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Assaying Tetracyclines by HPLC
Using the Allure' Biphenyl Stationary Phase
By Rick Lake,Pharmaceutical I nnovations Chemist, and Sherry Moyer, Innovations Chemist
• Superior selectivity and efficiency, using an Allure' Biphenyl column.
• Simplified analysis fo r high-throughput potency and stability-indicating assays.
• More easily achievable system suitab ility criter ia.
Tetracyclines are a widely used class of ant ibiotics
whose applications range from topical acne med ications for humans to premix feed additives for
livestock. Because of their widespread and liberal
use, tetracyclines are manufactured in large qua n­
tities, which generates th e need for a large number
of potency and stability-indicating assays. These
assays must be comp leted at regular intervals, in a
timely manner, over extended periods of time.
Consequently, it is critical that simple, rugged, and
selective met hods be developed. By selecting a sta­
tionary phase that produces optimum selectivity,
less demand to prod uce selectivity is placed on the
mobile phase, and a simp le isocratic analysis is
possible. Among the stationary phases we tested,
the Allure" Biphenyl and Allure" PFP Propyl sta­
tionary ph ases showed the best performance
(Table 1 and Figure 1).
Developing a simple mobile phase for this applica­
tion was a major concern . Ideally, to achieve ion­
ization equilibri um, choose a mob ile phase pH 2
units from the analytes' pKa. But two units below
the pKa values for the tetracyclines (approximate­
ly 3.3) would be below the recommended pH limit
for traditional silica-based columns, pH 2.
Consequently, we chose a pH of 2.5, and we added
a buffer to maintain pH. Because tetracyclines
form chelates with metal ions, we chose a non­
metal organic salt - ammonium phosphate - and,
to minimize surface metal content, we used only
columns made from high-pur ity Type B silica.
Lastly,we chose aceton itrile as the organic solvent,
because of its eluting strengt h and limited effect on
pKa: increasing the organic composition increases
pKa for acidic analytes and decreases pKa for basic
analytes, bu t a small amount of acetoni trile lessens
the effect, relative to a larger amount of methanol.
We evaluated several silica-based statio nary phas­
es, using the mobile phase described above, UV
detection, and isocratic conditions . The first selec­
tion criteria we used was selectivity, which we
measured by analyzing oxytetracycline and tetra cycline (an impurity in oxytetra cycline form ula­
tions) and determining the USP resolution and
selectivity (a) between the two compo unds. The
Allure?' Biphenyl and Allure?' PFP Propyl sta­
tionary phases showed th e best performance
among the columns we tested (Table 1 and Figure
1). These results suggest that the Allure?" Biphenyl
2006 vol, 1
Figure 1 Tetracycline and oxytetracycline have low pKa values that
make the analysis a challenge .
1
Peak List Ret. Time (min.)
1. oxytetracycline 4.92
* degradation peak 5.66
2. tetracycline 7.10
Column: Allure~ Biphenyl
Cal. # : 9166565
Dimensions: 150x4.6 mm
Particle size: 5/l~
Pore size: 60A
lCPH0342
Peak List Ret. Time(min.)
1. oxytetracycline 5.94
* degradation peak 7.08
2. tetracycline 8.57
Column: Allure~ PFP Propyl
Cal. #: 9169565
Dimensions: 150 x4.6 mm
Particle size: 5/l~
Pore size: 60A
Sample:
Inj.:
Cone.:
Samp lediluent:
Conditions:
Mobile phase:
Flow:
Temp.:
Del.:
20/lL (Allure~ Biphenyl column)or 30/lL(Allure" PFP Propyl column)
100/lg/ mLeachcomponent
methanol
20mMammoniumphosphate (pH 2.5):acetonitrile,80:20
ImL/min.
ambient
UV @ 254 nm
Table 1 Among tested columns, Allure' Biphenyl and Allure' PFP
Propyl columns show the best combination of resolution and
selectivity for tetracycl ine and oxytetracycline.
Stationary USP Selectivity
Phase Resolution (a)
Allure" Biphenyl 5.28 1.61
Allure" PFP Propyl 4.49 1.59
Ultra CI8 3.31 1.50
Allure~ Basix NA 1.34
Ultra C8 NA 0.47
Ultra PFP NA NA
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