Analysis of Degradation Products
of Doxycycline in Solution Exposed
to Light and Elevated Temperatures
Using the Agilent 1200 Infinity Series
High Dynamic Range Diode Array
Detector Impurity Analyzer System
Application Note
Small Molecule Pharmaceuticals & Generics
Author
Abstract
A.G.Huesgen
Tetracyclines undergo epimerization and degradation if stored under inappropriate
Agilent Technologies, Inc.
conditions. Stability experiments, which are important for the determination of the
Waldbronn, Germany
expiry date, were done using doxycycline. Doxycycline was exposed to light and
elevated temperature for days. To determine decomposition products, the Agilent
1200 Infinity Series HDR-DAD Impurity Analyzer System was used. Using this
analyzer, it was possible to detect peaks with peak heights < 0.2 mAU.
DAD 1
97
60 mm
96
Percentage change for doxycycline
96.0054
95
94.4245
DAD 2
Clustered DAD
Area (%)
94
94.2277
93.7092
93
92
90.7169
91
90
3.7 mm
89
88
Peak at
start
After
17 hours
After
23 hours
After
33 hours
After
5.5 days
Introduction
Experimental
Tetracyclines are antiobiotics used in
case of bacterial infections. Typical
applications are prophlylaxis and
therapy of human and animal infections.
They are also used in animal feed as
a growth promoter. When exposed to
heat and light, tetracyclines undergo
epimerization and degradation. Often,
the degradation products have very
low antibiotic activity, and some are
toxic1,2,3. In the pharmaceutical industry,
stability tests must be done for all
pharmaceutical products on the market.
Recommendations from the International
Conference on Harmonization (ICH)4
include long-term studies, where the
product is stored at room temperature
and humidity conditions. In further
studies, the product was stored at more
extreme conditions.
Instrumentation
The1200 Infinity Series HDR-DAD Impurity
Analyzer system offers the possibility
to recognize degradation products at
very low levels, due to the low noise
behavior of this detector. In addition, the
main compound can be analyzed within
the same experiment due to the wide
dynamic range of the system.
In the following experiment, doxycycline
was dissolved and the solution was
exposed to light at 40 °C for several
days. From time to time, the solution was
analyzed to discover additional impurities
and the changes in the main compound
and impurities, which were present in the
starting solution.
Compound analyzed
Agilent 1200 Infinity Series HDR-DAD
Impurity Analyzer System:
•
•
•
Agilent 1290 Infinity DAD with
60‑mm cell (G4212A) (clustered)
OH
Agilent 1200 Infinity HDR-DAD Kit
(G2199AA)
Agilent 1290 Infinity Thermostatted
Column Compartment (G1316C)
•
Agilent 1290 Infinity Autosampler
G4226A
•
Agilent 1290 Infinity Thermostat
(G1330B)
Agilent 1290 Infinity Quaternary
Pump (G4204A)
O
HO
HO O
O
NH2
OH
H
H
H2C H OH H N(CH3)2
Agilent 1290 Infinity DAD with
3.7‑mm cell (G4212A) (clustered)
•
•
Doxycycline and impurities. Doxycycline
was purchased from Sigma-Aldrich,
Germany
•HCl
Methacycline
OH
O
CH3
HO
HO O
OH
O
NH2 •HCl
•½H2O
OH
•½CH3CH2OH
N(CH3)2
Doxycycline hyclate
OH
O
HO
HO O
O
NH2
OH
CH3
OH
N(CH3)2
6-Epidoxycycline
Chromatographic conditions
Parameter
Value
Column
Agilent ZORBAX Eclipse XDB-C8, 3 × 100 mm, 3.5 µm (p/n 961967-302)
HDR-DAD
Wavelength = 350, 254, 280 nm, reference = off, data rate = 20 Hz
Flow rate
0.8 mL/min
Mobile phases
A=water, B=Acetonitrile, C=1%TFA in water
Gradient
at 0 minutes A = 65 %, B = 15 %, C = 20 %,
at 5 minutes A = 37 %, B = 43 %, C=20 %,
at 6 minutes A = 20 %, B=60 %, C=20 %
Stop time
6 minutes
Post time
3 minutes
Column temperature
40 °C
Injection volume
1 µL, 40 °C, needle wash for 3 seconds
Sample preparation
A 20 mg sample of doxycycline was
dissolved in 1.5 mL water/ACN = 1:2,
1-µL injection volume contained 13.33 µg
of doxycycline.
2
Acquisition and evaluation
software
Agilent OpenLAB CDS ChemStation
Edition Rev.C.01.05
Results and Discussion
The high dynamic range (HDR) system
was configured during instrument
configuration. Both detectors were
clustered, and the delay volume of the
capillary connecting both detectors
was filled in. In the user interface,
the clustered detectors appear as one
detector (Figure 1).
For the analysis of doxycycline, three
wavelengths were selected: 230, 280,
and 350 nm, to ensure discovery of all
impurities. In the data file, all three
HDR‑DAD signals were stored as well as
the three signals of the 60-mm and the
three signals of the 3.7-mm cell (Figure 2).
Having dissolved 20 mg of doxycycline,
the followings experiments were done:
•
Analysis of the fresh solution
•
The solution was exposed to
daylight at 40 °C for 17 hours and
analyzed afterwards
•
Analysis after 23 hours exposure
•
Analysis after 33 hours exposure
•
Analysis after 5.5 days exposure
Clustered detectors
Figure 1. User interface in an Agilent OpenLAB CDS ChemStation.
800
600
400
200
0
DAD1 A, Sig = 230,10
DAD1 B, Sig = 350,10
DAD1 C, Sig = 280,10
LNG1 A, Sig = 230,10
LNG1 B, Sig = 350,10
LNG1 C, Sig = 280,10
SHT1 A, Sig = 230,10
SHT1 B, Sig = 350,10
SHT1 C, Sig = 280,10
1
DAD-HDR
60-mm cell
3.7-mm cell
2
Figure 2. Stored signals.
3
3
4
5
min
One prerequisite for decomposition
studies is that even very small
decomposition peaks are detectable.
Therefore, the noise level must be as
low as possible. For the analysis of
doxycycline, the P to P noise level was as
mAU
800
700
600
500
400
300
200
100
mAU
-0.042
-0.044
-0.046
-0.048
-0.050
-0.052
-0.054
-0.056
-0.058
Figure 4 shows chromatograms of the
fresh and the exposed solution after
5.5 days.
P to P noise~ 0.013mAU
Impurities 1, 2, and 3 were present from
the beginning. Visibility of Impurity 4
started after 17 hours. After 5.5 days and
exposure to light at 40 °C, Impurities 5 to
13 were detected.
Doxycycline
0.94 0.96 0.98 1.00 1.02 1.04 1.06 1.08 1.10 min
mAU
2.5
Peak height = 0.125 mAU
2
1.5
low as 0.013 mAU (Figure 3). This enabled
the detection of peaks with a peak height
below 0.2 mAU.
1.541
1
0.5
2.191
1.959
1.751
2.334
2.391
0
-0.5
1.6
1.8
2
2.2
2.4 min
0
1
2
3
4
5
min
1.5
2.0
2.5
3.0
3.5
min
Impurity 1
30
4.3
20
Impurity 4
10
4.4
4.5
4.6
4.7
4.8
Impurity 3
0
-10
1
2
3
4
5
min
Figure 4. Degradation of doxycycline after 5.5 days, overlay of start chromatogram and degradation chromatogram.
4
4.9
Impurity 13
Impurity 11
mAU
14
12
10
8
6
4
2
0
-2
Impurity 2
Impurity 12
Analysis directly after dissolution
Analysis after exposure to light at 40 °C for 5.5 days
Impurity 9
Impurity 10
Impurity 8
Impurity 7
Impurity 6
mAU
14
12
mAU 10
8
70 6
4
60
2
50 0
-2
40
Impurity 5
Figure 3. Analysis of doxycycline and determination of noise level.
5
min
Figure 5 shows the process of
degradation after 17 hours, 23 hours,
33 hours, and 5.5 days.
96
95
94
Area (%)
The percentage of doxycycline decreased
continuously, whereas Impurities 1
and 2 increased. Impurity 2 increased
continuously, whereas Impurity 1
stagnated after the first increase. After
17 hours, Impurity 4 occurred and
increased even further for the duration of
the exposure. After 5.5 days, additional
small impurities became visible.
93
92
91
90
89
88
Conclusion
Peak
at start
After
17 hours
After
23 hours
After
33 hours
After
5.5 days
Percentage changes for impurites
4.5
4
Area (%)
3.5
3
Peaks at start
2.5
After 17 hours
2
After 23 hours
After 33 hours
1.5
After 5.5 days
1
0.5
0
Impurity 1
Impurity 2
Impurity 3
Impurity 4
Additional small peaks after 5.5 days
0.25
0.2
Area %
Stability tests are frequently done in the
pharmaceutical industry to determine
the date of expiry for a pharmaceutical
product. To detect degradation products
at trace levels, the Agilent 1200 Infinity
Series HDR-DAD Impurity Analyzer
System was used for the decomposition
process of doxycycline. Doxycycline
was exposed to daylight at 40 °C over
5.5 days, and the degradation was
monitored. It was possible to detect
impurities with < 0.2 mAU of peak
height. After 5.5 days of exposure, the
percentage of doxycycline was reduced
by 5.3 %, while additional impurities
occurred and the amount of impurities
already present in the original solution
increased proportionally.
Percentage change for doxycycline
97
0.15
0.1
0.05
0
Impurity 5
Impurity 6
Impurity 7
Figure 5. Process of degradation.
5
Impurity
Impurity 9 Impurity 10 Impurity 11 Impurity 12 Impurity 13
References
1. R. Injac, V. Djordjevic-Milic,
B. Srdjenovic “Thermostability
Testing and Degradation Profiles
of Doxycycline in Bulk, Tablets,
and Capsules by HPLC” Journal of
Chromatographic Science, Vol. 45
October (2007).
2. L.A. Mitscher “The Chemistry of the
Tetracycline Antibiotics” Medical
Research Series, vol. 9. Marcel Dekker,
New York, NY, p. 123 (1978).
3. Y. Liang, M.B. Denton, R.B. Bates
“Stability studies of tetracycline
in methanol solution” Journal of
Chromatography A, 827: p.45–55
(1998).
4. HPLC and Pharmaceutical Stability
Studies, Microbac Laboratories, Inc.
(2011) http://www.microbac.com/
uploads/Technical%20Articles/pdf/
HPLC%20and%20Pharmaceutical%20
Stability%20Studies.pdf, accessed
February (2014).
www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc., 2014
Published in the USA, March 1, 2014
5991-4044EN