Accuracy and precisionTo ascertain

Accuracy and precision
To ascertain the effectiveness of the method, suitability tests
were performed on a freshly prepared mixture of standard stock solutions of ursolic acid, betulinic acid, stigmasterol and
lupeol spiked with preanalysed with identified extract of
Shankhpushpi botanicals. The repeatability of sample application
and measurement of peak area were expressed in terms of
RSD % and the % RSD s for intra- and interday analysis are depicted
in Table II. Intraday precision (% RSD) on the basis of content
of ursolic acid, betulinic acid, stigmasterol and lupeol were found
to be 0.02–0.10, 0.004–0.65, 0.0003–0.1 and 0.007–0.04, respectively,
whereas interday precision (% RSD) on the basis of
the content were found to be 0.005–0.12, 0.009–0.01, 0.006–
0.02 and 0.003–0.03, respectively.
Robustness of the method
The low values of RSD % as shown in Table III indicate the robustness
of the method.
Limit of detection and quantification
The LOQ and LOD were calculated from the equations LOD ¼
3.3 (SD/S) and LOQ ¼ 10 (SD/S). The LOQ and LOD are shown
in Table IV.
Specificity
The peak purity of ursolic acid, betulinic acid, stigmasterol and
lupeol was assessed by comparing the spectra of standard at
peak start, peak apex and peak end positions of the spots, i.e., r
(start, middle) ¼ 0.9973 and r (middle, end) ¼ 0.9979. Good correlation
(r ¼ 0.9994) was also obtained between the standard
and sample.
stock solutions of ursolic acid, betulinic acid, stigmasterol and
lupeol spiked with preanalysed with identified extract of
Shankhpushpi botanicals. The repeatability of sample application
and measurement of peak area were expressed in terms of
RSD % and the % RSD s for intra- and interday analysis are depicted
in Table II. Intraday precision (% RSD) on the basis of content
of ursolic acid, betulinic acid, stigmasterol and lupeol were found
to be 0.02–0.10, 0.004–0.65, 0.0003–0.1 and 0.007–0.04, respectively,
whereas interday precision (% RSD) on the basis of
the content were found to be 0.005–0.12, 0.009–0.01, 0.006–
0.02 and 0.003–0.03, respectively.
Robustness of the method
The low values of RSD % as shown in Table III indicate the robustness
of the method.
Limit of detection and quantification
The LOQ and LOD were calculated from the equations LOD ¼
3.3 (SD/S) and LOQ ¼ 10 (SD/S). The LOQ and LOD are shown
in Table IV.
Specificity
The peak purity of ursolic acid, betulinic acid, stigmasterol and
lupeol was assessed by comparing the spectra of standard at
peak start, peak apex and peak end positions of the spots, i.e., r
(start, middle) ¼ 0.9973 and r (middle, end) ¼ 0.9979. Good correlation
(r ¼ 0.9994) was also obtained between the standard
and sample.
Recovery studies
The results of content estimation and recovery studies of ursolic
acid, betulinic acid, stigmasterol and lupeol from botanicals of
Shankhpushpi extracts after spiking it with 100 and 200 ng/
spot of additional standards are listed in Table V.Discussion
Shankhpushpi is an ayurvedic herb and utilized as raw material
for the herbal formulation (mainly syrup, tablet and bhasma) development
in various parts of India for treatment of nervous
debility. The major ambiguity reflected with this plant is that
there is adaptation of four herbs CP, EA, CT and CD because of
similarity in morphological appearance of flower as a source of
Shankhpushpi (43). So, there is need of method related to
their routine quality control. The simplicity of the sample preparation
and the possibility of analysing several samples of herbal
products simultaneously in a short time make HPTLC the method
of choice (44, 45). There are some HPTLC methods related to
quality control of individual herbs (8, 46). The literature also reveals
the presence of other validated methods for quantification
of ursolic, oleanolic and betulinic acids and quantification of
lupeol and stigmasterol in plant extracts and formulation (34–
36). These methods are simple and robust for identification of
single herbs but lacking in purpose of differentiation among
Shankhpushpi botanicals due to the limitation of markers selection.
So, there is no report of simultaneous quantification of
ursolic acid, betulinic acid, stigmasterol and lupeol, which can
be utilized for purpose of differentiation among the four
Shankhpushpi botanical extracts. Hence we developed a simple
and precise method for quantification of these marker
compounds.
In this method, ursolic acid, betulinic acid, stigmasterol and
lupeol were quantified from Shankhpushpi botanical extracts
by the TLC densitometric method using HPTLC. The TLC densitometric
method was validated in terms of precision, repeatability,
and accuracy. The selected mobile phase (petroleum
ether:ethyl acetate:toluene; 7:2:1, v/v/v) well resolved ursolic
acid, betulinic acid, stigmasterol and lupeol. AS reagent was used to derivatize the chromatograms. Two modes of derivatization
were checked: spraying and dipping. More reproducible results
were obtained in the latter c

Results (Indonesian) 1:

Akurasi dan presisiUntuk memastikan efektivitas metode, kesesuaian tesdilakukan pada campuran disiapkan dengan segar standar saham solusi asam ursolat, asam betulinic, stigmasterol danlupeol berduri dengan preanalysed dengan ekstrak diidentifikasiTumbuhan Shankhpushpi. Pengulangan contoh aplikasidan pengukuran puncak daerah itu dinyatakan dari segiRSD % dan RSD % s untuk analisis intra - dan interday digambarkandalam tabel II. Intraday presisi (% RSD) berdasarkan kontendari ursolat asam, asam betulinic, stigmasterol dan lupeol yang ditemukanmenjadi 0.02-0,10, 0.004 – 0,65, 0,0003 – 0.1 dan 0,007 – 0,04, masing-masing,Sedangkan interday presisi (% RSD) berdasarkan pada contoh yangkonten yang ditemukan 0.005 – 0,12, 0.009-0,01, 0.006-0.02 dan 0.003 – 0.03, masing-masing.Ketahanan metodeNilai-nilai rendah RSD % seperti yang ditunjukkan dalam tabel III menandakan kekokohanmetode.Batas Deteksi dan kuantifikasiLOQ dan LOD dihitung dari persamaan LOD ¼3.3 (SD/S) dan LOQ ¼ 10 (SD/S). LOQ dan LOD ditunjukkandalam tabel IV.KekhususanKemurnian puncak asam ursolat, asam betulinic, stigmasterol danlupeol dinilai dengan membandingkan spektrum standarpuncak mulai, puncak puncak dan posisi akhir puncak tempat, yaitu, r(mulai, menengah) ¼ 0.9973 dan r (tengah, akhir) ¼ 0.9979. Korelasi baik(r ¼ 0.9994) juga diperoleh antara standarand sample.stock solutions of ursolic acid, betulinic acid, stigmasterol andlupeol spiked with preanalysed with identified extract ofShankhpushpi botanicals. The repeatability of sample applicationand measurement of peak area were expressed in terms ofRSD % and the % RSD s for intra- and interday analysis are depictedin Table II. Intraday precision (% RSD) on the basis of contentof ursolic acid, betulinic acid, stigmasterol and lupeol were foundto be 0.02–0.10, 0.004–0.65, 0.0003–0.1 and 0.007–0.04, respectively,whereas interday precision (% RSD) on the basis ofthe content were found to be 0.005–0.12, 0.009–0.01, 0.006–0.02 and 0.003–0.03, respectively.Robustness of the methodThe low values of RSD % as shown in Table III indicate the robustnessof the method.Limit of detection and quantificationThe LOQ and LOD were calculated from the equations LOD ¼3.3 (SD/S) and LOQ ¼ 10 (SD/S). The LOQ and LOD are shownin Table IV.SpecificityThe peak purity of ursolic acid, betulinic acid, stigmasterol andlupeol was assessed by comparing the spectra of standard atpeak start, peak apex and peak end positions of the spots, i.e., r(start, middle) ¼ 0.9973 and r (middle, end) ¼ 0.9979. Good correlation(r ¼ 0.9994) was also obtained between the standardand sample.Recovery studiesThe results of content estimation and recovery studies of ursolicacid, betulinic acid, stigmasterol and lupeol from botanicals ofShankhpushpi ekstrak setelah spiking dengan 100 dan 200 ng /tempat tambahan standar tercantum dalam tabel V.DiscussionShankhpushpi adalah ramuan Ayurveda dan dimanfaatkan sebagai bahan bakuuntuk pengembangan formulasi herbal (terutama sirup, tablet dan bhasma)di berbagai bagian India untuk pengobatan gugupkelemahan. Ambiguitas utama tercermin dengan tanaman ini adalah bahwaada adaptasi dari empat herbal CP, EA, CT dan CD karenakesamaan dalam morfologi penampilan bunga sebagai sumberShankhpushpi (43). Jadi, ada kebutuhan metode yang berkaitanpengendalian kualitas rutin mereka. Kesederhanaan preparasi sampledan kemungkinan untuk menganalisis beberapa sampel herbalproduk secara bersamaan dalam waktu singkat membuat HPTLC metodepilihan (44, 45). Ada beberapa metode HPTLC yang berkaitankontrol kualitas individu herbal (8, 46). Literatur juga mengungkapkankeberadaan metode lain divalidasi untuk kuantifikasiasam ursolat, oleanolic dan betulinic dan kuantifikasilupeol dan stigmasterol dalam ekstrak tumbuhan dan formulasi (34-36). metode ini sederhana dan kuat untuk identifikasitumbuhan satu tapi kurang dalam tujuan diferensiasi antaraShankhpushpi tumbuhan karena keterbatasan pilihan spidol.Jadi, ada tidak ada laporan dari kuantifikasi simultanasam ursolat, asam betulinic, stigmasterol dan lupeol, yang dapatdimanfaatkan untuk tujuan diferensiasi antara empatEkstrak botani Shankhpushpi. Oleh karena itu kami mengembangkan sederhanadan metode yang tepat untuk kuantifikasi penanda inisenyawa.Dalam metode ini, asam ursolat, asam betulinic, stigmasterol danlupeol yang diukur dari ekstrak botani Shankhpushpidengan metode densitometric TLC menggunakan HPTLC. TLC densitometricmetode disahkan dalam hal ketepatan, pengulangan,dan akurasi. Fase mobile dipilih (perminyakanEter: etil asetat: toluena; 7:2:1, v/v/v) juga diselesaikan ursolatasam, asam betulinic, stigmasterol dan lupeol. SEBAGAI reagen digunakan untuk derivatize chromatograms. Dua mode derivatisasiDiperiksa: penyemprotan dan mencelupkan. Hasil yang lebih direproduksiDiperoleh dalam c kedua