Process Validation of Liq-Biotic™ a Veterinary Herbal Product: Ensuring Consistency, Quality, and Efficacy in Herbal Formulations

Validation is the systematic process of gathering documented evidence to demonstrate that any method, procedure, or activity employed in production and testing consistently maintains the required level of compliance. According to the FDA, ensuring product quality necessitates meticulous and comprehensive attention to several critical factors, such as selecting appropriate quality methods, conducting thorough testing of both the process and the final product, and providing documentation that confirms ongoing compliance [1].

The concept of validation first emerged in the United States in 1978. Over time, its scope has expanded significantly to include a wide range of activities—from analytical techniques used in the quality assurance of medicinal components and finished products to digitized process control systems [2]. Among these, process validation is a key parameter of current Good Manufacturing Practices (cGMP). It is integral to the quality system, which aims to consistently produce products that meet their intended use. To achieve these objectives, process validation plays a crucial role [3-4].

The fundamental objective of pharmaceutical development is to ensure the reliable production of medicinal products that consistently meet their established therapeutic and performance specifications. This begins with the establishment of predefined objectives, articulated in a comprehensive Quality Target Product Profile (QTPP) [5]. The QTPP serves as a foundational, dynamic document that outlines all critical quality requirements necessary for ensuring the drug product’s safety and efficacy. As development progresses, the QTPP is periodically revised to incorporate emerging data, thereby providing a framework for assessing the criticality of specific product quality attributes [6].

These Critical Quality Attributes (CQAs) include physical, chemical, biological, or microbiological properties essential for maintaining product quality, safety, and efficacy within defined limits [7]. CQAs apply to drug substances, finished products, excipients, intermediates, and container-closure systems. Initially, CQAs are often based on prior knowledge and data from similar products, particularly during early development when comprehensive characterization may be limited. Their criticality is determined through risk-based assessments that evaluate potential impacts on safety and efficacy. In the context of herbal formulations, raw materials such as herbs, oils, or metabolites can also be designated as CQAs based on such risk evaluations [8].

Ayurvedic products are complex polyherbal formulations requiring intricate and multi-staged manufacturing processes.  Homogeneity within a batch and consistency between batches are goals of process validation activities. It is suggested that the comprehensive specification for the herbal substance must be followed by a description and validation of the manufacturing process for the herbal preparation [9]. Development of authentic analytical methods which can reliably profile the phytochemical composition and help in validation of manufacturing process is a major challenge to scientists. Prior standardization of formulation during its designing and development stage with respect to its bioactive marker compounds as a key feature of critical quality attribute, ensures the phyto-equivalence during the manufacturing of product on commercial scale. This will ensure the batch-to-batch consistency in quality & efficacy [10].

Liq-Biotic™, a phytogenic essential oils based gut function modulator is an herbal liquid formulation developed by Zenex Animal Health India Private Limited, Baddi. It is rich in essential oils (EO), such as ajwain oil, eucalyptus oil, menthol, camphor, and basil oil which are known for their stomachic, anti-bacterial, antiviral, antioxidant, immunomodulator, growth promoting and anti-toxigenic properties [11-14]. It is usually recommended for supporting gut health and natural immune system, prevention of diarrhoea, optimizing digestion, stabilizing intestinal microflora, stimulating appetite, improving feed conversion ratio (FCR) and weight gain [15-17]. Thymol, a secondary metabolite in ajwain oil (Figure 1); exhibits significant biological activities related to gut function modulator, hence the inclusion of the thymol in the formulation will add value to its efficacy [18]. As a part of manufacturing process validation pre requisites, the protocol was designed with the objective to validate the mixing time for the batch (1500 litres) of the Liq-Biotic™ in mixing tank of capacity 2000 litres. Mixing was done with the help of homogeniser at a speed of 1400rpm. The environmental conditions were considered as per pre-defined acceptance criteria prior to conducting the process validation study. A well-designed sampling plan defining all the locations with time intervals from where the samples were to be collected was prepared and sampling was done accordingly. In total 18 samples were collected from the different positions of mixing tank at the time interval of 05, 10 & 15 minutes (Table 1). Analytical method for the estimation of active content in the samples was developed as the integral part of the exercise at R&D. The method was validated on the basis of its selectivity, linearity, precision, accuracy, limit of detection and limit of quantification according to International Conference on Harmonization (ICH) guidelines [19-20]. Estimation of % active content thymol was carried out as per its validated analytical method. The process was supposed to be validated if percentage coefficient of variance (CV) is observed to be NMT 5 between the two extremes of percentage active content obtained after analysis.

Figure 1: Thymol

Table 1. Sampling Plan for Process Validation.

MATERIAL AND METHODS

Reagents and materials:

All the reagents and solvents were of AR or HPLC grade as per requirement. The active reference compound thymol was procured from the Sigma Aldrich, latest controlled samples of Liq-Biotic™ were obtained from the QA/QC department of Zenex Animal Health India Private Limited, Baddi.

Preparation of standard solution of thymol:

Accurately weighed around 5 mg of standard thymol was dissolved in 50 mL of methanol to obtain stock concentrations of 100 μg/mL. Stock solutions was further diluted to obtain the dilution range of 18–72 μg/mL and then injected in HPLC system in order to prepare the calibration graphs and quantification of bioactive.

Preparation of test solution:

For the quantification of thymol, Liq-Biotic™ (05g) was sonicated with 35 mL of HPLC grade methanol for 20 minutes and filtered. The final volume was made to 50 mL with methanol, filtered the solution through 0.45 μm membrane filter before injecting into HPLC system.

High Performance Liquid Chromatography Apparatus and Conditions:

Thymol content was analysed by High Performance Liquid Chromatography (WATERS, binary pump 515 with PDA 2996 detector, USA). The data was acquired on the Empower 3.0 controlling software. Separation was obtained on Phenomenex Luna C-18 column (250 mm x 4.6 mm, 5μm).

Selection and Optimization of chromatographic condition:

To optimize the RP-HPLC-PDA parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for thymol (Figure 1) was obtained by Water: Acetonitrile in 50:50, v/v ratio, as a mobile phase in isocratic mode. The mobile phase was filtered through 0.45 μm Millipore filter and degassed before use. The flow rate was adjusted to 1.0 mL/min. Injection volume was adjusted to 20 μL and detection was made at 275 nm.

VALIDATION OF THE METHOD:

The proposed method was validated for the determination of thymol using following parameters as per ICH guidelines:

Calibration:

The marker compound in the formulation was quantified using a calibration curve established with five dilutions of the standard. The corresponding peak area in formulation was plotted against the concentrations of the standard injected. Peak identification was achieved by comparison of both the retention time (RT) and UV absorption spectrum with those obtained for standard.

Table 2. Chromatographic parameter.

Table 3. Results of precision, linear regression analysis and their correlation coefficient for quantitative analysis of marker compound.

LOD: Limit of detection, LOQ: Limit of quantification

Table 4. Results from recovery analysis.

System Suitability:

The analytical results obtained by the method developed are only valid if the defined system suitability criteria are fulfilled. In this investigation, the experimental result (Table 2) indicates that the chromatographic system was suitable for intended analysis. Standard solution mixture containing known concentration of thymol was injected six times, separately. RSD values for peak area and retention time of standard suggested the reproducibility for these parameters. The low RSD values (Table 2) for tailing factor and theoretical plates suggested good peak symmetry of thymol and good efficiency of column.

Linearity:

Linear regression analysis was used to calculate the slope, intercept and /regression coefficient (r2) for calibration plot. Linearity was determined by using minimum three and up to five concentrations of the standard solution. Response was found to be linear in the concentration ranges investigated (Figure 2: d, Table 3).

Range:

Range is the interval between upper and lower concentration of analyte in sample for which it has been demonstrated that the analytical method has suitable level of precision, accuracy and linearity. The linear response was observed over a range of 18-72 ppm (Figure 2: d, Table 3).

(a)                                                                      (b)

(c)                                                                      (d)

(e)                                                                      (f

Figure 2. Chromatograms showing the resolution of marker compound in the formulation. (a) Chromatogram of standard thymol. (b) Chromatogram of sample Liq-Biotic™. (c) Overlay of the thymol chromatograms i.e., sample against standard. (d) Calibration plot for thymol standard. (e) Spectral scan of standard thymol. (f) Spectral scan of thymol in Liq-Biotic™.

Precision:

Three different concentrations of marker compound solution in triplicates were injected on three different times within the same day and repeating the same on three different days to record intra-day and inter-day variations in the results. The low % RSD values of intraday and inter-day (Table 3) for the marker compounds thymol reveals that the proposed method is precise.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

For determination of limits of detection and quantification, different dilutions of the marker were injected with mobile phase as blank and determined on the basis of signal to noise ratio 3:1 and 10:1 respectively. The LOD and LOQ for the standard compounds were calculated and tabulated (Table 3).

Selectivity:

The retention time of thymol and their counterpart in the formulation was 24.80 ±0.02 minute. The UV-Vis spectrum of marker compound was compared with its counterpart in formulation at three different positions, the peak start, peak centre and peak end. There was good correlation between spectra obtained at each of the three positions. The thymol peak was, therefore, not masked by any peak of other compound present in the formulation (Figures 2: e,f), which was indicative of peak purity.

Accuracy:

Recovery experiments were conducted to check for the presence of positive or negative interferences from other ingredients/excipients present in the formulation and to study the accuracy of the method. Recovery was determined by the standard addition method. Thymol standard was added to the formulation at two different concentrations, extraction and analysis was performed as described above. Recovery was calculated for each standard at each concentration (Table 4). The low value of relative standard deviation indicates that the proposed method is accurate.

RESULTS AND DISCUSSION

The manufacturing process of Liq-Biotic™ was evaluated through mixing time validation to demonstrate uniformity, ensure consistency of product quality, and establish the optimal mixing duration. Samples were collected in accordance with the approved sampling plan and analysed for thymol content using a validated RP-HPLC-PDA method. The thymol content was found to be within the range of 0.053% to 0.066% (Table 5).

The process demonstrated percent coefficients of variation (%CV) ranging from 1.10 to 3.39 at mixing intervals of 5, 10, and 15 minutes. A minimum %CV of 1.1 was achieved within 15 minutes of mixing, indicating satisfactory blend uniformity. As per established GMP guidelines and standard operating procedures for formulation mixing, a %CV of ≤5.0 is considered acceptable. Accordingly, the results confirm that a mixing time of 15 minutes is adequate and validated to achieve uniform and consistent product quality.

Table 5. Thymol content in Liq-Biotic™.

T1- Top 1, T2- Top 2 & T3-Top 3

L1- Bottom 1, L2-L Bottom 2 & L3- Bottom 3