JPC - Jurnal Kromatografi Planar - TLC modern (2020) 33:71 - 77

https://doi.org/10.1007/s00764-019-00006-y

KERTAS PENELITIAN ASLI

Pengembangan dan validasi metode HPTLC sederhana

untuk menentukan agen antivirus subtipe 4 hepatitis C baru dalam bentuk sediaan
tabletnya

Mahmoud M. Elkhoudary 1,2 & Basma M. Selim 2 & Randa A. AbdelSalam 3 & Ghada M. Hadad 3 & Alaa El-Gindy 3

Dipublikasikan secara online: 21 Februari 2020

# Akadémiai Kiadó, Budapest, Hongaria 2020

Abstrak
Metode kromatografi lapis tipis (HPTLC) berkinerja tinggi yang baru, sederhana, tepat, akurat dan selektif telah dikembangkan dan divalidasi untuk
penentuan ledipasvir dan sofosbuvir secara bersamaan dalam bentuk sediaan tabletnya. Kromatografi
pemisahan dilakukan pada lembaran aluminium Merck TLC dari silika gel 60 F 254 menggunakan etil asetat: heksana: metanol dengan perbandingan 8: 1,25: 0,75 (% v / v) sebagai
fase gerak diikuti dengan pengukuran densitometri pada 256 nm. Metode ini divalidasi dalam istilah
linearitas, akurasi, presisi, batas deteksi, batas kuantifikasi dan spesifisitas sesuai dengan pedoman International Conference onHarmonization (ICH).
Kurva kalibrasi ditemukan linier antara 60 hingga 1980 dan 45 hingga 3600 ng / band untuk ledipasvir dan sofosbuvir, masing-masing, dengan nilai
koefisien regresi yang sangat tinggi ( r 2> 0,9999) dengan residu linier dan homoscedastic. Batas deteksi dan penghitungan ditemukan masing-masing 16,5
dan 50 ng / band, untuk ledipasvir dan 13 dan 39,5 ng / band, untuk sofosbuvir. Studi perbandingan dilakukan antara metode HPTLC yang dikembangkan
dan metode kromatografi cair kinerja tinggi (HPLC) yang dilaporkan. Hasil kuantitatif dari kedua metode analisis tidak menunjukkan perbedaan yang
signifikan secara statistik, sedangkan metode HPTLC yang dikembangkan efektif waktu dan biaya.

Kata kunci HCV. HPTLC. Ledipasvir. Sofosbuvir. MPIVIROPACKPLUS

pengantar beberapa subtipe berdasarkan analisis filogenetik dan sekuens dari

seluruh genom virus [ 3 ]. Genotipe 4 adalah varian paling umum dari virus
Hepatitis C adalah masalah kesehatan global. Seperti yang dilaporkan Organisasi hepatitis C di Timur Tengah dan Afrika, khususnya Mesir [ 4 ].
Kesehatan Dunia (WHO), 3 - 4 juta orang baru terinfeksi virus hepatitis C (HCV)
setiap tahun dan 130 orang - 170 juta orang terinfeksi secara kronis. Selain itu, Tujuan akhir pengobatan HCV kronis adalah untuk mencapai pemberantasan virus jangka

lebih dari 350.000 orang meninggal setiap tahun akibat penyakit hati terkait panjang, yang paling baik diprediksi dengan mendorong tanggapan virologi berkelanjutan (SVR

hepatitis C [ 1 ]. HCV adalah virus RNA beruntai tunggal kecil, terbungkus, dan atau SVR24) yang didefinisikan sebagai HCVRNA tidak terdeteksi dalam darah 24 minggu

beruntai tunggal yang termasuk dalam famili Flaviviridae; Infeksi HCV dikaitkan setelah penghentian pengobatan [ 5 ]. Lebih dari 99% pasien yang mencapai SVR memiliki viral

dengan perkembangan karsinoma hepatoseluler (HCC) [ 2 ]. HCV diklasifikasikan load tidak terdeteksi selama setahun setelah pengobatan, menjadikan SVR sebagai indikator

menjadi tujuh genotipe dengan penyembuhan yang kuat [ 6 ]. SVR12, HCVRNA yang tidak terdeteksi dalam darah 12 minggu

setelah penghentian pengobatan, baru-baru ini juga telah terbukti mengidentifikasi mereka yang

sembuh dari HCV.

* Mahmoud M. Elkhoudary Ledipasvir (LED) dan sofosbuvir (SOF) keduanya mengarahkan agen
mme-87@hotmail.com antivirus [ 7 ] (Gambar. 1 ). SOF adalah prodrug nukleotida bertarget hati dari
trifosfat aktif GS-461203, yang telah disetujui untuk digunakan dalam genotipe
1
Departemen Kimia Farmasi, Fakultas Farmasi, Universitas Horus-Mesir, HCV 1 - 4. Ia bekerja sebagai penghambat RNA polimerase yang bergantung
Damietta Baru, Mesir pada RNA NS5B, yang bertindak sebagai terminator rantai [ 8 , 9 ]. LED adalah
2
Departemen Kimia Farmasi, Fakultas Farmasi, Universitas Sinai, Arish, inhibitor NS5A yang efektif melawan genotipe 1a, 1b, 4a, dan 5a dan (dengan
Sinai Utara, Mesir aktivitas lebih rendah) melawan genotipe 2a dan 3a. Mekanisme kerjanya yang
3
Departemen Kimia Analitik Farmasi, Fakultas Farmasi, Universitas tepat tidak diketahui, tetapi satu
Terusan Suez, Ismailia, Mesir
72
Gambar 1 Struktur a) ledipasvir (LED) dan b) sofosbuvir (SOF)
mekanisme yang disarankan adalah penghambatan hiperfosforilasi NS5A,
yang tampaknya diperlukan untuk produksi virus. Penghambat NS5A juga
dapat menyebabkan kesalahan perakitan virus dengan mendistribusikan
kembali lokalisasi sub-seluler protein. Inhibitor NS5A dan NS5B
menunjukkan efek aditif atau sinergis bila digunakan dalam kombinasi [ 7 ].
Kombinasi kedua obat ini adalah pengobatan yang efektif dan aman untuk
berbagai subtipe HCV 4, termasuk mereka dengan sirosis kompensasi [ 10 ].
Penapisan literatur mengungkapkan bahwa SOF ditentukan dengan
kromatografi cair kinerja tinggi fase terbalik (RP-HPLC) dalam bentuk sediaan
curah dan tablet, serta dengan kromatografi cair kinerja-ultra. - spektrometri
massa tandem (UPLC - MS / MS) hanya dalam plasma manusia [ 11 ] atau
sebagai standar internal (IS) dalam penentuan daclatasvir [ 12 ] atau dengan
metabolitnya [ 13 ] dan dalam plasma tikus dengan metabolitnya [ 14 ]. LED
ditentukan dengan spektrofotometri ultraviolet (UV) sederhana [ 15 ] dan
dengan RP-HPLC [ 16 ] dalam bentuk sediaan curah dan tablet. Baik SOF dan
LED telah diukur dengan metode spektrofotometri [ 17 ], elektroforesis kapiler [ 18
], RP-HPLC dalam bentuk sediaan tablet [ 19 ], UPLC - Metode MS / MS dalam
plasma manusia [ 20 ] atau dengan beberapa agen antivirus [ 21 ] atau dengan
metabolitnya dalam plasma tikus [ 22 ].
Karena penyebaran infeksi HCV di seluruh dunia dan oleh karena itu
pentingnya kombinasi LED dan SOF dalam farmasi, penting untuk
mengembangkan metode kromatografi yang sangat sensitif, sederhana dan
dapat diandalkan untuk analisis obat-obat ini dalam formulasi binernya.
Survei literatur mengungkapkan bahwa dua artikel diterbitkan membahas
pemisahan LED dan SOF menggunakan kromatografi lapis tipis kinerja
tinggi (HPTLC) [ 23 , 24 ]. Yang pertama mengembangkan uji indikator
stabilitas LED dan SOF dengan adanya produk degradasi [ 23 ]. Namun,
pemisahan SOF dilakukan pada
JPC-J Planar Chromat (2020) 33:71 - 77
Rf = 0,21 yang cukup dekat dengan garis start, tidak mencapai pemisahan yang
cukup dari komponen yang dianalisis terutama dalam jenis analisis yang
menunjukkan stabilitas. Selain itu, integritas perhitungan rentang linieritas yang
diusulkan, batas deteksi (LOD) dan batas kuantifikasi (LOQ) sangat diragukan.
Artikel kedua memisahkan dua bentuk sediaan biner, salah satunya berisi LED
dan SOF [ 24 ]. Akan tetapi, metode yang digunakan sistem pelarut kuaterner
untuk pemisahan obat-obat ini yang secara praktis sulit untuk dibuat dan
dipertahankan dengan komposisi yang konstan bila dibuat beberapa kali selama
proses analisis. Jadi, demi kesederhanaan dan reproduktifitas, tujuan kami
adalah untuk mengembangkan dan memvalidasi metode HPTLC untuk analisis
LED dan SOF dalam bentuk murni dan tablet gabungannya. Metode yang
dikembangkan bertujuan untuk menyediakan alternatif yang lebih sederhana dan
biaya yang relatif lebih rendah dari metode yang saat ini diterbitkan. Metode yang
diusulkan berhasil menentukan campuran obat secara sederhana, reliabel, dan
tervalidasi penuh sehingga menghemat waktu dan tenaga.
Eksperimental
Bahan
Sampel murni
LED dan SOF dibeli dari Beijing Mesochem Technology Co., Ltd., Beijing,
China, dengan klaim kemurnian
99,63 dan 99,80% masing-masing sesuai dengan sertifikat perusahaan.
Formulasi farmasi
MPIVIROPACK PLUS ® tablet salut selaput diberi label mengandung 400
mg SOF dan 90 mg LED (Batch No. 1633474), diproduksi oleh Marcyrl
Pharmaceutical Industries, El Obour City, The West Extension, Block
20.005, Mesir.
Bahan kimia dan reagen
Etil asetat, heksana, kalium dihidrogen ortofosfat, 1-natrium heksana
sulfonat, metanol dan etanol adalah kelas analitik, dibeli dari Sigma-Aldrich
(St Louis, MO, USA). Asetonitril, metanol, dan air HPLCgrade dibeli dari
Laboratorium Riedel-de-Haën
Bahan Kimia, Seelze, Jerman). Silika gel 60 F. 254 Pelat HPTLC dibeli dari
Merck (Darmstadt, Jerman).
Peralatan
Analisis HPTLC dilakukan dengan menggunakan sistem HPTLC CAMAG
terkomputerisasi (CAMAG, Muttenz, Swiss) yang terdiri dari sistem
pengiriman otomatis
JPC-J Planar Chromat (2020) 33:71 - 77
(TLC Linomat IV), UV densitometer (TLC Scanner II). dan jarum suntik
mikro (jarum suntik Linomat 659.004, Hamilton, Bonaduz, Swiss; CAMAG).
Data disimpan dan diproses dengan perangkat lunak yang sesuai (Cats 3
melalui antarmuka RS232).
Kondisi kromatografi
For optimal sensitivity of the HPTLC method, solutions of the testing
samples and standard were applied to the HPTLC
plates (aluminum plates precoated with silica gel 60 F 254) as
Fig. 2 2D and 3D HPTLC
chromatograms of laboratory-
prepared mixture of 400 ng/band LED
and 1600 ng/band SOF
73
pita daripada bintik-bintik menggunakan CAMAG Linomat IV. Pita memiliki
panjang 4 mm dan panjang 10 μ L sampel diaplikasikan ke setiap band. Pita
dipisahkan dengan jarak 8 mm dan 10 mm dari dasar pelat. Ruang
pengembangan sudah jenuh dengan fase gerak. Pelat HPTLC
dikembangkan dengan cara menaik dengan etil asetat: heksana: metanol
dengan perbandingan 8: 1,25: 0,75 (% v / v) sebagai fase bergerak. Setelah
dikembangkan pada jarak 8 cm, pelat HPTLC dikeringkan dengan udara
dan dipindai λ = 256 nm. Panjang dan lebar scan disesuaikan untuk
menutupi seluruh band.
74 JPC-J Planar Chromat (2020) 33:71 – 77

Preparation of standard solutions Limit of detection and limit of quantification

Stock standard solutions of LED and SOF were prepared separately by
dissolving 10 mg of LED and 30 mg of SOF in 100 mL volumetric flask then
completed to the mark with ethanol. 1 mL aliquots were transferred from
each stock solution and diluted with ethanol to 10 mL. The standard
working solutions were prepared by diluted stock standard solutions by
dilution with ethanol to reach concentration ranges of 60 –
According to the International Conference on Harmonization (ICH)
recommendations [ 25 ] the approach based on the S.D. of the response and
the slope was used for determining the limits of detection and quantification.
Specificity

1980 ng/band for LED and 45 – 3600 ng/band for SOF. The specificity of the method was performed by testing possible
interferences in the presence of placebo against standard compounds [ 25 ].

Preparation of sample solutions

MPIVIROPACK PLUS ® tablets were accurately weighed and the average

weight was calculated. Then 10 tablets were finely powdered in a mortar and Results and discussion
an amount equivalent to one tablet content (90 mg LED and 400 mg SOF)
was dissolved in ethanol then transferred into 100 mL volumetric flask. To Chromatographic methods
ensure complete extraction of the drug, it was sonicated for 15 min, left to
cool then the volume was completed to 100 mL ethanol and filtered through This technique offers an easy way to quantify on TLC plate directly by

0.45 μ m membrane filters (Millipore, Burlington, MA, USA). Further dilutions measuring the optical density of the separated bands. The amounts of

were performed with ethanol to reach the calibration range of each compounds are determined by comparing the peak area of the unknown

compound. band to a standard curve from reference materials chromatographed

simultaneously under the same conditions.

Experimental conditions, such as mobile phase and wavelength of

Method validation
Linearity
Separate solutions of each drug concentration were prepared by weighing
appropriate amount of each drug thenmaking the needed dilution as
described previously then each solution was applied to the HPTLC plate [ 25 ].
Triplicate applications were made for each solution. The plate was
developed using the previously described mobile phase. The peak area was
plotted against the corresponding concentration to obtain the calibration
graph.
detection were optimized to provide accurate, precise and reproducible results
for simultaneous determination of LED and SOF. The scanning wavelength
was 256 nm for both LED
and SOF, where the measured SOF peak signal ( λ max = 258 nm) was
sufficiently large to compensate for its inferior sensitivity
over LED peak signal. An acceptable difference between the Rf values of
the two compounds with minimum tailing were obtained by using the
mobile phase consisting of ethyl acetate:hexane:methanol in the ratio of
8:1.25:0.75 (% v/v).
Addition of methanol to this system was important to avoid tailing of the more
polar SOF and to improve its peak sharpness. The developed HPTLC method
gave a good resolution of LED and SOF with sharp and symmetrical peaks
(Fig. 2 ).

Precision
Table 1 Assay parameters and regression characteristics for determination of LED and SOF by
HPTLC method
Repeatability of the method was achieved by using three different
concentrations at three different levels for LED and SOF, then the samples Regression parameters LED SOF

were analyzed three times within the same day using our proposed HPTLC
Regression coefficient ( r 2) 0.9999 0.9999
method (intra-day) and on three different days (inter-day) [ 25 ].
Calibration range [ng/band] 60.00 – 1980.00 45.00 – 3600.00
Detection limit (LOD) [ng/band] 16.50 13.00

Quantification limit (LOQ) [ng/band] 50.00 Slope ± SD 39.50

Accuracy 7.30 ± 0.03 3.89 ± 0.05

Confidence limit of the slope 7.23 – 7.37 4.00 – 4.03

Known amounts of the studied compounds were added to a known Intercept ± SD 264.60 ± 36.60 123.00 ± 42.85
concentration of the commercial pharmaceutical tablets (standard addition Confidence limit of the intercept 170.63 – 358.63 − 8.73 – 72.87
method) within the pre-specified linearity range [ 25 ]. Number of points 7 7
JPC-J Planar Chromat (2020) 33:71 – 77 75

Table 2 Intra-day and inter-day precision

of LED and SOF standard solutions by Compound Concentration [ng/band] Intra-day precision Inter-day precision

HPTLC method
Recovery%± S.D. RSD% Recovery%± S.D. RSD%

LED 180.00 99.79 ± 0.56 0.56 99.09 ± 0.80 0.81

1260.00 99.76 ± 0.71 0.71 98.99 ± 0.37 0.77

1980.00 99.94 ± 1.18 0.79 98.75 ± 0.68 0.99

SOF 160.00 99.52 ± 0.75 0.46 97.79 ± 1.26 1.04

1200.00 99.63 ± 0.13 0.33 100.14 ± 0.05 0.85

3600.00 99.46 ± 0.04 0.54 99.64 ± 0.19 0.89

Validation of the method Precision

The developed method was validated in accordance with the ICH
guidelines. The parameters assessed were linearity, precision, accuracy,
limit of detection (LOD), limit of quantification (LOQ) and specificity.
Linearity and range
Three samples were prepared as described in the ‘ Experimental ’
section and analyzed for each drug on the same day and three different days
and the % RSD of the mean measured concentration was calculated. The
results of the repeatability and intermediate precision experiments are shown
in Table 2 . The developed method was found to be precise as the coefficient
of variation (RSD%) values for repeatability and intermediate precision studies
were < 2%, respectively.

The linearity of LED and SOF was evaluated by analyzing a series of

different concentrations of each compound. Seven concentrations were
chosen, ranging between 60 and 1980 ng/band for LED and 45 – 3600
ng/band for SOF. Each concentration was repeated three times to provide
information on the variation in peak area values between samples of the
same concentration. The assay was performed according to experimental
conditions previously established. The linearity of the calibration graphs was
validated by the high value of the regression coefficient ( r 2) ( Table 1 ) in
addition to linear and homoscedastic residuals.
Accuracy
Accuracy study was performed by addition of known amounts of LED and
SOF to a known concentration of the commercial tablets (standard addition
method). The resulting mixtures were assayed and the results obtained for
LED and SOF were compared with expected results. The excellent
recoveries of standard addition method (Table 3 ) (Fig. 3 ) indicate high
accuracy of the proposed method.

Table 3 Application of the standard

addition technique to the analysis of LED Claimed taken [ng/band] Authentic added [ng/band] Recovery%
and SOF by HPTLC method
LED 90.00 54.00 99.92

90.00 72.00 98.29

90.00 90.00 99.89

90.00 108.00 100.19

90.00 135.00 98.97

Mean 99.45

SD 0.80

RSD% 0.80

SOF 400.00 240.00 99.33

400.00 320.00 100.65

400.00 400.00 99.56

400.00 480.00 99.09

400.00 600.00 98.44

Mean 99.41

SD 0.81

RSD% 0.81
76 JPC-J Planar Chromat (2020) 33:71 – 77

Fig. 3 HPTLC densitogram showing the

separation of placebo (track 1) and of drug
product samples spiked with of 54:135
ng/band LED and 240:600

ng/band SOF (track 2:10) at 256

nm

Limit of detection and limit of quantification
LOD and LOQ of the proposed method were determined according to the
ICH recommendations based on the standard deviation method. The LOD
and LOQ of the proposed method were assessed practically and are shown
in Table 1 .
PLUS ® tablets. Seven replicates determinations were made. Satisfactory
results were obtained for each compound in a good agreement with the
label claims (Table 4 ). Results of HPTLC were compared with those
obtained by the reported HPLC method for determination of LED and SOF [ 19
]. Statistical comparison of the results was performed with regard to
accuracy and precision using Student ’ s t test and the F-

Specificity ratio at 95% confidence level (Table 4 ). There was no significant difference
between the results.

The chromatograms of both placebo and dosage form were compared, and
no interference from the matrix components was detected with the separated
LED and SOF peaks using the proposed method (Fig. 3 ). Conclusions

A new, simple, precise, accurate and sensitive HPTLCmethod has been

Analysis of pharmaceutical formulation developed and validated for the simultaneous determination of LED and
SOF in pharmaceutical dosage form. The proposed method could separate
The proposed method was successfully applied for the determination of LED and SOF using simpler and relatively lower cost mobile phase than
LED and SOF in MPIVIROPACK previously

Table 4 Application of HPTLC for

determination of LED and SOF in Mean found ± S.D. a
MPIVIROPACK PLUS ®

tablets Proposed HPTLC method Reported HPLC method [ 19 ]

SOF LED SOF LED

MPIVIROPACK PLUS ® tablets 99.26 ± 0.81 99.77 ± 0.72 99.87 ± 0.38 99.73 ± 0.38

t 0.21 0.04 (2.23) b

F 2.27 2.51 (4.28) b

PLUS® tablets
a Mean and S.D. where n = 7, percentage recovery from the label claim amount

b Theoretical values for t and F

JPC-J Planar Chromat (2020) 33:71 – 77
described HPTLC methods where both components are separated at
reasonable and reproducible Rf values (0.43 and
0.59 for LED and SOF, respectively). Comparative study was performed
between the developed HPTLC method and the reported HPLC method.
The quantitative results of the two analytical methods did not show
statistically significant difference whereas the developed HPTLC method is
more rapid, cost-effective and can analyze a large number of samples.
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