ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry
http://www.e-journals.net 2011, 8(4), 1504-1511

Practical Approach for the Determination of

Response Factors of Impurities in Drugs by HPLC

DHARMENDRA KUSHWAH*, HARESH B. PATEL,

PRAVEEN K. SINHA and PIJUSH K. JANA

Unimark Remedies Ltd., 337, Kerala Nalsarover Road, Vill. Kerala, India
Tal. Bavla, Dist. Ahmedabad, Gujarat-382220, India
[email protected]

Received 31 August 2010; Revised 28 January 2011; Accepted 28 February 2011

Abstract: For the determination of accurate quantity of impurities in the

samples authentic impurity standards or response factors at a given wavelength
must be known. In the presented work a convenient method for determination
of relative response factors of impurities has been described without using an
authentic impurities standard. An approach for the determination of response
factors of the impurities where impurity standard is physically not available
was developed and verified using different approaches. One such method was
developed and verified by RP-HPLC using Cosmosil C18 MS-II column at UV
238 nm. Two different approaches were employed and the verification of
correctness of approach was done using a known related substance of known
response factor.

Keywords: Response factor (RF), RP-HPLC, Montelukast sodium (MNT), Michaels adduct.

Introduction
Impurities arising from the manufacturing process or via degradation are required to control
in the drug substances and drug product. There are guidelines from the international
conference on harmonization1-2 (ICH) for reporting threshold, identification threshold and
quantification threshold of impurities in new drug substances and drug products. It is very
important to determine the actual reproducible values of impurities within these threshold
values. The most accurate method of quantifying the impurity is to use external standard of
the impurity, this is not always practically possible due to the non availability of ample
supply of impurities. It is very difficult to isolate or synthesize and to maintain the impurities
standard over several years as this also requires periodic reevaluation of impurities.
1505 D. KUSHWAH et al.

Alternatively, response factors (RF) of the impurities are used for the determination of
actual amount of impurities present in pharmaceuticals. The response factor is the ratio
between a signal produced by impurity and active pharmaceutical ingredient (API) under the
same detection condition. Response Factors (RF) are determined by the analysis of standards
and are used to calculate the concentrations of analytes in samples using the following
equation;
Response of Impurity
RF =
Response of API

No significant work has been done on the determination of response factors of impurities
in drug product and drug substances using HPLC and UV-Vis. Detector. Ping Sun et al.3 has
reported the determination of Relative Responses factor (RRF) of impurities in Paclitaxel in
authentic pure materials which was estimated using high performance liquid chromatography
equipped with ultraviolet and charged aerosol detectors3. Gregory et al.4 has reported the
determination of relative Responses factor (RRF) using NMR spectrophotometer in absence of
any authentic standards4. Liang et al.5 used chemiluminescent nitrogen detection in
pharmaceutical analysis for the determination of equimolar response for nitrogen present in
impurities5. A formula for the determination of relative response factors was derived for butter
oil fatty acids and a regression line representing the best fit of the equation was calculated by
Steen et al.6 using gas chromatography6.
F = a + b/(CN − c)
Where, F and CN are the relative response factor and the number of carbon atoms in the
fatty acid butyl esters, respectively and a and b are constants. Nussbaum et al.7 used
chemiluminescence nitrogen-specific detector for the determination of relative UV response
factors by HPLC for nifedipine. Influence of relative response factor in the determination of
organic micro contaminants with isotopically labeled standards was studied by Luigi et al. 8 in
the in the analysis of organic micro contaminant entails. Investigation of response factor
ruggedness for the determination of drug impurities using high-performance liquid
chromatography with ultraviolet detection was done by Bernard A. Olsen et al 9.
In the presented work two different approaches have been demonstrated for the
determination of response factors of unknown and unspecified impurities and the
verification is done using authentic impurity standard.
Experimental
Acetonitrile and methanol of HPLC grade were used of Rankem. Ultra pure millipore water
was used. Ammonium bicarbonate (AR grade) triethylamine (HPLC grade), trifluoroacetic
acid (LR grade) were obtained from Merck, In house prepared montelukast working
standard, sample and michael adduct impurity wee used.
Instrumentation and chromatographic conditions
The HPLC system (alliance, waters) consisting of UV / PDA detector empower software
was used. Analytical balance (Mettler Toledo) was used. HPLC analysis was conducted
using a Cosmosil MS-II, 250 mm x 4.6 mm, 5 µm particle size column. Mobile phase A
constituted of 0.01 M ammonium bicarbonate solution, added tritely amine 2 mL per litter
and pH adjusted to 7.3 with trifluoroaceic acid, buffer and 60% and acetonitrile 40%.
Methanol was used as mobile phase B and diluent. Program gradient elution (time/min.)
%B- 0/13, 45/87, 50/87, 53/13, 60/13 was used with UV detection at 238 nm, flow rate
2 mL/min., column oven temperature 40 °C and injection volume 10 µ L.
 Practical Approach for the Determination of Response Factor 1506

Results and Discussion

The IUPAC name of montelukast sodium compound with structure A is [1-[[[(1R)-1-[3-
[(E)-2-(7-chloroquinolin-2yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl] propyl]
sulfanyl]methyl]cyclopropyl]acetate commercially available as sodium salt. (Molecular
weight 608.17)

OH

S
N
O

Cl

HO
CH3
H3C
Na.
A
HO

OH

S
N O

Cl

HO
CH 3
H 3C

B
HO

OH

S
N O

Cl

HO
CH3
H3C

C
Figure 1. Structure of montelukast sodium compound (A); and impurity Micheal
adduct-1(C) and adduct -2 (D).
1507 D. KUSHWAH et al.

Compound with structure B and C are impurity micheal adduct present in two isomeric
forms micheal adduct-1 and micheal adduct-2. IUPAC name of both the impurities are;
1-[[[(1R)-2-[3-[(1R)-1-[[[1-(carboxymethyl)cyclopropyl]methyl]sulfanyl]-3-[2-(2-hydroxy
propan-2-yl)phenyl]propyl]phenyl]-1-(7-chloroquinolin-2-yl)ethyl] sulfanyl] methyl] cyclo-
propanecarboxylic acid and 1-[[[(1S)-2-[3-[(1R)-1-[[[1 (carboxymethyl) cyclopropyl]methyl]
sulfanyl]-3-[2-(2-hydroxypropan-2-yl)phenyl] propyl] phenyl]-1-(7-chloroquinolin-2-yl)ethyl]
sulfanyl] methyl] cyclopropane carboxylic acid. (Molecular weight 717.23)
Chromophoric structure of both the compounds is almost similar hence the molar
absorption coefficient of both the components will be almost the same at 238 nm. The UV
spectra of micheal adduct-1, micheal adduct-2 and montelukast are shown in Figure 2.

Figure 2. UV spectra of micheal adduct-1, 2 and montelukast

The response factors were determined by two different approaches. In approach-1 two
types of samples, sample A and sample B of montelukast sodium were selected. Sample A
contains very trace amount of michael adduct impurity and it was prepared by subsequent
purification and re-crystallization and sample B contains some higher amount of michael
adduct impurity. Sample A and sample B were blended in different compositions and then
individual sample A, sample B and different blended mixtures were injected separately.
Concentration of the main drug was adjusted in such a way that, it is within the acceptable
linearity range of HPLC. The area count and % Area for both micheal adducts were taken by
adding the response of both the peaks of micheal adduct-1, micheal adduct-2 added.
Theoretical % area was calculated as per formula given below;
Theoretical % area = {( A1 x R1) + (A2 x R2)} / 100
Where, A1 and A 2 are the area of impurity in sample A and sample B and R1 and R2 are
concentration of sample A and sample B respectively in the blended samples. (% area in B x
ratio added)}/100 RF values are calculated as per formula given below;
RF value = Theoretical % area / Actual % area
RF value was calculated for individual blended mixture and finally average RF value
was calculated. The average calculated, RF value obtained is 1.06 (Table 1). Representative
chromatograms are given in Figure 3 to Figure 5. Concentration of main drug was adjusted
such, that it is within the acceptable linearity range of instrument (HPLC).
 Practical Approach for the Determination of Response Factor 1508

Table 1. Determination of RF value of unknown impurity (Approach-1)

Area Total% Theoretical RF Mean RF
Sample Total
area % area value value
Peak 1 Peak 2 area
Sample A (100%) 1141 2153 3294 0.02
A + B (75:25) 30822 21610 52432 0.23 0.24 1.04
A + B (50:50) 55664 34392 90056 0.42 0.47 1.09 1.06
A + B (25:75) 91790 58103 149893 0.68 0.69 1.01
Sample B (100%) 92281 69770 162051 0.91
AU

Minutes
Figure 3. Chromatogram of sample A
AU

Minutes
Figure 4. Chromatogram of mixture of sample A and B (50:50)
AU

Minutes
Figure 5. Chromatogram of sample B
1509 D. KUSHWAH et al.

In approach-2 linearity was plotted with the total area of micheal adduct (1+2) against
the micheal adduct concentration in all five samples. Concentration of michael adduct was
calculated by multiplying % area with RF value calculated as per Table 1. Separately
linearity of montelukast standard was determined at lower level (concentration similar to the
concentration of micheal adduct (1+2). The montelukast standard solutions were prepared
similar to the concentration of michael adduct. RF value of micheal adduct (1+2) was
calculated using the slop method, as given below;
RF value = slope of standard / slope of impurity 5
RF value obtained with approach-2 is 1.05 (Table 2), which is very close to the value
obtained with approach-1 (Table 1).
Table 2. Determination of RF value of unknown impurity (Approach-1)
Impurity Montelukast standard
Sample Total area % Area Conc., µg/mL Area Conc., µg/mL
3112 0.506
Sample A (100%) 3294 0.02 0.424 9586 1.011
A + B (75:25) 52432 0.24 4.835 9401 2.022
A + B (50:50) 90056 0.43 9.918 43442 5.056
A + B (25:75) 149893 0.70 14.00 99154 10.112
Sample B (100%) 162051 0.91 19.224 150559 15.168
200244 20.224
Correlation 0.99316 0.99957
Intercept 863 -2286
Slope 9572 10009
RF value 1.05
Verification for RF value using impurity standard
For the verification of RF values obtained with approach-1 and approach-2, small amount of
impurity was isolated using preparative HPLC. Linearity of micheal adduct (1+2) was
performed with montelukast standard as per USP and ICH guidelines10,11 and RF value was
calculated using slope method (eq.5). The RF value obtained is 1.08 (Table 3) which is very
close and is within acceptable range with the RF values obtained with approach-1 and approach-2.
Representative chromatograms and linearity plots are given in Figure 6 and Figure 7.
Table 3. Verification for RF value using impurity standard
Impurity Montelukast standard
Area Total area Conc., µg/mL Area Conc., µg/mL
Peak-1 Peak-2
1539 1110 2649 0.388 1636 0.272
3031 2063 5076 0.775 4024 0.544
4451 5127 9578 1.550 7428 1.089
12497 11568 24065 3.100 17751 2.178
24344 24664 49008 6.201 34235 4.355
49762 47560 97322 12.402 66401 8.711
97809 96232 194041 24.803 149772 17.422
Correlation 0.99991 0.99852
Intercept 0.1077 0.2466
Slope 7875.24 8537.46
RF value 1.08
 Practical Approach for the Determination of Response Factor 1510

Minutes

Figure 6. Chromatogram of Impurities with standard montelukast sodium

Imp-F
Linearity Plot Michel Adduct
MNTD
200000

150000
Area
Area

100000

50000

0
0.000 5.000 10.000 15.000 20.000 25.000
Conc.
Conc.

Figure 7. Linearity plot of impurities and standard

Conclusion
Determination of correct quantified value of any impurity is very important in
pharmaceutical industry. The presented work describes two different approaches for the
calculation of RF values without using impurity standard in drug substances and drug
products. This method can be applied for any potential impurity present in sample.
Verification of the approaches using impurity standard further strengthens the accuracy and
correctness of the method.
Acknowledgment
Authors are highly thankful to the management of Unimark Remedies Ltd. for granting
permission for the publication of this work.
References
1. ICH, Q3A (R2) Impurities in New Drug Substances: Text and Methodology, Current
Step 4 version.
2. ICH, Q3B (R2) Impurities in New Drug Substances: Text and Methodology, Current
Step 4 version.
3. Ping Sun, Xiande Wang, Lori Alquier and Cynthia A. Maryanoff, J Chromatogr A,
2008, 1177(1), 87-91.
1511 D. KUSHWAH et al.

4. Gregory K Webster Ian Marsden, Cynthia A Pommerening, Christina M Tyrakowski

and Brian Tobias, J Pharm Biomed Anal., 2009, 49(5), 1261-1265.
5. Liang X, Patel H, Young J, Shah P and Raglione T, J Pharm Biomed Anal., 2008,
47(4-5), 723-730.
6. Steen S. Jacobsen, Claus C. Becker and Gunhild Hølmer, Chemometrics and
Intelligent Laboratory Systems, 1994, 23, 231-234.
7. Nussbaum M A, Baertschi S W and Jansen P J, J Pharm Biomed Anal., 2002, 27(6),
983-993.
8. Luigi Turrio-Baldassarri, Alessandro di Domenico, Annarita Fulgenzi, Cinzia La Rocca,
Nicola Iacovella, Fabrizio Rodriguez and Fabrizio Volpi, Microchimica Acta, 1996,
123(1), 45-53.
9. Bernard A. Olsen and Mark D. Argentine, J Chromatogr. A, 1997, 762(1-2), 227-233.
10. ICH, Q2 (R1) Validation of Analytical Procedures: Text and Methodology, Current
Step 4 version.
11. United States Pharmacopeia, <1225> Validation of Compendial Procedures, USP32,
NF-27, Volume 1, 2nd Supplement.