**1. Introduction**

Bisphosphonates belong to a unique class of drugs which are chemically stable analogues of the inorganic pyrophosphate anion, a secondary product of various biochemical processes. The concentration levels of pyrophosphate anion in blood are associated with the mechanism of

bone calcification [1]. Like pyrophosphate, bisphosphonates have high affinity for bone mineral and bind strongly to hydroxyapatite calcium in the bone. The skeletal accumulation of bisphosphonates (on the skeleton) depends highly on the disposal of hydroxyapatite binding sites. Non-nitrogen containing bisphosphonates are accumulated into newly formed adenosine triphosphate (ATP) analogues and inhibit ATP-dependent processes, leading to osteoclast apoptosis [2]. Conversely, nitrogen containing bisphosphonates inhibit the action of the enzyme farnesyl pyrophosphate synthase (FPPS) enzyme, which is involved in the mevalonate pathway [3]. These drugs have become the therapy of choice for the managemen<sup>t</sup> of various skeletal disorders such as several types of osteoporosis, hypercalcemia, Paget disease and malignancy metastatic to bone [4]. However, despite the well-recognized benefits of bisphosphonates, these drugs may cause also osteonecrosis of the jaw [5,6].

Bisphosphonates are both polar and ionic compounds and by using reversed phase liquid chromatography are eluted rapidly. The development of a chromatographic method for the analysis of bisphosphonates is a challenge for the analysts due to their high hydrophilicity. In addition to this, the lack of chromophores in some bisphosphonates structures necessitates the use of tedious and time-consuming derivatization procedures for their detection. A literature survey revealed that in most publications adequate retention of bisphosphonates is achieved by using ion-pairing agents in the mobile phase [7–10], anion-exchange chromatography [11] and in some cases pre-column [12] or post-column [13] derivatization procedures [14]. A fused core Ascentis Express HILIC column has been used to quantitate risedronate sodium in pharmaceuticals with PDA and tandem mass spectrometric detection [15]. Bisphosphonates in biological matrices have been quantified after their methylation with trimethylsilyl diazomethane by the use of liquid chromatography–mass spectrometric methods [16–21].

During the last twenty years, HILIC has been proved to be a promising technique for the analysis of polar substances. The separation mechanism in HILIC involves multiple factors, such as partitioning, normal phase/adsorption interactions, hydrogen bonding, reversed-phase and electrostatic interactions [22]. The significance of each of these mechanisms depends on the type of mobile phase and stationary phase that will be used. HILIC requires the use of highly organic mobile phases that contain an aprotic solvent (mainly acetonitrile) in combination with at least 3% of an aqueous salt solution and polar stationary phases so to facilitate chromatographic separation [23]. The major factors affecting retention in HILIC are the type of the stationary phase, the percentage content of water which is the strongest eluent, along with the concentration, pH and type of the aqueous solution of the salt [24]. Studies on the retention mechanism of compounds in HILIC are of grea<sup>t</sup> interest, since it is difficult to predict the effect of the operation parameters on the retention of substances. Bisphosphonates, due to their increased polarity, are perfect candidates to study their retention mechanism in HILIC. Up to now, only a limited number of publications have been reported for the analysis of bisphosphonates in HILIC [15]. This paper describes studies on the retention mechanism of two nitrogen-containing bisphosphonates, namely risedronate and zoledronate, and one non-nitrogen-containing bisphosphonate, namely tiludronate, on a polymeric zwitterion ZIC ®-pHILIC column. It is the first time that zwitterionic hydrophilic interaction liquid chromatography is used to study the retention of bisphosphonates. The zwitterionic hydrophilic interaction liquid chromatography used in this work is a unique form of HILIC, which involved the use of substrates containing zwitterionic functional groups. The key factors influencing the chromatography of these analytes were systematically investigated. A HILIC stability-indicating assay method coupled with photodiode array detection was further optimized and validated to quantitate risedronate in commercial film-coated tablets. Accelerated stability studies of risedronate were also conducted under stress conditions to demonstrate the selectivity of the procedure. The applicability of the method for the quantitation of risedronate was finally proven via the analysis of commercial film-coated tablets containing risedronate as the active ingredient.

### **2. Materials and Methods**

### *2.1. Chemicals and Reagents*

Risedronate sodium salt, hydroxy-(1-hydroxy-1-phosphono-2-pyridin-3-ylethyl)phosphinate; sodium and tiludronate disodium salt, [(4-chlorophenyl)sulfanyl-[hydroxy(oxido) phosphoryl]methyl]- hydroxyphosphinate; disodium, were obtained from Sigma-Aldrich, Germany. Zoledronic acid monohydrate, (1-hydroxy-2-imidazol-1-yl-1-phosphonoethyl)phosphonic acid; hydrate, was obtained from TGI Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). All bisphosphonates were of pharmaceutical purity grade. Solvents of HPLC grade were obtained from E. Merck, Germany. Ammonium formate, ammonium acetate, acetic acid and sodium pyrophosphate were purchased from Acros Organics part of Thermo Fischer Scientific (Geel, Belgium). Water was deionized and further purified by means of a Merck Millipore Synergy UV system (Darmstadt, Germany). Kinesis KX hydrophilic polytetrafluoroethylene (PTFE) syringe filters (diameter 13 mm, pore size 0.22 μm) were purchased from Kinesis Ltd, Cambridge shire, UK.

Commercial film-coated tablet labelled to contain 35 mg risedronate sodium (equivalent to 32.5 mg risedronic acid). Inactive ingredients of the tablet core consist of crospovidone A, cellulose microcrystalline, magnesium stearate and lactose monohydrate. Inactive ingredients of the film coating consist of hypromellose, titanium dioxide E171, hydroxypropyl cellulose, macrogol, iron oxide red (E172), colloidal anhydrous silica and iron oxide yellow (E172).
