Development of official assay method for loperamide hydrochloride capsules by HPLC

Abstract

Currently, the potentiometric titration and the high pressure liquid chromatography (HPLC) method were utilized in Korean Pharmacopoeia XII (KP XII) as well as other pharmacopoeias (USP, EP, BP) for determination of loperamide hydrochloride in raw materials and capsules, respectively. The research objective is to overcome the remaining drawbacks from current methods such as solubility of mobile phase (KP XII), less scientific approach (USP 43) or using paired-ion chromatography reagent which shows some limitations (BP2017 and other formulation monographs). The proposed method was optimized by Design of Experiment (DoE) tool to obtain the satisfied method for determination of loperamide hydrochloride. The optimal condition was performed on the common C18 column (150 mm × 4.6 mm; 5 ㎛) using isocratic elution with the mobile phase containing 40 mM of potassium phosphate monobasic (pH 3.0) and acetonitrile (56:44), at a flow rate of 0.7 mL/min. The optimized method was validated and met the requirements of the International Conference on Harmonization. The developed method was applied to determine loperamide hydrochloride in capsules and can be used to update the current monograph in KP XII.

1. Introduction

Loperamide hydrochloride (Fig. 1) is chemically named as 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1- l]-N,N-dimethyl-2,2-diphenylbutanamide; hydrochloride.¹ It is an μ-opioid receptor agonist and results in antidiarrheal action. The FDA approved loperamide to treat various form of diarrhea as the main indication² and chemotherapy-induced diarrhea (especially related to irinotecan) for the off-label uses.³

Fig. 1. Chemical structure of loperamide hydrochlorides

Currently, the method for determination of loperamide hydrochloride in raw material is potentiometric titration in Korean Pharmacopoeia (KP XII),⁴ United State Pharmacopoeia (USP 43)⁵ and European Pharmacopoeia (EP 10.0).⁶ For loperamide hydrochloride capsules, HPLC method is mentioned in KP XII, USP 43 and British Pharmacopoeia (BP 2017).⁷ However, there are still some limitations in these HPLC methods. In KP XII, the binary eluent containing high concentration of sodium phosphate dibasic at pH 7.0 (approximately 42 mM) and high percentage of methanol (70 %) leads to precipitation in precolumn step. Generally, when using 70 % of methanol, the concentration of potassium phosphate at pH 7.0 should not be more than 35 mM. In addition, the sodium salts is expected to be less soluble in comparison with others.⁸ Therefore, there is the solubility problem in the assay test in KP XII. In USP43, the method seems to be unscientific and not robust because of the unrepeatable pH when adding 20 drops of phosphoric acid to mobile phase. Besides, using cyano column with the large particle size (10 μm) and high flow rate (2.0 mL/min) also makes it not become an ideal approach. The method in BP2017 used paired-ion chromatography reagent (sodium octanesulfonate) for eluent and high flow rate (1.5 mL/min) which can cause damage and short column life. Furthermore, the monographs of relatives have some imperfections such as using paired-ion chromatography reagent with high flow rate (loperamide hydrochloride tablets in USP43), using C8 column with high capacity in result (loperamide hydrochloride oral solution in USP 43) and using the complex mobile phase with tetrahydrofuran, acetonitrile, ammonium dihydrogen phosphate and paired-ion chromatography reagent (decanesulfonic acid) (loperamide hydrochloride oral solution in BP 2017). Therefore, developing a simple and robust method for determination of loperamide hydrochloride in capsules to replace the conventional methods is necessary.

Traditionally, the development and optimization for analytical methods have been conducted by one factor at time (OFAT) approach which changes one of factors and constants the remainders. Its major disadvantages are excluding the interaction between factors and increasing the number of experiments which will take time, effort and resources. Design of experiment (DoE) tool was developed to overcome these problems. In this methodology, multiple factors are systematically varied and the results are used to create mathematical models. From these models, it is possible to predict the interaction between factor and find optimal condition.^9,10 Nowadays, application of design of experiment concept in analytical development becomes more popular with several types of concept^11,12 because of its efficiency and variety in design.

In this research, we aimed to seek the optimal condition for alternative HPLC method. The DoE concept was applied to optimize each factors and minimize the number of experiments. The final condition was also validated according to ICH guideline¹³ for the reliable and accurate method for quantitating loperamide hydrochloride in commercial capsules.

2. Experimental

2.1. Chemicals and reagents

Loperamide hydrochloride standard was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The capsule formulations were obtained from Youngilpharm and GLPharma. The HPLC-grade acetonitrile, methanol and ethanol were purchased from J.T.Baker (Avantor Inc. – Gyeonggi, Korea), Honeywell Burdick & Jackson (B&J – Ulsan, Korea), Daejung (Siheung, Korea), respectively. Potassium phosphate monobasic was supplied by Kanto Chemical Co. Inc. (Tokyo, Japan).

2.2. Chromatographic conditions

The proposed method was developed by Shimadzu HPLC system from Shimadzu Corporation (Kyoto, Japan) including a DGU-20A5R degasser, LC-20AD pumps, SIL-20A autosampler, CBM-20A communication bus module, CTO-20AC column oven and SPD-M20A 230V photodiode array detector. For the intermediate precision, Agilent 1100 series HPLC system was utilized. Purified water was newly prepared in laboratory. Other chemicals and reagents for preliminary experiments were analytical grade.

The column Luna C18(2) (150 × 4.6 mm ID, 5 µm) connected with Phenomenex C18 guard column (3 × 4 mm ID) was used throughout the development process. For comparison with the conventional method from other pharmacopoeias, the column INNO CN (250 × 4.0 mm ID, 5 µm) and Aegispak C8 (150 × 4.6 mm ID, 5 µm) were employed.

For optimal condition, the mobile phase contained 40 mM of potassium phosphate monobasic (pH 3.0) and acetonitrile (56:44). The temperature was 35 ℃. The flow rate was 0.7 mL/min. Injection volume was 10 μL. Loperamide hydrochloride was detected at 214 nm.

2.3. Sample Preparation

Stock standard solution of loperamide hydrochloride (1 mg/mL) was prepared in 70 % methanol. Final standard solution (10 µg/mL) was diluted by the same diluent.

Sample solution: Transfer the contents of 20 capsules as completely as possible and accurately weigh the amount of powder, equivalent to about 2 mg of loperamide hydrochloride. That required amount powder was transferred to a 20 mL volumetric flask, added with methanol 70 % (about 50 % of the flask capacity) and sonicated for 15 minutes. This solution was diluted to volume with methanol 70 %, mixed and filtered. Transfer 5 mL of this solution to a 50 mL volumetric flask, dilute with methanol 70 % and mix. After dilution, the final solution containing 10 µg/mL of loperamide hydrochloride was used as a sample solution.

2.4. Method development

The preliminary experiments were conducted to initially select a suitable mobile phase composition such as type of organic solvent (acetonitrile, ethanol, and methanol), buffer (type and pH) or mobile phase additives (phosphoric acid, acetic acid, formic acid, and trifluoroacetic acid). According to the design from DoE software, the HPLC condition was optimized easily to obtain the satisfied method.

2.5. Method validation

The final method was validated to meet the requirements of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The validation procedure included:

Specificity: The blank, loperamide hydrochloride standard solution (10 ppm), loperamide hydrochloride in capsules – sample solution (10 ppm) were injected to assess the analyte in presence of other components (mobile phase, excipients, impurities…)

System suitability: Standard solution was injected repetitively to check the stability of the system under the optimal condition.

Linearity and limit of detection (LOD)/limit of quantification (LOQ): The range of standard concentrations from 1 to 30 ppm were selected for linearity test. The LOD and LOQ were based on the signal-to-noise ratio calculated from the chromatograms of the diluted standard solution.

Precision: The repeatability (intra-day and interday) and intermediate precision (different HPLC system) were conducted for precision.

Accuracy: The spiked solutions containing standard (80 %, 100 %, 120 %) and sample from formulation were used for measurement.

Robustness: The robustness of this method was assessed by the small variations in percentage of acetonitrile (± 2 %), buffer concentration (± 2 mM), pH of buffer (± 0.2), flow rate (± 0.1 mL/min) and temperature (± 2 ℃).

2.6. Method application

The proposed method was applied to determine loperamide hydrochloride in capsules from Youngilpharm and GLPharma. The content of loperamide hydrochloride in capsule was calculated according to the following equation:

Loperamide hydrochloride (C₂₉H₃₃ClN₂O₂.HCl) (mg) = m × (A_T/A_S)

Where:

m is the amount of loperamide hydrochloride weighed for standard solution,

A_T is the peak area in the sample solution (μAU*s), A_S is the peak area in the standard solution (μAU*s).

3. Results and Discussion

3.1. Method development

Initially, the method was developed using the simple acidic mobile phase. The acidic additives (phosphoric acid, acetic acid, formic acid, and trifluoroacetic acid) and the organic solvents (acetonitrile, methanol, and ethanol) were surveyed. The best result of this approach was obtained by the mobile phase containing methanol/0.05 % trifluoroacetic acid (65/35). Besides that approach, another method using the mobile phase containing buffer at acidic pH was also developed. In comparison with the initial condition, the mobile phase containing the buffer gave the better result in peak shape as well as column efficiency and the robustness of procedure. Then, several types of buffer with the suitable pH were also checked to find out the most appropriate buffer (Table 1). The comparative chromatograms showed that the combination of potassium phosphate monobasic and acetonitrile were the suitable combination for optimization (Fig. 2).

Table 1. Buffer types used for preliminary experiments

Fig. 2. Comparative chromatogram of buffer type survey

Next step, the screening and optimization concept were designed by DoE. In screening, two-level full factorial design was selected with five factors (buffer pH, buffer concentration, acetonitrile ratio, temperature and flow rate). Four responses were considered including capacity (k’), tailing factor (Tf), number of theoretical plate (NTP) and pressure. The detail screening design and results was shown in the Table 2. The analysis indicated that the high temperature positively affected on both four responses. Therefore, the temperature was set at 35 ℃ and excluded for optimization step. In responses, the highest pressure was 1705 psi which is acceptable so the pressure was also unimportant response and can be excluded.

Table 2. Screening design and results

For optimization, the Box-Behnken design was created with 30 runs divided into 3 blocks. After screening, the range of each factor was adjusted to be smaller and suitable. The domain included buffer pH (2.0-3.0), buffer concentration (30-50 mM), acetonitrile ratio (35-55 %) and flow rate (0.6-1.0 mL/ min). From Box-Cox transformation, the capacity and tailing factor responses were transformed to the natural log and inverse square root (k = -1) transformations, respectively. Statistical parameters from ANOVA of both 3 responses demonstrated that the results met the requirements (Table 3). Statistical data showed p-value < 0.05 which confirmed model significance. The difference between predicted and adjusted R² was in reasonable agreement (within 0.2). The adequate precision in ANOVA was sufficiently high (>4) so the model can be used to navigate the design space. From model graphs, the effect of each factor and interactions between the chosen factors on each response can be demonstrated. After analyzing the result, capacity and tailing factor were mainly influenced by percentage of ACN in the mobile phase. To obtain the suitable range of capacity (k’= 2-5), percentage of ACN should not be lower than 40 %. For peak shape, the lower ACN proportion showed the better result in tailing factor. The 3D plots in Fig. 3. showed the interactions between the chosen factors affecting on the number of theoretical plate. The higher number of theoretical plate can be achieved when the low ACN percentage combined with low flow rate, high buffer pH and middle buffer concentration. The objectives of this optimization are minimizing the capacity and maximizing the tailing factor as well as the number of theoretical plate. After setting these goals in the software, in 100 found solutions, the most suitable solution was chosen (buffer pH 3.0; buffer concentration 40 mM; 44 % ACN in mobile phase and flow rate 0.7 mL/min).

Table 3. ANOVA statistical parameters from optimization results

(*) The difference between predicted and adjusted R²

Fig. 3. Interactions between the chosen factors affecting on the number of theoretical plate.

Finally, the recommended condition was applied for a confirmation test (n = 6). The results were reached the goals with satisfied parameters of capacity (k’= 3.465), tailing factor (T_f= 1.147) and number of theoretical plate (NTP = 11537).

3.2. Validation

3.2.1. Specificity and system suitability

As illustrated in Fig. 4, peak of loperamide was eluted with reasonable capacity and good peak shape. Through the PDA detector, the presence of impurities in the main peak can be easily determined by purity index and similarity curve including similarity (SI) and threshold (t). In Fig. 5, the similarity index (SI) is not lower than the threshold index (t) at each sampling point. In addition, the minimum purity index which is the threshold subtracted from the similarity, is positive value for loperamide peak of standard and sample solution. It proved that no contaminated content is detected at position of loperamide peak in both standard and sample chromatogram.

Fig. 4. Typical chromatograms of blank (a), standard solution (b) and sample solution (c). Condition: Column Luna C18(2) (150 × 4.6 mm I.D., 5 μm), mobile phase containing 40 mM potassium phosphate monobasic buffer pH 3.0 and acetonitrile (56:44, v/v), flow rate 0.7 mL/min, injection volume 10μL, detection at 214 nm.

Fig. 5. Similarity curve and purity index of standard solution (a) and sample solution (b).

The system suitability of the proposed method was assessed based on relative standard deviation (RSD) of the retention time, peak area and the value of tailing factor as well as number of theoretical plates (Table 4).

Table 4. System suitability data (n = 6)

NTP: Number of Theoretical Plate

3.2.2. Linearity and LOD/LOQ

The sensitivity of the proposed method was determined by calculating the ratio of signal-to-noise. The LOD and LOQ were 0.02 µg/mL and 0.06 µg/ mL, respectively [n = 6]. The linearity was evaluated with the range from 1 to 30 µg/mL. The proposed method showed good linearity along this range with a correlation coefficient (R² ) > 0.9996 for both 6 sets. Statistical parameters of ANOVA (p = 0.05) showed the regression linearity of the method (Table 5).

Table 5. Linearity and sensitivity results

SD: Standard deviation

3.2.3. Precision and accuracy

The repeatability of method was confirmed by the RSD% of both intraday and interday precision (RSD% <1.05 % and < 1.06 %, respectively). For intermediate precision, the method was validated using another system (AGILENT 1100 series) with RSD% of peak area in 6 injections was 0.13 %.

The recovery of loperamide in each spiked samples in accuracy test was in the range of 98.77-101.28 % and the RSD% for each concentration was less than 1.26 %.

The obtained results of precision and accuracy test are shown in detail (Table 6).

Table 6. Results of repeatability precision and accuracy in validation

3.2.4. Robustness

The robustness of the proposed method was proven with small deliberate variations in the ratio of acetonitrile (± 2 %), buffer concentration (± 2 mM), pH of buffer (± 0.2), flow rate (± 0.1 mL/min) and temperature (± 2 ℃). The results of method were not influenced (except changes in retention time). In both cases, RSD% of retention time and peak area (n = 6) were not more than 0.47 % and 0.82 %, respectively. Peak shape and column efficiency were also confirmed with tailing factor < 1.15 and number of tailing factor > 11000.

3.3. Application

The proposed method was successfully applied on commercial products. The amount of loperamide hydrochloride present in 2 different capsules A and B (n = 6) was 99.49 % (RSD = 1.46 %) and 99.83 % (RSD = 1.83 %), respectively (Table 7).

Table 7. Content of Loperamide hydrochloride in capsules for application (n = 6)

4. Conclusions

The proposed HPLC method was considerably improved to effectively determine loperamide hydrochloride in capsules in comparison with conventional methods of other pharmacopoeias (Table 8). The developed method used the most popular column (C18) for convenient application and avoided using paired-ion chromatography reagent which can cause column consumption. Moreover, under DoE concept, HPLC conditions were optimized to obtain the best results in peak shape, column efficiency in reasonable running time with lowest pressure for long column life. The optimal method was successfully validated and obviously proved to be accurate, robust and sensitive. Therefore, this method is appropriate to frequently utilize in quality control and well worth replacing the current method in Korean Pharmacopoeia XII.

Table 8. Comparison with conventional methods from other pharmacopoeias for determination of Loperamide HCl formulation

NTP: Number of Theoretical Plate