Validated chromatographic approach for determination of two ternary mixtures in newly approved formulations for helicobacter pylori eradication: assessment of greenness profile and content uniformity

Abstract

A new, sensitive, and rapid isocratic reversed phase chromatographic method (RP-HPLC-UV) was developed for simultaneous separation of two newly co-formulated antiulcer mixtures; Amoxicillin, Vonoprazan and Clarithromycin [Mixture (I)], and Amoxicillin, Lansoprazole and Clarithromycin [Mixture (II)]. Analytical separation was performed using a Promosil C₁₈ column and ultraviolet detection at 210 nm. The separation was achieved within only 8 min. For both mixtures, an aqueous solution, composed of (Acetonitrile: Methanol: 0. 2 M phosphoric acid) within ratio of (30: 30: 40) adjusted to final pH 3.0, was the mobile phase. This method was validated as per the International Conference on Harmonization guidelines. The linearity ranges of these proposed method of the (Mixture (I)) were 25.0-400.0 µg/mL Amoxicillin, 0.5-8.0 µg/mL Vonoprazan, and 12.5-200.0 µg/mL Clarithromycin. And the linearity ranges of the (Mixture (II)) were 10.0-300.0 µg/mL Amoxicillin, 0.3-9.0 µg/mL Lansoprazole and 5.0-150.0 µg/mL Clarithromycin. This method was firstly applied for effective separation of Amoxicillin, Vonoprazan and Clarithromycin [Mixture (I)]. It fulfilled good repeatability, sensitivity, and accuracy (R.S.D. < 2.0%). The mean recoveries of the analytes in their Tri-Pak formulations were acceptable. The greenness of the developed chromatographic methods was assessed using an Eco-scale method and it was applied for content uniformity testing as per the United States Pharmacopoeia (USP) and the acceptance value of Amoxicillin, in Mixture (I) was 2.88, the acceptance values for Amoxicillin, Lansoprazole in Mixture (II) were 2.592, 2.424, respectively.

Keywords: Amoxicillin; Capsule; Clarithromycin; Content uniformity; Eco-scale; Lansoprazole; Liquid chromatographic method; Tri-Pak; Vonoprazan.

Fig. 1

a Typical chromatogram for the separation of AMOX (200.0 µg/mL, 2.5 min), VPZ (4.0 µg/mL, 3.3 min), and CLA (100.0 µg/mL, 7.6 min), in synthetic mixture (I). Chromatographic system: Promosil C₁₈ column (250 mm × 4.6 mm. 5 μm). Mobile phase mobile phase consisting of ACN: MeOH: 0.2 M OPA (30: 30: 40) at pH 3.0. Flow rate; 1.0 mL/min, UV detection at 210 nm. b Typical chromatogram for the separation of AMOX (200.0 µg/mL, 2.5 min), LAN (6.0 µg/mL, 5.01 min), and CLA (100.0 µg/mL, 7.5 min), in synthetic mixture (II). Chromatographic system: Promosil C₁₈ column (250 mm × 4.6 mm. 5 μm). Mobile phase mobile phase consisting of ACN: MeOH: 0.2 M OPA (30: 30: 40) at pH 3.0. Flow rate; 1.0 mL/min, UV detection at 210 nm

Fig. 2

a Typical chromatogram for the separation of AMOX (100 µg/mL, 2.5 min), VPZ (2.0 µg/mL, 3.3 min), and CLA (50.0 µg/mL, 7.6 min), in combined Tri-Pak dosage form. Chromatographic system: Promosil C₁₈ column (250 mm × 4.6 mm. 5 μm). Mobile phase mobile phase consisting of ACN: MeOH: 0.2 M OPA (30: 30: 40) at pH 3.0. Flow rate; 1.0 mL/min, UV detection at 210 nm. b Typical chromatogram for the separation of AMOX (100.0 µg/mL, 2.5 min), LAN (3.0 µg/mL, 5.01 min), and CLA (50.0 µg/mL, 7.5 min), in combined Tri-Pak dosage form. Chromatographic system: Promosil C₁₈ column (250 mm × 4.6 mm. 5 μm). Mobile phase consisting of ACN: MeOH: 0.2 M OPA (30: 30: 40) at pH 3.0. Flow rate; 1.0 mL/min, UV detection at 210 nm