Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Quantitative proton nuclear magnetic resonance for the structural and quantitative analysis of atropine sulfate Shi Shen a,b , Jing Yao a , Yaqin Shi a,∗ a b
National Institute for Food and Drug Control, Beijing 100050, People’s Republic of China National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 3 August 2013 Received in revised form 19 October 2013 Accepted 23 October 2013 Available online 7 November 2013 Keywords: Atropine sulfate qNMR VT-NMR Conformational isomer Reference standard
a b s t r a c t This study assessed a general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of atropine sulfate (Active Pharmaceutical Ingredient, API) as reference standard. The spectra were acquired in D2 O using maleic acid as the internal standard. Conformational behaviors of tropane ring were observed and studied by means of NMR and ROESY experiments at different temperature, which showed that the azine methyl group was at equilibrium for axial and equatorial conformations at room temperature. Signal delay and monitor signals of qNMR experimentation were optimized for quantification. The study reported here validated the method’s linearity, range, limit of quantification, stability and precision. The results were consistent with the results obtained from mass balance approach. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As a part of drug quality control process, the reference standard is widely used for qualitative and quantitative analysis. Unlike chromatography, quantitative 1 H NMR does not require a high purity reference standard for accurate quantification of the test compound of interest, because selected functional group(s) being observed, e.g. the nucleus of a hydrogen atom, has a molar response coefficient of 1 regardless of the compound, assuming that the proton does not exchange with the deuteriums of the solvent [1]. Therefore, QNMR is especially applicable for content determination of substances lack of ultraviolet adsorption [2–4] and highly suitable to evaluate the purity determination of primary reference standards [5–7] as well as the quality of drugs [3,8]. Atropine sulfate is a type of tropane alkaloids with description of benzene acetic acid ␣-(hydroxyl methyl)-8-methyl-8azabicyclo[3.2.1]oct-3-yl ester, sulfate (2:1) mono-hydrate, as shown in Fig. 1, and it is a competitive antagonist for the muscarinic acetylcholine receptors and classified as an anticholinergic drug. Sharma et al. [9] have reported a quantitative 1 H NMR method for analysis of atropine sulfate using N-methyl as monitor signal for quantification. In the study reported in this article, we found that atropine sulfate does not exist in the single unique conformation, but in conformational equilibrium observed from the variable
∗ Corresponding author. Tel.: +86 10 67095861; fax: +86 10 67095861. E-mail addresses: [email protected] (S. Shen), [email protected] (Y. Shi). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.036
temperature NMR spectra. Our finding suggests that Sharma’s method have limitation in the choice of monitor signals for quantification. In this report, we studied various conformational structures of atropine sulfate using methods of 1D and 2D spectroscopic experiments, such as VT-NMR, APT and ROESY techniques. Here, we describe a modified quantitative 1 H NMR method to determine the purity of atropine sulfate. The results are consistent with the result from mass balance approach. The method further confirms that the qNMR is a rapid, convenient and accurate technique for the value assignment of atropine sulfate as a reference standard. 2. Experimental 2.1. Materials Atropine sulfate 97.1% (determined by mass balance approach) was provided by Puri Pharmaceutical Factory, Henan, China (Batch No. 20110603); maleic acid 99.78% (standard for quantitative NMR) was purchased from Fluka Analytical, USA (Lot. BCBB7987V); deuterated solvent, D2 O (99.8%) was purchased from J&K Chemical, Japan. 2.2. Sample preparation Calibrated GilsonTM syringes (1 mL and 100 L) were used for the volume measurements and Micro-balance Mettler Toledo MX5 (Mettler-Toledo GmbH, Switzerland) was used for weight measurement.
S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
119
For conformational analysis, i.e. 1 H NMR, APT, VT-NMR and ROESY experiments, approximately 10 mg of atropine sulfate was dissolved in 600 L D2 O. For determination of the performance, i.e. linearity and range of 6 analytes containing from 6.988 to 56.104 mg atropine sulfate were dissolved in 1.0 mL D2 O. Sample weights for qNMR were approximately 27.00 mg (0.04 mol/L). The internal standard of maleic acid, 4.64 mg (0.04 mol/L) was added to each analyte. 2.3. NMR spectroscopy All of the 1 H, APT NMR spectra and the two-dimensional experiments, i.e. 1 H, 13 C heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), 1 H–1 H correlation spectroscopy (COSY) and rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) were performed at 298 K using Bruker Avance spectrometer at 500.13 MHz proton frequency with 5 mm dual-core probe and BVT23000 temperature control unit. Variable-temperature NMR (VT-NMR) experiments
2.773 2.603
4.975 4.967 4.959
6.284
Fig. 1. Structure of atropine sulfate.
N-methyl
Maleic acid protons
H-3 N-methyl
Fig. 2.
1
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5 ppm
3.028
5.5
0.999 2.010 1.033 1.011
6.0
1.000
6.5
0.969
7.0
5.031
7.5
H NMR of mixture of atropine sulfate and maleic acid in D2 O (500 MHz, 298 K).
Fig. 3. The variable-temperature 1 H NMR spectra of atropine sulfate in D2 O (the red rectangle indicates the signals of non-dominant conformation parts). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
Table 1 Linearity, range, precision and stability of atropine sulfate calculated by NMR spectroscopy. Linearity and range
Repeatability
Stability
Sample
mstd (mg/ml)
mx (mg/ml)
Ax /Astd
Sample
mstd (mg/ml)
mx (mg/ml)
Px (%)
No.
Content (%)
Time interval (h)
Px (%)
1 2 3 4 5 6
4.873 4.919 4.604 4.736 4.677 4.767
6.988 13.546 28.456 35.043 41.108 56.104
0.4755 0.9198 2.065 2.482 2.958 3.989
1 2 3 4 5 6
4.595 4.604 4.916 4.625 4.746 5.007
28.538 28.456 27.782 27.769 28.807 27.715
97.1 97.2 97.6 96.2 97.4 97.1
1 2 3 4 5 6
97.1 97.0 97.5 96.6 97.3 97.4
0 1 2 4 6 8
97.3 97.0 97.3 97.3 97.4 97.3
R2 RSD (%)
Precision
0.9999 –
– 0.50
were implemented under the temperature of 298 K, 313 K, 323 K, 333 K, 343 K and 353 K, respectively. The experiments were carried out with the following conditions that have been optimized for qNMR: 30◦ pulse of 12.75 s, 64 K data points, line broadening of 0.3, 32 scans and receiver gain of 161. The repetition delay was 25 s and it was determined by the T1 value of the longest relaxing maleic acid nuclei 3.8 s (D1 > 5T1 ) [10], which was calculated by the inversion recovery pulse program [11]. All processing and spectra handling have been performed using Topspin 1.3 or 2.1 program suites. Calibration of the chemical shift scale was performed by adjusting the residual H2 O 1 H signal to 4.70 ppm at 298 K. The purity of analyte Px was calculated by Eq. (1) [5]: Px =
Ax Nstd Mx mstd P , Astd Nx Mstd m std
(1)
where Mx and Mstd are the molecular weights of the analyte (676.86) and internal standard (116.07), respectively, m is the weighed mass of the investigated sample, mstd and Pstd are the weighed mass and the purity of the standard, Nstd and Astd correspond to the number of spins and the integrated signal area of a (typical) NMR line of the standard. 3. Results and discussions 3.1. Conformational analysis of atropine sulfate Atropine sulfate is an ester formed by scopolamine alcohol (3␣hydroxy-tropane, tropine) and racemic tropic acid. As shown in Fig. 2, the 1 H NMR spectrum (D2 O) of atropine sulfate exhibited some impurities signals around signals of tropane ring (except for
– 0.34
– 0.12
H-3 at ı 4.96) under room temperature, especially the obvious signal at ı 2.77 nearby N-methyl signal at ı 2.60, which suggested the solution conformations. The variable-temperature 1 H and APT (attached proton test) NMR experiments of atropine sulfate were carried out in order to verify the coexistence of a pair of conformer on the NMR time scale. Increasing the temperature from 298 to 353 K alter the 1 H NMR spectrum, especially the high-field signals of tropane protons (except for H-3) and N-methyl protons appear to undergo coalescence at 333 K, and regain decoalescence when the temperature was set to 298 K, and the results was shown in Fig. 3. The same phenomenon can be observed from variable-temperature APT spectrum. Hu et al. [12] have determined the conformation of atropine by molecule calculations, which showed that preferred conformation of piperidine ring in tropane was described as the chair. The ROESY correlations were observed as follows: from N-CH3 (ı 2.60) to H2 -6 and H2 -7 (ı 2.01, 1.88), from N-CH3 (ı 2.77) to H-2a and H-4a (ı 2.34). The results clearly verify that the preferred conformation of N-methyl group occupies the equatorial position, as shown in Fig. 4a, and the minor conformation of N-methyl group occupies the axial position, as shown in Fig. 4b. The 1 H and 13 C chemical shifts of the pairs of signals in tropane ring were assigned using COSY, HSQC and HMBC experiments (data not shown) and the assignments are consistent with Feeney’s literature [13]. According to the results above, we can conclude that the NMR minor signals (ı 2.77) do not indicate the impurities of atropine sulfate, but represent the N-methyl axial isomer which presented in the equilibrium mixture. Therefore only the signal of N-methyl in the equatorial position (ı 2.60) for quantification described as Sharma’s method [9] is not accurate; the quantitative method for atropine sulfate was revised
Fig. 4. Conformational analysis of atropine sulfate based on ROESY correlations.
S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
121
Table 2 Statistical results of the developed QNMR method and established method. Sample
1 2 3
mx (mg/ml)
mstd (mg/ml)
28.538 27.715 27.782
Mean (%) RSD (%)
4.595 5.007 4.916
Developed QNMR method
Reported QNMR approach
Nx /Nstd
Ax /Astd
Px (%)
1
1.036 0.923 0.948
97.0 97.0 97.6
– –
Nx /Nstd
Ax /Astd
Px (%)
1/3
2.972 2.668 2.735
92.8 93.5 93.8
97.2 0.36
93.4 0.55
with the signal of H-3 for quantification and validated in this paper.
93.4% if N-methyl (ı 2.60) was selected as monitor signal [9], which was lower than the exact content, as shown in Table 2.
3.2. Quantitative NMR method
4. Conclusions
In this study, the internal standard chosen was maleic acid. It has high aqueous solubility and the chemical shift of the methine of maleic acid provides a well-separated signal (6.28 ppm) without any interference from atropine sulfate in the integration region [14]. In qNMR, singlet signal is usually used for quantification, however sets of single signals of azine methyl from tropane ring were observed at 2.77 and 2.60 ppm, respectively. Therefore, the triplet at 4.96 ppm, originating from the proton in the 3-position of the atropine ring, was used as monitor signal for quantification (S/N ≥ 250), as shown in Fig. 2.
The study developed a simple, reliable qNMR method to determine the content of atropine sulfate (API), which revised the method described as literature [9], based on the results of a pair of conformer of atropine sulfate coexisted in D2 O by variable temperature NMR and ROESY experiments. Comparing the qNMR method with the mass balance approach, the content of atropine sulfate was almost identical, which indicated that the qNMR method could be the complementary with the mass balance approach for the value assignment of the reference standard.
3.3. Validation
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2013.10.036.
3.3.1. Linearity and range The calibration curves showed linearity over concentration ranges (w/w) from 1.434 to 11.769 mg for atropine sulfate per mg of maleic acid, as shown in Table 1. The correlation coefficient obtained with linear regression curve (y = 0.3394x − 0.0195) was 0.9999, which suggested that the selected method had excellent linearity over these concentration ranges. 3.3.2. Limit of quantification According to literature [10,15], signal-to-noise ratio (S/N) at least 150 is required for the target uncertainty of 1%, and it should be greater than 150:1 for 1 H NMR to achieve accurate quantification. The S/N in the spectra could be influenced by variations of the pulse flip angle, the number of scans, and the broadening (lb) [15]. LOQ was assessed by studying the S/N and investigated using analyte with the minimum concentration (5.54 mg/mL) of atropine sulfate, and the lowest S/N observed was 248. 3.3.3. Precision and stability Precision tests performed using the sample at the concentration of 0.04 mol/L showed that the relative standard deviation (% RSD) of intra-day was 0.50%. Therefore, the system precision is considered to be satisfactory. The stability was determined using the same sample at 0, 1, 2, 4, 6, 8 h, respectively, and the relative standard deviation (% RSD) values was 0.12%, as shown in Table 1. 3.4. Sample analysis The established analytical method was applied for the calibration of atropine sulfate (API). The result (97.2% calculated in C34 H48 N2 O10 S, n = 3, RSD 0.36%) is consistent with that obtained from mass balance approach. The content of the analyte would be
Appendix A. Supplementary data
References [1] G. Maniara, K. Rajamoorthi, S. Rajan, G.W. Stockton, Method performance and validation for quantitative analysis by 1 H and 31 P NMR spectroscopy. Applications to analytical standards and agricultural chemicals, Anal. Chem. 70 (1998) 4921–4928. [2] U. Holzgrabe, R. Deubner, C. Schollmayer, B. Waibel, Quantitative NMR spectroscopy – applications in drug analysis, J. Pharm. Biol. Anal. 38 (2005) 806–812. [3] J.A. Donarski, D.P.T. Roberts, A.J. Charlton, Quantitative NMR spectroscopy for the rapid measurement of methylglyoxal in manuka honey, Anal. Methods 2 (2010) 1479–1483. [4] C.K. Larive, D. Jayawickrama, L. Orfi, Quantitative analysis of peptides with NMR spectroscopy, Appl. Spectrosc. 51 (1997) 1531–1536. [5] S.-Y. Liu, C.-Q. Hu, A comparative uncertainty study of the calibration of macrolide antibiotic reference standards using quantitative nuclear magnetic resonance and mass balance methods, Anal. Chim. Acta 602 (2007) 114–121. [6] T. Schoenberger, Determination of standard sample purity using the highprecision 1 H-NMR process, Anal. Bioanal. Chem. 403 (2012) 247–254. [7] G.F. Pauli, qNMR – a versatile concept for the validation of natural product reference compounds, Phytochem. Anal. 12 (2001) 28–42. [8] U. Holzgrabe, M. Malet-Martino, Analytical challenges in drug counterfeiting and falsification – the NMR approach, J. Pharm. Biol. Anal. 55 (2011) 679–687. [9] R. Sharma, P.K. Gupta, A. Mazumder, D.K. Dubey, K. Ganesan, R. Vijayaraghavan, A quantitative NMR protocol for the simultaneous analysis of atropine and obidoxime in parenteral injection devices, J. Pharm. Biomed. Anal. 49 (2009) 1092–1096. [10] S.K. Bharti, R. Roy, Quantitative 1 H NMR spectroscopy, Trends Anal. Chem. 35 (2012) 5–26. [11] S. Berger, S. Braun, 200 and More NMR Experiments – A Practical Course, WileyVCH Verlag GmbH & Co. KGaA, Weinheim, 2004, pp. 160–164. [12] W.-X. Hu, L.-H. Yun, S.-S. Li, Conformational analysis of anticholinergic drugs. Molecular mechanic study of atropine alkaloids from Solanaceae, Bull. Acad. Mil. Med. Sci. 16 (1996) 101–107 (in Chinese). [13] J. Feeney, R. Foster, E.A. Piper, Nuclear magnetic resonance study of the conformations of atropine and scopolamine cations in aqueous solution, J. Chem. Soc. [Perkin 2] 15 (1977) 2016–2020. [14] T. Rundlof, M. Mathiasson, S. Bekiroglu, B. Hakkarainen, T. Bowden, T. Arvidsson, Survey and qualification of internal standards for quantification by 1 H NMR spectroscopy, J. Pharm. Biol. Anal. 52 (2010) 645–651. [15] F. Malz, H. Jancke, Validation of quantitative NMR, J. Pharm. Biol. Anal. 38 (2005) 813–823.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Quantitative proton nuclear magnetic resonance for the structural and quantitative analysis of atropine sulfate Shi Shen a,b , Jing Yao a , Yaqin Shi a,∗ a b
National Institute for Food and Drug Control, Beijing 100050, People’s Republic of China National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 3 August 2013 Received in revised form 19 October 2013 Accepted 23 October 2013 Available online 7 November 2013 Keywords: Atropine sulfate qNMR VT-NMR Conformational isomer Reference standard
a b s t r a c t This study assessed a general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of atropine sulfate (Active Pharmaceutical Ingredient, API) as reference standard. The spectra were acquired in D2 O using maleic acid as the internal standard. Conformational behaviors of tropane ring were observed and studied by means of NMR and ROESY experiments at different temperature, which showed that the azine methyl group was at equilibrium for axial and equatorial conformations at room temperature. Signal delay and monitor signals of qNMR experimentation were optimized for quantification. The study reported here validated the method’s linearity, range, limit of quantification, stability and precision. The results were consistent with the results obtained from mass balance approach. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As a part of drug quality control process, the reference standard is widely used for qualitative and quantitative analysis. Unlike chromatography, quantitative 1 H NMR does not require a high purity reference standard for accurate quantification of the test compound of interest, because selected functional group(s) being observed, e.g. the nucleus of a hydrogen atom, has a molar response coefficient of 1 regardless of the compound, assuming that the proton does not exchange with the deuteriums of the solvent [1]. Therefore, QNMR is especially applicable for content determination of substances lack of ultraviolet adsorption [2–4] and highly suitable to evaluate the purity determination of primary reference standards [5–7] as well as the quality of drugs [3,8]. Atropine sulfate is a type of tropane alkaloids with description of benzene acetic acid ␣-(hydroxyl methyl)-8-methyl-8azabicyclo[3.2.1]oct-3-yl ester, sulfate (2:1) mono-hydrate, as shown in Fig. 1, and it is a competitive antagonist for the muscarinic acetylcholine receptors and classified as an anticholinergic drug. Sharma et al. [9] have reported a quantitative 1 H NMR method for analysis of atropine sulfate using N-methyl as monitor signal for quantification. In the study reported in this article, we found that atropine sulfate does not exist in the single unique conformation, but in conformational equilibrium observed from the variable
∗ Corresponding author. Tel.: +86 10 67095861; fax: +86 10 67095861. E-mail addresses: [email protected] (S. Shen), [email protected] (Y. Shi). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.036
temperature NMR spectra. Our finding suggests that Sharma’s method have limitation in the choice of monitor signals for quantification. In this report, we studied various conformational structures of atropine sulfate using methods of 1D and 2D spectroscopic experiments, such as VT-NMR, APT and ROESY techniques. Here, we describe a modified quantitative 1 H NMR method to determine the purity of atropine sulfate. The results are consistent with the result from mass balance approach. The method further confirms that the qNMR is a rapid, convenient and accurate technique for the value assignment of atropine sulfate as a reference standard. 2. Experimental 2.1. Materials Atropine sulfate 97.1% (determined by mass balance approach) was provided by Puri Pharmaceutical Factory, Henan, China (Batch No. 20110603); maleic acid 99.78% (standard for quantitative NMR) was purchased from Fluka Analytical, USA (Lot. BCBB7987V); deuterated solvent, D2 O (99.8%) was purchased from J&K Chemical, Japan. 2.2. Sample preparation Calibrated GilsonTM syringes (1 mL and 100 L) were used for the volume measurements and Micro-balance Mettler Toledo MX5 (Mettler-Toledo GmbH, Switzerland) was used for weight measurement.
S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
119
For conformational analysis, i.e. 1 H NMR, APT, VT-NMR and ROESY experiments, approximately 10 mg of atropine sulfate was dissolved in 600 L D2 O. For determination of the performance, i.e. linearity and range of 6 analytes containing from 6.988 to 56.104 mg atropine sulfate were dissolved in 1.0 mL D2 O. Sample weights for qNMR were approximately 27.00 mg (0.04 mol/L). The internal standard of maleic acid, 4.64 mg (0.04 mol/L) was added to each analyte. 2.3. NMR spectroscopy All of the 1 H, APT NMR spectra and the two-dimensional experiments, i.e. 1 H, 13 C heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), 1 H–1 H correlation spectroscopy (COSY) and rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) were performed at 298 K using Bruker Avance spectrometer at 500.13 MHz proton frequency with 5 mm dual-core probe and BVT23000 temperature control unit. Variable-temperature NMR (VT-NMR) experiments
2.773 2.603
4.975 4.967 4.959
6.284
Fig. 1. Structure of atropine sulfate.
N-methyl
Maleic acid protons
H-3 N-methyl
Fig. 2.
1
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5 ppm
3.028
5.5
0.999 2.010 1.033 1.011
6.0
1.000
6.5
0.969
7.0
5.031
7.5
H NMR of mixture of atropine sulfate and maleic acid in D2 O (500 MHz, 298 K).
Fig. 3. The variable-temperature 1 H NMR spectra of atropine sulfate in D2 O (the red rectangle indicates the signals of non-dominant conformation parts). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
Table 1 Linearity, range, precision and stability of atropine sulfate calculated by NMR spectroscopy. Linearity and range
Repeatability
Stability
Sample
mstd (mg/ml)
mx (mg/ml)
Ax /Astd
Sample
mstd (mg/ml)
mx (mg/ml)
Px (%)
No.
Content (%)
Time interval (h)
Px (%)
1 2 3 4 5 6
4.873 4.919 4.604 4.736 4.677 4.767
6.988 13.546 28.456 35.043 41.108 56.104
0.4755 0.9198 2.065 2.482 2.958 3.989
1 2 3 4 5 6
4.595 4.604 4.916 4.625 4.746 5.007
28.538 28.456 27.782 27.769 28.807 27.715
97.1 97.2 97.6 96.2 97.4 97.1
1 2 3 4 5 6
97.1 97.0 97.5 96.6 97.3 97.4
0 1 2 4 6 8
97.3 97.0 97.3 97.3 97.4 97.3
R2 RSD (%)
Precision
0.9999 –
– 0.50
were implemented under the temperature of 298 K, 313 K, 323 K, 333 K, 343 K and 353 K, respectively. The experiments were carried out with the following conditions that have been optimized for qNMR: 30◦ pulse of 12.75 s, 64 K data points, line broadening of 0.3, 32 scans and receiver gain of 161. The repetition delay was 25 s and it was determined by the T1 value of the longest relaxing maleic acid nuclei 3.8 s (D1 > 5T1 ) [10], which was calculated by the inversion recovery pulse program [11]. All processing and spectra handling have been performed using Topspin 1.3 or 2.1 program suites. Calibration of the chemical shift scale was performed by adjusting the residual H2 O 1 H signal to 4.70 ppm at 298 K. The purity of analyte Px was calculated by Eq. (1) [5]: Px =
Ax Nstd Mx mstd P , Astd Nx Mstd m std
(1)
where Mx and Mstd are the molecular weights of the analyte (676.86) and internal standard (116.07), respectively, m is the weighed mass of the investigated sample, mstd and Pstd are the weighed mass and the purity of the standard, Nstd and Astd correspond to the number of spins and the integrated signal area of a (typical) NMR line of the standard. 3. Results and discussions 3.1. Conformational analysis of atropine sulfate Atropine sulfate is an ester formed by scopolamine alcohol (3␣hydroxy-tropane, tropine) and racemic tropic acid. As shown in Fig. 2, the 1 H NMR spectrum (D2 O) of atropine sulfate exhibited some impurities signals around signals of tropane ring (except for
– 0.34
– 0.12
H-3 at ı 4.96) under room temperature, especially the obvious signal at ı 2.77 nearby N-methyl signal at ı 2.60, which suggested the solution conformations. The variable-temperature 1 H and APT (attached proton test) NMR experiments of atropine sulfate were carried out in order to verify the coexistence of a pair of conformer on the NMR time scale. Increasing the temperature from 298 to 353 K alter the 1 H NMR spectrum, especially the high-field signals of tropane protons (except for H-3) and N-methyl protons appear to undergo coalescence at 333 K, and regain decoalescence when the temperature was set to 298 K, and the results was shown in Fig. 3. The same phenomenon can be observed from variable-temperature APT spectrum. Hu et al. [12] have determined the conformation of atropine by molecule calculations, which showed that preferred conformation of piperidine ring in tropane was described as the chair. The ROESY correlations were observed as follows: from N-CH3 (ı 2.60) to H2 -6 and H2 -7 (ı 2.01, 1.88), from N-CH3 (ı 2.77) to H-2a and H-4a (ı 2.34). The results clearly verify that the preferred conformation of N-methyl group occupies the equatorial position, as shown in Fig. 4a, and the minor conformation of N-methyl group occupies the axial position, as shown in Fig. 4b. The 1 H and 13 C chemical shifts of the pairs of signals in tropane ring were assigned using COSY, HSQC and HMBC experiments (data not shown) and the assignments are consistent with Feeney’s literature [13]. According to the results above, we can conclude that the NMR minor signals (ı 2.77) do not indicate the impurities of atropine sulfate, but represent the N-methyl axial isomer which presented in the equilibrium mixture. Therefore only the signal of N-methyl in the equatorial position (ı 2.60) for quantification described as Sharma’s method [9] is not accurate; the quantitative method for atropine sulfate was revised
Fig. 4. Conformational analysis of atropine sulfate based on ROESY correlations.
S. Shen et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 118–121
121
Table 2 Statistical results of the developed QNMR method and established method. Sample
1 2 3
mx (mg/ml)
mstd (mg/ml)
28.538 27.715 27.782
Mean (%) RSD (%)
4.595 5.007 4.916
Developed QNMR method
Reported QNMR approach
Nx /Nstd
Ax /Astd
Px (%)
1
1.036 0.923 0.948
97.0 97.0 97.6
– –
Nx /Nstd
Ax /Astd
Px (%)
1/3
2.972 2.668 2.735
92.8 93.5 93.8
97.2 0.36
93.4 0.55
with the signal of H-3 for quantification and validated in this paper.
93.4% if N-methyl (ı 2.60) was selected as monitor signal [9], which was lower than the exact content, as shown in Table 2.
3.2. Quantitative NMR method
4. Conclusions
In this study, the internal standard chosen was maleic acid. It has high aqueous solubility and the chemical shift of the methine of maleic acid provides a well-separated signal (6.28 ppm) without any interference from atropine sulfate in the integration region [14]. In qNMR, singlet signal is usually used for quantification, however sets of single signals of azine methyl from tropane ring were observed at 2.77 and 2.60 ppm, respectively. Therefore, the triplet at 4.96 ppm, originating from the proton in the 3-position of the atropine ring, was used as monitor signal for quantification (S/N ≥ 250), as shown in Fig. 2.
The study developed a simple, reliable qNMR method to determine the content of atropine sulfate (API), which revised the method described as literature [9], based on the results of a pair of conformer of atropine sulfate coexisted in D2 O by variable temperature NMR and ROESY experiments. Comparing the qNMR method with the mass balance approach, the content of atropine sulfate was almost identical, which indicated that the qNMR method could be the complementary with the mass balance approach for the value assignment of the reference standard.
3.3. Validation
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2013.10.036.
3.3.1. Linearity and range The calibration curves showed linearity over concentration ranges (w/w) from 1.434 to 11.769 mg for atropine sulfate per mg of maleic acid, as shown in Table 1. The correlation coefficient obtained with linear regression curve (y = 0.3394x − 0.0195) was 0.9999, which suggested that the selected method had excellent linearity over these concentration ranges. 3.3.2. Limit of quantification According to literature [10,15], signal-to-noise ratio (S/N) at least 150 is required for the target uncertainty of 1%, and it should be greater than 150:1 for 1 H NMR to achieve accurate quantification. The S/N in the spectra could be influenced by variations of the pulse flip angle, the number of scans, and the broadening (lb) [15]. LOQ was assessed by studying the S/N and investigated using analyte with the minimum concentration (5.54 mg/mL) of atropine sulfate, and the lowest S/N observed was 248. 3.3.3. Precision and stability Precision tests performed using the sample at the concentration of 0.04 mol/L showed that the relative standard deviation (% RSD) of intra-day was 0.50%. Therefore, the system precision is considered to be satisfactory. The stability was determined using the same sample at 0, 1, 2, 4, 6, 8 h, respectively, and the relative standard deviation (% RSD) values was 0.12%, as shown in Table 1. 3.4. Sample analysis The established analytical method was applied for the calibration of atropine sulfate (API). The result (97.2% calculated in C34 H48 N2 O10 S, n = 3, RSD 0.36%) is consistent with that obtained from mass balance approach. The content of the analyte would be
Appendix A. Supplementary data
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