TY - JOUR
AU1 - Burmaoglu, Rifat, Emre
AU2 - Saglik Aslan,, Serap
AB - Abstract A method has been developed and validated for analysis of zoledronic acid (ZOL) and its related substances by ion-pair reversed-phase high performance liquid chromatography with evaporative light scattering detection (ELSD). Chromatographic separation was achieved with gradient elution by using a C18 column, mobile phase containing 12 mM ammonium acetate buffer and 35 mM n-pentylamine, whose pH value is 7.0, and 5% acetonitrile. The mobile-phase flow rate was 1.0 mL/min. The calibration plot was linear in the range from 0.4 mg/mL to 6.0 mg/mL for ZOL and from 6.25 μg/mL to 100 μg/mL for its related substances. ZOL and its related substances, namely imidazole-1-yl-acetic acid, phosphate, phosphite and degradation products did not interfere with each other. The method was rapid, linear, accurate and reproducible. The high performance liquid chromatographic method that has been developed to determine the related substances and assay of ZOL can be used simultaneously to evaluate the quality of regular samples. It can be also used to test the stability samples of ZOL. Introduction ZOL, a bisphosphonic acid, is an inhibitor of osteoclastic bone resorption. zoledronic acid (ZOL) is chemically designated as (1-hydroxy-2-imidazole-1-yl-phosphonoethyl) phosphonic acid monohydrate. ZOL is a white crystalline powder. Zometa® (ZOL) injection is the registered product of Novartis Pharmaceutical Corporation (innovator), which is commercially available as a sterile liquid concentrate solution in vials for intravenous infusion. Each 5-mL vial contains 4.264 mg of ZOL monohydrate, corresponding to 4 mg ZOL on an anhydrous basis. The marketing approval for ZOL in Europe and the USA has been given in the year 2002 (1). It is a potent inhibitor of osteoclastic bone resorption and is clinically used for the treatment of malignant and benign bone diseases, e.g., osteoporosis, Paget’s disease or metastases of solid tumors (2–5). It is a new third generation bisphosphonate (6). Due to the chemical nature of ZOL, its chromatographic separation is challenging. Since bisphosphonates contain two phosphoric acid groups, they are ionic and highly polar. Thus, they show no retention on conventional reversed-phase liquid chromatographic (RP-LC) stationary phases such as RP-8 or RP-18. Furthermore, the ability to complex ubiquitous metal ions such as Ca2+ and their tendency to form multiply charged species gives rise to poor peak shape, baseline disturbance and irreproducible chromatograms (7). Bisphosphonates do not have the strong chromophores typically used for UV detection in HPLC methods (8). Many methods have been developed to analyze bisphosphonates on the basis of derivatization. Clodronate (9, 10), etidronate (11) and pamidronate (12, 13) were analyzed by gas chromatography with the aid of isobutylchloroformate and BSTFA [N, O-bis-(trimethylsilyl) trifluoroacetamide] as a marker. Liquid chromatographic analyses of bisphosphonates have been given in two different derivatization reactions. The first reaction involves the introduction of fluorogenic labels, coupled to the amino group of several bisphosphonates (14–23). The second derivatization reactions are based on forming complex reactions (24–30). Direct detection methods of bisphosphonates are especially based on ion exchange chromatography with refractive index detector (31), conductivity detector (32), electrochemical detector (33) and mass spectrometry detector (34). There are some pharmacopoeial methods for assaying alendronate active substance and its related substances in the European Pharmacopoeia (35) and there is a derivatization reaction described in the US Pharmacopoeia (36). Jiang et al. (37) determined ibandronate, a member of bisphosphonates, and its degradation products by RP-LC with evaporative light scattering detection (ELSD). In another article, Jiang et al. (38) used RP-LC with UV–visible detector to determine ZOL and its related compounds namely, imidazole-1-yl-acetic acid and unidentified-related compounds. Rao et al. (39) developed a stability indicating ion exchange RP-HPLC method with UV–visible detector. In this method, forced degradation study was conducted in order to give information on drug’s inherent stability and help in the validation of analytical methods to be used in stability studies. This paper dealt with ZOL and its related substances namely, imidazole and imidazole-1-yl-acetic acid with the other degradation products. In a review article by Jiang et al. (40), an ion-pair reversed-phase high performance liquid chromatographic method (ion-pair RP-HPLC) coupled with ELSD has been established, for the routine assay of ZOL and its related substances in bulk material and pharmaceutical dosage forms. Its related substances include remaining imidazole-1-yl-acetic acid in the synthesis of ZOL and other possible impurities of decomposition such as phosphoric acid and phosphate. Xie et al. (41) developed a simple analysis in which four bisphosphonates (alendronate, pamidronate, etidronate and ZOL) are determined simultaneously by RP-LC using n-amylamine as volatile ion-pair agent. Assay of these four important members of bisphophonates have been successfully analyzed using this method which was not developed for related substances. This paper describes a simple, rapid and selective gradient RP-HPLC method with ELSD to determine ZOL (Figure 1a) assay and its related substances namely imidazole-1-yl-acetic acid (Figure 1b), phosphate (Figure 1c), phosphite (Figure 1d) and the other possible degradation products in a single analysis with high precision and accuracy. The method validation was performed and fuled the International Conference on Harmonization (ICH) guidelines. The method determined degradation products by studying forced degradation study with drug substance powder and drug product solution. Degradation products were well resolved to each other. Therefore this method can be used for both routine and stability purposes. Figure 1. View largeDownload slide Structures of the analytes involved in the study: (a) zoledronic acid, (b) imidazole-1-yl-acetic acid, (c) phosphate and (d) phosphite. Figure 1. View largeDownload slide Structures of the analytes involved in the study: (a) zoledronic acid, (b) imidazole-1-yl-acetic acid, (c) phosphate and (d) phosphite. Materials and Methods Chemicals and reagents HPLC grade water, ultra gradient HPLC grade acetonitrile (Sigma-Aldrich, Germany), ultra gradient grade methanol (Sigma-Aldrich, Germany), ion- pair grade n-amylamine (Acros Organics, Belgium), ammonium acetate (Merck KGaA, Darmstadt, Germany), hydrogen peroxide (J.T. Baker, the Netherlands) and glacial acetic acid (Sigma-Aldrich, Germany). Milli-Q grade water. From an in-house Millipore system (Millipore, Billerica, MA). Mannitol (Merck KGaA, Darmstadt, Germany), sodium citrate trihydrate (Merck KGaA, Darmstadt, Germany). Apparatus HPLC system—A Waters 2695 Alliance HPLC system equipped with Waters 2424 ELSD and Waters 2489 UV–Visible detector controlled by Empower 2 software (Waters Corp., Milford, MA). Photostability Chamber—An Atlas Sunset CPS (+) and Atlas Suncool Cooler Unit (Chicago, USA). Columns—Ace-5 C8 column (15 cm × 4.6 mm id, 5 μm particle size, 100 Å pore size; Hichrom, Aberdeen, Scotland), Zorbax RX C8 column (25 cm × 4.6 mm id, 5 μm particle size, 80 Å pore size; Agilent, Chicago, USA), Hichrom C18 column (25 cm × 4.6 mm id, 5 μm particle size, 150 Å pore size; Hichrom, Reading, UK). Standards Phosphoric acid (J.T. Baker, the Netherlands), phosphorous acid (Riedel de Haen, Germany), imidazole-1-yl-acetic acid (Auspure, China), ZOL (Eczacibasi-Zentiva Chemical Products, Tekirdag, Turkey), zoledronate sodium (Eczacibasi-Zentiva Chemical Products,Tekirdag, Turkey). Chromatographic conditions Separation conditions—Flow rate, 1.0 mL/min; measurement parameters, gain, 5; data rate, 10; time constant, slow; mode, cooling; drift tube temperature, 50°C; pressure, 40 psi; column thermostat temperature, 30°C; mobile-phase A, aqueous solution containing 12 mM ammonium acetate buffer and 35 mM n-amylamine, pH adjusted to 7.0 with acetic acid; mobile phase B, gradient grade acetonitrile (Table I). Table I. Gradient elution profile Time, min A (%) B (%) 12 mM ammonium acetate buffer containing 35 mM n-amylamine, pH = 7.0 Acetonitrile 0 95 5 6 85 15 14 85 15 16 95 5 20 95 5 Time, min A (%) B (%) 12 mM ammonium acetate buffer containing 35 mM n-amylamine, pH = 7.0 Acetonitrile 0 95 5 6 85 15 14 85 15 16 95 5 20 95 5 Table I. Gradient elution profile Time, min A (%) B (%) 12 mM ammonium acetate buffer containing 35 mM n-amylamine, pH = 7.0 Acetonitrile 0 95 5 6 85 15 14 85 15 16 95 5 20 95 5 Time, min A (%) B (%) 12 mM ammonium acetate buffer containing 35 mM n-amylamine, pH = 7.0 Acetonitrile 0 95 5 6 85 15 14 85 15 16 95 5 20 95 5 Solutions Standard solutions for the assay of ZOL Standard solution 1:40 mg ZOL reference standard, accurately weighed to 0.01 mg, was dissolved in 10.0 mL water. Standard solution 2:50 mg ZOL reference standard, accurately weighed to 0.01 mg, was dissolved in 10.0 mL water. Standard solution 3:60 mg ZOL reference standard, accurately weighed to 0.01 mg, was dissolved in 10.0 mL water. Standard solutions for the related substances Stock standard solution: 125 mg imidazole-1-yl-acetic acid reference standard, 150 mg phosphoric acid and 125 mg phosphorous acid, accurately weighed to 0.1 mg, dissolved in 250.0 mL water. Standard solution A: 1.25 mL stock standard solution diluted to 50 mL with water (the concentration of each component was 12.5 μg/mL). Standard solution B: 2.50 mL stock standard solution diluted to 50 mL with water (the concentration of each component was 25.0 μg/mL). Standard solution C: 5.0 mL stock standard solution diluted to 50 mL with water (the concentration of each component was 50.0 μg/mL). Sample solutions Sample solution (for drug substance): 50 mg sample to be analyzed, accurately weighed to 0.1 mg, dissolved in 10 mL water. This sample was used to study assay and related substances determination for drug substance. Sample solution (for drug product): concentrated infusion solution containing ZOL equivalent to 4 mg per 5 mL was directly injected. Solution for forced degradation study About 3% (v/v) H2O2 solution: 10 mL of 30% (v/v) concentrated solution was diluted to 100 mL with water. Results HPLC method development A typical sample chromatogram spiked with the related substances is given in Figure 2. Elution order and relative retention times with respect to ZOL peak are; imidazole-1-yl-acetic acid (Rt: 3.77 min), (RRt ~0.6); phosphate (Rt: 4.43 min), (RRt ~0.7); phosphite (Rt: 4.99 min), (RRt ~0.8) and ZOL (Rt: 5.92 min), (RRt 1,0). Gradient elution profile is also listed in Table I. Effects of organic modifiers; methanol and acetonitrile are illustrated in Figure 3. pH value of mobile phase was determined by investigating the capacity factors of all interested peaks in three different pH values as of 4.5, 4.8 and 7.0. Three different HPLC columns were evaluated to achieve adequate peak performance parameters. ELSD parameters such as gas flow rate, drift tube temperature and nebulizer mode were evaluated carefully with different settings to yield best response of the interested peaks. Figure 2. View largeDownload slide A typical sample chromatogram spiked with the related substances, elution order and relative retention times with respect to zoledronic acid peak; imidazole-1-yl-acetic acid (Rt: 3.77 min), (RRt ~ 0.6); phosphate (Rt: 4.43 min), (RRt ~ 0.7); phosphite (Rt: 4.99 min), (RRt ~ 0.8); zoledronic acid (Rt: 5.92 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 2. View largeDownload slide A typical sample chromatogram spiked with the related substances, elution order and relative retention times with respect to zoledronic acid peak; imidazole-1-yl-acetic acid (Rt: 3.77 min), (RRt ~ 0.6); phosphate (Rt: 4.43 min), (RRt ~ 0.7); phosphite (Rt: 4.99 min), (RRt ~ 0.8); zoledronic acid (Rt: 5.92 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 3. View largeDownload slide The chromatograms representing primary peak (zoledronic acid) when methanol and acetonitrile were used as the organic modifier in the mobile phase, respectively. The chromatograms were obtained with Hichrom C18 column. (a) The chromatogram was obtained with the usage of methanol as the organic modifier. (b) The chromatogram was obtained with the usage of acetonitrile as the organic modifier. Figure 3. View largeDownload slide The chromatograms representing primary peak (zoledronic acid) when methanol and acetonitrile were used as the organic modifier in the mobile phase, respectively. The chromatograms were obtained with Hichrom C18 column. (a) The chromatogram was obtained with the usage of methanol as the organic modifier. (b) The chromatogram was obtained with the usage of acetonitrile as the organic modifier. Method validation A typical untreated sample chromatogram including the related substances is given in Figure 4. The related substances were not spiked to the sample solution. Elution order and relative retention times with respect to ZOL peak are; phosphate (Rt: 4.578 min), (RRt ~0.77); phosphite (Rt: 5.225 min), (RRt ~0.88) and ZOL (Rt: 5.960 min), (RRt 1,0). Figure 4. View largeDownload slide A typical untreated sample chromatogram including the related substances, the related substances were not spiked to the sample solution. Elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.578 min), (RRt ~ 0.77); phosphite (Rt: 5.225 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.960 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 4. View largeDownload slide A typical untreated sample chromatogram including the related substances, the related substances were not spiked to the sample solution. Elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.578 min), (RRt ~ 0.77); phosphite (Rt: 5.225 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.960 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. A typical sample chromatogram of oxidative degradation (3% H2O2) including the related substances was shown in Figure 5. Elution order and relative retention times with respect to ZOL peak are; imidazole-1-yl-acetic acid (Rt: 3.975 min), (RRt ~0.66), phosphate (Rt: 4.592 min), (RRt ~0.77); phosphite (Rt: 5.223 min), (RRt ~0.87) and ZOL (Rt: 5.975 min), (RRt 1,0). Figure 5. View largeDownload slide A typical sample chromatogram of oxidative degradation (3% H2O2) including the related substances. Elution order and relative retention times with respect to zoledronic acid peak; Imidazole-1-yl-acetic acid (Rt: 3.975 min), (RRt ~ 0.66), phosphate (Rt: 4.592 min), (RRt ~ 0.77); phosphite (Rt: 5.223 min), (RRt ~ 0.87); zoledronic acid (Rt: 5.975 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 5. View largeDownload slide A typical sample chromatogram of oxidative degradation (3% H2O2) including the related substances. Elution order and relative retention times with respect to zoledronic acid peak; Imidazole-1-yl-acetic acid (Rt: 3.975 min), (RRt ~ 0.66), phosphate (Rt: 4.592 min), (RRt ~ 0.77); phosphite (Rt: 5.223 min), (RRt ~ 0.87); zoledronic acid (Rt: 5.975 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Typical sample chromatograms of water hydrolysis by reflux, photo degradation and thermal degradation at 100°C including the related substances were given in Figures 6–8, respectively. Comparison table representing % difference of area in degraded sample with respect to area of the untreated sample solution was prepared (Table II). Figure 6. View largeDownload slide A typical sample chromatogram of water hydrolysis by reflux including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.578 min), (RRt ~ 0.77); phosphite (Rt: 5.227 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.959 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 6. View largeDownload slide A typical sample chromatogram of water hydrolysis by reflux including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.578 min), (RRt ~ 0.77); phosphite (Rt: 5.227 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.959 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 7. View largeDownload slide A typical sample chromatogram of photo degradation including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.576 min), (RRt ~0.77); phosphite (Rt: 5.220 min), (RRt ~0.88); zoledronic acid (Rt: 5.954 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 7. View largeDownload slide A typical sample chromatogram of photo degradation including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.576 min), (RRt ~0.77); phosphite (Rt: 5.220 min), (RRt ~0.88); zoledronic acid (Rt: 5.954 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 8. View largeDownload slide A typical sample chromatogram of thermal degradation at 100°C including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.560 min), (RRt ~ 0.78); phosphite (Rt: 5.199 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.876 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 8. View largeDownload slide A typical sample chromatogram of thermal degradation at 100°C including the related substances, elution order and relative retention times with respect to zoledronic acid peak; phosphate (Rt: 4.560 min), (RRt ~ 0.78); phosphite (Rt: 5.199 min), (RRt ~ 0.88); zoledronic acid (Rt: 5.876 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Table II. Comparison table representing % difference of area in degraded sample with respect to area of the untreated sample solution Area Peak eluted at Rt 3.7 min. Imidazole-1-yl-acetic acid Phosphate Phosphite Peak eluted at Rt 5.7 min. Zoledronic Acid Peak eluted at Rt 7.7 min. Peak eluted at Rt 8.5 min. Untreated sample 1,508 124,529 22,371 3,066 23.029.281 104,860 Water Hydrolysis 2,114 125,601 21,447 2,969 21.898.283 96,291 % Difference 40.2 0.8 −4.1 −3.2 −4.9 −8.2 Thermal degradation at 100°C 2,115 137,117 25,020 3,599 25.969.996 120,473 % Difference 40.3 10.1 11.8 17.4 12.8 14.9 Photo degradation by UV 1,164 133,682 21,913 4,226 22.936.312 89,884 % Difference −22.8 7.4 −2.0 37.8 −0.4 −14.3 Oxidative degradation 6869 205,461 23,323 29,440 22.429.565 72,034 52,289 % Difference Formed after degradation 65.0 4.3 860.2 −2.6 −31.3 Formed after degradation Area Peak eluted at Rt 3.7 min. Imidazole-1-yl-acetic acid Phosphate Phosphite Peak eluted at Rt 5.7 min. Zoledronic Acid Peak eluted at Rt 7.7 min. Peak eluted at Rt 8.5 min. Untreated sample 1,508 124,529 22,371 3,066 23.029.281 104,860 Water Hydrolysis 2,114 125,601 21,447 2,969 21.898.283 96,291 % Difference 40.2 0.8 −4.1 −3.2 −4.9 −8.2 Thermal degradation at 100°C 2,115 137,117 25,020 3,599 25.969.996 120,473 % Difference 40.3 10.1 11.8 17.4 12.8 14.9 Photo degradation by UV 1,164 133,682 21,913 4,226 22.936.312 89,884 % Difference −22.8 7.4 −2.0 37.8 −0.4 −14.3 Oxidative degradation 6869 205,461 23,323 29,440 22.429.565 72,034 52,289 % Difference Formed after degradation 65.0 4.3 860.2 −2.6 −31.3 Formed after degradation View Large Table II. Comparison table representing % difference of area in degraded sample with respect to area of the untreated sample solution Area Peak eluted at Rt 3.7 min. Imidazole-1-yl-acetic acid Phosphate Phosphite Peak eluted at Rt 5.7 min. Zoledronic Acid Peak eluted at Rt 7.7 min. Peak eluted at Rt 8.5 min. Untreated sample 1,508 124,529 22,371 3,066 23.029.281 104,860 Water Hydrolysis 2,114 125,601 21,447 2,969 21.898.283 96,291 % Difference 40.2 0.8 −4.1 −3.2 −4.9 −8.2 Thermal degradation at 100°C 2,115 137,117 25,020 3,599 25.969.996 120,473 % Difference 40.3 10.1 11.8 17.4 12.8 14.9 Photo degradation by UV 1,164 133,682 21,913 4,226 22.936.312 89,884 % Difference −22.8 7.4 −2.0 37.8 −0.4 −14.3 Oxidative degradation 6869 205,461 23,323 29,440 22.429.565 72,034 52,289 % Difference Formed after degradation 65.0 4.3 860.2 −2.6 −31.3 Formed after degradation Area Peak eluted at Rt 3.7 min. Imidazole-1-yl-acetic acid Phosphate Phosphite Peak eluted at Rt 5.7 min. Zoledronic Acid Peak eluted at Rt 7.7 min. Peak eluted at Rt 8.5 min. Untreated sample 1,508 124,529 22,371 3,066 23.029.281 104,860 Water Hydrolysis 2,114 125,601 21,447 2,969 21.898.283 96,291 % Difference 40.2 0.8 −4.1 −3.2 −4.9 −8.2 Thermal degradation at 100°C 2,115 137,117 25,020 3,599 25.969.996 120,473 % Difference 40.3 10.1 11.8 17.4 12.8 14.9 Photo degradation by UV 1,164 133,682 21,913 4,226 22.936.312 89,884 % Difference −22.8 7.4 −2.0 37.8 −0.4 −14.3 Oxidative degradation 6869 205,461 23,323 29,440 22.429.565 72,034 52,289 % Difference Formed after degradation 65.0 4.3 860.2 −2.6 −31.3 Formed after degradation View Large A typical placebo chromatogram of drug product is given in Figure 9. Placebo peaks were sodium cation came out from sodium citrate used as one of excipients (Rt: 2.046 min), mannitol (Rt: 2.419 min), citrate anion (Rt:8.335 min). A typical drug product sample chromatogram spiked with the related substances is shown in Figure 10. Elution order and relative retention times with respect to ZOL peak are; sodium cation (Rt: 2.048 min), (RRt ~0.36); mannitol (Rt: 2.419 min), (RRt ~0.43); imidazole-1-yl-acetic acid (Rt: 3.934 min), (RRt ~0.7); phosphate (Rt: 4.345 min), (RRt ~0.8); phosphite (Rt: 5.014 min), (RRt ~0.9) and ZOL (Rt: 5.651 min), (RRt 1,0). Figure 9. View largeDownload slide A typical placebo chromatogram of drug product. Placebo peaks were sodium cation (Rt: 2.046 min), mannitol (Rt: 2.419 min), citrate anion (Rt:8.335 min). The chromatogram was obtained with Hichrom C18 column. Figure 9. View largeDownload slide A typical placebo chromatogram of drug product. Placebo peaks were sodium cation (Rt: 2.046 min), mannitol (Rt: 2.419 min), citrate anion (Rt:8.335 min). The chromatogram was obtained with Hichrom C18 column. Figure 10. View largeDownload slide A typical drug product sample chromatogram spiked with the related substances, elution order and relative retention times with respect to zoledronic acid peak; sodium cation (Rt: 2.048 min), (RRt ~ 0.36); mannitol (Rt: 2.419 min), (RRt ~0.43); imidazole-1-yl-acetic acid (Rt: 3.934 min), (RRt ~ 0.7); phosphate (Rt: 4.345 min), (RRt ~ 0.8); phosphite (Rt: 5.014 min), (RRt ~ 0.9); zoledronic acid (Rt: 5.651 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. Figure 10. View largeDownload slide A typical drug product sample chromatogram spiked with the related substances, elution order and relative retention times with respect to zoledronic acid peak; sodium cation (Rt: 2.048 min), (RRt ~ 0.36); mannitol (Rt: 2.419 min), (RRt ~0.43); imidazole-1-yl-acetic acid (Rt: 3.934 min), (RRt ~ 0.7); phosphate (Rt: 4.345 min), (RRt ~ 0.8); phosphite (Rt: 5.014 min), (RRt ~ 0.9); zoledronic acid (Rt: 5.651 min), (RRt 1.0). The chromatogram was obtained with Hichrom C18 column. LOD and LOQ values, linear regression results for ZOL and its related substances, results of the accuracy, intra-day and inter-day precision studies are presented in Table III. Table III. Limit of detection, limit of quantitation, linearity, accuracy and precision results of proposed method Validation parameter Zoledronic acid Imidazole-1-yl-acetic acid Phosphate Phosphite Acceptance criteria LOD 0.9 μg/mL 1.0 μg/mL 0.75 μg/mL 0.50 μg/mL LOQ 1.7 μg/mL 1.5 μg/mL 2.5 μg/mL 1.5 μg/mL Linearitya  # of level 5 5 5 5  Range 0.39–5.96 mg/mL 6.25–100 μg/mL 6.25–100 μg/mL 6.25–100 μg/mL  A 1.453226e+006 1.132516e+001 1.989733e+001 2.715598e+001  B N/A 2.886326e+003 2.886326e+003 3.314965e+003  C −1.50828e+005 −7.504469e+003 −1.783851e+004 −1.346553e+004  r 0.9986 0.9998 0.9997 0.9999 r ≥ 0.99  RSS 1.61276e+011 3.854776e+007 2.146366e+008 1.061195e+008 Accuracy  Concentration 1 3.99 mg/mL 12.5 μg/mL 12.5 μg/mL 12.5 μg/mL For zoledronic acid recovery: % 98.0–102.0 For related substances recovery: 80.0%, 120.0  Recovery 1 (%) 101.3 103.1 103.1 103.1  Concentration 2 5.01 mg/mL 25.0 μg/mL 25.0 μg/mL 25.0 μg/mL  Recovery 2 (%) 100.9 104.4 104.4 104.4  Concentration 3 5.97 mg/mL 50.0 μg/mL 50.0 μg/mL 50.0 μg/mL  Recovery 3 (%) 99.5 105.6 105.6 105.6 Precision  Intra-day For zoledronic acid RSD%: Not less than 2%  Average 100.9 103.5 92.8 101.5  RSD % 0.4 2.1 3.3 3.0  Inter-day For related substances RSD%: Not less than 10%  Average 100.1 109.4 103.8 93.7  RSD % 0.8 1.1 2.6 2.9 Validation parameter Zoledronic acid Imidazole-1-yl-acetic acid Phosphate Phosphite Acceptance criteria LOD 0.9 μg/mL 1.0 μg/mL 0.75 μg/mL 0.50 μg/mL LOQ 1.7 μg/mL 1.5 μg/mL 2.5 μg/mL 1.5 μg/mL Linearitya  # of level 5 5 5 5  Range 0.39–5.96 mg/mL 6.25–100 μg/mL 6.25–100 μg/mL 6.25–100 μg/mL  A 1.453226e+006 1.132516e+001 1.989733e+001 2.715598e+001  B N/A 2.886326e+003 2.886326e+003 3.314965e+003  C −1.50828e+005 −7.504469e+003 −1.783851e+004 −1.346553e+004  r 0.9986 0.9998 0.9997 0.9999 r ≥ 0.99  RSS 1.61276e+011 3.854776e+007 2.146366e+008 1.061195e+008 Accuracy  Concentration 1 3.99 mg/mL 12.5 μg/mL 12.5 μg/mL 12.5 μg/mL For zoledronic acid recovery: % 98.0–102.0 For related substances recovery: 80.0%, 120.0  Recovery 1 (%) 101.3 103.1 103.1 103.1  Concentration 2 5.01 mg/mL 25.0 μg/mL 25.0 μg/mL 25.0 μg/mL  Recovery 2 (%) 100.9 104.4 104.4 104.4  Concentration 3 5.97 mg/mL 50.0 μg/mL 50.0 μg/mL 50.0 μg/mL  Recovery 3 (%) 99.5 105.6 105.6 105.6 Precision  Intra-day For zoledronic acid RSD%: Not less than 2%  Average 100.9 103.5 92.8 101.5  RSD % 0.4 2.1 3.3 3.0  Inter-day For related substances RSD%: Not less than 10%  Average 100.1 109.4 103.8 93.7  RSD % 0.8 1.1 2.6 2.9 aFor related substances, the quadratic regression equation is y = Ax2 + Bx + C, where y is the analyte peak area, x is the analyte concentration in μg/mL. For zoledronic acid assay, the linear regression equation is y = Ax + C, where y is the analyte peak area, x is the analyte concentration in mg/mL. A and C stand for slopes and y-intercept, respectively. RSS is residual sum of squares. Table III. Limit of detection, limit of quantitation, linearity, accuracy and precision results of proposed method Validation parameter Zoledronic acid Imidazole-1-yl-acetic acid Phosphate Phosphite Acceptance criteria LOD 0.9 μg/mL 1.0 μg/mL 0.75 μg/mL 0.50 μg/mL LOQ 1.7 μg/mL 1.5 μg/mL 2.5 μg/mL 1.5 μg/mL Linearitya  # of level 5 5 5 5  Range 0.39–5.96 mg/mL 6.25–100 μg/mL 6.25–100 μg/mL 6.25–100 μg/mL  A 1.453226e+006 1.132516e+001 1.989733e+001 2.715598e+001  B N/A 2.886326e+003 2.886326e+003 3.314965e+003  C −1.50828e+005 −7.504469e+003 −1.783851e+004 −1.346553e+004  r 0.9986 0.9998 0.9997 0.9999 r ≥ 0.99  RSS 1.61276e+011 3.854776e+007 2.146366e+008 1.061195e+008 Accuracy  Concentration 1 3.99 mg/mL 12.5 μg/mL 12.5 μg/mL 12.5 μg/mL For zoledronic acid recovery: % 98.0–102.0 For related substances recovery: 80.0%, 120.0  Recovery 1 (%) 101.3 103.1 103.1 103.1  Concentration 2 5.01 mg/mL 25.0 μg/mL 25.0 μg/mL 25.0 μg/mL  Recovery 2 (%) 100.9 104.4 104.4 104.4  Concentration 3 5.97 mg/mL 50.0 μg/mL 50.0 μg/mL 50.0 μg/mL  Recovery 3 (%) 99.5 105.6 105.6 105.6 Precision  Intra-day For zoledronic acid RSD%: Not less than 2%  Average 100.9 103.5 92.8 101.5  RSD % 0.4 2.1 3.3 3.0  Inter-day For related substances RSD%: Not less than 10%  Average 100.1 109.4 103.8 93.7  RSD % 0.8 1.1 2.6 2.9 Validation parameter Zoledronic acid Imidazole-1-yl-acetic acid Phosphate Phosphite Acceptance criteria LOD 0.9 μg/mL 1.0 μg/mL 0.75 μg/mL 0.50 μg/mL LOQ 1.7 μg/mL 1.5 μg/mL 2.5 μg/mL 1.5 μg/mL Linearitya  # of level 5 5 5 5  Range 0.39–5.96 mg/mL 6.25–100 μg/mL 6.25–100 μg/mL 6.25–100 μg/mL  A 1.453226e+006 1.132516e+001 1.989733e+001 2.715598e+001  B N/A 2.886326e+003 2.886326e+003 3.314965e+003  C −1.50828e+005 −7.504469e+003 −1.783851e+004 −1.346553e+004  r 0.9986 0.9998 0.9997 0.9999 r ≥ 0.99  RSS 1.61276e+011 3.854776e+007 2.146366e+008 1.061195e+008 Accuracy  Concentration 1 3.99 mg/mL 12.5 μg/mL 12.5 μg/mL 12.5 μg/mL For zoledronic acid recovery: % 98.0–102.0 For related substances recovery: 80.0%, 120.0  Recovery 1 (%) 101.3 103.1 103.1 103.1  Concentration 2 5.01 mg/mL 25.0 μg/mL 25.0 μg/mL 25.0 μg/mL  Recovery 2 (%) 100.9 104.4 104.4 104.4  Concentration 3 5.97 mg/mL 50.0 μg/mL 50.0 μg/mL 50.0 μg/mL  Recovery 3 (%) 99.5 105.6 105.6 105.6 Precision  Intra-day For zoledronic acid RSD%: Not less than 2%  Average 100.9 103.5 92.8 101.5  RSD % 0.4 2.1 3.3 3.0  Inter-day For related substances RSD%: Not less than 10%  Average 100.1 109.4 103.8 93.7  RSD % 0.8 1.1 2.6 2.9 aFor related substances, the quadratic regression equation is y = Ax2 + Bx + C, where y is the analyte peak area, x is the analyte concentration in μg/mL. For zoledronic acid assay, the linear regression equation is y = Ax + C, where y is the analyte peak area, x is the analyte concentration in mg/mL. A and C stand for slopes and y-intercept, respectively. RSS is residual sum of squares. In robustness study, system suitability parameters obtained from unchanged and changed conditions were performed. Statistical evaluation results of the developed method are given in Tables IV and V in comparison with the published method. Table IV. Comparison results of assay of zoledronic acid in active substance and finished product Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 100.8 99.9 4.04 4.02 2 101.2 100.4 4.06 4.14 3 100.9 100.0 4.04 4.06 4 101.0 100.7 4.05 4.12 5 101.5 100.7 4.07 4.10 6 100.2 100.6 4.01 4.07 Average 100.9 100.4 4.05 4.09 SD 0.44 0.35 0.02 0.04 % RSD 0.44 0.35 0.51 1.07 95% CI 100.5:101.4 99.1:101.0 4.02:4.07 4.04:4.13 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.657) Pass (0.628) Pass (0.565) Pass (0.904) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.172) Pass (0.073) Two sample t-test for the mean (P > 0.05) Pass (0.062) Pass (0.082) Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 100.8 99.9 4.04 4.02 2 101.2 100.4 4.06 4.14 3 100.9 100.0 4.04 4.06 4 101.0 100.7 4.05 4.12 5 101.5 100.7 4.07 4.10 6 100.2 100.6 4.01 4.07 Average 100.9 100.4 4.05 4.09 SD 0.44 0.35 0.02 0.04 % RSD 0.44 0.35 0.51 1.07 95% CI 100.5:101.4 99.1:101.0 4.02:4.07 4.04:4.13 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.657) Pass (0.628) Pass (0.565) Pass (0.904) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.172) Pass (0.073) Two sample t-test for the mean (P > 0.05) Pass (0.062) Pass (0.082) Table IV. Comparison results of assay of zoledronic acid in active substance and finished product Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 100.8 99.9 4.04 4.02 2 101.2 100.4 4.06 4.14 3 100.9 100.0 4.04 4.06 4 101.0 100.7 4.05 4.12 5 101.5 100.7 4.07 4.10 6 100.2 100.6 4.01 4.07 Average 100.9 100.4 4.05 4.09 SD 0.44 0.35 0.02 0.04 % RSD 0.44 0.35 0.51 1.07 95% CI 100.5:101.4 99.1:101.0 4.02:4.07 4.04:4.13 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.657) Pass (0.628) Pass (0.565) Pass (0.904) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.172) Pass (0.073) Two sample t-test for the mean (P > 0.05) Pass (0.062) Pass (0.082) Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 100.8 99.9 4.04 4.02 2 101.2 100.4 4.06 4.14 3 100.9 100.0 4.04 4.06 4 101.0 100.7 4.05 4.12 5 101.5 100.7 4.07 4.10 6 100.2 100.6 4.01 4.07 Average 100.9 100.4 4.05 4.09 SD 0.44 0.35 0.02 0.04 % RSD 0.44 0.35 0.51 1.07 95% CI 100.5:101.4 99.1:101.0 4.02:4.07 4.04:4.13 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.657) Pass (0.628) Pass (0.565) Pass (0.904) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.172) Pass (0.073) Two sample t-test for the mean (P > 0.05) Pass (0.062) Pass (0.082) Table V. Comparison results of determination of imidazole-1-yl-acetic acid in active substance and finished product Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 102.9 102.4 99.1 99.4 2 100.0 101.0 98.9 101.0 3 101.9 100.0 99.8 98.6 4 99.2 98.6 101.4 100.2 5 100.6 101.8 101.1 98.6 6 96.9 100.8 99.4 96.6 Average 100.3 100.8 99.95 99.1 SD 2.11 1.35 1.06 1.53 % RSD 2.10 1.34 1.06 1.54 95% CI 100.5:101.4 99.1:101.0 98.8:101.1 97.5:100.7 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.898) Pass (0.840) Pass (0.240) Pass (0.716) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.367) Pass (0.505) Two sample t-test for the mean (P > 0.05) Pass (0.627) Pass (0.277) Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 102.9 102.4 99.1 99.4 2 100.0 101.0 98.9 101.0 3 101.9 100.0 99.8 98.6 4 99.2 98.6 101.4 100.2 5 100.6 101.8 101.1 98.6 6 96.9 100.8 99.4 96.6 Average 100.3 100.8 99.95 99.1 SD 2.11 1.35 1.06 1.53 % RSD 2.10 1.34 1.06 1.54 95% CI 100.5:101.4 99.1:101.0 98.8:101.1 97.5:100.7 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.898) Pass (0.840) Pass (0.240) Pass (0.716) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.367) Pass (0.505) Two sample t-test for the mean (P > 0.05) Pass (0.627) Pass (0.277) Table V. Comparison results of determination of imidazole-1-yl-acetic acid in active substance and finished product Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 102.9 102.4 99.1 99.4 2 100.0 101.0 98.9 101.0 3 101.9 100.0 99.8 98.6 4 99.2 98.6 101.4 100.2 5 100.6 101.8 101.1 98.6 6 96.9 100.8 99.4 96.6 Average 100.3 100.8 99.95 99.1 SD 2.11 1.35 1.06 1.53 % RSD 2.10 1.34 1.06 1.54 95% CI 100.5:101.4 99.1:101.0 98.8:101.1 97.5:100.7 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.898) Pass (0.840) Pass (0.240) Pass (0.716) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.367) Pass (0.505) Two sample t-test for the mean (P > 0.05) Pass (0.627) Pass (0.277) Active substance Finished product Developed method Reference method Developed method Reference method N % % mg/5 mL mg/5 mL 1 102.9 102.4 99.1 99.4 2 100.0 101.0 98.9 101.0 3 101.9 100.0 99.8 98.6 4 99.2 98.6 101.4 100.2 5 100.6 101.8 101.1 98.6 6 96.9 100.8 99.4 96.6 Average 100.3 100.8 99.95 99.1 SD 2.11 1.35 1.06 1.53 % RSD 2.10 1.34 1.06 1.54 95% CI 100.5:101.4 99.1:101.0 98.8:101.1 97.5:100.7 Alpha level 0.05 0.05 Probability (P > 0.05) Pass (0.898) Pass (0.840) Pass (0.240) Pass (0.716) Two sample standard deviation test (f-test) (P > 0.05) Pass (0.367) Pass (0.505) Two sample t-test for the mean (P > 0.05) Pass (0.627) Pass (0.277) Discussion HPLC method development (a) Choice of ELSD—Since all the bisphosphonates are strongly polar and ionic, ion-pair RP-LC has been regarded as a useful technique for separation of these compounds. ELSD offered near-universal detection of the compounds covered in this study and it was chosen due to its ability to detect drug substance and its ionic related substances simultaneously which cannot be possible with conventional UV detector. (b) Choice of n-amylamine as ion-pair reagent—Different tetraalkylammonium salts are most commonly selected as ion-pairing agents in separation of bisphosphonates, but they are not compatible with ELSD because of their non-volatile properties. For this purpose, a volatile mobile-phase additive as ion-pair reagent was chosen to obtain reasonable retention times of interested peaks and low system noise. All the bisphosphonates are strongly polar and ionic, it is difficult to give the bisphosphonates sufficient retention on a hydrophobic stationary phase such as C18 column, and so the application of an ion-pairing agent can be very helpful. In reversed-phase LC-ELSD, volatile buffers and ion-pairing agents are required for the ELSD. n-amylamine enabled adequate separation in reasonable time. It has a lower boiling point and water solubility; moreover, it is easily evaporable and could yield steady baseline. Therefore, it is compatible with ELSD. (c) Effect of concentration of n-amylamine—The retention time of ZOL and their related substances increased with increasing n-amylamine concentration. Low concentration of n-amylamine has given poor resolution whereas high concentrations have given prolonged retention and caused undesired baseline drift and increase in background noise. Therefore, the optimum concentration was 35 mM as it enabled adequate separation in reasonable time and low system noise. (d) Effect of organic modifier—Initial attempt to use methanol as organic modifier resulted in a chromatogram with deformation of primary peak, changing the chromatographic conditions such as percentage of methanol could not ameliorate the peaks shape, methanol was therefore replaced with acetonitrile in an attempt to improve the peaks. Because preliminary results showed that ZOL peak deformation was significantly improved when acetonitrile was used as an organic modifier, the retention time of the four compounds was measured at constant n-amylamine concentration (35 mM) and pH (7.0). An increase in the retention times of all compounds was noted with a decrease in the percentage of acetonitrile. Satisfactory separation of four compounds was achieved when the acetonitrile ratio was 5%, which was used for the initial percentage of the gradient elution. (e) Effect of the mobile-phase pH—Due to the fact that the mobile-phase pH affected the ionization of the primary substance; pH has a key role to separate ZOL and its related substances. The capacity factors, k, were investigated at three different pH values (pH of 4.5, 4.8 and 7.0) at fixed n-amylamine (35 mM) concentration and initial acetonitrile percentage (5%). It was noticed that retention times of all interested peaks increased with increasing pH. This phenomenon was assessed by examining the capacity factors of the peaks with varying pH. The choice of pH 7.0 served a successful separation and good peak shapes. At this pH, capacity factors were found as 0,94, 1,31, 1,70 and 3,88 for imidazol-1-yl-acetic acid, phosphate, phosphite and ZOL, respectively. (f) Choice of the HPLC column—In order to choose a column which can give adequate separation and proper peak shape, some most-common type of columns including Ace 5 C8, Zorbax RX C8 and Hichrom C18 were investigated. The best results of parameters were achieved with the Hichrom C18 column. (g) Optimization of ELSD—The response of ELSD varies greatly with the driving gas flow-rate. Only the optimum sized droplets contribute to the signal, and the remainder of the sample contained in the smaller size droplets is wasted, so it is very important to control the gas flow-rate. When the gas flow rate is too low, large droplets are formed, resulting in spikes and noisy signals. Whereas, if the gas flow rate is too high, the droplets will decrease in size, which results in decreased signal response. In order to adjust the proper flow rate of nebulizer gas (high purity nitrogen), a solution containing all relevant impurity peaks was injected and the peak heights were checked. Optimum gas flow rate was determined to be 40 psi. The drift tube temperature also affects the response at low temperature; poor response is gained due to the poor mobile-phase evaporation. On the contrary, small droplets are formed at high temperature contributing poor response at ELSD. To obtain an optimum signal-to-noise (S/N) ratio which yields a good signal, a solution contains all interested impurity peaks was injected and the S/N ratios were checked. Optimum S/N ratio was detected at 60°C. Detector has ability to heat or cool the nebulizer source to adjust the signal quality. To contribute evaporation, the source is heated or to stay the compounds stable during spraying, the source is cooled down. The source can be used in off mode. It was evident that the cool mode was the most productive option to yield high S/N ratio. Method validation Validation of the developed method was conducted by following the latest ICH guidelines (42, 43) with respect to linearity, assay accuracy, LOD, LOQ and precision by two analysts. Results of the validation showed that the developed HPLC method to determine the related substances and assay of ZOL can be used to evaluate the quality of regular samples as well as the stability samples of ZOL. (a) Specificity and forced degradation study—To investigate the specificity of the method, related substances and main substance were injected and checked for any interference. For this purpose, related substances and ZOL solutions were injected separately and it was observed that there was no interference at the retention time of each substance. To determine the degradation product arises from ZOL and the inherent stability, solution containing ZOL was exposed to some forced degradation conditions for 24 h such as oxidation with 3% H2O2 at room temperature, boiling in reflux, keeping under UV light, keeping in an oven at 100°C. The photostability was carried out by exposing the solid-state sample to light illumination for not less than 1.2 million lux hours and UV energy for not less than 200 Wh/m2. Difference in area was calculated as follows: %Difference=[(Ad−Au)/Au]×100Ad: Area of interested peak in the chromatogram obtained from degraded sample. Au: Area of interested peak in the chromatogram obtained from untreated sample. A successful separation of ZOL and its known related substances and degradation products showed that the developed method is useful for stability studies. Drug product (4 mg ZOL/vial) and its placebo solution were injected and successful separations from placebo peaks were obtained. (b) LOD and LOQ—To determine the LOD and LOQ for ZOL and all three related substances, S/N approach described in ICH guideline (42, 43) was applied. It was performed by comparing measured signals from samples with known concentrations from low to high with those of blank samples. The validation showed that S/N values for LOD of all of the compounds were approximately 3, and the S/N values for LOQ were approximately 10. The experimental s/n values for LOD were 2.8, 3.2, 2.6, 1.5 for ZOL, imidazol-1-yl-acetic acid, phosphate and phosphite, respectively. The experimental S/N values for LOQ were 8.6, 9.6, 17.5, 11.2 for ZOL, imidazol-1-yl-acetic acid, phosphate and phosphite, respectively. (c) Linearity—To determine the linearity of the method, triplicate injections of samples at five different concentrations were carried out. For ZOL assay, the levels are 0.4 mg/mL, 0.8 mg/mL, 1.6 mg/mL, 4.0 mg/mL, 5.0 mg/mL and 6.0 mg/mL. Linear regression analysis gave a correlation coefficient (r) of 0.9972 for ZOL. For related substances, the range of linearity was between 6.25 μg/mL and 100.0 μg/mL including five different concentrations and three injections for each. The quadratic regression analysis gave a correlation coefficient (r) of 0.9998, 0.9997 and 0.9999 for imidazole-1-yl-acetic acid, phosphate and phosphite respectively. (d) Accuracy—Accuracy was assessed via a recovery study. Recoveries of ZOL were studied and overall recovery was found to be 100.5% at the concentration range between 4.0 mg/mL and 6.0 mg/mL. Recoveries of imidazole-1-yl-acetic acid were studied and overall recovery was found to be 104.4% at the concentration range between 12.5 μg/mL and 50.0 μg/mL. (e) Precision and intermediate precision—The method precision and intermediate precision for ZOL and related compounds was evaluated through the RSD of recovery of six independent samples. (f) Solution stability—In the solution stability study, standard and sample solution containing related substances spiked at their respective limit concentrations of 25 μg/mL were (0.5% for each) injected. The solutions were stored at ambient laboratory temperature in a clear volumetric flask exposed to room light. The study was conducted for 48 h. The initial area for corresponding compound and the area after 48th hour were compared with each other and it was observed that, under the mentioned conditions none of the related substance area was increased. Therefore, it was concluded that, the standard and sample solutions are stable for up to 48 h when exposed to regular laboratory lighting at room temperature. (g) Robustness—Small variation in the method parameters were made to demonstrate the robustness of the method. It should show the reliability of an analysis with respect to deliberate variations in method parameters. As usual; the flow rate of mobile phase, column thermostat temperature, and pH of the mobile phase were varied in a realistic range and the system suitability parameters were checked. It was proved that, the system suitability parameters stayed within the limits. Comparison of the developed method with the published method—the developed and validated method was compared with the method published by Rao et al. (39) by means of f and t-tests. The published method could analyze the assay of ZOL and imidazole-1-yl-acetic acid. The comparisons of assay of ZOL and specified related substance namely, imidazole-1-yl-acetic acid spiked to the sample solutions at the specification limits of 0.5% (25 μg/mL) were made. The methods were applied on both active substance and finished products. Statistical evaluations were done at 95% confidence level (alpha is 0.05) using Minitab® 16 software. For each group containing six independent measurements, the normality values were checked and they were found to be above 0.05 which means the series had good probability. Subsequently, f and t-tests were performed and it was demonstrated that the analysis results obtained from the developed and published methods gave similar results. The assessments were made by hypothesis test and the P-values above 0.05 indicated that the standard deviations and means of two groups were not significantly different. Conclusion The analytical method described in this paper is suitable for identification and quantification of ZOL and its related substances namely imidazole-1-yl-acetic acid, phosphate and phosphite. The method is suitable for active pharmaceutical ingredient of ZOL and pharmaceutical form of ZOL. This method was found to be linear in the range of 0.4–6.0 mg/mL for ZOL and is linear in the range of 6.25–100 μg/mL for related substances. It was also found to be accurate, precise and repeatable in the range of 80% and 120% of the specified concentrations which are 5.0 mg/mL for ZOL and 25 μg/mL for related substances. The method has been demonstrated to be sensitive, with an LOQ of 1.7 μg/mL, 1.5 μg/mL, 2.5 μg/mL and 1.5 μg/mL for ZOL, imidazole-1-yl-acetic acid, phosphate and phosphite, respectively. The method is specific and robust, and therefore suitable for routine analysis of ZOL and related substances in QC laboratories simultaneously. The proposed method has a single run which is more practical, economical and also it needs less time for quality control analysis in pharmaceutical industry. This method has been demonstrated to be stability indicating because it can separate degradation peaks from ZOL and accurately quantifies the contents of the main compounds in stability samples. References 1 Novartis Europharm Limited . ; Zometa ® Summary of Product Characteristics ( 2003 ), Novartis Europharm Limited Wimblehurst Road, Horsham, West Sussex, RH12 5AB UK. 2 Fleisch , H. ; Introduction to bisphosphonates. History and functional mechanisms ; Der Orthopade , ( 2007 ); 36 : 103 – 104 . Google Scholar Crossref Search ADS PubMed 3 Scriba , G.K.E. ; Bisphosphonate im Überblick ; Pharmazie in Unserer Zeit , ( 2000 ); 29 : 50 – 56 . 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TI - Determination of Zoledronic Acid and Its Related Substances by High Performance Liquid Chromatography with Evaporative Light Scattering Detection
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmy078
DA - 2019-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/determination-of-zoledronic-acid-and-its-related-substances-by-high-76CtyipbaB
SP - 33
VL - 57
IS - 1
DP - DeepDyve
ER -