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Rapid separation of tetracycline derivatives and their main degradation products by capillary zone electrophoresisElectrophoresis 2001, 22, 2775–2781 Carmen García-Ruiz1
Rapid separation of tetracycline derivatives
Antonio L. Crego1
Jose L. Lavandera2
and their main degradation products by capillary
Maria L. Marina1
A mixture of five tetracycline (TC) derivatives: minocycline (MC), demeclocycline (DMCTC), doxycycline (DC), and sancycline (SC), as well as each TC derivative from its main degradation product were separated by capillary zone electrophoresis (CZE).
The influence of the pH and the concentration and nature of the background electrolyte (BGE) on the separations was investigated. Ethylenediaminetetraacetic acid (EDTA; 1mM) was used as additive in a 25 mM phosphate buffer (pH 2.3) because this BGE enabled the rapid separation of the TC derivatives and of each TC derivative from its respective degradation product in less than 6 min. After optimization of the separation conditions, the analytical characteristics of the method were investigated. The para-meters involved were linearity, precision (repeatability and reproducibility), and limitsof detection (LODs). LODs obtained for the five TC derivatives studied were about3 mg/mL. Finally, the CZE method developed was applied to study the stability of TCderivatives and to analyze the TC derivative content in three different pharmaceuticalpreparations.
Keywords: Tetracyclines / Method development / Optimization
The physicochemical characteristics of TCs (ionogenicand water-soluble substances) make them suitable for Tetracyclines (TCs) are an important group of broad electrophoretic analysis. Thus, in recent years different spectrum antibiotics widely used for both humans and modes of capillary electrophoresis (CE) have been investi- animals . TCs are structurally related compounds with gated to separate TCs and their impurities, such as capil- multiple functional groups with acid-base properties, lary zone electrophoresis (CZE) [5–15], micellar electro- which presence confers them an amphoteric character.
kinetic chromatography [16, 17], nonaqueous CE [18– Most of these compounds exhibit an isoelectric point 21], and capillary electrochromatography [6, 22]. CZE between 4 and 6. In acid solution and upon storage, has been the most widely used mode of CE for separating TCs experience degradation reactions leading to pro- TCs since in 1992 the first CE method for resolving TC ducts that are often isomers from the original precursor.
from its degradation products was reported . Although The most important degradation products are those in this work an acid pH was used (pH 3.9), in general, CZE obtained by epimerization, dehydration, and combined methods for the analysis of TCs employ buffers at basic epimerization-dehydration reactions . On the other pHs (from 8.5 to 12.25) in order to avoid sample adsorp- hand, commercial samples usually contain significant tion on the capillary [8–15]. In these procedures, different amounts of impurities (degradation products or different kinds of additives such as ethylenediaminetetraacetic TC derivatives obtained in the synthesis) [3, 4] with only acid (EDTA), methanol, Triton X-100, or methyl-b-cyclo- minor structural differences among them but differing dextrin (methyl-b-CD) were used to improve the separa- widely in their pharmacological activities . Therefore, tion selectivity, obtaining analysis times of about 15 min analytical methods to perform the rapid analysis of TC antibiotics are needed in order to control their impuritiesin pharmaceutical preparations.
Although different separations of TC derivatives havebeen performed, the separation of multicomponent mix-tures has been scarcely reported and shorter analysistimes are desirable in order to use analytical methodolo- Correspondence: Dr. Maria Luisa Marina, Dpto. Química Analí-
gies for routine analysis. In fact, as far as we know, only tica, Facultad de Química, Universidad de Alcalá, 28871 Alcalá one CZE method has been reported on the separation of five TCs including TC, doxycycline (DC), and minocycline E-mail: [email protected]
(MC) in about 8 min . This method used a buffer at pH 3with 40% methanol and Abbreviations: DC, doxycycline; DMCTC, demeclocycline; MC,
minocycline; SC, sancycline; TC, tetracycline
TCs (TC, DC, MC, and demeclocycline (DMCTC)) was not WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 Electrophoresis 2001, 22, 2775–2781 achieved using phosphate buffer at pH 7.5 because of the five TC antibiotics employed (DC, MC, and TC) under these conditions three of them (MC, DC, and TC) were supplied by Sigma (St. Louis, MO, USA); DMCTC were practically overlapped . Also, the separation of was from Fluka (Buchs, Switzerland), and SC was sup- DMCTC from MC or DC has only been performed using plied by Hovione Sociedade Quimica (Lisbon, Portugal).
basic pH values with analysis times higher than 14 min Figure 1 shows the basic structure of these compounds.
[12–14]. On the other hand, some of the TCs have not Three pharmaceutical preparations were analyzed in this been separated still from their degradation products. As work: MC in capsules, which contain MC (100 mg) with an example, no reports have been found on the separa- corn starch and magnesium stearate as excipients; DC tion of sancycline (SC) from other TC derivatives or from in capsules, which contain DC with lactose, magnesium stearate, sodium lauryl sulfate, corn starch and alginicacid as excipients; DC in suspension, which contains In this work, the rapid separation of five TCs (TC, MC, DC with saccharin sodium, sodium hydroxide, calcium DMCTC, DC, and SC) and of each of these TCs and chloride, antifoam AF, apeline, eritrosine, carmine solu- its main degradation product by CZE is presented. The tion, sodium metabisulfite, butylparaben, propylparaben, influence of the pH, concentration and nature of the polyvinyl pyrrolidone, veegum K, 70% sorbitol, glycerin, background electrolyte (BGE), and the presence of aroma of raspberry, and water. The three pharmaceutical EDTA on the separations have been investigated. After preparations were acquired in chemists in Madrid optimization of the separation conditions, the analytical characteristics of the method have been investigatedand a study of the stability of the five TCs has beenperformed.
2 Materials and methods
An HP3D CE system (Hewlett-Packard, Waldbronn, Ger- many) equipped with an on-column diode array detectorand an HP 3D-CE Chemstation software was used.
Separations were performed on fused-silica capillaries of 50 mm ID and 375 mm OD, purchased from Composite Metal Services (Worcester, England). Capillaries had a total length of 33.5 cm and 25 cm to the detector. Capil-lary temperature was adjusted to 257C. Finally, detection Figure 1. Chemical structures of the TC derivatives
was performed at 265 nm. Electrolytic solutions were degassed in an ultrasonic bath KM from Raypa (Barce-lona, Spain). A model 654 pH-meter from Metrohm (Heri-sau, Switzerland) was employed to adjust the pH of the 2.3 Procedure
Solutions of 200 mM formic acid or 100 mM phosphate 2.2 Reagents and samples
buffer were prepared either by adding appropriate ali-quots of concentrated formic acid or by dissolving the All reagents employed were of analytical grade. Sodium appropriate amount of phosphate salt into water. The dihydrogen phosphate dihydrate, trisodium phosphate different pHs for 100 mM phosphate buffers were dodecahydrate, dimethyl sulfoxide (DMSO), sodium hy- adjusted as required by adding aliquots of 100 mM phos- droxide (NaOH) and hydrochloric acid (HCI) were supplied phoric acid solution. The concentrations 25, 50, and from Merck (Darmstadt, Germany); disodium hydrogen 75 mM, in phosphate buffer were obtained by diluting phosphate dodecahydrate and EDTA disodium salt di- the 100 mM solution with water. The pH desired for the hydrate were from Panreac (Barcelona, Spain); formic diluted solutions was adjusted with 1 M or 1 M NaOH.
acid was obtained from Riedel-de Haën (Seelze, Ger- All electrolyte solutions were filtered prior use through many); acetonitrile was from Lab Scan (Dublin, Ireland); 0.45 mm pore size disposable nylon filters from Scientific water used to prepare solutions was purified through a Resources (Eatontown, NJ, USA). Standard solutions Milli-Q system from Millipore (Bedford, MA, USA). Three were prepared by dissolving each TC antibiotic in DMSO Electrophoresis 2001, 22, 2775–2781 Figure 2. Separation of a mix-
ture of the five TCs studied at
acid, neutral, and basic pHs.
BGE, 100 mM phosphate buffer;
injection by pressure, 30 mbar
for 2 s of sample followed by
30 mbar for 2 s of BGE; capillary,
33.5 cm (25 cm to the detec-
tor)650 mm ID; temperature,
257C; applied voltage, 15 kV;
current intensity, * 40 mA; UV
detection, at 265 nm. I, impurity.
to achieve the desired concentration. In order to obtain we investigated the effect of pH using 100 mM phosphate the degradation products of these compounds a solution buffer (acid, neutral, and basic pHs corresponding to of 0.1% phosphoric acid with acetonitrile (50:50, appar- the pKa’s of the phosphoric acid, pH 2.3, 7.2, and 12.2).
ent pH 2.6) was employed. The solid products were As shown in Fig. 2, the separation of a mixture of the five dissolved in this solution and sonicated for 6 h before the TCs was only possible at acid pH for which the best analysis of the diluted solutions in water. Sample solu- efficiency and the shortest analysis time were obtained.
tions for MC and DC were prepared dissolving in 10 mL At pH 2.3 four concentrations of phosphate buffer were of DMSO the total content of one capsule. These solu- then considered (25, 50, 75, and 100 mM). A 25 mM phos- tions (10 000 mg/mL) and the suspension of DC (50 mg/ phate buffer gave the lowest values of migration times 5 mL = 10 000 mg/mL) were diluted in DMSO in order to and current intensity (~ 40 mA) without a significant loss obtain a final concentration of 50 mg/mL. These solutions in the resolution among the five TCs studied due to the were analyzed by the CZE method. Before first use, a new small selectivity changes obtained when the concentra- capillary was rinsed with 1 M NaOH for 30 min, 0.1 M tion of BGE was increased. Therefore, a 25 mM phosphate HCI for 5 min followed by 30-min rinse with BGE.
buffer was selected. On the other hand, results showed Between consecutive injections the capillary was only that only with a 25 mM phosphate buffer a resolution conditioned with the BGE for 4 min, but when the pH or higher than 1.5 (baseline resolution) was obtained for the nature of the BGE was changed, before first injection each TC derivative and its respective degradation pro- a conditioning for 30 min with the new BGE was made.
duct (the resolution between MC and its degradation pro- This conditioning method was used to obtain reproduci- duct decreased when increasing the buffer concentra- ble migration times. Injections were made by pressure: 30 mbar for 2 s of sample followed by 30 mbar for 2 sof BGE to improve the shape of the peaks.
In order to improve the resolutions obtained, the influenceof the nature of the BGE on the separation was investi-gated. Formic acid and phosphate buffer without or with 3 Results and discussion
EDTA were compared. Results showed that resolutions 3.1 Method development
increased when 25 mM phosphate buffer (pH 2.3) insteadof 200 mM formic acid (pH 2.2) was used. Moreover, The separation of TC antibiotics can be highly influenced the addition of 1 mM EDTA to 25 mM phosphate buffer by the pH of the medium due to the fact that these enabled an improvement in the resolutions corresponding substances are amphoteric compounds with isoelectric to the separation of TC and DMCTC and of each TC points ranging between 4 and 6. In a preliminary study, derivative and its respective degradation product.
Electrophoresis 2001, 22, 2775–2781 Finally, a more exhaustive study on the influence of the pH(ranging from 2.3 to 5.3) was achieved when using 25 mMphosphate buffer with 1 mM EDTA. Table 1 shows theresolutions obtained for the separation of the five TCsstudied at different acid pHs (from 2.3 to 4.3). It can beobserved that only at pH 2.3 it was possible to obtaina baseline resolution of the five TCs whereas at pH 3.1,3.7, or 4.3 only four of them were separated. On the otherhand, at pH 5.3 no peaks were detected for tetracyclinesbecause of their comigration with the electroosmoticflow (i.e., with the peak of DMSO, which was the samplesolvent). In addition, the analysis time increased whenincreasing pH values. Table 1 also shows that only atpH 2.3 it was possible to perform the separation of eachTC derivative and its respective degradation product,while at pH 3.1 and 3.7 no resolution was observed for Figure 3. CZE of a mixture of five TCs. BGE, 25 m
DC and its degradation product, and at pH 4.3 neither phosphate buffer (pH 2.3) with 1 mM EDTA. Other condi- DC nor MC were resolved from their respective degrada- Table 1. Resolution obtained at different acid pHs for
each two consecutive TCs in a mixture of thefive studied and for each TC derivative and itsmain degradation product BGE, 25 mM phosphate buffer-1 mM in EDTA. Experimen-tal conditions: injection by pressure, 30 mbar for 2 s ofsample followed by 30 mbar for 2 s of BGE; capillary,33.5 cm (25 cm to the detector)650 mm ID; temperature,257C; applied voltage, 15 kV; current intensity, 40 mA; UVdetection, at 265 nm.
In conclusion, the study performed enabled to select25 mM phosphate buffer (pH 2.3) with 1 mM EDTA as themost appropriate conditions for the rapid separation ofTC derivatives studied and their degradation products.
Under these selected conditions, the electropherogramof a mixture of the five TCs studied in this work has been Figure 4. Separation of each TC derivative and its main
obtained (Fig. 3). On the other hand, Fig. 4 shows the degradation product by CZE. Experimental conditions electropherograms obtained for the five TC derivatives Electrophoresis 2001, 22, 2775–2781 Table 2. Analytic characteristics of the CZE method
X = concentration of theTC derivate in mg/mL) a) Measured from six consecutive injections of a solution of the same degraded TC derivativeb) Measured from two consecutive injections with three different capillariesc) Calculated from the peak height based on a signal-to-noise ratio of 3 t1, migration time corresponding to the degradation product of the TC derivative, t2, migration timecorresponding to the TC derivative, A1, peak area corresponding to the degradation product of theTC derivative, A2, peak area corresponding to the TC derivative, BGE, 25 mM phosphate buffer (pH2.3)-1 mM EDTA, other experimental conditions as in Table 1.
and their respective degradation products when the one of the most important criteria for evaluating the above-mentioned conditions were used. It can be ob- analytical method performance, and its numerical value served that all TCs studied show a main degradation pro- is the relative standard deviation (RSD). The repeatability duct, their epimeres. In order to show if the TC derivatives in the migration time, peak area, and ratio between the were stable during the time of the analysis, the percen- peak area of the degradation product generated and tage of degradation of each TC derivative studied, when the peak area of the TC derivative (value proportional dissolved in the 25 mM phosphate buffer at pH 2.3, was to % degradation) was determined as the RSD obtained measured. Degradations lower than 3% were obtained for six consecutive injections of the degraded TC deriva- after 60 min in phosphate buffer, therefore, the degrada- tives. Table 2 shows that acceptable levels of precision tion of the TCs during the analysis by CZE (less than 6 min) were obtained for the developed method in terms of repeatability (RSDn = 6 from 0.2 to 2.6% for migrationtimes, from 0.6 to 4.2% for peak area, and from 0.9to 2.7% for the ratio between peak areas), since in all 3.2 Analytical characteristics of the method
cases RSDs calculated were lower than 5%. The repro- After the development of the method for the rapid analy- ducibility of the method was measured as the RSD sis of TCs, its analytical characteristics were examined, obtained for two consecutive injections with three differ- the results are shown in Table 2. The parameters involved ent capillaries (n = 6). Acceptable RSD values were ob- were linearity, precision (repeatability and reproducibility), tained for migration times (RSD < 2.5%), while only for and limit of detection (LOD). The linearity study showed DC a value of RSD 5 2.5%), while only for DC a value of that linear relationships with good correlations in all cases RSD & 7% was obtained for the ratio between peak (R2 4 0.99) were obtained for the variation of the peak areas (Table 2). Finally, an evaluation of the sensitivity area or peak height as a function of the concentration of was made by determining the values of the LOD calcu- TC derivative (working range from 10 to 100 mg/mL), using lated from the peak height based on a signal-to-noise at least six standard solutions (each solution was injected ratio of 3. The noise was estimated as the largest devia- tion of detector signal from baseline measured in a sec-tion of about 5 min in the absence of analyte (1610–4 AU).
The precision of the CZE method described was mea- LODs for the five TC derivatives ranged from 2.3 to sured as repeatability and reproducibility. Precision is Electrophoresis 2001, 22, 2775–2781 3.3 Application of the CZE method to study
the stability of TCs and for the analysis
of TC derivatives in pharmaceutical
First, a stability study was performed measuring thedegradation of the TCs dissolved in DMSO as an illustra-tion of the applicability of the optimized CZE methodfor the analysis of TCs. The percentage of degradationof each TC derivative was calculated from the ratiobetween the peak area of the degradation product gen-erated and the total peak area (the addition of the areasof the peaks of the TC derivative undegraded and itsdegradation product). The percentages of degradationobtained for TC, DMCTC, and SC were 3.6, 3.9, and3.4%, respectively, when the solution was freshly pre-pared whereas for MC and DC peaks due to degradationproducts were not observed in these conditions. How-ever, the degradation of these products was relativelyfast (from 6.2% for DC to 12.4% for MC in 26 h). Finally,the percentages of degradation after five days were:29.2% for TC, 29.0% for DMCTC, 17.5% for MC, 9.4%for SC, and 6.1% for DC. From these results it can beobserved that only DC was quite stable after five days.
Figure 5. Analysis of MC and DC in three pharmaceutical
Second, the CZE method developed was applied to the preparations by CZE. Experimental conditions as in Fig. 3.
analysis of MC and DC in three pharmaceutical prepara-tions. Table 3 shows the content of these TC derivativesfound by CZE in the three pharmaceutical preparations 4 Concluding remarks
studied (two capsules and one suspension). The bestresults were obtained for the suspension of DC. The worst An analytical method has been developed to separate by results were obtained for the capsules of DC and MC CZE five TC derivatives (TC, MC, DMCTC, DC, and SC). A probably due to the difficulty in recovering the sample study on the influence of the pH, concentration and nat- from the capsule. Good precision measured as RSD (%) ure of the BGE, and the use of additives enabled to select was achieved for the measured concentrations (RSD 25 mM phosphate buffer (pH 2.3) with 1 mM EDTA in order values from 0.49% to 1.85%). Finally, Fig. 5 shows the to achieve two objectives: (i) the separation of a mixture of electropherograms obtained for the three pharmaceutical the five TC derivatives, and (ii) the separation of each TC preparations using the CZE method developed in this derivative from its main degradation product (epimere). In work. It can be emphasized that any interferent peak both cases, the separations were performed in less than 6 min. The CZE method was evaluated by means of its line-arity, precision (repeatability and reproducibility), andLODs. Good linearity was obtained for all the derivatives Table 3. Content of two TC derivatives found by CZE in
studied (working range from 10 to 100 mg/mL, R2 4 0.99).
The repeatability of migration times and peak areas mea-sured as RSD was lower than 5%. The reproducibility of migration times and peak areas ratio between the epimereand the TC derivative using different capillaries was acceptable. LODs were about 3 mg/mL for the five TCs stu- Two applications of the CZE method were performed. A stability study of these compounds dissolved in DMSO label, concentration of the TC derivative specified on the label of the pharmaceutical preparation was performed to check the reliability of the method employed. The results obtained enabled to conclude that CE, averaged concentration (n = 5) if the TC derivative when the solutions of these compounds are freshly pre- Electrophoresis 2001, 22, 2775–2781 pared, the method can be employed due to the short ana-  Tavares, M. F. M., McGuffin, V. L., J. Chromatogr. A 1994, lysis time required for the analysis. In addition, the devel-  Van Schepdael, A., Saevels, J., Lepoundre, X., Kibaya, R., oped method was applied to the analysis of two of the TC Gang, N. Z., Roets, E., Hoogmartens, J., J. High Resol. derivatives studied (DC and MC) in pharmaceutical pre- Chromatogr. 1995, 18, 695–698.
 Van Schepdael, A., Kibaya, R., Roets, E., Hoogmartens, J., Chromatographia 1995, 41, 367–369.
The authors thank GlaxoWellcome Research and Devel-  Van Schepdael, A., Van den Bergh, I., Roets, E., Hoogmar- opment (Madrid, Spain) for the kind gift of sancycline tens, J., J. Chromatogr. A 1996, 730, 305–311.
employed in this work. The authors further thank Mrs. A.  Li, Y. M., Van Schepdael, A., Roets, E., Hoogmartens, J., J. Liq. Chromatogr. Rel. Technol. 1997, 20, 273–282.
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