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 Table of Contents  
Year : 2013  |  Volume : 4  |  Issue : 3  |  Page : 160-165  

Method development for Lawsone estimation in Trichup herbal hair powder by high-performance thin layer chromatography

1 Department of Pharmacy, Sumandeep Vidyapeeth, Pipariya, Waghodiya, Gujarat, India
2 Vasu Research Centre (A Division of Vasu Healthcare Pvt. Ltd.), 896/A, G.I.D.C, Makarpura, Vadodara, Gujarat, India

Date of Web Publication21-Aug-2013

Correspondence Address:
Bhavna R Solanki
Vasu Research Centre (A Division of Vasu Healthcare Pvt. Ltd.) 896 /A, G.I.D.C., Makarpura, Vadodara - 390 010, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-4040.116780

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A simple, specific, accurate, precise and robust high-performance thin-layer chromatographic method has been developed and validated for estimation of Lawsone in Trichup herbal hair powder (coded as a THHP), polyherbal formulation. The chromatographic development was carried out on aluminum plates pre-coated with silica gel 60F 254 and good resolution was achieved with Toluene: Ethyl acetate: Glacial acetic acid (8:1:1 v/v/v) as mobile phase. Lawsone detection was carried out densitometrically at 277 nm and obtained retardation factor value was 0.46 ± 0.02. The method was validated with respect to specificity, linearity, accuracy, precision and robustness. The calibration curve was achieved to be linear over a range of 5-60 μg/ml and regression coefficient was obtained 0.998. Accuracy of chromatographic method was evaluated by standard addition method; recovery was obtained 99.25 ± 0.61% . The peak purity of Lawsone was achieved 0.999 r. Relative standard deviation for intraday and inter-day precision was 0.37-0.56% and 0.42-0.55%, respectively. The limit of detection and limit of quantification of the Lawsone were found to be 1.08 μg/m land 3.28 μg/ml, respectively. This result shows that the method was well validated. In the present study, the Lawsone content was found 0.322 ± 0.014% in THHP. This study reveals that the proposed high performance thin layer chromatography method is accurate, fast and cost- effective for routine estimation of Lawsone in polyherbal formulation.

Keywords: Henna, high-performance thin-layer chromatography, Lawsone, Trichup herbal hair powder, validation

How to cite this article:
Patel MM, Solanki BR, Gurav NC, Patel PH, Verma SS. Method development for Lawsone estimation in Trichup herbal hair powder by high-performance thin layer chromatography. J Adv Pharm Technol Res 2013;4:160-5

How to cite this URL:
Patel MM, Solanki BR, Gurav NC, Patel PH, Verma SS. Method development for Lawsone estimation in Trichup herbal hair powder by high-performance thin layer chromatography. J Adv Pharm Technol Res [serial online] 2013 [cited 2023 Mar 27];4:160-5. Available from: https://www.japtr.org/text.asp?2013/4/3/160/116780

  Introduction Top

Lawsonia alba Lamk. (belongs to family Lythraceae) most commonly known as "Henna" invites attention of the investigators world-wide for its cosmetic and pharmacological profile ranging from dying to anti-inflammatory and anti-cancer activities. [1],[2] Henna has also antifungal activity against Malassezia species (causative organism of dandruff). [3] L. alba is an annual flowering plant, which has been used since the Bronze age to dye hair. L. alba is a much branched shrub that grows mainly in Middle East of Africa. [3],[4] In several parts of the world, it is traditionally used in various festivals and celebrations.

The principle coloring compound of Henna is "Lawsone," a red-orange colored compound present in dried leaves in a concentration of 1-1.5% w/w. [5],[6],[7] Chemically, it is called as 2-hydroxy-1,4 napthaquinone [Figure 1] having molecular formula C 10 H 6 O 3 , molecular weight 174.15 and melting point 190°C. Lawsone is mainly responsible for the colorant property of henna leaves. It is practically insoluble in water at 0.2%, soluble in dichloromethane (DCM), methanol, acetone, isopropyl alcohol, chloroform, ethyl acetate and diethyl ether. Lawsone is proposed to be used as a non-oxidizing hair coloring agent at a maximum concentration of 1.5% in the finished cosmetic product. [8]

Trichup herbal hair powder (THHP) is an advanced herbal blend that contains ayurvedic hair rejuvenating herbs including henna as one of the major ingredient. It is claimed to be ideal for all hair types and helps to treat dandruff, split ends and hair fall. The product has also claimed to restructure and repair the damaged hair shaft. THHP also contains other key ingredients such as Acacia concinna (Shikakai), Sapindus mukorosii (Arishtak), Eclipta alba (Bhringraj), Centella asiatica (Mandukparni), Indigofera tentoria (Nilpushpa), Citrus acida (Nimbuk), Emblica officinalis (Amalaki), Azadirachta indica (Neem), Glycyrrhiza glabra (Yashtimadhu), Rosa centifolia (Gulab), Vetiveria zizanioides (Ushir), Withania somnifera (Ashwagandha), Trigonella foenum graecum (Methi), Aloe vera (Kumari) and Nardostachy jatamansi (Jatamansi).

The literature review reveals that there are few methods available for estimation of Lawsone by high-performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC). [9],[10] Reported mobile phases did not show resolved spectra for Lawsone from the other herbal compounds in polyherbal THHP. Therefore, an attempt has been made to develop the method, which can resolve the spectra for Lawsone from other herbal compounds and can easily quantify for routine quality control analysis. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) Q2 (R1) guideline. [11]
Figure 1: Chemical structure of Lawsone

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  Materials and Methods Top


THHP is manufactured and marketed by Vasu Healthcare Pvt. Ltd. Raw materials were also procured from Vasu Healthcare Pvt. Ltd, Vadodara, India. Lawsone standard (97% pure) was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India. Purity and structure of standard compound were confirmed by HPLC and infrared spectral analysis. Analytical reagent grade solvents, i.e., DCM, ethyl acetate, acetic acid and toluene were obtained from Merck Specialties Pvt. Ltd., Mumbai, India.


A CAMAG HPTLC system equipped with a sample applicator Linomat V, Hamilton Syringe 100 μL, twin trough plate development chamber, CAMAG thin layer chromatography (TLC) Scanner IV, stationary phase pre-coated Merck silica gel 60F 254 , winCATS and integration software (Switzerland).

Preparation of Standard Stock Solution

Accurately weighed Lawsone (10 mg) in was taken in 10 ml volumetric flask and approximately 8 ml DCM was added and sonicated for 5 min. The volume was made up to 10 ml with the same to obtain 1000 μg/ml standard stock solution.

Preparation of Working Standard for Calibration Curve

Aliquots of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml of 1000 μg/ ml standard stock solution was transferred to 10 ml of volumetric flasks and made up to the mark with DCM to get concentration of 5, 10, 20, 30, 40, 50, 60 μg/ml.

Sample Preparation

Accurately weighed 300 mg of THHP was transferred to a test-tube and 10 ml of water was added. It was mixed on a cyclone mixture for 20 min. The whole suspension was transferred to a pre-washed separating funnel and 10 ml of DCM was added. The contents in the separating funnel was mixed for 20 min with occasionally release of pressure and allowed to settle down for 15 min. After separation, the lower DCM layer was collected in 10 ml volumetric flask and made up to the volume with 10 ml DCM. From that, an aliquot of 0.5 ml was taken and transferred to 10 ml volumetric flask and made up to mark with DCM and this was used as test solution.

Chromatographic Conditions

The samples were spotted in the form of 8 mm band with 100 μl Hamilton syringe on pre-coated silica gel aluminum plate 60F 254 (0.2 mm thickness) using a CAMAG Linomat V applicator. Band length was 8 mm and constant application rate was 200 nl/s. The plates were developed until the solvent rose to 8 cm. Solvent system toluene: Ethyl acetate: Glacial acetic acid (8:1:1 v/v/v) in CAMAG glass twin through chamber previously saturated for 15 min with solvents at room temperature (25 ± 2). Subsequent to the scanning, TLC plates were air dried and scanning was performed on a CAMAG TLC Scanner IV equipped with winCATS software version at 277 nm by absorbance measurement mode.

Optimization of High-performance Thin-layer Chromatography Method

Different solvents in different combinations were tried and good resolution, sharp and symmetrical peak was achieved with mobile phase toluene: Ethyl acetate: Glacial acetic acid in the proportion of 8:1:1 (v/v/v), respectively. Based on the solubility, stability and recovery of Lawsone, sample preparation was finalized using water and DCM as solvents. Lawsone was showed the retardation factor (Rf ) value 0.46, which was visualized at 277 nm wavelength [Figure 2] and [Figure 3].
Figure 2: Chromatogram of 50 μg/ml Lawsone Standard

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Figure 3: Chromatogram of Trichup herbal hair powder sample

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Statistical Analysis

Statistical calculations were carried out with the Microsoft Excel 2007 for Windows software package. Average, sum, standard deviation (STDEV), regression (RSQ) for statistical calculation and scattered chart were used for linearity; P > 0.05 was considered to be significant.

Method Validation

The proposed method was validated as per the ICH guidelines in terms of its linearity, accuracy, specificity, intraday and inter-day precision, robustness and ruggedness, limit of detection (LOD) and limit of quantification (LOQ).

System Suitability

A system suitability test was performed by applying six replicate spots of Lawsone working standard solution of 50 μg/ml concentration and verifying percentage relative standard deviation (RSD) of peak areas.


The specificity of the method was ascertained by analyzing the placebo of formulation. Lawsone spot in formulation was confirmed by comparing the Rf value and spectra of the spot with that of standard. The peak purity of Lawsone was assessed by comparing the spectra at three different levels, that is, peak start (S), peak apex (M) and peak end (E) positions of the spot.


Three replicate of 5, 10, 20, 30, 40, 50 and 60 μg/ml of Lawsone standard solution were applied on TLC plate for linearity study. TLC plates were developed under the above established conditions. Calibration curve of linearity was constructed for peak area signal against concentration.


Accuracy was assessed by standard addition method. Standard was added at three different levels to the sample solution (containing 4.6421 μg/ml concentration of Lawsone). The results were expressed as a percentage of Lawsone recovered in samples.


The precision of the method was determined by inter-day and intraday precision. An Intraday precision was determined by applying three sets of different concentrations (5 μg/ ml, 30 μg/ml and 60 μg/ml) on the same day and inter-day precision by applying three sets of different concentrations on different days.


Lawsone working standards (5, 10, 20, 30, 40, 50, 60 μg/ ml) were applied to detect the LOD and LOQ. The LOD and LOQ were calculated using equation LOD = 3.3 × σ/S and LOQ = 10 × σ/S, where, σ is SD of y-intercepts of the calibration curve (n = 5) and S is the slope of the corresponding calibration curve.


Robustness of the method was evaluated by making small deliberate changes in various chromatographic conditions. Mobile phase ratio was varied within 2% (toluene: Ethyl acetate: Glacial acetic acid 7.6:1.2:1.2 and 8.4:0.8:0.8 v/v/v). The wavelength was varied in the range of ± 2% (272 nm and 282 nm). Furthermore, there was changed the time from spotting to chromatography (0, 30 and 60 min) and from chromatography to scanning (0, 20 and 40 min). Mobile phase saturation time was varied viz. 15, 30, 60 min.


Stability of prepared standard and samples were evaluated. The standard and sample were stored at room temperature and analyzed at 0, 2, 6, 12, 24 and 48 h intervals. The percentage degradation was evaluated.

  Results and Discussion Top

Developed and validated HPTLC method for estimation of Lawsone in THHP was found to be simple, accurate, precise and robust. Different mobile phase were tried and good resolution was achieved with toluene: Ethyl acetate: Glacial acetic acid (8:1:1 v/v/v). Sample preparation was optimized by using water and DCM as solvents. Lawsone was detected at 0.46 Rf value at 277 nm by absorbance mode [Figure 2] and [Figure 3]. The developed method was validated as per ICH guideline Q2 (R1). Percentage RSD of peak areas was found to be 0.48, which indicates the suitability of system for the further validation parameters [Table 1]. There was the absence of the peak at Rf 0.46 in the placebo, indicates the method is specific as none of the excipients interfered with the analytes of interest [Figure 4], [Figure 5] and [Figure 6]. The peak purity of Lawsone was achieved r (S, M) =0.999 and r (M, E) =0.999 [Figure 7]. A calibration graph was constructed covering concentration range of 5-60 μg/ml and established RSQ coefficient was 0.998 [Table 2] and [Figure 8] and [Figure 9]. The accuracy data were expressed in terms of the percentage recoveries of Lawsone in samples. The recovery results were achieved 99.11 ± 1.20, 98.73 ± 0.60, 99.93 ± 0.68% for 20, 25 and 30 μg/ml spiked concentration respectively, which satisfying the acceptance criteria of study [Table 3]. The percentage RSD for intraday and inter-day precision was obtained 0.37-0.56% and 0.42-0.55%, respectively, which reveals that the proposed method is precise [Table 4] and [Table 5]. The LOD and LOQ values were achieved 1.08 μg/m land 3.28 μg/ml, respectively [Table 6]. The method was found to be robust with change of ± 2% mobile phase composition, mobile phase volume, development distance, saturation time of mobile phase, incubation time (for spotting to development and development to scanning) and wavelength [Table 7]. In prepared standard and sample, the Lawsone was found to be stable up to 48 h and showed the degradation < 2% [Table 8]. This denotes that the Lawsone is stable in standard and sample solution for at least 3 days at room temperature. In the present study, Lawsone was found to be 0.32 ± 0.014% in THHP [Table 9].
Table 1: System suitability

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Table 2: Linearity for lawsone standard

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Table 3: Accuracy study results

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Table 4: Intraday precision

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Table 5: Inter‑day precision

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Table 6: LOD and LOQ

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Table 7: Robustness of the method

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Table 8: Stability study

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Table 9: Quantification of Lawsone in THHP

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Figure 4: 3D graph for specificity. 1 ‑ Lawsone std. 50 μg/ml, 2 ‑ Henna, 3 ‑ Henna, 4 ‑ THHP, 5 ‑ THHP, 6 ‑ Placebo, 7 ‑ Placebo, 8 ‑ Placebo, 9 ‑ Placebo

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Figure 5: Overlay spectrum of standard Lawsone and sample

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Figure 6: Overlay spectrum of standard Lawsone and placebo

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Figure 7: Peak purity of Lawsone standard and Trichup herbal hair powder

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Figure 8: 3D graph for linearity. 1‑5 μg/ml, 2‑10 μg/ml, 3‑20 μg/ ml, 4‑30 μg/ml, 5‑40 μg/ml, 6‑50 μg/ml, 7‑60 μg/ml

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Figure 9: Calibration curve for Lawsone

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  Conclusion Top

The HPTLC method was developed and validated as per ICH guidelines and the method was found to simple, precise, robust, accurate reproducible and economic, thus can be used for routine quality control determination of Lawsone in poly herbal formulation.

  References Top

1.Bosoglu A, Birdane F, Solmaz H. The effect of henna (Folium lawsoniae) paste in ringworm in calves. Indian Vet J 1998;75:83-4.  Back to cited text no. 1
2.Kamal M, Jawaid T. Pharmacological activities of Lawsonia inermis Linn: A review. Int J Biomed Res 2010;1:37-43.  Back to cited text no. 2
3.Fariba B, Hassan R, Homeyra E. In vitro study of the effects of henna extracts (Lawsonia inermis) on Malassezia species. Jundishapur J Microbiol 2010;3:125-8.  Back to cited text no. 3
4.Varghese KJ, Silvipriya KS, Resmi S, Jolly CI. Lawsonia inermis (Henna): A natural dye of various therapeutic uses-A review. Inventi Impact: Cosmeceuticals. Vol. 2010. Article ID-Inventi: Cc/3/10, 2010. Available from: http://www.inventi.in/Article/cc/3/10.aspx. [Cited on 2013 Apr 28].  Back to cited text no. 4
5.Jain VC, Shah DP, Sonani NG, Dhakara S, Patel NM. Pharmacognostical and preliminary phytochemical investigation of Lawsonia inermis L. Leaf. Rom J Biol Plant Biol 2010;55:127-33.  Back to cited text no. 5
6.Chaudhary G, Goyal S, Poonia P. Lawsonia inermis Linnaeus: A phytopharmacological review. Int J Pharm Sci Drug Res 2010;2:91-8.  Back to cited text no. 6
7.Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 18 th ed. Pune: Nirali Prakashan; 2002. p. 1-4.  Back to cited text no. 7
8.Borade AS, Kale BN, Shete RV. A phyto pharmacological review on Lawsonia inermis (Linn.). Int J Pharm Life Sci 2011;2:536-41.  Back to cited text no. 8
9.Gallo FR, Multari G, Giambenedetti M, Federici E. Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry. Phytochem Anal 2008;19:550-9.  Back to cited text no. 9
10.Dhiman A, Sharma K, Goyal J, Garg M, Sharma A. Determination of Lawsone content in leaves of Lawsonia inermis Linn. Int J Pharm Sci 2012;1:17-20.  Back to cited text no. 10
11.Validation of analytical procedures: Text and Methodology, Q2(R1). International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use. ICH harmonised tripartite guideline. Current Step 4 version, Parent Guideline dated 27 October 1994.  Back to cited text no. 11


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]

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