spectroscopic techniques

Rapid Quantitative Determination of Cipro oxacin in

Pharmaceuticals by Use of Solid-State FT-Raman
Spectroscopy
STAVROULA G. SKOULIKA and CONSTANTINOS A. GEORGIOU*
Chemistry Laboratory, Agricultural University of Athens, 75 Iera Odos, 1185 5 Athens, Greece

FT-Raman spectroscopy based on band intensity or band area measurem ents was used for the quantitative determination of cipro oxacin in pharmaceutical solid dosage form s. Univariate calibration
was used for quantitative analysis. Bands observed at 1708 , 1624,
1548, 1493, 1273, 1253, 1238, 1024, 805, 787, 752, 718 , 665, and 638
cm 2 1 were used. Calibration curves were linear in the concentration
range of 3100% w/w with correlatio n coef cients of 0.990.996 and
0.991 0.9993 for band intensity and band area measurements, respectively. Precision ranged from 011 and 0.412% relative standard deviation (RSD) (n 5 3) for band intensity and band area
measurements, respectively, and results were in good agreem ent
with the results obtained by the current United States Pharmacopoeia (USP 24) and National Formulary (NF 19) method. Multivariate calibration was also used for quantitative analysis. Multiple
linear regressio n using the intensities of the 1545 and 1272 cm 2 1
bands gave results in accordan ce with those obtained by the current
United States Pharmacopoeia (USP 24) and National Formulary
(NF 19) method. As measurement takes just 30 s using the analytical
readout from a single band, the proposed method can be used to
replace tedious and time-consuming methods.
Index Headings: Cipro oxacin; Pharmaceuticals; Solids; FT-Raman; Quantitative; Determination.

INTRODUCTION
Cipro oxacin hydrochloride (Fig. 1) (1-cyclopropyl-6 uoro-1,4-dihydro-4-oxo -7-(piperazin-1-yl) quinoline-3carboxylic acid hydrochloride) is a uoroquinolone antibacterial agent with a wide spectrum of activity including Enterobacteriace, Pseudomonas aeruginosa, Haemophilus, Neisseria spp., staphylococci, and some other
Gram-positive bacteria.1,2 Cipro oxacin hydrochloride is
a pale yellow crystalline powder. It is soluble in water,
slightly soluble in acetic acid and m ethanol, very slightly
soluble in dehydrated alcohol, and practically insoluble
in acetone, acetonitrile, ethyl acetate, hexane, and methylene chloride. 2
Various methods based on spectrophotom etry, 3 6 ow
injection analysis, 7 uorim etry, 6 sequential injection analysis,8 and high performance liquid chromatography 9,10
have been developed for the quantitative determination
Received 13 Januar y 2001; accepted 4 May 2001.
* Author to whom correspondence should be sent.

Volume 55, Number 9, 2001

of cipro oxacin in pharm aceutical formulations. All these

methods require extensive manual sample pretreatment
and are tim e consuming. Therefore, the developm ent of
rapid analytical methods that do not require dissolution
of cipro oxacin solid dosage forms is highly desirable.
Raman spectroscopy has great potential in pharmaceutical analysis. The ability to analyze the structure of drug
substances in aqueous environments, the capability to
sample through glass, and the increased sensitivity to
sym metric stretches and highly polarizable bonds renders
Raman spectroscopy a powerful tool for the examination
of pharmaceuticals.11 Raman spectroscopy has only been
used for the qualitative characterization of drug substances.11,12 One of the useful features of Raman spectroscopy
is the ability to carry out direct m easurements on solids. 13
This capability is important in the current application.
The Raman spectrum of cipro oxacin hydrochloride
hexahydrate has been previously reported,14 but no application of Raman spectroscopy to the quantitative determination of cipro oxacin in pharmaceutical solid dosage forms has been described.
In this work, a solid-state FT-Raman m ethod for cipro oxacin determination is presented. The analysis of solid dosage forms was achieved through univariate or multivariate calibration, using band area and/or band intensity measurements. This work is part of an ongoing project in our laborator y concerning formulation analysis by
FT-Raman spectroscopy.1517
EXPERIMENTAL
Sam ple and Standard Preparation and Chemicals.
The cipro oxacin hydrochloride standard of 99.99% purity was a kind offer from Flavine, Spain. Cipro oxacin
hydrochloride hexahydrate was obtained through local
manufacturers. The purity of this substance was determined using the current United States Pharmacopoeia
(USP 24) and National Form ulary (NF 19) method. 10
Acetonitrile, phosphoric acid, and potassium brom ide of
analytical reagent grade were purchased from Merck.
Maize starch of analytical reagent grade was purchased
from Cerestar. Comm ercial form ulations were obtained

0003-7028 / 01 / 5509-1259$2.00 / 0
q 2001 Society for Applied Spectroscopy

APPLIED SPECTROSCOPY

1259

F IG . 1.

Cipro oxacin hydrochlor ide FT-Raman spectrum acquire d at 0.51 W excitation intensity in the range of 3200 500 cm 2 1.

through local m anufacturers. All chemicals were used

without further puri cation.
For quantitative determinations, 300 mg discs of 120
mm diameter were prepared. These discs contained cipro oxacin hydrochloride hexahydrate in the range of 3
100% and were used as standards for the preparation of
calibration curves. To prepare the aforementioned discs,
the appropriate amount of cipro oxacin hydrochloride
hexahydrate was mixed with maize starch and hom ogenized in a m ortar for approximately 4 min. The powder
was then pressed in an IR-die cast for 10 min at 1000
psi using a press. Comm ercial formulations containing
cipro oxacine hydrochloride hexahydrate were ground in
the m ortar for approximately 4 m in and the resulting
powder was treated in the same manner.
For the of cial high-performance liquid chromatography (HPLC) measurem ents, solutions described in the
current United States Pharm acopoeia (USP 24) and National Formulary (NF 19) method 10 were prepared.
TABLE I.
Strong
Medium
Weak
Ver y weak

Cipro axacin hydrochloride Raman bands (cm 2 1).

1624 (100)
1384 (78), 301 6 (44), 2987 (22), 1548 (31), 1467
(38), 1344 (29), 1273 (25), 752 (27)
3086 (15), 170 8 (14), 1493 (19), 1415 (16), 1316
(15), 1298 (16), 1253 (12), 1238 (14), 1225 (12),
1195 (12), 1024 (17), 638 (16)
1143 (8), 1050 (9), 805 (4), 787 (7), 718 (10), 704
(7), 665 (7), 538 (4), 498 (7)

% Relative intensities to the 1624 cm 2 1 band are shown in parentheses.

1260

Volume 55, Number 9, 2001

Apparatus. FT-Raman spectra were recorded with a

Nicolet 750 FT-Raman spectrometer equipped with a Nd:
YAG laser source that emits at 1064 nm . A CaF 2 beamsplitter, an Indium -Galium Arsenate (InGaAs) detector
and 1808 backscattering geometry were used in the spectrometer. A m otorized positioner focuses the laser beam
on the sample and a manual side-to-side adjuster allows
sam ple adjustment for maximum optical ef ciency. To
ensure that the spectrometer is ne tuned and the detector
signal m aximized, an optical bench alignm ent was performed before each batch of measurem ents. To acquire
Raman spectra, the discs were placed in a laboratorymade accessor y. 18 Spectra in the range of 50 4000 cm 2 1
were accum ulated from 100 scans collected during 3 m in
at a resolution of 4 cm 2 1 . The laser power intensity at the
sam ple was 0.51 W.
H igh-perform ance liquid chrom atographic analysis
was carried out using a Jasco PU-98 Pum p and a Jasco
UV-970 UV/vis detector equipped with a Hypersil BDS
C18, 5 mm , 4.6 3 250 mm column. The ow rate was
1.5 m L/min and detection was at 278 nm.
RESULTS AND DISCUSSIO N
Raman Spectrum of Cipro oxacin Hydrochloride.
Raman bands of the cipro oxacin hydrochloride standard
spectrum (Fig. 1) acquired in this study are sum marized
in Table I, where the classi cation of strong, medium,
weak, and very weak bands is based on the % intensities
relative to the 1624 cm 2 1 band. It should be noted that

TABLE II.
Band (cm 2 1 )

Cipro oxacin calibration data at 0.51 W excitation intensity (band intensityband area vs. % w/w concentration, n 5
Range for two-point
baseline correction

Equation of calibration graph

A. Band intensity calibration curve

1708
17271689
1624
1650 1592
1548
15671523
1493
1509 1484
1273
12821261
1253
12571247
1238
12431232
1024
10331014
805
809 800
787
794 771
752
759 736
718
723709
665
671 657
638
645 628

BI
BI
BI
BI
BI
BI
BI
BI
BI
BI
BI
BI
BI
BI

B. Band area calibration curve

1708
17271689
1624
1650 1592
1548
15671523
1493
1509 1484
1273
12821261
1253
12571247
1238
12431232
1024
10331014
805
809 800
787
794 771
752
759 736
718
723709
665
671 657
638
645 628

BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA

5
5
5
5
5
5
5
5
5
5
5
5
5
5

Correlation
coef cient

7).

Detection limit,
(% w/w)
% RSD (n 5 3)
a

(0.1 6 0.1) 1 (0.048 6 0.002) 3 C

(1 6 1) 1 (0.43 6 0.02) 3 C
(0.3 6 0.3) 1 (0.110 6 0.005) 3 C
(0.1 6 0.1) 1 (0.032 6 0.002) 3 C
(0.2 6 0.2) 1 (0.076 6 0.002) 3 C
(0.02 6 0.04) 1 (0.013 6 0.001) 3 C
(0.01 6 0.07) 1 (0.024 6 0.001) 3 C
(0.01 6 0.1) 1 (0.054 6 0.003) 3 C
(0.01 6 0.02) 1 (0.011 6 0.001) 3 C
(0.09 6 0.08) 1 (0.022 6 0.001) 3 C
(0.5 6 0.4) 1 (0.110 6 0.006) 3 C
(0.2 6 0.1) 1 (0.032 6 0.002) 3 C
(0.11 6 0.08) 1 (0.022 6 0.001) 3 C
(0.4 6 0.3) 1 (0.06 6 0.01) 3 C

0.994
0.994
0.994
0.991
0.996
0.994
0.993
0.994
0.993
0.991
0.991
0.99
0.991
0.99

6.3
7.0
8.2
9.4
7.9
9.2
8.8
5.6
5.5
10.9
10.9
9.4
10.9
15

0.3 6.8
1.57.0
1.39
0 10
2.710
3.810
2.510
2.710
1.511
2.0 10
0.110
2.611
1.9 9.7
3.0 9.6

(2 6 3) 1 (1.13 6 0.05) 3 C
(10 6 10) 1 (6.9 6 0.2) 3 C
(5 6 4) 1 (2.2 6 0.1) 3 C
(1 6 1) 1 (0.35 6 0.02) 3 C
(1 6 1) 1 (0.76 6 0.02) 3 C
(0.2 6 0.2) 1 (0.089 6 0.005) 3 C
(0.0 6 0.2) 1 (0.162 6 0.003) 3 C
(0.5 6 0.7) 1 (0.65 6 0.02) 3 C
(0.0 6 0.1) 1 (0.054 6 0.002) 3 C
(0.4 6 0.6) 1 (0.25 6 0.01) 3 C
(3 6 3) 1 (0.97 6 0.05) 3 C
(1 6 1) 1 (0.23 6 0.02) 3 C
(0.5 6 0.4) 1 (0.14 6 0.01) 3 C
(2 6 2) 1 (0.55 6 0.03) 3 C

0.996
0.999
0.997
0.993
0.999
0.995
0.9993
0.999
0.996
0.996
0.996
0.991
0.995
0.995

8
4.3
5.5
8.6
3.9
6.7
3.7
3.2
5.6
7.2
9.3
13
8.6
10.9

2.310
1.0 11
1.611
3.18
1.9 7
2.711
1.312
1.510
1.9 11
1.0 7
0.4 7
1.4 11
3.79
2.4 9

5
5
5
5
5
5
5
5
5
5
5
5
5
5

Calculated as three times the standard deviation of the intercept divided by the slope.

relative band intensities depend on the instrument response 19 and that spectra were not corrected for the instrument response.
Bands at 1708, 1624, 1575, and 1384 cm 2 1 have been
attributed to the n(COOH), n(C5O), and antisym metric
and sym m etric n(O C O ) vib ration s, resp ectively. 1 4
These bands are shifted in the case of cipro oxacin hydrochloride and hydrates.14 Lee et. al. assigned the band
around 1624 cm 2 1 to the C5O stretch of different modes
belonging to in-phase and out-of-phase vibrations. 20 The
band around 1500 cm 2 1 is attributed to vibrations of the
benzene ring. 21
Bands in the region of 1300 1250 cm 2 1 are assigned
to in-plane CH deform ations. 22 The bands obser ved at
1273 and 1238 cm 2 1 are probably assigned to various
modes of the phenyl groups. 23 Bands at 718, 665, and
638 cm 2 1 are attributed to the 5CH deformation vibrations. 24 Bands around 771 cm 2 1 are characteristic of the
substituted benzene ring deform ation m odes. 25 The 1024
cm 2 1 band is characteristic of the n(CC) skeletal conformation stretching modes, 26 while the 752 cm 2 1 band is
attributed to methylene rocking m odes. 27 The band at 805
cm 2 1 could be attributed to the CH out-of-plane bending
mode. 28
Q uantitative Analysis of Cipro oxacin Formulations. Quantitative analysis by Raman spectroscopy can
be achieved by univariate calibration utilizing the analytical signal resulting from a single band. For quantitative analysis of cipro oxacin hydrochloride formulations,
the 1708, 1624, 1548, 1493, 1273, 1253, 1238, 1024,
805, 787, 752, 718, 665, and 638 cm 2 1 bands were used.

These bands were chosen because maize starch, which

was used as a diluent for the preparation of the calibration
discs, does not interfere with them spectrally (Fig. 2,
spectrum a). 29 Band intensities and band areas were calculated employing a two point baseline correction utilizing the range mentioned in Table II.
Spectra acquired during calibration are shown in Fig.
2, while calibration data at 0.51 W excitation intensity
are presented in Table II. The use of excitation intensities
higher than 0.51 W resulted in the thermal decom position
of calibration and sample disks. The calibration curves
present excellent linearity with correlation coef cients in
the range of 0.99 0.996 and 0.9910.9993 for band intensity and band area m easurements, respectively.
The linear range was found to be 3100% w/w for all
bands. Detection limits were in the range of 5.515 and
3.213% w/w for band intensity and band area measurements, respectively. Precision values presented in Table
II were determined by rem oving the disc from the laboratory-made accessory, turning it by 608, and then replacing it between the acquisition of successive spectra.
Precision ranged from 0 11 and 0.4 12% RSD (n 5 3)
for band intensity and band area measurements, respectively. The proposed method includes the procedural step
of hom ogenizing the sam ples in the mortar so the precision is affected not only by sample positioning changes
but also by the degree of homogenization.
Results of the analysis of cipro oxacin hydrochloride
formulations by band intensity and band area measurements are shown in Tables III and IV respectively. As
shown by the experimental t-values, results obtained by
APPLIED SPECTROSCOPY

1261

F IG . 2. FT-Ram an spectra acquired from discs of: (a) 0, (b) 10, (c) 30, (d ) 50, (e) 70, ( f ) 90, and (g) 100% w/w cipro oxacin hydrochlor ide
measured in the range of (A) 180 0 900, (B) 820 730, and (C ) 730 610 cm 2 1 .

the proposed method com pare well to those acquired

through the current United States Pharmacopoeia (USP
24) and National Form ulary (NF 19) method.10 For quantitative analysis, the use of band areas is believed to give
more accurate results than the use of band intensities,30
as parameters in uencing the shape and position of Raman bands are m ore likely to affect the intensity than the
area of a band. However, as shown in Table III, the use
of intensity measurements gives results of equal accuracy.
Relative differences between results obtained through the
FT-Raman and the current United States Pharm acopoeia
(USP 24) and National Formular y (NF 19) m ethods10 are
in the range of 29.9 16.0% and 216.120.4% for band
intensity and band area m easurements, respectively.
The reproducibility of the homogenization procedure
carried out while preparing discs for the acquisition of
Raman spectra (see sample preparation), was examined.
Am ong the parameters that in uence the intensity of the
Raman signal detected from solids are particle size and
packing density. To achieve a uniform particle size for
calibration and standard disks, samples were ground and
then pressed in the IR-die cast along with the calibration
disks. A critical step in the analysis is grinding and m ixing. It was found that approxim ately 4 m in of grinding
was required to achieve a relative standard deviation in
the range 0 11% and 0.4 12% RSD (n 5 3) for band
intensity and band area measurements, respectively. Oth1262

Volume 55, Number 9, 2001

er factors that might affect Raman spectra are the applied

pressure while preparing the discs and the amount of time
allowed for this step. It should be emphasized that the
laser beam probes only the surface of the prepared discs.
Therefore, any differences in the drug concentration between the surface and the bulk of the disc will result in
gross analytical errors. A study of the in uence of the
previously m entioned factors has been conducted by testing how the intensity and the area of the bands are affected by the applied pressure in the range of 1000 3000
psi and the tim e allowed in the press in the range of 3
40 min. The experiments have been run in duplicate and
it has been proved that there is no relationship between
band intensity or area and the applied pressure or the time
allowed for pressing. During these experiments it was
found that Raman bands are not shifted regardless of the
pressure applied and the time allowed during pelleting.
This proves that either there is no water loss during pelleting or, if there is any water loss, the bands are not
shifted. To achieve suf cient mechanical stability of the
resulting discs, we selected a pressure of 1000 psi and
we allowed 10 m in for this step.
To select a suitable diluent for preparing standard cipro oxacin calibration disks, various substances, along
with potassium brom ide, were used. Among them were
maize starch and cellulose, which are the excipients commonly used in the preparation of cipro oxacin formula-

(1.699)
(1.699)
(1.609)
(0.000)
(1.145)
(1.699)
(1.699)
(1.699)
(0.849)
(1.699)
(1.699)
(0.849)
(1.609)
(0.849)
0.2
0.2
0.1
0.1
0.3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
2.2
2.2
2.3
2.4
2.2
2.2
2.2
2.2
2.3
2.2
2.2
2.3
2.3
2.3
(1.429)
(0.981)
(2.135)
(2.107)
(0.130)
(0.104)
(0.130)
(0.981)
(0.981)
(0.202)
(0.303)
(0.450)
(1.169)
(1.862)
0.4
0.3
0.3
0.6
0.4
0.5
0.4
0.3
0.3
0.6
0.4
0.5
0.4
0.4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Cipro oxacin concentration as determined through the current United States Pharmacopoeia (USP 24) and National Formulary (NF 19) m ethod 10
(n 5 3): a 5.0 6 0.1, b 5.4 6 0.2, c 5.15 6 0.06, d 5.3 6 0.2, e 4.77 6 0.03, f 4.87 6 0.01, and g 2.40 6 0.04 m g/tab 3 10 2 .
ttheoretical value for 95% con dence level: 2.776.
Manufacturer: a Bayer Hellas, b Pharmaten LTD, c Bros LTD, d Help SA, e Demo SA, f Medinova SA, and g Chrispa SA.

5.2
4.7
4.5
5.6
4.9
4.9
4.9
4.7
4.7
4.8
4.8
5.0
4.6
5.3
(2.126)
(0.977)
(1.551)
(0.734)
(1.456)
(0.993)
(1.598)
(0.600)
(2.030)
(0.257)
(2.030)
(2.030)
(1.626)
(1.598)
0.3
0.3
0.3
0.4
0.2
0.4
0.4
0.2
0.4
0.2
0.4
0.4
0.5
0.4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4.4
4.6
4.5
4.6
4.6
5.0
4.4
4.7
4.3
4.8
4.3
4.3
4.3
4.4
(1.609)
(1.441)
(0.961)
(2.191)
(2.252)
(1.162)
(2.324)
(1.609)
(1.550)
(2.402)
(1.930)
(1.190)
(1.609)
(1.917)
0.5
0.3
0.3
0.6
0.5
0.4
0.4
0.5
0.4
0.3
0.5
0.7
0.5
0.6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5.8
5.6
5.5
6.1
6.0
5.6
5.9
5.8
4.9
5.8
5.9
5.8
5.8
6.0
(1.982)
(1.204)
(0.860)
(1.867)
(1.867)
(1.499)
(1.499)
(1.927)
(1.927)
(1.867)
(1.204)
(0.718)
(0.144)
(1.580)
0.3
0.5
0.5
0.6
0.6
0.4
0.4
0.4
0.4
0.6
0.5
0.6
0.6
0.6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5.5
5.5
5.4
5.8
5.8
5.5
5.5
5.6
4.7
5.8
5.5
5.4
5.2
5.7
(2.324)
(0.775)
(1.550)
(0.822)
(0.000)
(0.643)
(0.961)
(0.000)
(1.937)
(0.775)
(0.000)
(0.480)
(0.643)
(0.612)
0.4
0.4
0.4
0.6
0.5
0.5
0.3
0.3
0.4
0.1
0.4
0.3
0.5
0.2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6.0
5.2
5.0
5.7
5.4
5.2
5.2
5.4
4.9
5.3
5.4
5.3
5.6
5.5
(1.960)
(1.096)
(0.420)
(1.960)
(1.699)
(0.775)
(1.699)
(1.681)
(2.191)
(2.101)
(1.699)
(1.074)
(0.570)
(1.139)
0.7
0.3
0.4
0.7
0.5
0.2
0.5
0.4
0.3
0.4
0.5
0.8
0.6
0.6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5.8
5.2
5.1
5.8
5.5
5.1
5.5
5.4
4.6
5.5
5.5
5.5
5.2
5.4
1708
1624
1548
1493
1273
1253
1238
1024
805
787
752
718
665
638

Labentrol 250 mg g
Bivorilan 500 mg f
Ufexil 500 mg e
Forterra 500 mg d
Ladinin 500 m g c
Citrovenot 500 mg b
Ciproxin 500 mg a
Band (cm 2 1 )

Cipro oxacin concentration, (m g/tab 6 SD) 3 10 2 2 (n 5 3) found during the analysis of seven formulations accompanied by the experimental t-value in parentheses.

Results of the analysis of cipro oxacin formulations by band intensity measurements.

TABLE III.

tions. The use of potassium brom ide results not only in

slightly lower correlation coef cients (r 5 0.980.99),
but also in calibration curves that deviate from linearity
in the high concentration range. W hile preparing calibration disks using potassium brom ide, we noticed an increase in the disk thickness with increasing cipro oxacin
hydrochloride hexahydrate content. This decreases the
surface concentration probed by the laser beam resulting
in the observed curvature of the calibration curve in the
high concentration range. M aize starch and cellulose gave
sim ilar analytical gures of merit. Results presented in
this paper were obtained with the use of maize starch.
The powder granule size and the density of disc packaging also affect the reproducibility of Raman spectra
acquired from solid sam ples. Techniques used to m inimize the in uence of these parameters involve 30,31 (1)
spectra normalization by use of internal or external standards, (2) the use of data treatment techniques such as
data smoothing and background subtraction, and (3) the
use of special sampling techniques and accessories. The
slurry technique, the step-n-repeat sampling accessory, or
any accessory that allows the acquisition of m ultiple
spectra from different sam ple regions are som e of the
sampling techniques that have been used for potentially
inhomogeneous samples.32 The rotating sample container
is another approach that provides spatial averaging of
spectra.30 In this work none of the above or any other
special sampling technique has been used. Sam ple preparation was simple: four min of hand grinding and ten
min of pressing in the IR-die cast.
A normalization procedure with the use of an internal
standard in order to compensate for variations in excitation intensity, sample orientation, and temperature uctuations, which strongly in uence Raman signals,11 was
undertaken. An internal standard should not interact with
the sample and perturb or overlap the analyte spectrum .
Internal standards should be strong emitters so that a
small quantity added to samples and standards will produce signals of adequate intensity. Sodium sulfate, which
emits at 993 cm 2 1 , was chosen according to these criteria.
The minim um concentration that could be used for the
analysis was 10% w/w, as at lower levels the 993 cm 2 1
band was dif cult to distinguish from the baseline. The
normalization procedure degraded the linearity of calibration curves resulting in correlation coef cients in the
range of 0.80.91 for all bands. The precision achieved
in the measurem ent of the internal standards band height
was 26% RSD (n 5 15). This is probably the reason why
the internal standard procedure did not work. It should
be m entioned here that, due to the failure of the internal
standard procedure, calibration data should be generated
daily. However, as pre-prepared calibration disks are
used, daily calibration using a single band readout does
not add a big overhead on the analysis time.
The stepwise m ultiple linear regression method that we
had successfully used for the quantitative analysis of liquid samples 17 was also applied. The selection of the m ost
signi cant bands was performed through the forward and
backward selection procedures by the m ultiple regression
module of the Statistica software from StatSoft, Inc. The
method demands that the number of independent variables be less than N21, where N is the number of dependent variables. As we used seven standards, up to ve
APPLIED SPECTROSCOPY

1263

(1.699)
(0.849)
(1.699)
(0.000)
(0.849)
(1.699)
(1.293)
(0.849)
(1.699)
(0.000)
(1.699)
(0.000)
(1.145)
(1.699)
0.2
0.2
0.2
0.1
0.2
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.3
0.2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
2.2
2.3
2.2
2.4
2.3
2.2
2.1
2.3
2.2
2.4
2.2
2.4
2.2
2.2
(0.104)
(1.558)
(0.981)
(1.148)
(0.736)
(1.471)
(1.862)
(0.981)
(2.241)
(0.375)
(1.471)
(0.242)
(1.282)
(1.207)
0.5
0.3
0.3
0.8
0.4
0.2
0.4
0.3
0.1
0.6
0.2
0.5
0.5
0.1
6
6
6
6
6
6
6
6
6
6
6
6
6
6

Volume 55, Number 9, 2001

Cipro oxacin concentration as determined through the current United States Pharmacopoeia (USP 24) and National Form ulary (NF 19) method 10
(n 5 3): a 5.0 6 0.1, b 5.4 6 0.2, c 5.15 6 0.06, d 5.3 6 0.2, e 4.77 6 0.03, f 4.87 6 0.01, and g 2.40 6 0.04 mg/tab 3 10 2 .
ttheoretical value for 95% con dence level: 2.776.
Manufacturer: a Bayer Hellas, b Pharm aten LTD, c Bros LTD, d Help SA, e Demo SA, f Medinova SA, and g Chrispa SA.

4.9
4.6
4.7
5.4
4.7
4.7
5.3
4.7
5.0
5.0
4.7
4.8
4.5
4.8
(2.126)
(0.172)
(1.280)
(1.917)
(1.456)
(2.052)
(0.779)
(1.971)
(1.666)
(0.130)
(1.456)
(0.588)
(0.747)
(2.030)
0.3
0.3
0.5
0.5
0.2
0.7
0.6
0.5
0.8
0.4
0.2
0.5
0.3
0.4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4.4
4.8
4.4
4.2
4.6
5.6
4.5
4.2
4.0
4.8
4.6
4.6
4.9
4.3
1708
1624
1548
1493
1273
1253
1238
1024
805
787
752
718
665
638

5.3
5.1
5.3
5.7
5.3
4.5
5.5
5.2
5.3
5.3
5.0
5.7
5.4
5.4

6
6
6
6
6
6
6
6
6
6
6
6
6
6

0.3
0.1
0.3
0.6
0.3
0.8
0.5
0.2
1.1
0.5
0.1
0.6
0.3
0.3

(1.644)
(1.225)
(1.644)
(1.994)
(1.644)
(1.074)
(1.699)
(1.550)
(0.471)
(1.019)
(0.000)
(1.994)
(2.191)
(2.191)

5.4
5.1
5.4
5.8
5.2
4.8
5.5
5.1
5.4
5.3
5.1
5.4
5.4
5.4

6
6
6
6
6
6
6
6
6
6
6
6
6
6

0.3
0.4
0.3
0.7
0.5
0.6
0.6
0.3
0.5
0.4
0.3
0.4
0.7
0.1

(0.000)
(1.162)
(0.000)
(0.952)
(0.643)
(1.644)
(0.274)
(1.441)
(0.000)
(0.387)
(1.441)
(0.000)
(0.000)
(0.000)

6
6
6
6
6
6
6
6
6
6
6
6
6
6

0.3
0.5
0.5
0.8
0.4
0.3
0.6
0.5
0.6
0.3
0.4
0.6
0.5
0.7

(1.982)
(0.860)
(1.548)
(2.267)
(1.499)
(1.982)
(1.867)
(1.204)
(1.867)
(1.982)
(0.214)
(1.006)
(0.172)
(2.096)

6
6
6
6
6
6
6
6
6
6
6
6
6
6

0.5
0.3
0.3
0.6
0.5
0.4
0.4
0.5
0.4
0.3
0.5
0.7
0.5
0.6
5.7
5.6
5.8
6.0
5.7
4.9
5.9
5.6
5.8
5.5
5.5
5.9
5.7
5.7
5.5
5.4
5.6
6.2
5.5
4.8
5.8
5.5
5.8
5.5
5.2
5.5
5.1
6.0

(1.287)
(1.441)
(2.402)
(1.917)
(1.287)
(1.550)
(2.324)
(0.965)
(1.937)
(0.961)
(0.643)
(1.428)
(1.287)
(1.096)

Labentrol 250 m g g
Bivorilan 500 mg f
Forterra 500 mg d

Ufexil 500 mg e

C 5 (0.2 6 1) 2 (3 6 1) 3 10 3 BI 1545
1 (6 6 1) 3 10 3 BI 1272 ,

Ladinin 500 mg c
Citrovenot 500 mg b
Ciproxin 500 mg a
Band (cm 2 1 )

Cipro oxacin concentration, (mg/tab 6 SD) 3 10 2 2 (n 5 3) found during the analysis of seven formulations accompanied by the experimental t-value in parentheses.

Results of the analysis of cipro oxacin form ulations by band area measurements.
TABLE IV.

1264

bands could be used to build the calibration model. Various combinations of the cipro oxacin bands were used.
The 1548, 1493, 1273, 1253, and 1238 cm 2 1 bands gave
the calibration model which best ts the data.
For band intensity measurements the calibration model
equation was

(r 5 0.997)

(1)

The application of the above model to the analysis of

the comm ercial formulations gave 4.9 6 0.1, 5.5 6 0.7,
5.7 6 0.5, 5.0 6 0.1, 4.9 6 0.2, 5.1 6 0.3, and 2.5 6
0.4 3 10 2 mg/tab for Ciproxin 500 mg, Citrovenot 500
mg, Ladinin 500 mg, Forterra 500 m g, Ufexil 500 mg,
Bivorilan 500 mg, and Labentrol 250 mg form ulations,
respectively. The relative differences between these results and those obtained through the reference method 10
are in the range of 25.710.7%. Although these results
are slightly closer to those acquired through the reference
method, there is no clear advantage to using this calibration if one also takes into account the increase in the
analysis time imposed by the need to acquire a higher
spectra region.
CO NCLUSION
The proposed analytical method provides results that
are in accordance with those obtained through the current
United States Pharmacopoeia (USP 24) and National Formular y (NF 19) method,10 while it does not require dissolution of the pharmaceutical dosage forms. As m easurem ent takes just 30 s using the analytical readout from
a single band, the proposed method can be used to replace tedious and tim e-consuming methods. FT-Raman
spectroscopy can be exploited for the quantitative analysis of other solid dosage forms. A further goal should
be the noninvasivenondestructive quantitative analysis
of solid dosage forms using spectra acquired through
their PVC blister packages.
ACKNO WLEDG MENTS
We gratefully acknowledge support, in the form of a scholarshi p to
S.G.S., from the Greek State Scholarship Foundation. The authors are
indebted to Professor Moschos G. Polissiou for provision of the experimental facilities.

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APPLIED SPECTROSCOPY

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