Pharmaceutical Articles

Developing a Simple, Rugged HPLC Assay for Tetracyclines

by Rick Lake, Pharmaceutical Innovations Chemist, and Sherry Moyer, Applications Technician

Superior selectivity and efficiency, using an Allure™ Biphenyl column.
Simplified analysis for high-throughput potency and stability-indicating assays.
More easily achievable system suitability criteria.

Tetracyclines are an extensively used group of antibiotic medicines. Their usage is common in both humans and animals alike. Tetracyclines are administered to livestock in acute doses when illnesses are diagnosed, but in the U.S. they are sometimes administered at constant low level doses. When administered in this manner, they have been found to promote growth and increase feed efficiency, as well as treat diseases. The liberal and wide-spread use of tetracycline drug products calls for the manufacture of large numbers of drug batches. Each batch must be tested for quality and stability purposes, and this, in turn, translates into a large number of potency and stability-indicating assays. These assays must be completed in a timely and efficient manner, prior to individual batch release and throughout the shelf-life of designated production lots. A typical stability study will extend over a minimum of 6 months at accelerated conditions (40°C/25%RH) and up to 3 years at long-term conditions (25°C/60%RH). It is critical, therefore, that simple, rugged, and selective methods be developed for assaying tetracyclines.

When developing a stability-indicating method, selecting a stationary phase that provides optimum selectivity is a priority. Stability-indicating methods are developed not only to determine the amount of active ingredient, but also to determine, quantitatively, amounts of impurities and accumulation of degradation products. Impurities and degradants in a pharmaceutical sample often are structurally similar to the active ingredient, making analyte resolution and, therefore, column selectivity important factors in method development. In developing an application for tetracyclines, we evaluated the selectivity and system suitability of several silica-based HPLC stationary phases for the purpose of creating a simple and robust test method.

Selectivity

The effect and importance of selectivity, efficiency, and capacity are illustrated by the resolution equation (Figure 1). From the equation, it is evident that selectivity affects resolution to the greatest degree. Selectivity, in turn, is controlled mainly by two factors: 1) analyte interaction with the stationary phase and 2) mobile phase composition. For potency and stability assays, in which sample throughput and method ruggedness are critical, complex mobile phases and elution gradients can result in assays that are costly and laborious, both in preparation of materials and in instrument operation. By selecting a stationary phase that produces optimum selectivity, less need is placed on the mobile phase to produce selectivity, and a simple isocratic analysis often can be achieved.

Figure 1 The resolution equation indicates selectivity has the greatest influence on resolution.

Selecting a Mobile Phase

Selecting the appropriate mobile phase is another crucial step in method development. For our tetracyclines application, determining a simple mobile phase composition that offered acceptable results with all stationary phases, in order to evaluate the stationary phases, was a major concern. The mobile phase must be consistent and simple, as discussed, yet produce acceptable ionization of the target compounds. The surest way to produce ionization equilibrium is to use a mobile phase that is 2 pH units from the analytes’ pKa, if the latter values are known. pKa values for the tetracyclines are approximately pH 3.5. To produce complete ionization, then, pH 1.5 would be the target mobile phase pH for this application, but this is below the recommended lower pH limit for standard silica-based columns —pH 2. Operating a column at or outside a pH limit will limit its lifetime, and should be avoided for routine testing, like potency and stability assays. Consequently, we used a pH of 2.5 for this analysis, added a buffer to the mobile phase to maintain pH, and found this produced acceptable ionization and chromatography. Because tetracyclines form chelates, we chose a non-metal inorganic salt — ammonium phosphate — as the buffer, and used only columns made from high-purity Type B silica. This silica has a low surface metal content, relative to Type A silica, and is much less acidic. These features are especially favorable for analyzing basic compounds, like tetracyclines. By holding chelation to a minimum, we eliminated potential problems with mass balance and precipitation that ultimately could lead to increased instrument maintenance and low analyte responses. We chose acetonitrile as the organic solvent for producing appropriate retention factors, because of its dispersive properties, eluting strength, and limited effect on pKa. In general, increasing the organic composition of the mobile phase increases pKa for acidic analytes and decreases pKa for basic analytes. A small amount of a stronger eluter, like acetonitrile, rather than a larger amount of a weaker eluter, like methanol, will limit the effect on pKa. We mixed all mobile phases prior to introduction into the instrument, and adjusted the pH after preparing the mobile phase, to ensure that no shift in pH would occur on addition of organic modifiers.

Evaluating Stationary Phase Selectivity

We evaluated several commonly used silica-based stationary phases for their selectivity for tetracycline-like chemical structures, using test compounds tetracycline and oxytetracycline (Figure 2). Tetracycline is an impurity in oxytetracycline formulations, and this same procedure is used to evaluate selectivity in the system suitability part of the USP monograph for oxytetracycline. The two test compounds have very similar structures, and the analysis should be good indication of a phase’s selectivity, if all other conditions are kept equal.

We compared the stationary phases by analyzing oxytetracycline / tetracycline, using the isocratic mobile phase described above, and UV detection. The first selection criterion we used was selectivity, as measured by determining the USP resolution factor and the selectivity factor (α) between the two compounds. Of the columns tested, the Allure™ Biphenyl, Allure™ PFP Propyl, and Ultra C18 columns produced the best results (Figure 3 and Table 1). The results indicate that the Allure™ Biphenyl stationary phase exhibits π-π bonding with the ring portions of the tetracyclines, and the embedded polarity of the Allure™ PFP Propyl pentafluorophenyl propyl phase interacts with tetracycline moieties. Either of these separation mechanisms provided a greater retention capacity, compared to a mechanism based on hydrophobicity, as exhibited by the alkyl chain of the Ultra C18 phase (Figure 3).

Figure 2 Tetracycline and oxytetracycline have low pKa values that make the analysis a challenge.

tetracycline	oxytetracycline

Figure 3 Allure™ Biphenyl, Allure™ PFP Propyl, and Ultra C18 columns provide the best resolution and selectivity for oxytetracycline and tetracycline.

Peak List		Retention Time (min.)	USP Tailing	Resolution
1.	oxytetracycline	4.92	5.94	3.71
2.	degradation peak	5.66	7.08
3.	tetracycline	7.10	8.57	4.88

Allure™ Biphenyl	Allure™ PFP Propyl	Ultra C18

Column:	Allure™ Biphenyl (cat.# 9199565) Allure™ PFP Propyl (cat.# 9169565) Ultra C18 (cat.# 9174565)
Dimensions:	150 x 4.6 mm
Particle size:	5µm
Pore size:	60Å or 100Å (Ultra C18)

Sample:
Inj.:	20µL (30µL for Allure™ PFP Propyl)
Conc.:	100µg/mL each component
Sample diluent:	methanol

Conditions:
Mobile phase:	20mM ammonium phosphate (pH 2.5):acetonitrile, 80:20
Flow:	1mL/min.
Temp.:	ambient
Det.:	UV @ 254 nm

Table 1 Among tested phases, Allure™ Biphenyl, Allure™ PFP Propyl, and Ultra C18 show the best resolution and selectivity for tetracycline and oxytetracycline.
Stationary Phase	Resolution	Selectivity Factor (α)
Allure™ Biphenyl Allure™ PFP Propyl Ultra C18 Allure™ Basix Ultra C8 Ultra PFP	5.28 4.49 3.31 NA NA NA	1.61 1.59 1.50 1.34 0.47 NA

System Suitability

Tetracycline drug products are produced under cGMP protocols and, therefore, manufacturers are required to use validated or compendial methods, both of which require the completion of system suitability criteria. We used the three columns that showed the best selectivity for oxytetracycline / tetracycline in a system suitability test, which consisted of 6 replicate injections of 100µg/mL tetracycline (prepared in acetonitrile). We used capacity factors, resolution between tetracycline and a degradation product, and USP tailing factors to evaluate the columns. Under these conditions, the Allure™ Biphenyl column demonstrated the greatest selectivity (resolution and α values) and the best peak shape (USP tailing factor), while the Allure™ PFP Propyl column showed the greatest retention (capacity factor) (Figure 4 and Table 2).

Conclusion

Overall, three columns — Allure™ Biphenyl, Allure™ PFP Propyl, and Ultra C18 — showed excellent repeatability for analyses of tetracyclines, as seen by the RSD values in the system suitability test. Of the three, however, the Allure™ Biphenyl column demonstrated that, under these conditions, it can be the best choice for a simple, rugged assay. It exhibited high selectivity, good capacity, and the least peak tailing. If retention alone is desired, the Allure™ PFP Propyl column also is an excellent candidate.

Figure 4 Overall, the Allure™ Biphenyl column is the best choice for assaying the tetracycline antibiotics.

Peak List		Retention Time (min.)	USP Tailing	Resolution
Allure™ Biphenyl
1.	degradation peak	5.60
2.	tetracycline	7.05	1.05	3.92
Allure™ PFP Propyl
1.	degradation peak	7.08
2.	tetracycline	8.57	1.07	3.78
Ultra C18
1.	degradation peak	4.06
2.	tetracycline	5.11	1.22	4.21

Allure™ Biphenyl	Allure™ PFP Propyl	Ultra C18

Column:	Allure™ Biphenyl (cat.# 9199565) Allure™ PFP Propyl (cat.# 9169565) Ultra C18 (cat.# 9174565)
Dimensions:	150 x 4.6 mm
Particle size:	5µm
Pore size:	60Å or 100Å (Ultra C18)

Sample:
Inj.:	5µL
Conc.:	100µg/mL each component
Sample diluent:	methanol

Conditions:
Mobile phase:	20mM ammonium phosphate (pH 2.5):acetonitrile, 80:20
Flow:	1mL/min.
Temp.:	ambient
Det.:	UV @ 254 nm

Table 2 High selectivity, good capacity, and negligible peak tailing indicate that an Allure™ Biphenyl column can be the best choice for a simple, rugged system suitability assay for tetracycline.
Replicate	Peak Area	Retention Time (min.)	Capacity Factor (k’)	USP Tailing	Resolution*
Allure™ Biphenyl
1 2 3 4 5 6 Mean Std Dev %RSD	2570014 2504772 2516604 2520571 2475618 2469269 2509475 36397.394 1.450	7.075 7.069 7.099 7.064 7.116 7.046 7.078 0.025 0.357	5.675 5.669 5.699 5.664 5.716 5.646 5.678 0.025 0.445	1.06 1.06 1.06 1.05 1.06 1.05 1.06 0.005 0.489	3.94 3.93 3.94 3.92 3.94 3.92 3.93 0.010 0.250
Allure™ PFP Propyl
1 2 3 4 5 6 Mean Std Dev %RSD	2477528 2494068 2496213 2441825 2502128 2492068 2483972 22202.935 0.894	9.546 9.562 9.515 9.517 9.437 9.513 9.515 0.043 0.452	8.146 8.162 8.115 8.117 8.037 8.113 8.115 0.043 0.530	1.27 1.27 1.27 1.28 1.28 1.28 1.275 0.005 0.430	3.79 3.81 3.8 3.77 3.77 3.78 3.79 0.016 0.431
Ultra C18
1 2 3 4 5 6 Mean Std Dev %RSD	2386133 2402884 2406270 2364886 2422630 2416013 2399802.667 21171.757 0.882	5.125 5.149 5.137 5.124 5.157 5.112 5.134 0.017 0.329	3.725 3.749 3.737 3.724 3.757 3.712 3.734 0.017 0.453	1.21 1.21 1.21 1.21 1.21 1.22 1.212 0.004 0.337	4.21 4.21 4.22 4.24 4.22 4.19 4.215 0.016 0.390
*Tetracycline/degradation product.