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Research article

Received: 5 October 2013,

Revised: 28 November 2013,

Accepted: 17 January 2014

Published online in Wiley Online Library: 12 March 2014

(wileyonlinelibrary.com) DOI 10.1002/bmc.3161

Development and validation of an HPLC method


for tetracycline-related USP monographs
Emad M. Hussien*
ABSTRACT: A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride
and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 m, 150 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile
at a ow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation
studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast
degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling
the autosampler temperature at 4 C and using 0.1% H3PO4 as diluent. The robustness of the method was tested starting with
the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear
over the range 80120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50150% of the acceptance
criteria specied in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantication
for 4-epianhydrotetracycline hydrochloride was 0.1 g/mL, 20 times lower than the acceptance criteria. The method was
specic, precise, accurate and robust. Copyright 2014 John Wiley & Sons, Ltd.
Keywords: tetracycline hydrochloride; impurities; US Pharmacopeia monographs; reversed-phase HPLC; method development

Introduction

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Tetracycline hydrochloride is a broad-spectrum antibiotic used to


treat human and animal infections. Tetracycline hydrochloride
samples usually contain certain impurities that are structurally related to tetracycline and typically include 4-epianhydrotetracycline
hydrochloride. The latter can be formed when tetracycline hydrochloride samples are stored under adverse conditions of temperature and humidity (Lindauer et al., 1976). This compound has been
reported to be toxic (Aszalos, 1985) and should not be present in
levels that exceed the limits specied by the US Pharmacopeia
(USP) (US Pharmacopeial Convention, 2013).
The USP prescribes a chromatographic method for the assay of tetracycline hydrochloride and for the limit of 4-epianhydrotetracycline
hydrochloride impurity in the drug substance monograph (US Pharmacopeial Convention, 2013). The same method is used in many
tetracycline hydrochloride dosage-form monographs, including
oral suspension, capsules, tablets, injection, ointement, ophthalmic ointement and ophthalmic suspension. This method uses
dimethyl formamide as a diluent and in the mobile phase, which
is not desirable in terms of handling and environmental impact.
Some other USP tetracycline hydrochloride monographs refer to
the method prescribed in the general chapter <226 > for the
limit of 4-epianhydrotetracycline hydrochloride. This method
uses open-column technology that is characterized by slow
separation, which can affect the accuracy of determination of
4-epianhydrotetracycline hydrochloride impurity.
Several chromatographic methods were suggested in the
literature for separation and determination of tetracycline in the
presence of other antibiotics such as oxytetracycline, chlortetracycline, doxycycline etc. These methods include HPLC with UV
(Cinquina et al., 2003; Posyniak et al., 1998; Vias et al., 2004),
chemiluminescence (Santiago Valverde et al., 2007) and tandem
mass spectrometry detection (Blasco et al., 2009; Carrasco-Pancorbo

Biomed. Chromatogr. 2014; 28: 12781283

et al., 2008). Unfortunately, none of these methods considered the


detection and determination of tetracycline impurities. Some other
methods such as capillary electrophoresis (Tjrnelund and Honor
Hansen, 1996; Zhang et al., 1992) and HPLC with tandem mass
spectrometry (Jia et al., 2009) and chemiluminescence detection
(Pena et al., 2000) were proposed for separation and determination
of tetracycline and its major degradation products epitetracycline,
epianhydrotetracycline and anhydrotetracycline. Such instruments
are expensive and not always available in laboratories for routine
analysis. Thus, none of these methods can be recommended as a
compendium method for the assessment of tetracycline and its
degradates. Other chromatographic methods had been reported
for the separation and assessment of tetracycline hydrochloride
and its related compounds (Hendrix et al., 1993; Khan et al., 1990;
Wolfs et al., 1986; Tsuji and Robertson, 1976), but they are tedious
and employ older columns.
This paper describes a simple, fast, reliable reversed-phase
gradient HPLC method for modernizing USP tetracycline hydrochloride-related monographs and general chapter <226>. An
accelerated stress degradation study was performed, and results
showed that the degradants and impurities were well resolved
from the main peak, conrming that the method is stability* Correspondence to: Emad M. Hussien, Department of Pharmaceutical
Chemistry, National Organization for Drug Control and Research, 51
Wezaret Elzeraa, Giza, Egypt. Email: emadhussien@yahoo.com

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.

Copyright 2014 John Wiley & Sons, Ltd.

HPLC method for tetracycline-related USP monographs


indicating. The system suitability test was developed as a part of
a validation package for pharmacopeial methods. The method
was validated for the assay of tetracycline hydrochloride and limits
of 4-epianhydrotetracycline hydrochloride in pharmaceutical bulk
and oral suspension.

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.

System suitability solution


To detect as many impurities as possible, a system suitability solution that
contained 25 g/mL of each of the following compounds was used: tetracycline hydrochloride, epitetracycline hydrochloride, 4-epianhydrotetracycline
hydrochloride and anhydrotetracycline hydrochloride.

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.

Placebo solution. A 5.0 mL aliquot of the suspension placebo was


transferred to a 100 mL volumetric ask, diluted to volume with diluent,
and mixed. About 8.0 mL of this solution was diluted to 100.0 mL with
the diluent and mixed.
Linearity solutions for tetracycline hydrochloride oral suspension. A 125% solution was prepared by dissolving 12.5 mg of USP
tetracycline hydrochloride reference standard in 100.0 mL of placebo
solution. Four other solutions were diluted from the 125% solution with
the diluent (9.0 to 10.0, 8.0 to 10.0, 7.0 to 10.0 and 6.0 to 10.0 mL).
Accuracy solutions for tetracycline hydrochloride oral suspension. Accurately weighed 8, 10 and 12 mg samples of the USP
tetracycline hydrochloride RS, in triplicate were prepared at each
level and dissolved each in 100.0 mL of placebo solution.
Precision solutions for tetracycline hydrochloride oral suspension. Six sample solutions were prepared from the formulation.

Validation solutions for the limit of 4-epianhydrotetracycline


hydrochloride procedures
Solution A: stock sample solution (1.0 mg/mL). A 50 mL solution
was prepared in diluent at 10 times the concentration of the sample
solution (0.1 mg/mL).
Solution B: stock impurity solution (5.0 g/mL). An accurately
weighed 2.5 mg of USP 4-epianhydrotetracycline hydrochloride reference standard was dissolved in 500.0 mL of diluent.
Linearity solutions. The stock impurity solution was serially diluted
from 5.0 to 25.0 mL, from 15.0 to 50.0 mL, from 10.0 to 25.0 mL, from
5.0 to 10.0 mL and from 15.0 to 25.0 mL with diluent to prepare ve linearity solutions at 50% (1.0 g/mL), 75% (1.5 g/mL), 100% (2.0 g/mL),
125% (2.5 g/mL) and 150% (3.0 g/mL) levels.
Accuracy/precision solutions. The stock impurity solution and the
stock sample solution were diluted with diluent following the scheme
below to prepare nine spike solutions at three levels, in triplicate at each
level:

150% (3.0 g/mL) level 6.0 mL B and 1.0 mL A to 10.0 mL;


100% (2.0 g/mL) level 10.0 mL B and 2.5 mL A to 25.0 mL;
50% (1.0 g/mL) level 10.0 mL B and 5.0 mL A to 50.0 mL;
0% 1.0 mL A to 10.0 mL.

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.

Validation solutions for the assay procedures


Diluent. The diluent was prepared by mixing 2 mL of 85% phosphoric
acid with 2 L of water.

Biomed. Chromatogr. 2014; 28: 12781283

Acid-, base- and oxidizer-stressed samples. The samples were


prepared by dissolving 10 mg of USP tetracycline hydrochloride RS in
10 mL of 0.1 M hydrochloric acid, 0.1 M sodium hydroxide or 0.03%
hydrogen peroxide. The samples were protected from light at ambient
temperature for 24, 24 and 6 h, respectively. The samples were diluted
to 0.1 mg/mL with diluent before analysis.

Copyright 2014 John Wiley & Sons, Ltd.

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Linearity solutions for tetracycline hydrochloride drug substance. A


125% solution was prepared by dissolving 12.5 mg of USP tetracycline
hydrochloride reference standard in 100.0 mL of diluent. Four other
solutions were diluted from the 125% solution (9.0 to 10.0, 8.0 to 10.0,
7.0 to 10.0 and 6.0 to 10.0 mL).

Tetracycline hydrochloride forced degradation

E. M. Hussien

A heat- and humidity-stressed sample. A heat- and humidity-stressed


sample was prepared by storing 10 mg of USP tetracycline hydrochloride RS
in a humidity chamber set at 80 C and 80% humidity for 12 days. The samples were diluted to 0.1 mg/mL with diluent before analysis.
A light-stressed sample. The light-stressed sample was prepared by
exposing 10 mg of USP tetracycline hydrochloride RS to UV light
2
(200 W h/m ) for 7 h and then white light (1200 kx h) for 36 h in a
photostability chamber. The samples were diluted to 0.1 mg/mL with
diluent before analysis.

Results and discussion


Method development
Figure 1 shows the separation of tetracycline hydrochloride and its
isomers epitetracycline hydrochloride, 4-epianhydrotetracycline
hydrochloride and anhydrotetracycline hydrochloride on a C18
column (3 mm, 4.6 150 mm) using 0.1% H3PO4/ACN mobile
phase in the gradient mode of HPLC. The column temperature
was set at 50 C. Complete separation of the four compounds
was achieved within 8 min. Another new small peak was
observed at 4.5 min. All peaks were well resolved. Epitetracycline
hydrochloride and tetracycline hydrochloride were eluted at
3.4 and 4 min, respectively, whereas 4-epianhydrotetracycline
hydrochloride and anhydrotetracycline hydrochloride were
eluted later at 6.3 and 7.1 min, respectively.

The diluent and the oral suspension placebo solution showed


no interference with all the peaks of interest. The sample
solutions from three tetracycline hydrochloride bulk materials
(including the USP RS) and the oral suspension exhibited no
new impurity.
The specicity of the method was further studied by forced
degradation (Fig. 2). The samples stressed in solid state (light, heat,
and heat/humidity) were stable. After light exposure (International
Conference on Harmonization conditions) the samples showed
little change in the chromatographic prole. After a 13 day
exposure at 80 C the samples lost 2% weight but exhibited a small
new peak at the front tail of the main peak, and the
anhydrotetracycline peak level increased 0.5% in total detected
area (TDA) from 0.5 to 1.0% TDA. After a 13-day exposure at 80C
and 80% relative humidity the samples lost 3% weight and
exhibited a small new peak at the front tail of the main peak as
well as a 4-epiandyrotetracycline peak (1.7% TDA). In addition,
the anhydrotetracycline peak level increased 1.5% in TDA (from
0.5 to 2.0% TDA).
However, the samples stressed in solution were much less
stable. The sample placed in 0.1 M HCl for one day lost 5% weight
but exhibited no new peaks, and only the anhydrotetracycline
peak increased from 0.5 to 7.4% TDA. The sample in 0.1 M NaOH
for one day lost 10% weight and exhibited several small new
peaks and a large new peak before the epitetracycline. The
sample placed in 0.03% H2O2 for a few hours lost 10% weight
and exhibited several new peaks before the main peak. The main
peak in all the stressed samples and unstressed samples passed
the spectral peak purity check.

0.20

4-Epianhydrotetracycline

AU

0.30

Tetracycline

Epitetracycline

0.40

0.10

Anhydrotetracycline

A heat-stressed sample. The heat-stressed sample was prepared by


heating 10 mg USP tetracycline hydrochloride RS at 80 C for 12 days.
The samples were diluted to 0.1 mg/mL with diluent before analysis.

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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Figure 2. Typical chromatograms from forced degradation studies.

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Copyright 2014 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2014; 28: 12781283

HPLC method for tetracycline-related USP monographs


The robustness of the method was examined starting with the
maximum variations allowed in the system suitability section of
USP general chapter Chromatography <621>. Under each
condition, a system suitability solution was analyzed ve times.
The retention time, relative retention time, tailing factor, peak
area RSD and resolution were evaluated. The system suitability
requirements were met under most of the maximum variations
including temperature 10 C, ow rate +50%, initial acetonitrile
content +5%, phosphoric acid concentration 10%, detection
wavelength 3 nm and a second brand of column. However,
the anhydrotetracycline hydrochloride peak did not elute under
a ow rate of 50% and acetonitrile content of 5%. The elution
of the peak was observed and the system suitability requirements were met under a ow rate of 20% and the initial
acetonitrile content of 2%.
Solution stability. The solution stability of tetracycline and
4-epianhydrotetracyline was sensitive to both temperature and
light. 4-Epianhydrotetracyline solution in water (2.0 g/mL) was
stable for about 3 h when the autosampler temperature was
set at 4 C. Use of 0.1% phosphoric acid instead of water
increased its stability to about 6 h. Tetracycline hydrochloride
at 0.1 mg/mL prepared either in 0.1% H3PO4 or water was stable
101

% peak area change

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.

for 24 h. A dramatic degradation of 4-epianhydrotetracyline


hydrochloride and tetracycline hydrochloride was observed
when the autosampler temperature was set at room temperature. Figure 3 shows the stability of tetracycline hydrochloride
in 0.1% H3PO4 and in water at 4 C and ambient temperature.
In all experiments, solution stability was studied when each
procedure was validated. The solutions were tested immediately
after preparation.
Filter study. A 0.22 m hydrophilic syringe lter was used for
sample solutions containing the oral suspension placebo. The
lter study showed no effect of the lter on the concentrations.
Method validation
Linearity. Table 1 lists the four sets of linearity results: two for
assay and two for the limit of 4-epianhydrotetercycline hydrochloride. The results met the specied acceptance criteria.
Accuracy and precision of the assays. The precision of the
instrument was tested by ve replicate injections of standard
solution. The relative standard deviation (RSD) of the peak area
response was <0.5%. System suitability was performed during
each analysis. The accuracy and precision are presented in
Tables 2 and 3. They were examined at three levels 80%
(0.08 mg/mL), 100% (0.1 mg/mL) and 120% (0.12 mg/mL) of the
analysis concentration of 0.1 mg/mL. The results met the specied acceptance criteria.
Accuracy and precision of the limit procedures. The
precision of the instrument was tested by six replicate injections
of standard solution. The relative standard deviation (RSD) of
the peak area response was <1.5%. System suitability was
performed during each analysis. The accuracy and precision
are presented in Table 4. They were examined at three levels
50% (1.0 g/mL), 100% (2.0 g/mL), and 150% (3.0 g/mL) of
the limit concentration of 2.0 g/mL when 0.1 mg/mL of
tetracycline hydrochloride exists. The results met the specied
acceptance criteria.
Quantitation limit. The quantitation limit was determined
based on signal-to-noise ratios by comparing measured signals from
samples with known concentrations of 4-epianhydrotetracycline
hydrochloride with the signal from the baseline. The quantitation
limit at 10 times peak-to-peak noise was found to be 0.10 g/mL,
representing 5% of the limit level. The peak area RSD from six

Table 1. Linearity results for the assay and limit procedures


Component

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

Biomed. Chromatogr. 2014; 28: 12781283

Copyright 2014 John Wiley & Sons, Ltd.

1281

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

Acceptance criteria: average assay = 97.8 2%; precision


(RSD) 1%.
The assay value from the Certicate of Analysis of the
commercial sample is 97.8%.
The USP tetracycline hydrochloride monograph specication
is not less than 90%.

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
(%)

Tetracycline hydrochloride drug substance


Spike 50%
1.09
101.1
1.07
102.3
1.05
101.2
Spike 100%
2.18
101.0
2.14
101.0
2.11
100.7
Spike 150%
3.27
101.4
3.21
100.1
3.16
100.5
Mean
101.0
RSD (n = 9)
0.62%
Tetracycline hydrochloride oral suspension
50%
1.02
102.8
1.06
101.2
1.04
100.0
100%
2.03
98.7
2.12
100.2
2.09
99.6
150%
3.05
99.0
3.18
101.8
3.13
98.8
Mean
100.2
RSD (n = 9)
1.38%

for

4-

Average
(%)

101.6

100.9

100.7

101.3

99.5

99.7

Acceptance criteria: average recovery = 100 8%; precision


(RSD) 4%.

99.6

Conclusion
99.3

99.8

89.9
91.1
90.4
89.8
91.0
88.5
1.1

Acceptance criteria: average recovery = 100 2%; precision


(RSD) 2%.
The precision was measured on the second day after the oral
suspension was prepared. The concentration of 100 g/mL was
based on the label claim. The potency was 90% of label claim.

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injections of a 4-epianhydrotetracycline hydrochloride solution


at 0.10 g/mL was 2.4% and met the acceptance criteria in
the USP Separation Science Laboratory (Rockville, MD, USA)
validation guideline.

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Table 4. Accuracy and precision results


epianhydrotetracycline hydrochloride limit.

A simple and fast gradient reversed-phase HPLC method for the


modernization of the USP tetracycline hydrochloride monographs
and general chapter <226 > for the limits of 4-epianhydrotetracycline impurity was developed. The proposed method successfully demonstrates chromatographic separation of tetracycline
from the structurally related compounds epitetracycline, anhydrotetracycline, 4-epianhydrotetracycline and other possible degradation products. The compounds were eluted within 8 min with a
complete separation. The method is accurate, precise, specic, linear, sensitive and robust for the assay of tetracycline hydrochloride
and the limit of 4-epianhydrotetracycline hydrochloride in the
drug substance and oral suspension.

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.

Copyright 2014 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2014; 28: 12781283

HPLC method for tetracycline-related USP monographs

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1283

Biomed. Chromatogr. 2014; 28: 12781283

Copyright 2014 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/bmc

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