Value of Eclipse Plus C18 and ph Selectivity to Analyze Ae-Containing Drugs Application Pharmaceuticals Authors John W. Henderson Jr., William J. Long, and Cliff Woodward Agilent Technologies 285 Centerville Road Wilgton, DE 1988 USA Abstract Three common ae-containing drugs were extracted from their commercial forms and analyzed by HPLC under several ph conditions to take advantage of ph selectivity as a tool for optimizing resolution for method development. An Eclipse Plus C18 column was found to provide excellent selectivity across the ph range of 2.5 to 7. while providing good peak shape for the aes at phs near and far from the drug s pka values. This rugged chromatographic performance, due to Eclipse Plus enhanced ZORBAX base silica, which reduces silanol/analyte activity, and advanced bonding and endcapping makes Eclipse Plus C18 a good choice for exploiting ph selectivity for method development of aecontaining compounds. Introduction Reversed phase HPLC is frequently used for the analysis of nitrogen-containing compounds in the pharmaceutical industry. Compounds such as derivatives of plant alkaloids, antibiotics, and many biogenic substances contain aes, and many are known to have pharmacological effects. Bringing to market a novel drug can be quite expensive, but lucrative. Competition in the pharmaceutical industry demands increased productivity in all stages of drug development, from discovery to QA/QC of the final product. HPLC is a key tool to isolate and identify potential drugs, their degradants, impurities, metabolites, and other related compounds in each stage of drug development. Proper HPLC column selection has a direct effect on laboratory efficiency and productivity, ultimately reducing the time to market of the new drug. In this study we performed HPLC analysis on three ae-containing drugs from their commercial form+two as tablets and one as an ointment+to demonstrate how Eclipse Plus C18 columns are an ideal first choice in method development, for example, in the QA/QC stage of drug development. Changing the mobile phase ph is useful for changing selectivity and resolving peaks of interest from other peaks; however, ph can also influence peak shape. Many columns have active (ionized) silanols above ph 3. It is these silanols that can interact with the analytes. The ph deteres the ionization of the compound, especially aes, and thereby the degree of interaction with ionized silanols of the HPLC column. At mid-ph many columns exhibit peak tailing for weak bases and weak acids. Eclipse Plus C18, however, exhibited great peak shape for the aes across a ph range of 2.7 to 7. This makes Eclipse Plus C18 a compelling choice for selectivity experiments and method optimization.
Experimental Three Agilent 12 Series liquid chromatographs consisting of a binary pump, autosampler, thermostatted column compartment and diode array detector with a semi-micro flow cell were set up with identical methods except for the mobile phase: System 1 A:.1% formic acid (v/v) ph 2.7 B: acetonitrile with.1% formic acid (v/v) System 2 A: 2 mm sodium acetate ph 4.8 B: acetonitrile System 3 A: 2 mm sodium phosphate ph 7. B: acetonitrile The gradient program used in these three systems was identical, 1 to 1% B in 1 utes at 1 ml/. 254 nm and 23 nm UV were monitored. The analytical columns used were ZORBAX Eclipse Plus C18, 4.6 x 15 mm, 5 μm, at a temperature of 4 C. The ae-containing drugs analyzed were pioglitazone HCl tablets (Actos, Takeda Pharmaceuticals), dipyridamole tablets (Persantine, Boehringer Ingelheim Pharmaceticals), and lidocaine ointment (E. Fougera & Co.). The samples were prepared to contain 1 mg/ml active ingredient. For the two tablets, sample preparation entailed pulverization of the tablets with mortar and pestle, transfer to a vial, the addition of methanol, then ultrasonication for 1. The extract solution was then filtered through a.45 μm PFTE syringe filter into three autosampler vials, one for each LC system. The lidocaine extract was prepared similarly, with the ointment weighed to a vial, ultrasonicated with methanol, and filtered into autosampler vials. Chemical structures and pka values are found in Figure 1. Pioglitazone HCI pka: 5.8 and 6.4 CH 3 O O S H O HCl H 3 C H O C 2 H 5 C C 2 H 5 CH 3 OH Lidocaine pka: 7.9 HO OH OH Dipyridamole pka: 6.4 Figure 1. Structure and pka of three ae-containing drugs. 2
Results and Discussion itrogen containing drugs are often classified as weak acids or weak bases because they partially ionize in aqueous solutions. The amount of ionization depends on the ph of the aqueous solution and the pka of the compound. This relationship is described by the Henderson-Hasselbach equation: ph = pka + log [A-] / [HA] Where ph is the negative log of the hydrogen ion concentration, pka is the ph at which a weak acid is 5% A- and 5% HA (commonly called the ionization constant), and [A- ] and [HA] are the concentrations of the conjugate acid and base, respectively. Mobile phases at the pka should be avoided because the equal amounts of interconverting forms of a molecule result in broad peak shape. At one ph unit away from the pka, 95% of the molecules exist in one form, and 5% exist as the other form. At two ph units away, the ratio is 99 to 1. A good rule of thumb is to operate two ph units away from the pka. Additionally, interactions of ae groups (H+) with ionized silanols (SiO-) of the silica support (between ph 3 and 7) is a secondary mechanism of retention and contributes to peak tailing. For these reasons it is desirable to use low ph mobile phases. Generally the best peak shapes of bases are obtained at low ph when silanol activity is imized. Occasionally, however, intermediate ph methods are necessary or preferred. In many cases, by using a well-buffered mobile phase, where ph is controlled, selective interactions between the analyte and column can be enhanced. Dipyridamole is a heart disease drug; when in the bloodstream, it causes vasodilation and inhibits blood clotting. Figure 2 is the analysis of a dipyridamole tablet under various ph conditions using an Eclipse Plus C18 column, including mobile phases that are about one ph unit away from its pka. In spite of dipyridamoles dual species equilibrium at ph 7 and 4.8 (pka 6.4), excellent peak shape is found in all three separation conditions, near and away from the pka. Analysis of drugs in tablet form can be challenging because extracted excipients, degradants, or unknowns can interfere with the peak of interest. 16 ph 2.7 14 12 1 8 6 4 2 16 ph 4.8 14 12 1 8 6 4 2 2 4 6 8 1 16 ph 7. 14 12 1 8 6 4 2 USP tailing factor: 1.5 Column: ZORBAX Eclipse Plus C18, 4.6 x 15 mm, 5 µm P/ 95999-2 Mobile phases: top: formic acid,.1%; mid: acetate, 2 mm; bot: phosphate, 2 mm Gradient: 1-1% acetonitrile/1. F = 1 ml/. Temp: 4 C UV: 254 nm Unknown coalesces with analyte 2 4 6 8 1 USP tailing factor: 1. USP tailing factor: 1. 2 4 6 8 1 Figure 2. Great peak shape and selectivity variability of dipyridamole under three scouting conditions by Eclipse Plus C18. 3
The method used for Figure 2 is a gradient. At the usually preferred low ph mobile phase, an unknown, possibly a degradent, co-elutes with the dipyridomole peak (top chromatogram). This unknown is identified by an arrow in the middle and bottom chromatograms. Although tailing factor is not usually measured in gradient methods, here it is useful to see the overlapping peaks, as indicated by the larger tailing factor at low ph. At ph 4.8 and 7, selectivity changes and resolves the two peaks. The excellent tailing factors of the resolved peaks at higher ph are what is expected from a robust column and gradient method. Figure 3 is another pharmaceutical drug, pioglitazone, analyzed under the same conditions as in Figure 1. Pioglitizone improves blood sugar control in people with type 2 diabetes. Again, when analyzed at the usually preferred lower ph, an unknown happened to co-elute with the analyte, as indicated by the tailing peak. Adjusting the ph higher altered the selectivity to resolve pioglitazone from the unknown and consequently is shown as a more symmetrical peak. Pioglitazone has two pka s: 5.8 and 6.4. Retention shifts forward at ph 7 compared to ph 4.8, indicating that the compound is probably more polar at ph 6.4 than at ph 4.8. Retention at ph 4.8 is perhaps due to two positive charges and at ph 7 to one positive charge. Also, changing ionic strength of the gradient could play a role. The third drug, lidocaine cream, is a local anesthetic for skin. It is a nonconjugated, mono-benzenoid drug; thus, there was not much detection at 254 nm (data not shown), but at 218 nm, there was much stronger absorbance and sensitivity for lidocaine. Figure 4 shows the lidocaine chromatograms with the three different buffers. The downward sloping baseline of the acetic acid chromatogram is a due to acetate having some absorbance at Column: ZORBAX Eclipse Plus C18, 4.6 x 15 mm, 5 µm P/ 95999-2 Mobile phases: top: formic acid,.1%; mid: acetate, 2 mm; bot: phosphate, 2 mm Gradient: 1-1% acetonitrile/1. F = 1 ml/. Temp: 4 C UV: 23 nm 7 6 5 4 3 2 1 5 4 3 2 1 A: ph 2.7 formic acid B: ph 4.8 acetate USP tailing factor: 1.4 Unknown coalesces with analyte 2 4 6 8 USP tailing factor: 1. 2 4 6 8 C: ph 7. phosphate 8 6 USP tailing factor: 1.1 4 2 2 4 6 8 Figure 3. Great peak shape and selectivity variability of pioglitazone under three scouting conditions by Eclipse Plus C18. 4
Column: ZORBAX Eclipse Plus C18, 4.6 x 15 mm, 5 µm P/ 95999-2 Mobile phases: top: formic acid,.1%; mid: acetate, 2 mm; bot: phosphate, 2 mm Gradient: 1-1% acetonitrile/1. F = 1 ml/. Temp: 4 C UV: 23 nm 15 ph 2.7 formic acid 1 5-5 -1-15 -2-25 2 4 6 8 1 15 ph 4.8 acetate 1 5-5 -1-15 -2-25 2 4 6 8 1 15 ph 7. phosphate 1 5-5 -1-15 -2-25 2 4 6 8 1 Figure 4. Great peak shape and selectivity variability of lidocaine under three scouting conditions with Eclipse Plus C18. 218 nm. As the gradient changes to higher organic strength and less acetate, the UV absorbance decreases. The phosphate mobile phase has little or no absorbance at 218 nm and therefore its baseline is relatively flat. As noted with the previous examples, the ph differences have a strong influence on selectivity and resolution of the compounds, and peak shape is excellent across the ph range, even near lidocaine s pka of 7.9. Conclusions Eclipse Plus C18 has an enhanced ZORBAX base silica that reduces silanol/analyte activity and advanced bonding and endcapping to further reduce peak tailing. Since this phase is less prone to silanol interactions, it is useful for exploiting selectivity changes by changing ph or anion additives. Examples of ionizable nitrogen-containing drugs were eluted off an Eclipse Plus C18 column under the same organic gradient conditions with different ph buffers. The different mobile phase phs altered selectivity of the peaks; and good peak shape was maintained far from and near an analyte s pka values. For More Information For more information on our products and services, visit our Web site at www.agilent.com/chem. 5
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