Development of Liquid chromatographic methods for t

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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

CHAPTER - III
DEVELOPMENT OF
LIQUID CHROMATOGRAPHIC
METHODS FOR THE DETERMINATION
OF DRUG PRODUCTS BY USING
DIFFERENT DETECTORS.

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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

3.1. INTRODUCTION:
A different type of detectors has been used for the analysis of pharmaceutical drug
product. UV detector, Fluorescence detector, Refractive index detector are commonly used
detectors for the determination of drug product. In this chapter isocratic reversed-phase HPLC
method with combine UV and Fluorescence Detector has been developed for the simultaneous
determination of Drospirenone and Ethinylestradiol in tablet dosage form. Where as another
HPLC method is used for the determination of Metoprolol and Telmisartan in the tablet dosage
form by using UV detector.
Drospirenone is a synthetic progestin that is used in birth control, chemically
(6R,7R,8R,9S,10R,13S,14S,15S,16S,17S)-1,3',4',6,6a,7,8,9,10,11,12,13,14,15,15a,16-
hexadecahydro-10,13-dimethylspiro-[17H-dicyclopropa-6,7:15,16]cyclopenta [a]phenanthrene-
17,2'(5H)-furan]-3,5'(2H)-dione. Ethinylestradiol1 is chemically 19-Nor-17α-pregna-1,3,5(10)-
trien-20-yne-3,17-diol is a derivative of estradiol. Ethinyl estradiol is an orally bio-active
estrogen used in almost all modern formulations of combined oral contraceptive pills. For the
prevention of pregnancy 3 mg Drospirenone and 30 mcg Ethinylestradiol per tablet is used as an
oral contraceptive pill in women.
Metoprolol succinate chemically is 1- (isopropyl amino)- 3- [4- (2-methoxyethyl)
pehnoxy] propan-2-ol2. is a selective β receptor blocker used in treatment of several diseases of
1
the cardiovascular system, especially hypertension. Telmisartan chemically is 2-(4-{[4-Methyl-
6-(1-methyl-1H-1, 3-benzodiazol-2-yl)-2-propyl -1H-1, 3-benzodiazol-1-
yl]methyl}phenyl)benzoic acid3 . It is angiotensin II receptor antagonist, effective in the
treatment of hypertension. It is also effective when used alone or in combination with other drugs
for the treatment of high blood pressure.
A Fixed dose combination of 40 mg Telmisartan and 50 mg Metoprolol ER is available
commercially as tablets (Telmaxx, Glenmark pharmaceuticals), are widely used for the treatment
of cardiovascular disease. Extended release formula of Metoprolol is designed such that it will
delay the release of the Metoprolol in the body over a period of time. A drug of this formula may
have effects that last until the next dosage is taken. The two main aspects of drug products that

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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

play an important role in shelf life determinations are assay of active drug and degradants
generated during the stability study.
3.2 LITERATURE SURVEY:
In this section summarized some of the important analytical methods for the
determination of Drospirenone, Ethinylestradiol, Metoprolol succinate and Telmisartan as a
individual component or in combination with another drug substance.

Philip A.L., et. al. (1987)4
Philip A.L., reported HPLC method for the determination of Norgostimate and Ethinyl
Estradiol in Tablet dosage form. Norgestimate and ethinyl estradiol were extracted from the
tablet matrix with methanol containing an internal standard. An aliquot was chromatographed on
a 5-μm, reversed-phase column using Water: Tetrahydrofuran: Methanol solution (65:25:10
v/v/v) as the mobile phase. The selectivity of the chromatographic system for intact norgestimate
and ethinyl estradiol was demonstrated by resolving both compounds from various potential
degradation products of each compound. An essential property of the chromatographic system
was its ability to separate norgestimate as its syn and anti isomers. The method is linear,
quantitative, and reproducible.
Nguyen H.T., et. al. (1990) 5
Nguyen H.T., reported Dissolution testing of norethindrone:ethinyl estradiol,
norethindrone:mestranol, and norethindrone acetate:ethinyl estradiol combination tablets,
Dissolution was studied utilizing the rotating basket method at 100 rpm in 600 mL of 0.1 M HCl
and 0.02% sodium lauryl sulfate (SLS). Acidic medium with surfactant was also found to be
suitable for combination products of Norethindrone : Mestranol and Norethindrone acetate :
Ethinyl estradiol. A modified U.S.P. assay procedure, a reversed-phase high-performance liquid
chromatographic (HPLC) method with a mobile phase of Acetonitrile and Phosphate buffer, was
used to analyze Norethindrone acetate and Ethinyl estradiol concurrently. The Norethindrone and
Mestranol were analyzed separately in these combination products. The Norethindrone was
found to hydrolyze to some extent (approximately 20% in 8 h) in acidic dissolution medium at
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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

room temperature, but less so at 4 °C. The Norethindron was identified as the sole degradation
product of Norethindrone acetate hydrolysis and was also measured to account for the total
amount of Norethindrone acetate dissolved.
Prabhakar B., et. al. (1999)6
Prabhakar B., et. al. reported HPLC method for the Simultaneous estimation of Ethinyl
estraidol and Levonorgestrel from transdermal Patches The determination was carried out on a
Phenomenex, C-18, 250 x 4.6 mm, 10 micron column using a mobile phase of acetonitrile/ water
(55:45). The flow rate was 1.2 ml/minute. The eluent was monitored at 225 nm. Quantitation was
done using an external standard. The method was found to be reproducible, with good resolution
between ETES and LN. The detector response was found to be linear in the concentration range
of 1- 5 ppm for ETES and 5.25 ppm for LN. The sensitivity of the method was found to be 0.1
mg/ml.
Zhang F., et. al. (2004)7
Zhang F., et. al. reported liquid chromatography/positive electrospray tandem mass
spectrometry method for the Quantitation of 17 -ethinylestradiol in aquatic samples using
liquid-liquid phase extraction, dansyl derivatization. Analysis was performed using high-
performance liquid chromatography with positive electrospray ionization and tandem mass
spectrometry (HPLC/ESI-MS/MS). Deuterated 17α-ethinylestradiol was used as internal
standard and was added to samples before extraction. A limit of quantitation of 1 ng/L was
obtained using a 25 mL aqueous sample. The average recovery of EE spiked into a 25 mL
2
tapwater sample was 100%. This highly sensitive quantitation method is useful for measuring
low levels of EE in aqueous environmental samples.
2
Gogineni R.P., et. al. (2005)8
Gogineni R.P., reported Validated RP-HPLC method for the estimation of method for
the estimation of Drospirenone in formulation. Isocratic elution at a flow rate of 1.0ml/min was
employed on a symmetry C18 (250x4.6mm, 5µm in particle size) at ambient temperature. The
mobile phase consisted of methanol:1% Ortho phosphoric acid 54.5:45.5 (V/V). The UV
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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

detection wavelength was 252nm and 20µl sample was injected. The retention time for
Drospirenone was 8.355 min. The percentage RSD for precision and accuracy of the method was
found to be less than 2%. The method was validated as per the ICH guidelines. The method was
successfully applied for routine analysis of Drospirenone in tablet dosage form.

Bhaumik U., et. al. (2005)9
Bhaumik U., reported LC-Tandem-MS method for the Determination of Drospirenone
in Human Plasma. The analytical method consists in the extraction of plasma sample with
dichloromethane, followed by determination of drospirenone by LC–MS–MS using
levonorgestrel as internal standard. Separation was achieved on a Peerless cyano column with an
isocratic mobile phase consisting of methanol and ammonium formate buffer. Protonated ions
formed by a Turboionspray in the positive mode were used to detect analyte and IS. The MS–MS
detection was by monitoring the fragmentation for drospirenone and for levonorgestrel on a
triple quadrupole mass spectrometer.
Denisova T. A., et. al. (2008)10
Denisova T. A., reported Study compounds were separated using an Agilent 1100
chromatograph fitted with an injector and a variable wavelength UV detector. Columns were
Nova-Pak C18 (10 x4.6 mm, 4 µm). The suitability of the chromatography system was verified
by performing repeat injections. The method is successfully adopted for identification and
quantification of ethinylestradiol, levonorgestrel and gastodene in oral contraceptives.
Sohrabi M.R., et. al. (2010)11
Sohrabi M.R et. al. reported Simultaneous spectrophotometric determination of
cyproterone acetate and ethinyl estradiol in tablets using continuous wavelet and derivative
transform, In this study a zero crossing technique based on continuous wavelet transform (CWT)
as well as classical derivative spectrophotometry (CDS) is presented for simultaneous
determination of cyproterone acetate and ethinyl estradiol in binary mixtures and commercial
dosage of drug, without using prior chemical pre-treatment. Absorption spectra were recorded in
the wavelength range 200–400 nm. Absorbance data were subjected to various mother wavelets

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Development of Liquid chromatographic methods for the determination of Drug products by using different detectors.

from continuous wavelet transform family to find the optimum point of the wavelet signal
processing (Matlab 7.5) gaus 15 and morl wavelet functions with scaling factor, a = 70 and 3rd
derivative with Δλ = 10 nm, were selected. Optimum value of scaling factor was chosen to obtain
an appropriate calibration for each method. The validation of proposed methods was investigated
by several synthetic mixtures and obtained results were successfully compared among each
other.
Eduardo A.J., et. al. (2011)12
Eduardo A.J., et. al. reported Bioequivalence of Two Oral Contraceptive Drugs
Containing Ethinylestradiol and Gestodene in Healthy Female Volunteer, The ethinylestradiol
samples were injected into a Zorbax SB-C18, 4.6 x 50 mm, 3.5 µm column and a Applied
Biosystems Sciex API 5000 tandem mass spectrometer. The mobile a phase was methanol–water
(78:22, v/v), acetic acid glacial 0.2% (v/v), and the mobile phase B was a mixture of Acetonitrile
100% and acetic acid glacial 0.2% (v/v). The chromatographic condition was a gradient mode
performed at 35ºC and at a flow rate of 1 mL/min. for pump no. 1 and 0.5 mL/min. for pump no.
2. The mass spectrometer was operated with + ESI and MRM using the optimized transitions
530.3 → 171.1 for the ethinylestradiol derivate and 534.4 → 171.1 for the ethinylestradiol-d4
derivative.
Bujji B.N., et. al. (2011)13
Reported HPLC method for the Simultaneous analysis of Risperidone and Drospirenone
drugs in Pharmaceutical dosage form. Chromatographic analysis was carried out at ambient
temperature, separation was achieved by gradient elution of C18 column and the mobile phases
of risperidone is methanol, acetonitrile, sodiumdihydrogen phosphate (60:15:25 v/v) and
orthophospharic acid, methanol, acetonitrile (60:25:15 v/v) is the mobile phase of drospirenone.
These mobile phases are sonicated about five minutes, filtered through 0.45 µm nylon
membrane. The injection volume was 20 µl. The mobile phase flow rate is 1.0 ml/min for
resperidone and 1.0 ml/min for drospirenone, the analysis was carried out at 235 nm and 250 nm
wave length of respective drug.

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Sarat M., et.al., (2012)14
Sarat M., et. al., reported a HPLC method for the simultaneous determination of
desogestrel and ethinyl estradiol tablets. The chromatographic separation was achieved on a
Zorbax SB C-18, 4.6 x 250mm, and 5µ column. The gradient LC method employs solutions A
and B as mobile phase. The solution A milli-Q water and acetonitrile in the ratio of 90:10 and
degas and solution B contains a mixture of Milli Q water and acetonitrile in the ratio of 5:95 and
degas. The flow rate was 1.0 ml/min and the detection wavelength was 225 nm. In the developed
HPLC method, the resolution between Desogestrel, Ethinyl Estradiol and its potential impurities,
namely Imp-1, Imp-2 and Imp-3 was found to be greater than 3. The drug was subjected to stress
conditions of hydrolysis, oxidation, photolysis and thermal degradation.
Shen J., et. al. (2005)15
Shen J., reported HPLC method for the Determination Telmisartan in Human plasma and
its application to a pharmacokinetic study. The HPLC system was equilibrated with the mobile
phase consisting of Acetonitrile- 0.02 mol/L KH PO (50:50 v/v) at a flow rate of 1.2 mL/ min.
2 4
The injection volume was 20 µL and the chromatographic peaks were detected at an excitation
wavelength of 305 nm and an emission wavelength of 365 nm. The column temperature was
maintained at 25°C.
Parvin Z.M., et. al. (2006)16
Parvin Z.M., reported HPLC method for the Simultaneous determination of metoprolol,
propranolol and phenol red in samples from rat in situ intestinal perfusion studies. The mobile
phase was a mixture of 55% methanol and 45% of 0.05 M KH2PO4 aqueous solution adjusted to
pH 6, to which was added 0.2 % (v/v) triethylamine. The mobile phase was filtered through
sintered glass filter P5 (1-1.6 micron) (Winteg, Germany) and degassed in sonicator (Liarre,
Italy) under vacuum. The mobile phase was pumped in isocratic mode at a flow rate of 1 ml/min
at ambient temperature. The UV detection was accomplished at 227 nm and samples of 20 µl
were injected using Hamilton injector syringe (Hamilton, Bonaduz, Switzerland) onto the
column.

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Mariusz S., et. al. (2006)17
Mariusz S., et. al. reported Determination of Metoprolol and Hydrochlorothiazide by
derivative spectrophotometric method in pharmaceutical preparations. The third order
derivative absorption spectra at lambda approximately 281 nm were used for metoprolol and the
first order derivative spectra at lambda approximately 282 nm were used for
hydrochlorothiazide. No interferences were found between both determined constituents and
those of matrix. A good accuracy and precision of simultaneous determination of metoprolol and
hydrochlorothiazide were confirmed by statistical analysis.
Wankhede S. B., et. al. (2007)18
Wankhede S.B. et. al. reported RP-HPLC method for simultaneous estimation of
Telmisartan and Hydrochlorothiazide in tablet dosage form. Chromatography was performed on
a ODS Hypersil C18 (25 cm×4.6 mm I.D) column from thermo in isocratic mode with mobile
phase containing acetonitrile:0.05 M KH PO pH 3.0 (60:40). The flow rate was 1.0 ml/min and
2 4
the eluent was monitored at 271 nm. The selected chromatographic conditions were found to
effectively separate telmisartan (RT- 5.19 min) and hydrochlorothiazide (RT- 2.97 min).
Gupta K.R., et. al. (2008)19
Gupta K.R., et. al. reported new spectrophotometric method for simultaneous determination of
Metoprolol Tartarate and Hydrochlorthiazide in tablets. The wavelengths selected for method
were 257.8 nm, 282.9 nm and 315.0 nm. The absorbance difference at first two wavelengths was
used for determination of Metoprolol and the latter was used for determination of
Hydrochlorthiazide. The recovery value for the drugs from the tablet matrix was found to be
100.55% (Metoprolol) and 99.97% (comparison with standard) and 98.09% (E1%, 1cm) for
hydrochlorthiazide. The method has an advantage that hydrochlorthiazide can be estimated in
combination, as there is no interference of Metoprolol at 315.0 nm. The method was evaluated
statistically for its accuracy and precision.

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Sakarkar D. M., et. al. (2008)20
Sakarkar D. M., et. al. reported RP-HPLC method for simultaneous estimation of
Amlodipine and Metoprolol in tablet formulation. The determination was carried out on a
Kromasil C18 (250 x 4.6 mm, 5 µm) column using a mobile phase of 0.02 M phosphate buffer
solution: Acetonitrile (70:30v/v, pH 3.0). The flow rate was 1.0ml/min with detection at 221 nm.
The retention time for Amlodipine was 2.57 min and for Metoprolol 4.49 min.
Baranowska I., et. al. (2009)21
Baranowska I., et. al. reported RP-HPLC method for the determination of Sotalol,
Metoprolol, alpha-hydroxymetoprolol, Paracetamol and its glucuronide and sulfate metabolites
in human urine. Analyses were carried out on a reversed-phase LiChroCART Purospher C18e
column (125 mm x 3 mm, 5 microm particles) (Merck) with gradient elution as well as
spectrophotometric and fluorometric detection. Good resolution of the analyzed substances was
obtained within a time range of no longer than 15 min.

Kumar S., et. al. (2010)22
Kumar S., et. al. reported spectrophotometric method for simultaneous determination of
Metoprolol Tartrate and Ramipril. The method is based on the ultraviolet absorbance maxima of
the above two drugs at 209.5 nm and 222 nm, respectively. Both the drugs obeyed Beer's law in
the concentration range of 40-120 and 4-20 μg/ml, respectively. The proposed methods were
successfully applied for the simultaneous determination of both drugs in commercial capsule
preparations. The results of the analysis have been validated statistically and by recovery studies.

Mustafa C., et. al. (2010)23
Mustafa C., et. al. reported spectrophotometric method for the determination of
Metoprolol Tartrate in Pharmaceutical Dosage Forms on Complex Formation with Cu(II).
Spectrophotometric method has been developed for the assay of Metoprolol Tartrate , which is
based on the complexation of drug with copper(II) [Cu(II)] at pH 6.0, using Britton-Robinson
buffer solution, to produce a blue adduct. The latter has a maximum absorbance at 675 nm and

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obeys Beer’s law within the concentration range 8.5-70 mg/mL. Regression analysis of the
calibration data showed a good correlation coefficient (r = 0.998) with a limit of detection of
5.56 mg/mL. The proposed procedure has been successfully applied to the determination of this
drug in its tablets. In addition, the spectral data and stability constant for the binuclear copper(II)
complex of MPT (Cu MPT Cl ) have been reported.
2 2 2

Kumar V., et. al. (2011)24
Kumar V., et.al. reported RP-HPLC method for the estimation of Telmisartan in serum
samples, SHIMADZU HPLC system equipped with a reverse phase C18 column (250x4.6mm,
5μm in particle size), a LC-20AT prominence liquid chromatography, a 20μl injection loop and
SPD-20A prominence UV/VIS detector and running on Spinchrom CFR software. Isocratic
elution with Buffer:Acetonitrile (35:65) (V/V) (PH-4.5) was used at a flow rate of 1.0ml/min.
The mobile phase was prepared freshly and degassed by sonicating for 5 min before use.

James D.T., et. al. (2011)25
James D.T., et. al. reported estimation of Telmisartan in Human Plasma by Reversed
Phase Liquid Chromatography Coupled with Tandem Mass Spectrometry - A Bioequivalence
Study Application. Mass Spectrometry Model API 4000, UFLC model UFLC XR equipped with
a model LC-20ADXR a binary pump, SIL-20ACXR auto sampler was used to keep temperature
at 5°C, CTO-20AC column oven used to keep temperature at 35° C and CBM-20Alite system
controller. Detection was made at m/z 513.2/469.3 for Telmisartan and 344/193.8 for internal
standard using ESI negative ion spray ionization mode. Analyst 1.5.1 software was used for the
quantification. The stationary phase was Hypurity Advance C18, 50 X 4.6 mm, 5µm.

Varma D., et. al. (2012)26
Varma D., et. al. reported RP-HPLC Method for the Simultaneous Estimation of
Telmisartan and Hydrochlorothiazide in Pharmaceutical Dosage form. The quantification was
carried out using ProntoSIL C18-EPS 4.6X150mm, 3µm enhanced polar selectivity column and
mobile phase comprised of potassium dihydrogen phosphate buffer pH adjusted to 3.2 ± 0.5 with
Orthophosphoric acid and Aacetonitrile in proportion of ratio 55:45 and degassed under

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