Malaysian
Journal of Analytical Sciences Vol 21 No 3 (2017): 605 - 618
DOI:
https://doi.org/10.17576/mjas-2017-2103-10
DEVELOPMENT
AND VALIDATION OF A REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-ULTRAVIOLET
METHOD FOR SIMULTANEOUS DETECTION OF CAFFEINE AND PHENOLPHTHALEIN IN WEIGHT
REDUCING SUPPLEMENTS
(Pembangunan dan Pengesahsahihan
Kaedah Fasa Terbalik-Kromatografi Cecair Prestasi Tinggi Ultraungu bagi
Pengesanan Kafein dan Fenolftalein Dalam Suplemen Pengurangan Berat Badan)
Elham Mohammadi Nasr and Lai Chun Wong*
Department
of Pharmaceutical Chemistry, School of Pharmacy,
International
Medical University, 57000 Kuala Lumpur, Malaysia
*Corresponding author: laichunwong@imu.edu.my
Received: 13
May 2016; Accepted: 12 April 2017
Abstract
A reversed-phase high performance liquid chromatography analytical method
with variable wavelength detection was developed for the simultaneous detection
of caffeine and phenolphthalein in weight reducing supplements. Chromatographic
separation was achieved with a C18 reversed-phase column using a gradient
elution of methanol and an ammonium acetate buffer (pH 5; 25 mM), with the
detector wavelength set at 254 nm. The method was validated according to The
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use guidelines. The method was proven
to be linear over a concentration of 10 – 100 μg/mL for both caffeine and
phenolphthalein, with mean correlation coefficients of 1.000 and 0.999,
respectively. The limit of detection for caffeine and phenolphthalein was 0.77
μg/mL and 0.47 μg/mL respectively, while the limit of quantification for
caffeine and phenolphthalein was 2.35 μg/mL and 1.44 μg/mL, resspectively.
The percentage relative standard deviation for both intra and inter-day
precision was less than 2%. The mean recovery values were calculated as 105.84%
and 113.58% for caffeine and phenolphthalein. In addition, the method was
proven to be robust when deliberate changes in flow rate, buffer pH and organic
mobile phase composition were tested.
Keywords: caffeine, phenolphthalein,
high performance liquid chromatography, validation, weight reducing supplement
Abstrak
Satu kaedah
analitikal kromatografi cecair prestasi tinggi (KCPT) fasa terbalik dengan
pengesanan pada pelbagai panjang gelmbang telah dibangunkan untuk pengesahan kafein dan fenolftalein dalam
suplemen pengurangan berat badan. Pemisahan kromatografik dicapai dengan turus
fasa terbalik C18 menggunakan kecerunan elusi metanol dan penampan ammonium asetat
(pH 5; 25 mM), pada panjang gelombong l = 254 nm. Kaedah ini telah
ditentusahkan mengikut garis panduan harmonisasi persidangan antarabangsa
keperluan teknikal untuk pendaftaran famaseutikal bagi pengunaan manusia.
Kaedah ini dibuktikan linear dalam julat kepekatan larutan piawai kafein dan fenolftalein di antara 10 – 100 μg/mL dengan nilai pekali korelasi (R2)
1.000 dan 0.999 untuk masing – masing bagi kafein dan fenolftalein. Had
pengesanan bagi kafein dan fenolftalein masing – masing ialah 0.77 μg/mL and
0.47 μg/mL. Keputusan had kuantifikasi bagi kafein dan phenolphthalein masing –
masing ialah 2.35 μg/mL and 1.44 μg/mL. Peratus
relatif sisihan piawai bagi ketepatan intra dan antara hari yang diukur ialah
kurang dari nilai 2%. Nilai min pemulihan semula yang dikira bagi kafein dan
fenolftalein masing-masing ialah 105.84 dan 113.58%. Kaedah ini juga dibuktikan
teguh apabila perubahan dalam kadar aliran, pH panampan dan komposisi fasa
bergerak diuji.
Kata kunci: kafein, fenolftalein, kromatografi
cecair prestasi tinggi, pengesahsahihan, suplemen pengurangan berat badan
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