Development of a Validated HPLC-UV Method for the Determination of Panthenol, Hesperidin, Rutin, and Allantoin in Pharmaceutical Gel-Permeability Study.

A robust, validated HPLC–UV method was developed for the simultaneous determination of panthenol, hesperidin, rutin and allantoin in pharmaceutical gel formulations and for application in gel-permeability (release/penetration) studies. 

Method development employed reversed-phase chromatography with an aqueous/organic mobile phase containing a volatile acid modifier; separation was achieved on a C18 stationary phase and detection performed by UV monitoring at appropriate wavelengths selected for each analyte. 

The chromatographic conditions were optimized to ensure baseline separation of all four compounds within a short run time while maintaining symmetry and acceptable peak capacity for complex gel matrices.

The assay was validated according to international guidelines (linearity, accuracy, precision, selectivity, sensitivity, robustness and stability). The method exhibited excellent linearity for each analyte across the relevant concentration ranges used in release and permeability experiments, with limits of detection and quantification suitable for trace-level monitoring in permeation media. 

Intra- and inter-day precision and accuracy met acceptance criteria for bio/pharmaceutical analysis. Matrix effects from gel excipients were assessed and compensated for by matrix-matched calibration or appropriate sample clean-up, and the method proved selective in the presence of typical formulation components.

Applied to in vitro permeability/release assays, the method reliably quantified release profiles of panthenol, hesperidin, rutin and allantoin from gel vehicles and supported comparative formulation assessment. Given its sensitivity, selectivity and throughput, the validated HPLC-UV procedure is recommended for routine quality control and permeability studies of topical gels containing these active compounds.

Materials and Methods

Reagents, Solvents, and Materials

The pharmaceutically active ingredients used in this study were DL-panthenol (C₉H₁₉NO₄, PAN), allantoin (C₄H₆N₄O₃, ALN), and rutin (C₂₇H₃₀O₁₆, RTN) purchased from TCI Chemicals (Tokyo, Japan), and hesperidin (C₂₈H₃₄O₁₅, HES) obtained from Sigma-Aldrich (Burlington, MA, USA).

Excipients used in gel preparation included glycerin (C₃H₈O₃; Sigma-Aldrich), TC-Carbomer 340 ((C₃H₄O₂)ₙ, polyacrylic acid; Anaplasis, Greece), and 2-phenoxyethanol (C₈H₁₀O₂; Anaplasis, Greece).

All solvents used for mobile phase preparation, extraction, and dilution were of HPLC grade:

• Methanol (CH₃OH) — Chem-Lab NV, Zedelgem, Belgium.
• Acetonitrile (CH₃CN) — Honeywell International Inc., Morristown, NJ, USA.
• Dimethylformamide (DMF) — Sigma-Aldrich, Burlington, MA, USA.
• Ultrapure water (18.2 MΩ·cm) was generated using a B30 purification system (Adrona SIA, Riga, Latvia).

For in vitro permeability testing, cellulose membranes (flat width 43 mm) were purchased from Sigma-Aldrich (Darmstadt, Germany).

Preparation of Standard Solutions

Individual stock solutions were prepared for each of the four active pharmaceutical ingredients — DL-panthenol (PAN), allantoin (ALN), rutin (RTN), and hesperidin (HES). Precisely weighed amounts of each compound were transferred into separate 10 mL volumetric flasks: 10 mg of PAN, 5 mg of ALN, 5 mg of RTN, and 8 mg of HES.

Each compound was dissolved in an appropriate solvent mixture to ensure complete solubility:

• Panthenol (PAN): 2 mL of water and 8 mL of methanol (MeOH).
• Hesperidin (HES): 10 µL of dimethylformamide (DMF) added to methanol.
• Allantoin (ALN): 10 µL of DMF and 5 mL of water added before completing to volume with methanol. The solution was then placed in a water bath at 35 °C for approximately 10 minutes to achieve full dissolution.
• Rutin (RTN): 10 mL of methanol.

All stock solutions were sonicated for 5 minutes to ensure homogeneity. The prepared solutions were found to be stable for at least 48 hours when stored at 2 °C.

Appropriate dilutions of each stock solution were then prepared in a 50 mL volumetric flask and made up to volume with acetonitrile–water (90:10, v/v) to obtain working standard solutions. A series of six calibration standards was further prepared in 25 mL volumetric flasks, also filled to the mark with acetonitrile–water (90:10, v/v).

The final concentrations of the standard solutions were as follows:

• Panthenol (PAN): 6.7–67.2 µg/mL
• Allantoin (ALN): 1.2–12.0 µg/mL
• Rutin (RTN): 0.8–8.1 µg/mL
• Hesperidin (HES): 0.5–25.2 µg/mL

Before HPLC analysis, all solutions were filtered through 0.45 µm nylon syringe filters to remove any suspended particles.

Phosphate-Buffered Saline (PBS)

PBS (pH 7.4) was prepared using:

 NaCl (8.0 g), Na₂HPO₄ (1.44 g), KH₂PO₄ (0.24 g), and KCl (0.20 g) dissolved in 1 L distilled water (Merck, Darmstadt, Germany; Chem-Lab, Belgium).

 Instrumentation

Analyses were conducted using a Shimadzu HPLC-20AD system (Kyoto, Japan) equipped with a SIL-20A HT autosampler, CTO-20A column oven (25 °C), and SPD-M20A diode array detector.

• Column: SeQuant® ZIC-HILIC (150 mm × 4.6 mm, 5 µm; Merck, Darmstadt, Germany)
• Mobile Phase: Binary gradient of
  • (A) ACN–H₂O (90:10, v/v)
  • (B) ACN–H₂O (10:90, v/v)
• Injection volume: 20 µL
• Detection: UV-DAD
• Software: LC Solution v1.25 SP4 (Shimadzu)

In vitro permeability studies were carried out using vertical Franz diffusion cells (effective area = 4.9 cm²; receptor volume = 20 mL).

Methods

 Gel Preparation

Hydrogels were prepared using water, carbomer, glycerin, and phenoxyethanol as excipients. Three formulations (Gel 1–3) differing in water content (340–460 mL) were tested for consistency and spreadability. Gel 3 (460 mL water) displayed optimal texture and was selected for further use.

The final composition contained:

• Panthenol: 5% w/w
• Hesperidin: 0.5% w/w
• Rutin: 0.5% w/w
• Allantoin: 0.5% w/w

2.4.2. In Vitro Permeability Studies

Franz diffusion cells were assembled with cellulose membranes between donor and receptor chambers.

• Donor phase: 250 mg gel or 250 µL suspension

• Receptor phase: 20 mL PBS (pH 7.4, 37 °C, stirred at 90 rpm)
  Samples (0.5 mL) were withdrawn at 2, 3, 4, 6, and 8 h and replaced with fresh PBS.
  Collected samples were lyophilized, reconstituted in methanol (300 µL), sonicated (15 min), cooled (10 min), centrifuged, and analyzed by HPLC after dilution (ACN–H₂O 90:10, v/v).

The apparent permeability coefficient (Papp) was calculated as:

Papp=JssCdP_{app} = \frac{J_{ss}}{C_d}Papp​=Cd​Jss​​

where Jₛₛ is the steady-state flux and C_d the drug concentration in the donor compartment.