ABSTRACT:

Antituberculosis drug Isoniazid (INH) and toxic substance are determined by capillary
gas chromatography after precolumn derivatization with acetylacetone (AA),
trifluoroacetylacetone (FFA), ethyl chloroformate (ECF). Phenylhydrazine (PHZ) was used as an
internal standard. GC separation was carried on HP-5 column (30m X 0.32mm i.d) with flame
ionization detection. For AA, elution was carried out at initial column temperature 120C with
heating rate 50C/min upto 280 C, with nitrogen flow rate of 1.5 mL/min and split ratio was
20:1v/v. For ECF, initial column temperature of 150C for 1 min at a heating rate of 10C/min up
to 250C, nitrogen flow rate of 4 ml/min and a split ratio of 10:1, v/v. By using AA, the linear
calibration range for INH was observed 6.2-100 g/mL with detection limit of 3.1 g/mL
corresponding to 150pg reaching to the detector, similarly linear calibration curve for hydrazine
was 7.1-57 g/mL with detection limits of 2.3 g/mL. By using FAA, linear calibration ranges
for INH and HZ were determined to be 2.5-25 g/mL and 2.5- 21.2 g/mL respectively, with
detection limit (S/N = 3) corresponding 62.5 pg reaching to the detector. Finally by using TCF,
linear calibration ranges for INH and HZ were observed between 3.5 37.5 and 3.5 35mg/ml
with corresponding detection limits of 0.18 and 0.17 ng reaching the detector.
The 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 1.01.4%. The amount of recovery of INH from pharmaceutical preparations was 97% with RSD 3%.
The second method, using FAA applied for the determination of INH and HZ from
pharmaceutical preparations. The method was also used for determination of INH from blood of
tuberculosis patient. The amount of INH found in the blood serum of tuberculosis patients on
chemotherapy with INH based drugs was within 0.82-4.8 g/mL with relative standard deviation
(RSD) of 2-5.8%. The recovery percentage of INH from blood samples was found 98% with
RSD 2.5%. The last method was was subsequentlyapplied to the determination of INH and HZ in
pharmaceutical preparations, achieving a relative standard deviation (RSD) of 3.8 5.8%. The
recovery percentage of INH from isoniazid syrup was 98% with an RSD of 5.2%.

INTRODUCTION:
1

INH (isonicotinoylhydrazine) is the most potent and selective tuberculostic antibacterial

agent in the therapy of tuberculosis. It inhibits the growth of tubercle bacillus in vitro in
concentration less than 1 g/mL. It is also employed as a propylactic agent for use in persons
constantly exposed to tubercular patients. The INH gains access to all organs and all body fluids
including cerebrospinal, renders the drug of special value in treating tuberculosis meningitis and
other extra pulmonary forms of the disease. When used alone, it is at least equal to streptomycin
in the therapy of tuberculosis. It is believed to affect lipids, nucleic acids, glycolysis or mycolic
acid biosynthesis. The hydrazine (HZ) is a toxic substance and may be present in pharmaceutical
preparations of INH as one of the decomposition products.(1,2,3)

Various analytical techniques such as titrimetry , spectrophotometry, spectrofluorimetry,

atomic absorption, chemiluminescence, electroanalytical techniques, kinetic determination, flow
injection, thin layer chromatography, capillary electrophoresis, liquid chromatography (LC) and
LC-Mass spectrometry have been used for quantitation of INH in pharmaceutical preparations
and biological samples. The LC with UV detection is either carried out by measuring the natural
absorbance of INH at 263 nm or by precolumn derivatization with a suitable derivatizing
reagent. The determination limits for INH has been reported within 0.5-8.0 g/mL. The gas
chromatography (GC) of INH was carried out after derivatization with trifluoroacetic anhydride
or bis(trimethylsilyl)trifluoroacetamide or trifluoroacetone or ethyl chloroformate (ECF) and was
quantified by flame ionization detection (FID) or mass spectrometry. The use of acid anhydride
affected the performance of GC column.(1,2,3)

Acetylacetone (AA) is inexpensive chemical and could react with primary amino group
to form stable Schiff base with increase in carbon number of derivative and increase in FID
sensitivity. Acetylacetone has been used for HPLC determination of dopamine. Present work
examines capillary GC for the determination of isoniazid and hydrazine after derivatization with
acetylacetone. The volatile derivatives are easy to elute and separate from capillary GC column
with required sensitivity for the analysis of pharmaceutical preparations.(1)

-diketone reacted with amino compounds to form Schiff bases and addition of carbon
chain could enhance FID sensitivity. FAA has been used for GC determination of putresine and
cadverine from biological fluids. The present work examines GC determination of INH and HZ
after derivatization with FAA(trifluoroacetylacetone) using PHZ (Phenylhydrazine) as an internal
standard. The volatile derivatives are easy to elute and separate from capillary GC column with
required sensitivity for the desired application for the analysis of biological fluids.(2)

Alkyl chloroformates have been used as derivatizing reagents for gas chromatographic
(GC) determinations of amines, amino acids, amino alcohol and acids. Husek has reviewed
chloroformates in GC as a general-purpose derivatizing reagent. Husek and Simek have
reviewed alkyl chloroformates in sample derivatization strategies for GC analysis on a decade
use as an esterifying reagent. Quantitation was carried out within the limits of 2.5 25 mg/ml.
The use of a simple and inexpensive derivatizing reagent for the GC determination of INH and
HZ from a pharmaceutical preparation could be of analytical interest for quality control. The
present work examined the capillary GC determination of INH and HZ using PHZ as an internal
standard after derivatization with ethyl chloroformate (ECF) with FID. The reagent ECF may
prove to be an additional GC reagent for the selective determination of INH and HZ.(3)

EXPERIMENTAL:
I. Materials and Methods:

All the chemicals used were of reagent or pharmaceutical grade. Freshly prepared double
distilled water was used throughout the study. Pure INH was obtained from Nabi Qasim
Pharmaceuticals, Karachi, Pakistan. HZ (24%), PHZ, FAA and methanol were purchased from E.
Merck, Darmstadt, Germany. The percentage of HZ in HZ solution was determined by the
titrimetry.

(1,2,3)

Hydrochloric acid (37%), potassium chloride, acetic acid, sodium acetate,

ammonium acetate, sodium bicarbonate, sodium carbonate, ammonium chloride were also
obtained from E.Merck Germany.(1,2) Buffer solutions in the pH range 1-10 at unit interval were
prepared from hydrochloric acid (0.1 M) and potassium chloride (1 M) (pH 1 &2), acetic acid (1
M) and sodium acetate (1 M) (pH 3 to 6); ammonium acetate (pH 7), sodium bicarbonate (1 M)
and sodium carbonate (saturated) (pH 8 & 9), ammonium chloride (1 M) and ammonia (1 M)
(pH 10) also sodium hydroxide (pH 12) in ECF derivatization. (1,2,3) Freshly prepared INH
solution was prepared by dissolving 10.95 mg INH in methanol: water (1:1 v/v) and final volume
was adjusted to 10 mL. Acetylacetone e (1 % v/v) was prepared in 100 mL of ethanol:water (1:1
v/v).(1) FAA (1% v/v) was prepared in 100 mL of methanol: water (1:1 v/v).(2)
The spectrophotometric studies were carried out on double beam Hitachi 220 (Hitachi
(Pvt) Ltd. Tokyo, Japan) spectrophotometer with dual 1 cm cells. (1,2) GC studies were carried out
on Agilent model 6890 Network GC system (Agilent Technologies Inc. USA) with split/splitless
injector operated in split mode coupled with flame ionization detection (FID), hydrogen
generator (Parker Balston, Analytical Gas system H2-90, Parker Hannifin, Havorhill, MA, USA)
and pure nitrogen (British Oxygen Company, Karachi). The computer with Chemstation software
controlled the gas chromatograph was used throughout the study. Capillary column HP-5 (30 m
0.32 mm I.D) with film thickness of 0.25 m (J & W scientific GC columns, USA) was used
throughout the study. The Orion model 420A pH meter (Orion Research Inc. Boston, USA) with
glass electrode with combined reference electrode was used for pH measurements.(1,2,3)

II Gas Chromatographic Determination: using different derivatizing agents (AA, FFA, ECF)
Acetylacetone

Trifluoroacetylacetone

Ethyl Chloroformate

1. The solution(1 mL) containing

1. The solution (1 mL) containing I

1. To a solution (15 ml) containing

INH(2-40 g), HZ (2-22 g) and

NH(1-20 g), HZ(1-17 g) and

INH (1.515 mg), HZ (1.414.0 mg)

PHZ (10.9 g) was added 1 mL of

PHZ(5 g) was added 1 mL of

and PHZ (23.2 mg) was added a 0.4-

potassium chloride hydrochloric acid

potassium chloride-hydrochloric acid ml pyridine solution, followed by 0.5

buffer pH 2, 1 mL(1 % v/v) AA and

buffer pH 2, 1 mL of (1% v/v) FAA

ml of pure ECF and 1 ml of 1M

heated at 75 C for 15 min. The

and heated at 75C for 15 min. The

carbonate solution, pH 9.The

contents were cooled at room

solution was cooled at room

contents were sonicated for 15 min,

temperature and was added 1 mL of

temperature and were added 1mL of

and chloroform (0.5 ml) was added.

chloroform. Contents were mixed

chloroform. Contents were mixed

The mixture was mixed well and

well, layers were allowed to separate. well, layers were allowed to separate. layers were allowed to separate.
2. Exactly 0.5 mL of chloroform was 2. Exactly 0.5 mL of chloroform was 2. Then, 0.50 ml of extract layer (1
pipetted out and transferred to screw

pipetted out and transferred to screw

ml) was pipetted out to a screw-

capped vial. Solvent was evaporated

capped vial. Solvent was evaporated

capped vial. Solvent was evaporated

& re-dissolved in 0.2 mL of ethanol.

3. The solution (1 L) was injected

re-dissolved in 0.2 mL of methanol.

3. The solution (1 L) was injected

and redissolved in 0.2 ml of ethanol.

3. The solution (1 ml) was used and

on capillary GC column HP-5 (30 m

on capillary GC column HP-5 at a

eluted from a capillary GC column

0.32 mm id) with layer thickness

column temperature of 100C with

HP-5 at a column temperature of

0.25 m at column temperature 120

heating rate 30C up to 280C.

150C for 1 min with a programmed

C with programmed heating rate

The total run time was 7 min and

heating rate of 10C/min upto 250C

50C/min upto 280 C with total run

nitrogen flow rate was 1 mL/min.

with a total run time 11 min. The

time 6.2 min. The nitrogen flow rate

The split ratio was 20:1.

nitrogen flow rate was maintained at

was maintained at 1.5 mL/min

4. Injection port and detector

4. Injection port and detector

4ml/min.
4. Injection port and detector

temperatures were fixed at 200 C &

temperatures were fixed at 200C

temperatures were fixed at 250 and

300 C. Hydrogen flow rate and

and 300C respectively. Hydrogen

300C. Hydrogen and nitrogen flow

nitrogen as make up gas flow rate

and nitrogen flow rates were fixed

rates were fixed at 40 and 45 ml/min

were fixed at 40 and 45 L/min, FID

at 40 and 45 mL/min for FID det.
for FID detection. Split ratio 10:1v/v.
III. Determination of Isoniazid in Pharmaceutical Preparations:using different derivatizing
agents (AA, FFA, ECF)
Acetylacetone

Trifluoroacetylacetone

Ethylchloroformate

1.Ten tablets of each Remactal & Remister (Novartis Pharma

(Pak.) Ltd.) and Myrin P (Leaderle Laboratories Division,
Cyanamid Pak. Ltd. Karachi) were powdered and 53.89 mg
Remactal, 55.90 mg Remistar and 56.12 mg Myrin P were

1. 53.89 mg Remactal,
53.90 mg Remistar
and 57.12 mg Myrin P
methanol:water (1:1).

1. 60 mg Remactal,
90 mg Weyeth
Myambutaol, 90 mg
Remactal (450)
ethanol:water (1:1)

dissolved in 3 portions of each (6 mL) in ethanol:water (1:1)

2. The solution was filtered and final volume was adjusted to

2. adjusted to 50 mL

2. adjusted to 50 mL

25 mL with ethanol:water (1:1 v/v). Solution (1 mL) was

with methanol:water

with ethanol:water

transferred to screwcapped vial and 1 mL of chloroform was

(1:1 v/v)

(1:1 v/v)

3. 5 g of PHZ

3. PHZ (23.2 g).

4. powder (51 mg)

dissolved in
ethanol:water (1:1
v/v).

4. powder (80 mg)

dissolved in
ethanol:water (1:1
v/v).

added. Contents were mixed well and the layers were allowed
to separate.
3. The aqueous layer was collected and the analytical GC
procedure II was followed by addition of PHZ (10.09 g).
4. Ten tablets of isoniazid (Unexo Lab. (Pvt) Ltd. Lahore) were
thoroughly ground and a powder (50 mg) was dissolved in
ethanol:water (1:1 v/v). Solution was filtered and the final
volume was adjusted to 100 mL. 1mL was further diluted to 25
mL. Solution (1 mL) was taken and the GC determination was

GC deter.5 g of PHZ
as internal standard.

followed.
5. 5ml of well-mixed INH syrup was dissolved in
ethanol:water (1:1 v/v),filtered & volume was adjusted to 100
mL. Solution (1 mL) was again diluted to 25 mL and solution
(1 mL) was added PHZ (10.9 g) and processed as analytical
procedure II. Amounts of INH were evaluated from ratio of the
peaks of analyte internal standard & external calibration curve.

5. 5ml dissolved in
methanol:water (1:1
v/v)

5. 1ml dissolved in
ethanol:water (1:1
v/v)

Further diluted to 50
mL

Further diluted to
100 mL

PHZ is not added here

PHZ (23.2 g)

AA: Percentage recovery of isoniazid from INH tablets by spiked sample:

Isoniazid tablets were processed as determination of isoniazid in pharmaceutical
preparations and two portion of solution (1.0 mL each) were taken. A portion was processed as
GC determination and the other portion was added INH (25 g) and again processed as GC
determination. Linear calibration was used to measure an increase in the response with added
INH and % recovery of INH from isoniazid tablets solution was calculated.(1)
6

ECF: Determination of Isoniazid from INH syrup by standard addition method:

Isoniazid syrup was processed according to the procedure, and two portions of the
solution (1.0 ml each) were taken. A portion was processed and to the other portion was added
12.5 mg INH. Quantitation was carried out from a linear calibration, and an increase in the
response for INH was used to calculate the percent recovery of INH from an INH tablet solution.
(3)

FFA: Determination of Isoniazid from Blood samples:

Blood sample (5 mL) of various patients suffering from pulmonary tuberculosis was
collected 2-4 hr after the administration of the drug. The samples were collected from the
patients by vinipuncture with disposable syringe from Tuberculosis Hospital, Kotri. The blood
samples, was incubated for 1 hr at (30C) and centrifuged at 3000 g for 10 min. The supertenant
layer was collected and was added methanol twice in the volume. The mixed contents were
centrifuged at 3000 g for 10 min. The supertnant was transferred into a vial and 2 mL of
chloroform was added. The contents were mixed well and layers were allowed to separate. The
upper layer was pipetted out into vial and PHZ 5 g was added. The derivatization procedure II.
then was followed.(2)

FFA: Recovery of Isoniazid from Blood samples:

Blood samples (5 mL) collected from a healthy volunteer was processed as above and
blood serum was added INH (6 g) and processed as above using FAA as derivatizing reagent.
The amount of INH was calculated from external calibration curve and internal standard.
III Determination of Hydrazine from Isoniazid Formulations:
Acetylacetone

Trifluoroacetylacetone

Ethyl Chloroformate

1. Wellmixed INH 2 mL containing

1. 0.5 mL of well-mixed INH syrup

1. Well-mixed INH syrup (2 ml)

(10 mg/mL) INH was processed as

containing (5 mg) INH was

containing (10 mg/ml) INH was

GC determination

processed as analytical procedureII.

dissolved in ethanolwater (1:1,

v/v), 5 ml, and the whole solution
was processed, and the response for
HZ was noted. The amount of HZ
was evaluated through an external
calibration curve

2. Ten tablets of isoniazid (INH)

2. Ten tablets of isoniazed were

2. Ten tablets of Remactal (450)

were thoroughly ground and a

thoroughly ground the powder (1g,

containing 300 mg of INH tablet

powder (1.2 g, containing 654.6 mg

containing 545.5 mg INH) was

were thoroughly ground, and the

INH) was dissolved in ethanol:

dissolved in methanol: water (1:1

powder (0.5 g containing 187.5 mg

water (1:1 v/v) and the solution was

v/v) and the solution was filtered

INH) was dissolved in ethanol

filtered and adjusted to 25 mL.

and adjusted to 25 mL.

water (1:1, v/v) and the solution

filtered and adjusted to 10 ml.

3. The solution 2 mL was taken and

3. The solution 1 mL was taken and

3. A solution of 3 ml was taken and

PHZ (10.9 g) was added and the

the analytical procedure II was

the analytical procedure II was

GC determination was followed.

followed. The signals

followed.

The signals corresponding to HZ

corresponding to HZ and PHZ were

and PHZ were recorded.

recorded.

RESULTS AND DISCUSSION:

I. Acetylacetone:

Isoniazid (INH) condenses easily with acetylacetone (AA) to form acetylacetoneisonicotinyl hydrazone. For the selective and sensitive determination of INH and hydrazine (HZ)
capillary column gas chromatography was examined. Precolumn derivatization was carried out
with AA and elution was examined from the capillary column HP-5 (30 m 0.32 mm) with layer
thickness 0.25 m at an initial column temperature 120 C with heating rate 50 C/min up to 280
C/ min. The run time was 6.2 min. Nitrogen flow rate was 1.5 mL/min. The detection was
performed by FID. The derivatives of HZ and INH gave a single peak with retention times of 2.4
and 4.30 min. The derivatives separated completely from the derivatizing reagent AA. The
derivatization conditions were optimized for the quantitative determination of INH and HZ by
measuring average peak height/peak area (n = 3). The effects of pH, the concentration of
derivatizing reagent and reaction time at 70-80 C were examined. A solution 1 L was injected
with split ratio 20:1 and the condition which gave maximum response, was considered optimum.

The pH varied between 1-10 at unit interval and it was observed that derivatization
occurred in acidic media (pH 1-3) and a decrease into the response was observed above pH 3.
The optimal response was obtained at pH 2 (Fig. 1). The derivatizing reagent concentration
varied between 1-3 mL (1 % v/v) at an internal of 0.5 mL. The average peak height/peak area (n
= 3) was plotted against the amount of reagent solution added and a similar response was
obtained at the amount of 1 mL and above, thus 1 mL (1 % v/v) was used. Heating time varied
between 5 to 25 min at 75 C and same average peak height (n = 3) was obtained at selected
heating time of 10 to 20 min and 15 min.

Using the conditions the hydrazine compound PHZ was also examined. The compounds
formed derivative with AA, eluted separately (Fig. 2) and did not affect the determination of INH
and HZ. PHZ was therefore used as internal standard. The linear calibration curves at the
optimized conditions for the determinations of INH and HZ were obtained by measuring average
peak height/peak area (n = 3) with 6.2-100 g/mL and 7.1-57 g/ mL with coefficient of
determination (r 2) 0.9904 and 0.9939, respectively. The detection limits measured as three time
the background noise were obtained with INH 3.1 g/mL and HZ 2.3 g/mL corresponding to
3.1 ng and 2.3 ng/injection (1 L) and 0.15 and 0.12 ng reaching up to FID detection. Common
additives glucose, magnesium stearate, gum acacia, talcum, methylparabin, lactose and starch
when added twice the concentration of INH (109 g/mL) did not interfere. The method was
applied for the determination of INH from pharmaceutical preparations viz., Ramactal, Remister,
Myrin P, isoniazid tablets and isoniazid syrup. The results are summarized in Table-1. The
isoniazid tablets and isoniazid syrup were analyzed after dissolution of INH in ethanol:water (1:1
v/v). The tablets Remactal, Remister and Myrin P also contained rifampicin, pyrazinamide and
ethambutal together with INH. INH after derivatization with AA was extracted in chloroform and
pyrazinamide and ethambutol did not elute from GC column and did not interfere the
10

determination of INH. Rifampicin is extracted in chloroform together with INH derivative, but
did not elute as symmetrical peak and disturbs the base line due to on column decomposition.
However rifampicin separated completely, when extracted in chloroform prior to derivatization
of INH in aqueous-methanolic solution and did not affect the determination of INH. The %
relative deviations (RD) were obtained 0.66 to 4.0 % from the values labeled by the
manufacturer with relative standard deviation (RSD) within 1-3 % (Table-1).

The % of INH recovered from pharmaceutical preparations by standard addition was

calculated and the amount of recovery was 97 % with RSD 3 %. (Figs. 3 & 4). The presence of
HZ was checked in isoniazid syrup and isoniazid tablets. Isoniazid syrup 2 mL containing 10
mg/mL of INH and isoniazid tablets 1.2 g containing 654.6 mg INH were analyzed for HZ.

11

12

II. Trifluoroacetylacetone:
INH and HZ were conjugated easily with FAA to form trifluoroacetylacetone-isonicotinyl
hydrazone and bis(trifluoroacetylacetone) hydrazone. The presence of trifluoromethyl group in
FAA could enhance the volatility and thermal stability of the conjugated. Therefore, FAA was
selected as the derivatizing reagent for the selective and sensitive determination of INH and HZ
by capillary column GC. Precolumn derivatization was carried out, and the elution was executed
from the capillary column HP-5 at an initial column temperature 100C with heating rate 30C
up to 280C. The run time was 7min. Nitrogen flow rate was 1 mL/min. Each of the derivatives
INH and HZ gave a single peak with retention time of 6.5 min and 3.9 min respectively. The
derivatives were separated completely from the derivatizing reagent.
The derivatization conditions were optimized for the quantitative determination of INH
and HZ by measuring average peak height (n = 3). The effects of pH, the concentration of
derivatizing reagent and reaction time at 70-80C were examined. A solution of 1 L was
injected with split ratio 20:1 and the condition, which gave maximum response, was considered
optimum. The pH was varied between 1-10 at unit interval and was observed that derivatization
occurred in acidic media (pH 1-3) and a decrease into the response was observed above pH 3.
13

The optimal response was obtained at pH 2 (Figure 1). The derivatizing reagent concentration
was varied between 1-3 mL (1% v/v) at an internal of 0.5 mL. The average peak height (n = 3)
was plotted against the amount of reagent solution added and a similar response was obtained at
the amount of 1 mL and above, thus 1 mL was used. Heating time was varied between 5 to 25
min. at 75C at an interval of 5 min and same average peak height (n = 3) was obtained after
heating time of 10 to 20 min and 15 min was selected.
Under the same conditions the hydrazine compound: PHZ was also examined. The
compound formed derivative with FAA eluted separately (Figure 2) and did not affect the
determination of INH and HZ. PHZ was therefore used as an internal standard. At the optimized
conditions, linear calibration curves for the simultaneous determination of INH and HZ were
obtained by measuring average peak height (n = 3) with 2.5-25 g/mL INH and 2.5-21.2 g/mL
HZ with co-efficient of determination (r2) 0.9806 and 0.9827 respectively using (n = 5) points
calibration. The detection limits measured as signal to noise ratio 3:1 were obtained with 1.25
g/mL for INH and HZ corresponding to 62.5 pg reaching to the detector with split ratio 20:1.
Common additives glucose, magnesium stearate, gum acacia, talcum, methylparaben,
lactose and starch, when added twice the concentration of INH and HZ, did not interfere. The
method was applied for the determination of INH from pharmaceutical preparations: Ramactal,
Remister, Myrin, INH tablets and INH syrup. The hydrazine was determined from INH tablets
and INH syrup. The GC results are summarized in Table 1 and 2. The INH tablets and INH syrup
were analyzed after dissolution of INH in methanol-water (1:1 v/v) (Figure 3). The tablets
Remactal, Remster and Myrin contained rifampicin, pyrazinamide and ethambutal together with
INH. INH after derivatization with FAA was extracted in chloroform and pyrazinamide and
ethambutol remained in aqueous phase and did not interfere the determination of INH.
Rifampicin is extracted in chloroform together with INH derivative, but did not elute as
symmetrical peak, and disturbed the base line due to on column decomposition. However
rifampicin separated completely, when extracted in chloroform prior to derivatization of INH in
aqueous-methanolic solution and did not affect the determination of INH. The relative
percentage deviations were found 1 to 6.7% from the values labeled by the manufacturer with
relative standard deviation within 4.0-8.5% (Table 1) The RSD is slightly on higher side because
of the presence of multi- component system. HZ was checked for its presence in INH syrup and
14

INH tablets. INH syrup containing 5 mg of INH and INH tablets containing 18.18 mg INH were
analyzed for HZ and PHZ was added as internal standard. The signals corresponding to HZ, and
PHZ were recorded. The Table 2 indicated the presence of 1.0 g HZ/5 mg INH in INH syrup
and 5 g HZ/18.18mg INH in INH tablets. Blood samples of patients suffering from tuberculosis
were collected after 2-4 hr of chemotherapy with a single dose of 300 mg of INH. The blood
sample after deproteinization was extracted with chloroform to remove the presence of any
rifampicin. The INH after derivatization with FAA was determined. The amount of INH in the
blood was observed within 0.82-4.8 g/mL with RSD 2.0-5.8% (Table 3). The recovery
percentage of INH from the blood was examined by spiking the serum of healthy volunteer with
INH and the amount of INH recovered was evaluated from calibration curve. The amount of
recovery was 98% with RSD 2.5%. (Figure 4)

15

III Ethyl Chloroformate:

Optimization of reaction conditions and separation: The compounds INH, HZ and PHZ reacted
with ECF to form volatile products , and eluted from a capillary GC column, each as a single
peak. The reaction was carried out in methanol, acetonitrile, aqueousmethanol, aqueous
acetonitrile and an aqueous solution containing a pyridine base. A better GC response (average
peak height/peak area) was observed using an aqueous solution containing pyridine as the
reaction medium, and was selected. The effect of the pH on the derivatization was examined
between 1 10 at unit interval. It was observed that derivatization occurred at pH values above
6, and the maximum response was observed at pH 9 (Fig. 1). The carbonate buffer was used
since it covered the pH range satisfactorily, as has been reported for amino-containing
16

compounds. The reaction mixture was sonicated at room temperature (30C) for 5 20 min at an
interval of 5 min, and the optimum response was observed within 15 min. Chloroform was used
for the extraction of derivatives, as reported for related compounds.1 Ethanolic solutions of the
derivatives were injected with split-mode injection on a GC column HP-5 (30 m 320 mm i.d.),
and the elution and separation of INH, HZ and PHZ derivatives were examined. Each compound
eluted as a symmetrical single peak separately from the derivatizing reagent. Complete
separation was obtained under the optimized conditions (gas chromatographic determination).
The resolution factor (Rs) between adjacent peaks was >1.5, and capacity factors k, for the HZ,
PHZ and INH derivatives were calculated as 1.3, 5.1, and 7.1. The HZ derivative of ECF eluted
first, followed by PHZ and INH (Fig. 2).
Quantitation and validation: Linear calibration curves obtained for INH and HZ were 3.5 37.5
and 3.5 35 mg/ml, and the coefficients of the determinations (r2) were 0.9985 and 0.9958,
respectively. The linear regression equations were y = 0.3883x + 0.4126 and y = 0.4653x +
0.4195, respectively. The limits of detection (LOD) measured with a signal-to-noise ratio of 3:1
were 1.87 and 1.75 mg/ml, corresponding to 0.18 and 0.17 ng for INH and HZ, respectively,
reaching the detector. The reproducibility was checked by targeting the elution of a threecomponent mixture of HZ, PHZ and INH in terms of the elution time and the peak height (n = 6).
The relative standard deviation (RSD) turned out to be within 0.3 1.4 and 0.4 2.1%,
respectively. Intra-day variations (n = 4), against in terms of the elution time and the peak height,
were measured and the RSD was found to be 0.5 1.7 and 0.6 2.3%. Test solutions of INH and
HZ (n = 4) were analyzed, and the relative % error was obtained within 1 2%.
Sample analysis: The 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 (1:1, v/v), and after derivatization was analyzed
by GC. Tablets of Remactal, Remactal (450) and Weyeth myambutanol contained rifampicin or
ethambutanol, together with INH. INH after its derivatization with ECF was extracted in
chloroform, but ethambutanol remained in the aqueous phase; therefore it did not interfere with
the determination of INH. The rifampicin, however, was extracted in chloroform together with
the INH derivative, but did not elute from the GC column, indicating on-column decomposition,
which disturbed the base line and the response of INH derivative. Thus, rifampicin was extracted
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in chloroform prior to the derivatization of INH with ECF. A stable response was obtained, and
relative deviation from the labeled values of the manufacturer fell within 2 6.6% with an RSD
of 1 4.2% (n = 3) (Table 1). The recovery percentage of INH, calculated by the standard
addition method for INH syrup, was 98% with an RSD of 2% (n = 3). The amount of HZ as the
decomposition product in INH syrup and a Remactal (450) tablet was examined. HZ was found
to be 1.6 mg/10 mg INH and 14.7 mg HZ/tablet with 300 mg INH with an RSD of 2.5 3.5%.
The concentration of HZ in INH syrup and the Remactal (450) tablet was below the proposed
safe limit of 0.1%. For 10-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 HPLC method with UV detection,32 which indicated a linear calibration
range within 0.5 8.0 mg/ml INH with a 50-ml loop injector with an analyses time of 15 min, as
compared to the present method with linear calibration of 3.5 37.5 mg/ml INH with 1 ml
injection with a split ratio of 10:1, v/v and an analyses time of 11 min. The GC procedure is also
free from the problem of safe disposal the used mobile phase.

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CONCLUSION:
Simple GC procedure has been developed for the determination of INH from
pharmaceutical preparations and blood samples after precolumn derivatization with AA, FAA,
ECF. HZ was also determined from INH formulations and PHZ was as internal standard. For first
method using AA; detection limits were obtained at sub-ng level reaching to the detector. The
analysis of pharmaceutical preparation was obtained with RSD 1-3%. For second method using
FFA; detection limits were obtained at 62.5 pg reaching to the detector. The analysis of
pharmaceutical preparations and blood samples after chemotherapy were observed with RSD
3.2-5.8%. The last method that is using ECF; detection limit observed for HZ and INH
corresponded to 0.17 and 0.18 ng reaching the FID detector. The analyses of pharmaceutical
preparations were observed with an RSD of 1 4.2%.

REFERENCES:
1. M.Y.Khuhawar, L.A.Zardari and A.J Laghari, "Capillary Gas Chromatographic Determination
of Isoniazid in Pharmaceutical preparation by Pre-column Derivatization with Acetylacetone".
Available at: 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%20Acetylacetone
2. M.Y.Khuhawar, and L.A.Zardari, "Capillary Gas Chromatographic Determination of Isoniazid

in Pharmaceutical preparation and blood by Pre-column Derivatization with

Trifluoroacetylacetone". Available at:
http://www.fda.gov.tw/files/publish_periodical/2_8.pdf

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3. M.Y.Khuhawar, and L.A.Zardari, "Ethyl Chloroformate as a Deivatizing reagent for the Gas
Chromatographic Determination of Isoniazid and Hydrazine in Pharmaceutical preparations".
Available at: http://www.ncbi.nlm.nih.gov/pubmed/18997381

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