Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 69 No. 4 pp. 597ñ601, 2012
ISSN 0001-6837
Polish Pharmaceutical Society
QUANTITATIVE ANALYSIS OF NOFRLOXACIN BY 1H NMR AND HPLC
ANITA FR•CKOWIAK and ZENON J. KOKOT*
Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences,
Grunwaldzka 6 St., 60-780 PoznaÒ, Poland
Abstract: 1H NMR and developed previously HPLC methods were applied to quantitative determination of norfloxacin in veterinary solution form for pigeon. Changes in concentration can lead to significant changes in the
1
H chemical shifts of non-exchangeable aromatic protons as a result of extensive self-association phenomena.
This chemical shift variation of protons was analyzed and applied in the quantitative determination of norfloxacin. The method is simple, rapid, precise and accurate, and can be used for quality control of this drug.
Keywords: fluoroquinolone, norfloxacin, quantitative 1H NMR, HPLC
nation is normally obtained from the ratio between
the integration of a specific signal of the analyte and
the integration of a specific signal of the internal
standard, but it can also be obtained from the measurement of chemical shift displacements of selected
non-exchangeable protons (18). It was found that
chemical shifts of three non-exchangeable aromatic
protons changed remarkably as the concentration
was varied. Such chemical shift variations can be so
pronounced that they lead to 1D and 2D spectroscopic signatures that may be misleading. Lack of
consideration of the importance of such concentration effects can lead to unwanted controversies in
the design of self-replicating systems (19, 20).
In the present work, 1H NMR technique has
been used to determine the concentration of norfloxacin and compared to the HPLC method (21).
The fluoroquinolone norfloxacin, 1-ethyl-6fluoro-1,4-dihydro-4-oxo-7-(1- piperazinyl)-3-quinoline carboxylic acid (Fig. 1), is a synthetic broad
spectrum antibacterial drug, which is very much
active against many Gram positive and Gram negative bacteria (1). It is mainly used in the treatment of
urinary tract infections.
Several analytical methods have been developed for the determination of fluoroquinolones (2).
There are many HPLC methods (3ñ9) and other
techniques as TLC (10) and fluorimetry (11, 12) for
determination of norfloxacin described in the literature. NMR methods are also used for determination
of fluoroquinolones (13ñ17). Quantitative determi-
EXPERIMENTAL
Materials
Norfloxacin (Nfx) was obtained from SigmaAldrich Co. (USA). Enoxacin (Enx) was obtained
from Sigma-Aldrich Co. (USA). Nfx 10% solution
for pigeon was obtained from PFO VETOS
FARMA (Poland). Deuterium oxide 99.8 atom % D
was obtained from Armar Chemicals (Switzerland).
Deuterium chloride (DCl) 0.1 M in deuterium oxide,
Figure 1. Structure of norfloxacin
* Corresponding author: e-mail: [email protected], phone: +48 061 854 66 11, fax: +48 061 854 66 09.
597
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ANITA FR•CKOWIAK and ZENON J. KOKOT
99.8 atom % D was obtained from Armar Chemicals
(Switzerland). 3-(Trimethylsilyl)-1-propanesulfonic
acid sodium salt (DSS) was obtained from SigmaAldrich Co. (USA). Acetonitrile for use in HPLC
was obtained from Mallinckrodt Baker B.V. (The
Netherlands). Tetrahydrofuran CHROMASOLV for
HPLC use was obtained from Sigma-Aldrich Co.
(USA). Ortho-phosphoric acid 85% was obtained
from Merck (Germany). Mille Q water was used to
prepare all aqueous solutions.
Figure 2. 1H NMR spectrum of norfloxacin
Figure 3. Partial 1H NMR spectra of norfloxacin aromatic protons at different concentrations
Quantitative analysis of norfloxacin by 1H NMR and HPLC
599
Figure 4. Chromatogram of norfloxacin standard solution and norfloxacin 10% solution for pigeon
Table 1. Recovery study of norfloxacin 10% solution for pigeons.
1
Norfloxacin conc. [%]
Recovery [%]
H NMR
10.2 ± 0.2
102 ± 1.4
HPLC
9.8 ± 0.01
98 ± 0.1
Apparatus
A HPLC system Series 1200 Agilent
Technologies consisting of a binary pump G1312A,
autosampler HiP-ALS G1367B and DAD G1315D
detector. The analysis was performed using a
reversed phase column ñ RP Hypersil BDS, 4.6 ◊
150 mm, 5 µm Agilent Technologies, and detection
was set at 280 nm. The mobile phase consisted of
0.01 mol/L ortho-phosphoric acid ñ acetonitrile ñ
tetrahydrofuran (89/10/1, v/v/v) and the flow rate
was 1.0 mL/min (21).
1
H NMR measurements were performed on a
Varian Gemini 300VT spectrometer with operating
frequency 300 MHz. The samples were run in 5 mm
NMR tubes. The temperature during all experiments
was kept at 293 K; 100 scans were acquired using a
spectral width of 5 kHz and a pulse delay time of 1.0
s. DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) was used as the chemical shift reference (δ =
0 ppm).
Preparation of solutions
1
H NMR
Calibration curve was prepared using five different concentrations ranging from 3.05 to 8.04
mg/mL. All solutions were prepared in D2O with
addition of equimolar quantities of 0.1 mol/L DCl
solution and filtered through the 0.65 µm membrane
filters (Millipore, nitrocellulose). Each solution was
analyzed five times.
A hundred milliliters of Nfx 10% solution for
pigeon was evaporated to dryness. The residue on
evaporation was kneaded in an agate mortar and the
solution for 1H NMR analysis was prepared (4.98
mg/mL).
HPLC
Calibration curve for HPLC measurements was
prepared using solutions ranging from 0.508 to
2.976 µg/mL. All solutions were prepared in 0.02
mol/L ortho-phosphoric acid solution with addition
of 0.9 µg/mL enoxacin solution as internal standard
and filtered through the 0.65 µm membrane filters
(Millipore, nitrocellulose). Each solution was injected on HPLC column five times.
A hundred milliliters of Nfx 10% solution for
pigeon was evaporated to dryness. The residue on
evaporation was kneaded in an agate mortar and the
solution for HPLC analysis was prepared (1.54
µg/mL).
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ANITA FR•CKOWIAK and ZENON J. KOKOT
RESULTS AND DISCUSSION
Linearity
H NMR
The signal displacements induced by concentration changes follow a specific pattern. Chemical shift
values (δ) of the aromatic protons (H-8, H-5, H-2)
were plotted against the concentration. A linear graph
was obtained when these chemical shift values were
correlated with the concentration (c). The linear graph
with the highest correlation coefficient was used for
the calculations (linear graph of H-8, r = 0.999, y =
ñ0.0157 ± 0.0006x + 7.1113 ± 0.0034) and the linear
range was found to be from 3.05 to 8.04 mg/mL.
1
A novel quantitative 1H NMR spectroscopic
method was proposed in 2006 (18) for Nfx,
ciprofloxacin and enoxacin determination in aqueous solutions; the analysis was based on chemical
shift migration of aromatic protons as a function of
concentration. This methodology was used to quantitation of Nfx hydrochloride in the veterinary solution for pigeon and the results were compared with
the HPLC analysis.
Chemical shifts variations result from molecular aggregation leading to the formation of differently packed assemblies. In such assemblies, the number of molecules and the orientation of association
should vary as a function of the concentration,
which in turn should manifest in the altered chemical shifts.
1
H NMR spectrum (Fig. 2) shows three signals
of aromatic protons: H-8, H-5 and H-2. Two of them
ñ H-8 and H-5 are coupled with the fluorine atom;
3
JH-8,F = 13 Hz, 4JH-5,F = 6.5 Hz.
Validation of methods
Methods were validated according to the
guidelines of the International Conference of
Harmonization ICH (22).
Precision
H NMR
The precision of the assay was determined in
relation to repeatability (intra-day). In order to evaluate the repeatability of the method, five samples of
Nfx were determined at concentrations from 3.05 to
8.04 mg/mL. Repeatability of the chemical shift displacement was evaluated by variation coefficients
(CV) and it was lower than 2%.
HPLC
The calibration plot for Nfx: ANfx/AIS = f(c) was
obtained in the concentration range 0.508 to 2.976
µg/mL. Linear correlation coefficients were obtained:
r = 0.999, y = 1.1062 ± 0.0305x ñ 0.0077 ± 0.0578).
Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ values were calculated from the
regression equations, where LOD = 3.3 Sy/a and
LOQ = 10Sy/a. Sy denoted the standard deviation of
the calibration curve and a was the slope of the corresponding calibration curve. For 1H NMR method,
LOD was 0.11 mg/mL and LOQ was 0.38 mg/mL
and for HPLC method this parameters were: LOD =
0.08 µg/mL and LOQ = 0.28 µg/mL.
1
HPLC
The precision of the HPLC assay was determined in relation to repeatability (intra-day). It was
estimated by establishing the injection repeatability (intra-run precision) and was evaluated by variation coefficients (CV) obtained from five repetitions of analysis (n = 5). The CV was lower than
0.2%.
Accuracy
The accuracy of the procedure was ascertained
from assessing an agreement between the measured
and the theoretical concentration of analyzed samples (recovery). For 1H NMR method accuracy was
99.83% and for HPLC method 99.10%.
Quantitative analysis by 1H NMR and HPLC
Figure 2 shows typical 1H NMR spectrum of
Nfx HCl. Selected non-exchangeable protons H-8,
H-5 and H-2 are in the aromatic region between 6.99
to 8.80 ppm. The results revealed a strong concentration-dependent chemical shift variation in 1H NMR
spectra (Fig. 3). Moreover it can be observed that the
H-8 signal become broader and the doublet disappear. This can be an effect of intermolecular (coalescence or overlap) or intramolecular (hydrogen bond
OH∑∑∑O=C) dynamics (16). The proportionality in the
linear range (3.05ñ8.04 mg/mL) enabled the quantitation of Nfx HCl in aqueous solutions. The best correlation was for the proton H-8 (δ = 7.03 ppm, Fig.
2). The obtained results were compared with the previously developed HPLC method. Nfx HCl (tR = 10.6
min) chromatogram with enoxacin (tR = 8.4 min) as
internal standard is presented in Figure 4.
Quantitative analysis of norfloxacin 10% solution
for pigeons
The concentration of Nfx 10% solution for
pigeon was calculated from calibration equations for
Quantitative analysis of norfloxacin by 1H NMR and HPLC
both 1H NMR and HPLC methods. The results presented in Table 1 show very good recovery.
CONCLUSION
Nuclear magnetic resonance spectroscopy is
one of the most important and most widely used
analytical methods, which enables a precise determination of the amount of molecular structures of
substances.
Here a validated protocol for quantitative 1H
NMR analysis of Nfx is described and compared to
the HPLC method (previously developed).
This quantitative 1H NMR method is simple
and rapid. The statistical analysis proves the high
precision and accuracy of the method. The performed study demonstrates that both HPLC and 1H
NMR can be successfully applied to the quantitative
and qualitative analysis of Nfx and can be used for
quality control of the tested drug.
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Received: 9. 03. 2011