ARTICLE IN PRESS
G Model PBA-9152; No. of Pages 4
Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy Baogang Xie a,∗,1 , Aihong Liu b,1 , Xiuzhong Fang c , Ying Chen a , Haijun Zhong a a b c
School of Pharmaceutical Science, Nanchang University, Nanchang 330006, PR China Center of analysis and testing, Nanchang University, Nanchang 330047, PR China School of Science, Nanchang University, Nanchang 330006, PR China
a r t i c l e
i n f o
Article history: Received 21 May 2013 Received in revised form 3 July 2013 Accepted 8 July 2013 Available online xxx Keywords: Alendronate sodium tablet 1 H NMR Quantification Quality evaluation
a b s t r a c t Determination of alendronate is crucial to routine quality control of alendronate tablets. However, tedious sample treatment processes such as derivatization were generally required by chromatographic separation of alendronate as its high polarity and no chromophore in the molecular structure. Here, we describe the use of 1 H NMR for the quantification of alendronate sodium in tablets. Linearity, recovery, selectivity and sensitivity of the assay were validated to be satisfactory with quick sample preparation and acquisition. The contents of alendronate sodium in tablets from five manufacturers were determined, the results showed that all assayed tablets fell within the range of 90.0–110.0% of the label claim and the relative standard deviation was less than 6.0%. The method was also successfully employed for alendronate tablet dissolution assay in this study. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Alendronate sodium (sodium [4-amino-1-hydroxybutylidene] bisphosphonate, Fig. 1A), an important amino bisphosphonate compound, is commonly used for the prevention and treatment of a variety of diseases such as Paget’s disease of bone and osteoporosis [1–3]. Tablets are the most widely used dosage form of alendronate sodium in clinic [4]. To date, there are more than ten pharmaceutical manufactures that produce them in China. Thus, for the purpose of developing new formulation and routine quality control of alendronate tablets, it is desirable to have a rapid, simple and robust analytical method for determining alendronate in tablets. Many of the analytical methods have been established to apply the determination of bisphosphonates in pharmaceuticals and biological materials by RP-HPLC, ion-pair HPLC and ionchromatography [5]. Though high sensitivity could be shown for these HPLC based technology, the majority of these assays relied on derivatization of alendronate using either pre-column [6–8] or post-column techniques [9]. High resolution proton nuclear magnetic resonance (1 H NMR), permits rapid, multicomponent analysis of samples with minimal sample pretreatment and non-destruction. It has been employed to quantify organic diphosphates [10] and the main organic
∗ Corresponding author. Tel.: +86 791 86361839. E-mail addresses: [email protected], [email protected] (B. Xie). 1 These authors contributed equally to this work.
components of vinegars [11]. However, to the best of our knowledge, this technology has not been applied for determination of bisphosphonates in pharmaceuticals. Thus, this report describes the use of 1 H NMR for the quantification of alendronate sodium in tablets. The validated assay would be applied for rapid quality assessment of alendronate tablets.
2. Experimental 2.1. Chemicals and materials Samples of alendronate sodium tablet from five manufacturers were summarized in Table 1. The reference sample for alendronate sodium was purchased from China National Institutes for Food and Drug Control. 4-Aminobutyric acid was purchased from Aladdin. D2 O and sodium salt of (trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) were purchased from Sigma–Aldrich.
2.2. Preparation of standard solutions of alendronate sodium Stock solution of alendronate was prepared by dissolving 220.0 mg of alendronate sodium in 10.0 mL of distilled water. The solutions were diluted immediately before use with purified water, to obtain working solutions of 11.0, 5.5, 2.75, 1.38, 0.69 and 0.34 mg/mL for each analyte.
0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.006
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
2
Fig. 1. Structure of alendronate sodium (A) and 4-aminobutyric acid (B) as well as the 1 H NMR spectrum of solution spiked with alendronate sodium and 4-aminobutyric acid standard (C).
2.3. Preparation of alendronate sodium tablet samples
line-broadening factor. Spectra were manually phase and baseline corrected, and chemical shifts were referenced to the TSP signal.
Quantitative determination of alendronate sodium in tablet formulation was performed by dispersing two tablets in 10.0 mL (or 50.0 mL) distilled water to give assay concentration of 2.0 mg/mL (or 2.8 mg/mL) according to the claimed amount in labels. The aqueous solution of tablets was then mechanically shaken for 20 min and sonicated for 10 min to solubilize the drug. An aliquot of each sample was then transferred to a polypropylene tube and centrifuged at 10,000 rpm for 10 min. The supernatant was used for further 1 H NMR analysis. Three replicates for each manufacturer product were prepared. 2.4.
1H
NMR analysis
The sample was prepared for 1 H NMR by diluting 300.0 L standard solution or aqueous solution of tablets with 200.0 L 0.2 M sodium phosphate buffer (pH 7.4), and 50.0 L TSP in D2 O (2.40 mM). 1 H NMR spectroscopy of the samples was carried out on a Bruker AvanceII-600 MHz spectrometer (Germany). The spectra were measured with 32 scans over a spectral width of 12 ppm, an acquisition time of 4.5 s and a recycle delay of 5 s. The presaturation method was used to suppress the proton signal of water. The spectra were Fourier transformed with FT size of 64 k and 0.3 Hz
2.5. Validation of the assay method The accuracy of the method was tested by adding known quantities of alendronate sodium (0.8, 1.4, 2.0 mg/mL respectively) to the alendronate tablet solution (1.4 mg/mL). Each sample was assayed in duplicate. Recovery was calculated as: R (%) = (S1 − S0 ) × 100%/S, where S0 and S1 are the measurements before and after addition of standards and S is the amount of added alendronate sodium. The limit of detection (LOD) was defined as the lowest concentration of the drug resulting in a signal-to-noise ratio of 3:1. The limit of quantification (LOQ) was defined as the lowest concentration of drug resulting in that of 10:1. 2.6. Quantification of alendronate sodium in tablets After 1 H NMR analysis, peak areas at 2.00 and 0.0 ppm were calculated by integration respectively. Calibration curves were constructed by plotting the peak-area ratio of analyte to TSP against the known concentration of alendronate sodium. Concentrations of the analytes in unknown samples were calculated using the results of the regression analysis.
Table 1 The content of alendronate sodium in tablets from five manufacturers (mean ± SD, n = 3). Sample name
Manufacturer
Batch number
Claim (mg)/tablet
Measured amount (mg)/tablet
Fosamax Gubon Dongle Vpharm Anlun
Merck Sharp & Dohme Pty Ltd. CSPC Ouyi Pharmaceutical Co., Ltd. Zhuozhou Dongle Pharmacy Co., Ltd. Ventruepharm Pharmaceutical (Hainal) Co., Ltd. Beijing Winsunny Co., Ltd.
12507 152120401 20120401 20120601 20120405
70.0 70.0 10.0 70.0 70.0
72.8 71.9 9.63 69.3 72.3
± ± ± ± ±
3.4 3.7 0.38 3.9 3.0
Potency (%) 104.0 102.7 96.3 99.0 103.3
± ± ± ± ±
4.9 5.3 3.8 5.6 4.3
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
3
and the solution of tablet from Fosamax spiked with alendronate sodium standard was 2.00 ± 0.05 to 1.0. The results suggested that there were free of interfering peaks to quantification of alendronate by 1 H NMR. 4-Aminobutyric acid, a potential degradation product of alendronate [12], was commonly identified and quantified as a related substance in alendronate sodium tablets for quality control [13,14]. From Fig. 1C, we could see that the signals of it (1.9, 2.3, 3.0 ppm) were found to be well resolved with those of alendronate. 4Aminobutyric acid was not detected in tablets in the present study. The LOD for it in aqueous solution was determined to be 0.04 mg/mL, which indicated that the degradation of alendronate was less than 2.0%. 3.2. Linearity, LOD, LOQ and accuracy for alendronate Fig. 2. 1 H NMR spectrum of alendronate sodium standard (A), aqueous solution of tablet from Fosamax (B) and solution of tablet spiked with alendronate sodium (C).
2.7. Dissolution studies Dissolution tests were performed according to the appendix XC of Chinese Pharmacopoeia (2010). Briefly, 6 tablets (70.0 mg/tablet) produced by Ventruepharm Pharmaceutical Co., Ltd were introduced into 100 mL of distilled water in three of the beakers respectively, the rotational speed for the paddles was set at 100 rpm, and the temperature was kept constant at 37 ◦ C. An aliquot of 2.0 mL was withdrawn at 5, 10, 20, 30, 45 and 60 min, and filtered through a 0.45 m membrane. The removed volume was replaced each time with 2.0 mL of fresh medium. The concentration of alendronate sodium was determined by 1 H NMR analysis. 3. Results and discussion 3.1. Method selectivity From Fig. 2A, we could see that alendronate sodium gave two signals at 2.0 (4H) and 3.1 (2H) ppm with the ratio of 2.00 by peak integration area. Signals of pharmaceutical excipients in tablets from Gubon, Fosamax and Vpharm appeared between 3.2 and 5.5 ppm, two signals at 1.2, 3.7 ppm and no obvious signals of excipients were seen for Dongle and Anlun respectively (Fig. 3), which indicated that the formulations of Dongle and Anlun were different from other three manufacturers. More importantly, the ratio of peak area at 2.0 to 3.1 ppm for tablets from the five manufacturers
Calibration curves of the alendronate were linear over the low (0.34 mg/mL) and high concentration (11.0 mg/mL) range with r2 = 0.9998. The LOD and LOQ for it were determined to be 0.1 mg/mL and 0.3 mg/mL respectively. The precision of it calculated as the relative standard deviation at low and high concentrations were better than 2.5 and 1.5% for intra-day assays respectively. The results of the accuracy studies showed that alendronate in aqueous solution can be measured reliably using the 1 H NMR method, because obtained recoveries were satisfactory (98–102%). The precision and accuracy of developed 1 H NMR methods were close to those of pre-column derivatization based HPLC method [8]. 3.3. Quantification of alendronate sodium in tablets from five manufacturers Under our assay conditions, alendronate sodium generated a narrow, well-resolved, and stable signal in 1 H NMR spectra with no interference from pharmaceutical excipients of tablets (Fig. 3). The content of alendronate sodium in tablets from five manufacturers was summarized in Table 1. All assayed tablets fell within the range of 90.0–110.0% of the label claim and the relative standard deviation was less than 6.0%, fulfilling the Chinese Pharmacopoeia criteria for content uniformity of tablets [15]. 3.4. Dissolution studies Dissolution testing has long been employed by the pharmaceutical industry as the benchmark in evaluating the product quality and predicting in vivo drug release behaviour. To validate the assay method for the application of dissolution study on alendronate sodium tablet, products from one of the five manufacturers were used in this study. Our results suggested that more than 90% of the label claim was released within 5 min. The products tested met the Chinese Pharmacopoeia and FDA requirements for highly soluble pharmaceutical compounds [15,16]. 4. Conclusion
Fig. 3. Typical 1 H NMR spectrum of aqueous solution of tablets from five manufacturers. Sample 1: Anlun; Sample 2: Dongle; Sample 3: Gubon; Sample 4: Fosamax; Sample 5: Vpharm.
In the present study, a rapid, precise, and accurate method was developed and validated for the quantification of alendronate in tablets by 1 H NMR. The advantages of it were the quick sample preparation and acquisition. The assay was linear over a broad range of concentrations and applicable for samples with a concentration of alendronate down to 0.3 mg/mL. It was successfully employed for alendronate sodium tablet content uniformity and dissolution assays for quality evaluation purposes.
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
4
Acknowledgements This work was supported by the National Nature Sciences Foundation of China (Grant Number: 30960456) and the Analysis and Measurement Foundation of Nanchang University (Grant Number: 2010018).
[7]
[8]
References [9] [1] R.G. Russell, M.J. Rogers, Bisphosphonates: from the laboratory to the clinic and back again, Bone 25 (1999) 97–106. [2] M. McClung, B. Clemmesen, A. Daifotis, N.L. Gilchrist, J. Eisman, R.S. Weinstein, G.e.-H. Fuleihan, C. Reda, A.J. Yates, P. Ravn, Alendronate prevents postmenopausal bone loss in women without osteoporosis. A double-blind, randomized, controlled trial. Alendronate Osteoporosis Prevention Study Group, Ann. Intern. Med. 128 (1998) 253–261. [3] D. Hosking, C.E. Chilvers, C. Christiansen, P. Ravn, R. Wasnich, P. Ross, M. McClung, A. Balske, D. Thompson, M. Daley, A.J. Yates, Prevention of bone loss with alendronate in postmenopausal women under 60 years of age. Early Postmenopausal Intervention Cohort Study Group, N. Engl. J. Med. 338 (1998) 485–492. [4] A.E. Denker, N. Lazarus, A. Porras, R. Ramakrishnan, M. Constanzer, B.B. Scott, C. Chavez-Eng, E. Woolf, L. Maganti, P. Larson, K. Gottesdiener, J.A. Wagner, Bioavailability of alendronate and vitamin D-3 in an alendronate/vitamin D-3 combination tablet, J. Clin. Pharmacol. 51 (2011) 1439–1448. [5] 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. [6] W.F. Kline, B.K. Matuszewski, W.F. Bayne, Determination of 4-amino-1hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column
[10] [11]
[12]
[13]
[14]
[15] [16]
derivatization with 2,3-naphthalene dicarboxyaldehyde and highperformance liquid chromatography with fluorescence detection, J. Chromatogr. 534 (1990) 139–149. 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. S.K. Al Deeb, I.I. Hamdan, S.M. Al Najjar, Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine, Talanta 64 (2004) 695–702. M.J. Lovdahl, D.J. Pietrzyk, Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection, J. Chromatogr. A 850 (1999) 143–152. S. Lenevich, M.D. Distefano, Nuclear magnetic resonance-based quantification of organic diphosphates, Anal. Biochem. 408 (2011) 316–320. A. Caligiani, D. Acquotti, G. Palla, V. Bocchi, Identification and quantification of the main organic components of vinegars by high resolution 1 H NMR spectroscopy, Anal. Chim. Acta 585 (2007) 110–119. U. Holzgrabe, R. Deubner, C. Schollmayer, B. Waibel, Quantitative NMR spectroscopy—applications in drug analysis, J. Pharm. Biomed. Anal. 38 (2005) 806–812. Y. Jiang, Z. Xie, D.Q. Zhang, Separation of alendronate and its related substances by ion-pair reverse phase chromatography with ELSD, J. Anal. Sci. 22 (2006) 137–140. Z.Z. Yuan, L.N. You, H. Zhao, Determination of alendronate sodium and its related substance 4-aminobutyric acid by RP-HPLC, Chin. J. Spectrosc. Lab. 30 (2013) 757–762. Chinese Pharmacopeia, the 2010 Version. Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, Food and Drug Administration Center for Drug Evaluation and Research (CDER), 2000.
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy Baogang Xie a,∗,1 , Aihong Liu b,1 , Xiuzhong Fang c , Ying Chen a , Haijun Zhong a a b c
School of Pharmaceutical Science, Nanchang University, Nanchang 330006, PR China Center of analysis and testing, Nanchang University, Nanchang 330047, PR China School of Science, Nanchang University, Nanchang 330006, PR China
a r t i c l e
i n f o
Article history: Received 21 May 2013 Received in revised form 3 July 2013 Accepted 8 July 2013 Available online xxx Keywords: Alendronate sodium tablet 1 H NMR Quantification Quality evaluation
a b s t r a c t Determination of alendronate is crucial to routine quality control of alendronate tablets. However, tedious sample treatment processes such as derivatization were generally required by chromatographic separation of alendronate as its high polarity and no chromophore in the molecular structure. Here, we describe the use of 1 H NMR for the quantification of alendronate sodium in tablets. Linearity, recovery, selectivity and sensitivity of the assay were validated to be satisfactory with quick sample preparation and acquisition. The contents of alendronate sodium in tablets from five manufacturers were determined, the results showed that all assayed tablets fell within the range of 90.0–110.0% of the label claim and the relative standard deviation was less than 6.0%. The method was also successfully employed for alendronate tablet dissolution assay in this study. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Alendronate sodium (sodium [4-amino-1-hydroxybutylidene] bisphosphonate, Fig. 1A), an important amino bisphosphonate compound, is commonly used for the prevention and treatment of a variety of diseases such as Paget’s disease of bone and osteoporosis [1–3]. Tablets are the most widely used dosage form of alendronate sodium in clinic [4]. To date, there are more than ten pharmaceutical manufactures that produce them in China. Thus, for the purpose of developing new formulation and routine quality control of alendronate tablets, it is desirable to have a rapid, simple and robust analytical method for determining alendronate in tablets. Many of the analytical methods have been established to apply the determination of bisphosphonates in pharmaceuticals and biological materials by RP-HPLC, ion-pair HPLC and ionchromatography [5]. Though high sensitivity could be shown for these HPLC based technology, the majority of these assays relied on derivatization of alendronate using either pre-column [6–8] or post-column techniques [9]. High resolution proton nuclear magnetic resonance (1 H NMR), permits rapid, multicomponent analysis of samples with minimal sample pretreatment and non-destruction. It has been employed to quantify organic diphosphates [10] and the main organic
∗ Corresponding author. Tel.: +86 791 86361839. E-mail addresses: [email protected], [email protected] (B. Xie). 1 These authors contributed equally to this work.
components of vinegars [11]. However, to the best of our knowledge, this technology has not been applied for determination of bisphosphonates in pharmaceuticals. Thus, this report describes the use of 1 H NMR for the quantification of alendronate sodium in tablets. The validated assay would be applied for rapid quality assessment of alendronate tablets.
2. Experimental 2.1. Chemicals and materials Samples of alendronate sodium tablet from five manufacturers were summarized in Table 1. The reference sample for alendronate sodium was purchased from China National Institutes for Food and Drug Control. 4-Aminobutyric acid was purchased from Aladdin. D2 O and sodium salt of (trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) were purchased from Sigma–Aldrich.
2.2. Preparation of standard solutions of alendronate sodium Stock solution of alendronate was prepared by dissolving 220.0 mg of alendronate sodium in 10.0 mL of distilled water. The solutions were diluted immediately before use with purified water, to obtain working solutions of 11.0, 5.5, 2.75, 1.38, 0.69 and 0.34 mg/mL for each analyte.
0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.006
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
2
Fig. 1. Structure of alendronate sodium (A) and 4-aminobutyric acid (B) as well as the 1 H NMR spectrum of solution spiked with alendronate sodium and 4-aminobutyric acid standard (C).
2.3. Preparation of alendronate sodium tablet samples
line-broadening factor. Spectra were manually phase and baseline corrected, and chemical shifts were referenced to the TSP signal.
Quantitative determination of alendronate sodium in tablet formulation was performed by dispersing two tablets in 10.0 mL (or 50.0 mL) distilled water to give assay concentration of 2.0 mg/mL (or 2.8 mg/mL) according to the claimed amount in labels. The aqueous solution of tablets was then mechanically shaken for 20 min and sonicated for 10 min to solubilize the drug. An aliquot of each sample was then transferred to a polypropylene tube and centrifuged at 10,000 rpm for 10 min. The supernatant was used for further 1 H NMR analysis. Three replicates for each manufacturer product were prepared. 2.4.
1H
NMR analysis
The sample was prepared for 1 H NMR by diluting 300.0 L standard solution or aqueous solution of tablets with 200.0 L 0.2 M sodium phosphate buffer (pH 7.4), and 50.0 L TSP in D2 O (2.40 mM). 1 H NMR spectroscopy of the samples was carried out on a Bruker AvanceII-600 MHz spectrometer (Germany). The spectra were measured with 32 scans over a spectral width of 12 ppm, an acquisition time of 4.5 s and a recycle delay of 5 s. The presaturation method was used to suppress the proton signal of water. The spectra were Fourier transformed with FT size of 64 k and 0.3 Hz
2.5. Validation of the assay method The accuracy of the method was tested by adding known quantities of alendronate sodium (0.8, 1.4, 2.0 mg/mL respectively) to the alendronate tablet solution (1.4 mg/mL). Each sample was assayed in duplicate. Recovery was calculated as: R (%) = (S1 − S0 ) × 100%/S, where S0 and S1 are the measurements before and after addition of standards and S is the amount of added alendronate sodium. The limit of detection (LOD) was defined as the lowest concentration of the drug resulting in a signal-to-noise ratio of 3:1. The limit of quantification (LOQ) was defined as the lowest concentration of drug resulting in that of 10:1. 2.6. Quantification of alendronate sodium in tablets After 1 H NMR analysis, peak areas at 2.00 and 0.0 ppm were calculated by integration respectively. Calibration curves were constructed by plotting the peak-area ratio of analyte to TSP against the known concentration of alendronate sodium. Concentrations of the analytes in unknown samples were calculated using the results of the regression analysis.
Table 1 The content of alendronate sodium in tablets from five manufacturers (mean ± SD, n = 3). Sample name
Manufacturer
Batch number
Claim (mg)/tablet
Measured amount (mg)/tablet
Fosamax Gubon Dongle Vpharm Anlun
Merck Sharp & Dohme Pty Ltd. CSPC Ouyi Pharmaceutical Co., Ltd. Zhuozhou Dongle Pharmacy Co., Ltd. Ventruepharm Pharmaceutical (Hainal) Co., Ltd. Beijing Winsunny Co., Ltd.
12507 152120401 20120401 20120601 20120405
70.0 70.0 10.0 70.0 70.0
72.8 71.9 9.63 69.3 72.3
± ± ± ± ±
3.4 3.7 0.38 3.9 3.0
Potency (%) 104.0 102.7 96.3 99.0 103.3
± ± ± ± ±
4.9 5.3 3.8 5.6 4.3
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
3
and the solution of tablet from Fosamax spiked with alendronate sodium standard was 2.00 ± 0.05 to 1.0. The results suggested that there were free of interfering peaks to quantification of alendronate by 1 H NMR. 4-Aminobutyric acid, a potential degradation product of alendronate [12], was commonly identified and quantified as a related substance in alendronate sodium tablets for quality control [13,14]. From Fig. 1C, we could see that the signals of it (1.9, 2.3, 3.0 ppm) were found to be well resolved with those of alendronate. 4Aminobutyric acid was not detected in tablets in the present study. The LOD for it in aqueous solution was determined to be 0.04 mg/mL, which indicated that the degradation of alendronate was less than 2.0%. 3.2. Linearity, LOD, LOQ and accuracy for alendronate Fig. 2. 1 H NMR spectrum of alendronate sodium standard (A), aqueous solution of tablet from Fosamax (B) and solution of tablet spiked with alendronate sodium (C).
2.7. Dissolution studies Dissolution tests were performed according to the appendix XC of Chinese Pharmacopoeia (2010). Briefly, 6 tablets (70.0 mg/tablet) produced by Ventruepharm Pharmaceutical Co., Ltd were introduced into 100 mL of distilled water in three of the beakers respectively, the rotational speed for the paddles was set at 100 rpm, and the temperature was kept constant at 37 ◦ C. An aliquot of 2.0 mL was withdrawn at 5, 10, 20, 30, 45 and 60 min, and filtered through a 0.45 m membrane. The removed volume was replaced each time with 2.0 mL of fresh medium. The concentration of alendronate sodium was determined by 1 H NMR analysis. 3. Results and discussion 3.1. Method selectivity From Fig. 2A, we could see that alendronate sodium gave two signals at 2.0 (4H) and 3.1 (2H) ppm with the ratio of 2.00 by peak integration area. Signals of pharmaceutical excipients in tablets from Gubon, Fosamax and Vpharm appeared between 3.2 and 5.5 ppm, two signals at 1.2, 3.7 ppm and no obvious signals of excipients were seen for Dongle and Anlun respectively (Fig. 3), which indicated that the formulations of Dongle and Anlun were different from other three manufacturers. More importantly, the ratio of peak area at 2.0 to 3.1 ppm for tablets from the five manufacturers
Calibration curves of the alendronate were linear over the low (0.34 mg/mL) and high concentration (11.0 mg/mL) range with r2 = 0.9998. The LOD and LOQ for it were determined to be 0.1 mg/mL and 0.3 mg/mL respectively. The precision of it calculated as the relative standard deviation at low and high concentrations were better than 2.5 and 1.5% for intra-day assays respectively. The results of the accuracy studies showed that alendronate in aqueous solution can be measured reliably using the 1 H NMR method, because obtained recoveries were satisfactory (98–102%). The precision and accuracy of developed 1 H NMR methods were close to those of pre-column derivatization based HPLC method [8]. 3.3. Quantification of alendronate sodium in tablets from five manufacturers Under our assay conditions, alendronate sodium generated a narrow, well-resolved, and stable signal in 1 H NMR spectra with no interference from pharmaceutical excipients of tablets (Fig. 3). The content of alendronate sodium in tablets from five manufacturers was summarized in Table 1. All assayed tablets fell within the range of 90.0–110.0% of the label claim and the relative standard deviation was less than 6.0%, fulfilling the Chinese Pharmacopoeia criteria for content uniformity of tablets [15]. 3.4. Dissolution studies Dissolution testing has long been employed by the pharmaceutical industry as the benchmark in evaluating the product quality and predicting in vivo drug release behaviour. To validate the assay method for the application of dissolution study on alendronate sodium tablet, products from one of the five manufacturers were used in this study. Our results suggested that more than 90% of the label claim was released within 5 min. The products tested met the Chinese Pharmacopoeia and FDA requirements for highly soluble pharmaceutical compounds [15,16]. 4. Conclusion
Fig. 3. Typical 1 H NMR spectrum of aqueous solution of tablets from five manufacturers. Sample 1: Anlun; Sample 2: Dongle; Sample 3: Gubon; Sample 4: Fosamax; Sample 5: Vpharm.
In the present study, a rapid, precise, and accurate method was developed and validated for the quantification of alendronate in tablets by 1 H NMR. The advantages of it were the quick sample preparation and acquisition. The assay was linear over a broad range of concentrations and applicable for samples with a concentration of alendronate down to 0.3 mg/mL. It was successfully employed for alendronate sodium tablet content uniformity and dissolution assays for quality evaluation purposes.
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
G Model PBA-9152; No. of Pages 4
ARTICLE IN PRESS B. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2013) xxx–xxx
4
Acknowledgements This work was supported by the National Nature Sciences Foundation of China (Grant Number: 30960456) and the Analysis and Measurement Foundation of Nanchang University (Grant Number: 2010018).
[7]
[8]
References [9] [1] R.G. Russell, M.J. Rogers, Bisphosphonates: from the laboratory to the clinic and back again, Bone 25 (1999) 97–106. [2] M. McClung, B. Clemmesen, A. Daifotis, N.L. Gilchrist, J. Eisman, R.S. Weinstein, G.e.-H. Fuleihan, C. Reda, A.J. Yates, P. Ravn, Alendronate prevents postmenopausal bone loss in women without osteoporosis. A double-blind, randomized, controlled trial. Alendronate Osteoporosis Prevention Study Group, Ann. Intern. Med. 128 (1998) 253–261. [3] D. Hosking, C.E. Chilvers, C. Christiansen, P. Ravn, R. Wasnich, P. Ross, M. McClung, A. Balske, D. Thompson, M. Daley, A.J. Yates, Prevention of bone loss with alendronate in postmenopausal women under 60 years of age. Early Postmenopausal Intervention Cohort Study Group, N. Engl. J. Med. 338 (1998) 485–492. [4] A.E. Denker, N. Lazarus, A. Porras, R. Ramakrishnan, M. Constanzer, B.B. Scott, C. Chavez-Eng, E. Woolf, L. Maganti, P. Larson, K. Gottesdiener, J.A. Wagner, Bioavailability of alendronate and vitamin D-3 in an alendronate/vitamin D-3 combination tablet, J. Clin. Pharmacol. 51 (2011) 1439–1448. [5] 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. [6] W.F. Kline, B.K. Matuszewski, W.F. Bayne, Determination of 4-amino-1hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column
[10] [11]
[12]
[13]
[14]
[15] [16]
derivatization with 2,3-naphthalene dicarboxyaldehyde and highperformance liquid chromatography with fluorescence detection, J. Chromatogr. 534 (1990) 139–149. 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. S.K. Al Deeb, I.I. Hamdan, S.M. Al Najjar, Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine, Talanta 64 (2004) 695–702. M.J. Lovdahl, D.J. Pietrzyk, Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection, J. Chromatogr. A 850 (1999) 143–152. S. Lenevich, M.D. Distefano, Nuclear magnetic resonance-based quantification of organic diphosphates, Anal. Biochem. 408 (2011) 316–320. A. Caligiani, D. Acquotti, G. Palla, V. Bocchi, Identification and quantification of the main organic components of vinegars by high resolution 1 H NMR spectroscopy, Anal. Chim. Acta 585 (2007) 110–119. U. Holzgrabe, R. Deubner, C. Schollmayer, B. Waibel, Quantitative NMR spectroscopy—applications in drug analysis, J. Pharm. Biomed. Anal. 38 (2005) 806–812. Y. Jiang, Z. Xie, D.Q. Zhang, Separation of alendronate and its related substances by ion-pair reverse phase chromatography with ELSD, J. Anal. Sci. 22 (2006) 137–140. Z.Z. Yuan, L.N. You, H. Zhao, Determination of alendronate sodium and its related substance 4-aminobutyric acid by RP-HPLC, Chin. J. Spectrosc. Lab. 30 (2013) 757–762. Chinese Pharmacopeia, the 2010 Version. Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, Food and Drug Administration Center for Drug Evaluation and Research (CDER), 2000.
Please cite this article in press as: B. Xie, et al., Rapid determination of alendronate to quality evaluation of tablets by high resolution 1 H NMR spectroscopy, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.07.006
