Application of NMR spectroscopy in the quantification of Drug. and its Salt with chemical shift changes

Transcription

1 Chapter 2 22 Application of NMR spectroscopy in the quantification of Drug and its Salt with chemical shift changes 2.1 Introduction NMR spectroscopy is one of the most important and widespread analytical technique in academic and industrial research. NMR spectroscopy can simultaneously determine both identity and quantity. It enables a unique quantitative determination of the relative amount of molecular groups, offering a tool to quantify entire substances even in mixture. Most of the methodologies used the average integral intensities of NMR signals comparing them to those of external or internal standards. Several studies were found in the application of NMR for analytical purposes [1 & 2]. Some of these studies address the quantitative analysis of drugs [ 3 5]. Chemical shifts are indicators of the chemical surroundings of atoms and hence they can be used to monitor the changes due to intra or intermolecular interaction through covalent or non-covalent binding. They have been studied to understand aggregation [6-8], molecular recognition [9&10] and to determine the association constants [11&12]. In recent times, quantitative information is obtained based on the chemical shift displacements of selected protons offering quite elegant and reproducible NMR method [13]. The high filed NMR instruments give increased sensitivity and signal resolution leading to improved limits of quantification [14].

2 Chapter 2 23 Nevertheless, 1 H NMR techniques still remain quite insensitive, when compared to techniques employing UV or MS for detection with which ppm and sub ppm level quantifications are achievable. Salts of the drug molecules are prepared to enhance the solubility or stability during the drug development [15]. A quick analytical method is required to estimate the completion of the salt formation. To meet this requirement, the method development for estimation of quantity of the drug in presence of its salt is undertaken. Pioglitazone HCl ( Scheme 2.1) and Dexlansoprazole Na (Scheme 2.2) have been selected for the study where the former represents salt of basic drug while the latter is a salt of acidic drug. Interestingly, in either case the presence of drug had a significant effect on the chemical shifts of its corresponding salt. The NMR spectra of the drug, its salt and their mixture (1:1) are compared (Figures 2.1 and 2.2). Scheme 2.1 Pioglitazone Structure and numbering of Pioglitazone and HCl.

3 Chapter 2 24 Figure H NMR spectra (7.3 to 8.7 ppm) (a) Pioglitazone HCl Salt, b) Pioglitazone:Pioglitazone HCl Salt (50:50) and c) Pioglitazone N 1 2 N H 18 O 10 S N O CF 3 17 Dexlansoprazole (DEX) N 10 2 Na 1 N O S N O CF 3 21 Dexlansoprazole Sodium (DEX Na) Scheme 2 Structures of Dexlansoprazole and Dexlansoprazole Sodium

4 Chapter 2 25 Figure H NMR spectra (4.3 to 8.5 ppm) (a) Dexlansoprazole Na, (b) Dexlansoprazole: Dexlansoprazole Na (50:50) and (c) Dexlansoprazole. Careful comparison of the spectrum of the mixture with the spectra of drug and salt, the chemical shift in the mixture is in between the chemical shift of individual components indicative of an averaged environment. The ph variations due to the addition of salt to free acid (or base) or vice versa could be the potential reason for the observed significant displacement of chemical shifts. On the contrary, the separation technique such as HPLC will give only one peak at the retention time of drug for the both drug and the salt. Hence the quantification of drug in presence salt will be a challenge using HPLC technique. Other analytical methods such as titrimetry, gravimetry and instrumental techniques such as Atomic Absorption Spectroscopy, Ion Chromotography would require either more compound or time for the analysis. The advantage of differentiation

5 Chapter 2 26 provided by NMR, discussed above, makes this technique not only elegant and simple to practice but also a quick one. Hence an attempt was made to develop a quantitative NMR methodology to estimate drug in presence of salt. This method was evaluated with respect to parameters such as accuracy, precision, linearity and robustness. 2.2 Experimental Materials Pioglitazone, Dexlansoprazole and their corresponding salts were obtained from process chemistry group of Dr. Reddy s Laboratories Ltd. Hyderabad, India. Dimethyl Sulfoxide d6 (99.98% D) was used for the preparation of solutions. The peak of tetra methyl silane (TMS) at 0.00 ppm was used as the chemical shift reference. DMSO-d6 was purchased from Camebridge Isotope Laboratories Inc. (Andover, USA) Preparation of solutions Stock solutions of drug and salt were prepared ~0.1 M in DMSO-d6 (having 0.005% TMS). Eight levels of different compositions of drug and salt ranging between 100% to 0% were used for the calibration chart of the chemical shifts NMR measurements The NMR experiments were performed on a UNITY INOVA, Mercury Plus and Gemini NMR spectrometers with the operating

6 Chapter 2 27 frequencies , and MHz respectively. Chemical shifts are referenced to TMS, at 0.0 ppm. Aliquots of 0.7 ml from each calibration solution were transferred in 5 mm tubes and each 1 H NMR spectra was recorded in triplicate under constant temperature control. Adequate care is taken during the preparation of the solutions and temperature control in order to minimize systematic and random errors. The effect of temperature on the results was also evaluated by recording the 1 H NMR spectra at three different temperatures 303, 318 and 333 K.

7 Chapter Results and discussion The quantitative analysis of the salts of Pioglitazone and Dexlansoprazole are presented in the following sections. 2.4 Pioglitazone and Pioglitazone HCl salt The quantification of Pioglitazone salt (PIO HCl) in the presence of Pioglitazone base (PIO) has been presented Detector s response function and linearity To develop a quantitative NMR methodology, the chemical shifts assigned to pyridine moiety protons of PIO were considered as the detector s response. It is observed that the resonances of three protons viz., H3, H4 and H6 have moved downfield when the content of PIO HCl is more. The range of chemical shift change (in ppm) for H3, H4 and H6 are, , and ppm respectively. The responses from H3, H4 and H6 were selected to derive the detector response function. A plot of chemical shift values of these signals as a function of the percentage of PIO yielded a linear graph of the form given below. = m x + c (2.1) Where is the chemical shift (in ppm or Hz); x is the mole fraction of the base; m the slope; c the intercept (ppm or Hz). The 1 H chemical shifts are given in Table 2.1 while the relevant graphs are shown in Figures 2.3 and 2.4.

8 Chapter 2 29 Table H Chemical shifts of H3, H4 and H6 in ppm and hertz % of PIO H3 H4 H6 ppm Hz ppm Hz ppm Hz Figure 2.3 Linearity graph of H3, H4 and H6 in ppm.

9 Chapter 2 30 Figure 2.4 Linearity graph of H3, H4 and H6 in Hz It is reasonable to expect similar changes in the carbon ( 13 C) chemical shifts of C3, C4 and C6 signals as observed in the corresponding protons. The 13 C chemical shifts were determined from the HSQC data. A linear relationship was observed with the carbon signals of pyridine similar to the proton signals (Figures 2.5, 2.6 and 2.7). The 13 C Chemical shift data (500 MHz) is given in Table 2.2, while the relevant liner graphs are displayed in Figures 2.8 and 2.9. The proposed quantitative method utilizes Equation 2.1 which describes the chemical shift as a function of %base and can be applied to any proton (and / or 13 C) on pyridine moiety. However extension of this approach to other similar situations can be done after relevant verification of the model.

10 Chapter 2 31 Figure 2.5 HSQC spectrum of PIO Figure 2.6 HSQC spectrum of PIO + PIO HCl (50-50)

11 Chapter 2 32 Figure 2.7 HSQC spectrum of PIO HCl Table 2.2 Chemical shifts of C3, C4 and C6 in ppm and Hz C3 C4 C6 % of Base ppm Hz ppm Hz ppm Hz

12 Chapter 2 33 Figure 2.8 Linearity graph of C3, C4 and C6 ppm Figure 2.9 Linearity graph of C3, C4 and C6 in Hz

13 Chapter Assessment of the method accuracy The assay of samples with known amount of PIO at three concentration levels and three replicates at each concentration levels helped to evaluate the accuracy of the method. The results are summarized in Table 2.3 shown below. The recovery was found between % demonstrate that the method is accurate for the quantification. Table 2.3 Accuracy results with chemical shift data of H4 on 400MHz Known % of PIO Determined % of PIO %recovery Relative error (%) Assessment of the method sensitivity In the present method, sensitivity depends on the ability to accurately measure the small chemical shift changes ( min) which would correspond to minimum change in concentration (mole fraction) ( xmin).

14 Chapter 2 35 The response function can be used to calculate the method sensitivity parameters viz., min and Δxmin. = mx + c (2.1) 1 = mx1 + c 2 = mx2 + c 2 1 = m (x2 x1) min = m xmin The min is same as the digital resolution of the instrument used. This would help us to calculate the quantifiable minimum change in concentration ( xmin) which serves as a measure for the sensitivity of the method. xmin = min / m (2.2) It is clear from the above equation that the sensitivity of the method, described in terms of minimum change in concentration ( xmin), depends on the slope of the straight line and the digital resolution of the NMR instrument used. The digital resolution for a 400 MHz NMR instrument is 0.6 Hz (High-Field Indirect Detection NMR Probe Installation manual, Varian).

15 Chapter 2 36 The response functions of chemical shifts of H3, H4 and H6 at 293 K using 500, 400 and 200 MHz are shown in the Table 2.4. Table 2.4 Response functions of protons Instrument Proton Response function R 2 xmin b 500 MHz H6 δ = x H4 δ = x H3 δ = x MHz H6 δ = x H4 δ = x H3 δ = x MHz H6 δ = x H4 δ = x H3 δ = x a Chemical shift values expressed in Hz. b min = 0.6 Hz for all the instrument The response from H4 proton was selected for the calculations as it displayed maximum 1 H chemical shift displacement Measurement of precision The repeatability of the proposed method was assessed by performing measurements with six replicate preparations at 10%, 50% and 90% of PIO HCl to PIO and the relative standard deviation (%) of 0.02, 0.02 and 0.01 respectively. The results are shown is Table 2.5 which indicates that the method is found to be precise.

16 Chapter 2 37 Table 2.5 Precision results Precision H4 in Hz Sample No. at 90% at 50% at 10% Average SD % RSD Limits of detection and quantification The limits of detection and quantification (LOD and LOQ respectively) are not relevant to methods for quantifying the major components. However, LOD and LOQ are addressed with respect to the technical specifications of the NMR instrument. The higher the resolution of the spectrometer, one gets the lower the values of LOD and LOQ. The instrument resolution value of 0.6 Hz has been taken as the minimum chemical shift difference that we can detect on 400 MHz NMR instrument. The detection limit was assessed as about x min (Section 2.4.3) based on the instrument resolution 0.6Hz. The quantification limit of the method was found to be 0.6% of PIO in PIO HCl. Precision at limit of quantification was performed by adding PIO to PIO HCl and relative standard deviation was found to be 0.02% and

17 Chapter 2 38 the recovery was found to be between 98 and 101%. The results are presented in the Table 2.6 below. Table 2.6 Results of precision at LOQ Precision at LOQ Sample No. H4 in Hz Average SD %RSD Specificity Specificity refers to the ability to assess unambiguously the analyte of interest in presence of other components. The analysis was performed for PIO and PIO HCl individually. The chemical shifts for H3, H4 and H6 in PIO are 7.926, and ppm respectively. On the other hand for PIO HCl the chemical shifts for H3, H4 and H6 are 7.255, and ppm. From the data it is evident that the method is specific to the protons of the pyridine moiety The magnetic field effect With the higher-field spectrometers, we get spectra with better resolution and leading to accurate measurements. A 400MHz

18 Chapter 2 39 instrument would provide a 0.6 Hz resolution in frequency corresponding to a displacement of ppm. Separate calibration lines were constructed utilizing a 200, 400 and 500 MHz instruments using the same calibrators in order to evaluate the effect of the magnetic field. The values are shown in Table 2.7. The slope was found (H4) , and on 200, 400 and 500 MHz instruments respectively. The graphs are shown in Figure This slope measurement is one of the criteria in the evaluation of the sensitivity of the method. These values show that the sensitivity of the method on 500 MHz instrument is 150% more than the 200 MHz and 25% more than 400 MHz instrument. Table 2.7 Magnetic Field effect % of PIO H4 in Hz 500MHz 400MHz 200MHz

19 Chapter 2 40 Figure 2.10 Magnetic field effect for H4 at 500, 400 and 200 MHz The temperature effect The temperature fluctuations during the analysis can affect the results. The NMR data was collected at 298, 313 and 333 K at two different concentrations 87.14% and 20.66% of base to evaluate the effect of temperature on the chemical shifts of the signals. Results are summarized in Table 2.8. A maximum shift of 0.02 ppm was observed when the temperature was changed by 35 C. This observation shows that the temperature has only minimal affect on the analysis.

20 Chapter 2 41 Table 2.8 Temperature (K) Results - Temperature effect 87.14% of Base 20.66% of Base H6 H4 H3 H6 H4 H Figure 2.11 Chemical shifts of H3, H4 and H6 as a function of temperature.

21 Chapter Dexlansoprazole and its Sodium salt The quantification of Dexlansoprazole Sodium Salt (DEX Na) in the presence of Dexlansoprazole Acid (DEX) has been presented Detector s response function and linearity To develop a quantitative NMR methodology, the chemical shifts of H4, H7 and H11a were considered as the detectors response (Scheme 2 and Figure 2.2). The range of chemical shifts displacement (in ppm) for H4 & H7, H11a are and ppm respectively (Table 2.9). The responses from H4 and H11a were selected to derive the detector response function. A plot of chemical shift values of these signals as a function of the percentage of DEX yielded a linear graph (Figures 2.12 and 2.13) as observed in the study of Pioglitazone presented in the previous section Similar changes were observed for the carbon ( 13 C) chemical shifts of C4 and C11 signals as seen for the corresponding protons. The 13 C chemical shift data (500 MHz) as determined by HSQC experiment are given in Table The HSQC spectra are shown in Figures 2.14, 2.15 and The relevant linear graphs are displayed in Figures 2.17 and 2.18.

22 Chapter 2 43 Table 2.9 Chemical shifts of H11a and H4 (500MHz) % of DEX Na H11a H4 ppm Hz ppm Hz Figure 2.12 Linearity graph of H4 and H11a in ppm.

23 Chapter 2 44 Figure 2.13 Linearity graph of H4 and H11a in Hz. Figure 2.14 HSQC spectrum of DEX Na

24 Chapter 2 45 Figure 2.15 HSQC spectrum of DEX and DEX Na (50:50) Figure 2.16 HSQC spectrum of DEX Table 2.10 Chemical shifts of C4 and C11 in ppm C4 C11 % of DEX ppm Hz ppm Hz

25 Chapter 2 46 Figure 2.17 Linearity graph of C4 and C11 in ppm Figure 2.18 Linearity graph of C4 and C11 in HZ

26 Chapter Assessment of the method accuracy The assay of samples with known amount of DEX at three concentration levels and three replicates at each concentration levels helped to evaluate the accuracy of the method. The results are summarized in Table 2.11 shown below. The recovery was found between % demonstrate that the method is accurate for the quantification. Table 2.11 Known % of DEX Accuracy results with chemical shift data of H4 on 400 MHz Determined % of DEX % recovery Relative error (%)

27 Chapter Assessment of the method sensitivity The sensitivity of the method has been assessed as explained in the section Table 2.12 Response function Instrument Proton Response function R 2 xmin b 500 MHz 11Ha δ = x H4 δ = x MHz 11Ha δ = x H4 δ = x Measurement of precision The repeatability of the proposed method was assessed by performing measurements with six replicate preparations at 10%, 50% and 90% of DEX to DEX Na and the relative standard deviation (%) of 0.029, and respectively. The results are shown is Table 2.13, which indicates that the method is found to be precise.

28 Chapter 2 49 Table 2.13 Precision Results Precision Sample No. H11a in Hz at 90% at 50% at 10% Average SD % RSD Specificity The analysis was performed for DEX and DEX Na individually. From the data it is evident that the method is specific to the protons at H4 and H11a The magnetic field effect As discussed in Section 2.4.7, higher-field spectrometers will give better resolution and leading to accurate measurements. Separate calibration lines were constructed utilizing a 400 and 500 MHz.

29 Chapter 2 50 Table 2.15 % of DEX Magnetic field effect H11a 500MHz 400MHz 500MHz 400MHz ppm Hz ppm Hz ppm Hz ppm Hz H4

30 Chapter 2 51 Figure 2.19 Magnetic field effect for H4 at 500 and 400 MHz in Hz The temperature effect The temperature fluctuations during the analysis can affect the results. The NMR data was collected at 298, 313 and 333 K at DEX, DEX Na and DEX : DEX Na (50:50). Results are summarized in Table A maximum shift of 0.02 ppm was observed when the temperature was changed by 35 C. This observation shows that the temperature has only minimal affect on the analysis. Table 2.15 Temp (in C) Temperature effect DEX and DEX Na (50:50) DEX DEX Na

31 Chapter 2 52 Figure 2.20 Chemical shifts of H4 and H11a as a function of temperature. 2.6 Conclusion The NMR method developed for quantification of salt in presence of its corresponding acid/base has been demonstrated taking

32 Chapter 2 53 examples of PIO/PIO HCl and DEX/DEX Na as pairs representing acidic and basic slat respectively. The method has been evaluated in terms of parameters such as accuracy, precision and robustness. The accuracy achieved with a 500 MHz and 400 MHz instruments was 2.5 and 2.0 times respectively with respect to the accuracy on the 200 MHz instrument.

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