Acta Chromatographica 24(2012)4, 603–613

DOI: 10.1556/AChrom.24.2012.4.7

High-Performance Thin-Layer Chromatographic

Analysis of Psoralen in Marketed Formulations and
Manufactured Solid Lipid Nanoparticles (SLNs):
Validation of the Method
N. AKHTAR1, MD. FAIYAZUDDIN1,2,*, G. MUSTAFA1, Y. SULTANA1,
S. BABOOTA, AND J. ALI1
1Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University,
New Delhi-110062, India
2Faculty of Pharmacy, Integral University, Lucknow-226026, Uttar Pradesh, India
*E-mail: md.faiyazuddin@yahoo.co.in

Summary. A quantitative method using precoated silica gel-60 Lichrosphere high-

performance thin-layer chromatography (HPTLC) plates, automated band wise sample
application, and n-hexane:acetone:formic acid (2:1:0.025 v/v/v) as mobile phase, has
been developed and validated for the analysis of psoralen in marketed formulations and
novel solid lipid nanoparticles (SLNs). Densitometric analysis was performed at 250 nm
in absorbance mode. Compact bands of psoralen were obtained at Rf 0.32 ± 0.02. The
method was validated for linearity, precision, robustness, sensitivity, specificity, and re-
covery. Linearity (r2 = 0.995), limit of detection (8.0 ng band−1), limit of quantification
(18.1 ng band−1), recovery (98.06–99.64%), and precision (≤0.74) were satisfactory. Statis-
tical analysis established that the developed method for quantification of psoralen in
marketed formulation and from solid lipid nanoparticles is reproducible and selective.

Key Words: psoralen, Babchi oil, high-performance thin-layer chromatography, valida-

tion, solid lipid nanoparticles

Introduction
Psoralen (PS), 7H-furo [3,2-g] [I] benzopyran-7-one (Fig. 1), is a tricyclic fu-
rocoumarin obtained from different species of Rutaceae, Umbelliferae, and
Compositae families, as well as synthesized commercially having potent pho-
tosensitizing properties. It is the functional constituent of Babchi oil (BO;
Psoralea corylifolia) [1]. In photochemotherapeutics, psoralens play a consid-
erable role, particularly in skin disorders characterized by hyperprolifera-
tion such as psoriasis which is effectively treated by means of PUVA ther-
apy (psoralen-plus-UVA light) [2]. Psoralen applied topically makes the
skin much more light sensitive [3]. Modern photochemotherapy involves
oral or topical administration of a photosensitizing psoralen followed by
exposure to long-wavelength (320–400 nm) UVA irradiation [4]. Aforemen-
tioned, psoralens are a group of compounds that bind to DNA in the rapid
cells in the presence of UV light in the A band causing DNA crosslinking
and thus prevent cellular division [5–7]. A variety of methods has been re-
0231–2522 © 2012 Akadémiai Kiadó, Budapest

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604 N. Akhtar et al.

ported for the analyses of psoralens including high-performance liquid

chromatography [8–10] and gas chromatography [11]. However, to our
knowledge, no validated high-performance thin-layer chromatography
(HPTLC) method is available for estimation of psoralen in marketed formu-
lation and lipid nanoparticles. Therefore, the objective of this study was to
develop and validate a rapid, simple, sensitive, and reproducible HPTLC
method for the quantitative determination of psoralen in marketed formula-
tions, SLNs as well as in formulations containing psoralen as it facilitates
automated application and scanning in situ along with repeated detection
of the chromatogram using different parameters.

Fig. 1. Chemical structure of psoralen

Materials and Methods

Psoralen was purchased from Sigma–Aldrich Chemicals (Bangalore, India).
All other chemicals and reagents used were of analytical grade and were
purchased from Merck Chemicals (Mumbai, India). Precoated silica gel
Lichrosphere aluminum sheets 60 F254 (20 cm × 10 cm: 200-μm thickness)
were purchased from E. Merck (Darmstadt, Germany).

Calibration

A stock solution of 100 μg mL–1 of standard psoralen was prepared in

methanol. Different volumes of the stock solution (0.2, 0.4, 0.5, 1, 1.5, and
2 μL) were applied in triplicate to a plate to furnish 20, 40, 50, 100, 150, and
200 ng psoralen band−1, respectively. Peak area data and the corresponding
amounts were treated by linear least-square regression analysis.

Chromatography
Chromatography was performed, as reported previously [12, 13], on
20 cm × 10 cm aluminum HPTLC plates precoated with 200-μm layers of sil-
ica gel 60 F254 (E. Merck, Darmstadt, Germany). Samples were applied as
bands 3 mm wide and 6.2 mm apart by means of CAMAG (Deutschland,

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HPTLC Analysis of Psoralen 605

Switzerland) Linomat V sample applicator equipped with a 100-μL syringe.

The constant application rate was 90 nL s−1. Linear ascending development
with n-hexane:acetone:formic acid (2:1:0.025 v/v/v) as mobile phase was
performed in a 20 cm × 10 cm twin trough glass chamber (CAMAG) previ-
ously saturated with mobile phase for 20 min at room temperature
(25 ± 2 °C) and relative humidity 60 ± 5%. The development distance was
8 cm (development time 10 min), and 20 mL of mobile phase was used.
Densitometric analysis was performed at 250 nm with a CAMAG TLC
Scanner III using deuterium and tungsten lamp, operated by WinCATS
software (Version 1.2.0). The slit dimensions were 4 mm × 0.1 mm, and the
scanning speed was 20 mm s−1.

Method Validation
The developed method was validated as per ICH guidelines by determining
linearity, range, precision, robustness, limits of detection (LOD) and quanti-
fication (LOQ), specificity, and recovery.

Precision and Accuracy

Precision (inter- and intra-day) and accuracy of the assay were evaluated by
performing replicate analyses (n = 6) of QC samples at low, medium, and
high QC levels of 200, 400, and 2000 ng band−1. Inter-day precision and ac-
curacy were determined by repeating the intra-day assay on three different
days. Precision was expressed as the coefficient of variation (CV, %) of
measured concentrations for each calibration level whereas accuracy was
expressed as percentage recovery [(PS found/PS applied) × 100].

Robustness
Robustness was studied in triplicate at 400 ng band−1 by making small
changes to mobile phase composition, mobile phase volume, and duration
of mobile phase saturation and activation of TLC plates; the effect on the re-
sults were examined by calculation of relative standard deviation (RSD) (%)
and standard error (SE) of peak areas. Mobile phases prepared from n-
hexane:acetone:formic acid in different proportions (2.1:0.90:0.025 v/v/v
and 1.9:1.10:0.025 v/v/v) were used for chromatography. Mobile phase vol-
ume and duration of saturation investigated were 20 ± 2 mL (18, 20, and
22 mL) and 20 ± 10 min (10, 20, and 30 min), respectively. The plates were
activated at 60 ± 5°C for 2, 5, and 7 min before chromatography.

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606 N. Akhtar et al.

Limits of Detection (LOD) and Quantification (LOQ)

To estimate the limits of detection (LOD) and quantification (LOQ), blank
methanol was applied six times, and the standard deviation (σ) of the ana-
lytical response was determined. The LOD was expressed as 3.3σ/slope of
the calibration plot for psoralen, and LOQ was expressed as 10σ/slope of
the calibration plot.

Recovery Studies
Recovery was studied by applying the method to drug samples to which
known amounts of psoralen corresponding to 50, 100, and 150% of the
psoralen had been added. Each level was analyzed in triplicates. This was to
check the recovery of psoralen at different levels in the formulations. Re-
covery of the drug at different levels in the samples was determined.

Specificity
The specificity of the method was assessed by analyzing and comparing the
Rf values and spectra of the psoralen band from a sample with that from a
standard. The peak purity of the psoralen bands was assessed by comparing
spectra acquired at three different positions on the band—the peak start (S),
peak apex, (M), and peak end (E).

Analysis of Psoralen in Marketed Formulations

Two marketed formulations, (i) Roghan Babchi (Batch no. #U-212/78-01;
Hamdard Wakf Laboratories, India) and (ii) Bakuchi Tail (Batch no.
#25D/11/93-0023; Vyas Pharmaceutical, India), were taken to analyze
psoralen content by developed HPTLC method. Approximately 10 mL of
both the marketed products were transferred to a 100-mL volumetric flask
with 50 mL methanol. The mixture was sonicated for 30 min then diluted to
volume with methanol. The resulting solution was centrifuged at 1500 rpm
for 5 min and filtered, and 2 μL of the solution was applied to a plate for
analysis. The analysis was repeated in triplicate.

Analysis of Psoralen in Prepared SLNs

The SLN was prepared by hot aqueous titration, as published earlier [14]. In
brief, an accurately weighed quantity of SLNs (equivalent to approximately
10 mg psoralen) was extracted with 50 mL methanol by sonication for

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HPTLC Analysis of Psoralen 607

30 min. The resultant was centrifuged at 12,000 rpm for 15 min. The super-
natant was filtered, and the filtrate was dried to constant weight at room
temperature. The residue was redissolved in 100 mL methanol. The filtered
solution (2 μL) was applied to a TLC plate followed by development and
scanning. The analysis was repeated in triplicate. The possibility of interfer-
ence of excipients with the analysis was studied.

Results and Discussion

Development of Optimum Mobile Phase
TLC procedure was optimized with the intention of developing a cost effec-
tive validated assay for quantification of psoralen in marketed formulations
and prepared SLNs. Varying ratios of n-hexane, acetone, and formic acid
were investigated for their suitability as the mobile phase. Initially, n-
hexane and acetone in 3:1 v/v were tried as mobile phase. A well defined
peak of psoralen was obtained but this mobile phase was unable to give bet-
ter resolution and distinct bands. Addition of formic acid and reduction of
the quantity of highly polar hexane improved the band characteristics. A ra-
tio of 2.5:0.75:0.025 v/v/v for n-hexane, acetone, and formic acid gave good
resolution with Rf 0.32 for psoralen but showed band broadening at 250 nm.
Of these, n-hexane–acetone–formic acid (2:1:0.025 v/v/v) was found to fur-
nish sharp and well resolved symmetrical peaks, and so this mobile phase
was selected. Well-defined bands were obtained when the chamber was
saturated with mobile phase for 20 min at room temperature.

Calibration
Table I summarizes data for the calibration curves analyzed by linear regres-
sion. Linear regression data for calibration plots for PS (n = 6) were indica-
tive of a good linear relationship (correlation coefficient, r2 = 0.995) between
Table I. Linear regression data for the calibration curve (n = 6)

Parameter Values
Linear range (ng band−1) 20–200
Regression equation Y = 14.644x + 248.06
Correlation coefficient (r2) 0.9956
Slope ± SD 14.64 ± 0.94
Intercept ± SD 248.0 ± 2.34
Standard error of slope 0.54
Standard error of intercept 1.35
95% Confidence interval of slope 16.96–12.32
95% Confidence interval of intercept 242.25–253.86

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608 N. Akhtar et al.

peak area and amount in the range 20–200 ng band−1 (Fig. 2). The mean
values (±SD) of the slope and intercept were 14.64 ± 0.94 and 248.0 ± 2.34,
respectively. No significant difference was observed in the slopes of stan-
dard plots (analysis of variance (ANOVA), P > 0.05).
3500
y= 14.644x+ 248.06
3000 2
3091.1
R = 0.9956
2500 2544.3

2000
Area

1734.5
1500
1000 982.2
865.8
500 470.9
0
0 50 100 150 200 250
Concentration (ng spot−1)
Fig. 2. Calibration curve for standard psoralen (20−200 ng band−1)

Method Validation
Precision and Accuracy
Intra-day and inter-day precisions, as coefficient of variation (CV, %) and
the accuracy of the assay, determined for PS concentrations of 500 and
1000 ng per band, are summarized in Table II. Intra-day precision (n = 6)

Table II. Precision and accuracy of the method (n = 6)

Concentration
Nominal concentration Precisionb Accuracyc
Mean area founda
(ng band−1) (CV, %) (%)
(ng band−1)
Intra-day batch
200 (Low QC sample) 3169.4 195.5 1.97 99.1
400 (Medium QC sample) 6138.3 398.6 1.42 99.6
2000 (High QC sample) 23,760.1 2007.4 1.73 100.3
Inter-day batch
200 (Low QC sample) 3252.13 197.3 2.45 98.6
400 (Medium QC sample) 6256.38 402.8 1.54 100.7
2000 (High QC sample) 22,865.77 1998.4 1.74 99.9
aMeanfrom six determination (n = 6).
bPrecision
as coefficient of variation (CV, %) = [(standard deviation)/(concentration
found)] × 100.
cAccuracy = [(concentration found)/(nominal concentration)] × 100.

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HPTLC Analysis of Psoralen 609

was ≤1.97%, and inter-day precision was ≤2.45%. Intra-day and inter-day
accuracies were 99.1–101.3 and 98.6–99.9%, respectively. When the repeat-
ability of the method was studied by assaying six samples of PS, at the same
concentration under the same experimental conditions, the coefficient of
variation was 1.09%. These values are within the acceptable range, so the
method was accurate, reliable, and reproducible.

Robustness
The CV (%) and SE of the peak area were calculated, in triplicate, for
changes in mobile-phase composition, mobile-phase volume, and duration
of saturation and activation of TLC plates for 400 ng band−1. The low values
of % CV (<2.03) and SE (<15.01) obtained after introducing small deliberate
changes in the method indicated that the method was robust (Table III).
There was no significant variation in the slope values (ANOVA, P > 0.05).

Table III. Robustnessa of the method

Optimization condition % CV SE

Mobile phase [(n-hexane–acetone–formic acid)

1.35 15.01
(2.1:0.90:0.025 v/v/v and 1.9:1.10:0.025 v/v/v)]

Mobile-phase volume (18, 20, and 22 mL) 1.57 10.70

Duration of saturation (10, 20, and 30 min) 2.03 13.38

Activation of TLC plates (2, 5, and 7 min) 1.13 1.38

aThree replicates were taken in each analysis (n = 3) at QC level of 400 ng band−1.

LOD and LOQ

LOD and LOQ were 8.0 and 18.1 ng band−1, respectively, indicating ade-
quate assay sensitivity. The LOD and LOQ were determined from the slope
of the lowest part of the calibration plot.

Recovery Studies
When the method was used for analysis of PS after spiking with 50, 100, and
150% additional drug, recovery ranged from 98.06 to 99.64% as indicated in
Table IV. The RSD of recovery was ranged between 1.37 and 1.81%.

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610 N. Akhtar et al.

Table IV. Recovery studies of psoralen from samples of known concentration (n = 6)

Excess drug
Theoretical Added Detecteda Recoverya RSD
added to the SE
(ng) (ng) (ng) (%) (%)
analyte

50% 120 118.4 98.7 1.81 0.24

100% 80 160 158.4 99.0 1.56 0.26
150% 200 199.2 99.6 1.37 0.28
aMean from six determinants.

Specificity
The spectra of psoralen bands were found to be superimposed on each
other. The purity of the psoralen band was assessed by comparing spectra
acquired at the peak start, peak apex, and peak end positions of the band.
Good correlation (r2 = 0.999) was obtained between the spectra of standard
psoralen and the spectra of marketed formulations and prepared SLNs
(Fig. 3).

Fig. 3. Overlaid absorption spectra of standard psoralen and psoralen of marketed

formulations and SLNs by HPTLC scanner

Analysis of Psoralen in Marketed Formulations

Analysis of psoralen in two marketed formulations was quantified by de-
veloped and validated method of HPTLC. Two microliters (2 μL) of the fil-
tered solution was applied on to the TLC plate followed by development
and scanning. Quantity of psoralen in marketed formulations is depicted in
Table V. Chromatogram was obtained from one of the marketed formula-

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HPTLC Analysis of Psoralen 611

tions of psoralen; Bakuchi Tail, Lic. No. (Batch no. #25D/11/93-0023), Vyas
Pharmaceutical, Indore, India) was at analogous Rf as obtained from stan-
dard psoralen (Peaks 1–5 belong to different components present in the
formulation); Peak 3: psoralen (Rf: 0.32) (Fig. 4).

Fig. 4. Chromatogram obtained from Bakuchi Tail (Batch no. #25D/11/93-0023, Vyas
Pharmaceutical, Indore, India) (Peaks 1–5 belong to different components present in the
formulation); Peak 3: psoralen (Rf : 0.32)

Table V. Quantification of psoralen in SLNs and marketed formulations

Quantity of psoralen
Formulations Mean areaa ±SD
(μg mL−1)

SLNs 1 2434.1 ± 357.11 74.66 (w/v)

SLNs 2 2567.9 ± 108.33 79.20 (w/v)
Roghan Babchi 3653.1 ± 115.18 116.29 (v/v)
Bakuchi Tail 4122.7 ± 247.88 132.33 (v/v)

aMean of from three determinants (n = 3)

Analysis of Psoralen in Prepared SLNs

Analysis of psoralen in prepared SLNs was quantified by developed and
validated method of HPTLC. Quantity of psoralen in SLNs formulations is
depicted in Table V. Chromatogram obtained from psoralen SLNs
(200 ng band−1; Rf: 0.32 ± 0.02) was observed in similar fashion as that of
standard psoralen. There was no interference from excipients or other active
components (Fig. 5).

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612 N. Akhtar et al.

Fig. 5. Chromatogram obtained from psoralen solid lipid nanoparticles

(200 ng band−1; Rf: 0.32 ± 0.02).

Conclusion
HPTLC method for the determination of psoralen was developed using n-
hexane:acetone:formic acid (2:1:0.025 v/v/v) as mobile phase. Peak areas of
the densitogram were quantified by densitometer at 250 nm. Statistical
analysis of the data proved the method to be precise, specific, accurate, re-
peatable, and selective for the analysis of psoralen in marketed as well as in
developed SLNs formulations. Recovery values of psoralen were found to
be about 98.8%, which showed the reliability and suitability of the method.

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HPTLC Analysis of Psoralen 613

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Accepted by DA

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