Mabrouk et al., 2018, AJMS 1(2): 1-4 DOI:10.5455/ajms.

Arabian Journal of Medical Sciences

http://www.ajms.tk

Determination of Etidronate in Pharmaceutical Formulations by RP-HPLC

Method with Indirect UV Detection
Mokhtar Mabrouka,b, Sherin F. Hammada, Mohamed A. Abdelazizc, Fotouh R. Mansour* a,b
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Egypt
b
Pharmaceutical Services Center, Faculty of Pharmacy, Tanta University
c
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Egypt
Article Info Abstract
A reversed-phase HPLC method with indirect UV detection has been developed for the determination
Keywords: of the non-chromophoric bisphosphonate drug, etidronate disodium, in bulk and in tablet dosage
Etidronate;
forms. The method was based on adding sodium salicylate as a probe component to the mobile
Bisphosphonates;
Non-chromophoric drugs; phase. Analyte detection was achieved by monitoring the decrease in the eluent background signal
Pharmaceutical Tablets; at 210 nm. The method was validated according to the guidelines of the International Conference on
RP-HPLC;
Harmoinzation and showed adequate levels of accuracy, precision and specificity for routine quality
Indirect UV Detection
control purposes. The linearity of the method was studied over the concentration range of 50-300
Received Sep 22, μg/mL. Calibration plots of the area of negative analyte signals plotted against the concentration
Revised Sep 28, showed a high value of the correlation coefficient (0.999), indicating a good linearity of the proposed
Published Sep 29, 2018
method. The method was also successfully applied for the analysis of two other non-chromophoric

*Corresponding author: bisphosphonates; alendronate sodium trihydrate and clodronate disodium tetrahydrate. These results
fototuhrashed@pharm.tanta.edu.eg prove that the method is applicable for determination of non-chromophoric bisphosphonates.

1. Introduction was adequate for quality control purposes, conductivity detectors

Bisphosphonates (BIS) (Fig. 1) are bone resorption inhibi- are highly sensitive to small variations in temperature which would
tor drugs that are widely indicated in a variety of bone metabol- significantly affect the method reproducibility (Fernandes et al.,
ic disorders, including osteoporosis (Sambrook et al., 2005), 2007). In addition to conductivity detection, other techniques have
Paget’s disease (Lyles et al., 2001), and hypercalcemia of ma- been reported such as evaporative light-scattering detection (Jiang
lignancy (Stewart, 2005). Moreover, they have shown antipar- and Xie, 2005), flame photometric detection (Chester et al., 1981),
asitic activity (Martin et al., 2001; Szajnman et al., 2003) and mass spectrometric detection (Qin et al., 1994), inductively-coupled
were found useful in rheumatoid arthritis (Nugent et al., 1994). plasma detection (Kovacevic et al., 2004), and corona-charged aer-
osol detection (Chester et al., 1981). However, these detection sys-
OH OH OH tems are generally not common in most quality control laboratories.
O ONa O ONa O ONa
P P P
Owing to their accuracy, ease of operation, and ease of cou-
OH OH Cl pling with most chromatographic systems, spectrophotometers
H2N OH ONa ONa
are regarded the most common LC detectors in pharmaceutical
P H3C P Cl P
analysis laboratories. As a consequence, great efforts have been
OH OH OH
O O O devoted to the formation of chromophoric bisphosphonate-deriv-
Fig. 1: Alendronate Sstructures
Molecular odium of studied
Etidronabisphosphonate
te Disodium Cldrugs
odronafrom
te Disoleft
diumto atives that are readily detectable by conventional spectrophotom-
right (Alendronate Sodium, Etidronate Disodium, Clordronate Disodium).
eters (Al Deeb et al., 2004; Walash et al., 2012). Nevertheless,
All compounds are presented in anhydrous form.
this “chemical derivatization” approach is often very tedious and
Routine chromatographic analysis of most members of this class time-consuming which make it inconvenient for routine qual-
of compounds is complicated due to the lack of a readily detecta- ity control analyses. Therefore, indirect photometric detection
ble chromophore for direct photometric detection (Thompson et al., would be an appropriate alternative for routine analyses because
1994). Consequently, previous chromatographic analysis of BIS has it allows the detection of non-UV-absorbing compounds using
mostly relied on alternative detection modes. Conductivity detection standard HPLC hardware and with minimal sample manipulation.
(coupled to ion chromatography) has been frequently employed for the The aim of this work is to develop, optimize and validate a re-
analysis of BIS in pharmaceuticals e.g. alendronate (Fernandes et al., versed-phase liquid chromatographic procedure with indirect UV
2007; Tsai et al., 1992), pamidronate (Fernandes et al., 2007), etidro- detection for the determination of etidronate disodium (ETD), an
nate (Fernandes et al., 2007), clodronate (Fernandes et al., 2007), and important representative of this class of compounds, in pure and
ibandronate (Kumar et al., 2011). Although the sensitivity achieved tablet forms. This method is based on adding sodium salicylate as

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 Mabrouk et al., 2018, AJMS 1(2): 1-4 DOI:10.5455/ajms.7

a visualizing agent to the mobile phase and monitoring the decreas-
ing (negative) signals of the eluent upon injecting the analyte. In-
direct detection allows for eliminating the derivatization reactions,
which are necessary when UV or fluorescence detection is applied,
thus making the developed method easier to apply and more con-
venient for routine quantitative applications of ETD and other BIS.
tographed under the optimized conditions, ETD was found to elute
at 3.4 min (Fig. 2). Chromatographic conditions including methanol
ratio, pH of aqueous component, sodium salicylate concentration,
and temperature were optimized as to achieve the highest sensitivity.

2. Experimental
2.1. Chemicals and reagents
Etidronate disodium (99.5%), alendronate sodium trihydrate
(99.6%), and clodronate disodium tetrahydrate (99.5%) were gen-
erously provided by Sigma Pharmaceutical Industries (Quesna,
Menofyia, Egypt). Sulphuric acid (ACS reagent, 95.0-98.0%), sodi-
um salicylate (Purity ≥99.5%), and methanol (HPLC grade, >99.9%)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fig. 2: Typical chromatogram of etidronate disodium (0.25mg/mL) at
optimized conditions showing the negative etidronate peak at 3.4 min.
2.2. Equipment and chromatographic conditions
All chromatographic runs were performed on a Dionex UltiMate The effect of varying the percent methanol in the mobile phase
3000RS HPLC system (Thermo ScientificTM, DionexTM, Sunny- on the method sensitivity was investigated in the range (40-60) %
vale, CA, USA) equipped with an LPG-3400RS quaternary pump, a (Fig. 3). Below 40% the peaks are very broad, while above 60%
WPS-3000RS autosampler, a TCC-3000RS column thermostat, and the analyte peak elutes early, overlapping with the first perturba-
a DAD-3000RS diode array detector. Chromeleon 7 was the soft- tion peak in the chromatogram. It was found that increasing the
ware employed in data collection and processing. An Inertsil reversed methanol ratio in the mobile phase is associated with a significant
phase C18 column (5 µm particle size, 150 mm × 4.6 mm i.d.) was improvement in the method sensitivity until it reaches 50%. There-
used exclusively throughout this study. A mobile phase consisting of after, any increase in methanol ratio would result in a significant
methanol: aqueous sodium salicylate (200μM, pH 3.5) (50:50, v/v) decrease in sensitivity. A MeOH: aqueous sodium salicylate ratio
was delivered at a flow rate of 1.0 mL/min. The pH of the aque- of 50:50 (v/v) was, thus, considered the optimum on performing
ous sodium salicylate was adjusted using dilute sulphuric acid. The subsequent experiments. The effect of pH of aqueous sodium salic-
injection volume was 20 µL and UV detection was set at 210 nm. ylate on the method sensitivity was also studied. A maximum sen-
2.3. Calibration Standards sitivity was found at a pH value of 3.5 (Fig. 4). The concentration
of sodium salicylate was also an important optimization parameter.
An aqueous stock solution of ETD at 1.0 mg/mL was used to pre- Sensitivity greatly enhanced as the salicylate concentration de-
pare a series of assay standard solutions in the range 50-300 μg/mL creased to 200 µM. As the concentration fell below this level, there
by accurately transferring appropriate aliquots into 10mL volumetric was a little decrease in method sensitivity (Fig. 5). Accordingly, a
flask and completing to the volume with methanol. Likewise, assay salicylate solution of 200 µM was used throughout the rest of the
standard solutions of ALD and CLD were prepared. study. Variation of temperature also affects the method sensitivity,
2.4. Application to commercial etidronate tablets although to a lower extent compared to other factors. To achieve
the highest possible sensitivity in subsequent chromatographic runs,
Ten Etidron® tablets (each contains 200 mg etidronate disodium)
the temperature of the column thermostat was set at 35°C (Fig. 6).
were weighed and ground. A quantity of the powdered tablets con-
taining the equivalent of 100 mg ETD was transferred into a 100-mL
volumetric flask, sonicated with an appropriate amount of water for
10 min, and completed to the volume with water. The resulting solu-
tion was filtered through a 0.45 cellulose nitrate paper to prepare a
stock sample solution of ETD of 1 mg/mL claim. An assay solution
at 100 µg/mL claim was then prepared by accurately transferring 1
mL of the drug stock solution into 10 mL-volumetric flask and com-
pleting to the mark with methanol. A portion of the assay solution
was transferred to an HPLC vial for analysis, then the % claim was
calculated and checked for compliance with USP specifications.

3. Results and Discussion

3.1. Optimization of chromatographic conditions
The chromatographic system was first equilibrated with the mo-
bile phase containing sodium salicylate until a steady background
signal was obtained. ETD detection was achieved by monitoring the Fig. 3: Effect of MeOH ratio on method sensitivity. The method sensitivity
decrease in the UV absorbance of the eluent, which exhibited an ab- was calculated from the slope of the calibration curve at each %MeOH.
sorption maximum at 210 nm, because of the displacement of salic-
ylate anions by ETD at the detector cell. When samples are chroma-

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 Mabrouk et al., 2018, AJMS 1(2): 1-4 DOI:10.5455/ajms.7

3.2. Method validation

The proposed method has been validated according to ICH
guideline Q2 (R1)(ICH, 2005) with regard to linearity, range, limit of
detection, limit of quantitation, accuracy, precision, and specificity.
Linearity and range. The linearity of the method was studied
over the concentration range of 50-300 μg/mL. Calibration plots of
the area of negative analyte signals plotted against the concentration
showed a high value of the correlation coefficient (0.999), indicating
a good linearity of the proposed method. Regression parameters are
Fig. 4: Effect of mobile phase pH on method sensitivity. The method summarized in Table 1.
sensitivity was calculated from the slope of the calibration curve at each pH. Limits of detection and quantitation (LOD & LOQ). According to
ICH guideline Q2(R1)(ICH, 2005), detection and quantitation limits
can be calculated from the standard deviation of the response given
by blank samples, σ and the slope of the calibration curve, S so
that the LOD is calculated from the formula 3.3σ /S while the formu-
la 10 σ /S is used to calculate the LOQ. Detection and quantitation
limits of the proposed method are shown in Table 1. It is important
to notice that a considerably lower concentration limits for detection
and quantitation could be achieved by increasing injection volume to
50 µL without any signs of column overloading.
Accuracy. For evaluating the accuracy of the proposed method,
nine standard solutions of ETD evenly distributed at three concentra-
Fig. 5: Effect of salicylate anion concentration on method
sensitivity. The method sensitivity was calculated from the slope of tion levels (triplicates at 75, 175, and 275 µg/mL) were analyzed. The
the calibration curve at each salicylate concentration. mean % recovery (±SD) was found to be 100.41 (±1.54), demonstrat-
ing a good accuracy of the proposed method (Table 2).
Precision. The method precision was determined by analyzing
triplicates of standard solutions of ETD at three concentration levels
(75, 175, and 275 µg/mL) on the same day (Intraday) and on three
consecutive days (Interday). The method showed an acceptable level
of intraday and interday precision as reflected by the values of %RSD
(Table 2) that were not more than 1.93 and 1.96, respectively.
Specificity. A standard addition experiment was applied as fol-
lows: an appropriate aliquot of the ETD stock sample solution (i.e.
that was prepared from ten powdered Etidron® tablets at 1 mg/mL
Fig. 6: Effect of temperature variation on method sensitivity. The method as previously indicated in the experimental section) was accurate-
sensitivity was calculated from the slope of the calibration curve at each ly transferred into a 10mL volumetric flask containing exactly the
temperature.
equivalent of 1 mg ETD followed by dilution to volume with meth-
Table 1: Regression parameters of the proposed method for the anol. Another solution prepared by transferring an exactly equal ali-
determination of etidronate. quot from the same stock sample solution, but without spiking with
Linear- r a b Sy/x Sa Sb DL QL ETD, was used to serve as a control for calculations. A percentage
ity (n = 6) (10-1) (10-3) µg/mL µ g / recovery of 100.7% (±1.4, SD) establishes the absence of assay bias
µg/mL mL
due to the tablet excipients.
50-300 0.9991 22.3 3.71 1.66 1.55 8.0 8.2 24.7
3.3. Application to tablet dosage form
r: correlation coefficient, a: intercept, b: slope, Sy/x: residual standard deviation
of the regression line, Sa: standard error of intercept, Sb: standard error of The method was applied to the analysis of Etidron® tablets (each
slope, DL: detection limit (calculated), QL: quantitation limit (calculated). tablet contains 200 mg etidronate disodium). The assay test was
based on the analysis of triplicate preparations of a composite of 10
Table 2: Evaluation of accuracy, intraday and interday precision of the Etidron®tablets resulting in an average of 107.3% claim. This is still
proposed method for the determination of etidronate.
Table 3: Regression parameters and retention times of the proposed method
Concen- Accuracy and intraday precision Interday precision
with other bisphosphonates
tration Mean con- SD RSD R Mean con- SD RSD Drug tR Linearity r Regression DL QL
µg/mL centration* % % centration* % (min) µg/mL (n = 6) equation µg/mL µg/mL
µg/mL µg/mL
75 74.00 1.43 1.93 98.66 74.63 1.46 1.96 ALD 3.2 50 - 300 0.9980 Y=0.2375X+ 12.7 38.6
22.38
175 176.77 2.88 1.63 101.01 173.64 3.10 1.79
CLD 3.1 50 - 300 0.9979 Y=0.215X+ 14.1 42.6
275 279.26 3.47 1.24 101.55 275.70 3.93 1.34 19.79
Mean % 100.41
ALD: alendronate sodium trihydrate, CLD: clodronate disodium tetrahydrate,
R ± SD ± 1.54
tR: retention time, r: correlation coefficient, DL: detection limit (calculated),
R: Recovery, * n = 3 QL: quantitation limit (calculated).
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 Mabrouk et al., 2018, AJMS 1(2): 1-4 DOI:10.5455/ajms.7

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