A novel automated hydrophilic interaction liquid chromatography method using diode-array detector/electrospray ionization tandem mass spectrometry for analysis of sodium risedronate and related degradation products in pharmaceuticals
Introduction
Bisphosphonates are a class of pharmacological active chemical compounds that inhibit osteoclast action and bone resorption. These compounds were originally used as water softeners [1], [2]. From a clinical point of view, they are used for the treatment of osteoporosis, bone metastasis, Paget's disease and other conditions that feature bone fragility. Among bisphosphonates, the most popular first-line drugs are alendronate and risedronate. Pyrophosphate is a natural inhibitor of the mineralization process in bones which are protected by alkaline phosphatase. Several separation analytical methods for the determination of this very important group of chemical compounds have been reviewed [1], [3]. The reviews cover and critically discuss a wide selection of instrumental analytical techniques ranging from liquid and gas chromatography to electrophoretic, enzymatic and automated approaches. Liquid chromatography (LC) generally offers reliable methods characterized by sensitivity, ruggedness and accuracy. The separation efficiency of these techniques makes them a useful tool not only for assay purposes, but even for impurity profiling and metabolite analysis as well [1], [4], [5]. LC modes applied to the determination of bisphosphonates include reversed phase-high pressure liquid chromatography (RP-HPLC), ion-pair chromatography (ICP) and ion chromatography (IC). The majority of these assays employ pre- or post-column derivatization reactions [1], [3], [6]. RP-HPLC is based on the use of solid particulate (usually suitably functionalized/chemically modified silica or polymeric materials) or monolithic support as stationary phases. Mixtures of organic solvents with buffers containing acidic or basic additives employed as mobile phases have been widely applied to separate moderately hydrophilic and hydrophobic compounds [1], [7], [8], [9], [10], [11], [12]. In some cases, changing the pH of the mobile phase in RP-HPLC fails to separate mixtures of very polar organic compounds with ionic character. In these circumstances, IPC is one of the most popular approaches to achieve efficient separations of such species. Different IPC methods were developed for the determination of sodium risedronate (SR) in pharmaceutical formulations [13], [14] and in biological samples [15], [16]. However IPC presents some disadvantages. Ion pair reagents can never be washed fully from the column. Trace of the ion pair reagent can change selectivity when used for non-ion-pair applications, so the reproducibility becomes a problem. Column temperature such as the organic content of the mobile phase should be carefully controlled and using gradient elution is very difficult. Moreover IPC is not ideal because it reduces the high performance liquid chromatography-mass spectrometry (HPLC-MS) compatibility of the method [17]. For SR analysis, several procedures required derivatization protocols [1], [6], [18], whereas other methods were developed using alternative detection modes such as spectrophotometry or evaporative light-scattering detection (ELSD) [19], [20], [21].
Currently, the pharmaceutical industry is particularly interested in developing rapid procedures to cope with a large number of samples and reduce the time required for the delivery of results. For this purpose, various chromatographic strategies have been recently developed in reversed phase-liquid chromatography (RP-LC) and have also become available in the hydrophilic interaction liquid chromatography (HILIC) mode [22]. HILIC is characterized by the use of a hydrophilic stationary phase and a hydrophobic mobile phase. The mechanism of HILIC separation is somewhat complicated since it consists not only of hydrophilic partitioning interactions (the mobile phase-aqueous layer partitioning process) but also secondary electrostatic (attraction or repulsion) and hydrogen-bonding interactions [23]. HILIC has many well-known advantages such as alternative selectivity to RP-HPLC, good retention of hydrophilic compounds (compared with poor retention in RP), low back pressure thanks to the lower viscosity of the organic-rich mobile phases typically used and higher mass spectrometry (MS) sensitivity due to efficient spraying and desolvation of these liquid phases. Of many such phases currently available, HILIC phase has shown wide applicability. Several stationary phases have emerged, made specifically for HILIC approaches. Each stationary material display different retention characteristics and selectivity, requiring distinct buffer constitutions for optimal results [24], [25], [26]. Since there is a growing demand for fast separation with improved resolution, it becomes necessary the use of the new generation of columns and instrumentation, in order to fulfill these requirements. The two most promising strategies commercially available to reach fast HILIC separations are columns packed with sub-2 μm porous particles for ultra high pressure liquid chromatography (UHPLC) and superficially porous particles (fused core). The characteristic features of the fused core technology are good separation, resolution, sensitivity, ruggedness and competitive selectivities [27]. They have been used for the separation and identification of many molecules [23], [27]. However, this type of column has never been applied to SR analysis before. Fused-core particle technology is commonly presented as an alternative to sub-2 μm particles. Various columns of fine particles (2 μm) have been developed, but they are not economically feasible as they require costly UHPLC machine. In addition to this, small particle columns need rigorous filtration of the mobile phase to avoid blockage of 0.5 μm frits of the column [27].
To ensure the quality and safety of SR commercial formulations and the necessity to analyze daily a high number of samples, the current work focuses on the study of a novel, fast, selective and sensitive automated HPLC method with ultraviolet diode-array detector (UV-DAD)/tandem electrospray ionization mass spectrometer evaluating a fused core column (Ascentis Express HILIC). In this way SR and low levels of its degradation products were quickly determined in new dosage forms. The stability of the drug has been evaluated after stress test on granules for oral solution, effervescent tablets and placebos. The main degradation product of SR corresponding to N-oxide derivative was obtained after oxidation with H2O2 and was identified for the first time by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis using an electrospray ionization source (ESI) and an ion trap analyzer.
Section snippets
Materials
SR (87.3% equivalent 100.3% calculated on the dried substance) was purchased from Polpharma (Warsaw, Poland). SR USP (99.9%) was provided from Nova Chimica (Milan, Italy). Ammonium formiate was purchased from Carlo Erba (Milan, Italy). Formic acid was provided from Panreac (Barcelona, Spain). Sodium hydrogen carbonate was provided from Unichimica (Roma, Italy). Citric acid and sodium carbonate were purchased from Brenntag (Milan, Italy). Ethylenediaminetetraacetic acid disodium salt dehydrate
Chromatography
In order to obtain an adequate separation of the peak of SR from those of its known degradation products and from those of the formulation excipients, different RP-HPLC columns and mobile phases were considered focusing on C18 columns. RP-HPLC stationary phases that contain polar groups often succeed in retaining and resolving compounds that C18 phases do not because they can interact with analytes in different ways from those of C18 alkyl chains. Unsatisfactory separations were observed in the
Conclusions
The new type of stationary phase Ascentis Express HILIC (fused core particle technology) has shown real advantages in terms of retention and selectivity providing a good separation of SR and its degradation products in shorter time (less than 4 min) with lower cost. Furthermore the technique does not require derivatization steps, detrimental ion-pair reagents and gradient elution. The simplicity of the procedure allowed no complicated sample preparation, increased sampling rate and favored the
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