Development and Validation of RP-Chiral HPLC Method for Determination of (R)-Enantiomer Excess Content in Efavirenz

Article abstract of DOI:10.14233/ajchem.2020.22755

A simple, specific, linear, accurate and precise reverse phase chiral HPLC method was developed for the separation of efavirenz enantiomers by using the Lux Amylose-2 column containing amylose tris(5-chloro-2-methyl phenyl carbamate) as a stationary phase. The mobile phase consists of 0.1 % formic acid in water and acetonitrile (55:45, v/v). The flow rate was kept at 1.0 mL/min and the detection wavelength used 252 nm and the column temperature was set at 25 oC. The limit of detection was 0.01 mg/mL and the limit of quantification was 0.04 mg/mL. The linearity calibration curve of (R)-enantiomer was shown well from the range of 0.04 mg/mL to 0.4 mg/mL. The values of the correlation coefficient were 0.999 and 0.999 for (R)-enantiomer and (S)-efavirenz, respectively. The percentage recoveries of (R)-enantiomer from efavirenz drug substance were ranged from 93.5% to 107.5%. The results demonstrated that developed RP-chiral HPLC method was simple, precise, robust and applicable for the estimation of (R)-enantiomer in efavirenz API. This method was validated in as per ICH Q2 (R1) and USP validation of compendial methods <1225>.

Full text of DOI:10.14233/ajchem.2020.22755

Asian Journal of Chemistry; Vol. 32, No. 9 (2020), 2208-2212
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22755
Development and Validation of RP-Chiral HPLC Method for
Determination of (R)-Enantiomer Excess Content in Efavirenz
1,2
3
1,2
2
1,*,
1,*,
CH. RAMESH , DHARMASOTH RAMA DEVI , M.N.B. SRINIVAS , S. RADHA KRISHNA , NAGARAJU RAJANA
and K. BASAVAIAH
1Department of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam-530003, India
2Laurus Labs Ltd., Visakhapatnam-531021, India
3A.U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530003, India
*Corresponding authors: E-mail: nagarajrajana@gmail.com; klbasu@gmail.com
Received: 3 April 2020;
Accepted: 26 May 2020;
Published online: 20 August 2020;
AJC-20014
A simple, specific, linear, accurate and precise reverse phase chiral HPLC method was developed for the separation of efavirenz enantiomers
by using the Lux Amylose-2 column containing amylose tris(5-chloro-2-methyl phenyl carbamate) as a stationary phase. The mobile
phase consists of 0.1 % formic acid in water and acetonitrile (55:45, v/v). The flow rate was kept at 1.0 mL/min and the detection
wavelength used 252 nm and the column temperature was set at 25 ºC. The limit of detection was 0.01 mg/mL and the limit of quantification
was 0.04 mg/mL. The linearity calibration curve of (R)-enantiomer was shown well from the range of 0.04 mg/mL to 0.4 mg/mL. The
values of the correlation coefficient were 0.999 and 0.999 for (R)-enantiomer and (S)-efavirenz, respectively. The percentage recoveries of
(R)-enantiomer from efavirenz drug substance were ranged from 93.5% to 107.5%. The results demonstrated that developed RP-chiral
HPLC method was simple, precise, robust and applicable for the estimation of (R)-enantiomer in efavirenzAPI. This method was validated
in as per ICH Q2 (R1) and USP validation of compendial methods <1225>.
Keywords: Efavirenz, (R)-Enantiomer, RP-chiral HPLC, Validation.
chiral identity, enantiomeric impurity and chiral assay tests
are required for product specifications and quantify each enan-
tiomer of the chiral drug individually [11,12].
INTRODUCTION
Efavirenz is chemically (4S)-6-chloro-4-(2-cyclopropyl-
ethynyl)-4-(trifluoromethyl)-1H-3,1-benzoxazin-2-one and it′s
molecular formula is C14H9NO2ClF3 with molecular weight
315.675. Efavirenz is a non-nucleoside reverse transcriptase
inhibitor (NNRTIs) with activity against HIV and one of the
first AIDS drugs approved for once-daily dosing and most
marketed pharmaceutical. It is used in combination with other
antiretrovirals for the therapy of HIV [1-6]. The drug molecule
contains one chiral center and the configuration of molecule is
"S" (Fig. 1).
Most of the drugs exist as enantiomers, as the chemical
property of enantiomers is different in a chiral environment,
only one isomer is essential and having a positive effect while
another enantiomer may lead unwanted side effects on human
body [7-10]. According to the International Conference on
Harmonization (ICH) guidelines, The United States Food and
DrugAdministration (US FDA) and other regulatory agencies,
Hence, it is important to encourage all the chiral drug mole-
cules have to be separated and tested to remove the undesired
isomer from the preparation and as well as to find optimal treat-
ment and the right therapeutic control for the patient [13,14].
Chromatography on solid stationary phases is one of the most
powerful separation techniques as a wide spectrum of possi-
bilities offered by adjusting both the liquid mobile phase and
the solid stationary phase to achieve the separation [15,16]. A
literature survey reveals that few analytical methods have been
established for determination efavirenz and its related
substances in drug substance and estimation of (R)-enantiomer
by normal phase chiral HPLC [17-24]. However, the separation
of efavirenz enantiomers has not yet been carried out by RP-
chiral HPLC. The more volatile solvents such as hexane, heptane,
etc. in the normal phase methods can be avoided by developing
the chiral separations by RP-Chiral HPLC.
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Vol. 32, No. 9 (2020)
RP-Chiral HPLC Method for Determination of (R)-Enantiomer Excess Content in Efavirenz 2209
H
H
N
Preparation of mobile phase
N
O
O
Mobile phase-A: Pipetted 1.0 mL of formic acid into 1000
mL of Milli-Q water and mixed well, filtered through 0.45
µm filter paper and degassed.
Mobile phase-B: Used a filtered and degassed acetonitrile.
Diluent: Mixed 500 mL of water and 500 mL of aceto-
nitrile, filtered through 0.45 µm filter paper and degassed.
O
O
Cl
Cl
F
F
F
F
F
F
RESULTS AND DISCUSSION
(R)-Enantiomer
(S)-Efavirenz
Method development and optimization: To develop the
quantitative reverse-phase chiral HPLC method for (R)-enan-
tiomer content in efavirenz, several trials were taken up by
changing the columns, mobile phase and also different tempera-
tures of the column. This HPLC method aims to get symmetry
in the shape of the peak and to separate (R)-enantiomer and
(S)-efavirenz with adequate resolution.
The attempts of method development were focused in
reverse phases buffers such as ammonium acetate, formic acid,
methanol, acetonitrile, etc. Initial method development attempts
were made on different stationary phases such as cellulose
tris(3,5-dimethylphenylcarbamate), cellulose tris(3-chloro-4-
methylphenylcarbamate), cellulose tris-(4-methylbenzoate),
cellulose tris(4-chloro-3-methylphenylcarbamate) and amylose
tris(5-chloro-2-methylphenylcarbamate) on 5 µm silica-gel
with different composition of solvent mixtures.
In this method development, the stationary phase has
played a significant role in achieving the resolution between
(R)-enantiomer and (S)-efavirenz. Finally, the satisfactory peak
shape and resolution were achieved between two isomers on
amylose tris(5-chloro-2-methylphenylcarbamate) column (250
× 4.6 mm and 5.0 µm particle size) by using formic acid
solution and acetonitrile as mobile phase and a mixture of water
and acetonitrile as diluent. These Lux polysaccharide-based
chiral stationary phase columns was specially designed to resolve
the stereoisomeric pharmaceutical compounds. Efavirenz mole-
cule contains one chiral center and the absolute configuration
of molecule is (S), hence the separation of isomers was effec-
tively done by using the column of Lux Amylose-2. The
method development trials are given in Table-1.
Fig. 1. Chemical structure of (R)-enantiomer and (S)-efavirenz
EXPERIMENTAL
Active pharmaceutical ingredient of efavirenz and efavi-
renz racemic mixture was synthesized by process research
development of Laurus Labs Limited,Visakhapatnam. Formic
acid (AR grade) and acetonitrile (HPLC grade) were purchased
from the Sigma-Aldrich, India. High purity water was prepared
using a Millipore ReferenceA+ (Millipore, Milford, MA, USA)
purification system.
Instrumentation: HPLC system with make Shimadzu
LC-2010 CHT and weights measurements are taken by using
an analytical balance with make Mettler Toledo XS205 dual
range. Data were processed through LC solution software.
Chromatographic conditions: A column used is Lux
Amylose-2, 250 mm × 4.6 mm, 5 µm particle size (Pheno-
menex) as stationary phase with a mobile phase 0.1% Formic
acid in water and acetonitrile ( 55:45, v/v ) at a flow rate of 1.0
mL min-1 and UV detection wavelength at 252 nm and 20 µL
sample was injected for 15 min.
Preparation of system suitability solution: Weighed 10
mg of efavirenz racemic mixture standard and transferred into
100 mL volumetric flask, dissolved and diluted to volume with
diluent and mixed well.
Preparation of standard solution: Weighed 50 mg of
efavirenz standard and transferred into 100 mL volumetric
flask, dissolved and diluted to volume with diluent and mixed
well.
TABLE-1
RESULTS OF METHOD DEVELOPMENT
Trial
1
Column
Dimensions
Stationary phase
Mobile phase
Conclusion
Cellulose tris-(3,5-
dimethylphenylcarbamate)
Cellulose tris-(3-chloro-4-
methylphenylcarbamate)
5 mM Ammonium acetate
in water and acetonitrile
5 mM Ammonium acetate
in water and acetonitrile
5 mM Ammonium acetate
in water and acetonitrile
5 mM Ammonium acetate
in water and acetonitrile
5 mM Ammonium acetate
in water and acetonitrile
0.1 % Formic acid in
water and methanol
0.1 % Formic acid in
water and acetonitrile
Lux Cellulose-1 250 × 4.6 mm × 5.0 µ
Lux Cellulose-2 250 × 4.6 mm × 5.0 µ
Lux Cellulose-3 250 × 4.6 mm × 5.0 µ
Lux Cellulose-4 250 × 4.6 mm × 5.0 µ
No separation
2
3
4
5
6
7
No separation
No separation
No separation
No separation
Cellulose tris-(4-methylbenzoate)
Cellulose tris-(4-chloro-3-
methylphenylcarbamate)
Amylose tris-(5-chloro-2-
methylphenylcarbamate)
Amylose tris-(5-chloro-2-
methylphenylcarbamate)
Amylose tris-(5-chloro-2-
methylphenylcarbamate)
Lux Amylose-2
Lux Amylose-2
Lux Amylose-2
250 × 4.6 mm × 5.0 µ
250 × 4.6 mm × 5.0 µ
250 × 4.6 mm × 5.0 µ
Separation achieved but
peaks are broad
Separation achieved with
sharp peaks

2210 Ramesh et al.
Asian J. Chem.
TABLE-4
RESULTS OF PRECISION AT LOQ
Method validation: The described (R)-enantiomer
content in the efavirenz trial method was validated as per the
ICH guidelines.
Specificity: The specificity of the method is performed
by injecting the efavirenz system suitability solution (Fig. 2)
and efavirenz standard solution (Fig. 3) individually. The speci-
ficity determined by using resolution between (R)-enantiomer
and (S)-efavirenz. Baseline separation with a resolution of more
than 3.0 achieved and the results are reported in Table-2.
No. of preparation
(R)-Enantiomer
(S)-Efavirenz
1
2
3
4
5
5197
4808
4617
5265
5273
4674
4972.3
306.2
6.15
4865
4837
5132
4854
5228
5160
5012.6
178.9
3.57
6
Average
STDEV
%RSD
500
400
300
200
100
0
times and peak areas for enantiomer. The repeatability of the
method was determined by analyzing six replicate injections
containing efavirenz and (R)-enantiomer (each 0.2 mg/mL).
The study was performed by the analyst on a different day, using
different column and instrument and for two different days to
study inter-day variation, and also prepare different solutions
on different days. The % RSD of (R)-enantiomer found in the
spiked samples was calculated.
5.0
7.5
10.0
min
12.5
15.0
Fig. 2. System suitability chromatogram of (R)-enantiomer and (S)-efavirenz
The RSD (%) of peak area for (R)-enantiomer in the study
of repeatability is shown inTable-5. Results for method precision
(intra- and inter-day repeatability) are within 5.0%. These results
confirm that the method is highly precise.
500
250
0
TABLE-5
PRECISION AT SPECIFICATION LEVEL
No. of preparation
(R)-Enantiomer area
1
2
3
4
5
26692
26247
27267
27603
27998
26759
27094
647.6
2.4
5
6
7
8
9
10
11
12
13
14
15
min
Fig. 3. Standard chromatogram of (S)-efavirenz
6
TABLE-2
SPECIFICITY RESULTS
Average
STDEV
%RSD
Retention
time (min)
Plate
count
Tailing
factor
Name
Resolution
(R)-Enantiomer
(S)-Efavirenz
9.721
11.063
8652
9018
1.3
1.3
–
3.04
Accuracy: The accuracy of the method was carried out
by injecting a known concentration i.e. of (R)-enantiomer to
efavirenz. The accuracy was calculated in terms of recovery (%)
and the obtained chromatogram is shown in Fig. 4. It is observed
that the percentage recovery of (R)-enantiomer in bulk drug
samples ranged from 93.5% to 107.5% and results are reported
in Table-6.
Limit of detection (LOD) and quantification (LOQ):
The LOD and LOQ for efavirenz and (R)-enantiomer were
determined at a signal to noise ratio of 3:1 and 10:1, respect-
ively, by injecting a series of a dilute solution with the known
concentration of efavirenz and (R)-enantiomer. The precision
study was carried out at the LOQ level by injecting six replicate
injections individually and the % RSD for the area counts was
calculated. The limit of detection and limit of quantification
for (R)-enantiomer and efavirenz are reported in Table-3 and
precision at LOQ in Table-4.
20
15
10
5
Precision: Method reproducibility was determined by
measuring repeatability and intermediate precision of retention
TABLE-3
0
RESULTS LOD AND LOQ
-5
Conc.
(mg/mL)
(R)-
Enantiomer
(S)-
Efavirenz
8
9
10
11
min
12
13
14
Parameter
S/N ratio
LOD
LOQ
0.01
0.04
1072
5197
1295
4864
17
48
Fig. 4. Chromatogram of (R)-enantiomer at 0.2 mg/mL and (S)-efavirenz
at test sample concentration

Vol. 32, No. 9 (2020)
RP-Chiral HPLC Method for Determination of (R)-Enantiomer Excess Content in Efavirenz 2211
TABLE-6
TABLE-8
RESULTS OF ROBUSTNESS
RESULTS OF ACCURACY AT 0.2 mg/mL OF
(R)-ENANTIOMER w.r.t SAMPLE CONCENTRATION
Parameter
0.9
Resolution
No. of preparation
Area (%)
Recovery (%)
2.99
3.04
3.01
3.11
3.04
2.96
3.27
3.04
2.74
1
2
3
4
5
6
0.187
0.215
0.208
0.196
0.200
0.209
93.5
107.5
104.0
98.0
100.0
104.5
Flow rate (mL/min)
1.0
1.1
20
25
30
10
15
20
Column temperature
(°C)
Organic ratio
Linearity : Detector response linearity was assessed by
preparing eight calibration sample solutions of (R)-enantiomer
covering from 0.04 to 0.4 mg/mL with respect to sample
concentration in the diluent. The regression curve was obtained
by plotting area versus concentrations using the least square
method. The correlation coefficient, slope and Y-intercept of
the calibration curve was calculated and the obtained plots
are shown in Fig. 5. For (R)-enantiomer, linear calibration curve
was obtained ranging from LOQ, 50, 80, 100, 120, 150 and
200%. The correlation coefficient obtained is greater than 0.999.
The results indicate excellent linearity (Table-7).
flask at room temperature on a laboratory bench for 48 h. Content
of (R)-enantiomer was checked for every 12 h interval and
compared with the freshly prepared solution. No variation was
observed in the content of (R)-enantiomer for the study period
and this indicates efavirenz sample solutions prepared in
diluent were stable up to 48 h at room temperature.
Mobile phase stability was carried out by evaluating the
content of (R)-enantiomer in efavirenz, which were prepared
freshly at every 12 h interval for 48 h. The same mobile phase
was used during the study period. No variation was observed
in the content of (R)-enantiomer for the study period and it
indicates prepared mobile phase was found to be stable up to
48 h at room temperature.
60000
50000
40000
30000
20000
10000
0
Conclusion
A simple, specific, linear, accurate and precise reverse
phase chiral HPLC method was successfully developed, which
was capable of separating the undesired enantiomer from
efavirenz, column Lux Amylose-2 (250 mm × 4.6 mm, 5.0
µm) was found to be selective for enantiomers of efavirenz.
The developed and validated method can be used for the esti-
mation of (R)-enantiomer in efavirenz API. The developed
method is also stable and can be used for the quantitative deter-
mination of chiral impurity in efavirenz drug substance form.
0
0.1
0.2
0.3
0.4
0.5
Concentration (mg/mL)
Fig. 5. Linearity plot for (R)-enantiomer
CONFLICT OF INTEREST
TABLE-7
LINEARITY OF (R)-ENANTIOMER
The authors declare that there is no conflict of interests
regarding the publication of this article.
Level
Concentration (mg/mL)
Area
LOQ
50
80
100
120
150
200
0.04
0.10
0.16
0.20
0.24
0.30
0.40
5500
13291
20668
26247
31477
39193
51959
0.999
129972
1.30
REFERENCES
1. M.M. Bastos, C.C.P. Costa, T.C. Bezerra, F. de C. da Silva and N.
Boechat, Eur. J. Med. Chem., 108, 455 (2016);
https://doi.org/10.1016/j.ejmech.2015.11.025
2. R. Gaida, I. Truter and C. Grobler, South Afr. J. Psych., 21, a783 (2015);
https://doi.org/10.4102/sajpsychiatry.v21i3.783
3. B.M. Best and M. Goicoechea, Expert Opin. Drug Metab. Toxicol., 4,
965 (2008);
Correlation coefficient
Slope
% Y-intercept
https://doi.org/10.1517/17425255.4.7.965
4. J.C. Adkins and S. Noble, Drugs, 56, 1055 (1998);
https://doi.org/10.2165/00003495-199856060-00014
5. S.M.E. Vrouenraets, F.W.N.M. Wit, J. van Tongeren and J.M.A. Lange,
Expert Opin Pharmacother., 8, 851 (2007);
https://doi.org/10.1517/14656566.8.6.851
6. M. Maggiolo, J. Antimicrob. Chemother., 64, 910 (2009);
https://doi.org/10.1093/jac/dkp334
Robustness: To determine the robustness of method, the
flow rate was changed from 0.9 to 1.1 mL/min. The effect of
a change in the percent acetonitrile, column temperature at 20
and 30 ºC instead of 25 ºC were studied, and the other chromato-
graphic conditions were held constant stated previously and
obtained results of robustness are shown in Table-8.
7. A. Gogoi, N. Mazumder, S. Konwer, H. Ranawat, N.-T. Chen and G.-
Y. Zhuo, Molecules, 24, 1007 ( 2019);
Analytical solution stability: Solution stability was studied
by keeping the test solution in the tightly capped volumetric
https://doi.org/10.3390/molecules24061007

2212 Ramesh et al.
Asian J. Chem.
8. L.A. Nguyen, H. He, C. Pham-Huy and C. Drugs, Int. J. Biomed. Sci.,
2, 85 (2006).
9. N. Chhabra, M.L. Aseri and D. Padmanabhan, Int. J. Appl. Basic Med.
Res., 3, 16 (2013);
17. K. Tachibana and A. Ohnishi, J. Chromatogr. A, 906, 127 (2001);
https://doi.org/10.1016/S0021-9673(00)00955-9
18. U. Seshachalam, D.V.L. Narasimha Rao and K.B. Chandrasekhar,
Pharmazie, 63, 107 (2008);
https://doi.org/10.4103/2229-516X.112233
https://doi.org/10.1691/ph.2008.7208
10. S.W. Smith, Toxicol. Sci., 110, 4 (2009);
https://doi.org/10.1093/toxsci/kfp097
19. S.S. Pujeri, A.M.A. Khader and J. Seetharamappa, J. Food Drug Anal.,
21, 93 (2013);
11. A.G. Rauws and K. Groen, Chirality, 6, 72 (1994);
https://doi.org/10.1002/chir.530060206
12. S.K. Branch, J. Pharm. Biomed. Anal., 38, 798 (2005);
https://doi.org/10.1016/j.jpba.2005.02.037
https://doi.org/10.6227/jfda.2013210112
20. A.F. Aubry, D.S. Sebastian, R.C. Williams and R.J. Boucher, Chirality,
13, 193 (2001);
https://doi.org/10.1002/chir.1019
13. V. Famiglini and R. Silvestri, Molecules, 21, 221 (2016);
https://doi.org/10.3390/molecules21020221
21. G. Lavison and D. Thiébaut, Chirality, 15, 630 (2003);
https://doi.org/10.1002/chir.10263
14. A. Nag, Asymmetric Synthesis of Drugs and Natural Products, CRC
Press: Boca Raton (2018).
15. B. Li and D.T. Haynie, ed.: S. Lee, Chiral Drug Separation, In: Encyclo-
pedia of Chemical Processing, , CRC Press: Boca Raton (2005).
16. O. Coskun, North. Clin. Istanb., 3, 156 (2016);
https://doi.org/10.14744/nci.2016.32757
22. S.D. Sharma and G. Singh, Adv. Anal. Chem., 3, 29 (2013).
23. Efavirenz Monograph, USP42 NF37, p. 1554 (2019).
24. Efavirenz monograph, The International Pharmacopeia, edn 8 (2018).