Establishment of inherent stability on piracetam by UPLC/HPLC and development of a validated stability-indicating method

2.1 Chemicals

Piracetam was obtained as a gratis sample from jubilant organosys (Noida, India). Analytical reagent (AR) Sodium hydroxide and hydrogen peroxide were purchased from S.D. Fine-chem. Hydrochloric acid and acetonitrile (99.8%) was from Merck India (Mumbai). All other chemicals were of analytical grade.

2.2 Instrumentation

2.3 Degradation studies

Stress studies were performed under conditions of dry heat (thermal studies), hydrolysis (acidic, alkaline and neutral), oxidation, and photolysis, as mentioned in ICH Q1A (R2) (1–4). The approach suggested by Singh and Bakshi was adopted for these studies. A minimum of four samples were generated for every stress condition, viz., blank solution stored under normal conditions, the blank subjected to stress in the same manner as this drug (Piracetam), a zero time sample containing this drug (which was stored under normal conditions), and this drug solution subjected to stress treatment. Hydrolytic decomposition of Piracetam was conducted at 80 °C in 5 M HCl, water, and 0.5 M NaOH at a drug concentration of 2 mg mL⁻¹ until sufficient degradation (∼20% of the initial amount) of this drug was achieved. For oxidative stress studies, Piracetam was dissolved at a concentration of 3 mg mL⁻¹ in 30% H₂O₂ and kept for two days at room temperature. Photolytic studies of the dry drug and this drug in solution in acetonitrile at a concentration of 2 mg mL⁻¹ were performed by exposure to sunlight during the daytime (60,000–70,000 lux) for 2 d.

2.4 Chromatography and development of a stability-indicating method

UPLC was performed with a binary solvent delivery pump, an auto sampler and a PDA detector of Acquity UPLC system manufactured by Waters Corporation; Milford, Massachusetts, USA; data were acquired and processed using Empower software. An initial literature search revealed that some reported HPLC methods for Piracetam were developed on either C8 or C18 columns, using different temperature conditions. Peak shapes were not persuasive, and there was substantial tailing. So, attempts were made to develop a simple method on an advanced BEH C18 column, with possible lowering of retention time at 30 °C column temperature. Separations were achieved using isocratic elution. The mobile phase was filtered through 0.22 μm PTFE membranes and had degassed. The injection volume was 2 μL and the mobile phase flow rate was kept constant at 0.15 mL min⁻¹. The detection wavelength was 210 nm; PDA analysis was conducted to study the behavior at other wavelengths. First, UPLC studies were performed on all reaction solutions individually and then on a mixture of degraded drug solutions. Different conditions, for example pH, mobile phase composition, and column temperature were varied to obtain a reasonable separation between this drug and the degradation products. Methanol was avoided during the study because of its significant absorption at the detection wavelength of this drug between 210 and 215 nm. Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm × 150 mm) stainless steel analytical column was used as stationary phase. In order to determine the method is stability indicating, forced degradation studies were conducted on Piracetam powder. The analysis was carried out by UPLC with a PDA detector at a wavelength of 210. Two microliters of each of forced degradation samples was injected at regular intervals.

HPLC chromatographic analysis was performed at ambient temperature on a Phenomenex (C-18) analytical column with a mobile phase composed of buffer (10 mM Amm. Acetate buffer pH 5.0): acetonitrile (80:20, v/v) and was isocratically eluted at a flow rate of 0.9 mL min⁻¹. A small sample volume of 20 μL was used for each sample run, being injected into the HPLC system. The chromatogram was monitored with UV detection at a wavelength of 210 nm.

2.5 Validation of the method

The method was validated in accordance with ICH guidelines Q2 (R1). To establish linearity and range, a stock solution containing 1 mg mL⁻¹ drug in acetonitrile was diluted to yield solutions in the concentration range of 10–50 μg mL⁻¹. The solutions were injected in triplicate using water–acetonitrile as mobile phase and keeping the injection volume constant (2 μL). To assess precision, six injections of five different concentrations (10, 20, 30, 40, and 50 μg mL⁻¹) were made on the same day and intraday precision was determined as relative standard deviation. These studies were also repeated on different days to determine interday precision. Accuracy was evaluated by fortifying a mixture of decomposed reaction solutions with five known concentrations of this drug and recovery of the added drug was evaluated. The specificity of the method for this drug was established by a study of the resolution factor of this drug peak from the nearest other peak. Overall selectivity was established by the determination of purity for each degradation product peak by the use of PDA detector. Robustness was assessed by changing the composition of mobile phase. Method detection limit (MDL) and method quantification limit (MQL) were determined experimentally, by an analysis of samples spiked with decreasing concentrations of the analytes. MDL was defined as the smallest amount of an analyte that can be reliably detected or differentiated from the background for a particular matrix (by a specific method). MQL was calculated as the smallest amount of an analyte that can be reliably quantified with a certain degree of reliability within a particular matrix (by a specific method). In chromatography methods, the limits are often set based on the ratio between the analyte signal and the baseline noise (for example, MDL = Height/Noise ratio of 3, MQL = Height/Noise ratio of 10).

3.1 UPLC and HPLC studies on the stressed solutions

The forced-degradation study shows that Piracetam degraded under alkali stress condition. The specificity and selectivity of the method with the samples under these stresses were demonstrated through the evaluation of R_T, RR_T, resolution, and purity data for all peaks in the chromatograms (http://www.chem.agilent.com/Library/posters/Public/ASMS_2011_MP_181.pdf; Singh and Bakshi, 2002; Singh et al., 2006). Piracetam did not degrade under acid, oxidative, thermal & photolytic stress conditions. In a mixture of solution, only one degradation product was formed. The retention times (R_T) and relative retention times (RR_T) of this drug and the degradation products are given in Table 1 for UPLC and HPLC. This drug and degradation products carry the notations Deg01 and Piracetam in accordance with the sequence in which the peaks appeared from left to right on UPLC and HPLC chromatogram (mixture of stressed sample) (Fig. 2). Ultra-performance liquid chromatography (UPLC) is a new category of separation technique based upon well-established principles of liquid chromatography, which utilizes sub-1.7 μ particles for a stationary phase. These particles operate at elevated mobile phase linear velocities to affect a dramatic increase in resolution, sensitivity and speed of analysis.

3.2 Development and optimization of the method

3.2.1

3.2.1 UPLC

The best separation was achieved on the same column at 30 °C using the mobile phase acetonitrile: water (70:30) in an isocratic mode. The flow rate was kept at 0.15 mL min⁻¹ at a constant volume of 2 μL, and the detection wavelength was 210 nm. UPLC studies on Piracetam under different stress conditions suggested the following degradation behaviors:

Piracetam gradually degraded with time on heating at 80 °C in 0.5 M NaOH after 10 h. The drug showed ∼25% degradation in 0.5 M NaOH at 80 °C. A new peak emerged at Rt 2.05 min along with the drug peak at Rt 3.56 (RR_T 1.00). In a mixture of the stressed samples (Fig. 2(a)), the degradation product at RR_T 0.576 (with the normal drug peak at RR_T 1.0) pertained to Deg01. The drug was stable to acidic stress, and it was observed that the drug was not degraded by heating at 80 °C in 5 M HCl for 24 h. the drug shows stable behavior at neutral hydrolysis on heating with water at 80 °C for two days. No degradation was observed on exposure of the drug to 30% H₂O₂ for five days, showing that it was stable against oxidative stress. The drug was also stable during exposure to direct sunlight (∼60,000–70,000 lux) for two days. Finally, there was no significant degradation of solid piracetam on exposure to dry heat at 50 °C for two months, indicating that the drug was stable to thermal stress.

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Figure 2

Chromatogram showing separation of Piracetam and its degradation products in a mixture of stressed samples by (a) UPLC and (b) HPLC.

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3.3 Validation of developed stability-indicating method

The response for this drug was strictly linear in the concentration range between 10 and 50 μg mL⁻¹. The linearity study data are given in Table 2.

Regression equation $$\begin{array}{cc}\hfill & Y=5129X+2648(\text{UPLC}{r}^2=0.999\text{,intercept}=2648)\hfill \\ \hfill & Y=17958X+37368(\text{HPLC}{r}^2=0.999\text{,intercept}=37368)\hfill \\ \hfill & Y=\text{AUC,}X=\text{conc}.\text{in}\mathrm{\mu }\text{g}{\text{mL}}^{-1}\hfill \end{array}$$

The data obtained from precision experiments are given in Table 3 for intra and inter day precision studies. The %RSD. values for intraday precision study and for interday study were <1.0% confirming that the method was sufficiently precise. Excellent recoveries were made at each added concentration shown in Table 4. Fig. 2(a) and (b) shows that the method was sufficiently specific to this drug. The USP resolution factor for this drug peak was >2 from the nearest resolving peak. The method was found to be robust by varying the composition of mobile phase as shown in Table 5. Good separations were always achieved, indicating that the method remained selective for all components under the tested conditions as shown in Fig. 2(a) and (b). The influence of retention time for different degradation products (UPLC/HPLC) has been depicted in Fig. 2.

The MDL and MQL were found to be 0.180 μg mL⁻¹and 1.10 μg mL⁻¹ for UPLC and 0.500 μg mL⁻¹ and 1.700 μg mL⁻¹respectively for HPLC.

3.4 Comparative study on chromatographic performance

A comparative data on the chromatographic performance of HPLC (isocratic) and UPLC (isocratic) have been obtained by injecting a mixture of stressed solution of Piracetam (30 μg mL⁻¹). It is observed that the elution time of pure Piracetam and degradation products in UPLC was reduced by 6-fold compared to that of isocratic mode HPLC. The resolution and theoretical plates obtained for Piracetam and degradation products in UPLC showed comparatively a better separation efficiency than HPLC.