Paper-Capillary Gas Chromatographic Determination of Isoniazid and Hydrazine in Pharmaceutical Preparations and Blood

8/9/2019 Paper-Capillary Gas Chromatographic Determination of Isoniazid and Hydrazine in Pharmaceutical Preparations and Blood

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ABSTRACT:

Antituberculosis drug Isoniazid (INH) and toxic substance are determined by capillary

gas chromatography after precolumn derivatization with acetylacetone (AA),

trifluoroacetylacetone (A), ethyl chloroformate (!")# $henylhydrazine ($H%) was used as an

internal standard# &" separation was carried on H$' column (*m + *#mm i#d) with flame

ionization detection# or AA, elution was carried out at initial column temperature -*." with

heating rate *."/min upto 0* .", with nitrogen flow rate of -# m1/min and split ratio was

*2-v/v# or !", initial column temperature of -*." for - min at a heating rate of -*."/min up

to *.", nitrogen flow rate of 3 ml/min and a split ratio of -*2-, v/v# 4y using AA, the linear

calibration range for INH was observed 5#'-** 6g/m1 with detection limit of #- 6g/m1

corresponding to -*pg reaching to the detector, similarly linear calibration curve for hydrazine

was 7#-'7 6g/m1 with detection limits of # 6g/m1# 4y using AA, linear calibration ranges

for INH and H% were determined to be #' 6g/m1 and #' -# 6g/m1 respectively, with

detection limit (8/N 9 ) corresponding 5# pg reaching to the detector# inally by using :",

linear calibration ranges for INH and H% were observed between # 7# and # mg/ml

with corresponding detection limits of *#-0 and *#-7 ng reaching the detector#

:he first method using AA was applied for determination of INH and hydrazine from

pharmaceutical preparations and relative deviation of INH from labelled values was within -#*'

-#3;# :he amount of recovery of INH from pharmaceutical preparations was ;#

:he second method, using AA applied for the determination of INH and H% from

pharmaceutical preparations# :he method was also used for determination of INH from blood of

tuberculosis patient# :he amount of INH found in the blood serum of tuberculosis patients on

chemotherapy with INH based drugs was within *#0-3#0 ?g/m1 with relative standard deviation

(=8>) of '#0;# :he recovery percentage of INH from blood samples was found #;# :he last method was was subse@uentlyapplied to the determination of INH and H% in

pharmaceutical preparations, achieving a relative standard deviation (=8>) of #0 #0;# :he

recovery percentage of INH from isoniazid syrup was of #;#

INTRODUCTION:

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G'diCetone reacted with amino compounds to form 8chiff bases and addition of carbon

chain could enhance I> sensitivity# AA has been used for &" determination of putresine and

cadverine from biological fluids# :he present worC examines &" determination of INH and H%

after derivatization with AA(trifluoroacetylacetone) using $H% ($henylhydrazine) as an internal

standard# :he volatile derivatives are easy to elute and separate from capillary &" column with

re@uired sensitivity for the desired application for the analysis of biological fluids#()

AlCyl chloroformates have been used as derivatizing reagents for gas chromatographic

(&") determinations of amines, amino acids, amino alcohol and acids# HuseC has reviewed

chloroformates in &" as a general'purpose derivatizing reagent# HuseC and 8imeC have

reviewed alCyl chloroformates in sample derivatization strategies for &" analysis on a decade

use as an esterifying reagent# uantitation was carried out within the limits of # mg/ml#

:he use of a simple and inexpensive derivatizing reagent for the &" determination of INH and

H% from a pharmaceutical preparation could be of analytical interest for @uality control# :he

present worC examined the capillary &" determination of INH and H% using $H% as an internal

standard after derivatization with ethyl chloroformate (!") with I># :he reagent !" may

prove to be an additional &" reagent for the selective determination of INH and H%#()

EXPERIMENTAL:

I.Materials and Methods:

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All the chemicals used were of reagent or pharmaceutical grade# reshly prepared double

distilled water was used throughout the study# $ure INH was obtained from Nabi asim

$harmaceuticals, arachi, $aCistan# H% (3;), $H%, AA and methanol were purchased from !#

EercC, >armstadt, &ermany# :he percentage of H% in H% solution was determined by the

titrimetry# (-,,) Hydrochloric acid (7;), potassium chloride, acetic acid, sodium acetate,

ammonium acetate, sodium bicarbonate, sodium carbonate, ammonium chloride were also

obtained from !#EercC &ermany#(-,) 4uffer solutions in the pH range -'-* at unit interval were

prepared from hydrochloric acid (*#- E) and potassium chloride (- E) (pH - J), acetic acid (-

E) and sodium acetate (- E) (pH to 5)K ammonium acetate (pH 7), sodium bicarbonate (- E)

and sodium carbonate (saturated) (pH 0 J


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".:he solution(- m1) containing

INH('3* 6g), H% (' 6g) and

$H% (-*#< 6g) was added - m1 of

potassium chloride hydrochloric acid

buffer pH , - m1(- ; v/v) AA and

heated at 7 O" for - min# :he

contents were cooled at room

temperature and was added - m1 of

chloroform# "ontents were mixed

well, layers were allowed to separate#

".:he solution (- m1) containing I

NH(--* ?g), H%(-'-7 ?g) and

$H%( ?g) was added - m1 of

potassium chloride'hydrochloric acid

buffer pH , - m1 of (-; v/v) AA

and heated at 7P" for - min# :he

solution was cooled at room

temperature and were added -m1 of

chloroform# "ontents were mixed

well, layers were allowed to separate#

".:o a solution (- ml) containin

INH (-#- mg), H% (-#3-3#* m

and $H% (# mg) was added a *#

ml pyridine solution, followed by *

ml of pure !" and - ml of -E

carbonate solution, pH


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".:en tablets of each =emactal J =emister (Novartis $harma

($aC#) 1td#) and Eyrin $ (1eaderle 1aboratories >ivision,

"yanamid $aC# 1td# arachi) were powdered and &$.'( !)

Re!actal* &&.(+ !) Re!i,tar an- &."# !)Myrin Pwere

dissolved in portions of each (5 m1) in etanol:/ater 0":"1

". &$.'( !) Re!actal*

&$.(+ !) Re!i,tar

an- &2."# !)Myrin P

!etanol:/ater 0":"1.

". + !) Re!actal*

(+ !) 3eyet

Mya!4utaol* (+ !)

Re!actal 0%&+1

etanol:/ater 0":"1

#.:he solution was filtered and final volume was a-5u,te- to

#& !L/it etanol:/ater 0":" 6761# 8olution (- m1) was

transferred to screwcapped vial and - m1 of chloroform was

added# "ontents were mixed well and the layers were allowed

to separate#

#. a-5u,te- to &+ !L

/it !etanol:/ater

0":" 6761

#. a-5u,te- to &+ !L

/it etanol:/ater

0":" 6761

$. :he a@ueous layer was collected and the analytical &"

procedure II was followed by addition of P89 0"+.+( )1#

$. & ;) of P89 $. P89 0#$.# )1#

%. :en tablets of isoniazid (Fnexo 1ab# ($vt) 1td# 1ahore) were

thoroughly ground and aurter -ilute- to

"++ !L

P89 0#$.# )1

AA: Percenta)e reco6ery of i,onia?i- fro! IN8 ta4let, 4y ,


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Isoniazid (INH) condenses easily with acetylacetone (AA) to form acetylacetone'

isonicotinyl hydrazone# or the selective and sensitive determination of INH and hydrazine (H%)

capillary column gas chromatography was examined# $recolumn derivatization was carried out

with AA and elution was examined from the capillary column H$' (* m *# mm) with layer

thicCness *# 6m at an initial column temperature -* O" with heating rate * O"/min up to 0*

O"/ min# :he run time was 5# min# Nitrogen flow rate was -# m1/min# :he detection was

performed by I># :he derivatives of H% and INH gave a single peaC with retention times of #3

and 3#* min# :he derivatives separated completely from the derivatizing reagent AA# :he

derivatization conditions were optimized for the @uantitative determination of INH and H% by

measuring average peaC height/peaC area (n 9 )# :he effects of pH, the concentration of

derivatizing reagent and reaction time at 7*'0* O" were examined# A solution - 61 was inDected

with split ratio *2- and the condition which gave maximum response, was considered optimum#

:he pH varied between -'-* at unit interval and it was observed that derivatization

occurred in acidic media (pH -') and a decrease into the response was observed above pH #

:he optimal response was obtained at pH (ig# -)# :he derivatizing reagent concentration

varied between -' m1 (- ; v/v) at an internal of *# m1# :he average peaC height/peaC area (n

9 ) was plotted against the amount of reagent solution added and a similar response was

obtained at the amount of - m1 and above, thus - m1 (- ; v/v) was used# Heating time varied

between to min at 7 O" and same average peaC height (n 9 ) was obtained at selected

heating time of -* to * min and - min#

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Fsing the conditions the hydrazine compound $H% was also examined# :he compounds

formed derivative with AA, eluted separately (ig# ) and did not affect the determination of INH

and H%# $H% was therefore used as internal standard# :he linear calibration curves at the

optimized conditions for the determinations of INH and H% were obtained by measuring average

peaC height/peaC area (n 9 ) with 5#'-** 6g/m1 and 7#-'7 6g/ m1 with coefficient of

determination (r ) *#


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II. Trifluoroacetylacetone:

INH and H% were conDugated easily with AA to form trifluoroacetylacetone'isonicotinyl

hydrazone and bis(trifluoroacetylacetone) hydrazone# :he presence of trifluoromethyl group in

AA could enhance the volatility and thermal stability of the conDugated# :herefore, AA was

selected as the derivatizing reagent for the selective and sensitive determination of INH and H%

by capillary column &"# $recolumn derivatization was carried out, and the elution was executed

from the capillary column H$- at an initial column temperature -**P" with heating rate *P"

up to 0*P"# :he run time was 7min# Nitrogen flow rate was - m1/min# !ach of the derivatives

INH and H% gave a single peaC with retention time of 5# min and #< min respectively# :he

derivatives were separated completely from the derivatizing reagent#

:he derivatization conditions were optimized for the @uantitative determination of INH

and H% by measuring average peaC height (n 9 )# :he effects of pH, the concentration of

derivatizing reagent and reaction time at 7*-0*P" were examined# A solution of - ?1 was

inDected with split ratio *2- and the condition, which gave maximum response, was considered

optimum# :he pH was varied between ---* at unit interval and was observed that derivatization

occurred in acidic media (pH -') and a decrease into the response was observed above pH #

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:he optimal response was obtained at pH (igure -)# :he derivatizing reagent concentration

was varied between -- m1 (-; v/v) at an internal of *# m1# :he average peaC height (n 9 )

was plotted against the amount of reagent solution added and a similar response was obtained at

the amount of - m1 and above, thus - m1 was used# Heating time was varied between to

min# at 7P" at an interval of min and same average peaC height (n 9 ) was obtained after

heating time of -* to * min and - min was selected#

Fnder the same conditions the hydrazine compound2 $H% was also examined# :he

compound formed derivative with AA eluted separately (igure ) and did not affect the

determination of INH and H%# $H% was therefore used as an internal standard# At the optimized

conditions, linear calibration curves for the simultaneous determination of INH and H% were

obtained by measuring average peaC height (n 9 ) with #' ?g/m1 INH and #'-# ?g/m1

H% with co'efficient of determination (r) *#


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IIIthyl Chloroformate:

Optimization of reaction conditions and separation: :he compounds INH, H% and $H% reacted

with !" to form volatile products , and eluted from a capillary &" column, each as a single

peaC# :he reaction was carried out in methanol, acetonitrile, a@ueousmethanol, a@ueous

acetonitrile and an a@ueous solution containing a pyridine base# A better &" response (averagepeaC height/peaC area) was observed using an a@ueous solution containing pyridine as the

reaction medium, and was selected# :he effect of the pH on the derivatization was examined

between - -* at unit interval# It was observed that derivatization occurred at pH values above

5, and the maximum response was observed at pH < (ig# -)# :he carbonate buffer was used

since it covered the pH range satisfactorily, as has been reported for amino'containing16


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compounds# :he reaction mixture was sonicated at room temperature (*.") for * min at an

interval of min, and the optimum response was observed within - min# "hloroform was used

for the extraction of derivatives, as reported for related compounds#- !thanolic solutions of the

derivatives were inDected with split'mode inDection on a &" column H$' (* m Q * mm i#d#),

and the elution and separation of INH, H% and $H% derivatives were examined# !ach compound

eluted as a symmetrical single peaC separately from the derivatizing reagent# "omplete

separation was obtained under the optimized conditions (gas chromatographic determination)#

:he resolution factor (Rs) between adDacent peaCs was R-#, and capacity factors kS, for the H%,

$H% and INH derivatives were calculated as -#, #-, and 7#-# :he H% derivative of !" eluted

first, followed by $H% and INH (ig# )#

Quantitation and validation: 1inear calibration curves obtained for INH and H% were # 7#

and # mg/ml, and the coefficients of the determinations (r2) were *# was found to be *# -#7 and *#5 #;# :est solutions of INH and

H% (n 9 3) were analyzed, and the relative ; error was obtained within - ;#

Sample analysis: :he determination of INH in pharmaceutical preparations was also examined#

Analyses of INH tablets and INH syrup did not encounter any difficulty# INH was extracted from

INH tablets and INH syrup with ethanolwater (-2-, v/v), and after derivatization was analyzed

by &"# :ablets of =emactal, =emactal (3*) and eyeth myambutanol contained rifampicin or

ethambutanol, together with INH# INH after its derivatization with !" was extracted in

chloroform, but ethambutanol remained in the a@ueous phaseK therefore it did not interfere with

the determination of INH# :he rifampicin, however, was extracted in chloroform together with

the INH derivative, but did not elute from the &" column, indicating on'column decomposition,

which disturbed the base line and the response of INH derivative# :hus, rifampicin was extracted

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in chloroform prior to the derivatization of INH with !"# A stable response was obtained, and

relative deviation from the labeled values of the manufacturer fell within 5#5; with an =8>

of - 3#; (n = ) (:able -)# :he recovery percentage of INH, calculated by the standard

addition method for INH syrup, was of ; ( n = )# :he amount of H% as the

decomposition product in INH syrup and a =emactal (3*) tablet was examined# H% was found

to be -#5 mg/-* mg INH and -3#7 mg H%/tablet with ** mg INH with an =8> of # #;#

:he concentration of H% in INH syrup and the =emactal (3*) tablet was below the proposed

safe limit of *#-;# or -*'fold addition of glucose, magnesium stearate, gum acacia, talcum,

methylparaben, lactose and starch to INH did not affect its determination# Now, comparing the

results with the reported H$1" method with FB detection, which indicated a linear calibration

range within *# 0#* mg/ml INH with a *'ml loop inDector with an analyses time of - min, as

compared to the present method with linear calibration of # 7# mg/ml INH with - ml

inDection with a split ratio of -*2-, v/v and an analyses time of -- min# :he &" procedure is also

free from the problem of safe disposal the used mobile phase#

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CONCLUSION:

8imple &" procedure has been developed for the determination of INH from

pharmaceutical preparations and blood samples after precolumn derivatization with AA, AA,

!"# H% was also determined from INH formulations and $H% was as internal standard# or first

method using AAK detection limits were obtained at sub'ng level reaching to the detector# :he

analysis of pharmaceutical preparation was obtained with =8> -';# or second method using

AK detection limits were obtained at 5# pg reaching to the detector# :he analysis of

pharmaceutical preparations and blood samples after chemotherapy were observed with =8>

#'#0;# :he last method that is using !"K detection limit observed for H% and INH

corresponded to *#-7 and *#-0 ng reaching the I> detector# :he analyses of pharmaceutical

preparations were observed with an =8> of - 3#;#

RE>ERENCES:

-# E#U#huhawar, 1#A#%ardari and A#L 1aghari, V"apillary &as "hromatographic >etermination

of Isoniazid in $harmaceutical preparation by $re'column >erivatization with AcetylacetoneV#

Available at2 http://www.asianjournalofcheistr!.co.in/"ser/#earch$rticle.asp%&

'olue()*ssue()$rticle()+riteria(+apillar!,20-as,20+hroatoraphic

,20eterination,20of,20*soniai,20in,20haraceutical,20reparation

,20!,20recolun,20eriatiation,20with,20$cet!lacetone

2. E#U#huhawar, and 1#A#%ardari, V"apillary &as "hromatographic >etermination of Isoniazid

in $harmaceutical preparation and blood by $re'column >erivatization with

:rifluoroacetylacetoneV# Available at2

http://www.fa.o.tw/les/pulishperioical/28.pf

20
http://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.fda.gov.tw/files/publish_periodical/2_8.pdfhttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.asianjournalofchemistry.co.in/User/SearchArticle.aspx?Volume=&Issue=&Article=&Criteria=Capillary%20Gas%20Chromatographic%20Determination%20of%20Isoniazid%20in%20Pharmaceutical%20Preparation%20by%20Pre-column%20Derivatization%20with%20Acetylacetonehttp://www.fda.gov.tw/files/publish_periodical/2_8.pdf


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Paper-Capillary Gas Chromatographic Determination of Isoniazid and Hydrazine in Pharmaceutical Preparations and Blood