Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed.

OBJECTIVE: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga.
MATERIALS AND METHODS: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase.
RESULTS: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10-80 μg/mL in HPLC and 25-1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63-1.97%, 0.62-2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin.
CONCLUSIONS: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient.

Ammi visnaga Linn., annual herb is a dicot belonging to Apiaceae family. This plant commonly known as khella grows to approximately 120 cm in height, primarily in Egypt, other regions of the Middle East and the Mediterranean. A. visnaga L. has also been naturalized to parts of the southeastern America. Some of the major constituents of drug include furanochromones derivatives, mainly khellin (1.0%) and visnagin (0.3%).[1] Other constituents of khella include two furocoumarins namely xanthotoxin and ammidin.[2] The fruits of the plant have been used in Egyptian folk medicine as diuretics and for the treatment of kidney and bladder stones.[3] Khella also has been used for the traditional management of diabetes in Israel.[4] Khellin [Figure 1] is used as a spamolitic agent in the therapy of asthma and angina pectoris and recently its use has been proposed for the treatment of vitiligo[5] and psoriasis.[6] Studies on the photogenic and mutagenic activity of khellin have also been reported.[78] Khellin inhibits calcium influx without any difference related to the specific calcium channels. These actions on calcium influx and intracellular mobilization can contribute to its vasorelaxant action.[9] High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) are the widely accepted analytical techniques for their high accuracy, precision and reproducibility of results whereas, HPTLC has many advantages because of low operating cost and less time consuming. Various analytical methods for determining the khellin content in the plant samples have been reported including thin-layer chromatography (TLC),[10] gas chromatography,[11] ultraviolet (UV)-visible spectrophotometric determination,[12] Fluorometric determination,[13] capillary electrophoresis,[14] voltammetry,[15] HPLC[161718] and HPTLC.[19] UV-visible spectrophotometric methods are insufficient in determining an accurate measurement of the khellin due to interference from other constituents in the extracts. Moreover the separation of khellin is not good using these methods. The estimation of khellin, using capillary electrophoresis, voltametry also showed low resolution owing to poor reproducibility. There are few HPLC and HPTLC methods available for the analysis of khellin but these methods required lengthy run times and complicated gradient elution systems using solvent mixtures. With this background, simple, accurate, sensitive, rapid, and economic validated reversed phase (RP)-HPLC and HPTLC methods were developed for the quantification of this marker compound in the seeds of A. visnaga. These methods have been validated as per International Conference on Harmonisation (ICH) guidelines[20] similar to the methods reported by laboratory.[2122232425]

Figure 1

The chemical structure of khellin

Materials and Methods

Standard khellin was procured from Fluka, USA. The seed extract containing khellin was procured from Herb Pharm, USA. All other chemicals were of HPLC grade for HPLC analysis and analytical reagent grade for HPTLC analysis purchased from Merck Specialties Pvt. Ltd., Mumbai (India).

Preparation of khellin standard and sample solutions

A stock solution of standard khellin in methanol having concentration 1 mg/mL was for HPLC analysis and a concentration of 250 µg/mL was used for HPTLC analysis. Serially dilution of the above said stock solutions of khellin having different concentrations were used for linearity plot analysis.

Liquid herbal extract of khellin was dissolved in methanol to get proper dilution and filtered through a 0.20 µm nonsterile regenerated cellulose membrane (Sartorius AG, Germany) for quantification assay by HPLC and HPTLC.

Quantitative analysis of khellin by high-performance liquid chromatography

The HPLC system (SHIMADZU, Japan), consisted of a binary pump (model LC-10AT VP), a UV-visible detector (model SPD-10AVP), a rheodyne injector (model 77251) equipped with CLASS-VP software (version 6.14) was used for analysis. A reverse phase C-18 column (5 µm particle, 250 mm × 4.6 mm) from Lichrocart, Germany was used to separate khellin. The mobile phase consisted of methanol: Water in the ratio of 75:25 (v/v). The flow rate of the HPLC system was set at 1.0 mL/min and the run time was 10 min per sample. The applied volume of the sample was 20 µL and the detection of khellin was carried out at 247 nm.

Quantitative estimation of khellin by high-performance thin-layer chromatography

Sample solutions were applied on a semiautomatic TLC sampler Linomat V (CAMAG, Muttenz, Switzerland) controlled by winCATS software 1.4.4. The plates were developed in 20 cm × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland). A TLC scanner III was used for scanning the TLC plates. Precoated silica gel aluminium plates 60F254 (E. Merck, Darmstadt, Germany) with thickness 0.2 mm were used for all determinations. The plates were prewashed with methanol and activated at 60°C for 5 min prior to chromatography. Seven different concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.0 µL) of standard solution of khellin were applied on 20 cm × 10 cm TLC plate for the preparation of calibration curve of khellin. A constant application rate of 150 nL/s was employed for khellin with a band width of 5.0 mm. Linear ascending development was carried out in 20 cm × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland). The optimized chamber saturation time for mobile phase was 30 min at 25°C and relative humidity of 60%. The chromatogram was developed up to 85% of total TLC plate height. The scanning speed was employed at 20 mm/s khellin and the slit dimension was kept at 4.0 mm × 0.45 mm for khellin. Twenty millilitre of mobile phase consisted of ethyl acetate: toluene: Formic acid (5.5:4.0:0.5, v/v/v) was used per plate. The optimized chamber saturation time for mobile phase was 15 min at room temperature (25 ± 2° C) at relative humidity of 60 ± 5%. The plates were developed and scanned within 10 min using densitometric scanner III in the absorbance mode at 254 nm. The source of radiation was deuterium lamp emitting a continuous radiation between 200 and 400 nm. The data obtained were analyzed by winCATS software (CAMAG, Muttenz, Switzerland) to get linear regression equation.

Validation of chromatographic methods

Both the HPLC and HPTLC methods for the estimation of khellinwere validated as per the ICH guidelines[21] for linearity, accuracy, robustness, precision, limit of detection and quantification.

Linearity, precision and accuracy

For HPLC, linearity was evaluated by applying each concentration (10–80 µg/mL) for khellin in triplicates per sample and five such samples were evaluated (n = 3 × 5). For HPTLC, Linearity was evaluated by applying each concentration (25–1,000 ng/spot) for khellin in triplicates per sample and seven such samples were evaluated (n = 3 × 7) [Figure 2].

Figure 2

(a and b) Calibration plot of the khellin using different concentrations versus peak area for HPLC and HPTLC

For HPLC, system repeatability was determined by six replicate applications and six times measurement of a standard solution at the analytical concentration of 20, 40 and 50 µg mL-1 of khellin. The repeatability of sample application and measurement of peak area for active compound were expressed in terms of relative standard deviation (RSD). Method repeatability was obtained from RSD value by repeating the assay six times on the same day for intraday precision. Intermediate precision was assessed by the assay of three, six sample sets on different days (interday precision) and on different system (intersystem precision). The intraday, interday and intersystem variations for determination of khellin were carried out at three different concentration levels 20, 40 and 50 µg/mL.

For HPTLC, system repeatability was determined by six replicate applications and 6 times measurement of a standard solution at the analytical concentration of 200, 400 and 800 ng/spot of khellin. The intraday, interday and intersystem variations for determination of khellin were carried out at three different concentration levels 200, 400 and 800 ng/spot.

The accuracy of the methods was determined by doing recovery studies and the amount of the drug recovered was calculated on the basis of % RSD. For this, preanalyzed samples were spiked with 0, 50, 100 and 150% of the standard solution and the mixtures were reanalyzed by the proposed methods. The experiments were performed 6 times. This was done to check the recovery of the drug at different levels in the formulations. Recovery studies were carried out for the liquid samples of the khellin extract.

Robustness of the method

Robustness of the method was checked by introducing small changes in the mobile phase composition and the effects on the results were examined. Mobile phase having different compositions like methanol: Water (75:25 v/v) for HPLC and ethyl acetate: Toluene (6.5:3.5) for HPTLC were tried and chromatograms were analyzed. The amount of mobile phase was varied in the range of ± 0.2%. Robustness of the method was carried out at three different concentration levels 20, 40 and 50 µg/mL for HPLC and similarly, three different concentration levels 200, 400 and 800 ng/spot were used. The detection wavelength was also changed (±2 nm) and % RSD were determined and found to be less than 2%.

Results and Discussion

Optimized high-performance liquid chromatography method

For HPLC analysis, different trials were carried out using many solvents in different proportions. When mobile phase consisting of methanol: Water was used in the ratio of 65:35 v/v, a peak was observed at the Rt of 3.89 min for khellin with a poor resolution of peak. In order to improve the resolution of the peak a new mobile phase with the composition of methanol: Water was used in the ratio of 75:25 v/v. This new mobile phase helped in achieving very sharp peak at the Rt of 3.89 min for khellin with good resolution of more than one [Figure 3].

Figure 3

(a and b) high-performance liquid chromatography chromatogram of khellin in extract (a) and in standard (b)

Optimized high-performance thin-layer chromatography method

For HPTLC analysis, different trials were carried out using many solvents in different proportions. When mobile phase consisting of ethyl acetate: Toluene was used in the ratio of 6.5:3.5, a peak was observed with Rf of 0.65 for khellin. But it was found that the resolution of the peak was poor. In order to improve the resolution of the peak, a new mobile phase with the composition of ethyl acetate: Toluene: Formic acid was used in the ratio of 5.5:4.0:0.5 (v/v/v). This new mobile phase helped in achieving very sharp peak at the Rf of 0.65 for khellin with good resolution of more than one [Figure 4].

Figure 4

(a and b) High-performance thin-layer chromatography chromatogram of khellin in extract and standard

For HPLC, linearity was found between concentration range of 10–80 µg/mL for khellin with r2 ± standard deviation (SD) =0.999 ± 0.001. For HPTLC, linearity was found between concentration range of 25–1,000 ng/spot for khellin with r2 ± SD = 0.995 ± 0.001. Results of linearity assay were shown in Tables 1 and 2 for HPTLC and HPLC result respectively. The calibration curves of khellin using HPLC and HPTLC are presented in Figure 2. Both the calibration curve was linear over the various defined concentration ranges.

Table 1

Validation data for khellin by HPTLC

Table 2

Validation data for khellin by HPLC

Precision of the proposed HPTLC and HPLC methods were obtained by intraday, interday and intersystem variations for three different concentration levels are summarized in Tables 3 and 4. The low % RSD indicated that the method is precise for the analysis.

Table 3

Intermediate precision data of HPTLC for khellin

Table 4

Intermediate precision data of the proposed HPLC method of khellin

The method presented here yielded high khellin recovery and fractions that were readily analyzed by HPLC and HPTLC methods used for quantitative estimation. The results of the recovery studies of HPTLC and HPLC were depicted in Tables 5 and 6. These tables showing evidence that khellin could be recovered with higher efficiency by using both HPLC and HPTLC methods.

Table 5

Recovery data for khellin by HPTLC

Table 6

Accuracy as recovery data of the proposed HPLC method of khellin

Limit of detection and limit of quantitation

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank solution (methanol) was spotted six times following the same method as explained above. The signal to noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of reference solution until the average responses were approximately three or 10 times the standard deviation of the responses for six replicate determinations. For HPLC, the proposed method LOD and LOQ were calculated using signal to noise ratio method and found to be 3.1 and 9.4 µg/mL for khellin [Table 2]. For HPTLC, the proposed method LOD and LOQ were calculated using signal to noise ratio method and found to be 7.2 and 21.9 ng/spot for khellin [Table 1].

Specificity

The specificity of the proposed methods was established by analyzing standard drug and sample. The specificity of the newly proposed method was ascertained by superimposing the spectrum of both standard and sample peaks and confirmed for its purity [Figure 5]. The peak for khellin in the sample was confirmed by comparing Rt, Rf and UV spectra of peak with that of standard. The peak purity (90%) of khellin was assessed by comparing the spectra at three different levels that is peak start, peak apex and peak end positions of the spot. Purity of sample peak corresponding to khellin was determined by taking the spectra and by comparing it with that of standard. Figure 2 showed that both methods have good linear correlation with respect to various concentrations.

Figure 5

Superimposed overlay ultraviolet spectra of khellin in peaks of standard and extract at 247 nm

The result of robustness data as obtained by HPTLC [Table 7] and HPLC [Table 8] showed low % RSD (<1.54) indicating robustness of method.

Table 7

Robustness data of the proposed HPTLC method of khellin

Table 8

Robustness data of the proposed HPLC method of khellin

Analysis of samples

Figures 3 and 4 showed that, there was no interference in sample (having immediate impurities) analysis through HPLC and HPTLC respectively, with respect to have a good resolution between peaks.

Conclusions

HPLC and HPTLC methods were developed and validated for quantitative estimation of khellin and the contents of this marker present in A. visnaga seeds were quantified and found to be 3.04% w/v in HPLC and 0.37% w/v in HPTLC. These methods were found to be simple, rapid, accurate, specific and robust for the analysis of khellin in crude drug and can be adopted by any laboratory for the quality control of crude drugs and formulations that contains khellin as active marker.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.