The Study of Precision of ISO 3632-2 Method for Analysis of Herbal Medicines

Alex Nemtsov, Pavel Kirshkov

Department of Herbal Medicine, Nazarbayev University, Kazakhstan.

*Corresponding Author E-mail: alex.nemtsov@agrikavosh.ir

ABSTRACT:

Saffron is one of the most valuable medicinal herbs which application is getting more important in dietary supplements with approved medical indications. Therefore, the quality control method and the content of its API (Active Pharmaceutical Ingredient), namely, crocins and safranal, needs to be analyzed in a harmonized way. In current method of ISIRI 259-2:1391 and ISO 3632-2:2011 determination is based on specific absorption, E 1% 1cm, which is more a screening method than a reliable quantitative method to measure the secondary metabolites or active components in saffron. There are efforts for its artificial production or defraud as well. As saffron is considered one of the most strategic plants in Iran, and with regard to the growing market in pharma industry, Zardband has done intra-laboratory comparison on the assays obtained by methods of UV-Vis and HPLC-DAD. The reliable method for pharma industry concluded to be routine analysis using HPLC-DAD.

KEYWORDS: Crocin, HPLC-DAD, Picrocrocin, Saffron, Safranal.

INTRODUCTION:

The most popular spice which is able to provide the combination of color, taste, and aroma to the foods and beverages is saffron, the “Red Gold”. The stigmas of saffron contain more than 100 components but three main secondary metabolites include: crocins (the characteristic color of saffron); safranal (the characteristic odor of saffron), picrocrocin (the characteristic bitter taste of saffron) (1-5).

Saffron has received much attention by scientists in recent years regarding its therapeutic indications most of which are ascribed to crocins and safranal:

Antioxidant, improvement of mild and moderate depression, improvement of PMS, treatment of amenorrhea, age related macular degeneration, anti-obesity effect (weight loss promoter), prevention and treatment of cancer/anti-tumor, nervous system booster, aphrodisiac effect, antinociceptive/anti-inflammatory, cardiovascular and antiarteriosclerotic effect, digestion stimulant, skin whitening and radiating agent, UV absorbing agent (anti-solar) (6-10).

The quality of saffron is currently determined according to ISIRI 259:1391 (in Iran) and in international commercial agreements is determined according to the ISO 3632:2011, both of which classify saffron into three categories depending upon their physical and chemical characteristics. The three foremost parameters used to define the quality of saffron are color, taste, and aroma. These parameters are determined by UV−Vis (ISO 3632-2:2011), that is, the at 440nm (coloring strength), the at 257nm (the wavelength of picrocrocin maximum absorbance), and the at 330nm (the wavelength of safranal maximum absorbance). The problem is that: In pharmaceutical dosage it is very important to have accurate measurement of the effective marker. Since at 440nm many colorant can absorb the light and at 257nm and 330nm UV absorption do not give an accurate measurement of picrocrocin and safranal because the trans isomers of the crocetin esters also absorb at 250nm and the cis isomers of the crocetin esters absorb at 250 and 330nm as well, causing interference in the measurements, it will be impossible to calculate the precise safranal and crocins content in the product. The other important point in this determination is that there is no linear correlation between and HPLC measurement results and the third point is that most adulteration take place because of inaccurate test method of , so the need for establishment of new method in this industry seem to be unavoidable. Additionally, the ISO 3632-2:2011 does not classify saffron based on safranal content, the range values of safranal for the three different categories is the same; this means one of the main properties is not being valued. Saffron is only classified for its content of crocins and picrocrocin (11-15). In recent years there has been some publications in this regard mostly from Italian and Spanish authors. As the global production on a by-quantity basis is now dominated by Iran, which accounts for more than 90% of the annual harvest, it is a Must for us to be pioneer in standardization of new method and to establish new method as a regulation (16-20).

Therefore, the purpose of this work was to compare the value returned by different methods and validate a HPLC method for the independent determination of the crocetin esters, picrocrocin, and safranal using only a saffron solution prepared according to the ISO 3632:2011(21-25).

MATERIAL AND METHODS:

Samples and Reagents: Samples of saffron was purchased from Novin Saffron. The samples were of Category I according to ISO 3632:2011 Standards: Safranal with a purity of ≥88% was obtained from Sigma-Aldrich (Madrid, Spain), and crocins and picrocrocin, with a purity of ≥99%, were obtained from Medicinal Plants and Drugs Research Institute, Shahid Beheshti University. Solvents and Water and acetonitrile were obtained from Merck.

Saffron Extract Preparation: The saffron aqueous extracts were prepared according to ISO 3632:2011.

HPLC−DAD Analysis: A total of 20μL of each sample (saffron aqueous extracts) were injected into an Agilent 1200 HPLC chromatograph equipped with a 150 × 4.6 mm inner diameter, 5μm CNW C18 column. The mobile phase were gradient water (A) and acetonitrile (B) with the following ratio: 20% B, 0−5 min; 20− 80% B, 5−15 min; and 80% B, 15−20 min. The flow rate was 0.8 mL/min. The DAD detector was set at 250, 330, and 440 nm for picrocrocin, safranal, and crocins detection, respectively. All of the analyses were performed in duplicate, and two measurements were taken for each replicate.

UV-vis Analysis: The saffron samples were analysed using a Shimadzu UV-1800 spectrophotometer. The absorbance at 250, 330 and 440nm of the 1% aqueous solutions of saffron was valuated using a 1cm pathway quartz cell against double distilled water as blank.

The result reported once as specific absorption as (as defined in ISO 3632-2:2011) and once calculated against calibration curve according to standard solutions of crocins (2-13mg/L), picrocrocin (6-22mg/L) and safranal (2-13mg/L). The latter is defined as “modified ISO 3632-2:2011”.

RESULTS AND DISCUSSION:

Saffron samples, were analyzed by spectrophotometric analysis and HPLC-DAD in order to evaluate the quality of the saffron. The chromatogram of a typical saffron sample is shown in Fig. 1. The peak purity check shows that all peaks are pure. Given the importance of this issue in pharma industry, the HPLC-DAD is able to differentiate all three metabolites in one run analysis with no overestimation in comparison to UV-vis analysis(26-30).

Figure 1: Typical Chromatogram of saffron using HPLC-DAD method

The data reported in table 1 represents the comparison of saffron second metabolites content obtained by each method.

Table 1: Picrocrocin, safranal and crocins content of saffron determined by different methods.

Analyte	ISO 3632-2	Modified ISO 3632-2	HPLC-DAD (%)
icrocrocin	79.2	19.7%	4.7%
Safranal	31.5	4.4%	2.3%
Crocins	222.9	19.7%	8.7%

In both method requiring calibration curve (HPLC-DAD and modified ISO 3632-2:2011) good linearity were observed (R²>0.99).

It was shown previously that there is no correlation between the safranal content obtained by and HPLC-DAD (García-Rodríguez et al., 2017). The overestimation generated by cis-crocetin esters and other compounds which also absorbed at 330 and 440 nm, (It is crystal clear in Fig. 1 too)) may mislead pharmacist for selecting the right dosage(31-36).

CONCLUSIONS:

Although ISO 3632-2:2011 describes test methods for saffron, the assay method is somehow qualitative and does not return the content quantitatively. It is obvious that the quality of saffron for pharmaceutical application needs more precise method and it is an urge to establish new methods legally. The study shows that the chromatographic procedures are the most suitable to determine the secondary metabolites of saffron and also fraud detection (in case of synthetic dyes) because of peak impurities in UV-vis analysis. These analytical methods could be considered in order to evaluate saffron quality and therefore included in the ISIRI or ISO technical specifications. It is very important that Iran as the major producer of saffron stigmas in the world to take action and define pharmaceutical quality of saffron since the widespread use of saffron as supplement worldwide.

REFERENCES:

1. Seyed Mojtaba Mostafavi FP, Ahmad Rouhollahi, Mina Adibi, editor Electrochemical Study and Determination of Thiophene by Cobalt Oxide Nanoparticle Modified Glassy Carbon Electrode. 6th Aegean Analytical Chemistry Days (AACD), Denizli, Turkey; 2008.

2. Seyed Mojtaba Mostafavi SA, Iman Seyedi, Mohammad Hoseini. Excel for Engineers: Toranj Group Publication, Ltd.; 2009.

3. Seyed Mojtaba Mostafavi AR, Masoumeh Piryaei. Electrochemistry; principles, methods, and applications: Toranj Group Publication, Ltd.; 2009.

4. Seyed Mojtaba Mostafavi AM. Determination of molecular structure by identification of functional groups: Toranj Group Publication, Ltd., Isfahan, Iran; 2010.

5. Seyed Mojtaba Mostafavi OZ. Thermodynamics: an engineering approach: Mani Publication, Ltd, Isfahan, Iran; 2010.

6. Seyed Mojtaba Mostafavi BR. Nanomaterial Chemistry: Toranj Group Publication, Ltd.; 2010.

7. Omid Zabihi SMM, Fatemeh Ravari, Aminreza Khodabandeh, Amin Hooshafza, Karim Zare, Mehrab Shahizadeh. The effect of zinc oxide nanoparticles on thermo-physical properties of diglycidyl ether of bisphenol A/2, 2′-Diamino-1, 1′-binaphthalene nanocomposites. Thermochimica acta. 2011; 521(1-2): 49-58.

8. Seyed Mojtaba Mostafavi AR, Mina Adibi, Farshid Pashaee, Masoumeh Piryaei. Modification of Glassy Carbon Electrode by a Simple, Inexpensive and Fast Method Using an Ionic Liquid Based on Immidazolium as Working Electrode in Electrochemical Determination of Some Biological Compounds. Asian Journal of Chemistry. 2011; 23(12).

9. Seyed Mojtaba Mostafavi AR, Mina Adibi, Farshid Pashaee, Masoumeh Piryaei. Electrochemical Investigation of Thiophene on Glassy Carbon Electrode and Quantitative Determination of it in Simulated Oil Solution by Differential Pulse Voltammetry and Amperometry Techniques. Asian Journal of Chemistry. 2011;23(12): 5356-60.

10. Omid Zabihi AK, Seyed Mojtaba Mostafavi. Preparation, optimization and thermal characterization of a novel conductive thermoset nanocomposite containing polythiophene nanoparticles using dynamic thermal analysis. Polymer degradation and stability. 2012;97(1): 3-13.

11. Mojtaba Shamsipur AAMB, Mohammad Teymouri, Tahereh Poursaberi, Seyed Mojtaba Mostafavi, Parviz Soleimani, Fereshteh Chitsazian, Shahram Abolhassan Tash. Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6. Biodegradation. 2012;23(2): 311-8.

12. Seyed Mojtaba Mostafavi AR, Ali Akbar Miranbeigi. Handbook of Mineral Analysis: Mani Publication, Ltd, Isfahan, Iran; 2012.

13. Seyed Mojtaba Mostafavi MP, Ahmad Rouhollahi, Ali Mohajeri. Separation and Quantification of Hydrocarbons of LPG Using Novel MWCNT-Silica Gel Nanocomposite as Packed Column Adsorbent of Gas Chromatography. Journal of NanoAnalysis. 2014;1(01): 01.

14. Seyed Mojtaba Mostafavi MP, Ahmad Rouhollahi, Ali Mohajeri. Separation of Aromatic and Alcoholic Mixtures using Novel MWCNT-Silica Gel Nanocomposite as an Adsorbent in Gas Chromatography. Journal of NanoAnalysis. 2014;1(01):11.

15. Mostafavi SM. 3D Graphene Biocatalysts for Development of Enzymatic Biofuel Cells: A Short Review. Journal of Nanoanalysis. 2015;2(2): 57-62.

16. Sepideh Parvanian SMM, Meysam Aghashiri. Multifunctional Nanoparticle Developments in Cancer Diagnosis and Treatmen. Sensing and Bio-Sensing Research. 2016;1(2):22.

17. Mostafavi SM, editor Enhancement of mechanical performance of polymer nanocomposites using ZnO nanoparticles. 5th International Conference on Composites: Characterization, Fabrication and Application (CCFA-5); 2016: Iran University of Science and Technology.

18. Abolfazl Davoudiroknabadi SMM, Seyed Sajad Sajadikhah. An Introduction to Nanotechnology. Australia: Mikima Book Publication; 2016.

19. Abolfazl Davodiroknabadi SMM, Ali Asghar Pasban. Fundamentals of Nanostructure and Nanomaterial. 1 ed. Australia: Mikima Book Publication; 2016.

20. Asghar Pasban SMM, Hanieh Malekzadeh, Benyamin Mohammad Nazari, Quantitative Determination of LPG Hydrocarbons by Modified Packed Column Adsorbent of Gas Chromatography Via Full Factorial Design. Journal of Nanoanalysis. 2017;4(1):31-40.

21. Somayyeh Heidari MI, Seyed Mojtaba Mostafavi. A Validated and Rapid HPLC Method for Quantification of Human Serum Albumin in Interferon beta-1a Biopharmaceutical Formulation. MedBioTech Journal. 2017;1(01):29.

22. Seyed Mojtaba Mostafavi KB, Masoud Amanlou. A new attempt to introduce efficient inhibitors for Caspas-9 according to structure-based Pharmacophore Screening strategy and Molecular Dynamics Simulations. Medbiotech Journal. 2017;01(01):1-8.

23. Masoud Amanlou SMM. In sillico screening to aim computational efficient inhibitors of caspase-9 by ligand-based pharmacophore modeling. Medbiotech Journal. 2017;01(01):34-41.

24. Seyed Mojtaba Mostafavi AAP, Masoumeh Piryaei, Saeed Sadeghpour, Majid Masoumi, Ahmad Rouhollahi, Ali Akbar Miran Beigi. Acidity removal of Iranian heavy crude oils by nanofluid demulsifier: An experimental investigation. Journal of Nanoanalysis. 2017:10-7.

25. Aida Badamchi Shabestari BAA, Maryam Shekarchi, Seyed Mojtaba Mostafavi. Development of Environmental Analysis for Determination of Total Mercury in Fish Oil Pearls by Microwave Closed Vessels Digestion Coupled with ICP-OES. Ekoloji. 2018;27(106):1935.

26. Mahya Bayat SMM. Investigation of Interleukin 2 as Signaling Molecule in Human Serum Albumin. The Pharmaceutical and Chemical Journal. 2018;5(02):183-9.

27. Samira Eissazadeh SMM, Masoumeh Piryaei, Mohammad Sadegh Taskhiri. Application of Polyaniline Nanostructure Based Biosensor for Glucose and Cholesterol Detection. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2019;10(1):150.

28. Samira Eissazadeh MP, Mohammad Sadegh Taskhiri, Mostafavi SM. Improvement of Sensitivity of Antigen-Antibody Detection of Semen Through Gold Nanoparticle. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2019;10(1):144.

29. Samira Eissazadeh MP, Seyed Mojtaba Mostafavi. Measurement of Some Amino Acid Using Biosensors Based on Silicon-Based Carbon Nanotubes. Journal of Computational and Theoretical Nanoscience. 2019;16:1.

30. Aida Badamchi Shabestari SMM, Hanieh Malekzadeh. Force Degradation Comparative Study on Biosimilar Adalimumab and Humira. Revista Latinoamericana de Hipertensión. 2019;13(06):496-509.

31. Seyed Mojtaba Mostafavi SE, Masoumeh Piryaei. Comparison of Polymer and Ceramic Membrane in the Separation of Proteins in Aqueous Solution Through Liquid Chromatography. Journal of Computational and Theoretical Nanoscience. 2019;16(1):157-64.

32. Amir Ahmadipour PS, Seyed Abdalreza Mostafavi. Assessment of empirical methods for estimating potential evapotranspiration in Zabol Synoptic Station by REF-ET model. Medbiotech Journal. 2019;03(01):1-4.

33. Saeed Jafari SAM. Investigation of nitrogen contamination of important subterranean water in the plain. Medbiotech Journal. 2019;03(01):10-2.

34. Z. Man AGE, Seyed Mojtaba Mostafavi, A. Surendar. Fuel oil characteristics and applications: economic and technological aspects. Petroleum Science and Technology. 2019;37(09):1041-4.

35. Seyed Mojtaba Mostafavi HM, Mohammad Sadegh Taskhiri. In Silico Prediction of Gas Chromatographic Retention Time of Some Organic Compounds on the Modified Carbon Nanotube Capillary Column. Journal of Computational and Theoretical Nanoscience. 2019;16(01):151-6.

36. Seyed Mojtaba Mostafavi AE. Mercury determination in work place air and human biological samples based on dispersive liquid-liquid micro-extraction coupled with cold vapor atomic absorption spectrometry. Analytical Methods in Environmental Chemistry Journal. 2019;2(04):49-58.

37. Niraj Vyas, Sangita Panchal. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Nebivolol and Indapamide in Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 98-102.

38. Gandla. Kumara Swamy, N. Ravindra, S. Sowmya. A New RP-HPLC Method Development and Validation for the Simultaneous Estimation of Amlodipine and Valsartan in Tablet Dosage Forms. Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 103-107.

39. Gandla. Kumaraswamy, N. Ravindra, B. Jyothsna. Development and Validation of a Reversed-Phase HPLC Method for Simultaneous Determination of Aspirin, Atenolol and Amlodipine in Capsules Dosage Forms. Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 116-120.

40. D. Narmada, P.V. Murali Krishna, Shaik Mohammad Yusuf, B. Ranganayakulu, K. Uday Praveen, P. Raja Abhilash. RP-HPLC method development and validation for estimation of Glibenclamide in tablet dosage form. Asian J. Pharm. Ana. 4(3): July-Sept 2014; Page 125-128.

41. Gandla Kumaraswamy, Gandla Lalitha, N. Ravindra, K.B. Pradeesha. Simultaneous Estimation of Amlodipine, Atenolol and Hydrochlorothiazide in Bulk and Tablet Dosage Form by RP-HPLC Method. Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 131-136.

42. CH. Naveen Kumar, N. Kannappan. Development and validation of Bioanalytical Method for Quantification of phenytoin in rat brain tissues as per ICH guidelines. Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 147-150.

43. G. Kumaraswamy, M.A. Zeeshan Hamza, R. Suthakaran. Development and Validation of RP-HPLC for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium Tablets. Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 151-155.

44. Mukund Nagarnaik, Anil Dhakulkar, Arun Sarjoshi, Girish Pandya. A Study of Validation and Uncertainty in real samples of Nicotine Polacrilex Gum by Reverse Phase HPLC. Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 156-161.

45. Sufiyan Ahmad, Ansari Sajjad, Md. Rageeb Md. Usman, Mohammed Imran, Rashid Akhtar. Development and Validation of RP- HPLC Method for Simultaneous Estimation of Metformin and Miglitol in Bulk and Dosage Form. Asian J. Pharm. Res. 2017; 7(3): 139-147.

46. Kavita wagh, Sandeep Sonawane, Santosh Chhaajed, Sanjay Kshirsagar. Development of RP-HPLC method for separation of atorvastatin calcium, amlodipine besylate and azilsartan medoxomil and its application to analyze their tablet dosage forms. Asian J. Pharm. Res. 2017; 7(3): 148-154.

Received on 31.05.2020 Modified on 20.06.2020

Asian J. Pharm. Ana. 2020; 10(3):125-128.

DOI: 10.5958/2231-5675.2020.00022.8