Quantitative determination of apigenin from apium graveolens l by HPLC – PDA method

Vietnam Journal of Science and Technology 58 (6A) (2020) 307-318 doi:10.15625/2525-2518/58/6A/15665 QUANTITATIVE DETERMINATION OF APIGENIN FROM APIUM GRAVEOLENS L BY HPLC-PDA METHOD Le Nguyen Tuong Vi1, 2, Le Tien Dung1, 2, *, Nguyen Cuu Khoa2, 3, *, Nguyen Ngoc Tuan1, 4, Quach Tong Hung1, 2, Nguyen Thi Phuong3, 5, Dang Thi Le Hang3, Nguyen Van Chinh3, Nguyen Tran Thuy Quynh3 Department of Pharmaceutical Biochemistry, Institute of Applied Materials Science, VAST, No 01B, TL29 Street, District 12, Ho Chi Minh City, Viet Nam Graduate University of Science and Technology, Vietnam Academy of Science and Technology, No 18, Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam Department of Materials and Pharmaceutical Chemistry, Institute of Applied Materials Science, Vietnam Academy of Science and Technology, No 01B, TL29 Street, District 12, Ho Chi Minh City, Viet Nam Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, No 12, Nguyen Van Bao Street, Go Vap District, Ho Chi Minh City, Viet Nam Inorganic Technology, Ho Chi Minh City University of Food Industry, No 140, Le Trong Tan Street, Tan Phu District, Ho Chi Minh City, Vietnam * Email: inpcdung@yahoo.com; nckhoavnn@yahoo.com Received: November 2021; Accepted for publication: 24 January 2021 Abstract In this paper, apigenin was isolated from the ethyl acetate extract and performed of column chromatography with solvent system n-hexane: ethyl acetate (100 %  1: 1) of Apium graveolens L to obtain fractions Fraction was applied on column chromatography with nhexane: ethyl acetate (2:1) The quantitative process of apigenin in Apium graveolens L by HPLC - PDA method was investigated using reverse phase column VDSpher PUR 100 C18 (25 cm × 4.6 mm, m), wavelength at 335 nm, the mobile phase was 0.1 % acetic acid: acetonitrile (60:40) with isocratic elution, flow rate mL/min; injection volume 20 µL; and column temperature 25 ℃ Validation results showed that the process had high specificity, linearity, repeatability with RSD = 1.000 %, accuracy recovery with 99.97 %, LOD was 0.1 μg/mL and the LOQ was 0.3 μg/mL Keywords: apigenin, Apium graveolens L., HPLC, anti-cancer Classification numbers: 1.1.1, 1.1.3 INTRODUCTION Apium graveolens L is a species of the family Apiaceae which are widely grown in the world and has been migrated to Viet Nam with the common name celery [1, 2] Chemical Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al composition analysis from the EtOH extract of Apium graveolens L revealed the presence of flavonoids, alkaloids, steroids and glycosides [3] such as apiumetin, celereoside, apiin, apigenin, luteolin, chrysoeriol 7-O-glucoside, 4,5-dihydro-3-butyl-phthalide, butyl-phthalide, etc [4] Flavonoid in celery can be able to inhibit the cell cycle, reduce oxidative stress, induce apoptosis and stimulate the immune system [5] One of the most prominent benefits of celery that has been studied is anti-cancer They have been studied from celery, including: apigenin, polyacetylenes, phthalides [6, 7] In addition, celery affects for lower blood pressure [8], lowering blood lipids [9], diuretic [10], antioxidant [11] In the flavonoids group of celery, apigenin is the most interesting compound There is an increasing evidence that apigenin is effective against various types of cancer both in vitro and in vivo cell lines in mice All results showed that apigenin has strong anticancer properties on a number of different human cancers and can be combined with many other chemotherapy agents [7] Recently, to follow trends of requirements for standardization of pharmaceutical quality, it is necessary to build a process of determination the number of bioactive compounds or specific substances that occupy a large proportion in medicinal herbs In another report, apigenin was quantified using Micellar Electrokinetic Chromatography with UV detector [12] Due to the attractive pharmacological properties of apigenin, it was evaluated as potential cancer chemopreventive agent propitious That is the reason why an analytical method was required, which allows quantification of apigenin in blood and tissues In a previous study [13], High performance liquid chromatography (HPLC) method was used to measure the steady-phase apigenin levels in tissues of mice receiving apigenin in their diet In addition, in the other report, apigenin and luteolin were simultaneously quantificated in ethanol extract of Clerodendrum serratum (Linn.) leaves by using RP-HPLC method This study set the stage for both these flavonoids could be reliably used in future as a chemotaxonomic marker for the standardization of C serratum extract that are used in herbal remedies [14] In this paper, HPLC method using reverse phase column C18 was also undertaken However, in this report, apigenin was extracted from Apium graveolens L via both of reports, we can evaluate the amount of apigenin in which herbal was higher So on, our study can give decision on herbal selection for better efficiency apigenin extracted for herbal remedies We consider choosing HPLC method to quantify apigenin because its characteristics are simple, of high sensitivity, and high accuracy Besides, PDA detector performed high selectivity and large linear range In the last two decades, the trend of using medicinal products of herbal origin for the prevention and treatment of diseases has become popular The World Health Organization has emphasized that ensuring the quality of these drugs must be based on appropriate and modern analytical techniques Therefore, we conducted the study "Quantitative determination of apigenin from Apium graveolens l by HPLC-PDA method" The results of this study would contribute to the development of standards for ingredients containing apigenin in celery for the quality management of medicinal herbs in the market MATERIALS AND METHODS 2.1 Reagents, chemical and plant materials Standard apigenin (99 %) was purchased from Sigma-Aldrich, Germany, batch number BCCC0074 A stock standard solution (20 ppm) was prepared in methanol, conserved at oC and used within months Acetonitrile, methanol, acetic acid, distilled water used for HPLC 308 Quantitative determination of apigenin from Apium graveolens L by HPLC-PDA method (Merck, Germany) n-hexane, ethyl acetate (EtOAc), methanol (MEOH), and ethanol (EtOH) were of analytical grade (Chemsol, Viet Nam) Thin layer chromatography (TLC) was performed on silica gel 60 F254 (Merck, Germany), column chromatography (CC) was performed on silica gel (240 - 430 mesh, Merck, Germany) Celery (Apium graveolens L.) was collected in Duc Trong district, Lam Dong province, Viet Nam The celery (20 kg) was dried at 50 °C to yield 1.5 kg of dried celery Dried celery samples were stored in the Department of Pharmaceutical Biochemistry - Institute of Applied Materials Science - Vietnam Academy of Science and Technology 2.2 Extraction and isolation The dried celery material (1.5 kg) was extracted with 80 % EtOH for hours with the ultrasonic waves at 40 °C, the ratio of material and solvent was 1:10 Extract liquid was evaporated off the solvent under reduced pressure (800 mmBar), to obtain 980 g of crude extract To isolate apigenin with purity ≥ 95 %, the crude extract was furnished on column chromatography with n-hexane, EtOAc, MeOH to obtain n-hexane fractions (184 g), EtOAc (320 g) and MeOH (280 g), respectively From the EtOAc fraction (320 g), the chromatography of the forward phase column with the solvent system with increasing polarity of n-hexane: EtOAc (100 %  1:1) to obtain fractions Fraction was chosen to chromatography column (250 - 360 mesh, India) with mixture n-hexane: EtOAc (2:1) to obtain 0.92 g of crystal clean pale yellow powder, that was re-checked on silica gel 60 F254 (Merck) TLC with the solvent n-hexane : EtOAc (1.5:1); the result was a yellow circle when showing with 10 % H2SO4 reagent in EtOH (Rf = 0.32) This compound structure was identified by analyzing and comparing 1H-NMR, 13C-NMR spectrum with its reference data 2.3 Apigenin qualitative by thin layer chromatography (TLC) Use a 60 F254 thin layer chromatography with the solvent n-hexane: EtOAc (1.5:1) Prepare a standard solution by dissolving the standard in methanol Prepare a sample solution at a concentration of about μL Deploy the chromatogram, then remove the plate to dry at room temperature Observed in ultraviolet light at 254 nm, apigenin trace at Rf = 0.32 2.4 The qualitative and quantitative process of apigenin All test samples and standards were injected into HPLC-PDA systems separately, recording the chromatogram, retention time, and peak area After surveying the maximum absorption wavelength (λmax) of apigenin by scanning apigenin standard samples on the UV-Vis spectrophotometer, the result of λmax of apigenin was recorded to use in quantitative procedures Chromatography conditions: VDSpher PUR 100 C18 reverse phase column (25 cm × 4.6 mm, µm); mobile phase: 0.1 % acetic acid: acetonitrile (60:40) with isocratic elution; detecting wavelength is 335 nm; flow rate mL/min; injection volume 20 µL; column temperature 25 ℃ All samples were filtered through a 0.22 μm filter before injecting into the HPLC system The retention time of apigenin found 9.48 minutes Standard sample preparation: Prepare 20 ppm apigenin standard in methanol using volumetric flask The solution was filtered through a 0.22 µm filter 309 Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al Sample preparation: Prepare 20 ppm celery extract in methanol using volumetric flask The solution was filtered through a 0.22 µm filter The process of quantification of apigenin was evaluated by examining the criteria: specificity, system compatibility, linearity, repeatability, accuracy, limit of detection (LOD) and limit of quantitation (LOQ) RESULTS AND DISCUSSION 3.1 Determination of the structure of isolated compounds a) b) Figure The TLC of two spots of apigenin extracted and apigenin standard doing with a mixture of n-Hexane : Ethyl acetate (1.5 : 1) a) TLC under UV light B b) TLC after spraying with acid H2SO4 10 % in ethanol The compound was a yellow powder, melting point: 345 - 350 ℃; 1H-NMR (methanol-d4, 500 MHz, TMS): 6.61 (1H, s, H-3), 6.23 (1H, d, J = Hz, H-6), 6.48 (1H, d, J = Hz, H-8), 7.87 (2H, d, J = Hz, H-2’, H-6’) and 6.95 (2H, d, J = Hz, H-3’ , H-5’); 13C-NMR (methanold4, 125 MHz, TMS): 166.32 (C-2), 103.87 (C-3), 183.93 (C-4), 163.23 (C-5), 100.14 (C-6), 166.02 (C-7), 95.05 (C-8), 159.45 (C-9), 105.34 (C-10), 123.31 (C-1’), 129.45 (C-2’, C-6’), 117.03 (C-3’, C-5’), 183.93 (C-4) The 1H-NMR spectrum of the isolated compound showed the presence of two aromatic couples combining meta at δ 6.21 and 6.77 corresponding to H-6 and H-8 protons, six aromatic protons in δ 7.93 (2H, d, J = Hz) and 6.92 (2H, d, J = Hz) for H-3 '/ H-5' and H-2 '/ H-6' protons of ring B, a single tip at δ 6.47 corresponding to proton H-3 characteristic for flavone structure In addition, two pairing signals at 6.21 (1H, d, J = Hz) and 6.77 (1H, d, J = Hz) showed a correlation of meta positions with each other This means that the two proton signals are in the same ring system (A ring) 13C-NMR spectrum showed the presence of twelve aromatic atoms; seven quaternary carbon atoms, five methine atoms, and quaternary carbon Values of 1H-NMR and 13C-NMR for all carbon atoms based on HSQC and HMBC correlation showed that the obtained compound's spectral data are consistent with 4,5,7-trihydroxyflavone, also known as apigenin The HMBC correlation between δH 6.48 with δC 100.14; 166.02; 159.45; 105.34 and proton δH 6.23 with carbon atoms at C 163.23; 105.34; 166.02 and 100.14 confirmed the position of δH 6.48 and 6.23 at H-8 and H-6 of ring A, respectively Another 310 Quantitative determination of apigenin from Apium graveolens L by HPLC-PDA method HMBC correlation between δH 6.95 with quaternary carbon sp2 at δC 123.31 and δH 7.87 with quaternary carbon sp2 at δC 162.74 confirmed the presence of the B ring system Olefinic protons at δH 6.61 give J correlations HMBC with δC 105.34 and 123.31 indicates that compound A is a group of flavones From the above analysis results and comparison with the reported reference, it is allowed to conclude that the compound is apigenin [15-17] 3.2 Quantification of apigenin in Apium graveolens L by using HPLC-PDA method 3.2.1 Quantification of apigenin by HPLC method Apigenin standard (99 % content) was provided by Sigma-Aldrich (Germany), batch number BCCC0074 was used as the standard Using HPLC-PDA method at 335 nm, VDSpher PUR 100 C18 column (250 4.6 mm, µm), mobile phase using a mixture of acetonitrile : 0.1 % CH3COOH solution (40:60), flow rate 1.0 mL/min, column temperature 25 oC The volume of sample to be injected was 20 μL Standard sample preparation: Dissolve mg of apigenin standard in 10 mL of methanol Aspirate mL dilute to 10 mL with methanol Sample preparation: Dissolve mg celery extract in 10 mL of methanol Aspirate mL dilute to 10 mL with methanol All samples were filtered through a 0.22 μm filter before injecting into the HPLC system Under the above conditions, Apigenin was qualitatively performed in the standard and test samples based on the retention time (tR) of apigenin peak in the chromatogram Table The qualitative results of apigenin using HPLC-PDA method Samples Retention time (tR) Average Blank sample There is no peak Standard sample Test sample 9.509 9.440 9.548 9.430 9.481 ± 0.056 Figure Chromatogram of apigenin in crude extract at 5000 ppm 311 Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al The results presented in Table show that the standard and test samples have similar retention time with a tR value of 9.481 ± 0.056 There are about ten main peaks in crude extract 5000 ppm, and the peak of apigenin (10th peak) appears at 9.445 (Figures and 3) Figure Chromatogram of apigenin in standard solution at ppm 3.2.2 Evaluation of apigenin quantitative procedure using HPLC method a) The system compatibility The apigenin standard sample was injected times into the chromatographic system and the chromatogram recorded The results in Table showed that the value of RSD of the retention time and peak area are both lower than % Evaluation criteria are within permissible limits Therefore, the method with presented conditions is highly compatible Table The results of system compatibility Standard sample Retention time tR (min) Peak area (mAU) Average SD RSD (%) 9.444 9.422 9.470 9.396 9.424 9.420 9.429 0.025 0.266 804853.5 797223.4 799668.2 806875.6 802236.7 810305.9 803527.2 4800.3 0.597 312 Tailing factor Theoretical disk 1.413 1.428 1.409 1.430 1.430 1.410 1.420 2865 2721 2814 2766 2645 2602 2735.5 Quantitative determination of apigenin from Apium graveolens L by HPLC-PDA method b) The specificity The injection of blank, standard and test sample into the chromatographic system was carried out according to the selected conditions Table shows the results of the specificity of the method Table Results of the specificity Sample Blank sample Retention time (tR) Peak area (mAU) 9.509 9.548 9.440 9.430 9.482 0.056 796013.2 799473.8 533451.3 535528.4 666116.675 151998.497 Standard Test sample Average SD The standard sample, blank sample, and the celery extract sample were analyzed under the same selected chromatographic conditions The results show that the peak response of the substances to be analyzed appears on the standard sample chromatogram, a corresponding peak appears in the sample chromatogram and at the same time no corresponding peak appears on the blank sample The apigenin sample peak is well-proportioned, sharp, with a small peak width Therefore, it can be concluded that this method guarantees selectivity and specificity c) Linear range A standard range of concentrations of ppm, ppm, ppm, ppm, ppm, 10 ppm in methanol and perform the analysis on HPLC systems was prepared The analytical results are presented in Table based on Microsoft excel software to plot the standard line graph Table Results of the construction of apigenin baseline using HPLC-PDA method Samples Standard concentrations (ppm) 10 Peak area (mAU) 164132.9 338734.1 668489.5 958543.0 1294758.7 1582270.1 The linear regression equation: y = 157454x + 20975 The correlation coefficient: R2 = 0.9994 Chromatographic analysis of the apigenin standards with the concentrations according to the above selected conditions The results in the concentration range indicate a linear relationship between the peak area and the analytical concentration in the range from ppm to 10 ppm The linear regression equation: y = 157454x + 20975 with correlation coefficient R = 0.9994 313 Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al Figure HPLC chromatogram of standard apigenin samples building calibration curve Figure Calibration curve for apigenin quantitative analysis by HPLC-PDA method d) Limit of detection (LOD), limit of quantitative (LOQ) 314 ( ⁄ ) ( ⁄ ) Quantitative determination of apigenin from Apium graveolens L by HPLC-PDA method SD: relative standard deviation of standard samples; a: slope of the calibration curve The results showed that the detection limit and quantitative limit of apigenin were 0.1 µg/mL and 0.3 µg/mL, respectively e) The repeatability To determine the repeatability of the method, separate tests were carried out for test samples in HPLC system at 100 % concentration The results of the survey are presented in Table The results in Table show that the repeatability of method is acceptable with RSD = 1.0 % (< %) Table Results of the repeatability of apigenin quantification by HPLC-PDA method Sample Peak area (mAU) Average SD RSD % 425042.1 424701.4 394793.0 430598.5 408745.7 417526.4 416901.2 13194.0 1.0 f) The accuracy The used procedure follows standard addition method The recovery rate was determined via calculating the amount of standard added into test sample in ratios of 80 %, 100 %, and 120 %, respectively, on a blank background The quantitation was conducted to define accuracy of apigenin Each concentration was repeated times under the same survey conditions The results are shown in Table The accuracy of the method is determined by the following formula: Accuracy or recovery rate (%) = × 100% Table Result of determining the accuracy of the method Sample % s addition Peak area Theoretical Actual Recovery concentr concentr rate (µg/ml) found (µg/ml) 649357.3 80 % 638348 3.99 99.77 % 3.92 98.02 % 650387.4 4.00 99.94 % 829792.6 5.14 102.74 % 5.05 101.07 % 100 % 816701.1 811135.8 5.02 100.37 % 964627.5 5.99 99.89 % 5.92 98.61 % 5.96 99.30 % 120 % 952570.4 959086.2 Average Average RSD % 99.24 % 1.07 101.39 % 1.20 99.27 % 0.64 99.97 % 0.97% 315 Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al In all tests, the RSD value was less than 2% and the average recovery rate was within the allowable limit of 98 % - 102 %, so the method achieved accuracy 3.2.3 Quantification of apigenin in Apium graveolens L It was quantified of samples of apigenin The sample processing followed the defined procedure and chromatography record followed the investigated parameters The result of the amount of apigenin is 93.72 % and in crude extract is 0.79 %, which are pretty higher in comparison with apigenin in celery raw material (0.24 %) [18] Figure The result of quantification of apigenin in Apium graveolens L in fraction Table Result of quantitation of apigenin in celery Apium graveolens L in fraction Sample Average SD Apigenin (%) 93.88 % 93.55 % 93.72 0.005 % CONCLUSIONS To quantify apigenin in celery samples, there are many methods such as HPLC, UPLC, etc We used the HPLC - PDA method with mobile phase solvent acetonitrile: 0.1 % CH3COOH solution (40:60), flow rate 1.0 mL/min, column temperature adjusted at 25 °C, wavelength at 335 nm, sample volume injected of 20 μL, and the retention time of 9.482 ± 0.056 The method achieves system compatibility, specificity, repeatability, trueness, linearity, low limit of detection and limit of quantitation The result of quantitation of apigenin in fraction Apium graveolens L collected in Duc Trong town, Lam Dong province, according to a built method is 93.72 % Therefore, this is a suitable procedure to quantify apigenin in samples of ingredients, semi-finished products, and products of celery products 316 Quantitative determination of apigenin from Apium graveolens L by HPLC-PDA method Acknowledgements The study was supported by Department of Science and Technology of Ho Chi Minh city (DOST), the decision number: “1301/QĐ-SKHCN” and the contract number: “123/2019/HĐQPTKHCN” CRediT authorship contribution statement LNTV: Project Administration, Methodology, Investigation, Writing-Original Draft Preparation; NCK: Conceptualization, Supervision, Funding acquisition; LTD: Conceptualization; NNT: Methodology, Validation; DTLH: Software; NVC: Investigation; NTTQ: Investigation; QTH: Formal analysis; NTP: Formal analysis Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper REFERENCES Lans C A - Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus, J Ethnobiol Ethnomed (45) (2006) Yarnell E - Botanical medicines for the urinary tract, World J Urol 20 (5) (2002) 28593 Khare C P - Indian medicinal plants: London, Springer Science Pub, 2008 Kooti W., Mansori E., Ghasemiboroon M Harizi M., Amirzargar A - Protective effects of celery (Apium graveolens) on testis and cauda epididymal spermatozoa in rat, Iran J Reprod Med 12 (5) (2014) 365-366 Mira Popovi Ç., Biljana Kaurinovi Ç., Svetlana Trivi Ç., Neda Mimica-Duki Ç., and Marija Bursa Ç - Effect of Celery (Apium graveolens) Extracts on Some Biochemical Parameters of Oxidative Stress in Mice Treated with Carbon Tetrachloride, Phytotherapy research, Phytother Res 20 (2006) 531-537 Atta A - Anti-nociceptive and antiinflammatory effects of some Jordanian medicinal plant extracts, J Ethnopharmacol 60 (2) (1998) 117-24 Zidorn C., Johrer K., Ganzera M., Schubert B., Sigmund E.M., Mader J., Greil R., Ellmerer E P., Stuppner H - Polyacetylenesfrom the apiaceae vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities, J Agric Food Chem 53 (7) (2005) 2518-23 Lans C A - Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus, J Ethnobiol Ethnomed (45) (2006) Teng C M., Lee L G., Ko S N - Inhibition of platelet aggregation by apigenin from Apium graveolens, Asia Pac J Pharmacol (85) (1985) 85-90 10 Al-Howiriny T., Alsheikh A., Alqasoumi S., Al-Yahya M., ElTahir K., Rafatullah S Gastric antiulcer, antisecretory and cytoprotective properties of celery in rats, Pharm Biol 48 (7) (2010) 786-793 11 Kooti W., Mansori E., Ghasemiboroon M Harizi M., Amirzargar A - Protective effects of celery (Apium graveolens) on testis and cauda epididymal spermatozoa in rat, Iran J Reprod Med 12 (5) (2014) 365-366 12 Głowacki Rafał; Furmaniak Paulina; Kubalczyk Paweł; Borowczyk Kamila Determination of Total Apigenin in Herbs by Micellar Electrokinetic Chromatography with UV Detection, Journal of Analytical Methods in Chemistry 2016 (2016) 1-8 317 Le Nguyen Tuong Vi, Le Tien Dung, Nguyen Cuu Khoa et al 13 Hong Cai, Delphine Raynaud, William P Steward, Andreas J Gescher - A simple HPLC method for the determination of apigenin in mouse tissues, Biomed Chromatogr 20 (10) (2006) 1038-1042 14 Shanmuga Sundaram Rajagopal, Babitha K Vazhayil, Liz Varghese, Mahadevan Nanjaian - Development and Validation of RP-HPLC Method for Simultaneous Determination of Apigenin and Luteolin in Ethanol Extract of Clerodendrum serratum (Linn.) Leaves, Asian Journal of Applied Sciences (2017) 52-60 15 Parabathina R K., Muralinath E., Swamy P L., Harikrishna V V S N., Sree K S - Role of certain flavonoids and vitamin-E against doxorubicin-induced oxiadative stress, J Chem Pharm Res (2011) 816- 834 16 Arora S., Vijay S., Kumar D - Phytochemical and antimicro-bial studies on the leaves of Spilanthes acmella, J Chem Pharm Res (2011) 145-150 17 VSP Chaturvedula, I Prakash - The aroma, taste, color and bioactive constituents of tea, J Chem Pharm Res (11) (2011) 2110-2124 18 Sung Bokyung, Chung Hae Young, Kim, Nam Deuk - Role of Apigenin in Cancer Prevention via the Induction of Apoptosis and Autophagy, Journal of Cancer Prevention 21 (4) (2016) 216-226 318 ... analysis by HPLC- PDA method d) Limit of detection (LOD), limit of quantitative (LOQ) 314 ( ⁄ ) ( ⁄ ) Quantitative determination of apigenin from Apium graveolens L by HPLC- PDA method SD: relative... is allowed to conclude that the compound is apigenin [15-17] 3.2 Quantification of apigenin in Apium graveolens L by using HPLC- PDA method 3.2.1 Quantification of apigenin by HPLC method Apigenin. .. 10 mL of methanol Aspirate mL dilute to 10 mL with methanol Sample preparation: Dissolve mg celery extract in 10 mL of methanol Aspirate mL dilute to 10 mL with methanol All samples were filtered