Quantification of Losartan Potassium Polymorphs Using Powder X-Ray Diffraction (2024)

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Abstract

The frequent occurrence of polymorphism of pharmaceutical solids has been known for a long time. Since pharmaceuticals, at some stage during the manufacturing process, are organic crystalline materials, polymorphism may affect these products during new drug development and formulation. In addition, sometimes the performance of different polymorphs of the same drug varies in bioavailability and clinical efficacy. Regulatory departments of some countries require pharmaceutical companies to deal with polymorphism of active ingredients before drug application. Therefore, the identification and specification of polymorphs have become an important part of the quality assurance process of pharmaceuticals. Equally, detection of unwanted solid forms in the developed one is required from a quality assurance point of view, and development of an accurate quantification method for analyzing polymorphic forms in pharmaceuticals has become an important aspect of drug development and manufacture (1–4).

The analytical techniques used for drug polymorphism are many, such as powder X-ray diffraction, infrared spectroscopy, Raman spectroscopy, thermal analysis, solid-state nuclear magnetic resonance, and microscopy (5–10), and each has its own characteristics. X-ray diffractometry (XRD), which is a powerful technique for characterizing pharmaceutical solids, is widely used for the identification of crystalline solid phases and offers a unique advantage in the quantitative analysis of mixtures (11). The powder X-ray diffraction analysis method, which has high accuracy and good resolution, with no damage to the sample, is simple to operate and widely used in polymorphic research.

Losartan potassium (LP) (Figure1), 2-butyl-4-chloro-1-[[2′-(1H-tetrazol-5-yl) [1,1′-diphenylbiphenyl]-4-yl] methyl]-1H-imidazole-5-methanol monopotassium salt, an antihypertensive drug (12–14), has several polymorphic forms, such as form I, form II, form III, form IV, form V, and amorphous (15–21). Form I is the one used as a medicine. Form III is a more stable anhydrous form at room temperature. Form I can be partially converted to form III at high temperature and humidity.

This study focused on three objectives:

1. preparing pure form I and III of LP by recrystallization methods;

2. characterizing the inherent nature of samples using differential scanning calorimetry (DSC), Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and powder X-ray diffraction (PXRD) to test the purity and the choice of quantification methods; and

3. developing a quantification calibration curve, which has been validated and checked for assay errors, for quantifying the amount of form III in polymorphic forms of LP using data obtained by PXRD.

Experimental

Materials

LP form I and LP form III were prepared as follows.

(a) Form I

To about 5 g of LP raw material obtained from Zhejiang Huahai Pharmaceutical Co., add 50 mL of n-propanol and shake. Heat in a water bath to reflux for 3 h. Next, distill until about 10 mL of liquid is left in the flask, and then allow to cool and stand for at least 13 h. Finally, filter to obtain the sample.

(b) Form III

To about 1 g of LP raw material obtained from Zhejiang Huahai Pharmaceutical Co., add 10 mL of acetone and 1 mL of water. Next, shake and heat to reflux in a water bath to dissolve, and then distill until about 2 mL of liquid is left in the flask. Allow to cool and evaporate to dryness.

The samples were determined by HPLC (22–24) and the determination results of chemical purity were all more than 99.5%.

All other reagents and solvents obtained from commercial suppliers were used as received.

Instrumentation

(a) DSC

A differential scanning calorimeter (DSC-Q2000; TA, USA) was used. About 3–5 mg of LP form I and form III samples were placed in an aluminum crucible, covered with aluminum sheet, and placed on the sample tray. The samples were heated from 40°C up to 300°C at a heating rate of 10°C/min under a nitrogen purge flow rate of 40 mL/min. The analysis software is TA Universal Analysis.

(b) (FT-IR)

The FT-IR spectra for each of the LP forms were obtained by averaging 32 scans performed using a Thermo Fisher Nicolet 6700 FT-IR spectrometer. About 2 mg of sample was gently ground with 200 mg of KBr and pressed into a 13 mm-diameter pellet with a hydraulic press at 900 MPa for 2 min. The spectrum for each sample was recorded over the 4000–400 cm⁻¹ spectral region at a resolution of 4 cm⁻¹.

(c) Raman spectroscopy

The Raman spectra for each of the LP forms were obtained using an Invia Rama laser Raman spectrometer (Renishaw, UK) equipped with Nd-YAG laser source at 532 nm wavelength. The spectrum for each sample was recorded over the 100–2000 cm⁻¹ spectral region at a resolution of 4 cm⁻¹. Data collection was performed by WiRE (Windows-based Raman Environment) software, version 7.2.

(d) PXRD

PXRD patterns for samples of different percentages of III/I were recorded at ambient temperature on a D8 Advance diffractometer (Bruker, Germany) that utilizes Cu Ka radiation (1.54 A°). The voltage and current were 40 kV and 40 mA, respectively. Samples were subjected to X-ray powder diffraction analysis in continuous mode with a step size of 0.02° and step time of 0.4 s over an angular range of 3–40° 2θ. Five hundred milligrams of powder mixture was loaded in a 25 mm holder made of poly methyl methacrylate (PMMA) and pressed by a clean glass slide to ensure coplanarity of the powder surface with the surface of the holder. Obtained diffractograms were analyzed with DIFFRAC plus EVA (ver. 9.0) diffraction software.

Results and discussion

Solid-State Characterization of Crystal Forms I and III

(a) Thermal analysis

The DSC curve (Figure2) of form I showed an endothermic peak at about 71°C, a subsequent endothermic peak at about 94°C, a small endothermic peak at about 239°C, and a sharp endothermic peak at about 275°C. Form III also had 4 endothermic peaks at about 81, 116, 239, and 274°C. According to the Heat of Fusion Rule, form III is more thermodynamically stable.

(b) FT-IR spectroscopy

The spectral region between 1800–400 cm⁻¹ is important in case of FT-IR for identification of forms I and III. Figure3 showed that the most characteristic peaks of form III were at 1643, 1028 cm⁻¹, and between 700–400 cm⁻¹, which differed from those of form I.

(c) Raman spectroscopy

Figure4 showed that the Raman spectra of forms I and III were practically the same, and the most characteristic peaks were at 993, 1280, 1496, and 1596 cm⁻¹. Form I and form III could not be effectively distinguished by using Raman spectroscopy.

(d) The PXRD analysis

The PXRD patterns (Figure5) for forms I and III showed several differences in their characteristic peaks that can be used to identify the two crystal forms. The characteristic peaks of LP form I corresponded well with the values reported in the WO patent 2010046804A2 (15). The PXRD pattern measured from powder sample is also in good agreement with the pattern calculated from the CIF files of LP form I. The characteristic peaks of LP form III corresponded well with the values reported in the CN patent 1612866 A (21).

Take the samples of LP forms I and III that have been placed at a temperature of 40 ± 2°C and a relative humidity of 75± 5% for 6 month and determine with PXRD. The PXRD patterns are shown in Figure6. Comparing with patterns of forms I and III in 0 months, form I has undergone crystal transformation, and the characteristic diffraction peaks of form III are generated. The crystal transformation phenomenon has not been observed in form III.

The Quantitative Analysis of Losartan Potassium forms I and III

(a) The choice of quantifiable characteristic peaks

In previous studies of the quantification of polymorphic forms of drugs by PXRD, the highest intensive peak (I/I₀=100%) was usually used to detect the amount of the different polymorphs in polymorphic mixtures (25). In this study, the highest peak (7.36° I/I₀=100%) of LP form I partially overlapped with the peak at 6.85 2θ, so the second highest peak (11.13° I/I₀=100%) was used to detect the polymorphic content of the mixtures. Form III had three major characteristic peaks (I/I₀≥60%) at 5.64°, 6.85°, 8.88° 2θ, and the highest peak (5.64° I/I₀=100%) was used for quantification (Figure7).

(b) Construction of the calibration curve

In general, the height and area of peaks are both considered for analysis. Peak shape and height vary more with changes in particle size, but the peak area tends to be less variable (26, 27). Therefore, in this study, peak area was used for the quantitative analysis. A linear relationship was demonstrated for the peak area of form III vs the weight percentage. The linear performance from different sets of parameters such as the absolute peak area of LP form III and the peak area ratio of LP form III and I [A_III/(A_III + A_I)].

The samples for construction of the calibration curve of LP form III in form I contained 1, 2, 5, 10, 20, and 50 w/w%. Before weighing, the two forms of LP were passed through a 100-mesh sieve to reduce the effect of particle size on the preferred orientation (28). The samples then were shaken with a vortex mixer for about 3 h to ensure full mixing. A qualitative crystal study of LP by PXRD showed that grinding did not induce a polymorph transformation between form III and form I. The change of peaks in intensity of these samples as a function of form III content is shown in Figure8.

The method that used the ratio of the 5.64° 2θ to the sum of peak parameters of 5.64° and 11.13° 2θ had the best performance. Figure9 showed that the calibration curve was quite linear over a wide range (1–50%, w/w) of form III, with a linear equation of y = 0.0074x-0.0069 and a high correlation coefficient (R²) of 0.9988. These results confirmed that PXRD is a very good method for quantifying LP mixtures of polymorphs (forms I and III).

(c) Validation of the analytical method

Any analytical method before being successfully utilized for quantification needs to be validated (29). The analytical method developed for quantifying the amount of LP form III in form I was validated using accuracy, precision, ruggedness, and LOQ (30). The results are provided in Table1.

The LOQ is the lowest concentration of the analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. For the instrumental method recording the noise at the baseline, the lowest concentration of test substance that is reliably determined can be calculated by comparing signal of sample at a known low concentration and signal of the blank. The concentration corresponding to the signal-to-noise ratio of 10:1 is generally accepted. The PXRD assay displayed a LOQ of 2.02% (w/w).

The curve showing the relationship between the actual and the predicted content (%, w/w) of form III in the mixture was plotted (Figure10) with an R² value of 0.9987, a fitted slope of 1.0024, and a small intercept of –0.0872.

(d) Estimation of assay error

Many potential assay errors, including instrument, inter-day, intra-day, sample position, and sample packing errors, which are associated with an PXRD quantitative analysis (25), may occur. So, all the assay errors were investigated in this work. The data from the assay error evaluation are shown in Table2.

Reproducibility of the instrument was tested by placing a single sample (5%, w/w) in the PXRD instrument and acquiring 6 data sets without removing the sample from the sample holder and instrument. Inter-day variability was determined for 6 days, a single sample (5%, w/w) was placed in the instrument, and the X-ray profile was recorded each day. Intra-day reproducibility was determined by using a single sample (5%, w/w) and acquiring 6 diffraction patterns. Effect of variation in position of sample holder within the instrument was examined by a single sample (5%, w/w) and randomly reposition the holder at 6 different positions. Variability from re-packing was investigated by a single sample (5%, w/w) and repeatedly pack the sample to the same holder 6 times.

Conclusions

In this study, a PXRD method was developed and validated for quantitative determination of LP forms I and III. Pure polymorphic forms were characterized before developing a quantification method. Careful and consistent sample preparation was needed to develop a robust calibration curve of quantitation. A 100-mesh sample particle size, step size of 0.02°, and step time of 0.4 s were selected in determining the proportions of the polymorphs in the LP mixtures. The calibration curve was found to be a linear fit across the range from 1–50% (w/w) with LOQ of 2.02%. Although the existence of the assay error, which is introduced by factors such as instrument, inter- and intra-day variation and sample packing, a systematic optimization reduced the size of errors. Therefore, this method was confirmed as an effective and practical method for the quantitative determination of LP polymorphs. Through this method, it would be possible to quantify the polymorphic mixture of LP in bulk drug samples.

Acknowledgments

This research was supported by the Major Program of Zhejiang Institute for Food and Drug Control (No : 2013–38-2).

References

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