Identification of furosemide photodegradation products in water-acetonitrile mixture

Article abstract of DOI:10.1248/cpb.c15-00122

The aim of this study was to identify the chemical structure of the photodegradation products of furosemide in a water-acetonitrile mixture (1:1). Furosemide solution was irradiated with a D65 fluorescent lamp and the products were isolated by preparative HPLC. The fractions were evaporated to dryness in vacuo. The purity of the photodegradation products was measured by HPLC. The purity of products 1, 3, and 4 was greater than 90%, whereas that of product 2 was 13%, therefore, photodegradation product 2 was unstable. We identified photodegradation products 1 and 3 as 4-chloro-5-sulfamoylanthranilic acid and 4-hydroxy-N-furfuryl-5-sulfamoylanthranilic acid, respectively, by LC/MS and NMR. Additionally, we assumed that photodegradation product 4 was methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-(methyleneamino)-5-sulfamoylbenzoate by LC/MS and NMR. This showed that furosemide underwent hydrolysis and substitution, and reacted with the acetonitrile under the light of a D65 fluorescent lamp. We were furthermore able to determine the elution times of the photodegradation products of furosemide by applying the Japanese Pharmacopoeia chromatographic method for related substances to the isolated products.

Full text of DOI:10.1248/cpb.c15-00122

Vol. 63, No. 8
Chem. Pharm. Bull. 63, 617–627 (2015)
617
Note
Identification of Furosemide Photodegradation Products in
Water–Acetonitrile Mixture
a,b
a
a
a
Shinji Katsura, Nobuo Yamada, Atsushi Nakashima, Sumihiro Shiraishi,
,
b
c
Takayuki Furuishi,* and Haruhisa Ueda
a
Research & Development Division, Teva Pharma Japan Inc.; 1–24–11 Taiko, Nakamura-ku, Nagoya 453–0801,
b
Japan: Department of Physical Chemistry, Hoshi University; 2–4–41 Ebara, Shinagawa-ku, Tokyo 142–8501,
Japan: and Department of Pharmaceutical Sciences, Nihon Pharmaceutical University; 10281 Komuro, Ina-machi
c
Kitaadachi-gun, Saitama 362–0806, Japan.
Received February 5, 2015; accepted May 29, 2015
The aim of this study was to identify the chemical structure of the photodegradation products of furose-
mide in a water–acetonitrile mixture (1:1). Furosemide solution was irradiated with a D65 fluorescent lamp
and the products were isolated by preparative HPLC. The fractions were evaporated to dryness in vacuo.
The purity of the photodegradation products was measured by HPLC. The purity of products 1, 3, and 4 was
greater than 90%, whereas that of product 2 was 13%, therefore, photodegradation product 2 was unstable.
We identified photodegradation products 1 and 3 as 4-chloro-5-sulfamoylanthranilic acid and 4-hydroxy-N-
furfuryl-5-sulfamoylanthranilic acid, respectively, by LC/MS and NMR. Additionally, we assumed that
photodegradation product 4 was methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-(methyleneamino)-5-sul-
famoylbenzoate by LC/MS and NMR. This showed that furosemide underwent hydrolysis and substitution,
and reacted with the acetonitrile under the light of a D65 fluorescent lamp. We were furthermore able to
determine the elution times of the photodegradation products of furosemide by applying the Japanese Phar-
macopoeia chromatographic method for related substances to the isolated products.
Key words furosemide; photodegradation; water–acetonitrile mixture; identification; Japanese Pharmaco-
poeia
3)
Furosemide
(4-chloro-N-furfuryl-5-sulfamoylanthranilic reported, to the best knowledge of the authors, no research
acid) is widely used as a diuretic in the treatment of edema. has yet been carried out with respect to determination of
It is available in solid and liquid dosage forms, but it is well photodegradation products of furosemide derived from water–
known that furosemide is unstable in aqueous and organic acetonitrile mixture.
solution. In aqueous solutions, furosemide is hydrolyzed to
Therefore, we have isolated and identified the photodegra-
saluamine (4-chloro-5-sufamoylanthranilic acid (CSA)) and dation products of furosemide in water–acetonitrile mixture
1,2)
furfuryl alcohol. Regarding organic solutions, Moore and by preparative HPLC, LC/MS and NMR. Furthermore, we
Sithipitaks reported a photodegradation pathway in oxygen- were able to determine the elution times of the photodegrada-
3)
free methanol. Furosemide degrades rapidly at acidic pH, tion products of furosemide by applying the Japanese Pharma-
although it is stable at alkaline pH under laboratory conditions copoeia chromatographic method for related substances to the
4
–6)
and diffuse daylight.
In addition, furosemide is a photosen- isolated products.
sitive drug and its degradation products and impurities have
7
–9)
been reported.
Asker and Ferdous also reported that furo-
semide is most stable at pH 7 and that the photodegradation
10)
of furosemide follows first-order kinetics. Because of such
backgrounds, numerous analytical methods have been devel-
11–13)
oped for assaying furosemide in pharmaceutical samples.
Furosemide related substances are determined by HPLC in
the purity test in the Japanese Pharmacopeia. In this method,
water–tetrahydrofuran–acetic acid mixture is used as mobile
phase and acetic acid/water–acetonitrile mixture is used to
dissolve the sample. On the contrary, recent progress in LC/
MS method has spurred a great deal of research into the
determination of related substances in purity test for active
pharmaceutical ingredients and drug formulations. We have
developed a new analytical method for determining the struc-
ture of furosemide impurities using by LC/MS method. In
this novel method, the mixture of formic acid/acetonitrile as
mobile phase and the mixture of water–acetonitrile as solvent
Fig. 1. Discoloration of Furosemide in Water–Acetonitrile Mixture
to dissolve the sample are selected, respectively. Although the (1:1) under D65 Fluorescent Lamp Light (600000lx·h)
photodegradation product of furosemide in methanol has been
(a) Before irradiation. (b) After irradiation.
*
To whom correspondence should be addressed. e-mail: t-furuishi@hoshi.ac.jp
©
2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
Fig. 2. Preparative HPLC Chromatogram of Photolyzed Furosemide Solution
(a) Photodegradation product 1. (b) Photodegradation product 2. (c) Photodegradation product 3. (d) Photodegradation product 4.
Experimental
of a vacuum evaporator (SD905, GL Sciences), an organic
Materials Furosemide was purchased from a commercial solvent recovery system (SC907, GL Sciences), a water chiller
source. Acetonitrile, methanol, tetrahydrofuran, acetic acid (CCM-1100, GL Sciences), and a nitrogen generator (NM910,
and formic acid were purchased from Wako Pure Chemical GL Sciences). The temperature of the heat block and chiller
Industries, Ltd. (Osaka, Japan) and Kanto Chemical Co., Inc. were 30 and 5°C, respectively, and the nitrogen flow rate was
(
Tokyo, Japan). The polytyrosine-1,3,6 LC/MS calibration 2.0–5.0L/min.
standard was purchased from Thermo Fisher Scientific Inc.
Purity of Furosemide Photodegradation Products The
Waltham, MA, U.S.A.). Dimethyl sulfoxide-d6 (DMSO-d6) photodegradation products (2mg) were dissolved in 2.2% ace-
(
containing 0.05% (v/v) tetramethylsilane (TMS) was pur- tic acid in water–acetonitrile mixture (1:1, 2mL). The sample
chased from Wako Pure Chemical Industries, Ltd. All re- solutions were analyzed by HPLC. The HPLC system con-
agents were commercially available and analytical grade.
sisted of a separation module (Alliance W2695, Waters, Mil-
Preparation of Furosemide Photodegradation Products ford, MA, U.S.A.) and a photodiode array detector (W2998,
Furosemide (1g) was dissolved in water–acetonitrile mixture Waters). The purity of the sample solutions was calculated by
(1:1) (100mL) and an aliquot (10mL) was transferred into a the area percentage method. The HPLC method conformed to
glass vial. The solution was exposed to D65 fluorescent lamp the Japanese Pharmacopoeia for substances related to furose-
light in a light stability cabinet (LTL200 D3 CJH, Nagano Sci- mide. A Mightysil RP-18 GP column (4.6mm i.d.×250mm,
ence, Japan) for 1 week at an illumination intensity of 4000lx. 5µm particle size, Kanto Chemical) was used. The photodiode
The temperature was 25°C and the relative humidity was 60%. array detector was set at 210–400nm. The temperature of
Isolation of Furosemide Photodegradation Products the column oven was 25°C. The mobile phase was water–
Water–acetonitrile mixture (1:1, 5mL) was added to a por- tetrahydrofuran–acetic acid (70:30:1) and the flow rate was
tion of the photolyzed furosemide solution (5mL). The solu- 1.25mL/min. The sample solution (20µL) was injected into
tion (2mL) was injected into a preparative HPLC system. the HPLC system and the run time was 60min.
The preparative HPLC system consisted of a UV-Vis detector
Mass Spectra of Furosemide Photodegradation Products
(
UV702, GL Sciences, Tokyo, Japan), a column oven (CO- The photodegradation products were dissolved in water–aceto-
705C, GL Sciences), an auto-sampler (G-Prep AS, GL Scienc- nitrile mixture (1:1) and the concentration was adjusted to
es), a fraction collector (FC204, GL Sciences) and a degasser 0.005mg/mL. The sample solutions were analyzed by LC/
GASTORR PG-12, GL Sciences). An Inertsil ODS-3 column MS. The LC/MS system consisted of an HPLC system
20mm i.d.×250mm, 5µm particle size, GL Sciences) and an (UltiMate3000, Dionex, Sunnyvale, CA, U.S.A.) and a mass
(
(
Inertsil ODS-3 guard column (20mm i.d.×50mm, 5µm par- spectrometer (LTQ Orbitrap Discovery, Thermo Fisher Sci-
ticle size, GL Sciences) were used. The detection wavelength entific, Waltham, MA, U.S.A.). The HPLC system consisted
was 272nm. The temperature of column oven was 30°C. The of a pump (HPG-3200RS, Dionex), an auto-sampler (WPS-
flow rate was 16mL/min. The mobile phases were 0.1% formic 3000RS, Dionex), a column compartment (TCC-3000RS, Di-
acid in water (solvent A) and acetonitrile (solvent B). The gra- onex), and a diode array detector (DAD-3000RS, Dionex). The
dient program was as follows: 0 to 30min, 50% A and 50% B; mass spectrometer consisted of a linear ion trap (LTQ XL,
30 to 35min, linear gradient from 50 to 10% A; 35 to 65min, Thermo Scientific) and a spectrometer (LTQ Orbitrap, Thermo
10% A and 90% B; 65 to 70min, linear gradient from 10% Scientific). An Inertsil ODS-3 column (2.1mm i.d.×150mm,
to 50% A; 70 to 90min, 50% A and 50% B. The fractions 5µm particle size, GL Sciences) was used. The wavelength
containing the photodegradation products were evaporated to of the diode array detector was 210–400nm. The temperature
dryness in vacuo. The vacuum evaporation system consisted of the column oven was 30°C. The mobile phases were 0.1%

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
619
Fig. 3. HPLC Chromatograms at 272nm and UV Spectra of Furosemide Photodegradation Products and Furosemide
(a) Furosemide. (b) Photodegradation product 1. (c) Photodegradation product 2. (d) Photodegradation product 3. (e) Photodegradation product 4. (f) Blank.
Table 1. Purity Data of Furosemide Photodegradation Products
Sample
a)
b)
Retention time (min)
Relative retention time
Purity (%)
Photodegradation product 1
5.94
12.54
3.44
0.32
0.68
0.19
0.49
0.74
94.9
13.0
86.4
99.5
98.5
Photodegradation product 2
Degradation product of photodegradation product 2
Photodegradation product 3
9.13
Photodegradation product 4
13.79
a) Relative retention to furosemide (retention time=18.53min). b) Calculated by area percentage method (except blank peaks).
formic acid in water (solvent A) and acetonitrile (solvent B). electrospray ionization (ESI) and negative ion mode, with a
The flow rate was 0.2mL/min. The gradient program was as resolution of 30000. The sheath gas flow rate and auxiliary
follows: 0 to 10min, 80% A and 20% B; 10 to 18min, linear gas flow rate were 50 and 10 arb. units, respectively. The ion
gradient from 80 to 65% A; 18 to 50min, 65% A and 35% B; spray voltage was 3kV. The capillary temperature was 330°C.
2
3
5
0 to 51min, linear gradient from 65 to 80% A; 51 to 75min, The MS and MS measurements were performed by collision-
8
0% A and 20% B. The sample solution (2µL) was injected induced dissociation (CID) with collision energy of 35eV. The
into the LC/MS system. The spectrometer was operated in spectrometer was calibrated with a polytyrosine-1,3,6 standard

6
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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
Fig. 4. UV Chromatograms of Furosemide and Furosemide Photodegradation Products at 272nm
(a) Furosemide. (b) Photodegradation product 1. (c) Photodegradation product 2. (d) Photodegradation product 3. (e) Photodegradation product 4.
and ultrapure helium was used.
Identification of Furosemide Photodegradation Prod-
ucts The photodegradation products (5mg) were dissolved in 1.5s. The number of scans for the H- and C-NMR was 32
were 5 and 2s, respectively, and the relaxation delay for the
1
1
1
13
1
13
H– H COSY, H– C HMQC, and H– C HMBC NMR was
1
13
1
1
DMSO-d containing 0.05% (v/v) TMS (0.6mL). The samples and 1000, respectively. The number of scans for the H– H
6
1
13
1
13
were analyzed by NMR (JNM-ECA-500, JEOL, Tokyo, COSY, H– C HMQC, and H– C HMBC NMR was 32.
Japan). The NMR spectrometer consisted of a 4mm CP-MAS
probe (JEOL), a 500MHz spectrometer (ECA-500, JEOL), Results and Discussion
and a compressor (SLP-07E-S53BP, Anest Iwata, Yokohama,
Preparation of Furosemide Photodegradation Products
1
13
Japan). One- and two-dimensional NMR spectra ( H-, C-, Bundgaard et al. and Yagi et al. reported that neutral furose-
1
1
1
13
H– H correlation spectroscopy (COSY), H– C heteronuclear mide (furosemide in its unionized form) is extremely suscep-
1
13
5,6)
multiple quantum correlation (HMQC), H– C heteronuclear tible to photodegradation in aqueous solution.
Rapid deg-
multiple bond correlation (HMBC)) were recorded at 25°C. radation was also observed in mixtures of water and organic
Tetramethylsilane (TMS) was used as an internal standard, solvents, such as methanol and acetonitrile. This suggests that
and all chemical shifts were assigned relative to its peak at furosemide is not ionized in water–acetonitrile mixture (1:1)
1
13
δ 0.00ppm. The relaxation delays for the H- and C-NMR and that the photodegradation of furosemide is accelerated by

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
621
1
Fig. 5. Negative Ion Mode ESI-MS Spectra of Photodegradation Products of Furosemide
(a) Photodegradation product 1. (b) Photodegradation product 2. (c) Photodegradation product 3. (d) Photodegradation product 4.
1
Table 2. MS Data of Furosemide and Furosemide Photodegradation Products
a)
Sample
Molecular ion
Accurate mass (m/z)
Elemental composition
−
Furosemide
[M−H]
[M−H]
[M−H]
329.0001
248.9741
555.0495
NI
C H ClN O S
1
2
10
2
5
−
Photodegradation product 1
Photodegradation product 2
Degradation product of photodegradation product 2
Photodegradation product 3
Photodegradation product 4
C H ClN O S
7 6 2 4
−
C H N O S
20 19 4 11 2
NI
NI
C H N O S
−
[M−H]
[M−H]
311.0337
352.0598
1
2
11
2
6
−
C H N O S
14 14 3 6
NI, not ionized. a) Calculated from the accurate mass.
2
3
Table 3. MS –MS Data of Furosemide and Furosemide Photodegradation Products
Precursor ion Product ion accu- Product ion elemental
Mass change
(Da)
Neutral loss
composition
n
Sample
MS
a)
b)
(m/z)
rate mass (m/z)
composition
Furosemide
2
3
2
3
2
3
2
329.00
285.01
248.97
204.98
555.05
511.06
311.03
285.0101
204.9842
204.9841
126.0114
511.0589
467.0696
267.0441
C H ClN O S
−44
−84
−44
−79
−44
−44
−44
−65
−42
−44
CO2
1
1
10
2
3
C H ClN O S
C H O
6
6
2
2
5
4
Photodegradation product 1
Photodegradation product 2
Photodegradation product 3
Photodegradation product 4
C H ClN O S
CO2
HNO2S
CO2
6
6
2
2
C H ClN
6
5
C H N O S
1
9
19
4
9
2
C H N O S
CO2
1
8
19
4
7
2
C H N O S
CO2
1
1
11
2
4
5
2
46.0403
C H NO
H NOS
1
2
8
3
2
3
352.06
310.05
310.0496
266.0599
C H N O S
C H O
1
2
12
3
5
2
2
C H N O S
CO2
1
1
12
3
3
a) Base peak ion monitored. b) Calculated from the accurate mass.

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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
Fig. 6. Fragmentation Patterns of Photodegradation Products 1 and 3 by CID with Collision Energy of 35eV
(a) Photodegradation product 1. (b) Photodegradation product 3.
1
13
Fig. 7. 500MHz H– C HMQC NMR Spectrum of Photodegradation Product 1 in DMSO-d at 25°C
6

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
623
1
13
Fig. 8. 500MHz H– C HMQC NMR Spectrum of Photodegradation Product 3 in DMSO-d at 25°C
6
acetonitrile. Figure 1 shows the color change of furosemide fractions were dried in vacuo to obtain 1 as yellow crystals, 2
in water–acetonitrile mixture (1:1) from colorless to yellow as brown crystals, and 3 and 4 as white crystals.
under D65 fluorescent lamp light (600000lx·h), suggesting
Purity of Photodegradation Products Figure 3 shows
that it underwent photodegradation.
the HPLC chromatograms and UV spectra of furosemide and
Isolation of Furosemide Photodegradation Products in its photodegradation products. Product 2 contained two large
Water–Acetonitrile Mixture (1:1) Figure 2 shows the pre- peaks. We assumed that the elution order of the photodegrada-
parative HPLC chromatogram of the photolyzed furosemide tion products was the same as for preparative HPLC. Thus,
solution. The furosemide photodegradation products were iso- the two peaks at 3.44 and 12.54min were assigned as the
lated by preparative HPLC. Products 1–4 had retention times product 2 degradation product and product 2, respectively.
of 8–10, 11–12, 13–15, and 25–26min, respectively. The HPLC
Changes in the UV spectra of furosemide in various sol-

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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
1
13
Fig. 9. 500MHz H– C HMQC Spectrum of Degradation Product of Photodegradation Product 2 in DMSO-d at 25°C
6
vents after exposure to fluorescent light and UV light were degradation product, respectively, because the UV spectrum
4
)
3)
reported by Rowbotham et al., Moore and Sithipitaks, and of the peak at 14.32min was similar to that of Fig. 2(c) (peak
10)
Asker and Ferdous. The UV spectra of photodegradation at 12.54min). In addition, product 1 and the degradation prod-
products 2 and 3 were similar to the spectrum of furosemide. uct of product 2 could not be separated under this LC condi-
However, the UV spectra of products 1 and 4 were different tion. To obtain a mass spectrum of product 1, a pure sample
from that of furosemide. The purity of photodegradation prod- was required.
1
ucts 1, 3 and 4 were greater than 90%, whereas that of pho-
Figure 5 shows the negative ion mode ESI-MS spectra of
todegradation product 2 was about 13% (Table 1). Therefore, the furosemide photodegradation products. The negative ion
1
photodegradation product 2 was unstable.
mode ESI-MS spectra show a deprotonated molecular ion
−
1
2,3
Mass Spectra of Furosemide Photodegradation Products ([M−H] ) and the MS and MS results are shown in Tables
Figure 4 shows the UV chromatograms of furosemide and its 2 and 3, respectively. Figure 6 shows the fragmentation pat-
photodegradation products at 272nm. The peaks at 14.32 and terns of photodegradation products 1 and 3 by CID with colli-
5
.08min were identified as photodegradation product 2 and its sion energy of 35eV. These results suggest that products 1 and

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
625
1
13
Fig. 10. 500MHz H– C HMQC Spectrum of Degradation of Photodegradation Product 4 in DMSO-d at 25°C
6
3
were CSA and 4-hydroxy-N-furfuryl-5-sulfamoylanthranilic sistent with products 1 and 3 being CSA and 4-hydroxy-N-fur-
acid, respectively. In addition, photodegradation product 4 was furyl-5-sulfamoylanthranilic acid. Furthermore, the carboxyl
2
identified by the MS spectrum as a product of the reaction group of the furosemide degradation products underwent H-D
between furosemide and acetonitrile.
exchange in DMSO-d6.
Figure 9 shows the 500MHz H– C HMQC NMR spec-
1
13
Identification of Furosemide Photodegradation Products
1
H-NMR has been used to identify and assay furosemide trum of the degradation product of photodegradation product
and its degradation product (CSA) in tablet and injection 2 in DMSO-d at 25°C. The degradation product of photodeg-
6
11,14)
1
13
formulations.
Figures 7 and 8 show the 500MHz H– C radation product 2 was identified as 4-hydroxy-2-(prop-2-yn-
HMQC NMR spectra of photodegradation products 1 and 3 in 1-ylamino)-5-sulfamoylbenzoic acid. However, photodegrada-
DMSO-d at 25°C, respectively. The NMR spectra were con- tion product 2 could not be identified by NMR and LC/MS.
6

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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
1
13
Fig. 11. 500MHz H– C HMBC Spectrum of Degradation of Photodegradation Product 4 in DMSO-d at 25°C
6
(a) Full spectrum. (b) Enlarged detail.
1
13
Figures 10 and 11 show the 500MHz H– C HMQC and DMSO-d (data not shown). We assumed that the product 4
6
1
13
H– C HMBC NMR spectra of photodegradation product 4 might be methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-
in DMSO-d at 25°C. Although the signal at δ 6.38–6.43ppm (methyleneamino)-5-sulfamoylbenzoate from the LC/MS and
6
1
was observed as a singlet in the H-NMR spectrum of prod- NMR results.
1
13
uct 4, H– C HMQC NMR in acetone-d6 confirmed that
We irradiated the related compounds methyl 4-hydroxyben-
1
the H-NMR signal of H-3 overlapped with that of H-2 in zoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate,

Vol. 63, No. 8 (2015)
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627
6
57–662 (1974).
Cruz J. E., Maness D. D., Yakatan G. J., Int. J. Pharm., 2, 275–281
1979).
Moore D. E., Sithipitaks V., J. Pharm. Pharmacol., 35, 489–493
1983).
Rowbotham P. C., Stanford J. B., Sugden J. K., Pharm. Acta Helv.,
1, 304–307 (1976).
Bundgaard H., Norgaard T., Nielsen N. M., Int. J. Pharm., 42,
17–224 (1988).
and butyl 4-hydroxybenzoate with D65 lamp light to deter-
mine whether they react with acetonitrile. The compounds
did not react with acetonitrile, suggesting that this reaction is
specific to furosemide.
2
)
(
3)
(
4
)
Conclusion
5
To identify the furosemide photodegradation products in a
water–acetonitrile mixture (1:1), we isolated the products by
preparative HPLC and identified their structures by LC/MS
and NMR. The results suggested that furosemide in water–
acetonitrile mixture (1:1) underwent hydrolysis and substitu-
tion of the Cl moiety with OH, and reacted with acetonitrile.
Furthermore, we were able to determine the elution times of
the photodegradation products of furosemide by applying the
Japanese Pharmacopoeia chromatographic method for related
substances to the isolated products.
5
)
2
6) Yagi N., Kenmotsu H., Sekikawa H., Takeda M., Chem. Pharm.
Bull., 39, 454–457 (1991).
7
)
Rapaka R. S., Roth J., Prasad V. K., Int. J. Pharm., 11, 237–247
1982).
(
8)
Miller J. H. M., Robert J. L., Sorensen A. M., J. Pharm. Biomed.
Anal., 11, 257–261 (1993).
Mizuma T., Benet L. Z., Lin E. T., J. Chromatogr. B: Biomed. Sci.
Appl., 718, 153–162 (1998).
9)
1
0) Asker A. F., Ferdous A. J., PDA J. Pharm. Sci. Technol., 50, 158–
62 (1996).
1) Hanna G. M., Lau-Cam C. A., J. AOAC Int., 76, 526–530 (1993).
1
Conflict of Interest Shinji Katsura, Nobuo Yamada,
1
Atsushi Nakashima and Sumihiro Shiraishi are employees of 12) Carda-Broch S., Esteve-Romero J., García-Alvarez-Coque M. C., J.
Teva Pharma Japan Inc.
Pharm. Biomed. Anal., 23, 803–817 (2000).
3) Ram V. R., Dave P. N., Joshi H. S., J. Chromatogr. Sci., 50, 721–726
2012).
4) Aboul-enein H. Y., Al-badr A. A., El-Din Rashed M. S., Spectrosc.
Lett., 12, 323–331 (1979).
1
(
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