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Introduction
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Present address: Department of Pharmaceutical Chemistry, National Organization for Drug Control and Research, 51 Wezaret Elzeraa, Giza, Egypt.
Visiting Scientist, Research and Development Laboratories, US Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD, 20852-1790,
USA
Abbreviations used: TDA, total detected area; USP, US Pharmacopeia.
Accuracy solutions for tetracycline hydrochloride drug substance. Accurately weighed 8, 10 and 12 mg samples of the commercial tetracycline hydrochloride (Spectrum Chemical, Shanghai,
China) in triplicate at the 80% (0.08 mg/mL) and 120% (0.12 mg/mL)
levels and six replicates at the 100% (0.1 mg/mL) level were prepared
and dissolved each in 100.0 mL of diluent.
Experimental
Precision solutions for tetracycline hydrochloride drug substance. The same six solutions at the 100% (0.1 mg/mL) level for
the accuracy study were used.
Materials
Tetracycline hydrochloride (lot L1H374), epitetracycline hydrochloride
(lot G0E261), and 4-epianhydrotetracycline hydrochloride (lot K0F299) were
USP Reference Standards (Rockville, MD, USA). Anhydrotetracycline
hydrochloride (lot SZBA299XV) was purchased from Sigma-Aldrich
(St Louis, MO, USA). Bulk tetracycline hydrochloride samples (lots
2 W3050 and 110M1693V) were obtained from Sigma-Aldrich (St Louis,
MO, USA) and Spectrum Chemical (Shanghai, China), respectively.
Tetracycline hydrochloride oral suspension (2.5 gram/100 mL) and placebo
were obtained from Care pharmacy (Rockville, MD, USA).
All reagents were of analytical grade. Water was obtained from a Milli-Q
water purication system (Millipore, Billerica, MA, USA).
Chromatographic conditions
Both Agilent HPLC 1100 and Waters 2695 Alliance systems were
controlled with Waters Empower 2 software. We tested both a Dionex
(Sunnyvale, CA, USA) Acclaim Polar Advantage II, 3 m, 4.6 150 mm
column and a Phenomenex (Torrance, CA, USA) Prodigy ODS-3, 3 m,
4.6 150 mm column. The column temperature was set at 50 C. The
autosampler temperature was kept at 4 C. Eluent components were
0.1% H3PO4 and acetonitrile. The proportion of acetonitrile was varied
from 15 to 40% in 7.5 min, back to 15% in 0.1 min, and held at 15% for
2.4 min. The ow rate was 1.0 mL/min. The detection wavelength was
280 nm. The injection volume was 10 L.
Sample solutions
The concentration for the sample solutions was set at 0.1 mg/mL. This
concentration worked for both the assay procedure and the limit of
4-epianhydrotetracycline hydrochloride procedure, so the same sample
solution was used for both the tests.
Standard solutions
The standard solution had the same concentration (0.1 mg/mL) as the
sample concentration for the assay procedures. However, the standard
solution for the limit procedure had a concentration corresponding to
that for the limit procedure. For example, the limit for tetracycline hydrochloride drug substance is 2.0%, so the standard solution concentration
was 2.0 g/mL of 4-epianhydrotetracycline hydrochloride.
Quantication limit solution. Based on the peak height and concentration from a standard solution, the quantication limit concentration
was estimated at 10 times the noise, 0.1 g/mL, equivalent to 5% of
the limit.
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1279
E. M. Hussien
0.20
4-Epianhydrotetracycline
AU
0.30
Tetracycline
Epitetracycline
0.40
0.10
Anhydrotetracycline
0.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Minutes
Figure 1. Typical HPLC chromatogram for system suitability solution containing 25 g/mL each of tetracycline hydrochloride, epitetracycline hydrochloride, 4-epianhydrotetracycline hydrochloride and anhydrotetracycline hydrochloride.
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100
(a)
(b)
99
98
97
(c)
96
95
10
15
20
25
30
Time, h
Figure 3. Stability of tetracycline hydrochloride solution containing 0.1
mg/mL at: (a) 4 C and 0.1% H3PO4 as diluent; (b) 4 C and water as
diluent; and (c) room temperature and 0.1% H3PO4 as diluent.
Concentration range
Regression line
Correlation coefcient
Intercept/nominal
(r )
Response (INR)
y = 17428x 2
y = 18305x + 23
1.000
0.999
0.1%
1.3%
y = 38878x 2
y = 36371x + 2
0.999
1.000
1.8%
2.2%
(g/mL)
Assay procedure
Drug substance
Oral suspension
Limit procedure
Drug substance
Oral suspension
73122
70120
13
13
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Acceptance criteria: r2 0.999 for assay and 0.99 for the limit.
INR = 2% for assay and 5% for the limit.
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E. M. Hussien
Table 2. Accuracy and precision results for drug substance
assay procedure
Level
Concentration
Assay
Average
RSD
(%)
(g/mL)
(%)
(%)
(%)
80
82.91
79.58
81.62
100.97
101.28
99.80
98.44
107.71
108.20
121.53
122.08
122.42
96.83
95.89
95.62
95.82
95.92
96.58
95.46
96.32
96.39
96.20
95.24
96.00
100
120
96.11
96.08
0.44
95.81
Table 3. Accuracy and precision results for the oral suspension assay procedures
Level
Accuracy
80%
100%
120%
Precision
100%
Concentration
(g/mL)
Recovery
(%)
78.87
80.49
79.89
99.67
96.37
100.94
120.93
117.06
121.69
99.8
99.7
99.3
99.4
99.1
99.3
99.2
100.2
100.1
100
RSD =
Average recovery
(%)
Concentraion
(g/mL)
Recovery
(%)
for
4-
Average
(%)
101.6
100.9
100.7
101.3
99.5
99.7
99.6
Conclusion
99.3
99.8
89.9
91.1
90.4
89.8
91.0
88.5
1.1
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Acknowledgment
The author thanks the US pharmacopeia (Rockville, MD, USA) for
the Visiting Science Program for pharmacopeial advancement.
The author is grateful to Dr Samir Wahab, director, and Dr Shane
Tan, at the Separation Science Laboratory, USP (Rockville, USA),
for valuable discussion about the tetracycline related monographs and manuscript revision. Special thank is extended to
Dr Zarima Kassymbek for her assistance in oral suspension measurements. The valuable advice and support of Alan Leeks and
Minli Liu are gratefully acknowledged.
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