Standard Analytical Methods of Bioactive Metabolitesfrom Lonicera japonica Flower Buds by HPLC-DAD and HPLC-MS/MS

Transcription

1 Standard Analytical Methods of Bioactive Metabolitesfrom Lonicera japonica Flower Buds by HPLCDAD and HPLCMS/MS Jongheon Shin College of Pharmacy Seoul National University Summary The standard analytical methods using HPLCDAD for 6 representative compounds (4 phenolics, 1 flavonoid, and 1 iridoid) derived from the flower buds of Lonicera japonica were studied. An optimal reversedphase chromatographic system consisting of a C 18 silica gel column and a mixture of methanol and water with.1% formic acid as the mobile phase was developed. Using the gradient solvent system, the standard samples were satisfactorily determined within 35 minutes after direct injection of the solution, and confirmed against the crude extract of the L. japonica flower buds with 7% aqueous ethanol. Various validation parameters such as specificity, linearity, detection limit, quantitation limit, accuracy, precision, and robustness were successfully obtained and individually validated. In addition, effectiveness of diverse extraction methods were compared to each other for the development of standard analytic method. Concentrations of standard components were measured for 21 commercial products from diverse sources. 1

2 Chemical structures of standard compounds HC H 3 CC HC 3,5Dicaffeoylquinic acid 3,5Dicaffeoylquinic acid methyl ester H C 2 H H H H 3 C CH 3 H H H CH 2 C 2,3,4Trihydroxy benzoic acid(i.s) TLC patternsilica plate EtAc : Me : H2 = 8.5 : 1 :.7 Detection reagent Ext. H2S methyl ester 3. FeCl Luteolin7Glc Ext. 6. I2 7. Extract Ext. 2

3 TLC patternds plate Me : H2 = 5.5 : 4.5 Detection reagent Ext. H2S methyl ester 3. FeCl Luteolin7Glc Ext. 6. I2 7. Extract Ext. Experimental perating conditions Column Column Temp. A stainless steel column 4.6mm in inside diameter and 15cm in length, packed with octadecyl silica gel for liquid chromatography(5μm in particle diameter) A : Methanol with.1% formic acid B : Water with.1% formic acid Mobile Phase Gradient system : min, A: %; min, A: 5%; 25 min, A: 5%; 25 min, A: %; 35 min, A: % Detector Flow rate :.3mL/min UV detector (wavelength : 25nm) 3

4 Specificity mau 1 6 DAD1A, Sig=25,4Ref=36,1 (RSK \1174.D) min 1. 2,3,4Trihydroxy benzoic acid(i.s) Luteolin7Glc 7. methyl ester Column : Shiseido CAPCELL PAK C18 UG1 Linearity and Range A Std /A IS 6 5 methyl ester C std /C IS Range(mg/L) Conversion equation Correlation coefficient(r) Chlorogenic aicd 1 ~ Y =.4381X ~ Y =.1438X ~ Y =.27926X ~ Y =.4118X ~ Y =.1476X methyl ester 1 ~ Y =.3492X

5 Detection limit and Quantitation limit methyl ester Detection limit(mg/l) Quantitation limit(mg/l) Experimetal procedure: 1) Ground powdered sample of Lonicera japonica flower buds, mg 2) Spike standard,, 1, and 15μg for and 16,, and 2μg for,,,, methyl ester. 3) Add 7% ethanol to make the volume to 1 ml, respectively. 4) Extract analyte by ultrasonication method for 6 min. 5) Filter an aliquot of the solution through.2 μm membrane. 6) Add μg of internal standard to 1 ml of the filtrate. 7) Concentrate filtrate containing internal standard. 8) Dissolve with % of aqueous methanol(with.1% formic acid), the mobile phase. 9) Inject μl of test solution to HPLC system through autosampler. 5

6 Accuracy methyl ester Spiked conc. (μg/ml) Recovery(%) RSD(%) Precision (Reproducibility) Peak area ratio(peak area/is area) Mean SD RSD(%) Retention time (min) Mean SD RSD(%) methyl ester

7 Precision (Intra / Interday) Mean (mg/l) Intraday SD RSD (%) Mean (mg/l) Interday SD RSD (%) methyl ester Robustness DAD1 A, Sig=25,4 Ref=36,1 (RSK \114.D) mau ,3,4Trihydroxy benzoic acid(i.s) Luteolin7Glc min 7. methyl ester Column : Agilent ZRBAX Eclipse XDBC18 7

8 Robustness mau 1 6 DAD1 A, Sig=25,4 Ref=36,1 (RSK \1194.D) 2 1 3,4 5, min 1. 2,3,4Trihydroxy benzoic acid(i.s) Luteolin7Glc 7. methyl ester Column : Phenomenex Gemini 5μ C18 Robustness column Columns Surface area/cm2 Pore size(ao) Analytes Theoretical plate(n) Agilent ZRBAX Eclipse XDBC ± ± ± phenomenex Gemini 5μ C ± ± ±76.37 Shiseido CAPCELL PAK C18 UG ± ± ± Capacity factor(k ) Agilent ZRBAX Eclipse XDBC ± ± ±.76 phenomenex Gemini 5μ C ± ± ±1. Shiseido CAPCELL PAK C18 UG ± ± ±.8 Separation factor(α) Agilent ZRBAX Eclipse XDBC ± ± ±. phenomenex Gemini 5μ C ± ±. 1.5 ±.1 Shiseido CAPCELL PAK C18 UG ±. 1. ± ±. Resolution(Rs) Agilent ZRBAX Eclipse XDBC ± ± ±.3 phenomenex Gemini 5μ C ± ±.. ±.13 Shiseido CAPCELL PAK C18 UG ± ± ±.1 8

9 Robustness column Columns Surface area/cm2 Pore size(ao) 35Dicaffeoyl quinic acid Analytes 35Dicaffeoyl quinic acid methyl ester Theoretical plate(n) Agilent ZRBAX Eclipse XDBC ± ± ±225.1 phenomenex Gemini 5μ C ± ± Shiseido CAPCELL PAK C18 UG ± ± ±.9 Capacity factor(k ) Agilent ZRBAX Eclipse XDBC ± ± ±.22 phenomenex Gemini 5μ C ± ±.66 Shiseido CAPCELL PAK C18 UG ±.8 6. ± ±.12 Separation factor(α) Agilent ZRBAX Eclipse XDBC ± ± ±. phenomenex Gemini 5μ C ± ±. Shiseido CAPCELL PAK C18 UG ±. 1.5 ± ±. Resolution(Rs) Agilent ZRBAX Eclipse XDBC ± ± ±.4 phenomenex Gemini 5μ C ± ±.2 Shiseido CAPCELL PAK C18 UG ± ± ±.2 Robustness temperature Teperature( ) Analytes Theoretical plate(n) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±12.89 Capacity factor(k ) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.64 Separation factor(α) 1.8 ±. 1. ±. 1.2 ± ±.1 1. ±. 1.4 ±. 1.7 ±. 1. ±. 1.6 ± ±. 1. ±. 1.8 ± ±. 1. ± ±. Resolution(Rs) 2.66 ± ±.4.93 ± ± ± ± ± ± ± ± ± ± ± ± ±.3 9

10 Robustness temperature Teperature( ) Analytes 35Dicaffeoyl quinic acid 35Dicaffeoyl quinic acid methyl ester Theoretical plate(n) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±11.59 Capacity factor(k ) 9.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.21 Separation factor(α) 1. ± ± ± ± ± ±. 1. ±. 1.6 ± ± ± ± ± ± ± ±. Resolution(Rs) ± ± ± ± ± ± ± ± ± ± ±.1 1. ± ± ± ±.11 Stability of analytes % Stability test(상온) 35Dicaffeoyl quinic acid 35Dicaffeoyl quinic acid methyl ester % Stability test(냉장) 35Dicaffeoyl quinic acid 35Dicaffeoyl quinic acid methyl ester 1 1 day day Analytes Temp.( ) Day Mean SD RSD (%) R.T R.T R.T Dicaffeoyl quinic acid R.T R.T Dicaffeoyl quinic acid methyl ester R.T

11 Crude sample Experimetal procedure: 1) Ground powdered sample of Lonicera japonica flower buds, mg 2) Extract analyte by sonication, or vortex, or reflux method for 6 min. 3) Filter an aliquot of the solution through.2 μm membrane 4) Add μg of internal standard to 1 ml of the filtrate 5) Concentrate this filtrate containing internal standard 6) Dissolve with % of aqueous methanol(with.1% formic acid), the mobile phase 7) Inject ul of test solution to HPLC system through autosampler Studies on Extraction Method Vortex (mg/l) Reflux(mg/L) Ultrasonication (mg/l) 24, ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.5 methyl ester ± ± ± 1.97 In addition, optimal solvent condition (7% Et) and extraction time (1 hr) were found using the ultrasonication method. 11

12 Studies on Extraction Solvent 1% Me 7% Me 1% Et 7% Et ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.5 methyl ester ± ± ±1.97 Extraction time: 6 min Extraction method: ultrasonication Studies on Extraction Time 1 min min min 6 min 9 min ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.85 methyl ester ± ± ± ±5.54 Extraction solvent: 7% Et Extraction method: ultrasonication 12

13 Crude sample I.S methyl ester 2,3,4THBA (I.S) Comparison of effectiveness by extraction methods Experimetal procedure: 1) Ground powdered sample of Lonicera japonica flower buds, mg 2) Extract analyte by sonication, or vortex, or reflux method for 6 min. 3) Filter an aliquot of the solution through.2 μm membrane 4) Add μg of internal standard to 1 ml of the filtrate 5) Concentrate this filtrate containing internal standard 6) Dissolve with % of aqueous methanol(with.1% formic acid), the mobile phase 7) Inject ul of test solution to HPLC system through autosampler 13

14 Comparison of concentration by sources (μg/g)( 25 (ug/g) 15 methyl ester 1 5 G1 G2 G3 G4 G5 G6 G7 G1: Hongcheon, gangwon do G2: Nakmoon, andong si G3: Goheung, jeonnam G4: Yangyang, gangwon do G5: Bonghwa, gyung book G6: Kyoyi, gangreung si G7: Buyeo, chung nam Comparison of concentration by sources (μg/g)( 5 (ug/g) methyl ester 1 G8 G9 G1 G11 G12 G13 G14 G8: Geojin eup, goseong gun G9: Uiseong, gyung book G1: Mt. seolak G11: Cheongsong, gyung book G12: Goonwi, gyung book G13: uljin gun, gyung book G14: Andong, gyung book 14

15 Comparison of concentration by sources (μg/g)( methyl ester (ug/g) 1 5 G15 G16 G17 G18 G19 G G21 G15: China G16: Milhyun(China) G17: Andong, gyungbook G18: Hanam Jeongju(onhyun), (China) G19: Sandong(China) G: Hanam(China) G21: Sandong(China) Comparison of concentration by sources (μg/g)( μg/g Chrologenic acid 3,5Dicaffeoyl quinic acid Luteolin7glucoside methyl ester Hongcheon Nakmoon Goheung Yangyang Bonghwa Kyoyi Buyeo Geojin eup Uiseong Mt. seolak Cheongsong

16 Comparison of concentration by sources (μg/g)( μg/g Chrologenic acid 3,5Dicaffeoyl quinic acid Luteolin7glucoside 3,5Dicaffeoyl quinic acid methyl ester Goonwi Uljin gun Andong China Milhyun(China) Andong Hanam (China) Sandong(China) Hanam(China) Sandong(China) MS(/MS) analysis parameters (Ion monitoring parameter) MW ms1 ms2 Collision energy Ion mode Positive Positive Positive Positive Positive methyl ester Positive 16

17 MS(/MS) analysis parameters <ESI source> Sheath gas flow rate(arb) Method 1 Method 2 method 3 Aux gas flow rate(arb) Spray voltage(kv) Capillary temp( ) Capillary voltage(v) Tube lens offest(v) <Ion optics> ctapole 1 offset(v) Lens voltage(v) ctapole 2 offset(v) ctapole RF amplitude(v pp) I.S methyl ester MSTIC Time (min) 1:, 2:, 3:, 4:, 5:, 6: methyl ester, 17

18 LC/MS/MS SRM mode Ion mode : Positive mode methyl ester Column : C18, 4.6 X 15 mm Mobile phase : A : Methanol with.1% formic acid B :Water with.1% formic acid Gradient system : min, A: %; min, A: 5%; 25 min, A: 5%; 25 min, A: %; 35 min, A: Flow rate :.3 ml/min MS(/MS) positive mode MS MS/MS 7 [M+H+] MS 9 MS/MS [M+H+]

19 MS(/MS) positive mode MS MS/MS [M+H+] MS [M+H+] MS/MS MS(/MS) positive mode MS [M+H+] MS/MS methyl ester 1 9 MS MS/MS [M+H+]

20 MS(/MS) positive mode 2,3,4Trihydroxy benzoic acid MS 9 MS/MS [M+H+]