- Email: [email protected]
Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form
Accepted Manuscript Title: Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form Author: Sharad Kharat Ajay Namdeo Piyush Mehta PII: DOI: Reference:
S1658-3655(16)30083-8 http://dx.doi.org/doi:10.1016/j.jtusci.2016.10.004 JTUSCI 343
To appear in: Received date: Revised date: Accepted date:
31-3-2016 7-9-2016 2-10-2016
Please cite this article as: S. Kharat, A. Namdeo, P. Mehta, Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form., Journal of Taibah University for Science (2016), http://dx.doi.org/10.1016/j.jtusci.2016.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form.
Sharad Kharat1†, Ajay Namdeo2, Piyush Mehta1
Dept. of Quality assurance, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune-38.
2
Dept. of Pharmacogonasy, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune-38.
us
Ac ce pt e
Abstract
d
M
an
†Corresponding author: Mr. Sharad Kharat Department of Quality assurance, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Kothrud, Pune 411038, Maharashtra, India. Email: [email protected]
cr
ip t
1
A simple and precise HPTLC methods were developed for the simultaneous estimation of the two anti-inflammatory drugs (Curcumin and Galangin). The method was tailored to analyze both drugs in their commercial dosage form (capsules) with no interference from ingredients. Chromatographic separation was performed over precoated TLC plates (60 F254, 20 × 10 cm, 250 µm thickness, Merck, Darmstadt, Germany) through linear ascending technique using nHexane: Ethyl acetate: Acetic acid: Methanol as a mobile phase. Detection and quantification was achieved at 404 nm through spectro-densitometric analysis. Analytical performance of the proposed HPTLC method was validated according to the ICH guidelines with respect to linearity, accuracy, precision, detection and quantitation limits, robustness and specificity. Calibration curves were linear in the limits of 80-450 and 200-1200 ng/spot for CU and GA, respectively with correlation coefficients (r2) >0.9998. The limits of detection were 18.31
Page 1 of 18
and 40.50 ng/spot for CU and GA respectively. The validated HPTLC method was successfully applied to the simultaneous determination of CU and GA in commercial polyherbal formulation.
ip t
Keywords- Curcumin, Galangin, HPTLC, Validation and Polyherbal formulation.
cr
1. Introduction
us
Natural products contains wide range of active chemical compounds including flavonoids, glycosides, triterpenoids, lipids, oils, steroids and organic acids which may be accountable for
an
its widespread pharmacological activity. The huge variety in chemical compounds pose a
precision and reproducibility [1,2].
M
challenge to analyze, authenticate and separate quickly with acceptable accuracy and
d
Various sophisticated analytical techniques are describe in the literature for analyze,
Ac ce pt e
authenticate and separate very complex chemical mixtures. Each technique has its own restrictions. For example, in liquid chromatography (LC), it is a complex function to change over from one mobile phase to another due to challenge of high back pressure and miscibility [3]. Chromatographic fingerprint investigation has proved to be a realistic and practical method for the quality assessment and species authentication of various traditional medicine. This chromatographic fingerprint of natural product can then be used to decide presence or absence of markers of concern and also the ratio of all detectable compounds. Even though high performance thin layer chromatography (HPTLC) has a few restrictions, such as the lower plate efficiency and narrow developing distance by comparison with GC and HPLC, it is still an valuable tool for quality assessment of natural products due to its ease, low cost and few requirement, and it has been profitably utilized to develop the chromatographic
Page 2 of 18
fingerprint for various natural products herbal drugs and commercial herbal formulations. Moreover, the colourful pattern and quantification at micron and nanogram levels help to differentiate various sample on the same plate [4,5,6].
ip t
Curcumin (CU) is well known polyphenolic moiety present in the rhizomes of the Curcuma longa Linn. belonging to family Zingiberaceae. (Fig. 1) It has a wide range of
cr
pharmacological and biological actions. This compound shows various therapeutic actions
us
including antioxidant, hepatoprotective, antirheumatic, antibacterial, antiprotozoal, antiinflammatory, anti-cancer, anti-HIV, neuro-protection and also active against several
an
malignancies. Additionally, safety of CU at very high doses has been verified in numerous human and animal investigation [7,8,9]. Numerous reports are found in the scientific
M
literature for the determination of CU, including UV spectrophotometer [10], HPTLC [11,12], high performance liquid chromatography (HPLC) [13,14] and LC-MS/MS [15] for
d
quantitative determination of CU in pharmaceutical dosage form, bulk drug and plasma.
Ac ce pt e
Moreover, some fluorimetric and NMR methods are also have been used for the rapid determination and quantitation of CU [16,17].
Galangin (GA) is a 3,5,7-trihydroxyflavone isolated from rhizomes of the Alpinia galanga belonging to family Zingiberaceae. (Fig. 2) Plant is commonly known as galangal. It is commonly known for its anti-inflammatory, antipyretic, abortifacient, aphrodisiac, carminative and emmenagogue properties. It is used in the treatment of various diseases such as heart diseases, kidney disorders, rheumatism, bronchitis, chronic enteritis and renal calculus. GA is also well known phytoconstituent of propolis. It shows good antibacterial activity against both gram positive and negative bacteria [18,19,20]. The identification and quantification of GA in various extract, in biological samples and formulations was addressed
Page 3 of 18
in some reports. Analytical methodology in these scientific reports involved the use of HPTLC-densitometric analysis [21], HPLC-UV [22], a reversed phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD) [23] and LC-MS/MS [24].
ip t
To the best of our information, no efforts have yet been made to examine this drug combination by any HPTLC method. The objective of present investigation is the
cr
development of a simple, precise and reproducible HPTLC method for the simultaneous
us
estimation of CU and GA in pure form and in commercial marketed formulation. As method validation is an essential constraint in analytical method development, the presented method
an
has been validated following the guidelines of the ICH.
2.1 Chemicals and reagents
M
2. Experimental
d
Standard curcumin and galangin were purchased from Natural Remedies Pvt. Ltd.,
Ac ce pt e
Bangalore, India. Herbal drug formulation (Antarth, Millenium Herbal Care Pvt. Ltd., Pune, India) used in this investigation was obtained from the local market. All other chemicals and reagents were of analytical grade.
2.2 Chromatographic conditions
The samples were spotted in the form of bands (6 mm width) with a Hamilton microliter syringe (100 µl) under controlled nitrogen stream using a Cammag Linomat V sample applicator (Switzerland). The slit dimension was kept at 5 × 0.45 mm and scanning speed 10 mm/s. Precoated TLC silica gel aluminum plates 60 F254 (20 × 10 cm, 250 µm thickness, Merck, Darmstadt, Germany) were utilized. Precoated TLC were pre-washed with methanol and activated at 110°C for 5 min prior to the sample application. Method development was
Page 4 of 18
executed in a Cammag twin trough glass chamber (20 × 20 cm) saturated with the mobile phase. The 10 ml mobile phase was composed of n-Hexane: Ethyl acetate: Acetic acid: Methanol (7:2:0.5:0.5 v/v/v). Prior to study, chamber was saturated with mobile phase for 30 min at room temperature (25 ± 5 °C) at relative humidity of 60 ± 5 %. Densitometric
ip t
scanning was achieved over Cammag TLC scanner III operated by win CATS software (V 1.44 CAMAG). The source of radiation used was UV spectrophotometer. The obtained bands
us
cr
were scanned at wavelength of 404 nm.
an
2.3 Preparation of Solutions
2.3.1 Standard Stock Solutions
M
A standard stock solutions of 1000 µg/mL was prepared by dissolving 10 mg of each marker in 10 ml of volumetric flask containing HPLC grade methanol. Prepared solution was further
d
diluted with HPLC grade methanol to obtained final concentrations of 100 µg/mL. These
Ac ce pt e
stock solutions were used for further studies.
2.3.2 Sample Stock Solution
A total of 5 capsule were weighed and powdered. To an accurately weighed quantity of the powder equivalent to 8.3 mg of CU and 10 mg of GA, methanol were added, sonicated for 15 min then filtered through whatman filter paper no.42 into a 10-mL volumetric flask.
3. Validation of the method The proposed analytical method was validated as per the International Conference on Harmonization (ICH) guidelines Q2 (R1) [25].
Page 5 of 18
3.1 Linearity Prepared standard stock solution was diluted to obtained linearity standard solutions containing CU and GA in the concentration range of 80-450 ng/spot and 200-1200 ng/spot, respectively. Three sets of such solutions were equipped. Every set was analyzed to obtained
ip t
a calibration curve. Standard deviation (SD), coefficient of determination (r2), slope and
cr
intercept of the calibration curves were estimated to determine linearity of the method.
us
3.2 Precision
The present method was validated for intraday and interday precision. Intraday precision was
an
determined by replicate the same method three times on the same day for three different concentrations of CU (100, 250 and 400 ng/spot) and GA (300, 700 and 1100 ng/spot). The
M
interday precision of the method was verify by performing similar method on different days under the same set of experimental situation. Repeatability of sample application and
Ac ce pt e
d
calculation of peak area for analyte were articulated in terms of % RSD.
3.3 Robustness
To determine the robustness of the present method, small changes in the chromatographic conditions were intentionally made, which may affect the performance of the method such as TLC plate development and developing distance, mobile-phase composition and chamber saturation time.
3.4 Specificity To verify specificity of the method, CU and GA standard and sample solution were applied to a HPTLC plate and the plate was developed and scanned as described above. The peak purity
Page 6 of 18
of CU and GA was measure by evaluating the spectra of marker compounds at peak apex, peak start and peak end positions of the spot.
3.5 Accuracy
ip t
Accuracy was estimated through the % recoveries of known quantity of combination of CU and GA added to solutions of marketed preparation. Marketed preparation was spiked with
cr
the known quantity of standard markers and the % ratios between the recovered and expected
us
concentrations were calculated. The analyzed samples were spiked with 80, 100 and 120 % of
an
500 ng/spot of both CU and GA. Accuracy was calculated from the following equation,
M
Spiked concertation − Mean concentration × 100 Spiked concertration
d
3.6 Limit of detection and Limit of quantitation
Ac ce pt e
The limit of detection (LOD) is the lowest amount of analyte that can be detected in a sample, but not necessarily quantified, under the stated experimental conditions. The limit of quantification (LOQ) was identified as the lowest amount of analyte that can be detected and quantified with acceptable accuracy, precision, and variability. LOD and LOQ are calculated by the signal to noise method.
4. Analysis of marketed product
To determine the content of CU and GA in capsule, to an accurately weighed quantity of the formulation equivalent to 8.33 mg of CU and 10 mg of GA, 7 mL methanol were added, sonicated for 15 min. The volume of resulting solution was made up to 10 mL with methanol. The % assay was determined. The possibility of chemical constituent’s interference with analysis was examined.
Page 7 of 18
5. Results and discussion Chromatographic separation were carried out on the standard solutions of CU and GA. Briefly, spots of standard samples were applied on the TLC plates. TLC plates were
ip t
developed by linear ascending development using various solvents like acetone, benzene, chloroform, ethyl acetate, methanol and toluene. Based on the observations of these primary
cr
trials, various binary, ternary and quaternary mixtures of solvents were tried to accomplish
us
optimum resolution between the CU and GA in their respective solvent mixtures under study. Initially, n-hexane and ethyl acetate in several ratios were attempted but inadequate
an
separation between CU and GA occurred even upon the addition of small quantity of acetic acid to the mobile phase. In these earlier examination (8:1.5:0.5), CU was clearly separated
M
however GA was not satisfactorily resolved. Again, addition of small quantity of acetic acid to the above mentioned mobile phase did not improve the separation. Conversely, addition of
d
methanol to the above mentioned mobile phase produced considerable perfection in both
Ac ce pt e
peak characteristics and separation. The optimized mobile phase composed of n-Hexane: Ethyl acetate: Acetic acid: Methanol (7:2:0.5:0.5 v/v/v) showed satisfactory resolution. Densitometric measurements were achieved with Cammag TLC scanner III operated by win CATS software (V 1.44 CAMAG). At wavelength of 404 nm (Fig. 3), optimized mobile phase showed highest sensitivity for quantification of both drugs.
Other chromatographic conditions like sample application rate and volume, sample application positions, chamber saturation time, total run length and distance between tracks were also optimized to give accurate, precise and reproducible Rf values, symmetrical peak shape and better resolution for the both drugs. The optimized chamber saturation time for the given mobile phase was found as 30 min at room temperature (25 + 5 °C) at relative humidity
Page 8 of 18
of 60 + 5 %. The total run length of the chromatogram run was approximately 80 mm. The distance between two tracks was 10 mm. The peak shape was more symmetrical and spots appeared more compact when the TLC plates were pre-treated with methanol and activated at 110°C for 5 min. After run the TLC plates were dried in air for 5-6 min. The slit dimension
ip t
and scanning speed was 5 × 0.45 mm and 10 mm/s respectively. The optimized
cr
chromatographic conditions are mentioned in Table 1.
us
5.1 Validation of the proposed method
Validation of the proposed methods was carried out in accordance with the International
M
5.1.1 Linearity
an
Conference on Harmonization (ICH) guidelines (2005).
The linearity of the presented HPTLC method was estimated by analyzing a series of
d
different concentrations for each of the both analytes. Under the optimized state mentioned
Ac ce pt e
above, the calculated peak areas at 404 nm were found to be proportional to concentrations of CU and GA respectively. Table 1 denotes the statistical parameters including linearity ranges, correlation coefficients, slopes, intercepts and % RSD of the slopes. Linearity graph of CU and GA was shown in Fig. 3 and 4 respectively.
5.1.2 Precision
The both intra-day and inter-day repeatability of the presented method was calculated for each drug. The outcomes of intra-day and inter-day repeatability are shown in Table 2. It shows % RSD which did not exceed 1 % for CU and GA representing the high level of precision of the given method.
Page 9 of 18
5.1.3 Robustness The parameters of the optimized methods were intentionally varied. Each factor selected to examine were charged at three levels (-1, 0 and 1). One time one factor was subjected to change and change in the responses of the both drugs were denoted. The summary of
ip t
robustness study was given in Table 3. The values of % RSD of the peak areas of both CU and GA under the analysis did not surpass 0.5 %. The satisfactory low values of % RSD of
us
cr
peak areas and unaffected Rf values suggested the robustness of the proposed method.
5.1.4 Specificity
an
The peak purity of CU and GA was assessed by comparing their respective spectra at peak apex, peak start and end positions of the peak. A good correlation (r2 value more than 0.999)
M
was acquired for both drugs. Good correlation values and satisfactory peak purity suggest no
Ac ce pt e
method is specific.
d
interference in the quantification of the CU and GA in sample solutions. This verifies that the
5.1.5 Accuracy
Results from accuracy studies, denoted in Table 4, were in acceptable range (94.68- 99.91 %), indicating the recovery of proposed method was good. Additionally, Low % RSD value proved the ruggedness of proposed method.
5.1.6 Limit of detection and Limit of quantitation The limit of detection (LOD) value for CU and GA were found to 18.31and 40.50 ng and limit of quantification (LOQ) value were 55.50 and 122.74 ng, respectively which shows the adequate sensitivity of the method (Table 1).
Page 10 of 18
5.2 Analysis of marketed product The developed HPTLC method was used for the assay of commercial formulation viz. Antarth®. The both drugs eluted at their specific Rf values. No interfering peaks were observed from any of the inactive ingredients. The % recovery and % RSD was given in
ip t
Table 5. It is clear from these observations that the proposed method is applicable for the analysis of the CU and GA in their combined commercial formulations with accuracy and
us
cr
precision (Fig. 6).
6. Conclusion
an
Present study illustrate a simple, accurate and precise HPTLC method for the simultaneous estimation of CU and GA. Additionally, the proposed method does not necessitate complex
M
treatment or sophisticated analytical unit usually associated with HPLC and LC-MS/MS procedures of analysis. Reliability was ascertain by testing a range of validation parameters
d
of the proposed method. At last, the proposed method was found sensitive and specific;
Ac ce pt e
hence, it can be suggested for the routine analysis of the cited phytochemicals either in bulk form or in their combined dosage forms.
7. References
1. Jyotshna, P. Srivastava, B. Killadi, K. Shanker, Uni-dimensional double development HPTLC-densitometry method for simultaneous analysis of mangiferin and lupeol content in mango (Mangifera indica) pulp and peel during storage, Food Chem. 176 (2015) 91-98.
Page 11 of 18
2. P. Mehta, R. Shah, L. Sathiyanarayanan, K. Mahadik, Pharmacokinetic profile of phytoconstituent(s) isolated from medicinal plants-a comprehensive review, J Tradit Complement Med. 5 (2015) 207-227.
ip t
3. A. Petruczynik, M. Waksmundzka-Hajnos, T. Plech, T. Tuzimski, M. Hajnos, G. Jozwiak, M. Gadzikowska, A. Rompala, TLC of alkaloids on cyanopropyl bonded stationary phases.
us
cr
Part II. Connection with RP18 and silica plates, J Chromatogr Sci. 46 (2008) 291-297.
4. D. Rathee, S. Rathee, P. Rathee, A. Deep, S. Anandjiwala, D. Rathe, HPTLC densitometric
an
quantification of stigmasterol and lupeol from Ficus religiosa, Arabian journal of chemistry.
M
8 (2015) 366-371.
5. A. Kamboj, A. Saluja, Development of validated HPTLC method for quantification of
d
stigmasterol from leaf and stem of Bryophyllum pinnatum, Arabian journal of chemistry.
Ac ce pt e
http://dx.doi.org/10.1016/j.arabjc.2013.10.006
6. Z. Sheikh, S. Shakeel, S. Gul, A. Zahoor, S. Khan, F. Zaidi, K. Usmanghani, A novel HPTLC method for quantitative estimation of biomarkers in polyherbal formulation, Asian Pac J Trop Biomed. 5 (2015) 955-959.
7. S. Sweetman, Martindale: The complete drug reference, 36th ed., Pharmaceutical Press, UK, 2009.
8. E. Park, C. Jeon, G. Ko, J. Kim, D. Sohn, Protective effect of curcumin in rat liver injury induced by carbon tetrachloride, J Pharm Pharmacol. 52 (2000) 437-440.
Page 12 of 18
9. P. Anand, A. Kunnumakkara, R. Newman, B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol Pharm. 4 (2007) 807-818.
ip t
10. K. Sharma, S. Agrawal, M. Gupta, Development and validation of uv spectrophotometric method for the estimation of curcumin in bulk drug and pharmaceutical dosage forms, Int J
us
cr
Drug Dev & Res. 4 (2012) 375-380.
11. K. Ashraf, M. Mujeeb, A. Ahmad, M. Amir, N. Mallick, D. Sharma, Validated HPTLC
an
analysis method for quantification of variability in content of curcumin in Curcuma longa L
(2012) S584-S588.
M
(turmeric) collected from different geographical region of India, Asian Pac J Trop Biomed. 2
d
12. V. Thakker, V. Shah, U. Shah, M. Suthar, Simultaneous estimation of gallic acid,
Ac ce pt e
curcumin and quercetin by HPTLC method, Journal of advanced pharmacy education & research. 1 (2011) 70-80.
13. W. Wichitnithad, N. Jongaroonngamsang, S. Pummangura, P. Rojsitthisak, A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts, Phytochem Anal. 20 (2009) 314-319.
14. G. Jayaprakasha, L. Rao, K. Sakariah, Improved HPLC method for the determination of curcumin, demethoxy-curcumin, and bis-demethoxy-curcumin, J Agric Food Chem. 50 (2002) 3668-3672.
Page 13 of 18
15. P. Ramalingam, Y. Ko, A validated LC-MS/MS method for quantitative analysis of curcumin in mouse plasma and brain tissue and its application in pharmacokinetic and brain distribution studies, J Chromatogr B Analy Technol Biomed Life Sci. 969 (2014) 101-118.
ip t
16. F. Wang, W. Hung, Y. Wang, Fluorescence enhancement effect for the determination of curcumin with yttrium(III)-curcumin-sodium dodecyl benzene sulfonate system, J Lumin.
us
cr
128 (2008) 110-116.
17. A. Goren, S. Cikrikci, M. Cergel, G. Bilsel, Rapid quantitation of curcumin in turmeric
an
via NMR and LC-tandem mass spectrometry, J Food Chem. 113 (2009) 1239-1242.
M
18. J. Patel, S. Ketkar, S. Patil, J. Fearnley, K. Mahadik, A. Paradkar, Potentiating
Ac ce pt e
med res. 4 (2015) 94-101.
d
antimicrobial efficacy of propolis through niosomal-based system for administration, Integr
19. D. Kaushik, J. Yadav, P. Kaushik, D. Sacher, R. Rani, Current pharmacological and phytochemical studies of the plant Alpinia galanga, Journal of chinese integrative medicine. 9 (2011) 1061-1065.
20. F. Chen, Y. Tan, H. Li, Z. Qin, H. Cai, W. Lai, X. Zhang, Y. Li, W. Guan, Y. Li, J. Zhang, Differential systemic exposure to galangin after oral and intravenous administration to rats, Chem Cent J. 9 (2015) 1-10.
Page 14 of 18
21. V. Kale, A. Namdeo, HPTLC densitometric evaluation by simultaneous estimation of galangin in Alpinia galanga and Alpinia officinarum, Der Pharmacia Lettre. 7 (2015) 158164.
ip t
22. L. Tao, Z. Wang, E. Zhu, Y. Lu, D. Wei, HPLC analysis of bioactive flavonoids from the
cr
rhizome of Alpinia officinarum, South african journal of botany. 72 (2006) 163-166.
us
23. S. Verza, C. Pavei, G. Borre, A. Silva, G. Ortega, P. Mayorga, Determination of galangin in commercial extracts of Alpinia officinarum by RP-HPLC-DAD, Lat Am J Pharm. 30
an
(2011) 576-579.
M
24. L. Kose, L. Gulcin, A. Goren, J. Namiesnik, A. Martinez-Ayala, S. Gorinstein, LCMS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum
Ac ce pt e
d
Hance) rhizomes, Ind Crops Prod. 74 (2015) 712-721.
25. International Conference on Harmonization, ICH Q2 (R1): Validation of Analytical Procedures: Text and Methodology, ICH Secretariat, Geneva, 2005.
Table 1 Linear regression data for calibration plots of the analyzed drugs using the proposed HPTLC methods. Parameters
Wavelength (nm)
Results CU
GA
404
404
Page 15 of 18
0.28 ± 0.02
0.48 ± 0.06
Linearity range (ng/spot)
80 - 450
200 - 1200
Correlation coefficient (r2)
0.9986
0.9991
Slope
19.51
10.86
Intercept
1431.60
440.18
% RSD of slope
0.45
0.78
LOD (ng/spot)
18.31
40.50
LOQ (ng/spot)
55.50
122.74
us
cr
ip t
Retention factor (Rf)
Concentration
Found ± SD a
RSD b
(ng/spot)
(ng/spot)
M
(%)
Intra-day precision
GA
100
91.67 ± 0.61
Ac ce pt e
CU
d
Analyte
an
Table 2 Intra-day and inter-day precision of CU and GA.
0.08
250
227.43 ± 0.72
0.77
400
360.46 ± 0.92
0.20
300
297.23 ± 0.88
0.56
700
677.73 ± 0.77
0.46
1100
1022.40 ± 0.87
0.33
100
97.57 ± 0.77
0.30
250
234.66 ± 0.87
0.45
400
365.56 ± 0.69
0.29
300
297.33 ± 0.94
0.26
Inter-day precision CU
GA
Page 16 of 18
686.46 ± 0.87
0.14
1100
1032.87 ± 0.74
0.42
a
Mean ± standard deviation for three determinations.
b
% relative standard deviation.
Table 3 Robustness evaluation of the method for CU. CU
GA
% RSD of Rf ± SD
% RSD of Rf ± SD
cr
Level
us
Factor
ip t
700
peak area
peak area
an
A: Development distance. -1
0.33
0.30 ± 0.06
0.23
0.50 ± 0.02
8
0
0.34
0.28 ± 0.02
0.33
0.48 ± 0.06
9
1
0.28
0. 31± 0.04
0.26
0.54 ± 0.05
M
7
7 8
-1
0.43
0.32 ± 0.04
0.40
0.51 ± 0.05
0
0.41
0.28 ± 0.02
0.36
0.48 ± 0.06
1
0.44
0.21 ± 0.01
0.41
0.43 ± 0.02
-1
0.17
0.27 ± 0.03
0.18
0.49 ± 0.07
0
0.17
0.28 ± 0.02
0.16
0.48 ± 0.06
1
0.18
0. 28 ± 0.04
0.19
0.51± 0.02
Ac ce pt e
6
d
B: Percentage of n-hexane in the mobile phase V/V.
C: Chamber saturation time. 20 30 40
Table 4 Accuracy studies of CU and GA at 80 %, 100 % and 120 % addition by the proposed TLC densitometric method. Analyte
Amount in
Amount added
Measured conc. %
%
Page 17 of 18
Recovery
RSD
200
160
340.87
94.68
0.04
200
200
396.55
99.13
0.01
200
240
438.22
99.59
0.01
600
480
1078.88
99.89
0.03
600
600
1187.68
98.91
0.01
600
720
1317.92
99.77
0.03
ip t
± S.D.
cr
GA
(µg)
us
CU
sample (µg)
Table 5 Application of the proposed HPTLC method for determination of the analyzed
CU
99.12 ± 0.27
GA
98.44 ± 0.33
% RSD
M
% Recovery
0.11
0.09
Ac ce pt e
d
Analyte
an
drugs in commercial formulation.
Page 18 of 18
S1658-3655(16)30083-8 http://dx.doi.org/doi:10.1016/j.jtusci.2016.10.004 JTUSCI 343
To appear in: Received date: Revised date: Accepted date:
31-3-2016 7-9-2016 2-10-2016
Please cite this article as: S. Kharat, A. Namdeo, P. Mehta, Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form., Journal of Taibah University for Science (2016), http://dx.doi.org/10.1016/j.jtusci.2016.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form.
Sharad Kharat1†, Ajay Namdeo2, Piyush Mehta1
Dept. of Quality assurance, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune-38.
2
Dept. of Pharmacogonasy, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune-38.
us
Ac ce pt e
Abstract
d
M
an
†Corresponding author: Mr. Sharad Kharat Department of Quality assurance, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Kothrud, Pune 411038, Maharashtra, India. Email: [email protected]
cr
ip t
1
A simple and precise HPTLC methods were developed for the simultaneous estimation of the two anti-inflammatory drugs (Curcumin and Galangin). The method was tailored to analyze both drugs in their commercial dosage form (capsules) with no interference from ingredients. Chromatographic separation was performed over precoated TLC plates (60 F254, 20 × 10 cm, 250 µm thickness, Merck, Darmstadt, Germany) through linear ascending technique using nHexane: Ethyl acetate: Acetic acid: Methanol as a mobile phase. Detection and quantification was achieved at 404 nm through spectro-densitometric analysis. Analytical performance of the proposed HPTLC method was validated according to the ICH guidelines with respect to linearity, accuracy, precision, detection and quantitation limits, robustness and specificity. Calibration curves were linear in the limits of 80-450 and 200-1200 ng/spot for CU and GA, respectively with correlation coefficients (r2) >0.9998. The limits of detection were 18.31
Page 1 of 18
and 40.50 ng/spot for CU and GA respectively. The validated HPTLC method was successfully applied to the simultaneous determination of CU and GA in commercial polyherbal formulation.
ip t
Keywords- Curcumin, Galangin, HPTLC, Validation and Polyherbal formulation.
cr
1. Introduction
us
Natural products contains wide range of active chemical compounds including flavonoids, glycosides, triterpenoids, lipids, oils, steroids and organic acids which may be accountable for
an
its widespread pharmacological activity. The huge variety in chemical compounds pose a
precision and reproducibility [1,2].
M
challenge to analyze, authenticate and separate quickly with acceptable accuracy and
d
Various sophisticated analytical techniques are describe in the literature for analyze,
Ac ce pt e
authenticate and separate very complex chemical mixtures. Each technique has its own restrictions. For example, in liquid chromatography (LC), it is a complex function to change over from one mobile phase to another due to challenge of high back pressure and miscibility [3]. Chromatographic fingerprint investigation has proved to be a realistic and practical method for the quality assessment and species authentication of various traditional medicine. This chromatographic fingerprint of natural product can then be used to decide presence or absence of markers of concern and also the ratio of all detectable compounds. Even though high performance thin layer chromatography (HPTLC) has a few restrictions, such as the lower plate efficiency and narrow developing distance by comparison with GC and HPLC, it is still an valuable tool for quality assessment of natural products due to its ease, low cost and few requirement, and it has been profitably utilized to develop the chromatographic
Page 2 of 18
fingerprint for various natural products herbal drugs and commercial herbal formulations. Moreover, the colourful pattern and quantification at micron and nanogram levels help to differentiate various sample on the same plate [4,5,6].
ip t
Curcumin (CU) is well known polyphenolic moiety present in the rhizomes of the Curcuma longa Linn. belonging to family Zingiberaceae. (Fig. 1) It has a wide range of
cr
pharmacological and biological actions. This compound shows various therapeutic actions
us
including antioxidant, hepatoprotective, antirheumatic, antibacterial, antiprotozoal, antiinflammatory, anti-cancer, anti-HIV, neuro-protection and also active against several
an
malignancies. Additionally, safety of CU at very high doses has been verified in numerous human and animal investigation [7,8,9]. Numerous reports are found in the scientific
M
literature for the determination of CU, including UV spectrophotometer [10], HPTLC [11,12], high performance liquid chromatography (HPLC) [13,14] and LC-MS/MS [15] for
d
quantitative determination of CU in pharmaceutical dosage form, bulk drug and plasma.
Ac ce pt e
Moreover, some fluorimetric and NMR methods are also have been used for the rapid determination and quantitation of CU [16,17].
Galangin (GA) is a 3,5,7-trihydroxyflavone isolated from rhizomes of the Alpinia galanga belonging to family Zingiberaceae. (Fig. 2) Plant is commonly known as galangal. It is commonly known for its anti-inflammatory, antipyretic, abortifacient, aphrodisiac, carminative and emmenagogue properties. It is used in the treatment of various diseases such as heart diseases, kidney disorders, rheumatism, bronchitis, chronic enteritis and renal calculus. GA is also well known phytoconstituent of propolis. It shows good antibacterial activity against both gram positive and negative bacteria [18,19,20]. The identification and quantification of GA in various extract, in biological samples and formulations was addressed
Page 3 of 18
in some reports. Analytical methodology in these scientific reports involved the use of HPTLC-densitometric analysis [21], HPLC-UV [22], a reversed phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD) [23] and LC-MS/MS [24].
ip t
To the best of our information, no efforts have yet been made to examine this drug combination by any HPTLC method. The objective of present investigation is the
cr
development of a simple, precise and reproducible HPTLC method for the simultaneous
us
estimation of CU and GA in pure form and in commercial marketed formulation. As method validation is an essential constraint in analytical method development, the presented method
an
has been validated following the guidelines of the ICH.
2.1 Chemicals and reagents
M
2. Experimental
d
Standard curcumin and galangin were purchased from Natural Remedies Pvt. Ltd.,
Ac ce pt e
Bangalore, India. Herbal drug formulation (Antarth, Millenium Herbal Care Pvt. Ltd., Pune, India) used in this investigation was obtained from the local market. All other chemicals and reagents were of analytical grade.
2.2 Chromatographic conditions
The samples were spotted in the form of bands (6 mm width) with a Hamilton microliter syringe (100 µl) under controlled nitrogen stream using a Cammag Linomat V sample applicator (Switzerland). The slit dimension was kept at 5 × 0.45 mm and scanning speed 10 mm/s. Precoated TLC silica gel aluminum plates 60 F254 (20 × 10 cm, 250 µm thickness, Merck, Darmstadt, Germany) were utilized. Precoated TLC were pre-washed with methanol and activated at 110°C for 5 min prior to the sample application. Method development was
Page 4 of 18
executed in a Cammag twin trough glass chamber (20 × 20 cm) saturated with the mobile phase. The 10 ml mobile phase was composed of n-Hexane: Ethyl acetate: Acetic acid: Methanol (7:2:0.5:0.5 v/v/v). Prior to study, chamber was saturated with mobile phase for 30 min at room temperature (25 ± 5 °C) at relative humidity of 60 ± 5 %. Densitometric
ip t
scanning was achieved over Cammag TLC scanner III operated by win CATS software (V 1.44 CAMAG). The source of radiation used was UV spectrophotometer. The obtained bands
us
cr
were scanned at wavelength of 404 nm.
an
2.3 Preparation of Solutions
2.3.1 Standard Stock Solutions
M
A standard stock solutions of 1000 µg/mL was prepared by dissolving 10 mg of each marker in 10 ml of volumetric flask containing HPLC grade methanol. Prepared solution was further
d
diluted with HPLC grade methanol to obtained final concentrations of 100 µg/mL. These
Ac ce pt e
stock solutions were used for further studies.
2.3.2 Sample Stock Solution
A total of 5 capsule were weighed and powdered. To an accurately weighed quantity of the powder equivalent to 8.3 mg of CU and 10 mg of GA, methanol were added, sonicated for 15 min then filtered through whatman filter paper no.42 into a 10-mL volumetric flask.
3. Validation of the method The proposed analytical method was validated as per the International Conference on Harmonization (ICH) guidelines Q2 (R1) [25].
Page 5 of 18
3.1 Linearity Prepared standard stock solution was diluted to obtained linearity standard solutions containing CU and GA in the concentration range of 80-450 ng/spot and 200-1200 ng/spot, respectively. Three sets of such solutions were equipped. Every set was analyzed to obtained
ip t
a calibration curve. Standard deviation (SD), coefficient of determination (r2), slope and
cr
intercept of the calibration curves were estimated to determine linearity of the method.
us
3.2 Precision
The present method was validated for intraday and interday precision. Intraday precision was
an
determined by replicate the same method three times on the same day for three different concentrations of CU (100, 250 and 400 ng/spot) and GA (300, 700 and 1100 ng/spot). The
M
interday precision of the method was verify by performing similar method on different days under the same set of experimental situation. Repeatability of sample application and
Ac ce pt e
d
calculation of peak area for analyte were articulated in terms of % RSD.
3.3 Robustness
To determine the robustness of the present method, small changes in the chromatographic conditions were intentionally made, which may affect the performance of the method such as TLC plate development and developing distance, mobile-phase composition and chamber saturation time.
3.4 Specificity To verify specificity of the method, CU and GA standard and sample solution were applied to a HPTLC plate and the plate was developed and scanned as described above. The peak purity
Page 6 of 18
of CU and GA was measure by evaluating the spectra of marker compounds at peak apex, peak start and peak end positions of the spot.
3.5 Accuracy
ip t
Accuracy was estimated through the % recoveries of known quantity of combination of CU and GA added to solutions of marketed preparation. Marketed preparation was spiked with
cr
the known quantity of standard markers and the % ratios between the recovered and expected
us
concentrations were calculated. The analyzed samples were spiked with 80, 100 and 120 % of
an
500 ng/spot of both CU and GA. Accuracy was calculated from the following equation,
M
Spiked concertation − Mean concentration × 100 Spiked concertration
d
3.6 Limit of detection and Limit of quantitation
Ac ce pt e
The limit of detection (LOD) is the lowest amount of analyte that can be detected in a sample, but not necessarily quantified, under the stated experimental conditions. The limit of quantification (LOQ) was identified as the lowest amount of analyte that can be detected and quantified with acceptable accuracy, precision, and variability. LOD and LOQ are calculated by the signal to noise method.
4. Analysis of marketed product
To determine the content of CU and GA in capsule, to an accurately weighed quantity of the formulation equivalent to 8.33 mg of CU and 10 mg of GA, 7 mL methanol were added, sonicated for 15 min. The volume of resulting solution was made up to 10 mL with methanol. The % assay was determined. The possibility of chemical constituent’s interference with analysis was examined.
Page 7 of 18
5. Results and discussion Chromatographic separation were carried out on the standard solutions of CU and GA. Briefly, spots of standard samples were applied on the TLC plates. TLC plates were
ip t
developed by linear ascending development using various solvents like acetone, benzene, chloroform, ethyl acetate, methanol and toluene. Based on the observations of these primary
cr
trials, various binary, ternary and quaternary mixtures of solvents were tried to accomplish
us
optimum resolution between the CU and GA in their respective solvent mixtures under study. Initially, n-hexane and ethyl acetate in several ratios were attempted but inadequate
an
separation between CU and GA occurred even upon the addition of small quantity of acetic acid to the mobile phase. In these earlier examination (8:1.5:0.5), CU was clearly separated
M
however GA was not satisfactorily resolved. Again, addition of small quantity of acetic acid to the above mentioned mobile phase did not improve the separation. Conversely, addition of
d
methanol to the above mentioned mobile phase produced considerable perfection in both
Ac ce pt e
peak characteristics and separation. The optimized mobile phase composed of n-Hexane: Ethyl acetate: Acetic acid: Methanol (7:2:0.5:0.5 v/v/v) showed satisfactory resolution. Densitometric measurements were achieved with Cammag TLC scanner III operated by win CATS software (V 1.44 CAMAG). At wavelength of 404 nm (Fig. 3), optimized mobile phase showed highest sensitivity for quantification of both drugs.
Other chromatographic conditions like sample application rate and volume, sample application positions, chamber saturation time, total run length and distance between tracks were also optimized to give accurate, precise and reproducible Rf values, symmetrical peak shape and better resolution for the both drugs. The optimized chamber saturation time for the given mobile phase was found as 30 min at room temperature (25 + 5 °C) at relative humidity
Page 8 of 18
of 60 + 5 %. The total run length of the chromatogram run was approximately 80 mm. The distance between two tracks was 10 mm. The peak shape was more symmetrical and spots appeared more compact when the TLC plates were pre-treated with methanol and activated at 110°C for 5 min. After run the TLC plates were dried in air for 5-6 min. The slit dimension
ip t
and scanning speed was 5 × 0.45 mm and 10 mm/s respectively. The optimized
cr
chromatographic conditions are mentioned in Table 1.
us
5.1 Validation of the proposed method
Validation of the proposed methods was carried out in accordance with the International
M
5.1.1 Linearity
an
Conference on Harmonization (ICH) guidelines (2005).
The linearity of the presented HPTLC method was estimated by analyzing a series of
d
different concentrations for each of the both analytes. Under the optimized state mentioned
Ac ce pt e
above, the calculated peak areas at 404 nm were found to be proportional to concentrations of CU and GA respectively. Table 1 denotes the statistical parameters including linearity ranges, correlation coefficients, slopes, intercepts and % RSD of the slopes. Linearity graph of CU and GA was shown in Fig. 3 and 4 respectively.
5.1.2 Precision
The both intra-day and inter-day repeatability of the presented method was calculated for each drug. The outcomes of intra-day and inter-day repeatability are shown in Table 2. It shows % RSD which did not exceed 1 % for CU and GA representing the high level of precision of the given method.
Page 9 of 18
5.1.3 Robustness The parameters of the optimized methods were intentionally varied. Each factor selected to examine were charged at three levels (-1, 0 and 1). One time one factor was subjected to change and change in the responses of the both drugs were denoted. The summary of
ip t
robustness study was given in Table 3. The values of % RSD of the peak areas of both CU and GA under the analysis did not surpass 0.5 %. The satisfactory low values of % RSD of
us
cr
peak areas and unaffected Rf values suggested the robustness of the proposed method.
5.1.4 Specificity
an
The peak purity of CU and GA was assessed by comparing their respective spectra at peak apex, peak start and end positions of the peak. A good correlation (r2 value more than 0.999)
M
was acquired for both drugs. Good correlation values and satisfactory peak purity suggest no
Ac ce pt e
method is specific.
d
interference in the quantification of the CU and GA in sample solutions. This verifies that the
5.1.5 Accuracy
Results from accuracy studies, denoted in Table 4, were in acceptable range (94.68- 99.91 %), indicating the recovery of proposed method was good. Additionally, Low % RSD value proved the ruggedness of proposed method.
5.1.6 Limit of detection and Limit of quantitation The limit of detection (LOD) value for CU and GA were found to 18.31and 40.50 ng and limit of quantification (LOQ) value were 55.50 and 122.74 ng, respectively which shows the adequate sensitivity of the method (Table 1).
Page 10 of 18
5.2 Analysis of marketed product The developed HPTLC method was used for the assay of commercial formulation viz. Antarth®. The both drugs eluted at their specific Rf values. No interfering peaks were observed from any of the inactive ingredients. The % recovery and % RSD was given in
ip t
Table 5. It is clear from these observations that the proposed method is applicable for the analysis of the CU and GA in their combined commercial formulations with accuracy and
us
cr
precision (Fig. 6).
6. Conclusion
an
Present study illustrate a simple, accurate and precise HPTLC method for the simultaneous estimation of CU and GA. Additionally, the proposed method does not necessitate complex
M
treatment or sophisticated analytical unit usually associated with HPLC and LC-MS/MS procedures of analysis. Reliability was ascertain by testing a range of validation parameters
d
of the proposed method. At last, the proposed method was found sensitive and specific;
Ac ce pt e
hence, it can be suggested for the routine analysis of the cited phytochemicals either in bulk form or in their combined dosage forms.
7. References
1. Jyotshna, P. Srivastava, B. Killadi, K. Shanker, Uni-dimensional double development HPTLC-densitometry method for simultaneous analysis of mangiferin and lupeol content in mango (Mangifera indica) pulp and peel during storage, Food Chem. 176 (2015) 91-98.
Page 11 of 18
2. P. Mehta, R. Shah, L. Sathiyanarayanan, K. Mahadik, Pharmacokinetic profile of phytoconstituent(s) isolated from medicinal plants-a comprehensive review, J Tradit Complement Med. 5 (2015) 207-227.
ip t
3. A. Petruczynik, M. Waksmundzka-Hajnos, T. Plech, T. Tuzimski, M. Hajnos, G. Jozwiak, M. Gadzikowska, A. Rompala, TLC of alkaloids on cyanopropyl bonded stationary phases.
us
cr
Part II. Connection with RP18 and silica plates, J Chromatogr Sci. 46 (2008) 291-297.
4. D. Rathee, S. Rathee, P. Rathee, A. Deep, S. Anandjiwala, D. Rathe, HPTLC densitometric
an
quantification of stigmasterol and lupeol from Ficus religiosa, Arabian journal of chemistry.
M
8 (2015) 366-371.
5. A. Kamboj, A. Saluja, Development of validated HPTLC method for quantification of
d
stigmasterol from leaf and stem of Bryophyllum pinnatum, Arabian journal of chemistry.
Ac ce pt e
http://dx.doi.org/10.1016/j.arabjc.2013.10.006
6. Z. Sheikh, S. Shakeel, S. Gul, A. Zahoor, S. Khan, F. Zaidi, K. Usmanghani, A novel HPTLC method for quantitative estimation of biomarkers in polyherbal formulation, Asian Pac J Trop Biomed. 5 (2015) 955-959.
7. S. Sweetman, Martindale: The complete drug reference, 36th ed., Pharmaceutical Press, UK, 2009.
8. E. Park, C. Jeon, G. Ko, J. Kim, D. Sohn, Protective effect of curcumin in rat liver injury induced by carbon tetrachloride, J Pharm Pharmacol. 52 (2000) 437-440.
Page 12 of 18
9. P. Anand, A. Kunnumakkara, R. Newman, B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol Pharm. 4 (2007) 807-818.
ip t
10. K. Sharma, S. Agrawal, M. Gupta, Development and validation of uv spectrophotometric method for the estimation of curcumin in bulk drug and pharmaceutical dosage forms, Int J
us
cr
Drug Dev & Res. 4 (2012) 375-380.
11. K. Ashraf, M. Mujeeb, A. Ahmad, M. Amir, N. Mallick, D. Sharma, Validated HPTLC
an
analysis method for quantification of variability in content of curcumin in Curcuma longa L
(2012) S584-S588.
M
(turmeric) collected from different geographical region of India, Asian Pac J Trop Biomed. 2
d
12. V. Thakker, V. Shah, U. Shah, M. Suthar, Simultaneous estimation of gallic acid,
Ac ce pt e
curcumin and quercetin by HPTLC method, Journal of advanced pharmacy education & research. 1 (2011) 70-80.
13. W. Wichitnithad, N. Jongaroonngamsang, S. Pummangura, P. Rojsitthisak, A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts, Phytochem Anal. 20 (2009) 314-319.
14. G. Jayaprakasha, L. Rao, K. Sakariah, Improved HPLC method for the determination of curcumin, demethoxy-curcumin, and bis-demethoxy-curcumin, J Agric Food Chem. 50 (2002) 3668-3672.
Page 13 of 18
15. P. Ramalingam, Y. Ko, A validated LC-MS/MS method for quantitative analysis of curcumin in mouse plasma and brain tissue and its application in pharmacokinetic and brain distribution studies, J Chromatogr B Analy Technol Biomed Life Sci. 969 (2014) 101-118.
ip t
16. F. Wang, W. Hung, Y. Wang, Fluorescence enhancement effect for the determination of curcumin with yttrium(III)-curcumin-sodium dodecyl benzene sulfonate system, J Lumin.
us
cr
128 (2008) 110-116.
17. A. Goren, S. Cikrikci, M. Cergel, G. Bilsel, Rapid quantitation of curcumin in turmeric
an
via NMR and LC-tandem mass spectrometry, J Food Chem. 113 (2009) 1239-1242.
M
18. J. Patel, S. Ketkar, S. Patil, J. Fearnley, K. Mahadik, A. Paradkar, Potentiating
Ac ce pt e
med res. 4 (2015) 94-101.
d
antimicrobial efficacy of propolis through niosomal-based system for administration, Integr
19. D. Kaushik, J. Yadav, P. Kaushik, D. Sacher, R. Rani, Current pharmacological and phytochemical studies of the plant Alpinia galanga, Journal of chinese integrative medicine. 9 (2011) 1061-1065.
20. F. Chen, Y. Tan, H. Li, Z. Qin, H. Cai, W. Lai, X. Zhang, Y. Li, W. Guan, Y. Li, J. Zhang, Differential systemic exposure to galangin after oral and intravenous administration to rats, Chem Cent J. 9 (2015) 1-10.
Page 14 of 18
21. V. Kale, A. Namdeo, HPTLC densitometric evaluation by simultaneous estimation of galangin in Alpinia galanga and Alpinia officinarum, Der Pharmacia Lettre. 7 (2015) 158164.
ip t
22. L. Tao, Z. Wang, E. Zhu, Y. Lu, D. Wei, HPLC analysis of bioactive flavonoids from the
cr
rhizome of Alpinia officinarum, South african journal of botany. 72 (2006) 163-166.
us
23. S. Verza, C. Pavei, G. Borre, A. Silva, G. Ortega, P. Mayorga, Determination of galangin in commercial extracts of Alpinia officinarum by RP-HPLC-DAD, Lat Am J Pharm. 30
an
(2011) 576-579.
M
24. L. Kose, L. Gulcin, A. Goren, J. Namiesnik, A. Martinez-Ayala, S. Gorinstein, LCMS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum
Ac ce pt e
d
Hance) rhizomes, Ind Crops Prod. 74 (2015) 712-721.
25. International Conference on Harmonization, ICH Q2 (R1): Validation of Analytical Procedures: Text and Methodology, ICH Secretariat, Geneva, 2005.
Table 1 Linear regression data for calibration plots of the analyzed drugs using the proposed HPTLC methods. Parameters
Wavelength (nm)
Results CU
GA
404
404
Page 15 of 18
0.28 ± 0.02
0.48 ± 0.06
Linearity range (ng/spot)
80 - 450
200 - 1200
Correlation coefficient (r2)
0.9986
0.9991
Slope
19.51
10.86
Intercept
1431.60
440.18
% RSD of slope
0.45
0.78
LOD (ng/spot)
18.31
40.50
LOQ (ng/spot)
55.50
122.74
us
cr
ip t
Retention factor (Rf)
Concentration
Found ± SD a
RSD b
(ng/spot)
(ng/spot)
M
(%)
Intra-day precision
GA
100
91.67 ± 0.61
Ac ce pt e
CU
d
Analyte
an
Table 2 Intra-day and inter-day precision of CU and GA.
0.08
250
227.43 ± 0.72
0.77
400
360.46 ± 0.92
0.20
300
297.23 ± 0.88
0.56
700
677.73 ± 0.77
0.46
1100
1022.40 ± 0.87
0.33
100
97.57 ± 0.77
0.30
250
234.66 ± 0.87
0.45
400
365.56 ± 0.69
0.29
300
297.33 ± 0.94
0.26
Inter-day precision CU
GA
Page 16 of 18
686.46 ± 0.87
0.14
1100
1032.87 ± 0.74
0.42
a
Mean ± standard deviation for three determinations.
b
% relative standard deviation.
Table 3 Robustness evaluation of the method for CU. CU
GA
% RSD of Rf ± SD
% RSD of Rf ± SD
cr
Level
us
Factor
ip t
700
peak area
peak area
an
A: Development distance. -1
0.33
0.30 ± 0.06
0.23
0.50 ± 0.02
8
0
0.34
0.28 ± 0.02
0.33
0.48 ± 0.06
9
1
0.28
0. 31± 0.04
0.26
0.54 ± 0.05
M
7
7 8
-1
0.43
0.32 ± 0.04
0.40
0.51 ± 0.05
0
0.41
0.28 ± 0.02
0.36
0.48 ± 0.06
1
0.44
0.21 ± 0.01
0.41
0.43 ± 0.02
-1
0.17
0.27 ± 0.03
0.18
0.49 ± 0.07
0
0.17
0.28 ± 0.02
0.16
0.48 ± 0.06
1
0.18
0. 28 ± 0.04
0.19
0.51± 0.02
Ac ce pt e
6
d
B: Percentage of n-hexane in the mobile phase V/V.
C: Chamber saturation time. 20 30 40
Table 4 Accuracy studies of CU and GA at 80 %, 100 % and 120 % addition by the proposed TLC densitometric method. Analyte
Amount in
Amount added
Measured conc. %
%
Page 17 of 18
Recovery
RSD
200
160
340.87
94.68
0.04
200
200
396.55
99.13
0.01
200
240
438.22
99.59
0.01
600
480
1078.88
99.89
0.03
600
600
1187.68
98.91
0.01
600
720
1317.92
99.77
0.03
ip t
± S.D.
cr
GA
(µg)
us
CU
sample (µg)
Table 5 Application of the proposed HPTLC method for determination of the analyzed
CU
99.12 ± 0.27
GA
98.44 ± 0.33
% RSD
M
% Recovery
0.11
0.09
Ac ce pt e
d
Analyte
an
drugs in commercial formulation.
Page 18 of 18