Instrumentation

Agilent 1260 Infinity II with a DAD detector and quaternary pump was the tool utilized for development and validation enabled with Agilent's Openlab EzChrom software. The Aczet analytical balance, Digital Systronic pH meter, Labman ultrasonicator, and Millipore vacuum filter pump (XI 5522050) were used for the method development with a nylon filter (for filtration) of Merck Millipore (0.22-μm).

Materials and reagents

The pharmaceutical-grade working standard of hydrocortisone butyrate (purity ≥ 99.80%) was obtained as a gift sample from Aadhaar Life Sciences Pvt. Ltd (Solapur, Maharashtra, India). Impurities, including Hydrocortisone-21-Butyrate (purity = 99.10%) purchased from Sigma Aldrich, India, Hydrocortisone 3-methyl enol ether 17-butyrate (purity = 98.93%) and hydrocortisone 17, 21-methylorthobutyrate (purity = 99.61%) from SimSon Pharma Ltd., India. All chemicals, such as HPLC-grade Acetonitrile (ACN), were procured from SD Fine Chemicals, Mumbai, India. Milli-Q water was obtained from Merck, India, for the current study. All weighing was done using calibrated NABL scales, and samples were produced in Class A glassware.

Chromatographic conditions

The mobile phase consisted of a combination of ACN (30% v/v) and purified water (70% v/v), as well as a mixture of ACN and water (95:5% v/v). A flow rate of 1.2 mL/min was maintained, and detection was performed at 254 nm (Additional file 1: Fig. S7) with an injection volume of 10 µL. The column temperature was kept at 35 °C, allowing a tolerance of ± 2 °C as per robustness guidelines.

Preparation of mobile phase

Mobile Phase A* 70% Purified Water: 30% ACN.

Mobile Phase A* was prepared by accurately measuring 700 mL of purified water and 300 mL of ACN, which were then mixed in a suitable container. The resulting mobile phase was filtered through a 0.45 µm nylon membrane and sonicated to degas.

Mobile phase B 5% Purified Water: 95% ACN.

Mobile Phase B was prepared by accurately measuring 50 mL of purified water and 950 mL of ACN, which were mixed in a suitable container. The resulting mobile phase was filtered through a 0.45 µm nylon membrane and sonicated to degas.

*Note: If the system suitability requirement is not met, changes to the chromatographic conditions can be made according to the robustness test from method validation. However, only one change can be made at a time.

Preparation of diluent

Throughout, ACN is used as diluent, pre-filtered through 0.45 μm nylon membrane after a brief sonication.

Preparation of standard stock solutionPreparation of HCB standard stock solution (1000 µg/mL)

The drug portion was first dried using standard conditions (78 °C and 3 h) before use. Then, 25 mg of HCB reference standard was weighed and transferred into a 25 mL volumetric flask (VF). The drug was made soluble by adding the necessary amount of diluent and then sonicated. Once equilibrium was reached, the volume was adjusted to 25 mL using diluent.

Preparation of working standard solution (200 µg/mL)

The stability of the working solution was observed for 4 days when stored at room temperature. 2.0 mL of stock standard was transferred into a 10 mL volumetric flask (VF). The volume was adjusted using diluent after degassing.

Preparation of HCB lotion formulation for assay (200 µg/mL)

A sample of 2.0 mL was transferred directly into a 10 mL volumetric flask (VF) using a TC (to contain) pipette. The pipette was rinsed quantitatively using a small quantity of diluent (less than 1 mL). The volume was then adjusted with diluent, and the sample was vortexed for approximately 1 min.

Forced degradation study

Following the ICH guidelines Q1A(R2) and Q1B [20], degradation studies were conducted on HCB-17. The chromatogram was analyzed based on the drug peak area and the presence of any additional/secondary peaks. Any variations in peak area or the emergence of secondary peaks were considered indicators of degradation.

Photolytic degradation

An excess quantity of the drug was exposed to UV (254 nm) and white light for a total of 1.2 million lux hours in a petri dish over a 24-h period. Afterwards, a stock solution was prepared by dissolving 10 mg of the drug in a 10 mL volumetric flask. The drug was made soluble by adding the necessary quantity of diluent and then sonicated. Once equilibrium was reached, the volume was adjusted to 10 mL using diluent. Subsequently, the solution was filtered through a 0.45 µm syringe filter before injection.

Thermal degradation

The thermal property of the drug was determined by placing an excess quantity of the sample in an oven at approximately 80 °C for 4 h. Subsequently, 10 mg of this sample was weighed and transferred into a 10 mL volumetric flask. Around 5 mL of diluent was used to dissolve the stressed solution, and the volume was adjusted to the mark using diluent. Later, the solution was filtered through a 0.45 µm syringe filter before injection.

Acid degradation

For acid degradation, 10 mg of the drug was accurately weighed and transferred into a 10 mL volumetric flask. To dissolve the drug, 5 mL of diluent was added, and the volume was made up to the mark with diluent. This solution is referred to as the stock solution.

Subsequently, 1 mL of the stock solution was transferred into a 10 mL volumetric flask, and 1 mL of a 200 µL of a 1N hydrochloric acid (HCl) solution was added. The solution was refluxed with diluent at 60 °C for 30 min to induce acid degradation. After refluxing, the solution was neutralized using a 200 µL solution of 1N sodium hydroxide (NaOH) and adjusted to the mark with diluent. Finally, the solution was filtered through a 0.45 µm syringe filter before injection.

Alkali degradation

For alkali degradation, 10 mg of the drug was accurately weighed and transferred into a 10 mL volumetric flask. To dissolve the drug, 5 mL of diluent was added, and the volume was made up to the mark with diluent. This solution is referred to as the stock solution.

Subsequently, 1 mL of the stock solution was transferred into a 10 mL volumetric flask, and 1 mL of a 100 µL of a 1N sodium hydroxide (NaOH) solution was added. The solution was refluxed with diluent at 60 °C for 30 min to induce alkali degradation. After refluxing, the solution was neutralized using 100 µL of a 1N hydrochloric acid (HCl) solution and adjusted to the mark with diluent. Finally, the solution was filtered through a 0.45 µm syringe filter before being ready for injection.

Peroxide degradation

For peroxide degradation, 10 mg of the drug was accurately weighed and transferred into a 10 mL volumetric flask. To dissolve the drug, 5 mL of diluent was added, and the volume was made up to the mark with diluent. This solution is referred to as the stock solution.

Subsequently, 1 mL of the stock solution was transferred into a 10 mL volumetric flask, and 1 mL of a 3% hydrogen peroxide (H2O2) solution was added. The solution was refluxed with diluent at 60 °C for 30 min to induce oxidative degradation. Afterward, the volume was made up to the mark, and the solution was filtered through a 0.45 µm syringe filter before injection.

Method validation

The developed method was validated as per ICH Q2 (R1) and USFDA [21] guidelines, considering parameters like Linearity and Range, Specificity (blank, placebo, standard and sample solution), Accuracy, Precision, Robustness, Ruggedness, Limit of Detection (LOD) and Limit of Quantitation (LOQ).

Solution stability

To know how long the prepared solution is stable, the working standard was analyzed at different time point, and it was confirmed that it was stable for up to 4 days. This was proved by analyzing the same at 4 different time points. Stability data is shown in (Table 1).

Table 1 Solution stability of hydrocortisone butyrate

The cumulative RSD after 4 days of keeping the VF at room temperature in a dry place away from light was 0.2%. The specification limits are 2% and therefore, the working standard is stable for at least 4 days.