GC-ECD Determination of Lindane

and Its Impurity a-HCH in Pharmaceutical

Products
Zorica Vujic
1
, Silvana Matic
2
, Vesna Kuntic
1
, Sote Vladimirov
1
, Snezana Uskokovic-Markovic
1,&
1
Faculty of Pharmacy, University of Belgrade, P.O.Box 146, 11000 Belgrade, Serbia; E-Mail: snezaum@pharmacy.bg.ac.rs
2
Hemofarm Concern, Zorka Pharma, S

abac, Serbia
Received: 30 March 2010 / Revised: 15 June 2010/ Accepted: 30 June 2010
Online publication: 21 July 2010
Abstract
A method was developed to determine lindane (c-HCH), the widely used organochlorine
pesticide, and its impurity a-HCH in bulk and pharmaceutical products (shampoo, gel and
emulsion). The system was able to successfully resolve the lindane peak from a-HCH and
interfering components. The method displays an excellent linearity over the concentration
range 0.515.0 lg mL
-1
for lindane and 0.0050.15 lg mL
-1
for a-HCH and a precision
better than 2.5% from intra- and inter-day analyses. LOQ and LOD were determined to be
0.45 and 0.15 lg mL
-1
for lindane and 0.005 and 0.002 lg mL
-1
for a-HCH.
Keywords
Gas chromatography-electron capture detection
Pharmaceutical products
Shampoo, gel and emulsion
Lindane
Introduction
Lindane, C
6
H
6
Cl
6
, the common name
for the c-isomer of hexachlorocyclo-
hexane (HCH), belongs to the organo-
chlorine pesticide class [1]. Being an
insecticide, larvicide and acaricide, lin-
dane is used topically for the treatment
of scabies and pediculosis in patients
[2]. As an active substance in pharma-
ceutical products, lindane is obtained
from lindane raw bulk which is ac-
quired from technical grade HCH.
Technical HCH contains eight isomers,
but the a-isomer (a-HCH) is a major
constituent and consequently the main
manufacturing impurity in lindane raw
bulk. Also, a-HCH can be realised as a
degradation product in lindane bulk
under improper storage conditions.
a-HCH has no insecticidal properties,
but shows signicant carcinogenic,
neurotoxic and hepatotoxic eects on
humans [3].
Gas chromatography (GC), com-
bined with electron capture detection
(ECD) and mass spectrometry (GC
MS), is the technique usually employed
to determine HCH isomers in human
liquids and tissues, food and milk, plant
materials and environmental samples
[48]. Although the list of the GC-ECD
methods that are available for detecting,
measuring, and/or monitoring HCH
isomers in dierent matrices is extensive
and exceed the number of stated refer-
ences, there is no data concerning the
simultaneous determination of lindane
and a-HCH in pharmaceutical dosage
forms. Thus, the principal goal of this
investigation was to set up the optimal
chromatographic conditions for the
separation and determination of both
isomers (c- and a-HCH) in lindane bulk
and dosage forms.
Experimental
Instrumentation
and Conditions
Shimadzu GC-9A chromatograph
equipped with integrator Data Apex
CSW 32, capillary glass column, 3.0 m
length 9 0.3 mm i.d 9 2.5 lm lm
2010, 72, 581584
DOI: 10.1365/s10337-010-1684-9
0009-5893/10/09 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH
Limited Short Communication Chromatographia 2010, 72, September (No. 5/6) 581
thickness, with stationary phase 10%
OV-1 on Chromosorb W support was
used. The detection was performed by
means of ECD, Ni
63
Nuclide Quantity
370 MBq. Oven temperature: 230 C,
injector temperature: 280 C, detector
temperature: 280 C. The nitrogen car-
rier gas ow: 30 mL min
-1
.
Chemicals and Solvents
Analytical standards of lindane and
a-HCH were obtained from Fluka
(Buchs, Switzerland). Lindane bulk was
obtained from Rallis India (Bombay,
India). The formulated products: Gamex
shampoo (1 mL containes 10 mg of
lindane), Gamex gel (1 g contains 3 mg)
and Gamex emulsion (1 mL contains
3 mg) were from Zorka Pharm (S

abac,
Serbia). n-Hexane, acetone and internal
standard diethyl-phtalate (IS) were from
Merck (Darmstadt, Germany). Placebos
of shampoo, gel and emulsion (ingredi-
ents without active substance) were
acquired from Zorka Pharm.
Standards
A stock solution of lindane (c = 0.01
mg mL
-1
) was prepared in acetone by
weighing 10.0 mg of lindane standard
into a 100-mL volumetric ask. A
1.0 mL portion of this solution was
transferred into a 10-mL volumetric
ask and diluted to volume with hexane.
A stock solution of a-HCH (c =
0.001 mg mL
-1
) was prepared in ace-
tone by weighing 10.0 mg of a-HCH
standard into a 100-mL volumetric ask.
A 1 mL portion of this solution was
transferred into a 100-mL volumetric
ask and diluted to volume with hexane.
A stock solution of IS (c = 5 mg
mL
-1
) in acetone was prepared.
For laboratory mixture, the stock
solutions of lindane (2 mL), a-HCH
(0.1 mL) and IS (2 mL), were transferred
into a 10-mL volumetric ask and
diluted to volume with hexane.
For the calibration curves, working
standard solutions were prepared to ob-
tain concentrations of 0.5020.0 lg mL
-1
for lindane and 0.0050.20 lg mL
-1
for
a-HCH. IS concentration was 0.05
mg mL
-1
.
Sample Preparation
A 50.0 mg sample of bulk lindane was
accurately weighed and transferred into
a 50-mL volumetric ask and lled to
volume with acetone. A 1 mL aliqout
of this solution was transferred into a
100-mL volumetric ask and diluted to
volume with hexane. Then 1 mL of this
solution was transferred into a 10-mL
volumetric ask, 1 mL of IS solution
was added, and then the mixture was
diluted to volume with hexane.
The equivalent of 1.0, 3.0 and 4.0 g
of shampoo, gel and emulsion, respec-
tively, was weighed in a 100-mL volu-
metric ask. About 30 mL of acetone
was added, followed by sonicating and
shaking for 10 min, and completion to
volume with the same solvent. The
solution was ltered through a 0.45 lm
Milipore membrane lter. A 1 mL por-
tion was transferred into a 10-mL volu-
metric ask and diluted to volume with
hexane. Then 1 mL of this solution was
transferred into a 10-mL volumetric
ask, and 1 mL of IS was added, with
dilution to volume with hexane.
For the determination of method accu-
racy, the same amount of placebos was
spiked with standard lindane or a-HCH
solution in a 100-mL volumetric ask to
obtain spiked concentrations 0.81.2 lg
mL
-1
and0.0080.012 lg mL
-1
for lindane
and a-HCH, respectively.
Fig. 1. GC-ECD chromatograms: a laboratory mixture, c
lindane
= 2.0 lg mL
-1
, c
a-HCH
=
0.02 lg mL
-1
; b raw bulk; c Gamex shampoo; d Gamex gel; e Gamex emulsion
582 Chromatographia 2010, 72, September (No. 5/6) Limited Short Communication
Results and Discussion
Method Optimisation
Optimum conditions were established by
a number of preliminary experiments.
The experimental settings described in
the Experimental part allow excellent
separation of lindane and its impurity,
Fig. 1a. Chromatographic parameters
such as: resolution (2.02), retention
factor (8.04 and 7.72 for lindane and
a-HCH, respectively) tailing factor (1.01
and 0.89), and the number of theoretical
plates (2,256 and 2,270), were calculated,
showing good system suitability and
satisfactory column eciency.
Method Validation
The selectivity of the method was inves-
tigated by observing potential interfer-
ences between lindane and its impurity
with excipients of products. None of the
formulation ingredients under examina-
tion generated chromatographic peaks
which interfere with either component.
Linear relationships of the peak area
of lindane/IS and a-HCH/IS as the
function of the concentration were ob-
tained over the range 0.515.0 lg mL
-1
for lindane (regression equation: y =
7740 x + 0.0046) and 5150 ng mL
-1
for a-HCH (y = 77.5 x + 0.0012) at the
constant concentration of IS. LOQ and
LOD were determined to be 0.45 and
0.15 lg mL
-1
for lindane and 0.005 and
0.002 lg mL
-1
for a-HCH.
The precision of the method was
determined by repeatability (intra-day)
and reproducibility (inter-day), Table 1,
showing low values for relative standard
deviation(RSD). Since the lowest value of
RSD was for 1 lg mL
-1
, this concentra-
tion was chosen for sample analysis (bulk
and pharmaceutical formulations).
The proposed methods, when used
for estimation of lindane or a-HCH from
pharmaceutical dosage forms after spik-
ing placebo with known amounts of
lindane or a-HCH, aorded a medium
recovery of 100% for lindane and
100.5% for a-HCH with RSD below
2.5%, conrming the accuracy of the
method.
Analysis of Bulk Lindane and
Pharmaceutical Formulations
The validated method was applied to
assay lindane and a-HCH in bulk. The
peaks of bulk carried the characteristic
of standard lindane and a-HCH
(Fig. 1b). The validity of the proposed
method was tested for pharmaceutical
preparations by analysing: shampoo, gel
and emulsion. Peaks of impurity were
not detected. No interference originating
from the matrix was observed, as well as
no reduction in response after running
seven analyses (Fig. 1ce). The good
recovery and low RSD (Table 1) conrm
the suitability of the proposed method.
Conclusion
The described GC-ECD method indi-
cates acceptable accuracy and precision
and can be used for simultaneous
determination of lindane and its
impurity a-HCH in bulk drug and
pharmaceutical products in routine
control analysis. Since there was no
interference of excipients and ingredi-
ents in the examined products, no
additional extraction or separation
procedures were required.
Acknowledgments
We acknowledge the nancial support
from the Ministry of Science and Envi-
ronment of the Republic of Serbia No
142072.
References
1. The Pesticide Fact Book (1985) Noyes
Data Corp. Park Ridge, NJ, USA,
pp 475483
2. Ferner R (1996) Martindales Extra Phar-
macopoeia. The Royal Pharmaceutical
Society, London, p 1437
3. Toxicological prole for alpha-, beta-,
gamma-, and delta-hexachlorocyclohexane
Table 1. Precision of the GC-ECD method of lindane and its impurity a-HCH and assay in bulk and formulations: shampoo, gel and suspension
Compound Taken
(lg mL
-1
)
Intra-day Inter-day
Found
(lg mL
-1
)
Recovery
(%)
RSD
(%)
Found
(lg mL
-1
)
Recovery
(%)
RSD
(%)
Lindane 1.0 0.98 98.5 0.9 0.98 98.0 1.3
7.0 7.18 102.5 2.1 7.28 104.2 2.3
12.0 12.10 100.8 1.7 11.90 99.2 1.9
a-HCH 0.01 0.0104 104.0 2.2 0.0105 105.0 2.3
0.05 0.0509 101.8 2.4 0.0512 102.4 2.5
0.12 0.1180 98.3 1.9 0.1240 103.3 2.0
Bulk 1.0 Lindane 0.998 99.8 1.4
a-HCH < LOD
Gamex shampoo 1.0 Lindane 1.006 100.6 0.9
a-HCH n.d.
Gamex gel 1.0 Lindane 1.005 100.5 1.5
a-HCH n.d.
Gamex emulsion 1.0 Lindane 1.001 100.1 2.9
a-HCH n.d.
n.d. not detected
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