Download method development and validation for analysis of

“METHOD DEVELOPMENT AND VALIDATION FOR ANALYSIS OF RISEDRONATE SODIUM AND
CALCIUM CARBONATE USING RP-HPLC TECHNIQUE IN PHARMACEUTICAL DOSAGE FORM”
A) BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Need for method development:
Numerous methods are required to document the identity, strength, quality, purity and potency of drug
substance and drug product. Methods are developed continuously in chromatographic science to estimate the
concentration of the active ingredient in drug substance or drug product.1 Risedronate sodium is available along
with calcium carbonate in the form of tablet dosage form (ACTONEL). The solubility profile of the drug is, it
comes under BCS class III. 2 Soluble in pH 7.0 potassium phosphate dibasic solution, 0.1 N NaOH and water; very
slightly soluble in 0.1 N Hcl , practically insoluble in isopropanol.3 A method can be developed using HPLC with
more retention for simultaneous estimation of risedronate sodium and calcium carbonate.
Need for using RP- HPLC:
High performance liquid chromatography provides reliable quantitative precision and accuracy along with
linear dynamic range (LDR) sufficient to allow the determination of the API and related substances in the same run
using a variety of detectors and can be performed on fully automated instrumentation.1
RP- HPLC method is used to elute the active constituent. About 75% of current HPLC analyses are
performed using reverse phase. This is due to safety considerations using non polar solvents but also to the
differences in sample preparation procedures required for normal phase versus reverse phase HPLC. In reverse
phase HPLC, the filtrate from this preparation may be injected directly on to the column. Dissolved excipients from
the dosage form are generally much more polar than the components of interest and are not retained by stationary
phase and consequently do not interfere with the analyses.1
Need for validation:
The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended
purpose4. According to International Conference on harmonization (1990) Q2A document, Validation of analytical
procedure is performed on various parameters such as specificity, linearity, accuracy, precision, limit of detection,
limit of quantitation, range, and robustness.
6.2 Review of Literature:
Bisphosphonates is a class of chemical compounds finding extensive medical applications against bone
disorders including osteoporosis, Pagets’ disease, etc. Non-N-containing members include etidronate, clodronate
and tiludronate, while N-containing bisphosphonates include active pharmaceutical compounds such as
pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate and zoledronate. This article covers 20
years of analytical research on this group of compounds, focusing on bioanalytical and pharmaceutical QC
applications. A wide range of analytical techniques is presented and critically discussed including among others
liquid and gas phase separations, electrophoretic, electroanalytical, automated and enzymatic approaches.5
.
High-performance liquid chromatography (HPLC) separation methods suitable for such molecules cannot
be used in tandem with mass spectrometry (MS) due to high non-volatile salt content; at the same time the sample
preparation, in biological fluids, is also a challenging problem. In the past ion-pair chromatography was mainly
used in the case of HPLC-MS of biphosphonates, but no application to quantitative pharmacokinetic (PK) studies
has been presented. In this study, after preliminary tests with ion-pair chromatography showing a poor sensitivity, a
combined derivatization of the amino group and the biphosphonate has been developed and tested in a PK study.
Using this analytical approach they(Tarcomnicu I, Silvestro L, Savu SR, Gherase A, Dulea C) were able to fully
validate the quantitation of alendronate in the range of 6.667-4860.0 ng/ml in urine (sample volume 2.0 ml); each
analytical run was 5.0 min long. 6
A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was
developed and validated by(Kyriakides D, Panderi) for the determination of risedronate in pharmaceutical dosage
forms. The determination was performed on a BDS C18 analytical column (250 mm × 4.6 mm i.d., 5 μm particle
size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium
(pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min−1. The ultraviolet
(UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as
internal standard and risedronate were 4.94 and 5.95 min, respectively. 7
An HPLC assay for the determination of risedronate in human urine was developed and validated by(Vallano P.T,
Shugarts S.B, KlineW.F et.al) Risedronate and the internal standard were isolated from 5-ml urine samples in a
two-part procedure. First, the analytes were precipitated from urine along with endogenous phosphates as calcium
salts by the addition of CaCl at alkaline pH. The precipitate was then dissolved in 0.05 M ethylene glycol-bis(baminoethyl ether)-N,N,N9,N9 -tetraacetic acid and subjected to ion-pair solid-phase extraction using a Waters HLB
cartridge (1 ml, 30 mg) with 1-octyltriethylammonium phosphate as the ion-pair reagent. Following extraction, the
analytes were initially separated from the majority of co-extracted endogenous components on a Waters X-Terra
RP18 (4.6350 mm, 3.5 mm) column. The effluent from the X-Terra was ‘‘heart-cut’’ onto a Phenomenex Synergi
Polar RP (4.63150 mm, 4 mm) column for final separation. UV detection (l5262 nm) was used to quantitate
risedronate in the concentration range of 7.5–250 ng/ ml.8
An analytical method for determination of risedronate, a member of bisphosphonates, is described for the
routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at
alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was
obtained by using a reversed-phase column (Hypersil ODS-2 C18, 4.6mm×250 mm, 5_m). The mobile phase was
an aqueous solution of buffer (contained 1.5mM EDTA-2Na, 1mM sodium etidronate, 11mM sodium phosphate
and 5mM tetrabutylammonium bromide as ion-pair reagent) – methanol (88:12, v/v) adjusted to pH 6.75 using 1M
NaOH. The flow rate was 1mlmin−1. UV detection (λ = 262 nm) was used to quantitate risedronate in the
concentration range of 10–500 ng ml−19
A simple, sensitive, rapid and accurate spectrophotometric method was developed for the determination of
risedronate, a bisphosphonate drug important for the treatment of a variety of bone diseases, in raw material and
pharmaceutical formulations. the proposed method is based on complex formation between risedronate and cu (II)
ions in acetate buffer of pH5.5.
The optimum conditions for this reaction were ascertained and a
spectrophotometric method was developed for the determination of risedronate in concentration range of 2-40
µg/mL with detection limit of 0.03 µg/mL (9.51 × 10-8 mol/L). the molar absorbtivity was 8.00 × 103l/mol/cm. the
method was successfully applied for the determination of risedronate in tablet dosage form .10
An HPLC assay for the determination of risedronate in human urine was developed and validated
by(Vallano P.T, Shugarts S.B, KlineW.F et.al) Risedronate and the internal standard were the mobile phase. The
proposed method was extensively validated according to ICH guidelines for the assay determination. 11
A method is based on RP HPLC with indirect UV detection was developed for the determination of
phosphates and phosphites as impurities in sodium risedronate. RP separation of the phosphates and phosphites was
achieved by adding tetrabutylammonium hydroxide as an ion pairing agent in the mobile phase. Potassium
hydrogen phthalate was added to the mobile phase as an ion chromophore in order to obtain high background
absorption of the mobile phase. Seperation was performed on a C18 column using a mixture of pH 8.2
buffer(containing 0.5 m M tetrbutyl ammonium hydroxide and 1mM phthalate) and acetonitrile (95+5, v/v) as the
mobile phase, with indirect uv detection at 248nm.12
Macedoniolatile organic amine was used as the mobile phase addictive during the separation of four
bisphosphonates (alendronate, pamidronate, zoledronic acid and etidronate). An isocratic liquid chromatography
method with evaporative light-scattering detection (ELSD) was developed for these bisphosphonates which are not
retained on non-polar column and lack chromophore for detection. The analytes have sufficiently separated from
each other on a Phenomenex C18 column. The effects of mobile phase composition and instrumental parameters of
ELSD were studied.13
Simple, fast and uniform method has been developed for the quantitative determination of bisphosphonates in
the quality control of pharmaceutical preparations. The method is based on ion-exchange liquid chromatography
with conductivity detection. Separation is performed on a Waters IC-PAK Anion column using 2 mM nitric acid or
25 mM succinic acid as the mobile phase. Retention of the bisphosphonates can be influenced by pH and the anion
concentration of the mobile phase. The analysis of ionized solutes by high-performance liquid chromatography
(HPLC) may be facilitated by the use of ion-pairing agents. 14
Bisphosphonate compounds have been studied as a class of potential drugs for the treatment of
various bone diseases. However, the analyses of these compounds are problematic because most of them do not
contain strong chromophores. Based on the unique structures of these compounds, they(Sean X. Peng*, Ray
Takigiku) have employed a capillary electrophoresis (CE) technique for the characterization of these compounds in
pharmaceutical dosage formulations. In this study, two CE methods were developed for the determination of a
bisphosphonate compound, 2-thioethane-1,1-bisphosphonic acid. The first method involved the use of an uncoated
column, a phosphate buffer, and hydrostatic injection with direct UV absorbance detection.15
Bisphosphonic acids and their salts can be detected after their liquid chromatographic separation by postcolumn indirect fluorescence detection (IFD). After separation the analyte is combined with the highly fluorescent
Al3+–morin (2′, 3, 4′, 5, 7-pentahydroxyflavone) solution and fluorescence decreases because of the formation of
the nonfluorescent Al3+–bisphosphonate complex. The decrease in fluorescence is proportional to the amount of
bisphosphonate present. Separation of the multivalent anionic bisphosphonate analytes from other anions and
sample matrix is achieved on a strong base anion-exchange column with a strong, basic eluent. The post-column
reaction variables, which influence IFD, are identified and optimized for the detection of the bisphosphonates after
separation on the anion exchanger. 16
6.3 Objective of study:
The objective of is to develop a method for simultaneous estimation of Risedronate sodium and calcium carbonate
using RP-HPLC.
The following are the steps that are to be followed to achieve good retention in the chromatogram:
METHODOLOGY:
RP-HPLC method development strategy:

Arrangement of formulation:
The formulation is available in the form of tablets as marketed product ACTONEL.

Investigating the molecule
The molecular structure, solubilities, polarity and affinity of risedronate and calcium carbonate are studied
to select an appropriate column.

Selection of column
The reverse phase column suitable for the properties of the molecule is set such as BDS C18, ODS, C18 etc.

Choosing initial mobile phase pH
In order to maximize the retention, the mobile phases for reversed phase HPLC of polar or ionizable
compounds should be chosen to minimize the charge on the molecule. Charged molecules are highly
solvated and have a greater affinity for the mobile phase over the stationary phase

Scouting pH conditions

Changing stationary phase selectivity

Investigating addition of ion pair agents to the mobile phase
With silica-based HPLC phases, sometimes it is impossible to operate at a pH where the analyte is
adequately retained. For risedronate with the two highly acidic phosphate groups, this condition would
require pH values significantly below pH 1 where the stationary phase is readily hydrolyzed, destroying the
HPLC column. One solution is to add an ion pair (IP) reagent to the mobile phase. IP reagents have polar
groups that interact with functional groups of opposite charge on the analyte, and hydrophobic groups that
interact with the reversed phase stationary phase
Validation strategy:
Validating the method for various validation parameters given in ICH guidelines.

Specificity
“Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected
to be present. Typically these might include impurities,
degradants, matrix, etc. Lack of specificity of an individual procedure may be compensated
by other supporting analytical procedure” 4

Linearity
“The linearity of an analytical procedure is its ability (within a given range) to
obtain test results which are directly proportional to the concentration (amount) of analyte in the sample”.4

Accuracy
“The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted
either as a conventional true value or an accepted reference value and the value found”.4

Precision
“The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series
of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed
conditions. Precision may be considered at three levels; repeatability, intermediate precision and reproducibility.”4

Range
“The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of
analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and linearity.”4

LOD(limit of detection)
“The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact value.4

LOQ(limit of quantitation)
The quantitation limit of an individual analytical procedure is the lowest concentration of analyte in a sample which
can be quantitatively determined with suitable precision and accuracy.”4

Robustness
Robustness testing means to evaluate the ability of a method to perform effectively in a typical laboratory
environment and with acceptable variations. Robustness definitions have been widely harmonized among
international drug authorities, which is mainly the merit of the International Conference on Harmonisation (ICH).4
B) 7.MATERIALS AND METHODS:
7.1 Source of Data:
Data is obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of
institution, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and
Journals.
7.2 Method of Collection of Data:
Data will be collected from the following step wise experimental procedures proposed in the study:
This is to verify that the method performance is not affected by typical changes in normal experiments. Therefore,
the variation in method conditions for robustness should be small and reflect typical day-today variation.
1. HPLC conditions
a. HPLC column (lot, age, brand)
b. Mobile-phase composition (pH ± 0.05 unit)
c. HPLC instrument (dwell volume, detection wavelength ± 2 nm, column
temperature ± 5◦C, flow rate)
d. Ion pair reagents
2. Sample preparation
a. Sample solvent (pH ± 0.05 unit)
b. Sample preparation procedure (shaking time, different membrane filters)
c. HPLC solution stability
Built-in Robustness in Method procedure taking into account factors like dilution error, mobile phase, ion pair
reagents, buffers etc.
Validation Process:
Some of the following components will be considered for the process validation:
a. Analytical test procedures.
b. Instrument calibration.
c. Critical support systems.
d. Raw materials.
e. Equipment.
f. Manufacturing stages.
g. Product design.
7.3 Does the study require any investigation or interventions to be conducted on patients or the human or
animals? If so please describe briefly:
No
7.4 Has ethical clearance been obtained from your institute
Not applicable
C) 8. LIST OF REFERENCES:
1. Ahuja S and Scypinski S. Hand book of modern pharmaceutical analysis. United states of America:
Academic press; 2001.
2. Mihoci M. Importance of formulation pH for therapeutic efficacy and formulation drug product. Division
III, Generics research and development Zagreb, Teva Pharmaceutical industries Ltd.
http://www.hfd-fg.hr/4-hrvatskikongres/Hotel%20Ambasador/Petak/Dvorana%20B/Istrazivanje%20i%20razvoj/4%20U-IR4%201530%20M.%20Mihoci.pdf (Retrieved on 2011 May 21)
3. Product monograph pr ACTONEL. Risedronate sodium.
http://www.sanofi-aventis.ca/products/en/actonel.pdf
(Retrieved on 2011 May 21)
4. Ermer J and J.H M.B Miller. Method validation in pharmaceutical analysis.
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim; 2005 .
5. Constantinos KZ and Paraskevas DT. Determination of bisphosphonate active pharmaceutical ingredients
in pharmaceuticals and biological material: A review of analytical methods. J Pharm and Biomed Anal
2008;48(3,4):483-96.
6. Tarcomnicu I, Silvestro L, Savu SR, Gherase A and Dulea C. Development and application of a highperformance liquid chromatography-mass spectrometry method to determine alendronate in human urine. J
Chromatogr A 2007;1160(1-2):21-33.
7. Kyriakides D and Panderi I. Development and validation of a reversed-phase
ion-pair high-performance
liquid chromatographic method for the determination of risedronate in pharmaceutical preparations. Anal
Chimica Acta 2007;584:153-59.
8. Vallano PT, Shugarts SB, KlineWF, Woolf E.J, Matuszewski BK. Determination of risedronate in human
urine by column-switching ion-pair High- performance liquid chromatography with ultravioletdetection. J
Chromatogr B 2003;794:23–33.
9. Hui-Juan Jia, Wei Li and Zhao K. Determination of risedronate in rat plasma
samples by ion-pair high-
performance liquid chromatography with UV detector. Anal Chimica Acta 2006;562:171–75.
10. Walash M.I, Metwally M.E.S,
Eid M and El-ShahenyR.N.
Spectrophotometric determination of
Risedronate in Pharmaceutical Formulations via Complex Formation with Cu (II) Ions: Application to
Content Uniformity Testing. Int J Biomed sci 2008; 4(4):303-09.
11. Austin A , radha Krishna T, Donald M and Omowunmi S. Stability indicating ion-pair HPLC method for
the determination of risedronate in a commercial formulation.
J liq chromatogr and Rel Technol 2004; (27):2799-813.
12. Breuzovska K, Dimitrovska A, Kitanovski Z, Petrusevska J, Ribarska JTand Jolevska ST. Development of
an ion-pair reversed-phase HPLC method with indirect UV detection for determination of phosphates and
phosphites as impurities in sodium risedronate. J AOAC Int 2010; 93(4):1113-20.
13. Zan Xie, Ye Jiang and Di-qun Zhang. Simple analysis of four bisphosphonates simultaneously by reverse
phase liquid chromatography using n-amylamine as volatile ion-pairing agent. J of Chromatogr A 2006;
1104(1-2):173-78.
14. Hartigh J, Langebroeka R and Vermeija P. Ion-exchange liquid chromatographic analysis of
bisphosphonates in pharmaceutical preparations. J Pharm Biomed Anal 1993;11(10):977-83.
15. Sean X. Peng, Ray T, Burton D and Larry L. Powell. Direct pharmaceutical analysis of bisphosphonates by
capillary electrophoresis. J of Chromatogr B 1998;709(1, 8) :157-60.
16. Michael J. Lovdahla and Donald J. Pietrzyk. Anion-exchange separation and determination of
bisphosphonates and related analytes by post-column indirect fluorescence detection. J of Chromatogr A
1999;850(1-2):143-52.