paper-capillary gas chromatographic determination of isoniazid and hydrazine in pharmaceutical...
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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
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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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