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CHAPTER 5 IDENTIFICATION OF THE ETHAMBUTOL AND OF ITS STEREOISOMERS BY SFC-MS/MS AND CD DETECTOR
Chapter 5
Prajesh Prajapati: Institute of R & D, GFSU Page 98 of 121
Identification of the Ethambutol and of its stereoisomers by SFC-MS/MS and
CD detector
ABSTRACT
A simple, specific, accurate and precise SFC-MS/MS method (using
TurboIonSpray probes) is described for the determination of Ethambutol and its
stereoisomers using mobile phase Dichloromethane: Methanol: Formic acid
(70:30:0.1 v/v/v) at flow rate of 0.3 mL/min. The aim of this study is to identify
chiral impurity present in formulation by CD detector and determined using
MS/MS. The EMB was determined using a C18 reverse-phase fused-core column
(Inertsil ODS- C18, 150mm×4.6mm, 5 µm). The method uses the principle of
circular dichroism was validated according to international guidelines in the
working range of 2 to 20µg/mL with accuracy (%RE) of EMB of -1.73% and
6.05%. In this study we have separated two enantiomers of optically active
Ethambutol i.e. levorotatory and optical inactive meso compound.
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5.1. INTRODUCTION
Ethambutol (2,2’-(Ethane-1,2-diyldiimino)dibutan-1-ol) (Fig. 5.1) has
been assorted by WHO as the first-line anti-tubercular drug for prevention and
intervention of tuberculosis [1, 2]. Ethambutol has three structural isomers, among
them one optically active isomer (+) dextrorotatory ethambutol is used for the
treatment of tuberculosis [3]. Its other optically active isomer (-) levorotatory
ethambutol is not used in treatment as it causes blindness and third isomer is
optically inactive and is much less active then (+) isomer [3-5]. Earlier studies on
ethambutol shows that different methods based on spectrophotometric [6, 7],
Florescence Probe [8], Ion Pair HPLC [9], HPLC [10-12], LC-MS [13], GC [14],
GC-MS [15], Spectrofluorimetry [16] HPTLC [17], RP-HPTLC [18] estimations
are depicted for estimation of EMB in human plasma or pharmaceutical dosage
form. A very few methods have been reported for separations of these
enantiomers by using Gas-Liquid Chromatography [19], HPLC [20] and HPLC
using CD detector [21]. Among the methods used for separation of enantiomers
of ethambutol, they took longer time for sample preparations and identification.
So in this context, we describe the simple, rapid and selective SFC-MS/MS
method for identification of enantiomers of EMB without any complexation and
pre-derivatization.
Fig. 5.1. Molecular structure of Ethambutol (EMB).
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In tuberculosis, first line agents have to be taken for longer periods at a
prescribed time interval and hence the quality of the product should be
maintained. As (-) isomer may cause the blindness so we have to quickly identify
the levorotatory isomer in pharmaceutical dosage forms.
5.2. Materials and methods
5.2.1 Materials
Ethambutol (EMB) standard was incurred as gratis samples from Sunij Pharma
Pvt. Ltd. (Vatva GIDC, Ahmedabad) and other stereoisomers have been
synthesized as described by R.G. Wilkinson et al [5]. Tablet containing EMB
were procured from local market. Dichloromethane (HPLC Grade) and Methanol
(HPLC Grade) – Lichrosolv®- was purchased from E. Merck (India) Ltd.,
Mumbai. Whatman filter paper no. 42 (0.45 µm) was used to filter the solutions.
5.2.2 Instruments:
5.2.2.1 Supercritical Fluid Chromatograph
A JASCO-2000 series (Japan Spectroscopic Co. Ltd., Hachioji, Japan) of
supercritical fluid chromatograph was exercised for separation of the drug which
consist of two pumps (PU-2080 and PU-2080 CO2), which were precisely able to
deliver the flow rate ranging from 0.001 to 10 mL/min for both Supercritical CO2
and modifier. The system is attached to back-pressure regulator (BP-2080), which
maintained pressure electronically that allowed the flow rate and pressure to be
controlled independently. For injecting our sample accurately in to the column, an
external loop having capacity of 20 μL was outfitted with rheodyne injector. The
temperature of the column was thermostatically controlled in a column oven
(Jasco-CO-2060), while inbuilt with a cooling circulator. Detection of analyte was
done by using CD detector (Jasco-CD-2070). The effluent coming from the SFC
was injected in the MS/MS for identification of drug.
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5.2.2.2 Mass spectrometer
An AB Sciex ((Toronto, Canada) QTRAP-4500 series mass spectrometer was
used in present investigation. It was equipped with exclusive TurboV™ source
contained TurboIonSpray probes which provide advanced linear ion trap
technology for highest level of sensitivity. The TurboV ion chamber has
embedded ceramic heater technology with improved gas dynamics. Data
acquisition and integration were done by windows-based analyst software.
5.2.2.3 SFC/MS/MS conditions
Fused silica column (inertsil ODS-C18, 150mm×4.6mm, 5 µm) protected
by precolumn filter cartridges was used for analysis of EMB. After optimization,
mobile phase consisting of Dichloromethane: Methanol: Formic acid (70:30:0.1
v/v/v) was used at flow rate of 0.3 mL/min and Supercritical CO2 was flowed at 2
mL/min.
While injecting the effluent from the SFC interface independent
parameters and heated nebulizer parameters were optimized. These were
(arbitrary units if not specified) CAD: High, CUR: 20, ISV: 5500. The optimized
value for MS/MS analyses were as follows: ESI positive ion mode; capillary
voltage, 3.5 kV; cone voltage, 40 V; Gas 1 (nebulizing gas) and Gas 2 (cone gas)
were set to 50 units each and the source temperature was set at 550°. High-purity
nitrogen was used as nebulizer and cone gas.
The injection volume and column temperature were set at 20 µL and 35
◦C, respectively. Full-scan SFC–MS/MS spectra were obtained by scanning from
m/z 100 to 500.
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5.2.3. Method:
5.2.3.1. Preparation of modifier phase:
A blend of 30 ml Methanol and 70 ml of Dichloromethane with 0.1% of formic
acid was filtered through 0.45 µm filter paper. After filtration, blend was
sonicated for 10 min to degas the mixture and used as modifier.
5.2.3.2. Preparation of EMB Stock Standard:
The EMB stock standard was prepared by dissolving 10 mg of EMB in 10 mL
mobile phase containing Dichloromethane: Methanol: Formic acid (70:30:0.1
v/v/v). This solution was kept in refrigerator at 2°C - 8°C.
5.2.3.3 Detection of possible enantiomers of EMB from dosage form:
For the detection of possible enantiomers of EMB from dosage form, 20 tablets
were weighed and make uniform powder. To sample solution, weigh powder
equivalent to 10 mg of EMB was transferred to a clean and dry 10 ml of
volumetric flask containing 5 ml of modifier phase as diluting solution and shaken
thoroughly to extract the drug from the excipients and then sonicated for 10 min
for complete dissolution of drug (standard addition can be done to make sure the
complete integrity of final concentration). The solution was allowed to cool at
room temperature and then the volume was made up to the mark with the same
diluting solution. The solution was filtered through Whatman filter paper (No.
42), sonicated for 10 min and appropriate aliquots of this solution was transferred
to 10 ml volumetric flask and diluted up to the mark with the mobile phase to give
a solution containing 20 µg/mL EMB. This solution was used for the detection of
possible enantiomers of EMB.
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5.2.4. Method validation
The method was validated by using international guidelines [23-25] for
selectivity, specificity and precision and accuracy.
5.2.4.1 Selectivity
To assess the selectivity of the proposed method, spiked and non-spiked samples
were analyzed by the SFC/MS/MS system.
5.2.4.2 Specificity
The specificity of this method was determined by comparisons of SFC/MS/MS
chromatograms of EMB at LOQ to those of six samples extracted from tablets.
5.2.4.3 Precision and accuracy
Precision and accuracy were determined by measuring the concentrations of
analyte in five replicates of QC samples at three different concentrations for three
separate batches. Assay precision was calculated using the relative standard
deviation (%RSD). Accuracy is defined as the relative deviation in the calculated
value (E) of a standard from that of its true value (T). It was calculated by using
the formula RE% = (E − T)/T × 100.
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5.3. RESULTS AND DISCUSSION
The aim of this work was to furnish ecofriendly, specific alternate method for
Detection of possible enantiomers of EMB in FDC (fixed dosage combinations)
by SFC-MS/MS without pre or post derivatization method. Previous experiments
and from literature review, we found that when volatile acid (like, formic acid)
added to the mobile phase will increase the positive ion of the analyte, which is
most helpful in MS/MS [26-28]. Therefore, 0.1% formic acid was added to the
Methanol: Dichloromethane (30:70 v/v) modifier in the SFC/MS work.
5.3.1. Selection of mobile phase
For the selection of mobile phase, we have varied the concentration of modifier
methanol and dichloromethane ranging from 30% to 100% at a flow rates ranging
from 0.1-0.3 mL/min and Supercritical Carbon Dioxide (SC-CO2) with flow rates
from 1.5-2.0 mL/min and chromatograms were recorded.
Figure 5.2: Chromatogram of standard EMB in proposed mobile phase
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Amongst the all result obtained, the optimized system containing
Dichloromethane: Methanol: Formic acid (70:30:0.1 v/v/v) at 0.3 mL/min and
CO2 at 2 mL/min, was found to be satisfactory and gave well resolved peak for
EMB and its enantiomers (Figure 5.2).
5.3.2 SFC-MS/MS optimization
In mass spectrometer positive ionization mode in TurboIonSpray was used to
produce ions as EMB contains an amine groups in its molecular structure.
Supercritical fluid chromatograph under UV trace when observed, there is no
significant peak observed and mass spectra of drug is not showing unknown peak,
so we can say that our standard drug is pure and free from traces of impurity and
can be used for identification of these drugs in FDC as well as urine samples. The
mass spectra of the EMB revealed a base peak m/z 115.9 [M+H]+. So, parameters
like cone voltage and de-clustering potential were optimized to obtain the
protonated parent ion [M+H]+ (Figure 5.3). After optimization, the MRM
transition of m/z 240→60 was selected for EMB. The mono-isotopic masses of
EMB was 204 ion peak at 205.1 as it is protonated molecular ion. It was observed
that there is no extra peak in mass spectrometry for EMB when analyzed in
pharmaceutical dosage forms. So, this method can be used in determination of
EMB from pharmaceutical dosage form. Mass spectra obtain for all racemate
were compared and found similar spectra. Thus we can conclude that all the
separated peak is of EMB racemates.
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Figure 5.3: Full scan MS/MS spectrum of EMB
5.3.3 Method validation
5.3.3.1 Specificity
Specificity of the method was assessed by injecting sample extracted from tablet
of various manufacturer. All samples were found to have no from excipient.
Retention time for EMB was 2.7 min. Chromatograms of standard EMB and EMB
extracted from tablets are shown in Figure 5.4.
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Figure 5.4: Chromatograms of EMB (a) extracted from tablet matrix (b)
standard
5.3.3.2 Precision and accuracy
Precision and accuracy of the method was determined at three QC levels by
measuring three replicates of it. The results are shown in Table 5.1. Intraday
precision in terms of RSD was in range of 1.32 and 2.27 and Interday precision in
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terms of RSD was between 0.79 and 4.45. The accuracy (RE) was between -
1.73% and 6.05%. The results revealed good precision and accuracy.
Table 5.1 Precision and accuracy for determining EMB
Concentration 2(μg/mL) 10(μg/mL) 20(μg/mL)
Intraday
(n=3)
Mean concentration
found
1.95 10.16 20.20
Accuracy (%RE) -1.20 3.24 6.05
Precision (%RSD) 1.32 2.27 2.02
Interday
(n=3)
Mean concentration
found
1.92 10.52 20.37
Accuracy (%RE) -1.73 1.69 2.96
Precision (%RSD) 4.45 3.51 0.79
Table 5.2 Robustness for EMB
Operator Pharmaceutical
EMB (mg) in FDC
Mean ± S.D. (n=3) %RSD
1 Tablet 199.54 ± 0.56 0.28
2* Tablet 199.36 ± 1.24 0.62
3# Tablet 198.57 ± 0.96 0.48
*Conditions: [mobile phase- Dichloromethane: Methanol: Formic acid (80:20:0.1 v/v/v) flow-rate
0.2 mlmin-1, SC-CO2 2 mLmin-1 column temperature 38°C and UV detection, at 260 nm)] # Conditions: [mobile phase Dichloromethane: Methanol: Formic acid (60:40:0.1 v/v/v), flow-rate
0.4 mlmin-1, SC-CO2 2.5 mLmin-1 column temperature 35°C and UV detection, at 267 nm)]
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5.3.3.3 Robustness
Robustness of the method was determined by two operators (2 and 3) other than
operator writing this paper, using standard method as described in this paper for
pharmaceutical dosage form under different chromatographic conditions than
those used in the present method. The chromatographic conditions and the results
obtained are listed in the Table 5.2.
5.3.3.4 Comparison of marketed formulation with standard
There was absence of other recemates in tablet (fig 5.4) as compared with the
standard. So we can say that the marketed formulations are safe to use.
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