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A Sensitive Spectrofluorimetric Method for Curcumin Analysis

A Sensitive Spectrofluorimetric Method for Curcumin Analysis Curcumin (CUR), a natural polyphenolic compound extracted from the rhizomes of Curcuma longa, is used as a pharma- ceutical agent, spice in food, and as a dye. Currently, CUR is being investigated for cancer treatment in Phase-II clinical trials. CUR also possesses excellent activities like anti-inflammatory, anti-microbial, and anti-oxidant, therefore quality control is crucial. The present research work was to develop a new, simple, validated and time-saving rapid 96-well plate spectrofluorimetric method for the determination of CUR. The developed method was compared with routinely used high performance liquid chromatography (HPLC) technique. The developed method were found to be linear in the concentra- 2 2 tion range of 15 to 3900 ng/mL with R ≥ 0.9983 for spectrofluorimetric and 50-7500 ng/mL with R ≥ 0.9999 for HPLC method. Accuracy, intraday and interday precision was adequate, with RSD lower than the suggested limits. The limits for the detection and the quantification of CUR were 7 and 15 ng/mL for spectrofluorimetric, and 25 and 50 ng/mL for HPLC respectively. The Bland-Altman analysis demonstrated the similarities between the two methods. The 96-well plate method was successfully applied to determine CUR in solid lipid nanoparticles (SLNs) and chitosan nanoparticles (Chi- NPs). The developed spectrofluorimetric method can hence serve as a possible replacement for the HPLC method for the quantification of CUR in healthcare and food products. Keywords Curcumin · Spectrofluorimetric method · HPLC · Validation · Cost-effective · 96 well plates Introduction different shades.[3] Rhizomes of Curcuma longa are used as expectorants, antiseptics, blood purifiers, insecticides and Curcumin (CUR) is a yellow polyphenolic compound also used in the treatment of spleen disorders, rheumatism, extracted from the rhizomes of Curcuma longa, commonly bronchitis, cough and cold, hypotensive, cholera and syphi- known as turmeric. It is widely grown in India and other lis.[4–6] WHO and Food and Agriculture Organization have Asian countries for its use as a spice in cooking, dyes in approved CUR as a food additive.[7, 8] Since many years, textiles, food preservative and as a cosmetic agent for skin CUR has been utilized to treat jaundice, liver ailments and care.[1] For nutritional purposes, CUR is used in dairy several other medical purposes.[9] CUR has demonstrated products, beverages, cereals, mustard, food concentrations, chemopreventive properties in several kinds of cancer by sausages, pickles, ice cream, meat, fish, eggs, and bakery suppressing the tumorigenic activity of a wide variety of products.[2] CUR is also used as a coloring agent in food carcinogens. [10] industries, pharmacy, confectionery, and for dyeing wool, Presently, several phase II clinical trials are ongoing on silk, cotton and in combination with natural dyes to get CUR for the chemoprevention of cancer in humans. Clini- cal trials have indicated that the clinical safety of turmeric (CUR) has no dose-limiting toxicity up to 8–12 g/day in humans.[11–16] Apart from cancer, CUR plays a vital role Shaila A Lewis in treating various diseases which include skin, ulcers, s.lewis@manipal.edu parasitic infections, and auto-immune diseases.[9] Nutra- ceuticals like CUR can be a promising option as immunity Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher boosters and antidepressants for psychoneuroimmunol- Education (MAHE), 576104 Manipal, Karnataka, India ogy (PNI) response.[17] CUR has shown antiviral activity School of Pharmacy, Faculty of Health Sciences, University against a broad spectrum of viruses like HIV, HSV-2, HPV of Botswana, Gaborone, Botswana 1 3 1518 Journal of Fluorescence (2022) 32:1517–1527 viruses, influenza virus, Zika virus, hepatitis virus, adeno- intramolecular hydrogen transfer.[35] Recent studies done virus and is being investigated as a therapeutic option for on the estimation of concentration of CUR using fluorim - the management of COVID-19 infection.[18] CUR has the etry in liposomal formulations and plasma showed that the potential to be a promising tool for increasing immunity as method is reliable for the detection of CUR.[36] A major well as improving the psychological well-being of COVID challenge in the method is sample processing time, and the infected patients and healthcare workers.[17]. volume of solution required for analysis is more. Different analytical methods like UV-Spectrophotometry 96-well plates can be easily accommodated in all com- [19–21], ultra-performance liquid chromatography (UPLC) mon instruments like HPLC, GC, LC-MS and can be used [22], thin layer chromatography (TLC) [23], high perfor- for applications such as sample collection and biological mance liquid chromatography (HPLC) and LC-MS have assays.[37, 38] The advantages of 96-well plates are that been reported in literature for the estimation of concentra- sample processing is easy, requires fewer reagents as the tion of CUR in biological as well as pharmaceutical matri- well volume is small and requires fewer plates to run an ces.[24–27] UV-Spectrophotometry is easy to use and gives experiment because more samples can be placed within the extremely accurate readings, but it takes time to analyze plate when compared over conventional sample holders. each sample, and its sensitivity is often inadequate at low [39]. sample concentrations. The TLC is a chromatographic tech- The main challenge with CUR is its instability at neutral nique where compounds can be analyzed based on the polar- or basic pH and also its poor absorption when taken orally ity of samples. Although it is easy to handle, it is not widely or applied topically.[40] CUR exists in the forms of dik- recommended due to its poor resolution. HPLC is extremely etone and keto-enol and thereby has three acidic protons, quick and efficient with high resolution, accuracy, and high two phenolic ones and one enolic proton[41], Fig. 1. Now- reproducibility. Despite its advantages, HPLC is costly, a-days, many formulations of CUR such as liposomes, solid requires higher quantities of solvents. The UPLC reduces the lipid nanoparticles (SLNs), chitosan nanoparticles, micelles, cost of operation and decreases the consumption of solvent phospholipid complexes, hydrogels, powder solutions, when compared with the HPLC, but the disadvantage is that solid dispersions and nanoemulsions have been reported to increase in pressure reduces the life of columns.[28] Chro- enhance its bioavailability.[42–44] The methods reported matography coupled with mass spectroscopy has evolved as in the literature for the estimation of these formulations are a significant analytical technique in sample analysis. Inspite UV-spectrophotometry, fluorescence spectrophotometry, of its sensitivity, the cost and maintenance of the instrument HPLC, and LC-MS.[43, 45, 46]. make its usage limited. [29] However, tandem mass spec- To the best of our knowledge, there is no such spectroflu - trometry provides precise structural information about ana- orimetric method reported for the estimation of concentra- lytes at low concentrations (nano to pictogram/mL).[30–32] tion of CUR using a 96-well plate. Therefore, there is a need Additional limitations of the reported techniques include the to develop a spectrofluorimetric method for the accurate and inability to quantify lower CUR concentrations, the tedious precise estimation of CUR in pure form and in formulations. extraction process, and long sample analysis time. The aim of the present work was to estimate CUR in pure The spectrofluorimetric method is preferred over other form and formulations by developing a suitable spectrofluo - analytical techniques because of its ease of processing, rimetric method. ICH guidelines Q2 (R1) guidelines were time-saving and cost-efficient nature. CUR emits low quan- followed to validate the developed analytical method. [47, tum yield (< 0.2) fluorescence by absorbing in the visible 48] region. Its emission properties depend highly on the polar- ity of its environment.[33, 34] The exciting photophysi- cal properties of CUR are responsible for the excited-state Fig. 1 Structure of CUR 1 3 Journal of Fluorescence (2022) 32:1517–1527 1519 Materials and Instrumentation solutions (100 µg/mL) were prepared in methanol, ethanol, and DMSO and the FI was measured using spectrofluorim - Materials eter microplate reader. To determine the effect of solvent ratios on the FI, CUR (100 µg/mL) was analyzed with CUR was obtained as a gift sample from Arjuna Natural increased solvent ratios of ethanol and DMSO. FI can vary Pvt Ltd, Kerala, India. Sulfobutyl ether-β-CD was gifted depending on the sensitivity of the analyzing instrument. by CyDex Pharmaceuticals, USA. Glyceryl Monostearate The variation in the FI caused by the changes in the instru- (GMS) and Chitosan were procured from Sigma-Aldrich, ment sensitivity was recorded by analyzing the solutions at St. Louis. Tween 80 was purchased from Merck Life sci- different instrument sensitivity levels (35 and 40). ences Pvt Ltd, Mumbai. Poloxamer 407 was procured as gift sample from Signet excipients Pvt Ltd, Mumbai. Acetic HPLC Method acid, ethanol, methanol, dimethyl formamide (DMF), ace- tone, chloroform, ethyl acetate and acetonitrile and dimethyl The estimation of concentration of CUR by HPLC method sulfoxide (DMSO) were procured from Spectrochem Pvt. was performed using UV detector at a wavelength of Ltd, Mumbai and Finar Ltd, Ahmedabad. All chemicals 426 nm. The efficient elution was obtained with Acetoni - used in the study were of analytical grade. Purified water trile (ACN) and 0.1% Acetic acid (AA) of pH-3.5 in a ratio used was collected from the Millipore Milli-Q Plus system of 40:60 as the mobile phase. The flow rate and injection (Millipore, USA). volume were set at 1.2 mL/min and 20 µl. The column and oven temperature were maintained at 35°C. The retention Instrumentation time was 17 min. The fluorescence measurements were performed by using Methodology Corning® 96-well flat-bottom plates and a Biotek FLx800 Spectrofluorimetric plate reader. The data was processed on Construction of Calibration Curve for Spectrofluorimetric Gen5 Software. The excitation and emission wavelengths Method for estimation of CUR were set at 485 and 528 nm. [49]. CUR was analyzed using HPLC system with a UV detector Stock solution (1000 µg/mL) of CUR was prepared by dis- (Shimadzu corporation) and a kinetex C18 LC column with solving 1 mg of CUR in 1mL of DMSO. To prepare working particle size 5 μm and dimensions 250 × 4.6 mm. solutions (15–3900 ng/mL), the stock solution was diluted with DMSO in a 96-well plate. The relative fluorescence intensity (RFI) of the solution was measured at 528 nm Experimental emission after an excitation at 485 nm against a solvent blank. The obtained fluorescence intensity (FI) of the sam- Development of New Method and Optimization ple was subtracted from the blank solvent reading to obtain corrected FI. The FI versus concentration of the CUR (ng/ For the estimation of concentration of CUR, a spectrofluo- mL) was plotted to obtain the calibration graph and corre- rimetric method has been developed using 96-well plates. sponding regression equation was derived. CUR in the desired concentrations was dissolved in organic solvent. From this, 200 µL of solution was transferred into Construction of Calibration Curve for HPLC Method the well of microplate, where successive serial dilutions were prepared and scanned using spectrofluorimeter micro - The HPLC method was adopted from the literature with plate reader. The obtained fluorescence intensity (FI) of the slight modifications.[50] Aliquots of CUR working solu- sample was subtracted from the blank solvent reading to tions over the range of 50–7500 ng/mL were prepared in obtain corrected FI. vials using methanol as the solvent. The concentration of The solvents for the development of the spectrofluori- CUR (ng/mL) versus area was plotted to get the calibration metric method for the detection of CUR were selected based graph and regression equation. on the compatibility of the plates, the exhibited FI, and the applicable solvent ratios. The solvents ethanol, methanol, Method Validation DMSO, DMF, chloroform, acetone, ethyl acetate, and ace- tonitrile were screened to check the FI of CUR. The desired The developed analytical method was validated as per ICH solvents for CUR estimation were selected based on their guidelines Q2 (R1).[51] It must be noted that all validation compatibility with the 96-well plate. Additionally, CUR 1 3 1520 Journal of Fluorescence (2022) 32:1517–1527 parameters for HPLC was followed same as the new method, analyzing it at two different times in a day. The interday pre - except for robustness. cision was determined after 24 h and the results confirmed adequate reliability. Measurements were reported as the Specificity relative standard deviation (%RSD).[57, 58]. Specificity of spectrofluorimetric and HPLC method was Robustness determined by analyzing the blank nanoparticles and CUR loaded nanoparticles in order to assess the possible interfer- Robustness is the ability of the method to provide unchanged ence of the formulation excipients in the analysis. [36, 52, results with deliberate variations. Robustness was examined 53] by evaluating the influence of small variation of method variables, including two different 96-well plates, solvent Linearity from two different manufacturers and stability of CUR in the solvent at room temperature for 48 h and the %RSD was A range of samples where the analyte concentration was calculated.[57–59]. directly proportional to the amount of sample was deter- mined to test the ability of the analytical method.[54] Limit of Detection and Limit of Quantification Samples for linearity ranging from 15 to 3900 ng/mL for spectrofluorimetry and 50-7500 ng/mL for HPLC were pre - Limit of detection (LOD) is the lowest amount of analyte pared from stock solution (1000 µg/mL). Triplicates of each in the sample that can be detected by the instrument but standard solution were analyzed using spectrofluorimeter not quantified. Limit of quantification (LOQ) is the lowest and HPLC. The calibration curve was obtained by plot- amount of analyte in the sample that can be detected and ting the FI versus concentrations for spectrofluorimetry and quantitatively determined[60]. peak area versus concentration for HPLC. The coefficient of The LOD and LOQ of the CUR was determined by S/N determination (R ) was then determined from the calibra- ratio method. The experiment was performed by measur- tion curve. ing signal with low concentrations of analytes of CUR with those of blank. S/N ratio 3:1 and 10:1 was considered Accuracy acceptable for estimating the detection and quantification respectively.[51]. The accuracy of the method is the closeness of the mea- sured value to the true value for the sample. [55] The accu- Method Comparison Study racy of the method was determined by the standard addition method. The study was performed at three levels 50%, 100% Method comparison study was performed to assess the effi - and 150%. The recovery samples (n = 6) were prepared and ciency of newly developed spectrofluorimetric method cor - then analyzed at their respective wavelengths using spec- related with HPLC for the estimation of CUR. A total of 60 trofluorimeter and HPLC. The % recovery for pure CUR samples were analyzed using developed spectrofluorimetry from the calibration curve was calculated by using follow- as well as HPLC. The collected data was tabulated, and the ing equation: mean result was calculated. The two means were then com- pared with each other to determine % standard deviation. [61] The Bland-Altman test was used to assess the compa- Ct rability of the method using SPSS 16.0. A graph was con- %Accuracy = *100 (1.1) Ca structed with the absolute difference between the two paired measurements plotted against their mean value. Precision Application of the Method in the Estimation of CUR in Solid Lipid Nanoparticles (SLNs) and Chitosan NPs Precision is defined as the closeness of an individual sample (Chi-NPs) subjected to multiple sampling measurements under speci- fied conditions.[ 56] Reproducibility was also determined CUR loaded SLNs containing glyceryl mono stearate, through precision studies. Repeatability was determined Tween 80 and Poloxamer 407 were prepared by the micro- by preparing six replicates of sample concentrations and emulsion dilution method.[62, 63] Chi-NPs were prepared measuring their intensity and peak area. An intraday preci- using sulfobutyl ether-β-CD as a cross linking agent fol- sion study was carried out by preparing a drug solution and lowed by ionic gelation method.[45, 46, 64] The obtained 1 3 Journal of Fluorescence (2022) 32:1517–1527 1521 nanoformulations were characterized with respect to par- ticle size, zeta potential, and polydispersity index (NanoZS, Malvern instruments, UK). To determine the amount of CUR in SLNs and Chi-NPS, 1mL of nanoparticle disper- sion was treated with methanol and then centrifuged. The obtained supernatant of SLNs and Chi-NPs was collected and then injected in the HPLC. The amount of CUR was measured from the obtained peak area. For the spectrofluorimetric method, the SLNs and Chi- NPs were extracted with DMSO, centrifuged and the Fig. 2 Optimization of Solvents (A) Based on Fluorescence obtained supernatant was poured into the each well of intensity (B) Based on solvent ratios (E – Ethanol and D – microplate and then analyzed with spectrofluorimeter. The DMSO) and (C) Based on effect of detector sensitivity on amount of CUR present in the NPs was calculated from the fluorescence obtained FI. values (Fig. 2C). Hence, we selected sensitivity 40 for fur- Statistical Analysis ther method development. All the trials were conducted in triplicates and the results Method Validation are presented as mean ± standard deviation. The comparison of the new spectrofluorimetric method and the HPLC was Validation of the Spectrofluorimetric Method carried out using the Bland-Altman Plot. Validation of the spectrofluorimetric method was done for analytical parameters under optimized conditions. For spec- Results and Discussion ificity, there was no fluorescence intensity shown for blank nanoparticles with spectrofluorimetric method. [36] Speci- CUR exhibited native fluorescence in organic solvents. The ficity for HPLC method was determined by comparing the aim of the present research is to enhance the FI in order to chromatograms obtained for blank nanoparticles and CUR develop a highly sensitive analytical method for CUR anal- loaded nanoparticles. The chromatogram (Fig. 3), confirms ysis. So, various experimental parameters were investigated that the excipients in the nanoparticles did not interfere in including plate compatibility, different solvents, solvent the CUR peak obtained for CUR loaded nanoparticles, evi- ratio and different sensitivity. dencing the specificity of the method. [53] In a previous study, the linearity concentration range for estimation of Selection of Operating Conditions CUR using spectrofluorimetric analysis was 50-500ng/mL with %accuracy of 80, 100 and 120% showed percent recov- The desired solvents for CUR estimation were selected eries ranging from 96.33 to 100.75%. The precision results based on their compatibility with the 96-well plate. Only from the literature showed an RSD of less than 8% which methanol, ethanol, and DMSO were found to be compat- was more than the present developed method of < 5% [36]. ible with the plate material, whereas the remaining solvents The results obtained from the regression analysis of pres- interacted with the plate material, leading to the degrada- ent developed spectrofluorimetric method showed linearity tion. Even though CUR ethanolic solutions showed higher over the concentration range of 15–3900 ng/mL and its R FI compared to DMSO and methanol, as shown in Fig. 2A. was 0.9983, as shown in Table 1; Fig. 4. The percentage DMSO was selected as the solvent for analysis due to its higher solubility. The combination of both ethanol and DMSO in different ratios showed an increase in FI with an increased DMSO, but these obtained values were not higher than those obtained with DMSO alone (Fig. 2B). Because CUR dissolved in DMSO could give fluorescence even at low concentrations (below 10 ng/ml), we selected DMSO alone for CUR estimation. Upon dilution with DMSO, the solution was observed under the different sensitivities (35 Fig. 3 Representative chromatograms profiles obtained for (A) Blank nanoparticles (B) CUR loaded nanoparticles and 40). At sensitivity 40, the solution exhibited high FI 1 3 1522 Journal of Fluorescence (2022) 32:1517–1527 Table 1 Analytical parameters of the developed spectrofluorimetric and HPLC methods Method Linear equation Linear range R LOD LOQ Intra-day RSD (%) Inter-day RSD (%) (n = 3) (n = 3) Spectro-fluorimetry 0.9983 7 ng/mL 15 ng/mL 4.763 4.645 Y = 71.781x + 2.8617 Y = 15–3900 ng/mL HPLC 50-7500 ng/mL 0.9999 25 ng/mL 50 ng/mL 0.305 0.799 Y = 100.71x – 1984 Fig. 4 HPLC chromatogram of CUR (A) bismethoxycurcumin, (B) desmethoxycurcumin and (C) curcumin accuracy for the different concentration levels (50, 100 and 1.702–2.662% respectively. [57, 58] The LOD and LOQ for 150%) was found to be 94.17, 103.16 and 99.81%, confirm- developed spectrofluorimetric method were found to be 7 ing the accuracy of the suggested method for the estimation ng/mL and 15 ng/mL and for HPLC it was found to be 25 of CUR. The results are tabulated in Table 2. Precision was ng/mL and 50 ng/mL respectively. determined by assessing intra-day and inter-day relative standard deviation. Results shown in Table 1indicated that Validation of the HPLC method inter-day and intra-day variability is reasonable and %RSD values were lower than 5% which were in acceptable range. The chromatogram exhibited three peaks, with the major [36] The robustness of spectrofluorimetric method deter - peak referred to as CUR and two minor peaks identified as mined using two different 96-well plates, solvent from two demothoxycurcumin and bismethoxy curcumin, which are different manufacturers and stability of CUR in solvent shown in Fig. 4. [65] showed a %RSD of 2.874–4.957%, 1.449–2.991% and The developed HPLC method was found to be linear over the range of 50-7500 ng/mL. The calibration curve plotted was found to be linear with a regression of 0.9999 Table 2 Accuracy results of different levels of CUR using Spectrofluo - shown in Fig. 5. Intra-day and inter-day precision results for rimetry and HPLC the estimation of CUR are shown in Table 1. The developed Levels Spectrofluorimetry HPLC method was found to be precise, as intra-day and inter-day Recovery (%) RSD (%) Recovery RSD (%) (%) batches showed RSD < 2% for HPLC and < 5% for spectro- 50% 94.17 5.6 97.06 1.04 fluorimetric method. The percentage accuracy ranged from 100% 103.16 12.84 95.92 0.57 97.06 to 97.88% with an RSD of less than 4% shown in 150% 99.81 9.4 97.88 3.15 Table 2, indicating a low variability and close agreement 1 3 Journal of Fluorescence (2022) 32:1517–1527 1523 between the experimental and theoretical concentration val- The developed spectrofluorimetric and HPLC method ues.[53] LOD and LOQ values for the CUR were found to was applied for the determination of CUR in Chi-NPs be 25 ng/mL and 50 ng/mL. and SLNs. Spectrofluorimetric analysis was carried out in 96-well plates with clear bottom. Particle size and Method Comparison Studies zeta potential for SLNs were found to be 542.40 nm and + 53.50 mV respectively, while Chi-NPs were 537.10 nm The comparability of the method was assessed statistically and − 17.70 mV. The amount of CUR found in Chi-NPs and by the Bland-Altman test using SPSS 16.0.[61] The Bland- SLNs was 2.12 ± 0.182 mg/mL and 2.48 ± 0.12 mg/mL by Altman plot gives the difference between a pair of measure - the spectrofluorimetric method and 3.36 ± 0.227 mg/mL and ments made with the two methods with respect to the mean 3.17 ± 0.141 mg/mL by HPLC method. The results obtained of this pair of measurements. The values which fall within from both the methods are nearly matching with each other, the “limits of agreement” summarize how well the two which indicate that the spectrofluorimetric method can be methods of measurements matched. If the two methods pro- used for the estimation of CUR. vide similar results, then the difference between them will Spectrofluorimetric method is an attractive option for be minimal, with an average nearly zero, and the limits of the pharmaceutical analysis and it can also be used for food agreement will be zero.[66]. quality analysis because of its less time for sample prepa- The differences between spectrofluorimetric and HPLC ration, quick analysis, no complex procedure and no much method were plotted against the mean of the two measure- manpower required.[67]. ments. Any possible relation between measurement error and true value can be evaluated by plotting differences against mean. Conclusions The plotted graph in Fig. 5 showed mean ± SD of 1588.9 ± 1279.75 ng/mL. The average of mean difference The proposed spectrofluorimetric method provides an inex- is 9.316 units which is showed as a center line in Fig. 6. As pensive, rapid, specific, sensitive, precise, reliable and accu- the mean difference is not zero, it indicates that the spectro- rate method for the analysis of CUR using spectrofluorimeter fluorimetric method measures 9.316 units more than HPLC. plate reader. The high sensitivity and analysis speeds are The agreement limits are from − 49.3 to 67.5 ng/mL. This the substantial advantages of this method when compared clearly demonstrates the potential of using HPLC and FLU with other methods. This systematically developed method method for the estimation of CUR. meets all criteria required as per ICH guidelines. This work 4.4 Application of the method in the estimation of also describes a validated spectrofluorimetric method for the CUR in Solid lipid nanoparticles (SLNs) and Chitosan determination of the CUR. Hence, it may be applied for the NPs (Chi-NPs). routine analysis of CUR in pure form and nanoformulations. Fig. 5 Calibration curve of CUR using Spectrofluorimetric (A) and HPLC method (B) 1 3 1524 Journal of Fluorescence (2022) 32:1517–1527 Fig. 6 Bland-Altman plot of HPLC and FLU method Funding Indian Council of Medical Research-Senior Research Fel- lowship (ICMR-SRF), Government of India to Anne Boyina Sravani Hence, the spectrofluorimetric method can serve as a viable [File No. 3/2/2/15/2020/NCD-III] and Manipal College of Pharmaceu- alternative method to replace the reported methods and high tical Sciences (MCOPS) and Manipal Academy of Higher Education cost HPLC methods. (MAHE). Acknowledgements The authors are grateful to Mr. Manas Kinra, Open access funding provided by Manipal Academy of Higher Educa- Research Scholar, Department of Pharmacology, Manipal College tion, Manipal of Pharmaceutical Sciences, for his technial assistance and Mani- pal Academy of Higher Education for the facilities and infrastructure. Data Availability Not Applicable. Author Contribution Anne Boyina Sravani: Conceptualization, Meth- Code Availability Not Applicable. odology, Validation, Investigation, Data Curation, Writing – Original Draft; Elizabeth Mary Mathew: Resources, Investigation, Formal Analysis, Software, Writing – Review and Editing; Vivek Ghate: Re- Declarations sources, Methodology, Writing – Review and Editing; Shaila Lewis: Conceptualization, Data Curation, Writing – Review and Editing, Conflict of Interest The authors have declared no conflict of interest. Supervision, Project Administration, and Funding Acquisition. All au- thors read and approved the final manuscript. Ethics Approval Not applicable as the study does not include any use of animals and humans. 1 3 Journal of Fluorescence (2022) 32:1517–1527 1525 Consent of Participate Not Applicable. Complemenatry Ther 24:13–18. https://doi.org/10.1089/ act.2017.29147.yhk 14. Passildas-Jahanmohan J, Eymard JC, Pouget M, Kwiatkowski F, Consent for Publication Not Applicable. Van Praagh I, Savareux L, Atger M, Durando X, Abrial C, Rich- ard DGCA (2021) Multicenter randomized phase II study com- This article is licensed under a Creative Commons Attribution 4.0 Inter- paring docetaxel plus curcumin versus docetaxel plus placebo in national License, which permits use, sharing, adaptation, distribution first‐line treatment of metastatic castration‐resistant prostate can- and reproduction in any medium or format, as long as you give appro- cer. Cancer Med 10:2332–2340 priate credit to the original author(s) and the source, provide a link to 15. Li YH, Niu YB, Sun Y, Zhang F, Liu CX, Fan LMQ (2015) Phase the Creative Commons licence, and indicate if changes were made. II A Trial of Curcumin Among Patients With Prevalent Subclini- The images or other third party material in this article are included in cal Neoplastic Lesions (Aberrant Crypt Foci). 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A Sensitive Spectrofluorimetric Method for Curcumin Analysis

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Springer Journals
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Copyright © The Author(s) 2022
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1053-0509
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10.1007/s10895-022-02947-w
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Abstract

Curcumin (CUR), a natural polyphenolic compound extracted from the rhizomes of Curcuma longa, is used as a pharma- ceutical agent, spice in food, and as a dye. Currently, CUR is being investigated for cancer treatment in Phase-II clinical trials. CUR also possesses excellent activities like anti-inflammatory, anti-microbial, and anti-oxidant, therefore quality control is crucial. The present research work was to develop a new, simple, validated and time-saving rapid 96-well plate spectrofluorimetric method for the determination of CUR. The developed method was compared with routinely used high performance liquid chromatography (HPLC) technique. The developed method were found to be linear in the concentra- 2 2 tion range of 15 to 3900 ng/mL with R ≥ 0.9983 for spectrofluorimetric and 50-7500 ng/mL with R ≥ 0.9999 for HPLC method. Accuracy, intraday and interday precision was adequate, with RSD lower than the suggested limits. The limits for the detection and the quantification of CUR were 7 and 15 ng/mL for spectrofluorimetric, and 25 and 50 ng/mL for HPLC respectively. The Bland-Altman analysis demonstrated the similarities between the two methods. The 96-well plate method was successfully applied to determine CUR in solid lipid nanoparticles (SLNs) and chitosan nanoparticles (Chi- NPs). The developed spectrofluorimetric method can hence serve as a possible replacement for the HPLC method for the quantification of CUR in healthcare and food products. Keywords Curcumin · Spectrofluorimetric method · HPLC · Validation · Cost-effective · 96 well plates Introduction different shades.[3] Rhizomes of Curcuma longa are used as expectorants, antiseptics, blood purifiers, insecticides and Curcumin (CUR) is a yellow polyphenolic compound also used in the treatment of spleen disorders, rheumatism, extracted from the rhizomes of Curcuma longa, commonly bronchitis, cough and cold, hypotensive, cholera and syphi- known as turmeric. It is widely grown in India and other lis.[4–6] WHO and Food and Agriculture Organization have Asian countries for its use as a spice in cooking, dyes in approved CUR as a food additive.[7, 8] Since many years, textiles, food preservative and as a cosmetic agent for skin CUR has been utilized to treat jaundice, liver ailments and care.[1] For nutritional purposes, CUR is used in dairy several other medical purposes.[9] CUR has demonstrated products, beverages, cereals, mustard, food concentrations, chemopreventive properties in several kinds of cancer by sausages, pickles, ice cream, meat, fish, eggs, and bakery suppressing the tumorigenic activity of a wide variety of products.[2] CUR is also used as a coloring agent in food carcinogens. [10] industries, pharmacy, confectionery, and for dyeing wool, Presently, several phase II clinical trials are ongoing on silk, cotton and in combination with natural dyes to get CUR for the chemoprevention of cancer in humans. Clini- cal trials have indicated that the clinical safety of turmeric (CUR) has no dose-limiting toxicity up to 8–12 g/day in humans.[11–16] Apart from cancer, CUR plays a vital role Shaila A Lewis in treating various diseases which include skin, ulcers, s.lewis@manipal.edu parasitic infections, and auto-immune diseases.[9] Nutra- ceuticals like CUR can be a promising option as immunity Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher boosters and antidepressants for psychoneuroimmunol- Education (MAHE), 576104 Manipal, Karnataka, India ogy (PNI) response.[17] CUR has shown antiviral activity School of Pharmacy, Faculty of Health Sciences, University against a broad spectrum of viruses like HIV, HSV-2, HPV of Botswana, Gaborone, Botswana 1 3 1518 Journal of Fluorescence (2022) 32:1517–1527 viruses, influenza virus, Zika virus, hepatitis virus, adeno- intramolecular hydrogen transfer.[35] Recent studies done virus and is being investigated as a therapeutic option for on the estimation of concentration of CUR using fluorim - the management of COVID-19 infection.[18] CUR has the etry in liposomal formulations and plasma showed that the potential to be a promising tool for increasing immunity as method is reliable for the detection of CUR.[36] A major well as improving the psychological well-being of COVID challenge in the method is sample processing time, and the infected patients and healthcare workers.[17]. volume of solution required for analysis is more. Different analytical methods like UV-Spectrophotometry 96-well plates can be easily accommodated in all com- [19–21], ultra-performance liquid chromatography (UPLC) mon instruments like HPLC, GC, LC-MS and can be used [22], thin layer chromatography (TLC) [23], high perfor- for applications such as sample collection and biological mance liquid chromatography (HPLC) and LC-MS have assays.[37, 38] The advantages of 96-well plates are that been reported in literature for the estimation of concentra- sample processing is easy, requires fewer reagents as the tion of CUR in biological as well as pharmaceutical matri- well volume is small and requires fewer plates to run an ces.[24–27] UV-Spectrophotometry is easy to use and gives experiment because more samples can be placed within the extremely accurate readings, but it takes time to analyze plate when compared over conventional sample holders. each sample, and its sensitivity is often inadequate at low [39]. sample concentrations. The TLC is a chromatographic tech- The main challenge with CUR is its instability at neutral nique where compounds can be analyzed based on the polar- or basic pH and also its poor absorption when taken orally ity of samples. Although it is easy to handle, it is not widely or applied topically.[40] CUR exists in the forms of dik- recommended due to its poor resolution. HPLC is extremely etone and keto-enol and thereby has three acidic protons, quick and efficient with high resolution, accuracy, and high two phenolic ones and one enolic proton[41], Fig. 1. Now- reproducibility. Despite its advantages, HPLC is costly, a-days, many formulations of CUR such as liposomes, solid requires higher quantities of solvents. The UPLC reduces the lipid nanoparticles (SLNs), chitosan nanoparticles, micelles, cost of operation and decreases the consumption of solvent phospholipid complexes, hydrogels, powder solutions, when compared with the HPLC, but the disadvantage is that solid dispersions and nanoemulsions have been reported to increase in pressure reduces the life of columns.[28] Chro- enhance its bioavailability.[42–44] The methods reported matography coupled with mass spectroscopy has evolved as in the literature for the estimation of these formulations are a significant analytical technique in sample analysis. Inspite UV-spectrophotometry, fluorescence spectrophotometry, of its sensitivity, the cost and maintenance of the instrument HPLC, and LC-MS.[43, 45, 46]. make its usage limited. [29] However, tandem mass spec- To the best of our knowledge, there is no such spectroflu - trometry provides precise structural information about ana- orimetric method reported for the estimation of concentra- lytes at low concentrations (nano to pictogram/mL).[30–32] tion of CUR using a 96-well plate. Therefore, there is a need Additional limitations of the reported techniques include the to develop a spectrofluorimetric method for the accurate and inability to quantify lower CUR concentrations, the tedious precise estimation of CUR in pure form and in formulations. extraction process, and long sample analysis time. The aim of the present work was to estimate CUR in pure The spectrofluorimetric method is preferred over other form and formulations by developing a suitable spectrofluo - analytical techniques because of its ease of processing, rimetric method. ICH guidelines Q2 (R1) guidelines were time-saving and cost-efficient nature. CUR emits low quan- followed to validate the developed analytical method. [47, tum yield (< 0.2) fluorescence by absorbing in the visible 48] region. Its emission properties depend highly on the polar- ity of its environment.[33, 34] The exciting photophysi- cal properties of CUR are responsible for the excited-state Fig. 1 Structure of CUR 1 3 Journal of Fluorescence (2022) 32:1517–1527 1519 Materials and Instrumentation solutions (100 µg/mL) were prepared in methanol, ethanol, and DMSO and the FI was measured using spectrofluorim - Materials eter microplate reader. To determine the effect of solvent ratios on the FI, CUR (100 µg/mL) was analyzed with CUR was obtained as a gift sample from Arjuna Natural increased solvent ratios of ethanol and DMSO. FI can vary Pvt Ltd, Kerala, India. Sulfobutyl ether-β-CD was gifted depending on the sensitivity of the analyzing instrument. by CyDex Pharmaceuticals, USA. Glyceryl Monostearate The variation in the FI caused by the changes in the instru- (GMS) and Chitosan were procured from Sigma-Aldrich, ment sensitivity was recorded by analyzing the solutions at St. Louis. Tween 80 was purchased from Merck Life sci- different instrument sensitivity levels (35 and 40). ences Pvt Ltd, Mumbai. Poloxamer 407 was procured as gift sample from Signet excipients Pvt Ltd, Mumbai. Acetic HPLC Method acid, ethanol, methanol, dimethyl formamide (DMF), ace- tone, chloroform, ethyl acetate and acetonitrile and dimethyl The estimation of concentration of CUR by HPLC method sulfoxide (DMSO) were procured from Spectrochem Pvt. was performed using UV detector at a wavelength of Ltd, Mumbai and Finar Ltd, Ahmedabad. All chemicals 426 nm. The efficient elution was obtained with Acetoni - used in the study were of analytical grade. Purified water trile (ACN) and 0.1% Acetic acid (AA) of pH-3.5 in a ratio used was collected from the Millipore Milli-Q Plus system of 40:60 as the mobile phase. The flow rate and injection (Millipore, USA). volume were set at 1.2 mL/min and 20 µl. The column and oven temperature were maintained at 35°C. The retention Instrumentation time was 17 min. The fluorescence measurements were performed by using Methodology Corning® 96-well flat-bottom plates and a Biotek FLx800 Spectrofluorimetric plate reader. The data was processed on Construction of Calibration Curve for Spectrofluorimetric Gen5 Software. The excitation and emission wavelengths Method for estimation of CUR were set at 485 and 528 nm. [49]. CUR was analyzed using HPLC system with a UV detector Stock solution (1000 µg/mL) of CUR was prepared by dis- (Shimadzu corporation) and a kinetex C18 LC column with solving 1 mg of CUR in 1mL of DMSO. To prepare working particle size 5 μm and dimensions 250 × 4.6 mm. solutions (15–3900 ng/mL), the stock solution was diluted with DMSO in a 96-well plate. The relative fluorescence intensity (RFI) of the solution was measured at 528 nm Experimental emission after an excitation at 485 nm against a solvent blank. The obtained fluorescence intensity (FI) of the sam- Development of New Method and Optimization ple was subtracted from the blank solvent reading to obtain corrected FI. The FI versus concentration of the CUR (ng/ For the estimation of concentration of CUR, a spectrofluo- mL) was plotted to obtain the calibration graph and corre- rimetric method has been developed using 96-well plates. sponding regression equation was derived. CUR in the desired concentrations was dissolved in organic solvent. From this, 200 µL of solution was transferred into Construction of Calibration Curve for HPLC Method the well of microplate, where successive serial dilutions were prepared and scanned using spectrofluorimeter micro - The HPLC method was adopted from the literature with plate reader. The obtained fluorescence intensity (FI) of the slight modifications.[50] Aliquots of CUR working solu- sample was subtracted from the blank solvent reading to tions over the range of 50–7500 ng/mL were prepared in obtain corrected FI. vials using methanol as the solvent. The concentration of The solvents for the development of the spectrofluori- CUR (ng/mL) versus area was plotted to get the calibration metric method for the detection of CUR were selected based graph and regression equation. on the compatibility of the plates, the exhibited FI, and the applicable solvent ratios. The solvents ethanol, methanol, Method Validation DMSO, DMF, chloroform, acetone, ethyl acetate, and ace- tonitrile were screened to check the FI of CUR. The desired The developed analytical method was validated as per ICH solvents for CUR estimation were selected based on their guidelines Q2 (R1).[51] It must be noted that all validation compatibility with the 96-well plate. Additionally, CUR 1 3 1520 Journal of Fluorescence (2022) 32:1517–1527 parameters for HPLC was followed same as the new method, analyzing it at two different times in a day. The interday pre - except for robustness. cision was determined after 24 h and the results confirmed adequate reliability. Measurements were reported as the Specificity relative standard deviation (%RSD).[57, 58]. Specificity of spectrofluorimetric and HPLC method was Robustness determined by analyzing the blank nanoparticles and CUR loaded nanoparticles in order to assess the possible interfer- Robustness is the ability of the method to provide unchanged ence of the formulation excipients in the analysis. [36, 52, results with deliberate variations. Robustness was examined 53] by evaluating the influence of small variation of method variables, including two different 96-well plates, solvent Linearity from two different manufacturers and stability of CUR in the solvent at room temperature for 48 h and the %RSD was A range of samples where the analyte concentration was calculated.[57–59]. directly proportional to the amount of sample was deter- mined to test the ability of the analytical method.[54] Limit of Detection and Limit of Quantification Samples for linearity ranging from 15 to 3900 ng/mL for spectrofluorimetry and 50-7500 ng/mL for HPLC were pre - Limit of detection (LOD) is the lowest amount of analyte pared from stock solution (1000 µg/mL). Triplicates of each in the sample that can be detected by the instrument but standard solution were analyzed using spectrofluorimeter not quantified. Limit of quantification (LOQ) is the lowest and HPLC. The calibration curve was obtained by plot- amount of analyte in the sample that can be detected and ting the FI versus concentrations for spectrofluorimetry and quantitatively determined[60]. peak area versus concentration for HPLC. The coefficient of The LOD and LOQ of the CUR was determined by S/N determination (R ) was then determined from the calibra- ratio method. The experiment was performed by measur- tion curve. ing signal with low concentrations of analytes of CUR with those of blank. S/N ratio 3:1 and 10:1 was considered Accuracy acceptable for estimating the detection and quantification respectively.[51]. The accuracy of the method is the closeness of the mea- sured value to the true value for the sample. [55] The accu- Method Comparison Study racy of the method was determined by the standard addition method. The study was performed at three levels 50%, 100% Method comparison study was performed to assess the effi - and 150%. The recovery samples (n = 6) were prepared and ciency of newly developed spectrofluorimetric method cor - then analyzed at their respective wavelengths using spec- related with HPLC for the estimation of CUR. A total of 60 trofluorimeter and HPLC. The % recovery for pure CUR samples were analyzed using developed spectrofluorimetry from the calibration curve was calculated by using follow- as well as HPLC. The collected data was tabulated, and the ing equation: mean result was calculated. The two means were then com- pared with each other to determine % standard deviation. [61] The Bland-Altman test was used to assess the compa- Ct rability of the method using SPSS 16.0. A graph was con- %Accuracy = *100 (1.1) Ca structed with the absolute difference between the two paired measurements plotted against their mean value. Precision Application of the Method in the Estimation of CUR in Solid Lipid Nanoparticles (SLNs) and Chitosan NPs Precision is defined as the closeness of an individual sample (Chi-NPs) subjected to multiple sampling measurements under speci- fied conditions.[ 56] Reproducibility was also determined CUR loaded SLNs containing glyceryl mono stearate, through precision studies. Repeatability was determined Tween 80 and Poloxamer 407 were prepared by the micro- by preparing six replicates of sample concentrations and emulsion dilution method.[62, 63] Chi-NPs were prepared measuring their intensity and peak area. An intraday preci- using sulfobutyl ether-β-CD as a cross linking agent fol- sion study was carried out by preparing a drug solution and lowed by ionic gelation method.[45, 46, 64] The obtained 1 3 Journal of Fluorescence (2022) 32:1517–1527 1521 nanoformulations were characterized with respect to par- ticle size, zeta potential, and polydispersity index (NanoZS, Malvern instruments, UK). To determine the amount of CUR in SLNs and Chi-NPS, 1mL of nanoparticle disper- sion was treated with methanol and then centrifuged. The obtained supernatant of SLNs and Chi-NPs was collected and then injected in the HPLC. The amount of CUR was measured from the obtained peak area. For the spectrofluorimetric method, the SLNs and Chi- NPs were extracted with DMSO, centrifuged and the Fig. 2 Optimization of Solvents (A) Based on Fluorescence obtained supernatant was poured into the each well of intensity (B) Based on solvent ratios (E – Ethanol and D – microplate and then analyzed with spectrofluorimeter. The DMSO) and (C) Based on effect of detector sensitivity on amount of CUR present in the NPs was calculated from the fluorescence obtained FI. values (Fig. 2C). Hence, we selected sensitivity 40 for fur- Statistical Analysis ther method development. All the trials were conducted in triplicates and the results Method Validation are presented as mean ± standard deviation. The comparison of the new spectrofluorimetric method and the HPLC was Validation of the Spectrofluorimetric Method carried out using the Bland-Altman Plot. Validation of the spectrofluorimetric method was done for analytical parameters under optimized conditions. For spec- Results and Discussion ificity, there was no fluorescence intensity shown for blank nanoparticles with spectrofluorimetric method. [36] Speci- CUR exhibited native fluorescence in organic solvents. The ficity for HPLC method was determined by comparing the aim of the present research is to enhance the FI in order to chromatograms obtained for blank nanoparticles and CUR develop a highly sensitive analytical method for CUR anal- loaded nanoparticles. The chromatogram (Fig. 3), confirms ysis. So, various experimental parameters were investigated that the excipients in the nanoparticles did not interfere in including plate compatibility, different solvents, solvent the CUR peak obtained for CUR loaded nanoparticles, evi- ratio and different sensitivity. dencing the specificity of the method. [53] In a previous study, the linearity concentration range for estimation of Selection of Operating Conditions CUR using spectrofluorimetric analysis was 50-500ng/mL with %accuracy of 80, 100 and 120% showed percent recov- The desired solvents for CUR estimation were selected eries ranging from 96.33 to 100.75%. The precision results based on their compatibility with the 96-well plate. Only from the literature showed an RSD of less than 8% which methanol, ethanol, and DMSO were found to be compat- was more than the present developed method of < 5% [36]. ible with the plate material, whereas the remaining solvents The results obtained from the regression analysis of pres- interacted with the plate material, leading to the degrada- ent developed spectrofluorimetric method showed linearity tion. Even though CUR ethanolic solutions showed higher over the concentration range of 15–3900 ng/mL and its R FI compared to DMSO and methanol, as shown in Fig. 2A. was 0.9983, as shown in Table 1; Fig. 4. The percentage DMSO was selected as the solvent for analysis due to its higher solubility. The combination of both ethanol and DMSO in different ratios showed an increase in FI with an increased DMSO, but these obtained values were not higher than those obtained with DMSO alone (Fig. 2B). Because CUR dissolved in DMSO could give fluorescence even at low concentrations (below 10 ng/ml), we selected DMSO alone for CUR estimation. Upon dilution with DMSO, the solution was observed under the different sensitivities (35 Fig. 3 Representative chromatograms profiles obtained for (A) Blank nanoparticles (B) CUR loaded nanoparticles and 40). At sensitivity 40, the solution exhibited high FI 1 3 1522 Journal of Fluorescence (2022) 32:1517–1527 Table 1 Analytical parameters of the developed spectrofluorimetric and HPLC methods Method Linear equation Linear range R LOD LOQ Intra-day RSD (%) Inter-day RSD (%) (n = 3) (n = 3) Spectro-fluorimetry 0.9983 7 ng/mL 15 ng/mL 4.763 4.645 Y = 71.781x + 2.8617 Y = 15–3900 ng/mL HPLC 50-7500 ng/mL 0.9999 25 ng/mL 50 ng/mL 0.305 0.799 Y = 100.71x – 1984 Fig. 4 HPLC chromatogram of CUR (A) bismethoxycurcumin, (B) desmethoxycurcumin and (C) curcumin accuracy for the different concentration levels (50, 100 and 1.702–2.662% respectively. [57, 58] The LOD and LOQ for 150%) was found to be 94.17, 103.16 and 99.81%, confirm- developed spectrofluorimetric method were found to be 7 ing the accuracy of the suggested method for the estimation ng/mL and 15 ng/mL and for HPLC it was found to be 25 of CUR. The results are tabulated in Table 2. Precision was ng/mL and 50 ng/mL respectively. determined by assessing intra-day and inter-day relative standard deviation. Results shown in Table 1indicated that Validation of the HPLC method inter-day and intra-day variability is reasonable and %RSD values were lower than 5% which were in acceptable range. The chromatogram exhibited three peaks, with the major [36] The robustness of spectrofluorimetric method deter - peak referred to as CUR and two minor peaks identified as mined using two different 96-well plates, solvent from two demothoxycurcumin and bismethoxy curcumin, which are different manufacturers and stability of CUR in solvent shown in Fig. 4. [65] showed a %RSD of 2.874–4.957%, 1.449–2.991% and The developed HPLC method was found to be linear over the range of 50-7500 ng/mL. The calibration curve plotted was found to be linear with a regression of 0.9999 Table 2 Accuracy results of different levels of CUR using Spectrofluo - shown in Fig. 5. Intra-day and inter-day precision results for rimetry and HPLC the estimation of CUR are shown in Table 1. The developed Levels Spectrofluorimetry HPLC method was found to be precise, as intra-day and inter-day Recovery (%) RSD (%) Recovery RSD (%) (%) batches showed RSD < 2% for HPLC and < 5% for spectro- 50% 94.17 5.6 97.06 1.04 fluorimetric method. The percentage accuracy ranged from 100% 103.16 12.84 95.92 0.57 97.06 to 97.88% with an RSD of less than 4% shown in 150% 99.81 9.4 97.88 3.15 Table 2, indicating a low variability and close agreement 1 3 Journal of Fluorescence (2022) 32:1517–1527 1523 between the experimental and theoretical concentration val- The developed spectrofluorimetric and HPLC method ues.[53] LOD and LOQ values for the CUR were found to was applied for the determination of CUR in Chi-NPs be 25 ng/mL and 50 ng/mL. and SLNs. Spectrofluorimetric analysis was carried out in 96-well plates with clear bottom. Particle size and Method Comparison Studies zeta potential for SLNs were found to be 542.40 nm and + 53.50 mV respectively, while Chi-NPs were 537.10 nm The comparability of the method was assessed statistically and − 17.70 mV. The amount of CUR found in Chi-NPs and by the Bland-Altman test using SPSS 16.0.[61] The Bland- SLNs was 2.12 ± 0.182 mg/mL and 2.48 ± 0.12 mg/mL by Altman plot gives the difference between a pair of measure - the spectrofluorimetric method and 3.36 ± 0.227 mg/mL and ments made with the two methods with respect to the mean 3.17 ± 0.141 mg/mL by HPLC method. The results obtained of this pair of measurements. The values which fall within from both the methods are nearly matching with each other, the “limits of agreement” summarize how well the two which indicate that the spectrofluorimetric method can be methods of measurements matched. If the two methods pro- used for the estimation of CUR. vide similar results, then the difference between them will Spectrofluorimetric method is an attractive option for be minimal, with an average nearly zero, and the limits of the pharmaceutical analysis and it can also be used for food agreement will be zero.[66]. quality analysis because of its less time for sample prepa- The differences between spectrofluorimetric and HPLC ration, quick analysis, no complex procedure and no much method were plotted against the mean of the two measure- manpower required.[67]. ments. Any possible relation between measurement error and true value can be evaluated by plotting differences against mean. Conclusions The plotted graph in Fig. 5 showed mean ± SD of 1588.9 ± 1279.75 ng/mL. The average of mean difference The proposed spectrofluorimetric method provides an inex- is 9.316 units which is showed as a center line in Fig. 6. As pensive, rapid, specific, sensitive, precise, reliable and accu- the mean difference is not zero, it indicates that the spectro- rate method for the analysis of CUR using spectrofluorimeter fluorimetric method measures 9.316 units more than HPLC. plate reader. The high sensitivity and analysis speeds are The agreement limits are from − 49.3 to 67.5 ng/mL. This the substantial advantages of this method when compared clearly demonstrates the potential of using HPLC and FLU with other methods. This systematically developed method method for the estimation of CUR. meets all criteria required as per ICH guidelines. This work 4.4 Application of the method in the estimation of also describes a validated spectrofluorimetric method for the CUR in Solid lipid nanoparticles (SLNs) and Chitosan determination of the CUR. Hence, it may be applied for the NPs (Chi-NPs). routine analysis of CUR in pure form and nanoformulations. Fig. 5 Calibration curve of CUR using Spectrofluorimetric (A) and HPLC method (B) 1 3 1524 Journal of Fluorescence (2022) 32:1517–1527 Fig. 6 Bland-Altman plot of HPLC and FLU method Funding Indian Council of Medical Research-Senior Research Fel- lowship (ICMR-SRF), Government of India to Anne Boyina Sravani Hence, the spectrofluorimetric method can serve as a viable [File No. 3/2/2/15/2020/NCD-III] and Manipal College of Pharmaceu- alternative method to replace the reported methods and high tical Sciences (MCOPS) and Manipal Academy of Higher Education cost HPLC methods. (MAHE). Acknowledgements The authors are grateful to Mr. Manas Kinra, Open access funding provided by Manipal Academy of Higher Educa- Research Scholar, Department of Pharmacology, Manipal College tion, Manipal of Pharmaceutical Sciences, for his technial assistance and Mani- pal Academy of Higher Education for the facilities and infrastructure. Data Availability Not Applicable. Author Contribution Anne Boyina Sravani: Conceptualization, Meth- Code Availability Not Applicable. odology, Validation, Investigation, Data Curation, Writing – Original Draft; Elizabeth Mary Mathew: Resources, Investigation, Formal Analysis, Software, Writing – Review and Editing; Vivek Ghate: Re- Declarations sources, Methodology, Writing – Review and Editing; Shaila Lewis: Conceptualization, Data Curation, Writing – Review and Editing, Conflict of Interest The authors have declared no conflict of interest. Supervision, Project Administration, and Funding Acquisition. All au- thors read and approved the final manuscript. Ethics Approval Not applicable as the study does not include any use of animals and humans. 1 3 Journal of Fluorescence (2022) 32:1517–1527 1525 Consent of Participate Not Applicable. Complemenatry Ther 24:13–18. https://doi.org/10.1089/ act.2017.29147.yhk 14. Passildas-Jahanmohan J, Eymard JC, Pouget M, Kwiatkowski F, Consent for Publication Not Applicable. 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Journal of FluorescenceSpringer Journals

Published: Jul 1, 2022

Keywords: Curcumin; Spectrofluorimetric method; HPLC; Validation; Cost-effective; 96 well plates

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