CHAPTER 5 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/55029/11/11_chapter 5.p… · Chapter 5 Prajesh Prajapati: Institute of R & D, GFSU Page 102 of 121 5.2.3. Method:

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.

Chapter 5


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).

Chapter 5


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.

Chapter 5


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.

Chapter 5


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.

Chapter 5


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.

Chapter 5


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

Chapter 5


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.

Chapter 5


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.

Chapter 5


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

Chapter 5


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)]

Chapter 5


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.

Chapter 5


REFERENCE:

[1] Treatment of Tuberculosis, Guidelines, 4th edition, World Health

Organisation. Available at:

http://whqlibdoc.who.int/publications/2010/9789241547833_eng.pdf, 2010.

(Accessed on 18th Feb, 2014).

[2] Ethambutol efficacy and toxicity: literature review and recommendations for

daily and intermittent dosage in children, WHO/HTM/TB/2006.365, 2006.

Available at:

http://whqlibdoc.who.int/hq/2006/who_htm_tb_2006.365_eng.pdf (Accessed

on 13th Nov, 2014).

[3] Lee, C.S., Benet, L.Z., Florey, K., eds. Analytical Profiles of Drug

Substances, Vol. 7. New York Academic Press, (1978) 231-249.

[4] Thomas, J.P., Baughn, C., Wilkinson, R.G., Shepherd, R.G. A new synthetic

compound with antituberculous activity in mice: Ethambutol (dextro-2,2’-

(ethylenediimino)-di-l-butanol) Am. Rev. Resp. Dis. 83:891-893, 1961.

[5] Wilkinson, R.G., Cantrall, M.B., Shepherd, R.G. Antituberculous agent. 111.

(+)-2,2’-(Ethylenediimino)-di-l-butanol and some analogs. J. Med. Pharm.

Chem. 5:835-845, 1962.

[6] Prema Gurumurthy, T N Gayathri, S Bhagavathy, P Venkatesan,

Standardization of the method for estimation of ethambutol in pharmaceutical

preparations and biological fluid, Indian Journal of Experimental Biology, 42

(2004), 68-73.

[7] Mohamed El-Sayed Mahrousa, Sensitive Spectrophotometric Methods for

Determination of Non UV Absorbing Ethambutol, Analytical Letters, 25 (2)

(1992) 269-280.

http://whqlibdoc.who.int/hq/2006/who_htm_tb_2006.365_eng.pdf

Chapter 5


[8] Wen-Ying Wu, Ji-Yuan Yang, Li-Ming Du, Hao Wu, Chang-Feng Li.,

Determination of ethambutol by a sensitive fluorescent probe, Spectrochimica

Acta Part A 79 (2011) 418–422.

[9] Z. Jiang, H. Wang, D.C. Locke, Determination of ethambutol by ion-pair

reversed phase liquid chromatography with UV detection, Analytica Chimica

Acta 456 (2002) 189–192.

[10] M. Breda*, R Marrari, E. Pianezzola, M. Strolin Benedetti, Determination of

ethambutol in human plasma and urine by high-performance liquid

chromatography with fluorescence detection, Journal of Chromatography A,

729 (1996) 301-307

[11] Philippe Cheneviera, Laurent Massiasa, Delphine Gueylarda, Robert Farinotti.

Determination of ethambutol in plasma by high-performance liquid

chromatography after pre-column derivatization, Journal of Chromatography

B, 708 (1998) 310–315.

[12] Ming Yan*, Tao Guo, Hongtao Song, Qingchun Zhao, and Yin Sui,

Determination of Ethambutol Hydrochloride in the Combination Tablets by

Precolumn Derivatization., Journal of Chromatographic Science, 45, (2007),

269-272.

[13] Zhilong Gong, Yousef Basir, David Chu, Melanie McCort-Tipton, A rapid

and robust liquid chromatography/tandem mass spectrometry method for

simultaneous analysis of anti-tuberculosis drugs—Ethambutol and

pyrazinamide in human plasma. Journal of Chromatography B, 877 (2009)

1698–1704.

[14] Bennett M. Richard, Joseph E. Manno’ and Barbara R. Manno, Gas

chromatographic determination of Ethambutol, Journal of Chromatography,

89 (1974) 80-83.

Chapter 5


[15] Mack R- Holdiness, Zafah H- Israili and Joseph B Justice, Gas

chromatographic-mass spectrometric determination of Ethambutol in human

plasma, Journal of Chromatography Biomedical Applications 224 (1981)

415-422.

[16] Abdel Maaboud I. Mohamed, Fardous A. Mohamed, Noha N. Atia, Seham

M. Botros. A novel spectrofluorimetric determination of four anti-TB drugs

in their pure and pharmaceutical dosage forms by quenching effect on the

fluorescence of NBS-phenothiazine product, Asian Journal of Biomedical and

Pharmaceutical Sciences; 3 (26) (2013) 21-27.

[17] A. Kenyon, T. Layloff, J. Sherma, Rapid screening of tuberculosis

pharmaceuticals by thin layer chromatography, J. Liq. Chrom. Rel. Technol.

24 (2001) 1479–1490.

[18] D.H. Shewiyo, E. Kaale, P.G. Risha, B. Dejaegher, J. Smeyers-Verbeke, Y.

Vander Heyden, Optimization of a reversed-phase-high-performance thin-

layer chromatography method for the separation of isoniazid, ethambutol,

rifampicin and pyrazinamide in fixed-dose combination antituberculosis

tablets, Journal of Chromatography A, 1260 (2012) 232– 238.

[19] Ye-Sook Kim, Jeong-Rok Youm, Man-Ki Park and Nam-Ho Paik,

Determination of the Optical Isomers of Ethambutol (Myambutol) and 2-

Amino-l-butanol by Gas-liquid Chromatography, Arch. Pharm, aes. 4(1), 1-

,~8(1981

[20] Blessington, B., Beiraghi, A. A study of the stereochemistry of ethambutol

using chiral liquid chromatography and synthesis. J. Chromatogr. 522:195-

203, 1990.

[21] B. Blessington, A. Beiraghi, T.W. Lo, A. Drake, and G. Jonas, Chiral hplc-cd

studies of the antituberculosis drug (+)-ethambutol, 4 (4), 227–229, 1992.

Chapter 5


[22] ICH Q2(R1). Text on Validation of Analytical Procedures: Methodology,

International conference on Harmonization of Technical Requirements for

Registration of Pharmaceutical for Human Use, Geneva: Jul 2000. Available

from:

http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Qu

ality/Q2_R1/Step4/Q2_R1__Guideline.pdf (Accessed on 13th Nov, 2014).

[23] AM Carlos, JG Crespo Afonso, Green Separation Processes-Fundamentals

and Applications, 2005 WILEY-VCH Verlag GmbH & Co. KGaA,

Weinheim.

[24] ICH Topic Q2, Validation of Analytical Procedures: Text and Methodology

International Conference on Harmonization Tripartite Guideline.

http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html

(accessed 15.11.14).

[25] Document No. SANCO/825/00 rev.8.1 Guidance Document on Residue

Analytical Methods. European Commission, Directorate General Health and

Consumer Protection.

http://ec.europa.eu/food/plant/protection/resources/guide_doc_825-

00_rev7_en.pdf (accessed on 15.11.14).

[26] Guidance for Industry: Bioanalytical Method Validation, 2001.

http://wwwfdagov/downloads/drugs/guidancecomplianceregulatoryinformatio

n/guidances/ucm070107pdf (accessed on 15.11.14)

[27] JD Pinkston; DT Stanton; D Wen, Elution and preliminary structure-retention

modeling of polar and ionic substances in supercritical fluid chromatography

using volatile ammonium salts as mobile phase additives., Journal of

Separation Science, 2004, 27, 115-23.

http://wwwfdagov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070107pdf

http://wwwfdagov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070107pdf

Chapter 5


[28] JD Pinkston, D Wen, KL Morand, DA Tirey, DT Stanton, Comparison of

LC/MS and SFC/MS for screening of a large and diverse library of

pharmaceutically relevant compounds, Analytical Chemistry, 2006,

78(21):7467-72.

[29] Prajesh Prajapati, Y.K. Agrawal, SFC–MS/MS for identification and

simultaneous estimation of the isoniazid and pyrazinamide in its dosage form,

The Journal of Supercritical Fluids, 95 (2014), 597-602.

http://dx.doi.org/10.1016/j.supflu.2014.09.012.

http://dx.doi.org/10.1016/j.supflu.2014.09.012

Documents

CHAPTER 5 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/55029/11/11_chapter 5.p… · Chapter 5 Prajesh Prajapati: Institute of R & D, GFSU Page 102 of 121 5.2.3. Method: