Determination of Carbamazepine

in Pharmaceutical Dosage Form Using GC-FID

2007, 66, S169–S172

Yucel Kadioglu&, Fatma Demirkaya

Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum, Turkey; E-Mail: yucel@atauni.edu.tr

Received: 28 January 2007 / Revised: 22 May 2007 / Accepted: 23 May 2007

Online publication: 15 August 2007

oxazepam, o-chlorooxazepam, N-meth-

yloxazepam, nitrooxazepam, carbamaze-
Abstract pine and carbamazepine-10,11-epoxide
with regard to decomposition and
The present work describes the methodology and validation of gas chromatography with flame misidentification. In another study, the
ionization detection (GC-FID) for the determination of carbamazepine with internal standard determination of the quantification of an-
(diazepam) in pharmaceutical preparations. The method was linear from 2–30 lg mL)1. The tiepileitic drugs such as carbamazepine,
RSD values for precision was less than 9%, accuracy (relative error) was better than 11% phenytoin, phenobarbital, primidone have
(n = 6). The developed method was successfully applied for the assay of pharmaceutical been examined by gas chromatography
dosage forms which do not require any preliminary separation or treatment of the samples. The used fused-silica capillary columns [32].
RSD values for Tegretol
tablets (200 mg) and Karberol
tablets (200 mg) was found to be CBZ has been generally analyzed by
4.03 and 3.25%, respectively. The results obtained from this method were compared with the HPLC and spectroscopic methods in
reference method (LC) reported in literature and no significant difference was found statistically. pharmaceutical preparations. The GC
method for determination together with
other drugs of CBZ in pharmaceutical
preparations has been reported.
Keywords The present work describes a newly
developed GC-FID method for determi-
Gas chromatography
nation of CBZ only with a simple sample
GC-FID
preparation without derivatization in
Pharmaceutical dosage form
pharmaceutical preparations using inter-
Carbamazepine
nal standard methodology. The devel-
oped method was validated by using
linearity, stability, precision, accuracy
and sensitivity parameters according to
Introduction tranquilizer, anticonvulsant and muscle International Conference on Harmoni-
relaxant [5, 6]. zation (ICH) guidelines [33]. The results
Carbamazepine (CBZ) (5-H-dibenz Several methods for determination of obtained of this new developed and vali-
[b,f]azepine-5-carboxamide) is an anti- CBZ in pharmaceutical preparations and dated method was statistically compared
convulsant used in clinical practice as first- standard solutions have been reported. to the reference method (LC) [16].
line treatment for generalized tonic–clonic These include spectrophotometric meth-
and partial seizures [1–3]. CBZ, poorly ods [7, 8], spectrofluorimetric methods [9–
soluble in aqueous media, has a high oral 12], Raman spectroscopy [13] and high- Experimental
bioavailability in humans [4]. Diazepam performance liquid chromatography
(DZ), (internal standard) 7-chloro-1,3- (HPLC) [14–20]. Gas chromatographic Chemicals and Reagents
dihydro-1-methyl-5-phenyl-2H-1,4-ben- methods have been published for deter-
zodiazepin-2-one is the most commonly mination of CBZ in human plasma/serum CBZ standard substance and diazepam
benzodiazepine drug used as hypnotic, [20–29] and pharmaceutical preparations have been kindly supplied by Novartis
[30–32]. Frigerio et al. [31] have examined Pharmaceutical Industry (Ankara,
gas chromatographic behavior of drugs of Turkey) and the Criminal Police Labo-
Application of Separation Techniques in Turkey

Limited Short Communication Chromatographia Supplement Vol. 66, 2007 S169

DOI: 10.1365/s10337-007-0327-2
 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

2 µg ml-1
5 µg ml-1
10 µg ml-1
(CBZ/DZ)
15 µg ml-1
20 µg ml-1
25 µg ml-1
CBZ 30 µg ml-1
Fig. 1. GC-FID chromatogram of standard solutions of CBZ contained ISI (10 lg mL)1)
ratories (Erzurum, Turkey), respectively.
Methanol and all other chemicals (ana-
lytical grade) were obtained from Merck
(Darmstadt, Germany). Pharmaceutical
dosage forms (Tegretol
, Karberol
)
containing CBZ were obtained commer-
cially from a pharmacy.
Stock and Standard Solutions
The stock solution of CBZ was prepared
to 50 lg mL)1concentration in metha-
nol. The standard solutions to 2, 5, 10,
15, 20, 25 and 30 lg mL)1 concentrations
and the quality control (QC) solutions to
2.5, 7.5 and 15 lg mL)1 concentrations
with 10 lg mL)1 ISI were prepared daily
from the stock solution; 10 lg mL)1 ISI
working solution was prepared from
100 lg mL)1 diazapam stock solution in
methanol. The solutions were stored at
4 C when not in use.
Instrumentation and
Analytical Conditions
An Agilent 6890N Network GC equipped
with a flame ionization detector (FID),
Agilent 7683 series autosampler and
Agilent chemstation software were used
to analyze the samples. Separation was
achieved using an HP-5, 30 m, packed
capillary column (5%-Phenyl)-meth-
ylpolysiloxane (0.320 mm · 25 lm, Agil-
ent Technologies, Palo Alto, CA, USA).
The split mode (5:1) was used with
S170
2,5
2 above. When the ramp rate was more or
1,5
1 peaks (analyte peak and matrix interfer-
0,5
0 in Fig. 1, good separation of CBZ from
0 5 10 15 20 25 30 35
ug/ml (CBZ)
DZ
nitrogen carrier gas at 2 mL min)1 flow
rate. Hydrogen and synthetic air were
used as auxiliary gases for the detector
(FID). The injector and detector tem-
peratures were set at 270 C. The oven
temperature programs: initial tempera-
ture 100 C, hold 1.7 min, hold 10 min at
220 C, ramp rate 100 C min)1, final
temperature 270 C, ramp rate 10 C
min)1; 1 lL solution was injected in to
the GC-FID system.
Preparation of Tablet Solutions
A total of 20 tablets containing CBZ (for
Tegretol
and Karberol
tablets) were
accurately weighed and powdered. One
tablet CBZ amount from this powder was
weighed. The powder of a CBZ tablet was
dissolved in methanol by using an ultra-
sonic bath for 15 min and the final con-
centration of solutions was 2 mg mL)1.
Results and Discussion
Method Development
The method development for the assay of
CBZ was based on its chemical proper-
ties. Methanol was used as solvent. The
GC-FID parameters used in the method
development were based on the boiling
point (193 C). Different temperature
programs were investigated for exception
of matrix interference. At the end of this
investigation, the best temperature pro-
Chromatographia Supplement Vol. 66, 2007
gram was selected for a good resolution
and thus for all experiments was used the
oven temperature program described
less 10 C min)1, a good resolution of the
ence peaks) was not obtained. As shown
internal standard was performed and no
further matrix interfering peaks at the
retention times of CBZ (tR = 6.1 min)
and ISI (tR = 13.9 min) were observed.
Chromatograms of standard and drug
sample gave very good peak shapes.
Linearity
The calibration curve (Fig. 1) was
established by plotting the ratio of the
peak areas of CBZ and ISI versus the
concentrations of CBZ samples. The
linear regression equation (with standard
error of intercept (Sa 0.0060) and slope
(Sb 0.0005) and correlation coefficient
(R) is y = 0.0661x-0.0129 (y the ratio of
peak areas of CBZ and DZ, x the con-
centration of CBZ) and 0.997, respec-
tively. The limit of detection (LOD
S/N = 3) and the limit of quantification
(LOQ S/N = 8) were found to be 0.75
and 1.00 lg mL)1, respectively. Both
accuracy and precision of these values
were well within the proposed criteria
(RSD% <20%).
Precision
Intra-day (repeatability) during the same
day and inter-day (intermediate preci-
sion) on different days (10 days) was
evaluated with six replicate of QC sam-
ples for precision. The RSD values for
intra-day and inter-day precision were
<9.0% for both (n = 6). Precision
studies of the GC-FID method showed
acceptable RSD values and relative errors
for accuracy for both the intra-day and
inter-day were <5.5 and <6.5%,
respectively (Table 1).
Accuracy/Recovery
The accuracy was determined by recovery
of known amounts of CBZ reference
standard adding the tablet samples at the
beginning of the process. In the first
recovery study, 20 lg mL)1 tablet solution
were added in 2.5, 7.5 and 15 lg mL)1
Limited Short Communication
Table 1. Precision and accuracy of the method for determination of CBZ (n = 6)
Intra-day
)1
Added lg mL Found ± SD < lg mL
4 2.63 ± 0.2
7.5 7.53 ± 0.4
15 14.72 ± 0.3
a
Average of six replicate determinations
b
Accuracyc (% relative error):(found-added/added) · 100
c
SD Standard deviation of six replicate determinations, RSD relative standard deviation
CBZ standard solutions with ISI. In the
second recovery study, 5, 10 and 15 lg
mL)1 tablet solutions were added in
5 lg mL)1 CBZ standard solution with
ISI. The percentage recovery of added
CBZ standard was calculated by
comparing the found and added concen-
trations (Cfound/Cadded · 100) in each
case. The mean recoveries for both
recovery studies ranged from 94.03 to
98.19% (for Tegratol
tablets) and from
92.0 to 98.65% (for Karberol
tablets).
The RSD values of recovery ranged from
0.75 to 3.50%.
Comparison
of Chromatographic
(LC and GC-FID) Methods
The suggested GC-FID method was
applied to the analysis of two dosage forms
containing CBZ without interference from
excipients encountered in pharmaceutical
preparations using internal standard
methodology. Also the developed and
validated GC-FID method was statisti-
cally compared with a reference method
(LC method) in the literature [16].
The linear concentration range of the
proposed and reference method was 2–30
and 0.2–1.7 lg mL)1, respectively. The
average recovery value for CBZ in
200 mg tablet composites ranged from
98.78 to 99.75% for the proposed method
and Cl (confidence interval) was found to
be 210.59–187.31 (for Tegretol
tablets)
and 205.41–186.84 (for Karberol
tab-
let). The RSD values obtained from
recovery studies ranged from 0.75 to
3.5%, which indicated high accuracy and
precision. In the reference method, the
average recovery value ranged from 101.4
and 99.7% for CBZ from 100 and 200 mg
tablet composites, respectively, and the
RSD values ranged from 2.59 to 3.00%.
The results obtained were compared
Limited Short Communication
Inter-day
)1
Precision RSD % b
Accuracy c
Found ± SDa lg mL)1
8.76 5.20 2.74 ± 0.2
4.69 0.40 7.14 ± 0.5
2.23 )1.86 15.95 ± 1.4
statistically by student’s t-test (for accu-
racy) and variance F-test (for precision)
with the reference method [16] at 95%
confidence level with five degrees of
freedom. The results showed that the
t-values (tc = 1.266 for Tegretol
tablets
and tc = 1.329 for Karberol
tablets)
and F-values (F = 1.266 for Tegretol

tablet and F = 1.329 for Karberol

tablet) were less than the tabulated values
(tt = 2.101) indicating that there was no
significant difference between the pro-
posed and reference methods (P >0.05).
Conclusion
In the present study, we report a highly
selective GC-FID method for determi-
nation of the CBZ of substances used to
be antiepileptic drugs without derivati-
zation in pharmaceutical preparations.
The GC-FID method has high recovery
and excellent reproducibility. For this
reasons, it can be used for the determi-
nation from pharmaceutical prepara-
tions of CBZ in routine quality control
measurement.
Acknowledgments
The authors are grateful to the University
of Ataturk for the financial support of
this work.
References
1. Bradley MK, Rene H, Levy RR, Matson
R, Meldrum B, Penry JK, Dreifuss FE
(eds) (1989) Antiepileptic drugs, 3rd ed.
Raven Press, New York, p 505
2. Olling M, Mensinga TT, Barends DM,
Groen C, Lake OA, Meulenbent J (1999)
Biopharmmaceutics and Drug Dispos
20:19–28
Chromatographia Supplement Vol. 66, 2007
Precision RSD %b Accuracyc
7.74 )10.50
7.18 )4.80
8.92 6.33
3. Lertratanangkoon K, Horing MG (1982)
Drug Metab Dispos 10:1–10
4. Jung H, Milan RC, Girard ME, Leon F,
Montoya MA (1997) Int J Pharm 152:37–44
5. Smith CM, Reynard AM (1995) Essentials
of pharmacology. W. B. Saunders, Phila-
delphia, p 226
6. Munson PL, Mueller RA, Breese GR
(1996) Principles of pharmacology. Chap-
man & Hall, USA, p 243
7. Jaffery NF, Ahmad SN, Jailkhani BL
(1983) J Pharmacol Met 9:33–39
8. Riad LE, Chen KK, Wagner WE,
Sawchuk RJ (1986) J Pharmacol Sci
75:897–900
9. Huang C, He Q, Chen H (2002) J Pharm
Biomed Anal 16:59–65
10. Fellenberg AJ, Pollard AC (1976) Clin
Chim Acta 69:429–431
11. Mansilla AE, Munoz de la Pena A,
Goicoechea HC (2004) Anal Chim Acta
506:161–170
12. Escandar GM, Gomez DG, Mansilla AE,
Munoz de la Pena A, Goicoechea HC
(2004) Anal Chim Acta 506:161–170
13. Auer ME, Griesser US, Sawatzki J (2003)
J Mol Struct 661:307–317
14. Cry TD, Matsui F, Sears RW, Curran
NM, Lovering EG (1987) Anal Chem
70:836–840
15. Mohammed EAH (2000) Il Farmaco
55:136–145
16. Walker ES (1988) J Assoc Anal Chem
71:523–525
17. Manoj Babu MK (2004) J Pharm Biomed
Anal 34:315–324
18. Law MW, Young CJ, Brain RA, Johnson
DJ, Hanson MA, Wilson CJ, Richards
SM, Solamon KR, Mabury SA (2004) E
Toxicol Chem 23:1431–1440
19. Gonzales-Barreiro C, Lores M, Casais
MC, Cela R (2003) J Choromatogr A
993:29–37
20. Jurgens U, May T, Hillenkotter K,
Rambeck B (1984) Therap Drug Monit
6:334–343
21. Sun L, Szafir I (1977) Clin Chem 23:1753–
1756
22. Worm K (2005) Int J Legal Med 77:41–45
23. Roger JD, Rogers Jr G, Sov A (1973) Clin
Chem 19:590–595
24. Friel P, Green J (1973) Clin Chim Acta
43:69–73
25. Lensmeyer GL (1977) Clin Toxicol 11:443–
454
26. Mashford ML, Ryan PL, Thomson WA
(1974) J Chromatogr A 89:11–15
27. Minkova G. Getova D (2001) Methods
Find Exp Clin Pharmacol 23:481–485
28. Moore FM, Simpson D, (1989) Clin Chem
35:1782–1784
S171
29. Tsatsakis AM, Psillakis TK, Tzatzarakis
M, Kourtopoulos H, Paritsis N (1997) Clin
Chim Acta 25:187–195
30. Burke JT, Thenot JP (1985) J Chromatogr
B 340:199–241
S172
31. Frigerio A, Baker KM, Belvedere G (1973)
Anal Chem 45:1846–1850
32. Chen K, Bashi HK (1991) Anal Toxic
15:82–85
Chromatographia Supplement Vol. 66, 2007
33. Validation of analytical procedures. In:
Proceedings of the international conference
on harmonization (ICH). Commission of
the European Communities (1996)
Limited Short Communication