In-Vitro EVALUATIONS OF LINCOMYCIN HYDROCHLORIDE CAPSULES: DEVELOPMENT, VALIDATION, AND COMPATIBILITY

1 ISSN Online: 3007-1941 ISSN Print: 3007-1933 IN-VITRO EVALUATIONS OF LINCOMYCIN HYDROCHLORIDE CAPSULES: DEVELOPMENT, VALIDATION, AND COMPATIBILITY Article Details A B S T R A C T Keywords: In-Vitro comparative evaluation, United State Pharmacopoeia (U.S.P.), pharmacopeia excipient selection Muhammad Imran Faculty of Sciences, The Superior University Lahore, Pakistan. Ali Imran Mallhi Faculty of Sciences, the Superior University Lahore, Pakistan Muhammad Atif* (Corresponding author) Faculty of Sciences, the Superior University Lahore, Pakistan Email: [email protected] Wajiha Khalid Faculty of Pharmaceutical Sciences (FPS), Riphah International University, Islamabad Rabia Faridi Department of Plant Breeding and Genetics, University of Agriculture Faisalabad, 38000. Pakistan Muhammad Rizwan Faculty of Sciences, the Superior University Lahore, Pakistan Maryam Aslam Department of Chemistry, Government College Women University Faisalabad This study reports for pharmaceutical formulation development, analytical validation, and In-Vitro comparative evaluation of generic Product Lincomycin 500mg hard gelatin capsules. The formulation was designed using Lincomycin as the active pharmaceutical ingredient, along with pharmacopeia excipients selected based on excipient compatibility studies conducted under accelerated conditions. The finished product met United State Pharmacopoeia (U.S.P.) Specifications for assay, dissolution, uniformity of dosage, and water content. Analytical method validation was performed using high-performance liquid chromatography (HPLC), and the method fulfilled all ICH and USP requirements for system suitability, specificity, accuracy, precision (repeatability and intermediate), robustness, and spiking recovery. Comparative dissolution studies were carried out in 0.1N HCl (pH 1.2), acetate buffer (pH 4.5), and phosphate buffer (pH 6.8) using USP Apparatus II. The test product demonstrated more than 85% drug release within 15 minutes in all media, and exhibited similarity factor (f2) values greater than 50 and difference factor (f1) values below 15 when compared with the reference product Novelink 500 mg Capsule. These results confirm the pharmaceutical equivalence of the test formulation and support its suitability for local production and regulatory approval. Muhammad Imran1, Ali Imran Mallhi2, Muhammad Atif*3, Wajiha Khalid4, Rabia Faridi5, Muhammad Rizwan6, Maryam Aslam7 https://msra.online/index.php/Journal/about https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) 2 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) 1. INTRODUCTION Lincomycin HCl is a semisynthetic derivative of the Lecin Base family having chemical name is methyl-[7chloro-6,7,8-trideoxy-6-trans-(1-methyl-4-propyl-l-2-pyrrollidin-carboxamido)-1-thio-l-threo-α-d-galactooctapyranoside Compendia name is Lincomycin with molecular formula is C₁₈H₃₄N₂O₆S· HCl and molecular weight is 443.00 g/mol, synthesized through the chemical modification of Lincomycin by coupling it with Llysine and formaldehyde, which enhances its solubility and bioavailability [1, 2] [3]. Classified as a secondgeneration Lincomycin, Lincomycin was developed to overcome the limitations of earlier agents like Lincomycin and chlortetracycline, particularly in terms of absorption, tissue distribution, and side effect profile [4]. The second-generation Lincomycin, including clindamycin and, were introduced to provide broader clinical utility and improve tolerability [5]. The history of Lincomycin begins with chlortetracycline (aureomycin), discovered in the late 1940s from Streptomyces aureofaciens, marking the advent of a new era in antimicrobial therapy [6]. Lincomycin quickly gained prominence due to their ability to inhibit a wide range of pathogens. Over time, structural modifications led to newer agents like clindamycin, , and Lincomycin, each with improved pharmacokinetic and pharmacodynamic characteristics [7]. These drugs are now used not only for infectious diseases but also for dermatological applications, such as acne and rosacea, due to their dual antimicrobial and anti-inflammatory actions [8]. Mechanistically, Lincomycin —acts as a bacteriostatic agent by inhibiting bacterial protein synthesis. It binds reversibly to the 30S ribosomal subunit, thereby preventing the attachment of aminoacyltRNA to the A site of the ribosome, effectively halting peptide elongation. This interruption disrupts bacterial replication and growth, accounting for its efficacy against a broad spectrum of gram-positive and gramnegative organisms[9]. Lincomycin demonstrates activity against several clinically relevant pathogens, including Cutibacterium acnes, Chlamydia trachomatis, Mycoplasma pneumoniae, and Rickettsia species [10]. This broad antimicrobial spectrum underpins its use in managing conditions like respiratory infections, sexually transmitted infections, and dermatologic disorders such as acne vulgaris[11]. One of the distinctive advantages of Lincomycin lies in its pharmacokinetics. It exhibits higher water solubility compared to its parent compound, resulting in more efficient oral absorption and greater bioavailability [12]. Once ingested, Lincomycin undergoes rapid hydrolysis in the gastrointestinal tract to release Lincomycin, which is the pharmacologically active moiety, confirming its role as a prodrug [13]. Plasma concentration studies reveal that only tetracycline is present systemically after oral administration of Lincomycin, supporting this metabolic transformation[14]. Additionally, Lincomycin provides better tissue penetration and has a longer half-life than older Lincomycin, which allows for once-daily or twice-daily dosing and improved patient compliance [15]. Gastrointestinal side effects such as nausea and abdominal discomfort, commonly observed with Lincomycin, appear to occur less frequently with Lincomycin, making it more suitable for long-term use[16]. A primary therapeutic application of Lincomycin is in the treatment of moderate to moderately severe acne vulgaris. Acne pathogenesis involves follicular occlusion, sebum overproduction, C. acnes colonization, and inflammation [17]. Lincomycin helps reduce C. acnes levels and exerts anti-inflammatory effects by inhibiting neutrophil chemotaxis and the release of inflammatory cytokines [18]. This dual action enhances its effectiveness in inflammatory acne, especially when topical treatments are insufficient [19]. Beyond acne, Lincomycin has been explored in the management of hidradenitis suppurativa (HS)—a chronic inflammatory skin disorder characterized by recurrent nodules, abscesses, and sinus tracts. Lincomycin, including Lincomycin, are among the first-line systemic treatments recommended in European S1 guidelines for mild to moderate HS [20]. Their efficacy is attributed to both antimicrobial effects and modulation of the immune response, although high-quality randomized trials are still needed to strengthen the evidence base [21]. Despite these benefits, Lincomycin, like all Lincomycin, carries a risk of adverse effects, notably photosensitivity reactions. Phototoxicity arises when the drug absorbs ultraviolet light, resulting in oxidative 3 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) damage to the skin [22]. While rare, this reaction necessitates preventive counseling for patients on sun exposure and photoprotection measures. Interestingly, Lincomycin appears to have a lower incidence of such reactions compared to earlier Lincomycin, although comprehensive epidemiological data are still evolving [23]. Continued research may further expand its application into areas such as resistant bacterial infections and immune-mediated inflammatory disorders. Figure 01 Chemical Structure of Lincomycin Hydrochloride 2. EXPERIMENTAL 2.1 Materials The formulation development of Lincomycin Hydrochloride (HCl) hard gelatin capsules was carried out using raw materials procured from authentic and reliable sources. The active pharmaceutical ingredient (API), Lincomycin Hydrochloride, was obtained from Pharmaceutical Grade, supplied as per the United States Pharmacopeia (U.S.P.) and British Pharmacopoeia (B.P), European Pharmacopoeia (Eur. Ph.) standards. A reference standard of Lincomycin HCl with a certified potency was also employed to ensure accurate quantitative analysis and serve as a primary benchmark for analytical validation. For excipients, Magnesium Stearate was used as a lubricant to reduce interparticle friction and improve powder flow during capsule filling, while Aerosil-200 (colloidal silicon dioxide) functioned as a glidant to prevent powder agglomeration and improve uniformity of capsule fill weight. The final dosage form was encapsulated in empty gelatin capsule shells, size #0, which are widely available and suitable for accommodating the required fill weight of the developed formulation. All excipients used were of pharmaceutical grade and complied with pharmacopeial standards. Their selection was based on prior compatibility studies, literature precedence, and established use in oral capsule formulations. 2.2 Methodology For qualitative and quantitative analysis, high-purity analytical-grade reagents and solvents were employed. The key chemicals included hydrochloric acid, purified water, sodium acetate, glacial acetic acid, monobasic potassium phosphate, sodium hydroxide, phosphoric acid, 2-methyl-2-propanol, di-potassium hydrogen orthophosphate, tetrabutylammonium hydrogen phosphate, sodium edetate, and sodium metabisulfite. Each reagent was procured from certified suppliers, and only analytical-grade materials were used to ensure accuracy, reproducibility, and compliance with pharmacopeial specifications. These reagents were primarily utilized for preparing mobile phases, buffers, and solutions required in highperformance liquid chromatography (HPLC), UV spectrophotometric analysis, dissolution studies, and Karl Fischer titration for moisture content analysis. The use of amber-colored volumetric flasks minimized lightinduced degradation of Lincomycin HCl during solution preparation, given the known photosensitivity of Lincomycin. The formulation and evaluation studies were performed using well-calibrated and validated laboratory instruments. The following equipment played a crucial role: 4 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Refrigerator – Used for the storage of reference standards, working standards, and sensitive reagents at controlled conditions to prevent degradation. HPLC system – Equipped with a stationary phase (HPLC Column: 4.6 mm × 25 cm, 5 µm packing, L-7/C8 Type) for chromatographic separation, quantification, and purity analysis of Lincomycin HCl. The selection of a C8 column ensured adequate retention and resolution of the analyte under optimized chromatographic conditions. UV-Visible Spectrophotometer – Employed for rapid determination of drug content and calibration curve construction during method validation. Dissolution Apparatus (USP Type I, 12-basket assembly) – Utilized for in vitro release testing to evaluate dissolution behavior of formulated capsules under simulated gastrointestinal conditions Karl Fischer Apparatus – Used for the determination of moisture content in raw materials and final formulations to ensure compliance with pharmacopeial limits. Glassware – High-quality volumetric flasks (amber-colored), beakers, graduated pipettes, and other standard laboratory glassware were used to prepare solutions with high accuracy. Each instrument was calibrated according to standard operating procedures (SOPs) before use. 2.2.1 Pre-formulation Studies Before the actual formulation, pre-formulation evaluations were conducted to study the physical and chemical properties of Lincomycin HCl and its interaction with excipients. These included: Organoleptic evaluation (appearance, color, and odor) of API Solubility profile in water and various buffers to guide dissolution method design. FTIR compatibility studies between the API and excipients (Magnesium Stearate, Aerosil-200) to confirm the absence of any chemical interactions that might affect drug stability. Micromeritic properties of the blend (bulk density, tapped density, angle of repose, compressibility index, Hausner’s ratio) to ensure suitability for capsule filling and good flow behavior 2.2.2 Formulation Development: The formulation of Lincomycin HCl capsules was developed by direct blending and encapsulation. The weighed quantities of API and excipients were geometrically mixed in a clean, dry stainless-steel container. Aerosil-200 was first blended with the drug to enhance uniformity and flow, followed by addition of magnesium stearate as a lubricant in the final mixing step. The final homogeneous blend was encapsulated into hard gelatin capsule shells, size #0, using a semiautomatic capsule filling machine. Filled capsules were stored in amber-colored containers to protect from light-induced degradation. Table# 01 Scale up of Tetracycline formulation Development: Sr, # RAW MATERIALS Role of Ingredients 1 Lincomycin HCl API 2 Colloidal silicon dioxide Glidant 3 pregelatinized starch (corn) Lubricant 4 Stearic acid Dissolution Enhancer 5 Empty Gelatin Capsule Shell#0 (Dark blue Cap and Light blue body) To enclose medicine 2.2.3 Post-formulation Assessment The developed capsules underwent various physicochemical evaluations: Weight Variation Test: Twenty Capsules were randomly selected and weighed individually and collectively to check compliance with pharmacopeial specifications. Disintegration Test: Conducted in purified water at 37 ± 0.5 °C to ensure that capsules disintegrated within the pharmacopeial time limit. Dissolution Testing: Performed using USP Dissolution Apparatus I (basket method) with appropriate dissolution media. Samples were withdrawn at defined intervals and analyzed by HPLC or UV spectrophotometer. Drug Content 5 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Uniformity: Capsules were assayed individually using validated HPLC methods to ensure uniform distribution of the drug. Moisture Content: Determined using Karl Fischer titration to confirm stability and prevent degradation due to excessive water content. 2.2.4 Analytical Method Validation: Quantitative estimation of Lincomycin HCl in the formulated capsules was carried out by HPLC method, optimized with a C8 column and a suitable mobile phase prepared from The method was validated in accordance with ICH guidelines as follows: 3. Linearity across a specified concentration range 4. Accuracy and Precision through recovery studies and repeatability testing 5. Specificity against excipients and potential degradation products 6. Robustness and Ruggedness by deliberate variations in method parameters 7. Complementary UV spectrophotometric analysis was employed for rapid estimation and cross-verification of results’ 3. RESULTS AND DISCUSSION Quantity of API was subjected to potency adjustment to in accordance with the API Contents and some adjustment of difference in quantity of API after potency adjustment were be deducted using Colloidal silicon dioxide. The results given as in the table as: Table 02 Formulation Development Sr, # RAW MATERIALS Role of Ingredients Percentage of Ingredients/cap. Scale mg/capsule 1 Lincomycin HCl API 98.33% 590.0* 2 Colloidal silicon dioxide Glidant 1.0% 6.0** 3 pregelatinized starch (corn) Lubricant 0.33% 2.000 4 Stearic acid Dissolution Enhancer 0.33% 2.000 Empty Gelatin Capsule Shell#0 (Dark blue Cap and Light blue body) To enclose medicine ---- 100 Total weight of Contents/ capsule 600mg Total weight of capsule with shell 700mg The pictorial view of the working is elaborated as: Step-1 Figure-02. Lincomycin (Final Mix) 6 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Step-2 Figure-03 Lincomycin formulation development (Active Raw Material/ API) Step-3 Figure-04 Lincomycin formulation development (at Intermediate/ After Encapsulation) Step-4 Figure-05 Lincomycin Finished Pharmaceutical Product (After Packaging) Packed in Alu-Pvc Foil 3,1 Adjustment and their outcomes in Analytical method Development (Contents By HPLC) In 10.0g of the Sodium hydroxide pellets in 90mL volumetric flasks and dilute up to the mark with water. As a mobile phase, add 8.1 mL of Phosphoric Acid in 600 mL of water and adjust with ammonium hydroxide to a pH of 6.0 Buffer solution……………………………….…….505.5 mL Acetonitrile……………………………….……..….97.2 mL Methanol… ………………………………………...97.2 mL Mix, shake well and degas by sonication and filter it through 0.45 µm membrane filter. 7 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Chromatographic Conditions: Column : L7/ C8 (4.6mm × 25cm) Detector  : 210 nm Flow rate : 1.0 mL / min Injection volume : 20 µl Column Temp. : 46 ºC Separately inject (provided 0.45µ disc filter) equal volumes of standard and sample preparations into the chromatograph and record the responses of the major peaks. The relative standard deviation for replicate injections of standard preparation should not be more than 2.0%, tailing factor NMT: 1.3. Calculate the percentage of Lincomycin by comparison of major peak responses of standard and sample solutions In 2.2mL Hydrochloric Acid (usually 37%) in 50mL water, now dilute up to 500mL with water and mix well. A reference solution of a commercial brand was also used for this adjustments. Tablet # 04 Product Description for in-vitro study both reference and test product Description Reference / Comparator Product Test Product Product Name Novelink 500mg Capsule Lecin 500mg Capsule Composition Each hard capsule contains: Lincomycin (U.S.P):…… 500 mg Each hard Capsule Contains: Lincomycin (U.S.P):…… 500 mg Lot # NL-3542 T-001 Figure-08 Reference Product used for in-Vitro Study/Bioequivalence Results in chromatogram are sequentially elaborated as further: Figure-06 HPLC Report/ Chromatogram for Lincomycin HCl Reference 8 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Figure-07 HPLC Report/ Chromatogram for Lincomycin Test Sample The recovery of Lincomycin between 95.0 to 105.0%; the RSD is of Lincomycin 2.0 %. and tailing factor is less the 0.7 The precision as well as the reproducibility of this method was satisfactory. Results of Comparative Dissolution Profile are as under: Figure-9 Graphically representation of CDP for reference and test product (pH 1.2) Figure-10 Graphically representation of CDP for reference and test product (pH 4.5) 30.00 80.00 45 60 75 Drug Release (%) Time Points (minutes) Acidic Medium (pH 1.2) (Lincomycin) 40.00 50.00 60.00 70.00 45 60 75 Drug Release (%) Time Points (minutes) Acetate Buffer (pH 4.5) (Lincomycin) 9 https://msra.online/index.php/Journal/about Volume 3, Issue 3 (2025) Figure-11 Graphically representation of CDP for reference and test product (pH 6.8) A comparative dissolution study was conducted between the test product, Lecin 500 mg Capsules, and the reference product, Novelink 500 mg Capsules. The difference factor (f1) was calculated to be 2.87%, and the similarity factor (f2) was 79.27%. These values fall within the acceptable regulatory limits (f1 < 15 and f2 > 50), indicating a high degree of similarity between the dissolution profiles of the two products. Both the test and reference formulations exhibited more than 75% drug release within 75 minutes in an acidic medium, which is a key requirement for immediate-release formulations. Additionally, the dissolution behavior was found to be comparable in acetate buffer (pH 4.5) and phosphate buffer (pH 6.8), further supporting the consistency of drug release across different physiological pH conditions. These findings confirm that the test product meets the regulatory criteria for dissolution profile similarity, providing strong evidence of in vitro bioequivalence. As such, the data support the conclusion that Lecin 500 mg Capsules perform similarly to Novelink 500 mg Capsules, and can be considered pharmaceutically equivalent in terms of dissolution behavior. 3.2 Excipient compatibility study A Description of the Drug-Excipient Samples is as under: Table-05 Description of Tetracycline formulation under the compatibility study: Sr, # RAW MATERIALS Role of Ingredients Percentage of Ingredients/cap. Scale mg/capsule 1 Lincomycin HCl API 98.33% 590.0* 2 Colloidal silicon dioxide Glidant 1.0% 6.0** 3 pregelatinized starch (corn) Lubricant 0.33% 2.000 4 stearic acid Dissolution Enhancer 0.33% 2.000 Empty Gelatin Capsule shell#0 (Dark blue Cap and Light blue body) To enclose medicine ---- 100 Total weight of Contents/ capsule 600mg Total weight of capsule with shell 700mg Drug Substance-Excipient Compatibility Study: Samples are prepared of drug substance and other ingredients as per Table#1 after one month at accelerated condition (temperature: 40°C±2°C, relative humidity 75%±5%). Drug substance compatibility is to be assessed through HPLC analysis of ingredients and drug substance in 30.00 35.00 40.00 45.00 45 60 75 Drug Release (%) Time Points (minutes) Phosphate Buffer (pH 6.8) (Lincomycin)