoylbuxahyrcanine (4)
H
H
O
OH N H
O
H
N -I sobutyroylbuxahyrcanine (I.S.)
Fig. 1. The structures of Buxus steroidal alkaloids.
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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Agilent 1260 Infinity HPLC system (Agilent Technologies, Singapore), equipped with binary pump, online degasser, auto sampler, and thermostatically controlled column compartment. The chromatography separation was performed on C-18 column (Merck, 4.6 55 mm I.D., 3 lm particle size) with a solvent flow rate of 0.5 mL/min at a temperature of 30 °C. The sample injection volume was set at 3 lL. Water (0.1% formic acid) and acetonitrile (also contained 0.1% formic acid) were used as the mobile phases A and B, respectively. The solvent gradient adopted was as follows: 5–75% B at 0–10 min, 75–95% B at 10–11 min, 5% B at 11.01– 16 min. This HPLC system was connected to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Singapore), equipped with an electrospray interface. The ESI source conditions were as follows: drying gas (N2) flow rate, 8.0 L/min; drying gas temperature, 300 °C; nebulizer, 40 psi; capillary voltage, 3000 V; cell accelerator voltage, 7 V; fragmentor, 135 V. LC–QQQ-MS mass spectra were recorded across the range from 100 to 800 m/z. Quantification was performed in comparison of an internal standard method. A steroidal alkaloid having similar skeleton as those of analytes was used an internal standard. Multiple Reaction Monitoring (MRM) mode was used for the quantification of standards with collision energy of 33–40 V and a solvent delay of 5 min. The dwell time was 200 ms. The fragment ions, listed in Table 1, allow the quantification by using one either the quantification ion, and the other ion as a qualifier. Extracted ion chromatograms (XICs) for the [M+H]+ ions of the target compounds were used for peak area determination. Peak area ratios of analytes/I.S. were used for quantification. The LOD and LOQ were estimated as 3 and 10 times of the noise level, respectively. Moreover, both were initially determined by diluting the known concentration of steroidal alkaloids standard till the mean instrumental responses were nearly three and ten times of the standard deviation of the responses for six manifold analyses. The intra-day variability study was measured by the injection of the same standard solution at four different times in the same day. The inter-day variability study was measured for three successive days by using three different concentrations of all standard steroidal alkaloids. All the operations, acquisition and analysis of data were controlled by Agilent Mass Hunter qualitative software Ver. B.04.00 and quantitative software Ver. B.05.00 (Agilent Technologies).
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3. Results and discussion
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3.1. Method optimization
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LC method was optimized to best chromatographic peak shapes and signal-to-noise ratios (S/N) of the analytes. Various gradients of acetonitrile–water containing 0.1% formic acid at a flow rate of 0.5 mL/min were used for optimizing the elution conditions in order to achieve the reliable quantification of these alkaloids. Under the gradient condition, as described previously, the internal standard is a baseline, separated from the other six compounds, while compounds 1 and 2 eluted at the same time segment and
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compounds 3–6 were also eluted at the same time segment. However, by virtue of the XIC mode in MRM, quantitative analysis could be achieved as the overlapping peaks produced different protonated [M+H]+ ions. Optimization of the MRM method was based on the selection of the transitions from the precursor ion to the different product ions for the selective and sensitive determination of the target steroidal alkaloids. Once the number of transitions was established, the influence of the dwell time on the sensitivity was checked, and 200 ms was found to be the best value. The transition with the highest signal intensity was used for the quantification of each analyte. Fragmentor voltage affects the sensitivity and fragmentation patterns of the analytes, significantly. Therefore, the voltage value was studied in the range of 50–250 V under optimized source conditions. In general, fragmentor voltage of 100 V provided minimal fragmentation in most of the analytes. Collision energies also affect the sensitivity and fragmentation pattern of the analytes significantly. MS–MS Spectra at different collision energies were studied in the range from 20 to 100 V under optimized source conditions. In general, collision energy of 33 V provided maximum intensity of fragmented ions. Fragmentor voltages and collision energies of all the analytes and internal standard are summarized in Table 1.
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3.2. Quantification and method validation
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LC–QQQ-MS quantification was carried out under MRM conditions by using the XICs of the protonated molecule of each steroidal alkaloid. Total ion chromatogram of standard compounds, obtained by MRM and qualifier and quantifier chromatogram of all standards of steroidal alkaloids, are shown in Figs. 2a and b. Peak area ratios of the analytes/I.S. were used for quantification. The linearity of the analytical response across the studied range was found to be excellent with correlation coefficients (r2) > 0.99 for all analytes. The values of linear calibration curves of 6 steroidal alkaloids are listed in Table 2. The developed method enables quantification of many targeted analytes in a single experiment and to filter out interfering compounds through selective and specific transitions chosen for each compound. Method precision was checked by intra-day and inter-day variability. The relative standard deviation (RSD) values, obtained from run-to-run and day-to-day precision studies are summarized in Table 3. The developed method was found to be precise with intra-day and inter-day variability and RSD values lie between 0.39–8.22% and 4.16– 25.49%, respectively. In addition, the RSD values of retention time were less than 0.12% and 0.38% in run-to-run and day-to-day analysis, respectively. Sensitivity of the method was evaluated by determining limits of detection (LOD) and quantification (LOD). Limits of detection and limit of quantification were 0.486– 8.08 ng/mL and 1.473–24.268 ng/mL, respectively, for all analyzed compounds. The LODs and LOQs for all analytes are presented in Table 2. Better signal-to-noise ratio allows quantification of steroidal alkaloids with lower Limits of Quantification (LOQ) as
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Table 1 Optimized LC–MS/MS acquisition method parameters for steroidal alkaloids. Analyte
1 2 3 4 5 6 I.S.
Time segment
1 1 3 3 3 3 2
Precursor ion (m/z)
386.3 372.3 370.3 507.2 521.2 563.4 473.3
MRM transitions (m/z) Identification
Quantification
119, 145, 135, 323, 307, 319, 340,
386 ? 119 372 ? 145 370 ? 84 507 ? 323 521 ? 307 563 ? 319 473 ? 340
105 131 84 444 325 440 410, 213
Fragmentor voltage (V)
Collision energy (V)
100 130 100 120 80 150 120
33 35 33 33 40 38 38
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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Fig. 2a. Total ion chromatogram of standard compounds obtained by MRM, qualifier and quantifier chromatogram of standards 1, 2 and 3.
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compared to previously reported quantification methods based on ESI-TOF-MS [17].
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3.3. Application to B. papillosa
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The optimized LC/QQQ-MS method was subsequently applied for the simultaneous qualitative and quantitative analysis of 6 steroidal alkaloids in the extract of leaves, roots and stem of B.
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papillosa. The quantified results obtained are summarized in Table 4. The results showed that compound 1 is present in all samples in a range of 16–28 lg/g and approx. 100 lg/g of compound 2 is present in leaves roots and stem extracts. Concentration of compound 3 in leaves was approximately three times higher than in roots and stem, while the concentration of compound 6 is four times higher in roots and stem, than in leaves. Compound 5 was present in very little amount in roots and stem and was absent
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Fig. 2b. Qualifier and quantifier chromatogram of standards 4, 5, 6 and I.S.
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in leaves, while compound 4 was negligible in concentrations in all samples. On the basis of these results, it could not be concluded that which part of the plant contains higher concentrations of steroidal alkaloids.
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4. Conclusion
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In this work, it was demonstrated that the liquid chromatography in combination with triple quadrupole mass
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spectrometry (LC–QQQ-MS) is an efficient method both for qualitative and quantitative analysis of steroidal alkaloids. Good precision and linearity was obtained, with significantly improved limits of detection for steroidal alkaloids. The excellent selectivity and sensitivity, as well as good linearity, allows identification and quantification of low levels of steroidal alkaloids in complex Buxus species extracts. Furthermore, the lower detection limits of the developed method can be used for the analysis of steroidal alkaloids in other medicinal plants as well as in herbal
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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Table 2 Calibration curve, correlation coefficient (r), instrumental LODs and instrumental LOQs for 6 analytes in a test range 0.1–1 (lg/mL). r2
Instrumental LODs (ng/mL)
Instrumental LOQs (ng/mL)
References
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1 2 3 4 5 6
y = 5.116x + 0.025 y = 1.974x + 0.125 y = 11.88x + 0.689 y = 0.977x + 0.25 y = 0.097x + 0.002 y = 0.296x + 0.022
0.996 0.992 0.998 0.989 0.991 0.995
8.008 7.61 5.165 5.102 0.486 3.789
24.268 23.062 15.65 15.461 1.473 11.481
[1] Duke JA. Handbook of medicinal herbs. 2nd ed. Boca Raton, FL: CRC Press; 2002. [2] Cordell GA. Introduction to alkaloids: a biogenetic approach. New York: Wiley; 1981. [3] Kiamuddin M, Hye HKMA. Pharmacological activity of an alkaloid from Sarcococca saligna. Pak J Sci Ind Res 1970;13. [4] Ata A, Andersh BJ. Buxus steroidal alkaloids: chemistry and biology. Alkaloids Chem Biol 2008;66:191–213. [5] Yan YX, Hu XD, Chen JC, Sun Y, Zhang XM, Qing C, et al. Cytotoxic triterpenoid alkaloids from Buxus microphylla. J Nat Prod 2009;72:308–11. [6] Mesaik MA, Halim SA, Ul-Haq Z, Choudhary MI, Shahnaz S, Ayatollahi SA, et al. Immunosuppressive activity of buxidin and E-buxenone from Buxus hyrcana. Chem Biol Drug Des 2010;75:310–7. [7] Atta-Ur-Rahman, Parveen S, Khalid A, Farooq A, Choudhary MI. Acetyl and butyrylcholinesterase-inhibiting triterpenoid alkaloids from Buxus papillosa. Phytochemistry 2001;58:963–8. [8] Tanaka H, Putalun W, Tsuzaki C, Shoyama Y. A simple determination of steroidal alkaloid glycosides by thin-layer chromatography immunostaining using monoclonal antibody against solamargine. FEBS Lett 1997;404:279–82. [9] Bianco G, Schmitt-Kopplin P, De Benedetto G, Kettrup A, Cataldi TR. Determination of glycoalkaloids and relative aglycones by nonaqueous capillary electrophoresis coupled with electrospray ionization-ion trap mass spectrometry. Electrophoresis 2002;23:2904–12. [10] Li SL, Lin G, Chan SW, Li P. Determination of the major isosteroidal alkaloids in bulbs of Fritillaria by high-performance liquid chromatography coupled with evaporative light scattering detection. J Chromatogr A 2001;909:207–14. [11] Shanker K, Gupta S, Srivastava P, Srivastava SK, Singh SC, Gupta MM. Simultaneous determination of three steroidal glycoalkaloids in Solanum xanthocarpum by high performance thin layer chromatography. J Pharm Biomed Anal 2011;54:497–502. [12] Grobosch T, Binscheck T, Martens F, Lampe D. Accidental intoxication with Veratrum album. J Anal Toxicol 2008;32:768–73. [13] Cataldi TR, Lelario F, Bufo SA. Analysis of tomato glycoalkaloids by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2005;19:3103–10. [14] Li HJ, Jiang Y, Li P. Characterizing distribution of steroidal alkaloids in Fritillaria spp. and related compound formulas by liquid chromatography–mass spectrometry combined with hierarchial cluster analysis. J Chromatogr A 2009;1216:2142–9. [15] Zhou JL, Xin GZ, Shi ZQ, Ren MT, Qi LW, Li HJ, et al. Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-offlight tandem mass spectrometry. J Chromatogr A 2010;1217:7109–22. [16] Li HL, Tang J, Liu RH, Lin M, Wang B, Lv YF, et al. Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multistage mass spectrometry. Rapid Commun Mass Spectrom 2007;21:869–79. [17] Zhou JL, Li P, Li HJ, Jiang Y, Ren MT, Liu Y. Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species. J Chromatogr A 2008;1177:126–37. [18] Musharraf SG, Ali A, Ali RA, Yousuf S, Rahman AU, Choudhary MI. Analysis and development of structure-fragmentation relationships in with anolides using an electrospray ionization quadrupole time-of-flight tandem mass spectrometry hybrid instrument. Rapid Commun Mass Spectrom 2011;25:104–14. [19] Musharraf SG, Goher M, Ali A, Adhikari A, Choudhary MI, Atta ur R. Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. Steroids 2012;77:138–48. [20] Musharraf SG, Goher M, Hussain A, Choudhary MI. Electrospray tandem mass spectrometric analysis of a dimeric conjugate, salvialeriafone and related compounds. Chem Cent J 2012;6. [21] Musharraf SG, Goher M, Shahnaz S, Choudhary MI, Atta ur R. Structurefragmentation relationship and rapid dereplication of Buxus steroidal alkaloids by electrospray ionization-quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2013;27:169–78. [22] Musharraf SG, Goher M, Wafo P, Kamdem RST. Electrospray tandem mass spectrometric analysis of duboscic acid, exploring the structural features of a new class of triterpenoids, dubosane. Int J Mass Spectrom 2012;310:77–80. [23] Choudhary MI, Shahnaz S, Parveen S, Khalid A, Majeed Ayatollahi SA, Atta Ur R, et al. New triterpenoid alkaloid cholinesterase inhibitors from Buxus hyrcana. J Nat Prod 2003;66:739–42. [24] Choudhary MI, Shahnaz S, Parveen S, Khalid A, Mesaik MA, Ayatollahi SA, et al. New cholinesterase-inhibiting triterpenoid alkaloids from Buxus hyrcana. Chem Biodivers 2006;3:1039–52.
293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368
Analyte
Intra-day variability (n = 4)
Inter-day variability (n = 3)
RSD (%) of peak area ratio
RSD (%) of retention time
RSD (%) of peak area ratio
RSD (%) of retention time
Conc. 2 lg/mL 1 1.0555 2 3.8970 3 7.7981 4 8.2258 5 3.5914 6 2.4158
0.1090 0.0270 0.0107 0.1197 0.0240 0.0187
25.4908 23.0421 15.8732 15.6232 21.7534 9.4786
0.1500 0.1641 0.3868 0.2292 0.2797 0.2873
Conc. 4 lg/mL 1 0.9506 2 2.0976 3 1.9449 4 2.4317 5 1.8872 6 0.3920
ND ND ND ND ND ND
19.1153 17.4734 13.6685 15.4660 23.6461 13.9492
0.2596 0.3441 0.3819 0.1531 0.2583 0.2196
Conc. 6 lg/mL 1 2.0162 2 2.1163 3 0.7192 4 1.9405 5 1.7002 6 1.3104
ND ND ND ND ND ND
22.1005 22.6698 19.4660 21.8091 20.2245 4.1615
0.1617 0.2958 0.3367 0.1264 0.2164 0.2151
ND = no drift.
Table 4 Contents of steroidal alkaloids in Buxus papillosa (lg/g). Analyte
Roots
Stem
Leaves
1 2 3 4 5 6
16.88916 106.9681 32.43682 nd 5.991689 23.45083
18.74735 116.0798 34.42218 nd 5.21344 24.37042
27.6107 108.4411 94.52262 nd 0.0 6.231151
nd = Not detected.
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formulations. Moreover, the short analysis time allow a large number of samples to be analyzed in high-throughput manner.
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Acknowledgements
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The authors are grateful to OPCW (Organization for the Prohibition of Chemical Weapons) for providing funds of the Project Account No. L/ICA/ICB/173673/12. Authors are also thankful to and Prof. Atta-ur-Rahman and Prof. M. Iqbal Choudhary (H.E.J. Research Institute of Chemistry, International Center for
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Calibration curve
Table 3 Precision of six steroidal alkaloids at the concentration of 2, 4 and 6 lg/mL, expressed as RSD (%).
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Analyte
y: Peak area ratios of the analytes/I.S.; x: concentrations of standards (lg/mL).
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Chemical and Biological Sciences, University of Karachi) for useful discussions.
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Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry
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Syed Ghulam Musharraf ⇑, Madiha Goher, Bibi Zareena
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H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
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Article history: Received 17 November 2014 Received in revised form 7 February 2015 Accepted 31 March 2015 Available online xxxx Keywords: Steroidal alkaloids Electrospray ionization mass spectrometry Buxus papillosa ESI-QQQ-MS/MS
a b s t r a c t Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486–8.08 ng/mL and 1.473–24.268 ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15 min run time. This paper describes the application of LC– QQQ-MS technique for steroidal alkaloids analysis in plant samples. Ó 2015 Published by Elsevier Inc.
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1. Introduction
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Buxus alkaloids represent a unique class of compounds possessing a triterpenoidal skeleton in which one or two nitrogen atoms are incorporated as side chains. About 100 species of genus Buxus are present in Africa, America, Asia and Europe [1]. The genus Buxus is among the richest source of steroidal alkaloids and more than 200 new steroidal alkaloids have already been reported [2]. Herbal formulations based Buxus species are widely used in Ayurvedic, Greco-Arabic and Chinese medicines in Asian countries due to their impressive biological activities. Extracts of genus Buxus have been used in the indigenous systems of medicine for the treatment of various disorders, such as malaria, rheumatism and skin infections. They also showed anticholinesterase [3], anti-HIV [4], immunosuppressive, and cytotoxicity activities [5,6]. Most of the biological activities of Buxus species are largely due to the steroidal alkaloids present therein. Buxus papillosa is widely distributed in Khyber Pakhtunkhwa (KPK) province of Pakistan. It is a well studied species of the genus Buxus with over 50 triterpenoidal and steroidal alkaloids. Many of the alkaloids are known for the various pharmacological activities, such as acetylcholinesterase and butyrylcholinesterase inhibitory activity [7]. Buxus alkaloids, also called alkamine, have nitrogen atoms, either at C-3 or C-20 or at both positions. On the basis of their basic
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⇑ Corresponding author. Tel.: +92 21 34824924 5, +92 21 4819010; fax: +92 21 34819018 9. E-mail address: [email protected] (S.G. Musharraf).
skeleton, Buxus alkaloids can be divided into the following groups: (1) derivatives of 9b,19-cyclo-4,4,14a-trimethyl-5a-pregnane: having a basic skeleton with a cyclopropane ring between C-9 and C19. (2) derivatives of 9(10–19)abeo,14a-trimethyl-5a-pregnane, resulting from the opening of the 9b-19-cyclopropane ring system. Numerous studies on quantitative and qualitative analysis of different steroidal alkaloids have been published. The identification of glycosides of solasodine steroidal alkaloid have been reported by using techniques such as thin layer chromatography (TLC) [8], capillary electrophoresis mass spectrometry (CE-MS) [9] and high-performance liquid chromatography (HPLC) [10]. An HPTLC method was developed for the determination of steroidal glycoalkaloids in Solanum xanthocarpum [11]. Similarly, method was developed for the simultaneous identification and analysis of Veratrum alkaloids [12]. Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum), have been identified by using optimized reversed-phase LC with electrospray ionization (ESI) and ion trap mass spectrometry (IT-MS) [13]. An HPLC–MS method for characterizing cevanine-type, veratramine-type, jervine-type and secosolanidine-type alkaloids in Fritillaria species has been developed [14,15]. Electrospray ionization multi-stage mass spectrometry was used to study the fragmentation of protoverine-type, germine type and zygadenin-type alkaloids, isolated from the Chinese herb, Veratrum nigrum L. [16]. However, only one report on the quantification of steroidal alkaloids through LC–ESI-TOF-MS method is found in literature used for quantification of steroidal alkaloids from Fritillaria species [17].
http://dx.doi.org/10.1016/j.steroids.2015.03.018 0039-128X/Ó 2015 Published by Elsevier Inc.
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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In continuation of our studies on the application of electrospray ionization mass spectrometry and high throughput dereplication strategy development [18–22], we describe here the development and validation of an LC–ESI-QQQ-MS/MS method for the quantification of steroidal alkaloids in B. papillosa with better limit of detection in short analysis time. This is the first report on the quantification of steroidal alkaloids in any plant using ESI-QQQ-MS.
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2. Experimental
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2.1. Chemicals and reagents
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Chemicals and solvents were of analytical reagent and HPLC grades, respectively, and purchased from Aldrich–Sigma (USA). Deionized water (Milli-Q), used throughout the study, was obtained from Millipore Milli Q Plus System (Bedford, USA). The steroidal alkaloid standards, cyclomicrobuxine (1), cyclomicrobuxinine (2), E-buxenone (3), N-benzoylbuxahyrcanine (4), buxidine (5), buxandrine (6), and N-Isobutyroylbuxahyrcanine (internal standards, I.S.) (Fig. 1) were obtained from the Molecular Bank facility of the Dr. Panjwani Center for Molecular Medicine and Drug Research (International Center for Chemical and Biological Sciences), University of Karachi. The isolation and characterization details of the analyzed steroidal alkaloids have been reported previously [23,24].
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2.2. Standard solution and calibration standards
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Stock standard solutions were prepared in methanol at a final concentration of 1 mg/mL. These solutions were stored at 4 °C. Working solutions, used for LC–QQQ-MS analysis, were obtained by diluting the stock solutions with acetonitrile (with concentrations between 0.1 lg/mL and 19 lg/mL). Stock solution (260 lg/ mL) of internal standard, I.S. (N-Isobutyroylbuxahyrcanine) was
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prepared in methanol and stored at 4 °C until use, and the final concentration of the internal standard was 0.5 lg/mL in the calibration standards range of 0.1–1 lg/mL and 12 lg/mL in the calibration range of 3–19 lg/mL, respectively.
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2.3. Sample preparation
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The plant B. papillosa (leaves, stems & roots) was collected from the Swat, Pakistan (Voucher # 9718). The plant material was identified by Mr. Mehboob ur Rehman, Plant taxonomist, the Department of Botany, Government Postgraduate Jahanzeb College, Swat. The dried leaves, roots and stems of B. papillosa were separately powdered to a homogeneous size, and sieved through a 400 lm mesh, followed by drying at 60 °C in the oven for 2 h. The dried leaves, roots and stem powder (500 mg) was pre-alkalized with 5 mL ammonia solution (33%) for 1 h, and immersed in 12 mL methanol overnight, then ultrasonicated for 1 h. After being filtered, the extract was concentrated to dryness in vacuum at 45 °C. 10 mL methanol was added in the residue and ultrasonicated for 5 min. Two working solutions for two different concentrations of internal standard were prepared by taking 100 lL of this reconstituted residue solution, and both were made up to 1 mL with acetonitrile containing a final concentration of 0.5 and 12 lg/mL internal standard respectively. The resultant solutions were centrifuged at 12,000 rpm for 10 min, the supernatants were filtered through Millipore filter (0.45 lm), and transferred to an autosampler vial for LC–QQQ-MS analysis.
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2.4. LC–QQQ-MS analysis
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Liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) in positive ionization mode, was used to detect the steroidal alkaloids. The LC analysis of the selected steroidal alkaloids was carried out using an
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O
H N
O
H
OH
H N H
H
Cyclomicrobuxine (1)
H
H
H
OH N H
H
H
E-Buxenone (3)
Cyclomicrobuxinine (2)
N
N H
O
H N H
H
H
Buxidine (5)
N H
O
N O
O
H O
N H HO
Buxandrine (6)
H
OH N H
H
OH
HO
N-Benzoylbuxahyrcanine (4)
H
H
O
OH N H
O
H
N -I sobutyroylbuxahyrcanine (I.S.)
Fig. 1. The structures of Buxus steroidal alkaloids.
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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Agilent 1260 Infinity HPLC system (Agilent Technologies, Singapore), equipped with binary pump, online degasser, auto sampler, and thermostatically controlled column compartment. The chromatography separation was performed on C-18 column (Merck, 4.6 55 mm I.D., 3 lm particle size) with a solvent flow rate of 0.5 mL/min at a temperature of 30 °C. The sample injection volume was set at 3 lL. Water (0.1% formic acid) and acetonitrile (also contained 0.1% formic acid) were used as the mobile phases A and B, respectively. The solvent gradient adopted was as follows: 5–75% B at 0–10 min, 75–95% B at 10–11 min, 5% B at 11.01– 16 min. This HPLC system was connected to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Singapore), equipped with an electrospray interface. The ESI source conditions were as follows: drying gas (N2) flow rate, 8.0 L/min; drying gas temperature, 300 °C; nebulizer, 40 psi; capillary voltage, 3000 V; cell accelerator voltage, 7 V; fragmentor, 135 V. LC–QQQ-MS mass spectra were recorded across the range from 100 to 800 m/z. Quantification was performed in comparison of an internal standard method. A steroidal alkaloid having similar skeleton as those of analytes was used an internal standard. Multiple Reaction Monitoring (MRM) mode was used for the quantification of standards with collision energy of 33–40 V and a solvent delay of 5 min. The dwell time was 200 ms. The fragment ions, listed in Table 1, allow the quantification by using one either the quantification ion, and the other ion as a qualifier. Extracted ion chromatograms (XICs) for the [M+H]+ ions of the target compounds were used for peak area determination. Peak area ratios of analytes/I.S. were used for quantification. The LOD and LOQ were estimated as 3 and 10 times of the noise level, respectively. Moreover, both were initially determined by diluting the known concentration of steroidal alkaloids standard till the mean instrumental responses were nearly three and ten times of the standard deviation of the responses for six manifold analyses. The intra-day variability study was measured by the injection of the same standard solution at four different times in the same day. The inter-day variability study was measured for three successive days by using three different concentrations of all standard steroidal alkaloids. All the operations, acquisition and analysis of data were controlled by Agilent Mass Hunter qualitative software Ver. B.04.00 and quantitative software Ver. B.05.00 (Agilent Technologies).
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3. Results and discussion
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3.1. Method optimization
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LC method was optimized to best chromatographic peak shapes and signal-to-noise ratios (S/N) of the analytes. Various gradients of acetonitrile–water containing 0.1% formic acid at a flow rate of 0.5 mL/min were used for optimizing the elution conditions in order to achieve the reliable quantification of these alkaloids. Under the gradient condition, as described previously, the internal standard is a baseline, separated from the other six compounds, while compounds 1 and 2 eluted at the same time segment and
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compounds 3–6 were also eluted at the same time segment. However, by virtue of the XIC mode in MRM, quantitative analysis could be achieved as the overlapping peaks produced different protonated [M+H]+ ions. Optimization of the MRM method was based on the selection of the transitions from the precursor ion to the different product ions for the selective and sensitive determination of the target steroidal alkaloids. Once the number of transitions was established, the influence of the dwell time on the sensitivity was checked, and 200 ms was found to be the best value. The transition with the highest signal intensity was used for the quantification of each analyte. Fragmentor voltage affects the sensitivity and fragmentation patterns of the analytes, significantly. Therefore, the voltage value was studied in the range of 50–250 V under optimized source conditions. In general, fragmentor voltage of 100 V provided minimal fragmentation in most of the analytes. Collision energies also affect the sensitivity and fragmentation pattern of the analytes significantly. MS–MS Spectra at different collision energies were studied in the range from 20 to 100 V under optimized source conditions. In general, collision energy of 33 V provided maximum intensity of fragmented ions. Fragmentor voltages and collision energies of all the analytes and internal standard are summarized in Table 1.
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3.2. Quantification and method validation
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LC–QQQ-MS quantification was carried out under MRM conditions by using the XICs of the protonated molecule of each steroidal alkaloid. Total ion chromatogram of standard compounds, obtained by MRM and qualifier and quantifier chromatogram of all standards of steroidal alkaloids, are shown in Figs. 2a and b. Peak area ratios of the analytes/I.S. were used for quantification. The linearity of the analytical response across the studied range was found to be excellent with correlation coefficients (r2) > 0.99 for all analytes. The values of linear calibration curves of 6 steroidal alkaloids are listed in Table 2. The developed method enables quantification of many targeted analytes in a single experiment and to filter out interfering compounds through selective and specific transitions chosen for each compound. Method precision was checked by intra-day and inter-day variability. The relative standard deviation (RSD) values, obtained from run-to-run and day-to-day precision studies are summarized in Table 3. The developed method was found to be precise with intra-day and inter-day variability and RSD values lie between 0.39–8.22% and 4.16– 25.49%, respectively. In addition, the RSD values of retention time were less than 0.12% and 0.38% in run-to-run and day-to-day analysis, respectively. Sensitivity of the method was evaluated by determining limits of detection (LOD) and quantification (LOD). Limits of detection and limit of quantification were 0.486– 8.08 ng/mL and 1.473–24.268 ng/mL, respectively, for all analyzed compounds. The LODs and LOQs for all analytes are presented in Table 2. Better signal-to-noise ratio allows quantification of steroidal alkaloids with lower Limits of Quantification (LOQ) as
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Table 1 Optimized LC–MS/MS acquisition method parameters for steroidal alkaloids. Analyte
1 2 3 4 5 6 I.S.
Time segment
1 1 3 3 3 3 2
Precursor ion (m/z)
386.3 372.3 370.3 507.2 521.2 563.4 473.3
MRM transitions (m/z) Identification
Quantification
119, 145, 135, 323, 307, 319, 340,
386 ? 119 372 ? 145 370 ? 84 507 ? 323 521 ? 307 563 ? 319 473 ? 340
105 131 84 444 325 440 410, 213
Fragmentor voltage (V)
Collision energy (V)
100 130 100 120 80 150 120
33 35 33 33 40 38 38
Please cite this article in press as: Musharraf SG et al. Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.03.018
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Fig. 2a. Total ion chromatogram of standard compounds obtained by MRM, qualifier and quantifier chromatogram of standards 1, 2 and 3.
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compared to previously reported quantification methods based on ESI-TOF-MS [17].
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3.3. Application to B. papillosa
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The optimized LC/QQQ-MS method was subsequently applied for the simultaneous qualitative and quantitative analysis of 6 steroidal alkaloids in the extract of leaves, roots and stem of B.
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papillosa. The quantified results obtained are summarized in Table 4. The results showed that compound 1 is present in all samples in a range of 16–28 lg/g and approx. 100 lg/g of compound 2 is present in leaves roots and stem extracts. Concentration of compound 3 in leaves was approximately three times higher than in roots and stem, while the concentration of compound 6 is four times higher in roots and stem, than in leaves. Compound 5 was present in very little amount in roots and stem and was absent
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Fig. 2b. Qualifier and quantifier chromatogram of standards 4, 5, 6 and I.S.
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in leaves, while compound 4 was negligible in concentrations in all samples. On the basis of these results, it could not be concluded that which part of the plant contains higher concentrations of steroidal alkaloids.
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4. Conclusion
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In this work, it was demonstrated that the liquid chromatography in combination with triple quadrupole mass
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spectrometry (LC–QQQ-MS) is an efficient method both for qualitative and quantitative analysis of steroidal alkaloids. Good precision and linearity was obtained, with significantly improved limits of detection for steroidal alkaloids. The excellent selectivity and sensitivity, as well as good linearity, allows identification and quantification of low levels of steroidal alkaloids in complex Buxus species extracts. Furthermore, the lower detection limits of the developed method can be used for the analysis of steroidal alkaloids in other medicinal plants as well as in herbal
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Table 2 Calibration curve, correlation coefficient (r), instrumental LODs and instrumental LOQs for 6 analytes in a test range 0.1–1 (lg/mL). r2
Instrumental LODs (ng/mL)
Instrumental LOQs (ng/mL)
References
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1 2 3 4 5 6
y = 5.116x + 0.025 y = 1.974x + 0.125 y = 11.88x + 0.689 y = 0.977x + 0.25 y = 0.097x + 0.002 y = 0.296x + 0.022
0.996 0.992 0.998 0.989 0.991 0.995
8.008 7.61 5.165 5.102 0.486 3.789
24.268 23.062 15.65 15.461 1.473 11.481
[1] Duke JA. Handbook of medicinal herbs. 2nd ed. Boca Raton, FL: CRC Press; 2002. [2] Cordell GA. Introduction to alkaloids: a biogenetic approach. New York: Wiley; 1981. [3] Kiamuddin M, Hye HKMA. Pharmacological activity of an alkaloid from Sarcococca saligna. Pak J Sci Ind Res 1970;13. [4] Ata A, Andersh BJ. Buxus steroidal alkaloids: chemistry and biology. Alkaloids Chem Biol 2008;66:191–213. [5] Yan YX, Hu XD, Chen JC, Sun Y, Zhang XM, Qing C, et al. Cytotoxic triterpenoid alkaloids from Buxus microphylla. J Nat Prod 2009;72:308–11. [6] Mesaik MA, Halim SA, Ul-Haq Z, Choudhary MI, Shahnaz S, Ayatollahi SA, et al. Immunosuppressive activity of buxidin and E-buxenone from Buxus hyrcana. Chem Biol Drug Des 2010;75:310–7. [7] Atta-Ur-Rahman, Parveen S, Khalid A, Farooq A, Choudhary MI. Acetyl and butyrylcholinesterase-inhibiting triterpenoid alkaloids from Buxus papillosa. Phytochemistry 2001;58:963–8. [8] Tanaka H, Putalun W, Tsuzaki C, Shoyama Y. A simple determination of steroidal alkaloid glycosides by thin-layer chromatography immunostaining using monoclonal antibody against solamargine. FEBS Lett 1997;404:279–82. [9] Bianco G, Schmitt-Kopplin P, De Benedetto G, Kettrup A, Cataldi TR. Determination of glycoalkaloids and relative aglycones by nonaqueous capillary electrophoresis coupled with electrospray ionization-ion trap mass spectrometry. Electrophoresis 2002;23:2904–12. [10] Li SL, Lin G, Chan SW, Li P. Determination of the major isosteroidal alkaloids in bulbs of Fritillaria by high-performance liquid chromatography coupled with evaporative light scattering detection. J Chromatogr A 2001;909:207–14. [11] Shanker K, Gupta S, Srivastava P, Srivastava SK, Singh SC, Gupta MM. Simultaneous determination of three steroidal glycoalkaloids in Solanum xanthocarpum by high performance thin layer chromatography. J Pharm Biomed Anal 2011;54:497–502. [12] Grobosch T, Binscheck T, Martens F, Lampe D. Accidental intoxication with Veratrum album. J Anal Toxicol 2008;32:768–73. [13] Cataldi TR, Lelario F, Bufo SA. Analysis of tomato glycoalkaloids by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2005;19:3103–10. [14] Li HJ, Jiang Y, Li P. Characterizing distribution of steroidal alkaloids in Fritillaria spp. and related compound formulas by liquid chromatography–mass spectrometry combined with hierarchial cluster analysis. J Chromatogr A 2009;1216:2142–9. [15] Zhou JL, Xin GZ, Shi ZQ, Ren MT, Qi LW, Li HJ, et al. Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-offlight tandem mass spectrometry. J Chromatogr A 2010;1217:7109–22. [16] Li HL, Tang J, Liu RH, Lin M, Wang B, Lv YF, et al. Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multistage mass spectrometry. Rapid Commun Mass Spectrom 2007;21:869–79. [17] Zhou JL, Li P, Li HJ, Jiang Y, Ren MT, Liu Y. Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species. J Chromatogr A 2008;1177:126–37. [18] Musharraf SG, Ali A, Ali RA, Yousuf S, Rahman AU, Choudhary MI. Analysis and development of structure-fragmentation relationships in with anolides using an electrospray ionization quadrupole time-of-flight tandem mass spectrometry hybrid instrument. Rapid Commun Mass Spectrom 2011;25:104–14. [19] Musharraf SG, Goher M, Ali A, Adhikari A, Choudhary MI, Atta ur R. Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. Steroids 2012;77:138–48. [20] Musharraf SG, Goher M, Hussain A, Choudhary MI. Electrospray tandem mass spectrometric analysis of a dimeric conjugate, salvialeriafone and related compounds. Chem Cent J 2012;6. [21] Musharraf SG, Goher M, Shahnaz S, Choudhary MI, Atta ur R. Structurefragmentation relationship and rapid dereplication of Buxus steroidal alkaloids by electrospray ionization-quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2013;27:169–78. [22] Musharraf SG, Goher M, Wafo P, Kamdem RST. Electrospray tandem mass spectrometric analysis of duboscic acid, exploring the structural features of a new class of triterpenoids, dubosane. Int J Mass Spectrom 2012;310:77–80. [23] Choudhary MI, Shahnaz S, Parveen S, Khalid A, Majeed Ayatollahi SA, Atta Ur R, et al. New triterpenoid alkaloid cholinesterase inhibitors from Buxus hyrcana. J Nat Prod 2003;66:739–42. [24] Choudhary MI, Shahnaz S, Parveen S, Khalid A, Mesaik MA, Ayatollahi SA, et al. New cholinesterase-inhibiting triterpenoid alkaloids from Buxus hyrcana. Chem Biodivers 2006;3:1039–52.
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Analyte
Intra-day variability (n = 4)
Inter-day variability (n = 3)
RSD (%) of peak area ratio
RSD (%) of retention time
RSD (%) of peak area ratio
RSD (%) of retention time
Conc. 2 lg/mL 1 1.0555 2 3.8970 3 7.7981 4 8.2258 5 3.5914 6 2.4158
0.1090 0.0270 0.0107 0.1197 0.0240 0.0187
25.4908 23.0421 15.8732 15.6232 21.7534 9.4786
0.1500 0.1641 0.3868 0.2292 0.2797 0.2873
Conc. 4 lg/mL 1 0.9506 2 2.0976 3 1.9449 4 2.4317 5 1.8872 6 0.3920
ND ND ND ND ND ND
19.1153 17.4734 13.6685 15.4660 23.6461 13.9492
0.2596 0.3441 0.3819 0.1531 0.2583 0.2196
Conc. 6 lg/mL 1 2.0162 2 2.1163 3 0.7192 4 1.9405 5 1.7002 6 1.3104
ND ND ND ND ND ND
22.1005 22.6698 19.4660 21.8091 20.2245 4.1615
0.1617 0.2958 0.3367 0.1264 0.2164 0.2151
ND = no drift.
Table 4 Contents of steroidal alkaloids in Buxus papillosa (lg/g). Analyte
Roots
Stem
Leaves
1 2 3 4 5 6
16.88916 106.9681 32.43682 nd 5.991689 23.45083
18.74735 116.0798 34.42218 nd 5.21344 24.37042
27.6107 108.4411 94.52262 nd 0.0 6.231151
nd = Not detected.
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formulations. Moreover, the short analysis time allow a large number of samples to be analyzed in high-throughput manner.
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Acknowledgements
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The authors are grateful to OPCW (Organization for the Prohibition of Chemical Weapons) for providing funds of the Project Account No. L/ICA/ICB/173673/12. Authors are also thankful to and Prof. Atta-ur-Rahman and Prof. M. Iqbal Choudhary (H.E.J. Research Institute of Chemistry, International Center for
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Calibration curve
Table 3 Precision of six steroidal alkaloids at the concentration of 2, 4 and 6 lg/mL, expressed as RSD (%).
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Analyte
y: Peak area ratios of the analytes/I.S.; x: concentrations of standards (lg/mL).
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Chemical and Biological Sciences, University of Karachi) for useful discussions.
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