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Quantitative Analysis of Twelve Marker Compounds in Palmijihwang-hwan using Ultra-Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry
Quantitative Analysis of Twelve Marker Compounds in Palmijihwang-hwan using Ultra-Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry
Natural Product Sciences. 2014. Sep, 20(3): 182-190
Copyright © 2014, The Korean Society of Pharmacognosy
  • Received : April 28, 2014
  • Accepted : May 29, 2014
  • Published : September 30, 2014
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About the Authors
Chang-Seob Seo
Hyeun-Kyoo Shin
hkshin@kiom.re.kr

Abstract
An ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS) method was established for quantitative analysis of twelve components, allantoin ( 1 ), morroniside ( 2 ), 5-hydroxymethyl-2-furfural (5-HMF) ( 3 ), loganin ( 4 ), coumarin ( 5 ), cinnamic acid ( 6 ), mesaconitine ( 7 ), cinnamaldehyde ( 8 ), hypaconitine ( 9 ), aconitine ( 10 ), alisol B ( 11 ), and alisol B acetate ( 12 ) in a Palmijihwang-hwan decoction. The twelve constituents were separated on a UPLC BEH C 18 column (2.1 × 100 mm, 1.7 μm) at a column temperature of 40 ℃ by gradient elution with 0.1% (v/v) formic acid in water and acetonitrile as the mobile phase. The flow rate was 0.3 mL/min and the injection volume was 2.0 μL. Calibration curves of all compounds were acquired with values of the correlation coefficient ≥ 0.99 within the test ranges. The limits of detection and quantification for all analytes were 0.01 - 4.53 ng/mL and 0.03 - 13.60 ng/mL, respectively. The concentrations of the compounds 1 - 9 and 12 were 72.83, 4389.00, 4859.00, 3155.17, 223.67, 33.50, 1.97, 518.00, 2.25, and 25.00 μg/g, respectively. However, compounds 10 and 11 were not detected.
Keywords
Introduction
Generally, traditional herbal medicines include many herbs and components. They have been widely used to prevent and treat various diseases associated with multiple targets and are suitable for long-term administration compared with chemically synthesized medicine. 1 - 3 Palmijihwang-hwan (PMJHH), also known as Ba-Wei-Di-Huang-Wan in Chinese and Hachimi-jio-gan in Japanese, is a well-known traditional Korean herbal formula, consisting of eight herbal medicines: Rehmanniae Radix Preparata, Dioscoreae Rhizoma, Corni Fructus, Moutan Cortex Radicis, Hoelen, Alismatis Rhizoma, Cinnamomi Cortex Spissus, and Aconiti Lateralis Radix Preparata. It has been used for preventing and treating aging including osteoporosis and kidney deficiency such as renal nephritis, diabetic nephropathy, nephritic syndrome, and glomerulonephritis. 3 , 4 In addition, the pharmacological effects of PMJHH have demonstrated a protective effect with respect to pancreatic dysfunction due to fibrosis and oxidative stress in type 2 diabetes, 5 improvement of agerelated unbalanced Ca metabolism, 4 and a renal protective effect through the influence of hypoxia-inducible factor. 6 As mentioned above, a number of biological activities of PMJHH have been reported. However, no quantitative analysis of the marker constituents for the quality control of PMJHH has been reported. Therefore, we performed qualitative and quantitative analyses to improve the quality control of PMJHH. In the present study, we improved the quality control of PMJHH through the quantitative analysis of twelve marker components: Allantoin ( 1 ) from Dioscoreae Rhizoma; morroniside ( 2 ) and loganin ( 4 ) from Corni Fructus; 5-hydroxymethyl-2-furfural (5-HMF) ( 3 ) from Rehmanniae Radix Preparata; coumarin ( 5 ), cinnamic acid ( 6 ), and cinnamaldehyde ( 8 ) from Cinnamomi Cortex Spissus; mesaconitine ( 7 ), hypaconitine ( 9 ), and aconitine ( 10 ) from Aconiti Lateralis Radix Preparata; and alisol B ( 11 ) and alisol B acetate ( 12 ) from Alismatis Rhizoma. This was achieved by using an accurate and precise ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS) method.
Experimental
General experimental procedures – The chromatographic analysis was performed using a Waters ACQUITY UPLC system (Milford, MA, USA) equipped with a pump, degasser, column oven, and autosampler. The 12 compounds were separated on an ACQUITY UPLC BEH C 18 column (2.1 × 100 mm, 1.7 μm). The LC-MS/MS system used was the Waters ACQUITY TQD LC-MS/MS system (Milford, MA, USA) with an electrospray ionization (ESI) source. Data were acquired and processed using Waters MassLynx software (version 4.1, Milford, MA, USA).
Chemicals and reagents – The reference standard compounds 1 , 3 , 5 and 6 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and compound 2 was obtained from Chengdu Biopurify Phytochemicals (Chengdu, China). Compounds 4 , 8 , 11 , and 12 were purchased from Wako Chemicals (Osaka, Japan) and compounds 7 , 9 , and 10 were obtained from ChemFaces (Wuhan, China). The purities of reference compounds 1 - 12 were greater than 98.0% according to HPLC analysis. The chemical structures of marker compounds 1 - 12 are shown in Fig. 1 . HPLC-grade methanol, acetonitrile and water were purchased from J.T. Baker (Phillipsburg, NJ, USA) and analytical reagent-grade, formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).
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Chemical structures of the 12 marker compounds in Palmijihwang-hwan.
Plant materials – The PMJHH herbal medicine, composed of eight herbal components was purchased from the Korean herbal market, Kwangmyungdang Medicinal Herbs (Ulsan, Korea). The origin of these herbal materials was confirmed taxonomically by Prof. Je Hyun Lee, Dongguk University, Gyeongju, Korea. Voucher specimens (2012-KE35-1 through KE35-8) have been deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine.
Preparation of standard solutions – Reference compounds 1 - 12 were accurately weighed and dissolved in methanol at a concentration of 1.0 mg/mL. Each standard stock solution was kept at 4 ℃ and used after serial dilution with methanol before LC-MS/MS analysis.
Preparation of PMJHH water extract and sample solutions – The PMJHH decoction composed of the eight crude herbal medicines, Rehmanniae Radix Preparata, Dioscoreae Rhizoma, Corni Fructus, Moutan Cortex Radicis, Hoelen, Alismatis Rhizoma, Cinnamomi Cortex Spissus, and Aconiti Lateralis Radix Preparata was mixed as indicated in Table 1 (5.0 kg; 101.25 g × 49.4) and extracted in a 10-fold mass of water at 100 ℃ for 2 h under pressure (1 kgf/cm 2 ) using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, Korea). The water extract was then filtered through a standard sieve (no. 270, 53 μm; Chung Gye Sang Gong Sa, Seoul, Korea) and lyophilized by freeze-drying (1239.6 g). The yield of PMJHH extract was 24.79%. For quantitative analysis of the soluble components by LC-MS/MS, 100 mg of the lyophilized PMJHH extract was dissolved in 5 mL of distilled water. Subsequently, the solution was diluted 100-fold and filtered through a 0.22 μm membrane filter before injection into the LC-MS/MS system.
Composition of Palmijihwang-hwan
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Composition of Palmijihwang-hwan
Liquid chromatographic conditions – The gradient elution of two mobile phase systems with 0.1% (v/v) formic acid in water (A) and acetonitrile (B) was as follows: 20 - 95% B for 0 - 14.0 min, 95 - 100% B for 14.0 - 15.0 min, and 100-20% B for 15.0-15.1 min, with a re-equilibrium time of 3 min. The flow rate was 0.3 mL/min, the column temperature was maintained at 45 ℃, and the injection volume was 2.0 μL ( Table 2 ).
Conditions for the LC-MS/MS analysis of Palmijihwanghwan
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a0.1% (v/v) formic acid in water bAcetonitrile
LC-MS/MS conditions – The MS conditions were as follows: capillary voltage 3.3 kV, extractor voltage 3.0 V, RF lens voltage 0.3 V, source temperature 120 ℃, desolvation temperature 300 ℃, desolvation gas 600 L/h, cone gas 50 L/h and collision gas 0.14 mL/min ( Table 2 ).
Calibration curves, limits of detection (LOD), and quantification (LOQ) – The calibration curves of compounds 1 - 12 were calculated by plotting the peak areas ( y ) versus the corresponding concentrations ( x , ng/mL) using standard solutions. For the calibration curves, four concentrations of each compound (10, 50, 100, and 500 ng/mL) were prepared and measured in triplicate. The LOD and LOQ data under the present chromatographic conditions were determined at signal-to-noise (S/N) ratios of 3 and 10, respectively.
Results and Discussion
Linearity, range, LOD, and LOQ – The linearity of the analytical method was assessed from the correlation coefficient ( r 2 ) of the calibration curves of each compound. As shown in Table 3 , the calibration curves of the twelve compounds showed good linearity with r 2 ≥ 0.9971 in four different concentration ranges. The LODs and LOQs of the tested 12 compounds were 0.01 - 4.53 ng/mL and 0.03 - 13.60 ng/mL, respectively.
Linearities, regression equation, correlation coefficients, LOD, and LOQ for 12 marker compounds
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ay: peak area of compounds; x: concentration (ng/mL) of compounds. bLOD = 3 × signal-to-noise ratio. cLOQ = 10 × signal-to-noise ratio.
Peak detection of marker compounds – Using the optimized MS conditions, the mass spectral data of the investigated compounds in both negative and positive ion modes are shown in Table 3 . Compounds 1 and 2 were detected in the negative ion mode [M – H] at m/z 157.0 and 405.3, respectively, while compounds 3 - 12 were detected using the positive ion mode [M + H] + at m/z 126.9, 391.3, 146.8, 148.9, 632.6, 132.8, 616.5, 646.6, 473.6, and 515.6, respectively ( Table 4 and Fig. 2 ). As shown in Fig. 2 , compound 1 tended to eliminate NH 2 CONH 2 to produce the fragmentation peaks m/z 97.0 [M– H – NH 2 CONH 2 ] . 7 Compounds 2 and 4 belong to the glucosides and are apt to lose the glucose to produce aglycone ions of m/z 243.0 [M– H – Glu] and m/z 229.0 [M + H – Glu] + , respectively. 8 Compounds 3 , 6 , and 8 were detected at m/z 108.8 [M + H – H 2 O] + , 130.9 [M + H –H 2 O] + , and 115.1 [M + H – H 2 O] + , respectively, by eliminating one H 2 O molecule from the each precursor ion. 9 - 11 Compound 5 contains an ion at m/z 103.1 corresponding to [M + H – CO 2 ] + , which is assigned to the loss of CO 2 from the pyrone ring system. 12 The MS fragmentations of compounds 7 , 9 , and 10 were detected at m/z 572.5 [M + H – AcOH] + , 556.4 [M + H – AcOH] + , and 586.2 [M + H – AcOH] + , with the loss of acetic acid from the parent ion [M + H] + at m/z 632.6, 616.5, and 646.6, respectively. 13 Compound 11 exhibited an [M + H] + ion at m/z 473.6. In addition, the characteristic MS fragmentation of compound 11 was observed at m/z 365.4 [M + H – 2H 2 O – C 4 H 8 O] + by eliminating two H 2 O and C 4 H 8 O molecules from the parent ion. 14 An ion in compound 12 was detected at m/z 437.5 [M + H – AcOH – H 2 O] + , which is assigned to the loss of acetic acid and H 2 O molecules from the parent ion. 15
Mass detection condition of the 12 marker compounds
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Mass detection condition of the 12 marker compounds
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Q1 (A) and Q3 (B) mass spectra of 12 standard compounds. Allantoin (1), morroniside (2), 5-HMF (3), loganin (4), coumarin (5), cinnamic acid (6), mesaconitine (7), cinnamaldehyde (8), hypaconitine (9), aconitine (10), alisol B (11), alisol B acetate (12).
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continued
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continued
Quantitative analysis of twelve marker compounds in PMJHH – The established analytical method using LC–MS/MS was applied to the quantitative analysis of the twelve marker compounds in the PMJHH extract ( Fig. 3 ). As shown in Table 5 , the amounts of the tested compounds 1 - 12 in the PMJHH extracts were detected to 4859.00 μg/g. In addition, compounds 2 - 4 , which are major constituents of Cornus officinalis and Rehmannia glutinosa , were found in concentrations of 4389.00, 4859.00, and 3155.17 μg/g, respectively, and were the most abundant compounds in comparison to the others in the PMJHH extract.
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Total ion chromatograms of 12 reference compounds (A) and Palmijihwang-hwan extract (B) by LC-MS/MS MRM mode. Allantoin (1), morroniside (2), 5-HMF (3), loganin (4), coumarin (5), cinnamic acid (6), mesaconitine (7), cinnamaldehyde (8), hypaconitine (9), aconitine (10), alisol B (11), alisol B acetate (12).
Contents of the 12 compounds in Palmijihwang-hwan (n = 3)
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aN.D. means not detected.
In conclusion, a rapid, accurate, and reliable LC-MS/MS method was established for the quantitative analysis of twelve marker compounds in the herbal formula, PMJHH. Consequently, this method has been successfully applied to the quantitative analysis for the quality control of PMJHH and could be helpful and valuable for the quality assessment of PMJHH or related herbal formulas.
Acknowledgements
This research was supported by a grant (no. K14030) from the Korea Institute of Oriental Medicine.
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