2260 Young Min Han1 ∗ Moonhee Jang2 ∗ In Sook Kim1 Seung Hyun Kim3 Hye Hyun Yoo1 1 Institute
of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Republic of Korea 2 National Forensic Service, Seoul, Republic of Korea 3 College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea Received February 18, 2015 Revised April 2, 2015 Accepted April 14, 2015
J. Sep. Sci. 2015, 38, 2260–2266
Research Article
Simultaneous quantitation of six major quassinoids in Tongkat Ali dietary supplements by liquid chromatography with tandem mass spectrometry Tongkat Ali (Eurycoma longifolia) is one of the most popular traditional herbs in Southeast Asia and generally consumed as forms of dietary supplements, tea, or drink additives for coffee or energy beverages. In this study, the liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of six major quassinoids of Tongkat Ali (eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)-epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone) was developed and validated. Using the developed method, the content of the six quassinoids was measured in Tongkat Ali containing dietary supplement tablets or capsules, and the resulting data were used to confirm the presence of Tongkat Ali in those products. Among the six quassinoids, eurycomanone was the most abundant quassinoid in all samples tested. The developed method would be useful for the quality assessment of Tongkat Ali containing dietary supplements. Keywords: Dietary supplements / Eurycoma longifolia / Liquid chromatography with tandem mass spectrometry / Quassinoids / Tongkat Ali DOI 10.1002/jssc.201500207
1 Introduction Eurycoma longifolia Jack (Simaroubaceae), commonly known as Tongkat Ali, is one of the most popular ethnobotanical plants found in Southeast Asia. Root extracts from this plant have been reported to have various beneficial functions such as antimalarial, antiulcer, antipyretic, and antimicrobial activities [1]. Especially, E. longifolia is believed to exert improving effects for erectile dysfunction, libido, male infertility, boosting athletic performance, bodybuilding, or reducing body fat [2, 3]. E. longifolia is, therefore, popularly used as forms of food, tea, dietary supplements, or drink additives for coffee or energy beverages. Phytochemical studies have shown that E. longifolia contains a variety of natural constituents, including quassinoids [4–8], cathinone alkaloids [9, 10], and squalene derivatives [11, 12]. Among them, quassinoids represent the major constituents of E. longifolia roots [13]. The quassinoids, indigenously found in the Simaroubaceae family, are a group of diterpenoids with diverse pharmacological properties such as anticancer, antimalarial, antipyretic, and cytotoxic activities [14–18]. Correspondence: Professor Hye Hyun Yoo, Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Gyeonggi-do 426–791, Republic of Korea E-mail: [email protected] Fax: +82-31-400-5958
Abbreviations: PTFE, Polytetrafluoroethylene; MRM, Multiple reaction monitoring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
With a growing demand for herbal products in the health supplements market, quality evaluation studies of raw materials and commercial products have been extensively conducted. Recently, E. longifolia extract has gained great popularity in the dietary supplement market because of its ability to improve sperm quality and its aphrodisiac properties, which is not limited to Southeast Asia. However, the chronic use of E. longifolia containing products is controversial in terms of safety and a recommended optimal dosage is unknown. Furthermore, it is unclear as to whether E. longifolia containing products actually contain E. longifolia extracts as described in their product content labels. The current available information on quality criteria for E. longifolia and its related products is limited. Therefore, it is worthwhile investigating their chemical composition and quantifying bioactive constituents for quality and safety concern. Several reports demonstrating analytical methods for E. longifolia extracts have been published. Chua et al. performed metabolite profiling of E. longifolia using LC–MS/MS-based techniques [13]. This study focused mainly on the characterization and identification of the metabolites rather than their quantification. Teh and his colleagues presented a LC– MS method for the determination of quassinoids in various batches of E. longifolia extract [19]. Furthermore, the LC– MS method may have some limitations when applied to dietary supplement products with various ingredients because ∗ Young Min Han and Moonhee Jang contributed equally to this work.
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2015, 38, 2260–2266
of lower sensitivity and selectivity. In this case, LC–MS/MS approaches could be needed for identification and quantification of bioactive constituents in complicated matrix. However, to our knowledge, a quantitative LC–MS/MS method for E. longifolia has not been published. In the present study, we developed and validated an LC–MS/MS method for the simultaneous determination of six quassinoids in E. longifolia plant material and dietary supplements. Authentic presence of the extract in commercial products was also verified using the developed method. Accordingly, major quassinoids are presented as suitable markers for the QC of E. longifolia containing products.
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Table 1. Precursor–product ion pairs used in multiple reaction monitoring
Eurycomanone 13,21-dihydroeurycomanone 13a(21)-epoxyeurycomanone 14,15-dihydroxyklaineanone Eurycomalactone Longilactone
Precursor ion
Product ion
CE (V)
409.1 411.2 425.2 397.2 349.2 367.2
391.2 393.1 407.1 361.1 313.1 349.1
10 10 10 10 1 10
2.4 LC–MS/MS
2 Materials and methods 2.1 Chemicals and materials The roots of E. longifolia were collected in Dak Lak province, Vietnam and authenticated by Dr. Bui Van Thanh in Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Vietnam. The reference standards of Eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone were isolated in a previous study [8]. HPLC-grade methanol, acetonitrile, and acetic acid were purchased from JT Baker (Phillipsburg, NJ, USA). Deionized water was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). Seven dietary supplements (four types of capsules and three types of tablets) claiming to contain Tongkat Ali extract were purchased from online markets.
2.2 Preparation of standard solutions Individual standard stock solutions of the six reference compounds (mentioned above) were prepared at a concentration of 1 mg/mL in water/methanol (30:70, v/v). Working standard solutions were prepared by serial dilution to appropriate concentrations.
2.3 Sample preparation The crude methanol extract of E. longifolia root was obtained as previously described [8]. The extract was dissolved in water/methanol (30:70, v/v) at a concentration of 10 mg/mL and filtered through a 0.45 m PTFE syringe filter for analysis. For Tongkat Ali products, tablets were pulverized with a mortar and pestle. For capsules, the contents were emptied and mixed. The samples were weighed (3 g) and dissolved in 30 mL of methanol. After sonication for 30 min, the sample solution was centrifuged for 5 min at 13 000 rpm. The supernatant was filtered through 0.45 m filter before analysis. The samples solutions were diluted appropriately for the calibration range before analysis. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The analysis was performed using an Agilent 1260 infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to 6460 triple quadrupole mass spectrometer (Agilent Technologies). Chromatographic separation was carried out using a C18 column (2.1 mm × 50 mm, 2.7 m, Agilent Technologies, Palo Alto, CA, USA). Gradient elution was performed with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile/water (90:10, v/v) (B) at a flow rate of 0.25 mL/min. The initial condition was 5% B, which was increased to 40% B over 7 min, and finally increased to 80% B at 8 min. Finally, the initial condition was restored and held for 4.5 min to re-equilibrate the system. The total run time was 12.5 min. The column oven was maintained at 40⬚C. The injection volume was 2 L. The mass spectrometer was operated in the ESI positive ion mode. Nitrogen was used as nebulizer gas at a pressure of 45 psi, a carrier gas at 10 L/min and 325⬚C, and a sheath gas at 12 L/min and 400⬚C. Detection of ions was performed in the multiple reaction monitoring (MRM) mode. The precursor–product ion pairs used in MRM mode were summarized in Table 1.
2.5 Method validation The developed method was validated for specificity, linearity, LOD, LOQ, accuracy (as recovery), and precision. Specificity was assessed to evaluate potential interference with the signals of the analytes. Linearity was determined by independent analysis of three sets of calibrators with six concentrations ranging from 50 to 1000 ng/mL. LOD and LOQ were evaluated by injecting a series of standard solutions with decreasing analyte concentrations. LOD and LOQ were defined as the lowest concentration that produces acceptable peak shape and an S/N of at least 3 and 10, respectively. Intra- and inter-day precision and accuracy were determined from six replicates of samples at three concentrations (100, 500, and 1000 ng/mL) on five different days. For accuracy and precision tests, known amounts of standard solutions were spiked to tablet and capsule samples and prepared using the method described above. Accuracy was represented as average recoveries (%) of the spiked analyte concentrations and precision was expressed as RSD% of repeated analysis. www.jss-journal.com
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J. Sep. Sci. 2015, 38, 2260–2266
Figure 1. Representative MS/MS spectra of (A) eurycomanone, (B) 13,21-dihydroeurycomanone, (C) 13␣(21)-epoxyeurycomanone, (D) 14,15-dihydroxyklaineanone, (E) eurycomalactone, and (F) longilactone
3 Results and discussion 3.1 Method development and validation In a preliminary study, the methanolic extract of Tongkat Ali was analyzed in Q1 scan mode using LC–MS/MS. Based on the resulting chromatogram, six major quassinoids, eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone, were chosen as marker compounds of E. longifolia extracts. The target compounds were favorably ionized in positive ion mode to yield the signals of the protonated molecular ion [M+H]+ . In the product ion scan, the fragment ions with neutral loss of H2 O were found to be the predominant product ion for all analytes (Fig. 1). This is thought to be due to the complicated and rigid C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
chemical structures of quassinoids. The MRM conditions were then optimized based on these results. Subsequently, the chromatographic separation was achieved on a C18 column with a gradient mobile phase by modifying the previously published methods [13, 19]. The LC–MS/MS detection using the MRM mode contributed to a decreased analytical run time through enhanced selectivity as well as improvement in sensitivity. The developed method allowed a rapid and efficient analysis; all analytes were eluted between 3.1 and 6.9 min, and their peak shape was satisfactory. Meanwhile, internal standard was not used in this method. The use of an internal standard is generally recommended in MS analysis. However, due to the unavailability of an appropriate internal standard for quassinoids [19], the quantitative method was established here using an external standard calibration method. www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2015, 38, 2260–2266
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Figure 2. Typical extracted ion chromatograms for (A) standard mixture, (B) methanol extract of E. longifolia, and (C) Tongkat Ali containing capsules and (D) tablets Table 2. Calibration curve data for six quassinoids
Analytes
Range (ng/mL)
Calibration curve equation
Correlation coefficient, R2
LOD (ng/mL)
LOQ (ng/mL)
Eurycomanone 13,21-dihydroeurycomanone 13a(21)-epoxyeurycomanone 14,15-dihydroxyklaineanone Eurycomalactone Longilactone
50–2000 50–2000 50–2000 50–2000 50–2000 50–2000
y = 41.1285x + 90.0088 y = 71.4604x + 94.7947 y = 39.1084x + 99.4949 y = 2.9760x + 17.0073 y = 3.4896x + 2.7047 y = 5.4847x + 2.0792
0.999 0.999 0.999 0.999 0.998 0.999
1 0.5 1 2 2 5
5 2 5 10 10 20
Figure 2 shows the representative chromatograms of six quassinoids in the standard mixture, methanolic extracts of E. longifolia, and Tongkat Ali containing capsules and tablets. When compared with the standard chromatogram (Fig. 2A), no significant interference with analyte detection was observed in any of the samples tested (Fig. 2B–D), suggesting high specificity of the present method. Good linearity was achieved for all analytes within the range of 50–1000 ng/mL. Average regression coefficients (R2 ) were greater than 0.998 for all analytes. LOD ranged from 0.5 to 5 ng/mL, and LOQ ranged from 2 to 20 ng/mL (Table 2). The accuracy and precision was evaluated by measuring the recovery of standardspiked samples. Recovery for capsules and tablets was within the range of 88.8–98.4 and 82.5–99.4%, respectively, and their RSD values ranged from 0.3 to 4.4% for capsules and from 0.6 to 7.9% for tablets (Table 3). These results indicate that the developed method is acceptable for accurate and precise determination of target compounds. Recently, Teh et al. presented a quantitative LC–MS method for the analysis of quassinoids in E. longifolia extract [19]. In their method, LC with a trap mass spectrometer
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
was used and chromatographic conditions were optimized; the mobile phase consisting of water/methanol (91:9)/0.1% acetic acid was used under isocratic elution. The resulting chromatogram showed a good separation but the peak shapes were broad. The LOD range was 30 to 100 ng/mL, and the total run time was more than 50 min. Meanwhile, our method adopted a gradient using the mobile phase consisting of acetonitrile and 0.1% formic acid with an LC triple-quadrupole mass spectrometer. Accordingly, our current LC–MS/MS method achieved a lower detection limit (0.5–5 ng/mL) and a shorter chromatographic analysis time (12.5 min). These compounds may also be easily detected at maximum absorption of UV radiation around 238 nm due to the presence of the chromophoric ␣,-unsaturated ketone in ring A of the quassinoids [20]. However, HPLC methods would be also difficult to apply for the analysis of mixtures of numerous ingredients because of chromatographic interferences. Taken together, the present method could facilitate the determination of quassinoids from dietary supplement products containing complicated matrix components (ingredients) or even low amounts of Tongkat Ali extracts.
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Table 3. Intra- and inter-day precision and accuracy for the determination of six quassinoids in capsules and tablets
Compound
Concentration Capsules spiked (ng/ml)
Tablets
Intra-day (n = 3)
Inter-day (n = 5)
Intra-day (n = 3)
Inter-day (n = 5)
Accuracy Precision Accuracy Precision Accuracy Precision Accuracy Precision (%recovery) (%RSD) (%recovery) (%RSD) (%recovery) (%RSD) (%recovery) (%RSD) Eurycomanone
100 500 1000 13,21-dihydroeurycomanone 100 500 1000 13a(21)-epoxyeurycomanone 100 500 1000 14,15-dihydroxyklaineanone 100 500 1000 Eurycomalactone 100 500 1000 Longilactone 100 500 1000
91.0 94.5 92.3 93.3 92.9 89.9 92.7 95.9 94.3 92.7 96.6 96.2 94.4 98.1 94.5 94.2 95.2 92.8
3.0 3.7 3.0 1.6 1.1 0.8 2.3 3.1 1.7 3.8 3.1 1.1 3.3 1.8 2.1 4.4 1.2 1.0
82.5 87.6 91.3 89.2 88.9 91.5 92.9 92.9 93.9 88.2 91.4 93.8 95.6 95.2 95.0 87.8 89.3 91.1
6.4 0.8 1.0 1.0 0.6 0.7 1.9 1.0 0.9 5.9 1.1 0.8 3.0 1.7 1.3 2.0 2.7 1.1
88.2 90.0 91.6 92.9 90.7 91.5 93.0 92.4 94.3 91.2 92.2 92.1 99.4 98.1 92.9 89.6 91.6 90.6
6.9 3.6 0.8 3.4 1.5 0.6 1.2 1.1 0.9 7.9 0.9 1.2 3.1 2.4 3.1 3.7 1.8 1.2
92.3 94.0 92.9 94.1 94.5 93.1 93.8 94.8 93.9 88.8 97.4 94.4 93.8 98.4 96.5 90.6 93.9 93.1
1.5 1.4 1.9 1.3 2.4 2.2 0.6 1.5 0.3 3.1 2.5 1.2 3.0 0.9 1.4 2.4 1.2 1.7
Table 4. Content of six quassinoids in the Eurycoma longifolia extract and dietary supplement products
Sample Code
Content (g/g)a)
A
B
C
D
E
F
CS-1 CS-2 CS-3 CS-4 TB-1 TB-2 TB-3
208.0 497.1 300.3 356.6 1601.5 18.8 ND
42.4 512.6 75.5 106.0 607.6 4.8
of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Republic of Korea 2 National Forensic Service, Seoul, Republic of Korea 3 College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea Received February 18, 2015 Revised April 2, 2015 Accepted April 14, 2015
J. Sep. Sci. 2015, 38, 2260–2266
Research Article
Simultaneous quantitation of six major quassinoids in Tongkat Ali dietary supplements by liquid chromatography with tandem mass spectrometry Tongkat Ali (Eurycoma longifolia) is one of the most popular traditional herbs in Southeast Asia and generally consumed as forms of dietary supplements, tea, or drink additives for coffee or energy beverages. In this study, the liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of six major quassinoids of Tongkat Ali (eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)-epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone) was developed and validated. Using the developed method, the content of the six quassinoids was measured in Tongkat Ali containing dietary supplement tablets or capsules, and the resulting data were used to confirm the presence of Tongkat Ali in those products. Among the six quassinoids, eurycomanone was the most abundant quassinoid in all samples tested. The developed method would be useful for the quality assessment of Tongkat Ali containing dietary supplements. Keywords: Dietary supplements / Eurycoma longifolia / Liquid chromatography with tandem mass spectrometry / Quassinoids / Tongkat Ali DOI 10.1002/jssc.201500207
1 Introduction Eurycoma longifolia Jack (Simaroubaceae), commonly known as Tongkat Ali, is one of the most popular ethnobotanical plants found in Southeast Asia. Root extracts from this plant have been reported to have various beneficial functions such as antimalarial, antiulcer, antipyretic, and antimicrobial activities [1]. Especially, E. longifolia is believed to exert improving effects for erectile dysfunction, libido, male infertility, boosting athletic performance, bodybuilding, or reducing body fat [2, 3]. E. longifolia is, therefore, popularly used as forms of food, tea, dietary supplements, or drink additives for coffee or energy beverages. Phytochemical studies have shown that E. longifolia contains a variety of natural constituents, including quassinoids [4–8], cathinone alkaloids [9, 10], and squalene derivatives [11, 12]. Among them, quassinoids represent the major constituents of E. longifolia roots [13]. The quassinoids, indigenously found in the Simaroubaceae family, are a group of diterpenoids with diverse pharmacological properties such as anticancer, antimalarial, antipyretic, and cytotoxic activities [14–18]. Correspondence: Professor Hye Hyun Yoo, Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Gyeonggi-do 426–791, Republic of Korea E-mail: [email protected] Fax: +82-31-400-5958
Abbreviations: PTFE, Polytetrafluoroethylene; MRM, Multiple reaction monitoring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
With a growing demand for herbal products in the health supplements market, quality evaluation studies of raw materials and commercial products have been extensively conducted. Recently, E. longifolia extract has gained great popularity in the dietary supplement market because of its ability to improve sperm quality and its aphrodisiac properties, which is not limited to Southeast Asia. However, the chronic use of E. longifolia containing products is controversial in terms of safety and a recommended optimal dosage is unknown. Furthermore, it is unclear as to whether E. longifolia containing products actually contain E. longifolia extracts as described in their product content labels. The current available information on quality criteria for E. longifolia and its related products is limited. Therefore, it is worthwhile investigating their chemical composition and quantifying bioactive constituents for quality and safety concern. Several reports demonstrating analytical methods for E. longifolia extracts have been published. Chua et al. performed metabolite profiling of E. longifolia using LC–MS/MS-based techniques [13]. This study focused mainly on the characterization and identification of the metabolites rather than their quantification. Teh and his colleagues presented a LC– MS method for the determination of quassinoids in various batches of E. longifolia extract [19]. Furthermore, the LC– MS method may have some limitations when applied to dietary supplement products with various ingredients because ∗ Young Min Han and Moonhee Jang contributed equally to this work.
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2015, 38, 2260–2266
of lower sensitivity and selectivity. In this case, LC–MS/MS approaches could be needed for identification and quantification of bioactive constituents in complicated matrix. However, to our knowledge, a quantitative LC–MS/MS method for E. longifolia has not been published. In the present study, we developed and validated an LC–MS/MS method for the simultaneous determination of six quassinoids in E. longifolia plant material and dietary supplements. Authentic presence of the extract in commercial products was also verified using the developed method. Accordingly, major quassinoids are presented as suitable markers for the QC of E. longifolia containing products.
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Table 1. Precursor–product ion pairs used in multiple reaction monitoring
Eurycomanone 13,21-dihydroeurycomanone 13a(21)-epoxyeurycomanone 14,15-dihydroxyklaineanone Eurycomalactone Longilactone
Precursor ion
Product ion
CE (V)
409.1 411.2 425.2 397.2 349.2 367.2
391.2 393.1 407.1 361.1 313.1 349.1
10 10 10 10 1 10
2.4 LC–MS/MS
2 Materials and methods 2.1 Chemicals and materials The roots of E. longifolia were collected in Dak Lak province, Vietnam and authenticated by Dr. Bui Van Thanh in Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Vietnam. The reference standards of Eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone were isolated in a previous study [8]. HPLC-grade methanol, acetonitrile, and acetic acid were purchased from JT Baker (Phillipsburg, NJ, USA). Deionized water was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). Seven dietary supplements (four types of capsules and three types of tablets) claiming to contain Tongkat Ali extract were purchased from online markets.
2.2 Preparation of standard solutions Individual standard stock solutions of the six reference compounds (mentioned above) were prepared at a concentration of 1 mg/mL in water/methanol (30:70, v/v). Working standard solutions were prepared by serial dilution to appropriate concentrations.
2.3 Sample preparation The crude methanol extract of E. longifolia root was obtained as previously described [8]. The extract was dissolved in water/methanol (30:70, v/v) at a concentration of 10 mg/mL and filtered through a 0.45 m PTFE syringe filter for analysis. For Tongkat Ali products, tablets were pulverized with a mortar and pestle. For capsules, the contents were emptied and mixed. The samples were weighed (3 g) and dissolved in 30 mL of methanol. After sonication for 30 min, the sample solution was centrifuged for 5 min at 13 000 rpm. The supernatant was filtered through 0.45 m filter before analysis. The samples solutions were diluted appropriately for the calibration range before analysis. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The analysis was performed using an Agilent 1260 infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to 6460 triple quadrupole mass spectrometer (Agilent Technologies). Chromatographic separation was carried out using a C18 column (2.1 mm × 50 mm, 2.7 m, Agilent Technologies, Palo Alto, CA, USA). Gradient elution was performed with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile/water (90:10, v/v) (B) at a flow rate of 0.25 mL/min. The initial condition was 5% B, which was increased to 40% B over 7 min, and finally increased to 80% B at 8 min. Finally, the initial condition was restored and held for 4.5 min to re-equilibrate the system. The total run time was 12.5 min. The column oven was maintained at 40⬚C. The injection volume was 2 L. The mass spectrometer was operated in the ESI positive ion mode. Nitrogen was used as nebulizer gas at a pressure of 45 psi, a carrier gas at 10 L/min and 325⬚C, and a sheath gas at 12 L/min and 400⬚C. Detection of ions was performed in the multiple reaction monitoring (MRM) mode. The precursor–product ion pairs used in MRM mode were summarized in Table 1.
2.5 Method validation The developed method was validated for specificity, linearity, LOD, LOQ, accuracy (as recovery), and precision. Specificity was assessed to evaluate potential interference with the signals of the analytes. Linearity was determined by independent analysis of three sets of calibrators with six concentrations ranging from 50 to 1000 ng/mL. LOD and LOQ were evaluated by injecting a series of standard solutions with decreasing analyte concentrations. LOD and LOQ were defined as the lowest concentration that produces acceptable peak shape and an S/N of at least 3 and 10, respectively. Intra- and inter-day precision and accuracy were determined from six replicates of samples at three concentrations (100, 500, and 1000 ng/mL) on five different days. For accuracy and precision tests, known amounts of standard solutions were spiked to tablet and capsule samples and prepared using the method described above. Accuracy was represented as average recoveries (%) of the spiked analyte concentrations and precision was expressed as RSD% of repeated analysis. www.jss-journal.com
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J. Sep. Sci. 2015, 38, 2260–2266
Figure 1. Representative MS/MS spectra of (A) eurycomanone, (B) 13,21-dihydroeurycomanone, (C) 13␣(21)-epoxyeurycomanone, (D) 14,15-dihydroxyklaineanone, (E) eurycomalactone, and (F) longilactone
3 Results and discussion 3.1 Method development and validation In a preliminary study, the methanolic extract of Tongkat Ali was analyzed in Q1 scan mode using LC–MS/MS. Based on the resulting chromatogram, six major quassinoids, eurycomanone, 13,21-dihydroeurycomanone, 13␣(21)epoxyeurycomanone, 14,15-dihydroxyklaineanone, eurycomalactone, and longilactone, were chosen as marker compounds of E. longifolia extracts. The target compounds were favorably ionized in positive ion mode to yield the signals of the protonated molecular ion [M+H]+ . In the product ion scan, the fragment ions with neutral loss of H2 O were found to be the predominant product ion for all analytes (Fig. 1). This is thought to be due to the complicated and rigid C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
chemical structures of quassinoids. The MRM conditions were then optimized based on these results. Subsequently, the chromatographic separation was achieved on a C18 column with a gradient mobile phase by modifying the previously published methods [13, 19]. The LC–MS/MS detection using the MRM mode contributed to a decreased analytical run time through enhanced selectivity as well as improvement in sensitivity. The developed method allowed a rapid and efficient analysis; all analytes were eluted between 3.1 and 6.9 min, and their peak shape was satisfactory. Meanwhile, internal standard was not used in this method. The use of an internal standard is generally recommended in MS analysis. However, due to the unavailability of an appropriate internal standard for quassinoids [19], the quantitative method was established here using an external standard calibration method. www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2015, 38, 2260–2266
2263
Figure 2. Typical extracted ion chromatograms for (A) standard mixture, (B) methanol extract of E. longifolia, and (C) Tongkat Ali containing capsules and (D) tablets Table 2. Calibration curve data for six quassinoids
Analytes
Range (ng/mL)
Calibration curve equation
Correlation coefficient, R2
LOD (ng/mL)
LOQ (ng/mL)
Eurycomanone 13,21-dihydroeurycomanone 13a(21)-epoxyeurycomanone 14,15-dihydroxyklaineanone Eurycomalactone Longilactone
50–2000 50–2000 50–2000 50–2000 50–2000 50–2000
y = 41.1285x + 90.0088 y = 71.4604x + 94.7947 y = 39.1084x + 99.4949 y = 2.9760x + 17.0073 y = 3.4896x + 2.7047 y = 5.4847x + 2.0792
0.999 0.999 0.999 0.999 0.998 0.999
1 0.5 1 2 2 5
5 2 5 10 10 20
Figure 2 shows the representative chromatograms of six quassinoids in the standard mixture, methanolic extracts of E. longifolia, and Tongkat Ali containing capsules and tablets. When compared with the standard chromatogram (Fig. 2A), no significant interference with analyte detection was observed in any of the samples tested (Fig. 2B–D), suggesting high specificity of the present method. Good linearity was achieved for all analytes within the range of 50–1000 ng/mL. Average regression coefficients (R2 ) were greater than 0.998 for all analytes. LOD ranged from 0.5 to 5 ng/mL, and LOQ ranged from 2 to 20 ng/mL (Table 2). The accuracy and precision was evaluated by measuring the recovery of standardspiked samples. Recovery for capsules and tablets was within the range of 88.8–98.4 and 82.5–99.4%, respectively, and their RSD values ranged from 0.3 to 4.4% for capsules and from 0.6 to 7.9% for tablets (Table 3). These results indicate that the developed method is acceptable for accurate and precise determination of target compounds. Recently, Teh et al. presented a quantitative LC–MS method for the analysis of quassinoids in E. longifolia extract [19]. In their method, LC with a trap mass spectrometer
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
was used and chromatographic conditions were optimized; the mobile phase consisting of water/methanol (91:9)/0.1% acetic acid was used under isocratic elution. The resulting chromatogram showed a good separation but the peak shapes were broad. The LOD range was 30 to 100 ng/mL, and the total run time was more than 50 min. Meanwhile, our method adopted a gradient using the mobile phase consisting of acetonitrile and 0.1% formic acid with an LC triple-quadrupole mass spectrometer. Accordingly, our current LC–MS/MS method achieved a lower detection limit (0.5–5 ng/mL) and a shorter chromatographic analysis time (12.5 min). These compounds may also be easily detected at maximum absorption of UV radiation around 238 nm due to the presence of the chromophoric ␣,-unsaturated ketone in ring A of the quassinoids [20]. However, HPLC methods would be also difficult to apply for the analysis of mixtures of numerous ingredients because of chromatographic interferences. Taken together, the present method could facilitate the determination of quassinoids from dietary supplement products containing complicated matrix components (ingredients) or even low amounts of Tongkat Ali extracts.
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Y. M. Han et al.
Table 3. Intra- and inter-day precision and accuracy for the determination of six quassinoids in capsules and tablets
Compound
Concentration Capsules spiked (ng/ml)
Tablets
Intra-day (n = 3)
Inter-day (n = 5)
Intra-day (n = 3)
Inter-day (n = 5)
Accuracy Precision Accuracy Precision Accuracy Precision Accuracy Precision (%recovery) (%RSD) (%recovery) (%RSD) (%recovery) (%RSD) (%recovery) (%RSD) Eurycomanone
100 500 1000 13,21-dihydroeurycomanone 100 500 1000 13a(21)-epoxyeurycomanone 100 500 1000 14,15-dihydroxyklaineanone 100 500 1000 Eurycomalactone 100 500 1000 Longilactone 100 500 1000
91.0 94.5 92.3 93.3 92.9 89.9 92.7 95.9 94.3 92.7 96.6 96.2 94.4 98.1 94.5 94.2 95.2 92.8
3.0 3.7 3.0 1.6 1.1 0.8 2.3 3.1 1.7 3.8 3.1 1.1 3.3 1.8 2.1 4.4 1.2 1.0
82.5 87.6 91.3 89.2 88.9 91.5 92.9 92.9 93.9 88.2 91.4 93.8 95.6 95.2 95.0 87.8 89.3 91.1
6.4 0.8 1.0 1.0 0.6 0.7 1.9 1.0 0.9 5.9 1.1 0.8 3.0 1.7 1.3 2.0 2.7 1.1
88.2 90.0 91.6 92.9 90.7 91.5 93.0 92.4 94.3 91.2 92.2 92.1 99.4 98.1 92.9 89.6 91.6 90.6
6.9 3.6 0.8 3.4 1.5 0.6 1.2 1.1 0.9 7.9 0.9 1.2 3.1 2.4 3.1 3.7 1.8 1.2
92.3 94.0 92.9 94.1 94.5 93.1 93.8 94.8 93.9 88.8 97.4 94.4 93.8 98.4 96.5 90.6 93.9 93.1
1.5 1.4 1.9 1.3 2.4 2.2 0.6 1.5 0.3 3.1 2.5 1.2 3.0 0.9 1.4 2.4 1.2 1.7
Table 4. Content of six quassinoids in the Eurycoma longifolia extract and dietary supplement products
Sample Code
Content (g/g)a)
A
B
C
D
E
F
CS-1 CS-2 CS-3 CS-4 TB-1 TB-2 TB-3
208.0 497.1 300.3 356.6 1601.5 18.8 ND
42.4 512.6 75.5 106.0 607.6 4.8
