electrospray ionization tandem mass spectrometry for analysis of sodium risedronate and related degradation products in pharmaceuticals

Journal of Chromatography A, 1365 (2014) 131–139

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

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 Tiziana Bertolini a , Lorenza Vicentini a , Silvia Boschetti a , Paolo Andreatta a , Rita Gatti b,∗ a b

E-Pharma Trento S.p.A., Research and Development, via Provina 2, 38123 Trento, TN, Italy Department of Pharmacy & Biotechnology, Alma Mater Studiorum – Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy

a r t i c l e

i n f o

Article history: Received 11 April 2014 Received in revised form 1 September 2014 Accepted 3 September 2014 Available online 16 September 2014 Keywords: Sodium risedronate HILIC fused core technology HPLC-ESI-MS Pharmaceutical formulations Degradation products Stability indicating

a b s t r a c t A simple, sensitive and fast hydrophilic interaction liquid chromatography (HILIC) method using ultraviolet diode-array detector (UV-DAD)/electrospray ionization tandem mass spectrometry was developed for the automated high performance liquid chromatography (HPLC) determination of sodium risedronate (SR) and its degradation products in new pharmaceuticals. The chromatographic separations were performed on Ascentis Express HILIC 2.7 ␮m (150 mm × 2.1 mm, i.d.) stainless steel column (fused core). The mobile phase consisted of formate buffer solution (pH 3.4; 0.03 M)/acetonitrile 42:58 and 45:55 (v/v) for granules for oral solution and effervescent tablet analysis, respectively, at a flow-rate of 0.2 mL/min, setting the wavelength at 262 nm. Stability characteristics of SR were evaluated by performing stress test studies. The main degradation product formed under oxidation conditions corresponding to sodium hydrogen (1-hydroxy-2-(1-oxidopyridin-3-yl)-1-phosphonoethyl)phosphonate was characterized by high performance liquid chromatography-electrospray ionization-mass tandem mass spectrometry (HPLC-ESI-MS/MS). The validation parameters such as linearity, sensitivity, accuracy, precision and selectivity were found to be highly satisfactory. Linear responses were observed in standard and in fortified placebo solutions. Intra-day precision (relative standard deviation, RSD) was ≤1.1% for peak area and ≤0.2% for retention times (tR ) without significant differences between intra- and interday data. Recovery studies showed good results for all the examined compounds (from 98.7 to 101.0%) with RSD ranging from 0.6 to 0.7%. The limits of detection (LOD) and quantitation (LOQ) were 1 and 3 ng/mL, respectively. The high stability of standard and sample solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed stability indicating method is suitable for the quality control of SR in new and commercial pharmaceutical formulations. © 2014 Elsevier B.V. All rights reserved.

1. 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.

∗ Corresponding author. Tel.: +39 051 2099707; fax: +39 051 2099734. E-mail address: [email protected] (R. Gatti). http://dx.doi.org/10.1016/j.chroma.2014.09.016 0021-9673/© 2014 Elsevier B.V. All rights reserved.

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

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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 [1,7–12]. 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) [19–21]. 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 (RPLC) 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 organicrich 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 [24–26]. 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 ␮m 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 ␮m particles. Various columns of fine particles (2 ␮m) 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 ␮m 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 H2 O2 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. 2. Experimental 2.1. 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 (EDTA) was obtained from Fluka (Milan, Italy). Acetonitrile Chromasolv® was purchased from Sigma–Aldrich (Milan, Italy). Purified water by a Milli-RX apparatus (Millipore, Milford, MA, USA) using 0.22 ␮m filters was used for the preparation of all solutions, buffers and mobile phase. 2.2. Solutions Routine SR standard solutions (0.2 mg/mL) used for the analysis of granules for oral solution, dilute SR solutions (0.7 ␮g/mL) in placebo (a mixture of the components without SR) used for degradation product analysis and SR standard solutions (0.18 ␮g/mL in placebo) used as reporting threshold solution for the system suitability test were prepared in citrate buffer (pH 5.5)/acetonitrile 50:50, v/v. Routine SR standard solutions (22 mg/mL) used for the analysis of effervescent tablets, dilute SR standard solutions (0.88 ␮g/mL) in placebo used for degradation product analysis and dilute SR standard solutions (0.22 ␮g/mL) in placebo used as reporting threshold solution for the system suitability test were prepared in a mixture of sodium hydrogen carbonate, ammonium formate buffer (pH 6.3; 0.03 M)/acetonitrile 50:50 (v/v). After filtration through 0.22 PVDF filter, SR standard solutions were injected immediately in the chromatograph. Buffer solution (pH 5.5) was prepared by adding citric (0.15 M) and sodium carbonate (0.03 M) to sodium hydrogen carbonate (0.3 M). Ammonium formate buffer solution (pH 6.3; 0.03 M) was prepared with sodium hydrogen carbonate 10 g/L (0.12 M) in ammonium formate buffer at pH 6.3 (0.03 M). The buffer solution was filtered through 0.22 ␮m filter. 2.3. Equipment The liquid chromatography equipment consisted of an Agilent series 1260 gradient unit, an Agilent series 1260 pumping system, an Agilent series 1260 Infinity thermostatted column compartment and an Agilent series 1260 UV-DAD detector (Agilent, Germany).

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Automatic injections of 10 ␮L volume were carried out using an Agilent series 1260 autosampler (Agilent, Germany). Data were collected on a PC equipped with the integration program OpenLAB CDS Chemstation Agilent. LC-MS method was performed using Finnigan LXQ Linear Ion Trap mass spectrometer instrument from Thermo Scientific (San Jose, CA, USA) equipped with an electrospray ionization (ESI) source and with Xcalibur software.

2.4. Chromatographic conditions HPLC separations were performed at 25 ± 2 ◦ C by diode array detection, setting the working wavelength at 262 nm (bandwidth 8 nm) and reference wavelength at 360 nm (bandwidth 100 nm). For routine analyses a Supelco, Ascentis Express HILIC 2.7 ␮m (150 mm × 2.1 mm, i.d.) stainless steel column (fused core) was chosen using a mobile phase consisting of ammonium formate buffer solution (pH 3.4; 0.03 M)/acetonitrile 42:58 and 45:55, v/v for the analysis of granules for oral solution and effervescent tablets, respectively, at a flow-rate of 0.2 mL/min. The column was directly interfaced with the mass spectrometer without flow splitting. The operative conditions of the ESI system are the following: 3.5 kV spray voltage (positive polarity), capillary temperature 270 ◦ C, sheath gas 22, auxiliary gas 4, sweep gas 1. The mass chromatograms were acquired in total ion current (TIC) in the range m/z 100–800, in selected ion monitoring mode (SIM) on m/z 284 and m/z 300 and in MS/MS mode (collision energy 35%) in the range m/z 80–300.

2.5. Stress test studies SR raw material, finished products (granules for oral solution and effervescent tablets) and appropriate placebo were stressed under conditions that usually cause degradation, included the following: dry heat (70 ◦ C), heating in neutral water solution (70 ◦ C for 2 h), acidic media (0.3 M HCl water solution heated at 70 ◦ C for 2 h), basic media (0.3 M NaOH water solution heated at 70 ◦ C for 2 h) and oxidating media (2% H2 O2 at room temperature for about 15 min). After exposure to the conditions mentioned above the solutions were allowed to cool to room temperature and neutralized with acid or base (when necessary).

2.6. Sodium hydrogen (1-hydroxy-2-(1-oxidopyridin-3-yl)-1phosphonoethyl)phosphonate (SR N-oxide) characterization The main degradation product corresponding to SR N-oxide was obtained after oxidation. Both SR and the degradation product were characterized as follows: SR: MW 283, ESI-MS (positive mode): m/z 284 [M+H]+ , HPLC-ESI-MS/MS data reported as m/z (relative abundance): 266 (17.93), 204 (64.87), 202 (90.05), 186 (100), 168 (1.98), 122 (27.72), 94 (0.98); SR N-oxide: MW 299, ESI-MS (positive mode): m/z 300 [M+H]+ , HPLC-ESI-MS/MS data reported as m/z (relative abundance): 282 (33.01), 220 (38.35), 218 (100), 138 (81.63), 110 (4.03).

2.7. Calibration graphs Standard and placebo solutions, fortified appropriately with SR (concentration ranges as in Table 2) were prepared in the same solvent mixture, as reported in Section 2.2. Each final solution was injected five times and SR peak-area was plotted against the corresponding SR concentration to obtain the calibration graphs.

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2.8. Analysis of pharmaceutical formulations 2.8.1. Sample preparation 2.8.1.1. Granules for oral solution and effervescent tablets. Twenty dosage units were finely ground and an amount of powder of each formulation equivalent to about 20 mg of SR was introduced in an appropriate 100 mL volumetric flask. 2.8.1.2. Assay procedure. Each amount of powder was dissolved with about 30 mL of buffer solution (pH 6.3 and 5.5 for effervescent tablets and granules for oral solution, respectively), adding 50 mL of acetonitrile, after ultrasonication for few minutes, and filling up to volume with the appropriate buffer solution. The final solutions were filtered through a 0.22 ␮m PVDF filter and injected in the UV-DAD chromatograph connected with the mass spectrometer. SR content in the samples was determined by external standardization. Each degradation product was quantified as percentage (area/area) of a SR standard solution (0.7 mg/mL). 3. Results and discussion 3.1. 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 analysis of effervescent tablets, owing to the interference of excipients with SR peak. So a stationary phase with enhanced polar retention and selectivity without ion-pairing was evaluated. The new type of stationary phase, Ascentis Express HILIC, is based on the recent fused core technology. Its particles are superficially porous and consist of a 1.7 ␮m solid core surrounded by a 0.5 ␮m porous silica layer. The benefit is the small diffusion path compared with the conventionally fully porous particles, thanks to this it was possible to obtain good separation, with lower back pressure. Costs and time analysis were reduced as well. In this study the chromatographic conditions were optimized and a mixture of formate buffer (pH 3.4; 0.03 M))/acetonitrile was used as eluent according to typical mobile phases [24]. Ammonium formate was chosen because of its compatibility with MS. Since mobile phase and flow were mass compatible the column was directly interfaced with the mass spectrometer without flow splitting. The chromatograms of SR reference substance with its UV spectrum and that of the solvent (blank) are presented in Fig. 1. In comparison with previous works [1,13–16] the analysis of SR and its degradation products is shorter (less than 4 min) (Figs. 1–3). Tailing factor values of SR and degradation products in both formulations are quite good: 0.92–1.73 (Table 1). Moreover HILIC stationary phase allowed to overcome other problems: the addition of an ion pair agent (tetrabutylammonium phosphate, tetrabutylammonium hydroxide or 1-octyltriethylammonium phosphate) and chelating agents (sodium pyrophosphate or EDTA) is not necessary to avoid the interactions of SR (a very strong chelator) with the metals in HPLC system [1,13–16]. Furthermore, HILIC presents the advantage to use volatile buffers with high percentage of organic solvent. The mobile phase is compatible with a LC-MS method, allowing not only the detection but even the identification of degradation products, such as SR N-oxide obtained by using H2 O2 . On the other hand some authors show a strong interaction peak in the chromatogram due to the presence of

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Fig. 1. Representative HPLC-UV-DAD separations: of SR (a) and blank (b). Detail: representative UV-DAD spectrum of SR. Chromatographic conditions and detection as described in the text.

sodium peroxide in the sample, whereas the degradation products could not be detected at the selected chromatographic conditions [14]. 3.2. Method validation The method was validated to show compliance with international requirements for analytical methods in the quality control of pharmaceuticals. For validation of the analytical method, the guide-lines of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use were followed [28]. Stress test studies were performed to provide further indications of the method specificity with respect to the degradation products of SR. 3.2.1. System suitability test Having optimized the efficiency of the developed chromatographic conditions the quality of the chromatography was

monitored not only for SR analysis [13] but even for degradation products by evaluating through system suitability tests: retention time, tailing factor and theoretical plates in samples. The parameters and the related acceptance limits, as defined in the company validation standard operating procedure (SOP), are reported in Table 1. The reporting threshold solution is used to verify S/N (signal to noise ratio). In all cases, the relative standard deviation (RSD) for the analyte peak area for five consecutive injections was <2.0%.

3.2.2. Selectivity The method selectivity was demonstrated overlapping standard solution and blank (solvent) (Fig. 1). There were no impurities or coelution in the sample chromatograms with the analyte peak due to placebo (Fig. 2 A and B). In particular injections of placebo of both formulations containing inactive ingredients showed no interference with SR peak. Furthermore, peak purity tests were performed using a photodiode array detector to confirm that the analyte

Table 1 System suitability test. Compound

Parameters

SR

tR (min)a N (USP)b N (Eur.Pharm.)b fa (10%)c fa (Eur.Pharm., USP)c S/Ne

3.17 13,133 12,557 1.25 1.45 50

3.25 7291 7225 1.44 1.73 20

tR (min)a N (USP)b N (Eur.Pharm.)b fa (10%)c fa (Eur.Pharm., USP)c

2.76 11,740 8180 1.16 1.44

2.89 8516 7760 1.46 1.74

2.7 ± 20%

tR (min)a N (USP)b N (Eur.Pharm.)b fa (10%)c fa (Eur.Pharm., USP)c

2.62 12,760 10,330 0.92 0.93

2.69 9439 8075 1.22 1.22

2.6 ± 20%

N-oxided

Unidentified peak

a b c d e

Retention time. Theoretical plate number. Asymmetry factor. SR threshold solution (0.1% of the theoretical content). S/N = signal to noise ratio.

Soluble granules for oral solution

Effervescent tablets

Acceptance limits 3.2 ± 20%

0.8–2.5 0.8–2.5 ≥10

0.8–2.5 0.8–2.5

0.8–2.5 0.8–2.5

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Fig. 2. HPLC-UV-DAD separation overlay of SR in granules for oral solution (A) and effervescent tablets (B): (a) sample, (b) placebo, (c) standard (SR 0.4%) and (d) blank solutions. Chromatographic conditions as reported in Fig. 1.

chromatographic peak is pure (purity tail and purity front ≥997) and not attributable to more than one component. 3.2.3. Stability of the solutions Injections of standard and sample solutions were performed at different times after their preparation. The solutions (n = 2 for each experiment) were stored at room temperature, in a vial, inside the autosampler device. The solutions were considered stable during the time if the fluctuation  (%) resulted ≤2.0. Standard solutions showed to be stable for 2 h and 30 min, effervescent tablet solutions for 48 h, while the solutions of granules for oral solutions for 58 h, after the preparation. 3.2.4. Stress test studies and characterization of SR N-oxide Stress test studies were carried out (Section 2.5) on SR, finished products and placebo of both formulations. There is no interference with the peaks of the SR and its degradation products (Figs. 3 and 4) due to placebo. After stress test SR peak decrease and some degradation products appear in the chromatograms.

The main degradation product of the raw materials and finished products was observed in the oxidative stress conditions by applying very mild conditions (few minutes with about 1% of H2 O2 at room temperature) and was identified by mass spectrometry as SR N-oxide. Both SR and SR N-oxide molecular weights were determined by means of ESI-MS and by means of HPLC-ESI-MS/MS were obtained structural informations (Fig. 5). By comparison of the fragmentation pathways of SR and SR N-oxide it can be noticed that every molecular fragment of SR N-oxide has a m/z value equivalent to the corresponding SR fragment plus the oxygen mass. It can be assessed that the oxidation occurs on the nitrogen of the pyridine ring. The structures of the identified products, reported in the scheme of the fragmentation pattern confirm the spectral data. SR and SR N-oxide molecular ions cannot be seen in HPLC-ESI-MS/MS spectra, because they were probably subjected to a complete fragmentation. In basic conditions, an unidentified degradation product was observed in both formulations as reported in Figs. 3 and 4. On the basis of the obtained data it can be assessed that the unidentified

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Fig. 3. HPLC-UV-DAD separations of SR and degradation products in granules for oral solution (a) after stress basic; peak: 1. unidentified degradation product; (b) after stress oxidation, peak: 1. SR N-oxide and (c) before stress test. Detail: representative UV-DAD spectrum of degradation products. Chromatographic conditions as reported in Fig. 1.

degradation product (ESI-MS (positive mode): MW137, m/z 138 [M+H]+ ) is due to the interaction of SR with the excipients and not to the instability of the drug substance in basic conditions. According to the peak purity values obtained for SR peak in all stressed samples, the presence of peaks co-eluted with SR is excluded and the peak of SR is considered spectrally

pure. The mass balance was verified in all the stress conditions. The results obtained with the solid thermal stress test (at 70 ◦ C) suggest that there are no incompatibility between SR and excipients of both formulations. The finished products stored in the solid form should be stable over time.

Fig. 4. HPLC-UV-DAD and HPLC-ESI-MS separations of SR and degradation products in effervescent tablets, stressed with H2 O2 : UV-DAD detection at  = 262 nm (a); SIM at 300 (b) and 284 (c) m/z, and stressed with NaOH: UV-DAD detection at  = 262 nm (d); SIM at 284 m/z (e) and XIC at 138 m/z (f). Chromatographic conditions as described in the text.

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137

Fig. 5. Explicative HPLC-ESI-MS/MS spectra of SR (A) and SR N-oxide (B). Detail: representative scheme of SR and SR N-oxide fragmentation pattern.

3.2.5. Linearity The linearity was determined as linear regression with the leastsquare method both on five standard solution and five placebo solutions spiked with SR having the following concentration levels: 50, 75, 100, 115 and 130% of the claimed SR content in the sample. Each level was prepared one time and each solution was injected five times. As an explicative example, the validating parameters of calibration curve of granules for oral solution (slope, intercept, determination coefficient, standard deviation of the slope and standard deviation of the intercept) are shown in Table 2. The slope and y-intercept of the standard and related placebo solutions are not significantly different. Analog data were found for peak areas and concentrations over the range of 0.09–0.27 mg/mL. The easy comparison between the calibration

curves confirms that the matrix did not interfere with the compound analysis. The reported data were obtained at concentration ranges lower than those previously described for direct SR analysis [13,14,19,20]. Satisfactory relationships were obtained in any case. 3.2.6. Limit of detection (LOD) and quantitation (LOQ) LOD and LOQ have been established by the determination of the signal to noise ratio of 3:1 and 10:1, respectively. Good data were obtained corresponding to about 1 (LOD) and 3 (LOQ) ng/mL (Table 2), equal to 0.007 and 0.015% of SR content in each formulations. The sensitivity of the method proved to be about 7 times higher than previous ion-pair UV-HPLC direct method [15] and more sensitive of others [13,14,19,20]. The good obtained data

Table 2 Data for calibration graphs (n = 5). Compound

Slopea (SD)b

y-Intercepta (SD)b

Determination Coefficient (r2 )

Concentration range (mg/mL)

SRc SRd SRe

45,554.3.(171.78) 45,154.4 (191.10) 25.557.(0.35)

43.949. (29.39) 66.562 (36.36) −0.299 (0.14)

0.9998 0.9998 0.9953

0.11/0.22 0.09/0.27

a b c d e

According to y = a x + b, where x = compound concentration, y = SR peak-area. SD = standard deviation. Standard solutions for SR assay. Placebo solutions for SR assay. Placebo solution for SR degradation product analysis.

Concentration range (␮g/mL)

LOD (ng/mL)

LOQ (ng/mL)

0.015–0.718

1

3

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Table 3 Repeatability and intermediate precision data of sample solutions of SR in granules for oral solution and effervescent tablets (SR content: 35 mg/unit) for area and retention time (tR ). Formulation

Mean area RSD (%)

Confidence (%)a

Mean tR (min) RSD (%)

Confidence (%)a

Mean mg/unit RSD (%)

Confidence (%)a

Repeatability (n = 6) analyst A/day 1 Granules for oral solution Effervescent tablets

7570 (0.7) 10,159 (0.6)

0.6 0.5

3.1 (0.1) 3.2 (0.2)

0.2 0.3

33.3 (0.5) 34.9 (0.5)

0.4 0.4

Analyst B/day 2 Granules for oral Effervescent tablets

7600 (0.6) 10,156 (1.1)

0.5 0.9

3.2 (0.1) 3.2 (0.1)

0.2 0.2

33.4 (0.4) 34.4 (1.0)

0.3 0.8

Intermediate precision (n = 12) Granules for oral Effervescent tablets

7585 (0.6) 10,158 (0.8)

0.4 0.5

3.1 (0.2) 3.2 (0.2)

0.2 0.2

33.3 (0.5) 34.6 (1.0)

0.6 0.6

a

% = confidence percentage (˛ = 0.05).

can suggest other useful method applications such as to biological samples [15,19] as far as they require higher sensitivity than pharmaceuticals.

of oral solution and recovery = 101%; RSD = 0.7% for effervescent tablets (Table 4)).

3.2.7. Precision The method precision was expressed as repeatability and intermediate precision. The repeatability and intermediate precision of the method were calculated on sample solutions to verify its applicability. To this end, twenty effervescent tablets and the content of twenty sachets were finely ground, separately. The repeatability was calculated employing six test sample solutions, each one prepared according to the analytical method starting from a homogeneous sample of finished product. The intermediate precision was determined with twelve sample solutions prepared changing the parameters time-analyst: six solutions were prepared by the analyst A in the day 1 and other six solutions were prepared by the analyst B in the day 2. The results expressed as RSD ≤0.7% (area) and 0.1% (tR ) for granules for oral solutions, and RSD ≤1.1% (area) and 0.2% (tR ) for effervescent tablets (Table 3) demonstrate a satisfactory precision of the described method. There was no significant difference between intra- and inter-day data.

3.2.9. Ruggedness The ruggedness of the method was evaluated varying some operating conditions, including different operators in the laboratory, changing the source of reagents and solvents, and changing the HILIC column (of the same type and manufacturer). The method proved to be rugged enough to allow routine laboratory use. Solution stability, intermediate precision, stress test and ruggedness can give an indication of the method robustness.

3.2.8. Accuracy The accuracy was based on the recovery of known amounts of analyte, spiking SR in placebo of both analyzed formulations. Fortified samples were prepared in triplicate at tree levels over a range 75–125% of the target concentration. Quantitative recoveries were obtained in each instance (recovery = 98.7%; RSD = 0.6% for granules

3.3. Determination of SR in new medicinal products The analytical method was developed and validated for the quantitative determination of SR in new pharmaceutical formulations: granules for oral (excipients: sucrose, maltodextrin, sodium hydrogen carbonate, flavor, acesulfame potassium) and effervescent tablets (excipients: sodium hydrogen carbonate, sodium carbonate, citric acid, sorbitol, flavor, acesulfame potassium). The method was applied for the analysis of some batches of these medicinal products and the results were found within the acceptance limits for both the content of SR (95–105% of the claimed content) and each degradation product (not more than the 0.2% with respect to the SR content), thus demonstrating that the products comply with the requirements of the European quality criteria.

Table 4 Recovery data of SR in granules for oral solution and effervescent tablets. Formulation

Compound

Level (%)

Spiked amount (mg/mL)

Mean recovery (%, n = 3)

Mean recovery (%, n = 9)

RSD (%)

Granules for oral solutiona

SR

75

0.131 0.142 0.139 0.167 0.191 0.172 0.229 0.214 0.227

99.8 100.3 98.8 100.4 98.7 99.0 99.2 99.0 99.1

99.6

99.4

0.6

0.154 0.187 0.148 0.226 0.224 0.238 0.287 0.281 0.271

99.9 100.7 98.9 100.4 101.0 100.5 100.3 99.6 99.4

99.8

100.1

0.7

100

125

Effervescent tabletsb

SR

75

100

125

a b

Placebo amount about 2200 mg. Placebo amount about 750 mg.

Recovery (%)

99.3

99.1

100.7

99.8

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This point out the capability of the method to provide reliable qualiquantitative data in comparison with others [13,14].

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4. 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 automation, which is fundamental for the industrial activity. The method presents a high sensitivity (LOD 1 ng/mL) suitable for the quantitation of the main impurity trace. SR N-oxide, corresponding to the main degradation product obtained after stress test was identified for the first time by HPLC-ESI-MS/MS. The validation procedure confirms that this technique provides reliable analyses of SR and degradation products. The proposed method is highly recommended for the quality control of many SR samples, including new or commercial pharmaceuticals and raw materials and is applicable to all conventional HPLC systems. References [1] C.K. Zacharis, P.D. Tzanavaras, Determination of bisphosphonate active pharmaceutical ingredients in pharmaceuticals and biological material: a review of analytical methods, J. Pharm. Biomed. Anal. 48 (2008) 483–496. [2] R.G. Russell, M.J. Rogers, Bisphosphonates: from the laboratory to the clinic and back again, Bone 25 (1999) 97–106. [3] R.W. Sparidans, J. den Hartigh, Chromatographic analysis of bisphosphonates, Pharm. World Sci. 21 (1999) 1–10. [4] S. Görög, Drug safety, drug quality, drug analysis, J. Pharm. Biomed. Anal. 48 (2008) 247–253. [5] M.S. Chang, Q.J.J. Zhang, T.A. El-Shourbagy, Drug Dev. Res. 68 (2007) 107–133. [6] T. Pérez-Ruiz, C. Martínez-Lozano, M.D. García-Martínez, A sensitive postcolumn photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates, J. Chromatogr. A 1216 (2009) 1312–1318. [7] W.F. Kline, B.K. Matuszewski, W.F. Bayne, Determination of 4-amino1-hydroxybutane-1,1-bisphosphonic acid in urine by automated precolumn derivatization with 2,3-naphthalene dicarboxyaldehyde and highperformance liquid chromatography with fluorescence detection, J. Chromatogr. 534 (1990) 139–149. [8] W.F. Kline, B.K. Matuszewski, Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection, J. Chromatogr. 583 (1992) 183–193. [9] L.E. King, R. Vieth, Extraction and measurement of pamidronate from bone samples using automated pre-column derivatization, high-performance liquid chromatography and fluorescence detection, J. Chromatogr. B 678 (1996) 325–330. [10] C. Apostolou, Y. Dotsikas, C. Kousoulos, G. Tsatsou, F. Colocouri, G.S. Soumelas, Y.L. Loukas, Application of a semi-automated 96-well format solidphase extraction, column-switching, fluorescence detection protocol for the

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