Burana-osot et al.

2015

Original Article

TJPS
The Thai Journal of Pharmaceutical Sciences
39 (4), October-December 2015: 127-140

Development and Validation of a Stability-Indicating

HPLC Method for Determination of Clorazepate
Dipotassium and Its Main Impurities in Bulk Drug
and Capsules
Jankana Burana-osot1,*, Chanokporn Sukonpan1 and Sooksri Ungboriboonpisal2
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Nakorn-pathom, 73000,
Thailand.
2
Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 11000,
Thailand.
Abstract
A simple isocratic stability-indicating HPLC method was developed and validated for determination of
clorazepate dipotassium in the presence of its main impurities; nordiazepam and 2-amino-5-chlorobenzophenone, in
bulk drug and capsules. The chromatographic analysis was performed on a Zorbax Eclipse XDB-C18 column (75
mm x 4.6 mm i.d., 3.5 µm) using a mobile phase consisting of 5 mM ammonium formate in methanol and 5 mM
ammonium formate in water (65: 35, v/v) at a flow rate of 0.7 mL/min and UV detection at 230 nm. The forced
degradation studies were performed under various conditions according to the ICH guidelines. The degradation
products from the studies were investigated by HPLC and, later, by tandem LC-MS. The validation tests including
specificity, linearity, accuracy, precision, LOD and LOQ were performed. The calibration curves of the drug and
the two related substances were linear in the concentration of 2 to 100 µg/mL (r2 = 0.9990), 2-50 µg/mL (r2 =
0.9995) and 0.4-25 µg/mL (r2 = 0.9993), respectively. The proposed method was proven to be suitable for the
quantitative determination and stability studies of clorazepate dipotassium in bulk drug and capsules.

Keywords: clorazepate dipotassium, capsules, forced degradation, isocratic HPLC, LC-MS, stability-indicating

Introduction
Clorazepate dipotassium (CZP), (3RS)-7-chloro-2-
oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-
carboxylate compound with potassium hydroxide (1:1), is
a soluble prodrug or drug precursor of long-acting
benzodiazepine derivatives (Figure 1). It is utilized for the
treatment of anxiety disorders, insomnia and alcohol
withdrawal as well as adjunctive therapy in the
management of epilepsy (partial seizures). Shortly after
orally administrated, CZP is degraded into an active non-
Correspondence to: Jankana Burana-osot polar compound, N-desmethyldiazepam (NDZP) or
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
nordiazepam (7-chloro-5-phenyl-1, 3-dihydro-2H-1, 4-
Silpakorn University, Nakorn-pathom, 73000, Thailand.
Tel: +66 34255800, Fax: +66 34255801 benzodiazepin-2-one) by losing one water and one CO2
Email: buranaosot_j@su.ac.th molecule [1-3]. The acidic condition in the stomach
makes CZP and NDZP further degraded into 2-amino-5-
Received: 13 March 2015 chlorobenzophenone ((2-amino-5-chlorophenyl) phenyl-
Revised: 19 May 2015 methanone or ACB) and glycine [2]. The British
Accepted: 28 July 2015 Pharmacopoeia (BP) and the United States Pharmacopeia
(USP) enlist ACB and NDZP (Figure 1) as impurities A
Academic Editor: Wanchai De-Eknamkul and B in the monograph and require the limit testing for

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128 Burana-osot et al.2015

both of them [4,5]. Titration and TLC procedures are HPLC-DAD and LC-MS Conditions
described in BP for the assay of an active pharmaceutical The HPLC system consisted of an Agilent 1100 series
ingredient (API) and impurities only in bulk drug, pump, an on-line solvent degasser, an autosampler, a
respectively [4] whereas the USP employs titration and photodiode-array detector (DAD) and a Chemstation
HPLC method for the determination of API in bulk form software version A.08.01 (Agilent Technologies, USA). A
and pharmaceutical dosage form respectively. Even reversed-phase column, 75 mm x 4.6 mm packed with 3.5
though HPLC is used for the determination of impurities; µm, Zorbax Eclipes XDB-C18 modified silica (Agilent
however, 3 different conditions are required for CZP and Technologies, USA) and a guard column, 20 mm x 3.9
each impurity [5]. These are complicated, time-consuming mm packed with 5 µm, C18 were used (Agilent
and produced a lot of chemical waste. Technologies, USA). The assay system was operated at
O O
ambient temperature. The separation was carried out
H H
N O N
Cl under isocratic elution with 5 mM ammonium formate in
OK. KOH methanol: 5 mM ammonium formate in water (pH 5) in
N Cl N
Cl
the ratio of 65:35 (v/v). The flow rate was 0.7 mL/min
NH2 O
and the wavelength was monitored at 230 nm. The
Clorazepate Dipotassium Nordiazepam 2-amino-5-chloro-benzophenone injection volume was 5 µL. The forced degradation
(MW 407.97) (MW 270.06) (MW 231.05) samples were analyzed using a DAD detector in scan
mode covering the range of 200-400 nm.
Figure 1 Chemical structures and the exact mass of To characterize the degradation products, all samples
clorazepate dipotassium and its impurities: nordiazepam from the forced degradation studies were subjected to a
and 2-amino-5-chlorobenzophenone. HPLC system (as described above) coupled to an Agilent
G2445D LC/MSD Trap SL mass spectrometer which
The non-pharmacopoeial method for the assay of
operated using MSD Trap software version 4.0 LC-MS.
CZP in bulk powder and dosage form has been reported
The column and the mobile phase as described above
including HPLC [6-7] and differential pulse polarographic
were used, but the flow rate was reduced to 0.4 mL/min.
method [8,9]. Though a stability-indicating HPLC method
Positive electron spray ionization (ESI) mode was used
has been reported, only ACB was studied [7]. The
with mass/charge (m/z) ratio in the range of 50-3000 m/z.
quantitative assay of CZP in the presence of its
The probe voltage was set to 7.0 kV, the capillary voltage
degradation products was performed by secondary
was at 3,500 V, the gas temperature was 325°C, and the
derivative UV spectrophotometric [10-12] and
nebulizer gas flow was 8.0 L/min.
spectrodensitometric method [13] which involved
extraction and derivatization procedure. A-bio-analytical
method for the determination of CZP and other Preparation of Stock and Standard Solutions
A stock solution of CZP in water was prepared at a
benzodiazepines in whole blood, serum and urine by LC,
concentration of 1 mg/mL. The solution vial was covered
LC-MS and GC-MS was reported as well [14-17].
with aluminum foil to protect it from light and stored at
However, these were non-stability-indicating techniques.
This work was conducted in order to develop a selective 4°C and found to be stable over one week. Working
and validate a stability-indicating HPLC-UV-MS method aqueous standards were prepared from a stock by the
for the determination of CZP in bulk and capsule appropriate dilution at 2, 5, 10, 25, 40, 50, 60 and 100
formulation after performing stress studies under a variety µg/mL. Two standard stock solutions of impurities; NDZP
of ICH recommended test conditions [18]. In addition, the and ACB, were prepared individually at a concentration of
proposed method could be applied to monitor the presence 1.0 mg/mL using water as solvent. A combined standard
of ACB and NDZP in CZP bulk and capsule formulation solution containing 50 µg/mL of CZP, 25 µg/mL of
in one chromatographic run. NDZP and 10 µg/mL of ACB was prepared in water for a
specificity test.
Materials and Methods
Preparation of Sample Solutions
Chemicals and Reagents
The contents of twenty capsules were weighed, mixed
Reference and working standard of CZP and its two
impurities; NDZP and ACB, were kindly supported by well, and an aliquot of powder equivalent to the weight of
Bureau of Drug and Narcotic, Department of Medical one capsule was accurately weighed into a 100 mL
Sciences, Ministry of Public Health, Thailand. Two volumetric flask and added a part of water. After
batches of a local commercially available capsule sonication for 15 min, the mixture was made up to volume
with water and then filtered. Suitable aliquot of the filtrate
formulation used were labeled to contain 5 mg of CZP.
was further diluted to yield starting concentration of 50
The HPLC-grade methanol and ammonium formate
(99.99%) were purchased from Merck (Darmstadt, µg/mL and filtered through a 0.22 µm nylon filter.
Germany). Other chemicals used were of analytical grade.
Forced Degradation Conditions
High purity water was prepared by using Milli-Q RO
Stress degradation studies of CZP were carried out
system (Millipore, Bedford, MA, USA).
under the conditions of acid and base hydrolysis,
oxidation and photolysis. Each degradation stress was
performed in an aqueous solution of the drug at the

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Burana-osot et al., 2015 129

concentration of 1 mg/mL. The experimental vials were One of the major aims for this study was to develop
wrapped in aluminum foil during the incubation in order the HPLC method that could determine CZP and its main
to avoid the effect of light except photolysis. The samples degradation products concomitantly. The condition from
from each stress experiment were taken at designated the drug assay monograph in the USP 29 was used as a
time, diluted to yield stated CZP concentration of about starting point for the assay development. According to
50 µg/mL, filtered through a 0.22 µm membrane and the chromatogram from the USP assay, peaks of both CZP
analyzed by HPLC along with a non-stressed sample. and NDZP were broad and asymmetry. In addition, the
Each stress studies were performed in triplicates. phosphate buffer using in the USP assay could cause the
The hydrolytic degradation studies were performed in ion suppression and salt precipitation in the MS ion
water, acidic and basic media at two different source. To avoid the MS problem, a volatilable salt as
temperatures, at room temperature and 80 °C (in a heat ammonium formate was employed. Moreover, methanol
block). The acidic hydrolysis was carried out in 0.01- 0.1 was used as an alternative solvent since an ammonium
N HCl solution while the basic degradation study was formate was insoluble in acetonitrile. An isocratic system
performed in 0.01- 0.1 N NaOH media. The samples at a with a proportion of 35:65 (v/v) 5 mM ammonium
designed time were taken out from the experimental vial formate in water: 5 mM ammonium formate in methanol
and adjusted the pH to 5 with 0.01-0.1 N NaOH or 0.01- provided a good resolution and a good peak shape. With
0.1 N HCl (as desirable). For oxidative stress studies, the the optimized system, the peaks of CZP, NDZP and ACB
samples were treated with 0.3-3.0% (v/v) H2O2 at room were observed at 1.8, 4.6 and 8.5 min, respectively
temperature and 80 °C (in a heat block). Susceptibility of (Figure 2). The overlaid DAD showed good UV
the drug to light was studied. Approximately 100 mg of absorbance of all monitored compounds at 230 nm and
active pharmaceutical ingredient powder of CZP was this wavelength has been chosen for detection. To verify
spread on a glass dish in a layer that was less than 2 mm the separation ability of the developed assay condition, it
in thickness. The samples were exposed to natural was tested on the bulk drug and capsules and the assay
sunlight for 4 days, weighed and prepared as previously results are shown in Figure 2(B) and (C), respectively. A
described. minor peak of NDZP was observed in the chromatograms
of the drug and the commercial sample. Additionally, no
Results and Discussion excipient interference was observed under this assay
condition.
Development and Optimization of the Method

Figure 2 Typical HPLC chromatograms of (A) a standard mixture solution of CZP, NDZP and ACB (B) CZP bulk drug
and (C) CZP capsules. All CZP solutions were prepared at a concentration of 50 µg/mL

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130 Burana-osot et al., 2015
Table 1 System suitability parameter.

Parameter k′ α Rs N USP tailing factor

clorazepate dipotassium 3.95 3,539 1.23

nordiazepam 12.23 3.10 17.78 7,835 1.11

2-amino-5-chlorobenzophenone 24.94 2.04 15.36 9,672 1.06

Preferable levels >2 > 2,500 < 1.5

HPLC Results of Forced Degradation Studies

The system suitability parameters listed in Table 1 Forced degradation was performed to provide the
was established by ten replicates. All parameters were stability-indicating properties. The chromatograms
satisfactory with good specificity for the stability obtained from the stressed samples are illustrated in
assessment of CZP, NDZP and ACB. In addition, the drug Figure 3. The degradation products were well separated
was also completely separated from all forced gradation from their parent drug. This result could verify the
products with the satisfactory resolution greater than 2. stability indicating power of the developed method. The
retention times of CZP and its degradation products from
each stress condition are summarized in Table 2.

Table 2 Forced degradation studies of CZP

Degradation mode Conditions % Area Degradation Retention time

of CZP products found (min)
Control none 99.90 NDZP 4.6

Hydrolysis
Neutral hydrolysis water , RT, 24 h 93.73 NDZP 4.6
water , 80°C, 10 min 68.40 NDZP 4.6
water , 80°C, 30 min 13.52 NDZP 4.6

Alkaline hydrolysis 0.1 N NaOH, RT, 24 h 95.21 NDZP 4.6

0.1 N NaOH, 80°C, 10 min 72.71 NDZP 4.6
0.1 N NaOH, 80°C, 30 min 34.49 Unknown I 2.8
NDZP 4.6
Unknown II 9.1
ACB 8.5
Acid hydrolysis 0.01 N HCl, RT, 10 min 11.80 NDZP 4.6
0.01 N HCl, RT, 30 min 0.00 NDZP 4.6
0.01 N HCl, 80°C, 5 min 0.00 NDZP 4.6
0.01 N HCl, 80°C, 30 min 0.00 Unknown I 2.8
NDZP 4.6
0.01 N HCl, 80°C, 120 min 0.00 Unknown I 2.8
NDZP 4.6
ACB 8.5
Oxidation 3% H2O2, RT, 24 h 87.44 NDZP 4.6
3% H2O2, 80°C, 10 min 73.65 NDZP 4.6
3% H2O2, 80°C, 30 min 14.80 NDZP 4.6

Photolysis Sunlight, 48 hr 0.00 NDZP 4.6

(solid state)
RT= room temperature

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Burana-osot et al., 2015 131

The data suggested that NDZP was the main The retention time, ion mass and major fragments of CZP
degradation product from all of the stress conditions. The and its degradation products are listed in Table 3.
results were confirmed by the mass spectra on LC-MS.

Table 3 LC-MS data for identification of CZP and its degradation products under m/z value for [M+H]+.

Compound LC LC-MS/MS MS MS/MS fragment ions

Retention time Retention time

(min) (min)

CZP 1.8 2.8 315.0 271.1

271.0 254.0, 243.0, 236.0, 226.0, 208.0,

193.0, 165.0 and 140.0*

NDZP 4.6 7.9 271.1 243.0, 226.0, 208.0, 193.0, 165.0

and 140.0*

ACB 8.5 14.9 232.0 185.9, 153.9* and 126.0

Unknown I 2.8 5.9 289.0 244.0, 232.0*, 216.1 and 154.0

Unknown II 9.1 15.3 271.2 253.9, 236.1 and 226.0*

* Most abundance MS/MS fragmentions

Due to the fragileness of the ester bond in the CZP structure were replaced by proton (Figure 4(A)).
structure, there were two ions at m/z value of 271.1 and According to the results from the stress studies, four
315.1 under the peak of CZP (MW 407.97) at 2.8 min. degradation products of CZP were observed. The main
Regarding to the fragmentation outcome from the ms/ms degradation product found in every stress sample at 7.9
experiment, the first ion was the [M+H]+ of NDZP (MW min was NDZP with 271.0 m/z value of [M+H]+ (Figure
270.71) which was occurred from the thermal degradation 4(B)). ACB is shown at 14.9 min with 232.0 m/z value
of CZP within the ionization chamber. The other ion was (Figure 4(C)). The other two unknown degradation
a molecular ion peak of the acid form of CZP. Two products were appeared at 5.9 min with m/z 289.0 and
potassium ions in the form of K and KOH in the CZP 15.3 min with m/z 271.2, respectively (Figure 4(D) and
(E).

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132 Burana-osot et al., 2015

Figure 3 HPLC chromatograms of (A) CZP in water at room temperature at initial (B) acid hydrolysis-degraded CZP at
room temperature for 30 min (C) acid hydrolysis-degraded CZP at 80°C for 5 min (D) acid hydrolysis-degraded CZP at
80°C for 30 min. (E) base hydrolysis-degraded CZP at 80°C for 5 min and (F) oxidative-degraded CZP at 80°C for 10
min.

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Burana-osot et al., 2015 133

Figure 4 MS profiles of (A) CZP at Rt 2.8 min, (B) NDZP at Rt 7.8 min, (C) ACB at Rt 14.9 min, (D) unknown I at Rt
5.9 min and (E) unknown II at Rt 15.3 min.

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134 Burana-osot et al., 2015

Acidic Condition peak appeared at 2.8 min (unknown I) (Figure 3(D)).

CZP was found to be very labile in acid. It Parallel LC-MS investigation, the loss of carbon dioxide
completely degraded to NDZP immediately after put in from the molecule could be accelerated by heat and the
0.1 N HCl. To be able to monitor the degradation proton in the acid medium. Therefore in 0.01 N HCl at
behavior of the drug in acid media, the acid strength of the 80°C at 5 min, only a peak of NDZP is shown (Figure
media was reduced for 10 times. At room temperature, 5(A) and (D)). A peak of unknown I with m/z value of
less than 12% was left after kept in 0.01 N HCl for 10 min 289.3 was observed after 30 min (Figure 5(C)). Increasing
and total drug was disappeared within 30 min (Figure in the mass value for 18 amu (m/z 271.0→m/z 289.3)
3(B)). NDZP was the only degradation product produced. suggested an addition of a water molecule. With the
When the temperature was raised up to 80°C, the longer exposure, the water adduct of NDZP was gradually
degradation of the drug increased dramatically. Total drug turn into ACB (m/z value of 232.0) (Figure 5(B) and (E)).
turned into NDZP within the first five min of incubation A loss of 57 amu suggested an addition of another water
(Figure 3(C)). After 60 min of incubation, three peaks molecule and, concomitantly, an elimination of glycine
were observed. The first two peaks at 4.6 and 8.5 min (m/z value of 75) to get this degradation. The proposed
corresponded to NDZP and ACB, respectively. The other acid hydrolysis degradation pathway is shown in Figure 6.

Figure 5 MS profiles for acid hydrolysis-degraded CZP at 80°C (A) at 5 min and (B) at 60 min and the MS/MS tandem
fragmentation patterns for the daughter ions at (C) m/z 289 (unknown I), (D) m/z 271.0 NDZP and (E) m/z 232.0 of ACB

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Burana-osot et al., 2015 135

O H O
H
N O N temperature for 24 h. Even at 80°C, less than 30% of the
- CO2

Cl N
OH
Cl N drug turned into NDZP (Figure 3(E)). At 30 min of
incubation, 43.2 % of the drug still left but four
degradation peaks were observed. On the TIC
Nordiazepam
Clorazepic acid
+
[M+H] = 315
+
[M+H-CO2 ] = 271 chromatogram (Figure 7), three peaks at 5.9 min (m/z
289.3) of unknown I, 7.9 min (m/z 271.1) of NDZP, and
+ H2O
14.9 min (m/z 232.0) of ACB were already found in the
chromatogram under the acid stress condition. An
Cl NH2 OH
O
additional peak (unknown II) was arisen at 15.3 min (m/z
+ H2O Cl
N 271.2) and had the same mass as NDZP dose. When they
O
NH2
were fragmentized in the MS/MS experiment, NDZP
NH2 O HO

2-Amino-5-chloro-benzophenone
Glycine
+
(Figure 7(C)) and unknown II (Figure 7(E)) showed a
[NDZP+H+H2O ] = 289
+
[NDZP+H+H2O-Gly ] = 232 different mass profile. NDZP produced six daughter ions
at m/z 243.0 ([MH-CO]+), 226.0 ([MH-NHCO-H2]+),
Figure 6 Proposed acid hydrolysis degradation pathway 208.0 ([MH-CO-Cl]+), 193.0 ([MH-NHCO-Cl]+), 165.0
of CZP. ([MH-CO-Ph]+) and 140.00 ([MH-CO-PhCN]+), while,
unknown II had only three major ions at m/z 253.0 ([MH-
Alkali Condition NH2]+), 236.1 ([MH-Cl]+) and 226.0 ([MH-NHCO-H2]+).
CZP was found to be more stable in alkali. 95% of
the drug was still unchanged after storage at room

Figure 7 MS/MS tandem profiles for basic hydrolysis-degraded of CZP at 80°C for 60 min (A). Fragmentation patterns
for the daughter ions; (B) m/z 289 (unknown I), (C) m/z 271.0 NDZP, (D) m/z 232.0 of ACB and (E) m/z 271.0
(unknown II).

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136 Burana-osot et al., 2015

Neutral Condition through the study of resolution factor of CZP peak from
10% of drug degraded to NDZP after 24 hours of the nearest resolving peak. Peaks were identified with
storage at room temperature. At 80 °C, 70% and 10% of retention times compared with standards and confirmed
the drug were left after 10 and 30 min of incubation, the characteristic spectra by DAD in both sample and
respectively, leading to the formation of NDZP. standard solutions. The peak purity value was greater than
the threshold value of 995 and thus establishing the
Oxidative Degradation Studies selectivity of the assay method. The mass detector also
The drug showed 12% degradation in 3% H2O2 at proved the mass purity for CZP, NDZP and ACB and thus
room temperature for 24 h, forming only NDZP and found confirmed the stability-indicating capacity of the
90% degradation of the drug at 80°C for 20 min (Figure developed method.
3(F)).
Linearity and Range
Photolytic Degradation Studies Linearity of system was established by analysis of
Color of the drug powder was changed from white to eight different concentrations ranging from 2 to 100
off-white after exposed to sunlight over 4 days. The µg/mL. Linear calibration plot for the related substances,
HPLC result showed that the drug was completely NDZP and ACB were obtained at concentration range of
degraded to NDZP. 2-50 and 0.4-25 µg/mL, respectively. Triplicate injections
were performed for each concentration and the peak area
versus concentration data was calculated by least-squares
Validation of the HPLC Method regression method. The correlation coefficient for CZP,
NDZP and ACB was 0.9990, 0.9995 and 0.9993,
Specificity respectively, indicating good linearity. The linearity
The specificity of the method was established studies data are summarized in Table 4.

Table 4 Linearity, LOD and LOQ data.

Parameter CZP NDZP ACB

Linearity

Calibration range (µg/mL) 2-100 2-50 0.4-25

Regression equation

Slope (S) 40.6206 69.7757 43.9667

Standard deviation of slope 0.1086 0.3972 0.2650

%RSD of slope 0.2673 0.5693 0.6028

Intercept -35.2680 8.3809 -10.5870

Correlation coefficients (R2) 0.9990 0.9995 0.9993

LOD (µg/mL) 0.18 0.07 0.05

LOQ (µg/mL) 0.62 0.24 0.18

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Burana-osot et al., 2015 137

Limit of Detection (LOD) and Limit of Quantitation from 50 to 150% of the specification level were spiked in
(LOQ) sample solutions. Three samples were prepared at each
The LOD and LOQ of CZP, NDZP and ACB were concentration. The recovery of added drug was calculated
determined at a signal-to-noise ratio of 3:1 and 10:1, by comparing the peak area of the test samples with that
respectively, by injecting a series of dilute solutions with of the standard solutions. The data obtained from 9
known concentrations. The results given in Table 4 samples is summarized in Table 5. The result indicated
suggested that this method could be used for monitoring that the proposed method was accurate and precise.
CZP’s stability. The results also indicated sensitivity of Method accuracy or linearity of method, determined by
the developed method for the degradation product plotting the amount of CZP found against the amount
determination at the low concentration. added over the range of 50-150 % of label amount,
Accuracy showed good linearity (y = 0.970x–0.85) with r2 = 0.9990.
The accuracy of the method was tested by The result was found to be satisfactory for intended
determination of CZP in the solution prepared by standard purpose and was adequate for routine analysis.
addition method. Three known amounts of CZP ranging

Table 5 Accuracy results: recovery data of CZP.

Spiked concentration Measured concentration* %Recovery %RSD

(µg/mL) (µg/mL)

22.8 23.1 101.32 0.11

45.6 44.7 98.30 0.07

91.2 89.4 98.05 0.11

Mean recovery (n=9) 99.38 1.79

* Mean value for three replicates of three different concentrations (n=9), and three injections for each replicate.

Precision analyzed in the same day to determine method precision

Precision was evaluated in term of repeatability and intermediate precision was performed on three
(system precision), method precision and intermediate different days by preparing and analyzing in six replicates
precision. To measure the repeatability, ten replicates of separated sample solution at the same concentration
(n=10) of the CZP standard solution and a sample solution level. The %RSD values for method precision and
at 100% of the target level were analyzed. The RSD% of intermediate precision were 0.13 and 1.31, respectively.
peak area response and retention time was calculated and The low RSD (< 2%) showed the suitability of the method
showed the satisfactory repeatability of the system (< for the determination of CZP in capsules. The precision
1%). Ten replicates (n = 10) of sample solutions were data is summarized in Table 6.

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138 Burana-osot et al., 2015

Table 6 Precision data.

CZP NDZP ACB

System precision (n = 10) RSD (%) RSD (%) RSD (%)

Standard Peak area 0.1069 0.0790 0.4506

Retention time 0.0607 0.0548 0.0180

Sample Peak area 0.0192 0.5157 Not found

Retention time 0.0156 0.0240 Not found

Actual Measured Concentration

Concentration

(µg/mL) (µg/mL) RSD (%)

Method precision (n = 10) 50 49.17 0.13

Intermediate precision

Day 1 (n = 6) 47.41 47.91 0.62

Day 2 (n = 6) 47.03 46.46 0.28

Day 3 (n = 6) 47.56 46.73 0.50

Total (n = 18) 47.33 46.33 1.31

Robustness Assays in Pharmaceutical Preparation

The SST parameters kept on unaffected over The proposed validated method was applied to
deliberate changes in the chromatographic conditions determine CZP in two difference batches of CZP
(variation of the ratio of methanol in mobile phase by capsules. Satisfactory results were obtained from batch I
+2%, the flow rate of mobile phase by +0.05% and the as the mean percentage found in good agreement with
wavelength of 230 nm by +2%), illustrating the label claimed. Batch II was found to be expired before the
robustness of the method. expiration date. Also, the discoloration of CZP in batch II
was observed. NDZP could be investigated while ACB
was non-detected in both batches. The results indicated
that this method could be adopted for determination of
CZP and monitored NDZP and ACB in capsules (Table
7).

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Burana-osot et al., 2015 139

Table 7 Determination of CZP in capsules.

CZP NDZP

% label amount* % RSD µg per capsule found* % RSD

Batch I 99.25 0.18 0.64 7.89

Batch II 88.82 0.19 15.21 0.56

* Mean value for three replicates (n=3), and two injections for each replicate.

Conclusion [6] L. Elrod Jr, D.M.Shada, and V.E. Taylor. High-

performance liquid chromatographic analysis of
A simple isocratic RP-HPLC method was clorazepate dipotassium and monopotassium in solid
successfully developed for the determination of CZP in dosage forms, J. Pharm. Sci. 70: 793-795 (1981).
the active ingredient and its pharmaceutical formulations.
The complete separation of the analytes were [7] M.M. Ellaithy, M. Abdelkawy, and R.M. Tolba.
accomplished within 10 min and found to be specific, Stability indicating HPLC assay for the analysis of
linear, precise and accurate. All validation parameters clorazepate dipotassium, Bull. Fac. Pharm. Cairo Univ.
were within the acceptance range. The m/z values and 40: 1-5 (2002).
fragmentation patterns obtained for the degradation
products through LC-MS studies helped to confirm the [8] C.W. Abruzzo, M.A. Brooks, S. Cotler, and S.A.
know degradation products; NDZP and ACB. The Kaplan. Differential pulse polarographic assay procedure
advantage of the method was the mobile phase developed and in vitro biopharmaceutical properties of dipotassium
could be used with both DAD and MS. This proposed clorazepate, J. Pharmacokinet. Biopharm, 4: 29-41
method might be applied for the routine quantitative (1976).
analysis of CZP in bulk drug and capsules as well as the
content uniformity test. It also has been used for the [9] S. Hanna, F. Diana, J. Slevinski, K. Veronich, and L.
stability study of CZP and monitored the related Lachman. Differential pulse polarographic determination
compounds as involved in both BP and USP. of clorazepate monopotassium and dipotassium, J. Pharm.
Sci. 67: 1723-1725 (1978).
Acknowledgements
[10] F.A. El-Yazbi, M.H. Barary, and M.H. Abdel-Hay.
The authors would like to thank Faculty of pharmacy, Determination of nitrazepam and dipotassium clorazepate
Silpakorn University, Thailand for financial support. in the presence of their degradation products using second
derivative spectrophotometry, Int. J. Pharm. 27: 139-144
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