Adaptable Organic Impurities Method for the Analysis of Diphenhydramine Hydrochloride Oral Solution

To start the standard development process and have earlier stakeholder engagement, USP is using a new approach for developing and sharing information with our stakeholders. Through a collaboration between USP’s Small Molecules Department and Global Analytical Development Laboratory, methods are being developed and validated for drug substances and drug products for which no monographs are currently available or a new approach for analysis is introduced. The Emerging Standards are intended to improve USP’s official standards elaboration process by increasing transparency and allowing for broader stakeholder participation by publishing on the USP website prior to formal notice and comment through publication in the Pharmacopeial Forum.

The method presented in this report is one of the three methods described in the white paper, Emerging Standards and Introduction of Adaptable Organic Impurities Procedures for Over-the-Counter Drug Products. The introduction of method operable design region (MODR) space, instead of single method parameters, allows for greater flexibility for end users. This means that users may find suitable conditions within the MODR space for testing new or marketed products. This flexibility is crucial, especially for OTC drug products, where varying formulations and compositions make a single compendial procedure impractical.

Certain commercial software, instruments, or materials may be identified in this document to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the software, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate.

This document is not a USP compendial standard and is intended to serve as a resource for informational purposes only. It does not reflect USP or USP’s Expert Body opinions of future revisions to the official text of the USP-NF. Parties relying on the information in this document bear independent responsibility for awareness of and compliance with any applicable federal, state, or local laws and requirements.

Diphenhydramine is in a class of medications called antihistamines. It works by blocking the action of histamine, a substance in the body that causes allergic symptoms. Diphenhydramine is used to relieve red, irritated, itchy, watery eyes; sneezing; and runny nose caused by hay fever, allergies, or the common cold. It is also used to relieve cough caused by minor throat or airway irritation. Additionally, diphenhydramine is used to prevent and treat motion sickness, and to treat insomnia (difficulty falling asleep or staying asleep). It may also be used to control abnormal movements in people who have early-stage Parkinsonian syndrome or who are experiencing movement problems as a side effect of a medication. Diphenhydramine is an active ingredient in various dosage forms such as tablets, capsules, and oral solutions.

The USP-NF contains monographs for Diphenhydramine Hydrochloride and Diphenhydramine Citrate drug substances^2,3, and several monographs for drug products including Diphenhydramine Hydrochloride Capsules⁴, Diphenhydramine Hydrochloride Injection⁵, Diphenhydramine Hydrochloride Oral Powder⁶, Diphenhydramine Hydrochloride Oral Solution⁷, Diphenhydramine Hydrochloride and Ibuprofen Capsules⁸, Diphenhydramine and Phenylephrine Hydrochlorides Tablets⁹, Diphenhydramine and Pseudoephedrine Capsules¹⁰, Diphenhydramine Citrate and Ibuprofen Tablets¹¹, and Acetaminophen, Diphenhydramine Hydrochloride, and Pseudoephedrine Hydrochloride Tablets¹². Including an organic impurity procedure for over-the-counter drug product monographs present a significant challenge as it would need to cover a large number of marketed products with various active ingredients and formulation compositions. Diphenhydramine Hydrochloride Oral Solution was selected in a pilot study for the development of adaptable organic impurities method within a design space.

USP General Chapter <1220> Analytical Procedure Life Cycle¹³ provides a framework for the implementation of the analytical procedure life cycle approach that is consistent with the quality by design (QbD) concepts described in the ICH guidelines. Stage 1 of the life cycle includes systematic procedure development experiments that result in an understanding of the effect of procedure parameters on the procedure performance. Design of Experiment (DOE), which includes statistical multi-variate analysis and modeling is an important tool in Stage 1. The knowledge acquired through DOE studies also enables the determination of robust operation regions for procedure parameters and, if desired, a method operable design region (MODR).

This document summarizes method development efforts assisted by the Fusion QbD® software including in silico robustness study followed by verification. It also describes a final procedure that may be suitable for the quantitation of four degradation products, diphenhydramine N-oxide, diphenhydramine related compound (RC) B, benzhydrol, and benzophenone in the presence of various impurities and excipients in Diphenhydramine Hydrochloride Oral Solution. A summary of validation data and representative chromatographic results are included.

3.1. Diphenhydramine Hydrochloride and Impurities Standards

USP Reference Standards for diphenhydramine hydrochloride, diphenhydramine N-oxide, diphenhydramine related compound (RC) A, diphenhydramine RC B, benzhydrol, benzophenone, bromodiphenhydramine hydrochloride, orphenadrine RC F (4-methyldiphenhydramine), and sodium benzoate were used. The chemical structures of diphenhydramine hydrochloride, related impurities, and common excipients found in diphenhydramine hydrochloride oral solutions are shown in Figures 1–3.

Figure 1. Diphenhydramine Hydrochloride
 

Figure 2a. Diphenhydramine N-oxide (hydrochloride salt)
(N-oxide)
 

Figure 2b. Diphenhydramine Related Compound A (hydrochloride salt)
(RC A)
 

Figure 2c. Diphenhydramine Related Compound B
(RC B)
 

Figure 2d. Benzhydrol
(BZH)
 

Figure 2e. Benzophenone
(BPH)
 

Figure 2f. Bromodiphenhydramine (hydrochloride salt)
(Br-DPH)
 

Figure 2g. 4-Methyldiphenhydramine (citrate salt)
(Methyl-DPH) (Orphenadrine Related Compound F)
 

Figure 2. Impurities

Figure 3. Sodium Benzoate

3.2. Samples

Six commercial Diphenhydramine Hydrochloride Oral Solutions and two mock (faux) formulations prepared for this study with help from the USP OTC Diphenhydramine Subcommittee were used to evaluate the methods described in this document.

3.3. Reagents

Trifluoroacetic acid (TFA) (LC/MS grade), acetonitrile (LC/MS grade), methanol (LC/MS grade), and isopropanol (Optima grade) were obtained from Fisher Chemical. Ultrapure water was obtained from a Milli-Q water purification system.

The goal was to develop an adaptable method for Organic Impurities (OI) targeting known degradation products of diphenhydramine present in various Oral Solution formulations. Three high-performance liquid chromatography (HPLC) procedures based on different column chemistries and mobile phase composition were proposed by the USP OTC Diphenhydramine Subcommittee as a starting point for further evaluation and optimization. Optimization of the method that is based on pentafluorophenyl (PFP) (L43) column chemistry and a mobile phase consisting of 0.1% TFA in water (Solution A), and acetonitrile (Solution B) delivered in a gradient mode is presented here. Fusion QbD^® software was utilized for the DOE in an optimization study to explore the MODR and to select adaptable chromatographic conditions, i.e., starting conditions and allowable changes.

4.1. LC Conditions Optimization Study

A solution used in the preliminary study included the following compounds: diphenhydramine hydrochloride, known process impurities and degradation products, i.e., diphenhydramine related compound A, diphenhydramine related compound B, diphenhydramine N-oxide, benzhydrol, benzophenone, methyldiphenhydramine, bromodiphenhydramine, diphenhydramine quaternary salt, and excipients found in different oral solution formulations, i.e., sodium benzoate, acesulfame potassium, saccharin, D&C Red No. 33, FD&C Red No. 40, and FD&C Blue No. 1. The study showed that all excipients except sodium benzoate elute at the beginning of the run and do not interfere with any analyte of interest. These excipients were excluded from the optimization study. An optimization study was performed to understand the effect of different method variables on peak resolution. HPLC conditions that were maintained constant included: aqueous mobile phase (Solution A: 0.1% TFA in water), detection wavelength (220 nm), injection volume (15 µL), and column brand/type (Waters XSelect HSS PFP). The initial ratio of Solution A and Solution B in the mobile phase was set at 70:30 as in the starting point method.

The effect of five method variables, initial hold time, gradient time, flow rate, column temperature, and acetonitrile: methanol ratio in Solution B, on a resolution between all peaks of interest was studied (Table 1). The software generated 32 runs for the design, with randomization and replicates considered. The injected solution contained 0.25 mg/mL of diphenhydramine hydrochloride, 1.875 µg/mL (0.75%) of diphenhydramine RC A, 0.75 µg/mL (0.3%) of diphenhydramine RC B, 11.25 µg/mL (4.5%) of diphenhydramine N-oxide, 7.5 µg/mL (3.0%) of benzophenone, 18.75 µg/mL (7.5%) of benzhydrol, 0.75 µg/mL (0.3%) of bromodiphenhydramine, 0.75 µg/mL (0.3%) of methyldiphenhydramine and 25 µg/mL (10%) of sodium benzoate in acetonitrile and water (2:8).

Table 1. LC Conditions Optimization Design

The chromatographic results were imported back into the software for data modeling, analysis, and visualization. The software guided the selection of method condition ranges that provide the largest area of continuous selectivity and robustness. In-silico robustness study was performed by the software with theoretical Monte Carlo simulations using the design of experiment (DOE)-derived models (Section 5.2). The areas in Figure 4 represent the 5-factor MODR within which the method is predicted to be robust by meeting all critical method attribute (CMA) response goals, including peak resolution of not less than (NLT) 1.0 between diphenhydramine and RC A, NLT 4.0 between diphenhydramine and N-oxide, and NLT 1.5 for all other pairs. The biggest effect on selectivity was seen from the strong solvent ratio, i.e., the percent of acetonitrile in Solution B. Based on this observation and to make the design space more manageable, the MODR was simplified from 5-factor to 2-factor (Figure 5). As flow rate, isocratic hold time, and column temperature had the minimal impact on selectivity, the conditions were fixed at 0.8 mL/min, 2.5 minutes, and 35°, respectively. The areas within the rectangles (gradient time 13.5 – 25 min and strong solvent ratio, acetonitrile and methanol, (70:30 – 85:15)) include method conditions that can each be independently adjusted while still meeting response goals according to the model prediction. The starting method is chosen as 19 min gradient time and Solution B consisting of acetonitrile and methanol at (80:20), which is near the center of the MODR.

Figure 4a. MODR at flow rate 0.6 mL/min represented by the unshaded areas

Figure 4b. MODR at flow rate 0.7 mL/min represented by the unshaded areas

Figure 4c. MODR at flow rate 0.8 mL/min represented by the unshaded areas

Figure 4. 5-factor MODR 

Figure 5. 2-factor MODR at flow rate 0.8 mL/min, 2.5 min initial hold time, 35° column temperature represented by the unshaded areas

4.2. In-silico Robustness Study

In-silico robustness was conducted by way of theoretical Monte Carlo simulations using the DOE-derived models. Maximum expected variation was set for each parameter studied, maintaining CMA response goals. A robustness capability metric, Cpk, was set at 1.33. The resulting unshaded region represents method conditions that meet mean performance and robustness (Figures 4 and 5).

4.3. Degradation Products

The objective of the proposed adaptable method is the quantitation of known degradation products of diphenhydramine in various Oral Solution formulations. Forced degradation and Identification of unknown degradants that may be formed in different formulations was out of the scope of this study. Refer to Section 6.2, Instruments and Method, for the final procedure condition.

5.1. Validation Strategy

The adaptable HPLC method from the Method Development section was validated as the OI procedure. The validation strategy and corresponding protocol for validation of the MODR were developed following a new ICH guideline Q14¹⁴, revised guideline ICH Q2(R2)¹⁵, and USP General Chapter <1225>¹⁶. The strategy included an evaluation of prior knowledge and data collected during the method development and risk assessment. The in-silico robustness study followed by verification and establishment of the System Suitability Test as a part of the control strategy was performed during the method development stage.

The reportable range was set at 0.1% as the low end for all degradation products. The high end of the reportable range was set at 0.3% for RC B, 3.0% for benzophenone (BPH), 4.5% for N-oxide, and 7.5% for benzhydrol (BZH). The validation protocol included an evaluation of specificity, linearity, Relative Response Factors (RRFs), lower range limit, and repeatability at the central point (starting conditions) and the edges (extreme conditions, i.e., allowable variations) of the area selected inside the MODR (Figure 5). Accuracy and intermediate precision were evaluated only at the starting conditions. This is due to the fact that the major chromatography factors affecting the recovery are resolution and peak shape which were modeled or evaluated and verified across the entire MODR. Another major factor for the recovery is sample preparation procedure which is unchanged across the design space. Sample preparation including a dilution of oral solution in diluent (acetonitrile and water 1:4) was found suitable for all formulations except the products containing carboxymethylcellulose (CMC) or xanthan gum. These excipients adversely affected chromatography. A cleaning procedure using isopropanol to remove CMC or xanthan gum from the sample was developed (refer to Section 5.3).

The adaptable method was found to be specific, linear, accurate, precise, and free from interferences for the samples evaluated.

5.2. Instruments and Method

The analysis of the Diphenhydramine Hydrochloride Oral Solution was performed using Waters Alliance and Agilent 1260 instruments with PDA/DAD detectors, and the results were processed using Empower (Waters software) and Fusion QbD® software. The Waters XSelect HSS PFP, 3.0-mm x 15.0-cm, 3.5-µm (L43) column was used. The analysis was performed at a column temperature of 35°, with a flow rate of 0.8 mL/min and 15 µL as the injection volume. The autosampler was kept at 25°. The detector was set at UV 220 nm (diphenhydramine RC B, benzhydrol, and diphenhydramine N-oxide) and UV 254 nm (benzophenone). The mobile phase contained 0.1% TFA in water (Solution A) and a mixture of acetonitrile and methanol, with 0.05% TFA (Solution B) delivered in a gradient mode. The gradient program is listed in Table 2. A summary of validation conditions is shown in Table 3.

Table 2. Gradient Program

Table 3. Validation Conditions

5.3. Solutions

Solution A (0.1% TFA): Transfer 1800 mL of water to a 2-L volumetric flask. Add 2.0 mL of TFA, dilute to volume with water, and mix well.

Solution B (0.05% TFA in acetonitrile and methanol): Mix 800 mL of acetonitrile, 200 mL of methanol, and 0.5 mL of TFA in a 1-L glass container (starting conditions). Mix 850 mL of acetonitrile, 150 mL of methanol, and 0.5 mL of TFA in a 1-L glass container (extreme conditions 1 and 2). Mix 700 mL of acetonitrile, 300 mL of methanol, and 0.5 mL of TFA in a 1-L glass container (extreme conditions 3 and 4).

Diluent (acetonitrile and water, 1:4): Mix 400 mL of acetonitrile and 1600 mL of water in a 2-L glass container.

Preparation of individual Stock solutions: Individual Stock solutions consisting of 125 µg/mL each of DPH HCl, RC B, RC A, BZH, BPH, and N-oxide were prepared by dissolving appropriate amounts of each standard in Diluent. Note: Stock solutions containing BZH, BPH, and RC B were prepared by first dissolving the standards in acetonitrile and then adding water so that the final solvent composition matches that of the diluent.

Preparation of Standard solution: A solution consisting of 1.25 μg/mL (0.5%) of each DPH HCl, RC B, BZH, BPH, and N-oxide was prepared by combining appropriate volumes of individual Stock solutions in Diluent.

Preparation of Sensitivity solution: A solution consisting of 0.25 μg/mL (0.1%) of DPH HCl was prepared by combining appropriate volumes of individual Stock solutions in Diluent.

Preparation of System suitability solution: A solution consisting of 0.25 mg/mL of DPH HCl, 2.5 μg/mL (1.0%) of each RC A and N-oxide by combining appropriate volumes of individual Stock solutions in Diluent.

Preparation of Sample solution (for formulations without xanthan gum or carboxymethylcellulose): A solution with nominal 0.25 mg/mL of DPH HCl from Diphenhydramine Hydrochloride Oral Solution was prepared as follows. Diluted a portion of Diphenhydramine Hydrochloride Oral Solution to a final DPH HCl nominal concentration of 0.25 mg/mL with Diluent.

Preparation of Sample solution (for formulations containing xanthan gum or carboxymethylcellulose): A solution with nominal 0.25 mg/mL of DPH HCl from Diphenhydramine Hydrochloride Oral Solution was prepared as follows. A portion of Diphenhydramine Hydrochloride Oral Solution was mixed with a volume of isopropanol to give a DPH HCl nominal concentration of 1.0 mg/mL. An aliquot of the solution was centrifuged at room temperature at 3500 rpm for 10 minutes. A portion of the supernatant was diluted with water to a final concentration of 0.25 mg/mL. The solution was filtered through a 0.45-µm pore-size nylon syringe filter discarding the first milliliter.

5.4. System Suitability Parameters, Validation Performance Characteristics, and Results

The system suitability was established at the starting and extreme conditions. The system suitability parameters and results are summarized in Table 4.

The validation protocol followed the strategy described in Section 6.1. Specificity, relative response factors (RRF), linearity, and repeatability were established for the starting conditions and each of the four extreme conditions. Whereas accuracy and intermediate precision were established at the starting conditions, only.

The reportable range was set at 0.1% as the low end for all degradation products. The method was validated in the range of 0.1% ‒ 0.3% for RC B, 0.1% ‒ 7.5% for BZH, 0.1% ‒ 4.5% N-oxide, and 0.1% ‒ 3.0% for BPH. The validation performance characteristics and results are summarized in Table 5.

Representative chromatograms of Diluent, Sensitivity solution, Standard solution, System suitability solution (starting conditions) are presented in Figures 6–9, and chromatograms of Sample solutions (starting or extreme conditions) are presented in Figures 10–13.

Table 4. Summary of System Suitability Parameters, Solutions, and Results for the Organic Impurities Test 

Table 5. Summary of Validation Performance Characteristics, Solutions, and Results for the Organic Impurities Test

Table 6. Relative Response Factor

RRF values of the impurities were calculated by dividing the slope of the linearity curve for each impurity by the slope of the linearity curve for diphenhydramine HCl.

Figure 6. Chromatogram of Diluent (starting conditions)

 
Figure 7. Chromatogram of Sensitivity solution (starting conditions)

 
Figure 8a. Chromatogram at 220 nm of Standard solution (starting conditions)

 
Figure 8b. Chromatogram at 254 nm of Standard solution (starting conditions)

Figure 8. Chromatogram of Standard solution (starting conditions)

 
Figure 9. Chromatogram of System suitability solution (starting conditions)

 
Figure 10a. Chromatogram at 220 nm (expanded scale) of Sample solution (Sample A)

 
Figure 10b. Chromatogram at 254 nm (expanded scale) of Sample solution (Sample A)
Figure 10. Chromatograms of Sample solution at starting conditions

 
Figure 11a. Chromatogram at 220 nm (expanded scale) of Sample solution (Sample D)

 
Figure 11b. Chromatogram at 254 nm (expanded scale) of Sample solution (Sample D)

Figure 11. Chromatograms of Sample solution at extreme conditions 1

 
Figure 12a. Chromatogram at 220 nm (expanded scale) of Sample solution (Sample G)

 
Figure 12b. Chromatogram at 254 nm (expanded scale) of Sample solution (Sample G)

Figure 12. Chromatograms of Sample solution at extreme conditions 3

 
Figure 13a. Chromatogram at 220 nm (expanded scale) of Sample solution (Sample H)

 
Figure 13b. Chromatogram at 254 nm (expanded scale) of Sample solution (Sample H)

Figure 13. Chromatograms of Sample solution at extreme conditions 4

USP acknowledges Johnson & Johnson, Proctor & Gamble, Sanofi, and Perrigo for their donation of sample materials and methods that supported the development of this standard.

USP would also like to acknowledge the expert volunteers who contributed to this work:
Donna Seibert, Robert Buice, Gail Reed, Xiaoping Wang, Gregory Webster, Kylen Whitaker, Jingyue Yang