IP-HPLC of Risedronate

By: Jacinth A. M. McKenzie, Chemfiles Volume 5 Article 5

Senior R&D Scientist, Supelco

Risedronate, the active pharmaceutical ingredient in Actonel®, is a bisphosphonate drug used for the treatment and prevention of osteoporosis.* In this report, analysis of risedronate is performed on an AscentisTM C18 column using ion pair reagents. When ion pair chromatography is required, it is imperative to use the highest purity reagents available that produce clean baselines and reproducible separations.

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Analytical challenge

Supelco’s technical service was contacted by a pharmaceutical customer who required a method to analyze risedronate. The requirements were basic: keep the injection to injection time between 5 and 6 minutes, use a standard 15 cm length HPLC column, isocratic flow conditions and UV detection. Upon inspection, it was apparent that the HPLC analysis of risedronate (Figure 1) using silica-based particles under reversed phase conditions would be challenging. First, the polar phosphate and hydroxyl groups in the molecule are not offset by significant hydrophobic regions, conditions not amenable to reversed phase retention. Second, the phosphate and hydroxyl groups are likely chelation sites with metal ions that may be present in the system. Third, the basic pyridine group can interact with silanols on the silica surface. Both silanol interactions and chelation can lead to poor peak shape and low analyte recoveries.

Figure 1 ......... Risedronate structure

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RP-HPLC method development strategy

  1. Choosing initial mobile phase pH
    In order to maximize the retention, the mobile phases for reversed phase HPLC of polar or ionizable compounds should be chosen to minimize the charge on the molecule. Charged molecules are highly solvated and have a greater affinity for the mobile phase over the stationary phase. Acidic compounds are less solvated below their pKa, while basic compounds are less solvated above their pKa. Estimations of pKa for risedronate were performed using ACD Labs software (Toronto, Canada). The results of calculations gave three pKa values, 0.32 and 0.94 for the acidic phosphate groups, and 5.07 for the nitrogen in the pyridine ring. The overall acidic character led us to start with acidic mobile phases.
  2. Scouting pH conditions
    At low pH, a gradient from 5 to 75% methanol over 20 minutes, 25 mM potassium phosphate (pH 2.5) at 1 mL/min on a 15 cm x 4.6 mm I.D., 5 μm Ascentis C18 column did not give adequate retention (k = 0.30, Figure 2). In the event that the ionization of the pyridine nitrogen plays a significant role in the solvation character of the molecule, we increased the mobile phase pH to 7 and ran a fast gradient from 5 to 19% methanol over 4 minutes, 25 mM potassium phosphate (pH 7) at 1 mL/min on a 15 cm x 4.6 mm I.D., 5 μm Ascentis C18 column. Retention was still not adequate (k = 0.21, Figure 3).

    Figure 2 ......... HPLC of risedronate at low pH, no ion pair, C18 column, gradient

    Figure 3 ......... HPLC of risedronate at pH 7, no ion pair, C18 column, gradient

  3. Changing stationary phase selectivity
    Within the population of reversed phase columns, C18 is by far the most commonly employed. However, other stationary phases exist that still operate via a reversed phase mechanism, but give different selectivity or enhanced retention compared to a C18. Two such phases are Discovery HS F5 (pentafluorophenyl phase) and Ascentis RP-Amide (embedded polar group phase). The above steps were repeated on 15 cm x 4.6 mm columns of these two phases. Neither column provided significant retention enhancement of risedronate under these conditions.
  4. Investigating ion pair chromatography
    With silica-based HPLC phases, sometimes it is impos sible to operate at a pH where the analyte is adequately retained. For risedronate with the two highly acidic phosphate groups, this condition would require pH values significantly below pH 1 where the stationary phase is readily hydrolyzed, destroying the HPLC column. One solution is to add an ion pair (IP) reagent to the mobile phase. IP reagents have polar groups that interact with functional groups of opposite charge on the analyte, and hydrophobic groups that interact with the reversed phase stationary phase. In this method tetrabutyl ammonium hydroxide (TBAH) was chosen as the IP reagent. TBAH enables the reversed phase retention of risedronate in two ways; the NH4 + groups of TBAH interact with the PO4 - groups of risedronate negating their charge and the butyl groups can interact with the stationary phase.

Although gradient elution is not used with ion pair HPLC, it is useful when scouting for optimum elution conditions. A fast gradient from 5 to 20% methanol over 5 minutes, held at 20% methanol for 10 minutes in 25 mM potassium phosphate containing 5 mM tetrabutyl ammonium hydroxide (pH 7 with phosphoric acid) at 1 mL/min was run on a 15 cm x 4.6 mm I.D, 5 μm Ascentis C18 column. Under these conditions, risedronate retention increased to 11 minutes (k = 6.02, Figure 4). Isocratic conditions of 25% methanol satisfied the customer‘s requirement of 5 to 6 minutes per injection (Figure 5).

Ion pair chromatography using TBAH and an Ascentis C18 HPLC column provided the customer with the desired results. Analysis was complete within 6 minutes and peak shape for this difficult compound was excellent.

Figure 4 ......... IP-HPLC of risedronate at pH 7, 5 mM TBAH, C18 column, gradient


Figure 5 ......... Desired isocratic analysis: IP-HPLC of risedronate at pH 7, 5 mM TBAH, C18 column


Column: Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles
Mobile phase: 75:25, 25 mM potassium phosphate dibasic containing 5 mM tetrabutyl ammonium hydroxide (TBAH), pH 7 with phosphoric acid : methanol
Flow rate: 1.0 mL/min
Temp.: 35 °C
Detection: UV at 262 nm
Sample: 20 μL, risedronate (100 μg/mL) in aqueous mobile phase component

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