chapter 6 analysis of diazepam using high...
Post on 20-Mar-2020
9 Views
Preview:
TRANSCRIPT
100
CHAPTER 6
ANALYSIS OF DIAZEPAM USING HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHIC METHOD
Introduction:
Diazepam is a drug belonging to benzodiazepine group (7-chloro-1-methyl-5-
phenyl-3H-1,4-benzodiazepine-2-one] known for its depression activity on the central
nervous system [M. Abid et al., 2006] with anticonvulsant, anxiolytic, sedative and
muscle relaxant properties [J. P. Hullihan et al., 1983]. It is also used in the treatment of
alcoholic withdrawal syndrome [M.Lucht et al., 2003] and in the treatment of
organophosphorus poisoning [T.C.Marrs, 2003]. Diazepam is one of the most widely
prescribed benzodiazepine for a variety of conditions particularly for anxiety, depression,
epilepsy and insomnia [M. Abid et al., 2006, L.P. Longo and B. Johnson, 2005].
Diazepam was first synthesized in 1955 by Leo Sterbatch [Uchibayashi. M, 2007,
Tauseef Mehdi, 2012] and marketed by Hoffmann-La Roche in 1963 in the brand name
as Valium [Tauseef Mehdi, 2012]. The mechanism of action of diazepam is attributed to
the production of the neurotransmitter gamma-aminobutyric acid (GABA) [Riss J et al.,
2008]. The synthesis, chemical neuroscience, pharmacology, drug metabolism, adverse
effects, dependence, clinical use and regulatory issues were all discussed by Nicholas E.
Calcaterra and James C. Barrow [Nicholas E. Calcaterra and James C. Barrow, 2014].
Diazepam in clinical dosage form as valium when given as therapy to patients proved to
improve health conditions and enabled the patients to think more clearly [H. Marlin
Beerman, 1964]. Clinical vigilance regarding withdrawal of benzodiazepines after a long
term prescription was urged for all consumers especially important among alcoholics
consuming high doses of benzodiazepines [P. Cushman and D. Benzer, 1980]. Diazepam
101
and its metabolites are useful in management of disturbed sleep [Coral H. Clarke and
A.N. Nicholson, 1978]. Also there was evidence that shows increased crash risk in
drivers who were using benzodiazepines like diazepam for some anxiety disorder [M.C.
Longo et al., 2001]. In cases of overdose of diazepam or other benzodiazepines
[Geoffrey K Isbister et al., 2004, B. Ukley and W. Hermann, 2012, C.Oti et al., 2010] it
may be necessary to carry out the analysis and confirm the concentration of the same in
the victim’s blood. Therefore, sensitive methods for the determination of benzodiazepine
are considered to be necessary. Changes in plasma concentrations of diazepam with
different ways of administration was reported in literatures [D.W.Sturdee, 1976] for
which, it is essential to adopt suitable method for analysis of the same.
When literature was surveyed it was found that using HPLC alone, there were
many methods available for quantitative determination of diazepam from various
biological and non biological samples using different procedures. They include;
extraction of diazepam from rat’s brain with SPE method [Mercolini L et al., 2009],
analysis of diazepam and its metabolites from human blood [Kabra PM et al., 1978], in
human plasma and urine [Azzam RM et al., 1998], extraction using ether from the blood
treated with aqueous ammonia [H.M.Stevens, 1985]. To detect over dosages and also to
determine therapeutic dosages, from serum diazepam using GC, chloroform extraction
was used [Lowell B. Foster and Christopher S. Frings, 1970]. Diazepam in
pharmaceutical preparations was quantified using HPLC with RP column in dosage
forms [M.Lazar et al., 2013], in tablet form along with propranolol hydrochloride with
RP system [Patel Satish A et al., 2011], in combined dosage forms along with imipramine
hydrochloride [P.Pydiratnam et al., 2013, VRB Vemula and PK Sharma, 2013] and from
102
injectable formulation together with degradation product, 2-methylamino-5-
chlorobenzophenone [F. M. Smith and N.O. Nuessle, 1982]. FTIR spectroscopic method
is also available for the quantification of diazepam in pharmaceuticals by extracting with
chloroform and measuring the area of the peak in the interval 1672-1682 cm-1
[Javier
Moros et al., 2006]. Similarly, among spectrophotometric methods, diazepam was treated
with alkaline dimethyl sulphoxide which produces reddish colour because of the
formation of charge transfer complex [Mohammad Sarwar, 2008]. Another method of
quantification of diazepam involved 1:1 ion-association complex formation with
bromocresol green at pH 3.5 and extraction of the dye into chloroform layer followed by
spectrophotometric measurements [S.Sadeghi et al., 2002]. With the help of derivative
spectroscopy diazepam was quantified in microemulsion gels in presence of propylene
glycol and Tween 80 [D.G.Dastidar and Biswanth Sa, 2009] and in presence of otilonium
bromide [Mannucci C et al., 1992]. Other simple methods include interaction of
diazepam with picric acid dinitrobenzoic acids to form products which is used for
spectrophotometric determination for some pharmaceutical preparations [W.F.El Hawary
et al., 2007]. Rapid Resolution Liquid Chromatography-Tandem Mass Spectrometry for
some benzodiazepines including diazepam and its metabolites has been reported for
clinical urine samples [Ren-Yu Hsu et al., 2013]. Molecularly imprinted solid phase
extraction protocol for estimation of diazepam from hair of postmortem samples has been
compared with SPE LC MS MS method [M.M.Ariffin et al., 2007]. Diazepam along with
its metabolites was determined from the hair samples of abusers using GCMS [Sooyeun
Lee et al., 2011]. Gas Chromatography was used for measurement of diazepam and its
metabolites in plasma samples using ECD [W. Loscher, 1982] and in whole blood using
103
GC with NPD detector with derivatization process [Horton Mccurdy et al., 1979].
Cerebrospinal fluid was used as an alternate testing matrix for monitoring concentration
of diazepam when blood was not available for analysis using GC MS was presented by
David A. Engelhar and Amanda J. Jenkins [David A. Engelhar and Amanda J. Jenkins,
2007]. Electrochemical quantification of diazepam in pharmaceutical products using
polarographic studies was also presented [M.G. Garcia et al., 1993]. Electrochemical
behavior of diazepam at the modified carbon paste electrode was done using cyclic
voltammetry and square voltammetry methods which gave linear range and very low
detection limits, successfully applied for the determination of diazepam in tablets and
human serum [M.R. Milani Hosseini and Ali Motahrian, 2015]. The methods for the
determination of diazepam from human plasma / blood involves the use of special
techniques like solid phase microextraction coupled with LC MS [M.Walles et al., 2004]
or coupled with GC MS [M.H.De Oliveira et al., 2005].
Thin Layer Chromatography (TLC) is basically used to monitor reaction products,
purity of the sample, active ingredient quantification [Naveen Bimal and Bhupinder
Singh Sekhon, 2013]. By applying spots on plates, usually made of silica gel as
stationary phase, the solvent was made to move on the stationary phase through capillary
action by keeping the stationary phase inside a mobile phase it was depending on the
polarity of solvent, the constituents of the spot start to separate. High Performance Thin
Layer Chromatography (HPTLC) is an enhancement over TLC as the developed plates
can be scanned under UV light and the corresponding spectrum of each separated spot
can be obtained. In HPTLC, simultaneous multisampling analysis on a single plate allows
a real in system calibration in contrast to other chromatographic techniques [P.D.Sethi,
104
1996]. HPTLC was preferred over HPLC in some studies due to simplicity, ease and low
cost [M.Bakavoli and M.Kaykhali, 2003]. HPTLC is proven to be a very useful
instrument for analysis of many types of samples in many areas [Naveen Bimal and
Bhupinder Singh Sekhon, 2013].
A number of HPTLC methods are also available for quantification of diazepam
from cold drinks which are adulterated for criminal motives [R.K.Sarin et al., 1998],
extracting the sample at pH 8.5 with diethyl ether and chloroform and scanning the
developed spots at 230 nm. Diazepam in tablets forms were analyzed with HPTLC by
extracting the active ingredient into methanol and densitometric measurement at 232 nm
using toluene and acetone as mobile phases [V.P.Machale et al., 2011]. HPTLC analyses
of diazepam along with imipramine using the zero crossing point and derivative spectra
were used for quantification from combined dosage form [Bhadani Shweta et al., 2013].
Using chloroform-isopropanol mixture, the acid hydrolyzed product of diazepam was
extracted from urine samples and it was quantified with HPTLC [R.Jain, 1993]. By
spraying with a reagent of sodium hydroxide and m-dinitrobenzene in dimethyl
sulphoxide, amongst other benzodiazepines, it was found that only diazepam produces
violet bands on TLC plates in biological samples [B.B. Daundkar et al, 2008]. In another
study by P.K.Salo et al, diazepam along with other benzodiazepines were separated with
dichloromethane and acetone (93:7) as one-dimensional mobile phase, toluene, acetone,
ethanol and 25% ammonia (70:20:3:1) for two dimensional separation, followed by
detection at 254 nm [P.K.Salo et al., 2007]. In most of the earlier works, solvent systems
used for TLC of diazepam involve the use of more harmful solvents like toluene or
chloroform as mobile phases.
105
Liquid-liquid extraction is the most commonly used method for extraction of
drugs from the matrices. Diazepam together with other drugs were extracted with less
toxic solvents from spiked blood samples using chlorobutane, ethyl acetate, diethyl ether,
heptane, hexane and isooctane solvents [ZhiBin Huang et al., 2015]. Extraction of
diazepam was carried with diethyl ether from blood [Haram K et al., 1976], with
chloroform from serum [Lowell B. Foster and Christopher S. Frings, 1970], with toluene
[Raisys VA, 1980] and with benzene from plasma [Loscher, 1982] have been described.
As a consequence of literature on diazepam [M. Abid et al, 2006, L.P. Longo 2005, J. P.
Hullihan et al., 1983] and considering the importance of diazepam analysis in various
samples [H. Marlin Beerman, 1964, P. Cushman, 1980, Geoffrey K Isbister et al., 2004,
B. Ukley et al., 2012, C.Oti et al., 2010], it is here, efficacy of diazepam extraction from
blood samples is performed bu HPTLC experiments using three different extraction
solvents namely, acetonitrile, chloroform and diethyl ether was checked in neutral, acidic
and basic conditions and determined diazepam using new HPTLC method employing an
optimized mobile phase, hexane and ethyl acetate (7:3)..
The developed new HPTLC method with and hexane ethyl acetate (7:3) was
successfully applied for the determination of diazepam present in forensic blood samples
and the samples of seized drug material and also present in some pharmaceuticals.
Diazepam was shown to be very stable in blood and tissues even for several months
under room temperature [B.Levine et al., 1983] and so blood was chosen to spike with
diazepam for its determination through the new method [Vani. N et al., 2013].
106
Objectives:
To develop new HPTLC method involving hexane and ethyl acetate (7:3) as
solvent system for the determination of diazepam present in blood and pharmaceutical
samples.
Experimental:
Materials and Methods:
Instrumentation: HPTLC system (Camag, Muttenz, Switzerland) fitted with auto sampler,
UV scanner and photo documentation system - loaded with Wincats software.
UV-visible double beam spectrophotometer (Chemito, India) and Fourier Transform Infra
Red spectrometer (Thermo Scientific, USA) were used.
Materials: Diazepam used here was recrystallised in methanol and purity was checked
with its melting point, UV and IR spectral analysis. AR grade acetonitrile, methanol,
chloroform, diethyl ether, Silica gel 60 F254 TLC plates (E.Merck) were used. The blood
sample used here was obtained from a local hospital.
Preparation of standard diazepam solution:
Standard stock solution of 1 mg/mL diazepam was prepared by dissolving 0.1 g of
diazepam in methanol and diluting the solution to 100 ml with methanol. This solution
was diluted or used as such to get different concentration of diazepam, used in calibration
and spiked blood sample preparations.
Extraction procedure for sample preparation:
2 ml blood sample was spiked with different volumes of diazepam stock solution
and each sample was vortex mixed for 5 min. Extraction was carried out thrice with 5 ml
of acetonitrile, centrifuged at 3000 rpm for 15 min., filtered through Whatmann 40 filter
107
paper containing silica and the filtrate contained in a beaker was evaporated on a water
bath maintained at 60°C. The residue remained was extracted with methanol and
transferred the extract into a 5 ml in a volumetric flask and diluted to the volume with
methanol. An aliquot of this was transferred into a labeled HPTLC glass vial and spotted
on the TLC plate so as to get 5 µg/spot. Similar extractions were carried out by making
spiked blood acidic with 1 ml 0.01M hydrochloric acid (pH 2), basic with 1 ml 0.1M
ammonia (pH 11). The same procedure was repeated with chloroform and diethyl ether
instead of acetonitrile. Blank blood samples without diazepam were processed in the
same way and it revealed a profile with negligible interference.
HPTLC method and chromatographic conditions:
The labeled vials with different concentration of diazepam solutions were kept in
Camag auto sampler ATS 4 and known volumes were spotted (band length, 6 mm) on the
plates along with different volumes of standard solution using the Wincats software.
These plates, that were washed with methanol and dried at 80°C before application of the
samples, were developed in a solvent chamber, previously saturated with hexane and
ethyl acetate (7:3) as mobile phase. Each plate was developed to a distance of 80 mm,
removed from the chamber, solvent was removed by drying with hot air and developed
spots on the plate was digitally documented using in built Camag documentation system
with camera by illumination at 254 nm.
After documentation, quantification was done with densitometric measurement at
230 nm followed by UV scanning with Camag TLC scanner 3. Using the following
parameters the densitogram (chromatogram) and the UV spectrum were recorded.
HPTLC instrument parameters
108
Slit dimension: 6.00 x 4.00 mm
Data resolution: 1 nm/step
Wavelength range: 200-400 nm
Lamp: Deuterium
Mode: Absorption
Scanning speed: 100 nm/sec
Method validation:
The following parameters were used to validate the developed HPTLC method.
Linearity and range:
From the stock solution of diazepam, appropriate dilutions were made to get a
series of solutions with concentrations ranging from 0.01 to 1 mg/mL.
20 μL of these
prepared solutions were spotted on three different plates so as to get a series of spots
having the concentration range, 0.2 - 20 μg/spot. Plates were developed as described
earlier and average peak areas were plotted against the corresponding concentrations of
diazepam to get its calibration curve and linear regression equation.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
LOD was calculated as the concentration at which signal to noise ratio was more
than three and LOQ was calculated as signal to noise ratio was more than ten.
Precision:
2.5, 1.25 and 0.5 ml of 1 mg/mL stock solution was added to 2 ml blood samples.
Extractions were carried with diethyl ether by making the solutions basic and
reconstituted in 5 ml methanol as explained earlier. The intra-day repeatability was
evaluated by spotting, 20 μL of each of the above solutions three times repeatedly on the
plates and then plates were scanned to know the peak areas after development in mobile
phase. Similarly inter-day reproducibility was evaluated by five times analysing these
109
sample solutions over a period of three days. Amount of diazepam was estimated by
applying the obtained peak area values to the regression line equations. Accuracy of the
method is reported as percent recovery and error.
Specificity:
In order to understand the interference for the determination of diazepam by new
HPTLC method from some commonly used benzodiazepines with diazepam, equal
volumes of 1 mg / mL solution of nitrazepam, chlonazepam, lorazepam,
chlordiazepoxide, alprazolam and clozapine were added with 1 mg/mL solution of
diazepam, one at a time and spotted on the TLC plate and developed it using the new
mobile phase as discussed earlier and they were individualized by calculating the Rf and
UV pattern.
Results and discussion:
Selection of mobile phase for thin layer chromatography:
Ethyl acetate together with ammonium hydroxide, methanol and with acetonitrile
were used as mobile phases for the quantification of diazepam from pharmaceuticals
[Bernard Fried and Joseph Sherma, 2005]. Frequently used benzodiazepine derivates
including diazepam were separated using Stahl’s triangle based on structure, polarity,
solubility and stability concepts to choose mobile phases after in situ hydrolysis to form
benzophenones [Hancu G et al., 2011]. Similarly, Subbiah Thangadurai et al. have
considered chloroform and methanol (97:3) as a good solvent system for the separation of
nine benzodiazepines among chosen ten solvent systems [Subbiah Thangadurai et al.,
2013] using TLC. By considering the toxic nature of toluene and chloroform mobile
phases used in earlier works for benzodiazepines [M.Bakavoli and M. Kaaykhali, 2003,
110
V.P.Machale et al., 2011, B.B. Daundkar et al., 2008], a new solvent system consisting of
hexane and ethyl acetate was developed here for diazepam to separate it from other
benzodiazepines as ethyl acetate was considered as greener solvent [Yao Shen et al.,
2015]. A mixture of these solvents was used in different proportions and a fraction of 7:3
v/v of hexane and ethyl acetate was giving a good separation with reproducible results
(SD = 0.09) having retention factor 0.34 (Rf = distance traveled by analyte/distance
traveled by mobile phase). This mobile phase was used for further studies.
Fig. 1: Variation of retention factor with change in fraction of hexane and ethyl acetate
After development with the mobile phase, the plates were viewed under UV light
which shows the black coloured spots in green background and the image was captured
with Camag Reprostar 3, photo documentation system. Commercially available TLC
plates are normally coated with fluorescent quenching materials so as to facilitate UV
detection [Caitlin Sullivan and Joseph Sherma, 2004]. The photo captured one such plates
and subsequently sprayed with Iodoplatinate reagent which gives blue violet colour spots
[Lukasz Komsa et al., 2014] are shown in Fig. 2. Overlaid densitograms for the
developed spots used in making calibration curve and their corresponding overlaid UV
spectra are shown in Fig. 3.
111
Fig. 2: Developed plate with photographed under UV light (left) and same plate after UV
scanning was sprayed with Iodoplatinate reagent (right) which gives violet coloured spots
for diazepam
Fig. 3: 3D display of densitogram of developed spots of diazepam on the TLC plate
showing Rf =0.34 (left) with inlay indicating concentration of each densitogram and their
overlaid UV spectra (right) with λ max = 233 nm
Selection of solvent for extraction:
The efficacy of extraction and percentage recoveries of the diazepam with three
different solvents and each solvent at three different pH conditions were experimented for
an intermediate concentration of diazepam, 5 μg/spot, the results obtained are given in
Table 1.
a – 2 µg/spot
b – 4 µg/spot
c – 6 µg/spot
d – 8 µg/spot
e – 10 µg/spot f – 12 µg/spot
g – 14 µg/spot
112
Table 1: Efficacy of extraction with different solvents
Solvent pH Filtration
speed
Precipitation
of blood
Filtrate Recovery
%
SD
Acetonitrile
2 Fast Yes No need for
separation
92.0 0.81
7 Slow Yes 91.3 1.15
11 Fast Yes 89.4 0.72
Diethyl
ether
2 Fast Slight Needs separation 84.6 0.55
7 Fast No 96.4 1.42
11 Fast No 98.6 0.95
Chloroform
2 Slow Slight
Needs separation
88.6 1.15
7 Slow No 90.1 0.85
11 Slow No 92.5 0.97
Ref. method
[St. Pierre
MV,
1987Using
ethyl acetate
7 Slow No
Needs separation
94.4
0.92
Acetonitrile is used as a deproteinization reagent for the extraction of
benzodiazepines from whole blood [Jessica L. Westland and Frank L. Dorman, 2013].
When acetonitrile was used as a solvent for the extraction of diazepam from the blood
sample, the blood was precipitating with the solvent and that enabled the filtration easier
and neat. But acetonitrile was miscible with water which makes serum to get mixed with
the filtrate, which was taking a longer time for the evaporation of the filtrate containing
diazepam. Further, extraction of diazepam from the blood sample using acetonitrile
solvent was yielding lower recoveries of diazepam which may be due to its trapping in
the precipitate (B.Levine, 1999) and it needed more volume of the solvent than the
specified 15 ml for its effective recovery. However, when diazepam was extracted with
acetonitrile from the blood sample that was made acidic was giving a good recovery
compared to the samples which were basic or normal.
113
When the solvent chloroform was used for the extraction of diazepam from the
blood sample, there was a formation of emulsion that was making the process of
extraction and filtration difficult. Recovery of diazepam into chloroform from the spiked
blood samples at three different pH conditions was found to be less compared to its
extraction either into acetonitrile or diethyl ether. However, diazepam extraction into
chloroform from the blood sample which was made acidic was less compared to the
blood samples that were normal and made basic.
When the solvent diethyl ether was used to extract diazepam from the blood
sample that was made acidic there was a slight precipitation but such precipitation was
not observed when attempted to extract the diazepam either from the normal blood
sample or the sample that was made basic. As the solvent diethyl ether was evaporating
quickly from the filtrate, time needed for its removal was less than 5 min on a water bath
at 60° C. There was a good diazepam extraction in to diethyl ether either from the normal
blood sample or from the sample that was made basic but its extraction from the sample
that was made basic was better. Diethyl ether was proved as a solvent of choice for
extraction of diazepam from fermented fruit juices [Lukasz Komsta et al., 2013] and by
considering the less toxic nature of diethyl ether, in the present study also, diethyl ether
has been used. This extraction process of diazepam was not involving more number of
extraction steps and also unlike other methods that involve special techniques [M.Walles
et al., 2004, M.H.De Oliveira et al., 2005]. A good recovery of diazepam, 98.6%
(%RSD=1.13) was an indication that the method developed here could be used in the new
HPTLC method for the accurate and quantitative determination of diazepam from the
blood samples.
114
Further, the method was compared with another thin layer chromatographic
procedure using ethyl acetate as extraction solvent and developed in a solvent chamber
consisting of chloroform-ethyl acetate-ethanol-ammonium hydroxide [St. M.V. Pierre,
1987], followed by densitometric measurement at 230 nm. Even though, a good recovery
of diazepam was obtained, 94.4% (%RSD=0.92), the spot of diazepam will move almost
to the solvent front (Rf=0.97) which makes it difficult to scan the spots since we get a
zig-zag pattern near the solvent front due to ammonium hydroxide, indicating better
validity of hexane and ethyl acetate (7:3) system.
Validation parameters:
The validation parameters established for the quantitative determination of
diazepam through the new method are given in Table 2. The relationship between the
concentration of diazepam in standard solutions and the corresponding peak responses at
230 nm was found to be linear in the range of 0.5-20 μg/spot of diazepam with
correlation coefficient of 0.998. The results obtained are shown in Fig.4. The values of
LOD and LOQ were found to be 0.2 and 0.5 μg/spot respectively.
Table 2: Summary of validation parameters
Parameter Value
Linearity range 0.5 – 20 μ g / spot
Correlation coefficient 0.998
Slope 32.19
Intercept 154.4
Limit of detection 0.2 µg
Limit of quantification 0.5 µg
115
Fig. 4: HPTLC calibration graph of diazepam
Precision and repeatability for the determination of diazepam in its three different
concentrations as indicated in Table 3 for spiked human blood sample extraction using
diethyl ether in basic media are reflecting a good robustness of the method.
Table 3: Accuracy and precision data of diazepam in spiked human blood
Actual
Concentration
[μ g]
Measured
concentration
[μ g]
Recovery
[%]
SD
μ g
RSD
[%]
Accuracy
Error
[%]
Intra-daya)
(n=3)
2 1.97 98.06 1.11 1.13 -1.5
5 4.84 96.32 0.91 1.50 -3.2
10 9.76 96.24 1.44 1.50 -2.4
Inter-dayb)
(n=3)
2 1.89 94.83 3.88 4.10 -5.5
5 4.75 95.13 3.93 4.13 -5.0
10 8.98 89.86 5.46 6.08 -10.2
a) mean for n =3 b) mean for n = 5
Evaluation of specificity of the new mobile phase for diazepam:
In addition to diazepam there are other benzodiazepines that were reported to be
most commonly prescribed (L.P. Longo, 2000). Therefore, workability of the new
solvent system for estimation of diazepam in presence of such benzodiazepines was
determined.
116
Fig. 5: UV spectra of benzodiazepines separated using the proposed mobile phase
[a]: UV spectrum of alprazolam with Rf = 0.02 and λ max = 227, 249, 285 nm
[b]: UV spectrum of nitrazepam with Rf = 0.05 and λ max = 278, 308, 225 nm
[c]: UV spectrum of clozapine with Rf = 0.08 and λ max = 233, 262, 295 nm
[d]: UV spectrum of lorazepam with Rf = 0.1 and λ max = 237, 257, 325 nm
[e]: UV spectrum of clonazepam with Rf = 0.13 and λ max = 306, 260, 222 nm
[f]: UV spectrum of chlordiazepoxide with Rf = 0.03 and λ max = 258, 316 nm
[g]: UV spectrum of diazepam with Rf = 0.34 and λ max = 233, 254, 310 nm
117
Each sample was individualized by UV pattern and Rf values as given in Fig. 4.
The results obtained are accounting for the effective separation of diazepam from other
benzodiazepines. Therefore, diazepam could be determined quantitatively even when it is
present along with other benzodiazepines.
Applications:
1. Postmortem blood sample: Earlier discussed method was employed to extract
diazepam from 2 ml of post mortem blood sample using diethyl ether in basic medium
and the extract was spotted on the TLC plate and developed with the new mobile phase.
A concentration of 2.981 µg/ml of blood was obtained with the new method which was
sufficient to produce toxic effects but it was less for causing fatalities [E.G.C. Clarke,
1986], but there was also a smothering effect as reported by the doctor in his post mortem
report.
2. Forensic drug sample: A white color powder sample was received to the laboratory
for the analysis of diazepam. A known quantity of the sample was extracted in methanol
and an aliquot was spotted on the TLC plate and quantified using the new mobile phase.
An average concentration of only 5.6% of diazepam was obtained and the remaining
material was being urea/other adulterants.
3. Pharmaceutical samples: The new method was applied to determine the diazepam
present in four different market samples; three samples were with single component and
the other one was with double components. Imipramine hydrochloride is a
benzodiazepine with antidepressant property which can be effectively separated from
diazepam with respect to Rf and UV absorption pattern (Rf = 0.05 and λ max = 251 nm).
Known amount of the values obtained were tabulated in Table 5. The results indicate
118
that the new HPTLC method was simple and specific for the estimation of diazepam in
its formulations.
Table 4: Analysis of diazepam in dosage forms
Sample Components Label claim
(mg/tablet)
Amount found
(mg/tablet)
% Assaya
01 Diazepam 2 1.94 97.0±0.06
02 Diazepam 5 4.92 98.4±0.05
03 Diazepam 10 9.88 98.8±0.16
04 Diazepam with
Imipramine HCl
2 2.07 103.5±0.10
a - mean ± standard deviation of five determinations
Conclusion:
HPTLC technique combined with sample application and densitometric scanning
has been proved to be sensitive, reliable and suitable for qualitative and quantitative
analysis of pharmaceuitical, environmental, toxicological, forensic and food samples
[P.K.Salo et al., 2007]. Therefore, in the present work, without using more toxic solvents
like toluene and chloroform [M.Bakavoli and M. Kaaykhali, 2003, V.P.Machale et al.,
2011, B.B. Daundkar et al., 2008], separation of benzodiazepines was carried out using
ethyl acetate and hexane solvents. Hexane and ethyl acetate in the ratio (7:3 v/v) was
found to be a very effective new mobile phase for HPTLC determination of diazepam
with Rf value 0.34 (SD=0.09). Using different techniques, samples containing diazepam
has been extracted using solvents like chloroform [Lowell B. Foster and Christopher S.
Frings, 1970], toluene [Raisys VA et al., 1980] and with benzene from plasma [Loscher,
1982]. Therefore, an attempt was made here to develop a method of extraction of
diazepam with diethyl ether in basic medium and it was found to be very specific without
119
the interference from common benzodiazepines [Vani. N et al., 2013]. As the results
were reproducible, specific, less time consuming and simple without use of special
techniques [M.Walles et al., 2004, M.H.De Oliveira et al., 2005], the method is suitable
for practice in any clinical or forensic laboratory for the satisfactory determination of
diazepam present in blood and pharmaceutical samples.
The results obtained from the study of optimization of extraction procedure,
selection of solvent system and quantification of diazepam from spiked blood samples
were published in JPC-Modern TLC, 26(4) (2013) 343-348.
120
Summary
A high performance thin layer chromatographic method combined with
densitometric measurement and UV scanning was used to optimize new mobile phase
consisting of hexane and ethyl acetate (7:3 v/v) for the estimation of diazepam in spiked
human blood, forensic and pharmaceutical samples. Efficacy and ease of extraction with
three solvents acetonitrile, diethyl ether and chloroform, each at three different pH
conditions were studied. Validation parameters for diazepam were found to be linear in
the range 0.5-20 μg/spot with r=0.998, LOD, 0.2 μg, LOQ 0.5 μg. Usefulness of the new
mobile phase for the separation of six common benzodiazepines namely nitrazepam,
clonazepam, lorazepam, chlordiazepoxide, alprazolam, clozapine from diazepam and
were identified through their retention factor and UV pattern.
121
References:
[1] M. Abid, H.J.Harishikeshavan and M.Asad, Indian Journal of Physiology
Pharmacology, 2006, Vol. 50, No. 2, pp 143-151.
[2] J.P.Hullihan, S.Spector, T.Taniguchi and J.K.Wang, British Journal of Pharmacology,
1983, Vol.78, No. 2, pp 321-327.
[3] M.Lucht, K.U.Kuenh, J.Armbruster, G.Abraham, M.Gaensicke, S.Barnow, H.Tretzel
and H.J.Freyberger, Alcohol and Alcoholism, 2003, Vol. 38, No. 2, pp 168-175.
[4] T.C.Marrs, Toxicological Reviews, 2003, Vol. 22, pp 75-81.
[5] L.P. Longo and B.Johnson, American Family Physician, 2000, Vol. 61, No.7, pp
2121-2128.
[6] Uchibayashi M, Yakugaku Zashi, 2007, Vol.127, No.1, pp 217-224.
[7] Tauseef Mehdi, British Journal of Medical Practitioners, 2012, Vol. 5, No. 1, a501.
[8] Riss J, Cloyd J, Gates J and Collins S, Acta Neurologica Scandinavica, 2008, Vol.
118, No.2, pp 69-86.
[9] Nicholas E. Calcaterra and James C. Barrow, ACS Chemical Neurosciences, 2014,
Vol, 5, No.4, pp 253-260.
[10] H. Marlin Beerman, American Journal of Psychiatry, 1964, Vol. 120, No. 9, pp 870-
874.
[11] P. Cushman, D. Benzer, Drug and Alcohol Dependence, 1980, Vol. 6, No. 6, pp
365-371.
[12] Coral H. Clarke and A.N.Nicholson, British Journal of Clinical Pharmacology, 1978,
Vol.6, pp 325-331.
122
[13] M.C. Longo, R.J. Lokan and J.M.White, Journal of Traffic Medicine, 2001, Vol. 29,
No. 1-2, pp 36-43.
[14] Geoffrey K Isbister, Luke O’Regan, David Sibbritt and Ian M Whyte, British Journal
of Clinical Pharmacology, 2004, Vol. 58, No. 1, pp 88-95.
[15] B. Ukley, W. Hermann, Der Nervnarzt, 2013, Vol. 84, No. 2, pp 223-225.
[16] C.Oti, D. Uncles, N. Sable and J. Willers, Anaesthesia, 2010, Vol. 65, No. 1, pp 110-
111.
[17] D.W. Sturdee, British Journal of Anaesthesia, 1976, Vol. 48, No.11, pp 1091-1096.
[18] Lowell B. Foster and Christopher S. Frings, Clinical Chemistry, 1970, Vol.16, No.3,
pp 177-179.
[19] Mercolini L, Mandrioli R, Iannello C, Matrisciano F, Nicoletti F and Raggi MA,
Talanta, 2009, Vol. 50, No. 1, pp 279-285.
[20] Kabra PM, Stevens GL and Marton LJ, Journal of Chromatography A, 1978, Vol.
150, No. 2, pp 355-360.
[21] Azzam RM, Notarianni LJ, Ali HM, Journal of Chromatography B: Biomedical
Sciences and Applications, 1998, Vol. 708, No. 1-2, pp 304-309.
[22] H.M.Stevens, Journal of Forensic Science Society, 1985, Vol. 25, No. 1, pp 67-79.
[23] M. Lazar, A. Mouzdahir, M. Zahouily, Asian Journal of Pharmaceutical Analysis
Medicinal Chemistry, 2013, Vol. 1, No. 3, pp 140-147.
[24] Patel Satish A, Patel Paresh U, Patel Shweta M, American Journal of PharmTech
Research, 2011, Vol. 1, No. 4, pp 355-365.
[25] P. Pydiratnam, T. Santosh Kumar, S. Satynarayana, International Journal of
Pharmaceutical Drug Analysis, 2013, Vol. 1, No. 1, 13-24.
123
[26] Venkata Raveendra Babu Vemula and Pankaj Kumar Sharma, International Journal
of Pharmacy and Pharmaceutical Sciences, 2013, Vol. 5, No. 3, pp 249-253.
[27] F.Mauriece Smith and Noel O. Nuessle, Analytical Letters, 1982, Vol. 15, No. 4, pp
363-371.
[28] Javier Moros, Salvador Garrigues, Miguel de la Guardia, Journal of Pharmaceutical
and Biomedical Analysis, 2006, Vol. 43, No. 4, pp 1277-1282.
[29] Mohammad Sarwar, Microgram, 2008, Vol. 6, No. 3-4, pp 53-71.
[30] S.Sadeghi, R.Takjoo, S.Hanghgoo, Analytical Letters, 2002, Vol. 35, No. 13, pp
2119-2131.
[31] Debabrata Ghosh Dastidar and Biswanth Sa, Pharmaceutical Science and
Technology, 2009, Vol. 10, No. 4, pp 1396-1400.
[32] Mannucci C, Bertini J, Cocchini A, Perico A, Salvagnini F and Triolo A, Journal of
Pharmaceutical Sciences, 1992, Vol. 81, No. 12, pp 1175-1157.
[33] W.F.El-Hawary, Y.M.Issa and A.Talat, Internatinal Journal Biomedical Science,
2007, Vol. 3, No. 1, pp 50-55.
[34] Ren-Yu Hsu, ShanAn Chan, Shu-Ling Lin, Tzuen-Yeuan Lin, Wei-Lan Chu, Ming-
[35] Ren Fuh, Journal of Food and Drug Analysis, 2013, Vol. 21, No. 4, pp 376-383.
[36] M.M.Ariffin, E.I.Miller, P.A.G.Cormack, R.A.Anderson, Analytical Chemistry,
2007, Vol. 79, No. 1, pp 256-262.
[37] Sooyeun Lee, Eunyoung Han, Sanghwan In, Hwakyung Choi, Heesun Chung and
Kyu Hyuck chung, Journal of Analytical Toxicology, 2011, Vol. 35, No. 5, pp 312-315.
[38] W. Loscher, Therapeutic Drug Monitoring, 1982, Vol. 4, No. 3, pp 315-318.
124
[39] Horton Mccurdy, E. Larry Slightom, Joan C Harril, Journal of Analytical
Toxicology, 1979, Vol. 3, No. 5, pp 195-198.
[40] David A. Engelhar and Amanda J. Jenkins, Journal of Analytical Toxicology, 2007,
Vol. 31, No. 9, pp 581-587.
[41] M.G. Garcia, A.Garcia, I.Gonzalez, Talanta, 1993, Vol. 40, No. 12, 1775-1779.
[42] Mohammad Reza Milani Hosseini and Ali Motahrian, RSC Advances, 2015, Vol. 5,
pp 81650-816593.
[43] M.Walles, W.M.Mullett, J.Pawliszyn, Journal of Chromatography A, 2004, Vol.
1025, No. 1, pp 85-92.
[44] M.H.De Oliveira, M.E.C.Queiroz, D.Carvalho, S.M.Silva, F.M.Lancas,
Chromatographia, 2005, Vol. 32, No. 3-4, pp 215-219.
[45] Naveen Bimal and Bhupinder Singh Sekhon, Pharmatechmedica, 2013, Vol. 2, No.4,
pp 323-333.
[46] P.D.Sethi, HPTLC Quantitative Analysis of Pharmaceutical Formulations, 1st Ed.,
CBS Publishers & Distributors, India, 1996.
[47] M.Bakavoli and M.Kaykhali, Journal of Pharmaceutical and Biomedical Analysis,
2003, Vol. 31, No. 6, pp 1185-1189.
[48] R.K.Sarin, G.P.Sharma, K.M.Varshney and S.N.Rasool, Journal of Chromatography
A, 1998, Vol. 822, No. 2, pp 332-335.
[49] V.P.Machale, A.T. Gatade, R.T.Sane, International Journal of Pharmaceutical
Research and Development, 2011, Vol. 3, pp 34-41.
[50] Bhadani Shweta, Patel Paresh, Modi Hiral, International Journal of Drug
Development and Research, 2013, Vol. 5, No.1, 91-98.
125
[51] R. Jain, Journal of Chromatography B: Biomedical Sciences and Applications, 1993,
Vol. 615, No. 2, pp 365-368.
[52] B.B. Daundkar, M.K.Malve, R.Krishnamurthy, Journal of Planar Chromatography-
Modern TLC, 2008, Vol. 21, No. 4, pp 249-250.
[53] P.K. Salo, S. Vilmunen, H. Salomies, R.A. Ketola, R. Kostiainen, Analytical
Chemistry, 2007, Vol. 79, Vol. 5, pp 2101-2108.
[54] ZhiBin Huang, Tianfang Yu, Lin Guo, Zebin Lin, ZiQin Zhao, Yiwen Shen, Yan
Jiang, Yonghong Ye, Yulan Rao, Toxicology Reports, 2015, Vol. 2, 785-791.
[55] Haram K, Sagen N, Brandt RD, International Journal of Gynecology and Obstetrics,
1976, Vol. 14, No. 6, pp 545-549.
[56] Raisys VA, Friel PN, Graff PR, Opheim KE and Wilensky AJ, Journal of
Chromatography B: Biomedical Sciences and Applications, 1980, Vol. 183, No. 4, pp
441-448.
[56] B. Levine, RV Blanke, JC Valentour, Journal Forensic Sciences, 1983, Vol. 28, No.
1, pp 102-115.
[57] Bernard Fried and Joseph Sherma, 4th
Ed., Marcel Dekkar Inc., New York, 2005.
[58] G.Hancu, Eniko Fulop, Aura Rusu, Eleonora Mircia and A. Gyeresi, Annalele
Universitatti din Bucresti: Chimie, 2011, Vol. 20, No. 2, pp 181-188.
[59] Subbiah Thangadurai, Ahilandam Dhanalakshmi, Madurai Veeran Suresh Kumar,
Malaysian Journal of Forensic Sciences, 2013, Vol. 4, No. 10, pp 47-53.
[60] Yao Shen, Bo Chen and Teris A. van Beek, Green Chemistry, 2015, Vol. 17, No. 7,
pp 4073-4081.
126
[61] Caitlin Sullivan and Joseph Sherma, Journal of Liquid Chromatography and Related
Technologies, 2004, Vol. 27, No. 13, pp 1993-2002.
[62] Lukasz Komsta, Monika Waksmundzka-Hajnos and Joseph Shema, TLC in Drug
Analysis, 1st Ed., CRC Press, Boca Raton, 2013.
[63] Jessica L. Westland, Frank L. Dorman, Journal of Pharmaceutical Analysis, 2013,
Vol. 3, No. 6, pp 509-517.
[64] B.Levine, Principles of Forensic Toxicology, 2nd
Ed., American Association for
Clinical Chemistry, Washington DC, 1999.
[65] St. M.V. Pierre and K.S. Pang, Journal of Chromatography, 1987, Vol. 421, No. 2,
pp 291-307.
[67] Vani. N, B.M. Mohan, G. Nagendrappa, Journal of Planar Chromatography-Modern
TLC, 2013, Vol. 26, No. 4, pp 343-348.
[66] E.G.C. Clarke, Clarke’s Isolation and Identification of drugs, 2nd
Ed.,
Pharmaceutical Press, London, 1986.
top related