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Novel RP-HPLC-DAD method for simultaneous determination of aminosalycilates and folic acid assay in proposed fixed dose combinations for treatment of inflammatory bowel disease

Malabsorption and deficiency of folic acid and its connection with risk of developing colorectal cancer is no novelty when it comes to patients with inflammatory bowel disease (IBD). IBDs representatives, Crohn’s disease and Ulcerative Colitis are chronic inflammatory conditions that affect gastrointestinal tract. Absorption of folic acid takes place mainly in small intestine with decreasing absorption gradient from jejunum to colon. Since small intestine is affected by Crohn’s disease, damaged tissue on sidewalls makes absorption of folic acid insufficient. Additionally, sulfasalazine and olsalazine, drugs used for treatment of IBD, inhibit the absorption of folic acid. To prevent consequences caused by folic acid deficiency additional intake of folic acid is mandatory to control its level in IBD patients. For diseases that require treatment with multiple drugs, such as IBD, fixed-dose combination (FDC) therapy, in which two or more active pharmaceutical ingredients (API) are formulated in a fixed proportional manner into a single dosage form, may offer help in improvement of therapeutic outcomes. To improve IBD patient adherence a FDC of aminosalycilate drug and folic acid is proposed and therefore single RP-HPLC-DAD method was developed and validated for simultaneous assay determination of all 4 representatives of aminosalicylate drugs (mesalazine, sulfasalazine, olsalazine and balsalazide) and folic acid. The method was developed on Agilent 1100 HPLC system using XBridge Phenyl column (150 mm x 4.6 mm, 3.5 µm) with gradient elution that was carried out at 25°C in a flow rate of 1 mL/min with 0.2% HCOOH water solution as mobile phase A and 0.2% HCOOH in methanol as mobile phase B. DAD detection of mesalazine, olsalazine, sulfasalazine and balsalazide was performed at 320 nm whilst folic acid was monitored on 285nm. High linearity was obtained with R2 ranging from 0.9997 to 0.9999. Accuracy was performed on three concentration levels with recovery percentage being in range of 98% to 102%. Inter- and intra-day precision showed low RSD values implying that method is precise enough for its intended use. Method remained robust in changes of mobile phase pH, gradient, flow rate and column temperature. To confirm the applicability, developed method was used to determine assay of commercially available products used in treatment of IBD. Results showed that assays of all analysed drugs were within the specification range of 95 to 105% of declared assay value. According to results of this study, developed and validated method is suitable for future research. This work has been supported in part by the Croatian Science Foundation under the project number [UIP-2017-05-3949]. This work has been supported in part by the European Union from the European Social Fund.

aminosalicylates, folic acid, inflammatory bowel disease, HPLC

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