INTRODUCTION:
Metronidazole is the most popular representative in
the group of medicines of 5-nitroimidazole derivatives1,2, different analytical techniques are used for their
determination3–8. Chemically,
metronidazole is 2-methyl-5-nitroimidazole-1-ethanol and has the structural
formula as shown on Figure 1.
Figure 1. Chemical structure of metronidazole
Based on the information about metronidazole side
effects9–12 and its interaction with alcohol13–15 we can
make the conclusion that metronidazole is a potential analyte in chemical
toxicological investigations.
Earlier we developed the procedures of metronidazole
determination by the methods of UV-spectrophotometry16 and
high-performance liquid chromatography17, and used them to study
metronidazole extraction18.
The method of gas-liquid chromatography (GLC)
with different types of
detection has the wide popularity in forensic toxicology; it is used for
screening and confirming investigations – with the purpose of analytes
detection, identification and determination19,20. But the method of
GLC is described for metronidazole
determination very seldom21–26.
The method of GLC with flame-ionization detection (FID)21 has been applied for metronidazole dosage forms analysis in the mixture
with miconazole nitrate, and the range of the method application is
50–6030mg/mL, but it is too high concentrations for
metronidazole determination in forensic toxicology, because we need to quantify
the medicine at the concentration level of 5–20 mg/mL9,15,19. The GLC/FID-procedures22,23 include the step of metronidazole conversion to trimethylsilyl
derivative, but under such conditions we may see too much peaks of foreign
compounds of the chromatograms, i. e. specificity of the procedure in relation
to the matrix components may be unsatisfied. The authors24 have
proposed the method of GLC
with mass spectrometric detection (MSD) for metronidazole determination in the presence of
ronidazole, ipronidazole and dimetridazole, but these compounds are not actual
for Ukraine (they are not registered in Ukraine) and other 5-nitroimidazole
derivatives are interested to study in the mixture with metronidazole.
The purpose of our paper is to develop GLC-procedures
of metronidazole quantification with flame-ionization and mass spectrometric detection, and carry out step-by-step validation27–29
of the developed procedures in
the variants of the method of calibration curve (MCC)30–33, method
of standard (MS)30,34 and method of additions (MA)30,35 to choose the optimal variant for further
application in analytical toxicology.
The present article is the continuation of scientific
researches of Analytical Chemistry Department of the National University of Pharmacy
in the area of investigations of 5-nitroimidazoles16–18,36–40.
MATERIALS AND METHODS:
Metronidazole was of pharmacopoeial purity. All
reagents were of analytical grade.
The GLC/FID-analyses were performed using gas
chromatograph HP 6890 Hewlett Packard; column – НР-1 Æ0.32mm ´ 30m, 0.25μm, 100% dimethylpolysiloxane;
temperature of the column thermostat – 70ºС (3 min.), increasing the
temperature with the rate of 40ºС/min. to 180ºС (keeping for 2
min.), increasing the temperature with the rate of 40ºС/min. to
250ºС (keeping for 3 min.); injector temperature – 280ºС; detector
temperature – 280ºС; volume rate of carrier gas (helium) – 1.5mL/min;
split mode – 1:2; the volume of injection – 2μL.
The GLC/MSD-analyses were performed using Agilent
6890N Gas Chromatograph; columns – 1) НР-5MS Æ0.25mm ´ 30m, 0.25μm, 5% diphenylpolysiloxan/ 95% dimethylpolysiloxan;
2) DB-17MS Æ0.25 mm ´ 30 m, 0.15μm, 50% diphenylpolysiloxan/50%
dimethylpolysiloxan; columns are connected sequentially through Deans switch;
temperature of the column thermostat – 70ºС (2 min.), increasing the
temperature with the rate of 45ºС/min. to 210ºС, increasing the
temperature with the rate of 6ºС/min. to 320ºС (keeping for 12.56
min.); detector – mass spectrometer Agilent 5973N MSD with turbo pump; transfer
line temperature – 280ºС; ion source temperature – 230ºС;
quadrupole temperature – 150ºС; ionisation mode – electron impact; electron
energy – 70eV; scanning range – 40 – 750m/z; threshold – 110; injector– Agilent
7683 Injector/Autosampler; injector temperature – 250ºС; splitless mode;
inlet carrier gas (helium) pressure: 1st column – 26.06 psi, 2nd column – 19.30 psi; the volume of injection – 1μL.
The stock solutions 1, 2 and 3 (100μg/mL) were
prepared by dissolving 50.0mg of metronidazole
in 50.00mL of 0.1 M NaOH solution and the solutions were
diluted to 500.0mL with the distilled water. The reference solution (8μg/mL)
was prepared by diluting 4.00mL of the stock solution 1 to 50.0mL with 0.01M NaOH
solution. The stock solution 2 was diluted with 0.01 M NaOH solution to
prepare the model solutions 1 – 7 having concentrations of 2; 4; 6; 8; 10; 12
and 14 μg/mL respectively.
The addition solution 1 (300μg/mL) was prepared
by dissolving 60.0mg of metronidazole
in 20.00 mL of 0.1 M NaOH solution and the solutions were
diluted to 200.0mL with the distilled water. The stock solution 3 was diluted
with 0.01 M NaOH solution to prepare the model solutions 8 – 13
having concentrations of 10; 10; 20; 30; 40; 40μg/mL respectively. The
model solutions 8.1 – 13.1 were prepared by diluting 10.00mL of the model
solution 8 – 13 to 50.0mL with 0.01 M NaOH solution. For preparing the model
solutions 8.2 – 13.2 10.00mL of the model solutions 8 – 13 were mixed with 1.00mL
of the addition solution 1 and diluted to 50.0 mL with 0.01M NaOH
solution.
a – metronidazole
b – secnidazole
c – tinidazole
d – ornidazole
e – nimorazole
Figure 3. Mass-spectra of 5-nitroimidazoles
RESULTS AND DISCUSSION:
The chromatographic conditions has been
chosen for metronidazole determination by the method of gas-liquid
chromatography in two variants of performance with flame-ionization and
mass-spectrometry detection with temperature program changing during the
analysis from 70°C to 250°C or 320°C respectively. Under proposed conditions metronidazole
and four other 5-nitroimidazole derivatives (registered in Ukraine) are
separated and detected without preliminary derivatization. In the method of
GLC/FID the retention time for metronidazole is 9.08 min, for tinidazole is
5.42 min, for ornidazole is 7.42 min, for secnidazole is 8.97 min, and for
nimorazole is 11.35 min. In the method of GLC/MSD the retention time for metronidazole
is 12.01 min, for secnidazole is 11.74 min, for ornidazole is 13.95 min, for
nimorazole is 14.81 min, and for tinidazole is 17.22 min.
The typical chromatograms of the mixture of metronidazole, secnidazole,
tinidazole, ornidazole and nimorazole are presented in Figures 2. Mass-spectra
of metronidazole, secnidazole, tinidazole, ornidazole and nimorazole obtained under
the proposed GLC/MSD-conditions are presented in Figure 3.
Method validation:
Validation of the developed procedure has
been carried out in the variants of the method of calibration curve30–33,
method of standard30,34 and method of additions30,35.
Such validation parameters as in process
stability, linearity/calibration model, accuracy and precision (repeatability)
have been estimated by model solutions according to Scheme 141,
which allows to assess the suitability of the actual analytical procedure for
further work.
The validation provides application of the
normalized coordinates that allows to calculate the validation characteristics,
which do not depend on the analyte and features of the method of analysis30,42.
The metronidazole concentration in the model solution
for the point of 100% in the normalized coordinates 
has been chosen as the concentration
provided the «signal/noise» ratio at the level of ≥40. For normalization
of the obtained experimental data the reference solution with the analyte
concentration of 
is used.
The analytical ranges D of the
method application are 25 – 125%, 25 – 150% and 25 – 175%; the number of concentration
levels g equals 5, 6 or 7 respectively in constant increments of 25%.
Acceptability criteria for validation
parameters have been formed on the basis of systematic application of “insignificance
concept”42 and proceeding from the value of extreme uncertainty 
for analytical toxicology, which equals
20%19,43.
In the MCC acceptability criteria
for linear dependence and precision have been found proceeding from the equality
of uncertainty of calibration curve plotting 
and uncertainty of analysis of the sample 
.
Acceptability criteria for validation
parameters have been calculated proceeding from two approaches30: Approach
1 – uncertainty of analyte quantification in model solutions 
is equal to uncertainty of sample
preparation procedure; Approach 2: uncertainty of analyte quantification
in model solutions is insignificant as compared with total
uncertainty 
.
Validation results:
Validation of the procedures has been carried out
within 3 different analytical runs using different batches of reagents and different
glassware; experiments have been performed by three different analysts. The
results obtained within one analytical run are presented in Tables 1 – 5, but results
of other analytical runs are at the same range of values. In process
stability of metronidazole in the model solution was verified by chromatographing
the reference solution immediately and in 1, 12, 24, 36 and 48 hours after its
preparation, and the systematic error 
was calculated and assessed (Table 1). In
process stability of metronidazole in model solutions is satisfied the
acceptability criteria for all periods of time, therefore we may use the
solutions of metronidazole at least for 48 hours after its preparation.
To determine linearity/calibration model
the model solutions 1 – 7 were analysed within 1 run, correlation coefficient 
, rest standard deviation 
and also absolute term 
(if it is necessary) were calculated and assessed
(Table 2).
The proposed methods are planned to be applied not only in the variant
of the method of calibration curve, but also in the variants of the method of standard and method of additives, which require the presence of the
directly proportional relationship between analyte content and analytical
signal within the specified range. Thus, it is necessary to confirm not only
the acceptable level of procedure linearity, but also to demonstrate the
insignificance of absolute term in the linear dependence of the form 
42.
The total results of validation allow to
point to the conclusion about acceptable accuracy and precision
of GLC/MSD-procedure of metronidazole quantitative determination in the
variants of MCC, MS and MA for all ranges of the method application within both
approaches of their estimation. As for GLC/FID-procedure accuracy and precision
are satisfied the requirements only in the variants of MCC and MA; the
method of standard can be applied only for the application range of 25 – 175%.
According to the validation results we may
recommend the developed procedures for further application in forensic
toxicology with the purpose of development of the methods of biological liquids
analysis for metronidazole quantification.
It should be noted that the procedures in
the variant of MCC are characterized by the best values of accuracy and
precision. In turn, the procedures in the variant of MS are characterized by
the worst values of systematic and random errors. For the variant of MA the
middle values of accuracy and precision are observed. Thus application of the
method of calibration curve is optimal for analysis, but for single
determinations the method of additions should be used.
CONCLUSIONS:
New procedures of metronidazole quantitative determination by the
methods of GLC/FID and GLC/MSD have been developed. Their validation by such
parameters as stability, linearity, accuracy and precision in the variants of
the method of calibration curve, method of standard and method of additions has
been carried out and acceptability for application has been shown.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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= 8 
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