Optimized Gas Chromatography Method for Quantifying Residual Solvents in Clindamycin Phosphate Pledgets: A Comprehensive ICH Q2(R1) Validation Study

Optimized Gas Chromatography Method for Quantifying Residual Solvents in Clindamycin Phosphate Pledgets: A Comprehensive ICH Q2(R1) Validation Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimized Gas Chromatography Method for Quantifying Residual Solvents in Clindamycin Phosphate Pledgets: A Comprehensive ICH Q2(R1) Validation Study Vivek Rayala, Abhilash Reddy Yaramala, Sudhir Maddela, Ratna Manjula Rayi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7626940/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Clindamycin phosphate (CP) is widely prescribed for the treatment of anaerobic and gram-positive bacterial infections. Accurate quantification of residual solvents, particularly isopropyl alcohol (IPA), is essential to ensure product safety and compliance with regulatory standards. This study aimed to formulate CP pledgets and to develop and validate a sensitive Gas Chromatography-Flame Ionization Detector (GC-FID) method for IPA determination. Methods: Chromatographic separation was achieved using an Rtx-1301 column under optimized GC-FID conditions, enabling selective detection of IPA without interference from the matrix. The method was validated in accordance with ICH Q2 (R1) guidelines, evaluating linearity, specificity, accuracy, precision, and robustness. Results: The method demonstrated excellent linearity from the LOQ to 200% of the specified limits, with correlation coefficients (R²) of 1.000. Recovery studies confirmed high accuracy (99.1–101.8%), while both method and intermediate precision yielded %RSD values below 15%. Chromatographic performance showed sharp peak resolution and reproducible separation without excipient interference, confirming specificity. Conclusion: The validated GC-FID method provides a reliable, accurate, and robust analytical tool for quantifying IPA in CP pledgets. Its high precision and specificity make it suitable for routine quality control and regulatory compliance, thereby ensuring product safety and efficacy. Clindamycin phosphate pledgets residual solvents isopropyl alcohol GC-FID method validation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Clindamycin Phosphate (CP), (Methyl 7-chloro-6,7,8-trideoxy-6-(1-methyl-trans-4-propyl-L-2- Pyrrolidine carboxamido)-1-thio-L-threo-α-D-galacto-octopyranoside 2-(dihydrogen phosphate)), is a widely used bacteriostatic agent. It is effective against a broad spectrum of anaerobic bacteria and gram-positive aerobes[ 1 ]. CP is commonly prescribed for the treatment of skin and soft tissue infections caused by erythromycin-resistant Staphylococcus aureus. CP, a member of the macrolide-lincosamide-streptogramin group B (MLS B ) family, has long been considered an effective alternative drug against S. aureus infections due to its proven safety, ease of administration, and favourable pharmacokinetic properties. The drug exerts its antibacterial effect by reversible binding to the 50S subunit of the ribosome, thereby precluding protein synthesis [ 2 ] The chemical structure of CP is depicted in Fig:1. CP has broad therapeutic applications, so its quality and safety are of the utmost importance. Pharmaceuticals must be analyzed for residual solvents, as they may be incorporated during the manufacturing of drug substances. Residual solvents are a critical quality attribute that must be controlled according to regulatory guidelines outlined in the International Council for Harmonisation (ICH) Q3C. Insightful analytical methods are requisite for detecting and quantifying these residual solvents to meet regulatory requirements and patient well-being. GC emerged as a preferred analytical technique for identifying and quantifying residual solvents in pharmaceuticals because of its substantial capabilities and sensitivity of capillary columns. GC can vary based on the type of column (capillary or wide-bore), chromatographic conditions, use of static headspace injectors, and Flame Ionization Detectors (FID). The variations depend on the specific application and sample preparation[ 3 ]. A few researchers worked on the determination of residual solvents in CP [ 4 , 5 , 6 , and 7 ] and none of them reported the determination of isopropyl alcohol (IPA) in CP pledgets using FID. The present investigation focuses on developing and validating an innovative GC-FID method that quantifies IPA in CP pledgets. The method was validated as per ICH guidelines. Methods Materials: CP was obtained as a gift sample from Glenmark Life Sciences Ltd. Ethanol was procured from Merck. IPA was purchased from Thermo Fischer Scientific. All other solvents used in the analysis are of GC grade. Instrumentation A GC (Agilent Technologies 6890N) equipped with FID connected to an Agilent G1888 Headspace sampler was used to develop and validate GC methods. It has a standard oven option for temperature ramping and split injection ports. Liquid sample injection was achieved via the G1890 auto-sampling unit from Agilent Technologies, using a 10 µL syringe. Waters Empower 3 Software was employed to process and quantify the peaks. Preparation of Diluent (Internal Standard Solution) Transfer ethanol (1.0 mL) into a volumetric flask (VF) of 1000 mL capacity filled to the mark with dimethyl sulfoxide (DMSO) and agitated to achieve homogeneity. Preparation of Standard Solution Transfer IPA (0.157 g ) into a VF (200 mL) containing diluent (50 mL). The flask was gently swirled to initiate mixing and diluted with diluent up to its volume. (This solution contains 785 µg/mL or 0.001 mL/mL of Isopropyl alcohol). Preparation of CP Pledgets: CP pledgets are topical formulations designed for the treatment of acne and other bacterial skin infections. These pledgets are made by impregnating fabric pads or similar carriers with a CP solution, ensuring convenient application and effective antibiotic delivery. CP was typically dissolved in a hydroalcoholic base (IPA). To the above solution, phosphate buffer was added to maintain the pH range of 5–6, and preservative (methylparaben) was added and mixed well. Polyethylene glycol was added to the above mixture and mixed well to obtain a homogeneous solution. Now, immerse the pledgets (fabric pads) in the solution for 2–3 minutes, ensuring uniform impregnation. Remove excess solution and package the pledgets in airtight, sterile containers to prevent evaporation or contamination. The formula for the preparation of CP pledgets is shown in Table 1 . The prepared pledgets were evaluated for physical and chemical characterization, drug content uniformity, microbiological evaluation, in-vitro release studies, and skin penetration studies. Table 1 Formula for the Preparation of CP Topical Solution 1%w/v Pledgets S. No. Ingredients Quantity 1. CP 1g 2. Purified water 70mL 3. IPA 20mL 4. PEG 10mL 5. Methyl Paraben 0.2g Preparation of Sample Stock Solution: Ten CP pledgets were randomly selected from a batch using sterile, clean forceps. The selected pledgets were transferred into a glass bottle, ensuring no contamination occurred during the transfer. Subsequently, add diluent (190 mL) to the bottle. The solution was stirred (30 minutes ) continuously to facilitate complete interaction between the pledgets and the diluent. (This solution contains about 0.025 mL/mL of isopropyl alcohol). (Note: Consider 10 mL from the 10 pledgets and 190 mL of diluent, making the total volume 200 mL.) Preparation of Sample Solution Transfer sample stock solution (4.0 mL) into a VF (100 mL). Swirl the flask gently, and diluted with diluent up to its volume, and mix well. Linearity Study: Accurately weigh and transfer 1570 mg of IPA into a VF (200 mL), containing diluent (50 mL). Swirl the flask gently, dilute with diluent up to its volume, and mix well. The resulting linearity stock solution contains 7850 µg/mL of IPA. Accuracy Study: The accuracy of the test method for determining IPA in CP pledgets was assessed by spiking IPA to the CP Topical Solution, which did not contain IPA. The study was performed at spike levels corresponding to 50% to 200% of the target concentration of 785 µg/mL, resulting in a concentration range from 393 µg/mL to 1572 µg/mL. Preparation of Accuracy Stock Solution The linearity stock solution was used as the accuracy stock solution for spiking to the placebo matrix. Preparation of Working Accuracy Sample Solutions: Accuracy sample solutions were prepared by carefully transferring ten pledgets (which did not contain IPA) into a suitable bottle. An appropriate volume of the accuracy stock solution was added to the bottle, followed by the volume of the diluent, and stirred (30 minutes ) continuously to achieve complete interaction between the IPA and the placebo matrix. After thorough mixing, the resulting solution was used for analysis. Specificity Study: The specificity of the test method was evaluated to ensure that it accurately quantifies isopropyl alcohol (IPA) without interference from the diluent or placebo. The study involves analyzing the diluent, placebo, and analyte (IPA) under optimized chromatographic conditions. The potential for interference was assessed by examining the chromatographic profiles of the diluent and placebo for any co-elution or significant peaks at the retention time of IPA. Preparation of Placebo Solution: Ten CP pledgets were taken in a suitable container(excluding those containing IPA). Add 190 mL of diluent to the container and stir (30 minutes). Accurately transfer the solution (4 mL) into VF (100 mL) diluted with diluent up to its volume and mix well. Precision Study: Precision is a key parameter in validating analytical methods, assessing results consistency across repeated measurements. Precision is typically assessed as method precision (MP (six replicates)) and intermediate precision (IP) was evaluated under diverse conditions. Robustness: Robustness was assessed by varying one parameter at a time while maintaining all other parameters constant. This study ensures that minor, deliberate changes in method conditions do not significantly affect the analytical performance. In the present investigation, the effect of changes to the initial temperature and carrier gas flow rate was studied Results and Discussion Optimization of Chromatographic Conditions: In this study, various chromatographic columns and conditions were assessed to optimize the separation and quantification of IPA in CP pledgets. Various column dimensions were tested, but the best separation was achieved on the RTX-1301 column (30 m × 0.53 mm, 3 µm). Several chromatographic parameters were systematically optimized to enhance both reproducibility and peak shape. Initially, methanol was used as the diluent at a column temperature of 50°C. Under these conditions, separation was achieved with methanol eluting at approximately 3 minutes and IPA at 5 minutes, as shown in Fig. 2. However, the method's reproducibility was not satisfactory, where inconsistent peak areas were observed across replicate injections. To improve reproducibility, the sample temperature was increased to 180°C, which resulted in a more consistent separation profile. While the separation improved, carryover of IPA was observed between injections. To mitigate this, the sample temperature was further raised to 240°C, eliminating carryover but causing a significant increase in the response intensity. The split ratio was modified from 1:1 to 1:10 to adjust for the higher response, which did not yield satisfactory reproducibility. The %RSD of the peak area remained above 15%, indicating that the method was not yet stable. Further trials were conducted by testing various diluents, including DMSO, dimethylacetamide (DMA), dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP), in addition to methanol. DMA, DMF, and NMP resulted in poor peak shapes shown in Fig. 3A (DMF), Fig. 3B (DMA), and Fig. 3C (NMP), whereas DMSO produced acceptable peak shapes shown in Fig. 3D. However, reproducibility remained a challenge, with significant variability in response from preparation to preparation, leading to high %RSD values. The incorporation of an internal standard (ethanol) in the DMSO diluent was found to improve both peak shape and reproducibility, as shown in Fig. 4. The presence of the internal standard allowed for more consistent responses across different preparations, and the %RSD was reduced to acceptable levels. Consequently, the method was validated using DMSO as the diluent and ethanol as the internal standard, ensuring reliable and reproducible results. The optimized chromatographic conditions mentioned in Table 2 were used to validate the developed method for quantification of IPA as per ICH guidelines., which demonstrated excellent precision, accuracy, and reliability, as detailed in subsequent sections of this study. System Suitability (SS): SS of the optimized GC-FID method, assessed by analyzing the standard solution (six replicates). Chromatographic parameters, including the number of theoretical plates (N), tailing factor (T), and resolution (Rs), were determined. The percent relative standard deviation (RSD) of IPA and other chromatographic performance criteria met the pre-defined acceptance limits outlined by the ICH, as detailed in Table 3, confirming the method's reliability and consistency. Table 3 System Suitability Results Solvent Retention time % RSD of area (6) T N Rs IPA 5.4 0.3 0.9 50086 2.07 Acceptance Criteria N/A NMT 15.0 NMT 2.0 NLT 2000 NLT 1.5 N/A-Not applicanle, NMT-Not more than, NLT- Not less than Linearity Study: The linearity study was assessed across a concentration range of 50%-200% of the specified concentration level of IPA. The results shown in Table 4 confirmed linearity between the analyte concentration and peak area ratio of IPA, meeting all predefined acceptance criteria. The correlation coefficient was 1.000, confirming the method's reliability and suitability across the linearity range. The %Y-intercept was 0.6%, indicating minimal deviation from linearity at the lower concentrations. The slope of the calibration curve shown in Fig. 5 is 0.001381, reflecting the sensitivity of the method to changes in IPA concentration. The %RSD for peak area ratios across all linearity levels ranged from 0.2% to 0.9%, depicting the methods precision throughout the tested range. Table 4 Linearity of IPA S. No. Percentage Level Conc (µg /mL) Peak Area (mean) 1 LOQ 393 0.54 2 75 590 0.81 3 100 785 1.09 4 125 983 1.35 5 150 1178 1.63 6 200 1570 2.17 Accuracy Study: The analytical method was assessed for the recovery of IPA at three concentration levels (50%, 100%, and 200%) of the target concentration. The percent recovery of IPA at each level is summarized in Table 5. The results showed excellent recovery across all the tested concentration levels, with mean recoveries falling within the 99.1% to 101.8% range. This confirms that the method can recover IPA with high reliability, regardless of the concentration tested. The low %RSD values across all concentration levels was found to be in the range of 0.2% to 0.6% indicating that the method is precise and reproducible. Therefore, this method is suitable for routine quantitative analysis of IPA. Table 5 Accuracy Results of IPA Accuracy Levels Concentration of Isopropyl alcohol (µg /mL) Percent Recovered Mean Percent Recovered %RSD R1-50% 393 98.54 99.1 0.6 99.67 99.06 R2-100% 785 100.83 100.6 0.4 100.84 100.19 R3-200% 1570 101.95 101.8 0.2 101.65 101.84 Mean Percent Recovered and %RSD Limits 95.0%-105.0% NMT 5.0% Specificity Study: The specificity of the test method confirms a precise match in retention times of the standard and the spiked sample matrix, and no interfering peaks of blank and placebo detected at the target solvent retention time, as shown in Figs. 6, 7, and 8. Precision Study: The method and intermediate precision of the analytical method of IPA were assessed by calculating the percent recovery for a series of six replicate sample preparations. The results in Table 6 indicate that the method is highly reproducible and reliable. Both the method precision and intermediate precision analyses showed minimal variation in recovery values, with mean recoveries of 50.3% and 51.6%, respectively. Whereas, the overall precision analysis (combining both the method precision and intermediate precision results, the overall %RSD for all twelve samples) was 1.6%, which is well below the acceptable limit of 5.0%. This further confirms the reliability of the method across different conditions. Table 6 Results of precision study of IPA Parameter IPA System precision (standard solution) (peak area) 0.3 Repeatability (intraday) (content ppm) 1.0 Intermediate precision (interday) (content ppm) 3.3 Cumulative (intraday and interday) (content ppm 4.3 Robustness Study: The robustness study determines the method’s ability to withstand deliberate variations in analytical parameters. The SS was assessed by analyzing standard solutions, altering column temperature and carrier gas flow rate other than the target method specifications. The results shown in Table 7 indicate that the method remains highly consistent and reliable even when subjected to deliberate changes in the initial column temperature. The low %RSD of 0.5% confirms the precision of the method under these conditions, and the resolution indicates good separation between peaks. Table 7 IPA Robustness Results Parameter Conditions %RSD (peak area (n = 6)) Rt N T Flow rate (±0.02) mL/min 4.3 0.8 4.6 699 1.0 4.5 0.5 5.4 50086 1.0 4.7 0.5 4.0 21804 1.0 Column Oven Temperature (±5 0 C) 45 0.5 4.7 21021 1.0 50 0.5 5.4 50086 1.0 55 1.0 3.8 22869 1.0 Rt-Retention Time Discussion The optimization process revealed that while methanol initially enabled separation, it failed to provide reproducibility. Increasing temperatures improved reproducibility but introduced issues such as carryover and excessively high response intensities, highlighting the need for a stable diluent. Among the tested solvents, DMSO proved to be superior in maintaining acceptable peak shapes. The use of ethanol as an internal standard was critical in addressing variability issues, reducing %RSD, and achieving reliable quantification. This approach enhanced reproducibility by correcting for variability between preparations. The validated method fulfilled all ICH requirements for analytical methods. System suitability parameters confirmed the robustness of the chromatographic system. Linearity was excellent across a wide concentration range, with a correlation coefficient of 1.000, and minimal deviation at low concentrations. Accuracy studies demonstrated high recovery rates, indicating the method’s suitability for quantitative analysis. Specificity studies confirmed the absence of matrix interference, ensuring reliable identification and quantification of IPA. Precision and intermediate precision studies highlighted the method’s reproducibility under varying conditions. Robustness results further established the reliability of the method under minor, deliberate variations in operating parameters. Overall, the developed GC-FID method using DMSO as diluent and ethanol as internal standard proved precise, accurate, specific, robust, and reproducible for the quantification of IPA in CP pledgets. Conclusion A robust and reliable GC-FID method was successfully developed and validated to quantify IPA in CP pledgets. The RTX-1301 column and the optimized chromatographic conditions ensured excellent separation and quantification of IPA without interference from the drug matrix or blank with a symmetric peak shape and retention time. The method was validated as per ICH Q2 (R1), ensuring linearity, accuracy, precision, specificity, and robustness. The validated method ensures regulatory compliance and meets stringent quality requirements, applicable to routine quality control. Declarations Acknowledgements: The authors are very much thankful to Glenmark Life Sciences Ltd, Hyderabad, for providing the gift sample of Clindamycin Phosphate, and Nirmala College of Pharmacy, Atmakuru, for their support to complete the work. Funding: No funding received Conflicts of Interest: No conflicts of interest References Brook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev. 2013 Jul;26(3):526-46. Deotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. Indian J Med Microbiol. 2010 Apr-Jun;28(2):124-6. Grodowska, K., & Parczewski, A. (2010). Analytical methods for residual solvents determination in pharmaceutical products Acta Poloniae Pharmaceutica- Drug Research, Vol. 67 No. 1 pp. 13-26. Liu, S. & Chen, G.-B & Hong, L.-Y. (2014). Determination of residual organic solvents in clindamycin phosphate by headspace gas chromatography. 3. 217-219. Hong-Mei, Z. & Zhi-Fang, X. & Ya-Ning, Z. & Quan-Xun, D. (2008). Determination of residual solvents in clindamycin phosphate by headspace gas chromatography. 33. 217-219. Jing, F.-m. (2009). Determination of residual solvents in clindamycin phosphate by headspace GC method . Journal of Pharmaceutical Analysis , 5(1), 45-50 Wang Jian, Wang Hongbo, Wang Zhijian. Determination of Nine Residual Organic Solvent in Clindamycin Palmitate Hydrochloride by GC. Chinese Journal of Modern Applied Pharmacy, 2012, 29(9): 829-833. Table 2 Table 2 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":49616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical Structure of Clindamycin Phosphate\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/a4b2d2b315340d61f81b2dfb.png"},{"id":92171672,"identity":"31b804c2-c437-40bd-bf68-6794ce572128","added_by":"auto","created_at":"2025-09-25 11:57:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypical Chromatogram with methanol as diluent\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/a7df6c482c1cfb8edb784ecc.png"},{"id":92168837,"identity":"eca99435-482e-462d-8aad-f4f0e13efbe1","added_by":"auto","created_at":"2025-09-25 11:33:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":730414,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/b58f39194fdfbae152778d90.png"},{"id":92170633,"identity":"2cd89b86-8852-4951-a7b1-4c248074ca7c","added_by":"auto","created_at":"2025-09-25 11:49:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78921,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/91a0c8664301aea6cf7d9ac4.png"},{"id":92168839,"identity":"c1cc0419-8719-409c-b716-d816e532cfc8","added_by":"auto","created_at":"2025-09-25 11:33:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinearity Plot of Isopropyl alcohol\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/06101224f45791ee45a5b359.png"},{"id":92168838,"identity":"9b11ed9b-0bc9-4406-9cab-2b85d8bcd46b","added_by":"auto","created_at":"2025-09-25 11:33:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypical chromatogram of Diluent\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/b9e76f6bdddc52f67dd1d49c.png"},{"id":92168834,"identity":"7835b68d-c820-42c6-8353-2ddc2b2d1e5e","added_by":"auto","created_at":"2025-09-25 11:33:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":75766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypical Chromatogram of placebo\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/1a7b79ad5f4c208a86024c7c.png"},{"id":92170134,"identity":"23d0e99d-0f55-421f-bd2f-755b71b118ee","added_by":"auto","created_at":"2025-09-25 11:41:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":86290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypical Chromatogram of IPA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/5c096b6abcacfc06c5074e47.png"},{"id":92820759,"identity":"2647d530-5df8-402f-8255-09c6b67289fb","added_by":"auto","created_at":"2025-10-06 01:46:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2180593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/a09c20c2-336e-4be4-8650-c0d973557315.pdf"},{"id":92168829,"identity":"6a97da1d-9849-41bc-9ede-8bf1ef6f4032","added_by":"auto","created_at":"2025-09-25 11:33:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20408,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7626940/v1/f3ee5b77001455d2476d4735.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimized Gas Chromatography Method for Quantifying Residual Solvents in Clindamycin Phosphate Pledgets: A Comprehensive ICH Q2(R1) Validation Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClindamycin Phosphate (CP), (Methyl 7-chloro-6,7,8-trideoxy-6-(1-methyl-trans-4-propyl-L-2- Pyrrolidine carboxamido)-1-thio-L-threo-α-D-galacto-octopyranoside 2-(dihydrogen phosphate)), is a widely used bacteriostatic agent. It is effective against a broad spectrum of anaerobic bacteria and gram-positive aerobes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. CP is commonly prescribed for the treatment of skin and soft tissue infections caused by erythromycin-resistant \u003cem\u003eStaphylococcus aureus.\u003c/em\u003e CP, a member of the macrolide-lincosamide-streptogramin group B (MLS\u003csub\u003eB\u003c/sub\u003e) family, has long been considered an effective alternative drug against \u003cem\u003eS. aureus\u003c/em\u003e infections due to its proven safety, ease of administration, and favourable pharmacokinetic properties. The drug exerts its antibacterial effect by reversible binding to the 50S subunit of the ribosome, thereby precluding protein synthesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The chemical structure of CP is depicted in Fig:1.\u003c/p\u003e\u003cp\u003eCP has broad therapeutic applications, so its quality and safety are of the utmost importance. Pharmaceuticals must be analyzed for residual solvents, as they may be incorporated during the manufacturing of drug substances. Residual solvents are a critical quality attribute that must be controlled according to regulatory guidelines outlined in the International Council for Harmonisation (ICH) Q3C. Insightful analytical methods are requisite for detecting and quantifying these residual solvents to meet regulatory requirements and patient well-being.\u003c/p\u003e\u003cp\u003eGC emerged as a preferred analytical technique for identifying and quantifying residual solvents in pharmaceuticals because of its substantial capabilities and sensitivity of capillary columns. GC can vary based on the type of column (capillary or wide-bore), chromatographic conditions, use of static headspace injectors, and Flame Ionization Detectors (FID). The variations depend on the specific application and sample preparation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A few researchers worked on the determination of residual solvents in CP [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, and \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and none of them reported the determination of isopropyl alcohol (IPA) in CP pledgets using FID.\u003c/p\u003e\u003cp\u003eThe present investigation focuses on developing and validating an innovative GC-FID method that quantifies IPA in CP pledgets. The method was validated as per ICH guidelines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials:\u003c/h2\u003e\u003cp\u003eCP was obtained as a gift sample from Glenmark Life Sciences Ltd. Ethanol was procured from Merck. IPA was purchased from Thermo Fischer Scientific. All other solvents used in the analysis are of GC grade.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInstrumentation\u003c/h3\u003e\n\u003cp\u003eA GC (Agilent Technologies 6890N) equipped with FID connected to an Agilent G1888 Headspace sampler was used to develop and validate GC methods. It has a standard oven option for temperature ramping and split injection ports. Liquid sample injection was achieved via the G1890 auto-sampling unit from Agilent Technologies, using a 10 \u0026micro;L syringe. Waters Empower 3 Software was employed to process and quantify the peaks.\u003c/p\u003e\n\u003ch3\u003ePreparation of Diluent (Internal Standard Solution)\u003c/h3\u003e\n\u003cp\u003eTransfer ethanol (1.0 mL) into a volumetric flask (VF) of 1000 mL capacity filled to the mark with dimethyl sulfoxide (DMSO) and agitated to achieve homogeneity.\u003c/p\u003e\n\u003ch3\u003ePreparation of Standard Solution\u003c/h3\u003e\n\u003cp\u003eTransfer IPA (0.157 g ) into a VF (200 mL) containing diluent (50 mL). The flask was gently swirled to initiate mixing and diluted with diluent up to its volume. \u003cem\u003e(This solution contains 785 \u0026micro;g/mL or 0.001 mL/mL of Isopropyl alcohol).\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of CP Pledgets:\u003c/h3\u003e\n\u003cp\u003eCP pledgets are topical formulations designed for the treatment of acne and other bacterial skin infections. These pledgets are made by impregnating fabric pads or similar carriers with a CP solution, ensuring convenient application and effective antibiotic delivery. CP was typically dissolved in a hydroalcoholic base (IPA). To the above solution, phosphate buffer was added to maintain the pH range of 5\u0026ndash;6, and preservative (methylparaben) was added and mixed well. Polyethylene glycol was added to the above mixture and mixed well to obtain a homogeneous solution. Now, immerse the pledgets (fabric pads) in the solution for 2\u0026ndash;3 minutes, ensuring uniform impregnation. Remove excess solution and package the pledgets in airtight, sterile containers to prevent evaporation or contamination. The formula for the preparation of CP pledgets is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The prepared pledgets were evaluated for physical and chemical characterization, drug content uniformity, microbiological evaluation, in-vitro release studies, and skin penetration studies.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFormula for the Preparation of CP Topical Solution 1%w/v Pledgets\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIngredients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eQuantity\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePurified water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIPA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePEG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMethyl Paraben\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Sample Stock Solution:\u003c/h2\u003e\u003cp\u003eTen CP pledgets were randomly selected from a batch using sterile, clean forceps. The selected pledgets were transferred into a glass bottle, ensuring no contamination occurred during the transfer. Subsequently, add diluent (190 mL) to the bottle. The solution was stirred (30 minutes ) continuously to facilitate complete interaction between the pledgets and the diluent. \u003cem\u003e(This solution contains about 0.025 mL/mL of isopropyl alcohol).\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(Note: Consider 10 mL from the 10 pledgets and 190 mL of diluent, making the total volume 200 mL.)\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of Sample Solution\u003c/h3\u003e\n\u003cp\u003eTransfer sample stock solution (4.0 mL) into a VF (100 mL). Swirl the flask gently, and diluted with diluent up to its volume, and mix well.\u003c/p\u003e\n\u003ch3\u003eLinearity Study:\u003c/h3\u003e\n\u003cp\u003eAccurately weigh and transfer 1570 mg of IPA into a VF (200 mL), containing diluent (50 mL). Swirl the flask gently, dilute with diluent up to its volume, and mix well. The resulting linearity stock solution contains 7850 \u0026micro;g/mL of IPA.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAccuracy Study:\u003c/h2\u003e\u003cp\u003eThe accuracy of the test method for determining IPA in CP pledgets was assessed by spiking IPA to the CP Topical Solution, which did not contain IPA. The study was performed at spike levels corresponding to 50% to 200% of the target concentration of 785 \u0026micro;g/mL, resulting in a concentration range from 393 \u0026micro;g/mL to 1572 \u0026micro;g/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Accuracy Stock Solution\u003c/h2\u003e\u003cp\u003eThe linearity stock solution was used as the accuracy stock solution for spiking to the placebo matrix.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Working Accuracy Sample Solutions:\u003c/h2\u003e\u003cp\u003eAccuracy sample solutions were prepared by carefully transferring ten pledgets (which did not contain IPA) into a suitable bottle. An appropriate volume of the accuracy stock solution was added to the bottle, followed by the volume of the diluent, and stirred (30 minutes ) continuously to achieve complete interaction between the IPA and the placebo matrix. After thorough mixing, the resulting solution was used for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSpecificity Study:\u003c/h2\u003e\u003cp\u003eThe specificity of the test method was evaluated to ensure that it accurately quantifies isopropyl alcohol (IPA) without interference from the diluent or placebo. The study involves analyzing the diluent, placebo, and analyte (IPA) under optimized chromatographic conditions. The potential for interference was assessed by examining the chromatographic profiles of the diluent and placebo for any co-elution or significant peaks at the retention time of IPA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Placebo Solution:\u003c/h2\u003e\u003cp\u003eTen CP pledgets were taken in a suitable container(excluding those containing IPA). Add 190 mL of diluent to the container and stir (30 minutes). Accurately transfer the solution (4 mL) into VF (100 mL) diluted with diluent up to its volume and mix well.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePrecision Study:\u003c/h2\u003e\u003cp\u003ePrecision is a key parameter in validating analytical methods, assessing results consistency across repeated measurements. Precision is typically assessed as method precision (MP (six replicates)) and intermediate precision (IP) was evaluated under diverse conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRobustness:\u003c/h2\u003e\u003cp\u003eRobustness was assessed by varying one parameter at a time while maintaining all other parameters constant. This study ensures that minor, deliberate changes in method conditions do not significantly affect the analytical performance. In the present investigation, the effect of changes to the initial temperature and carrier gas flow rate was studied\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eOptimization of Chromatographic Conditions:\u003c/h2\u003e\n \u003cp\u003eIn this study, various chromatographic columns and conditions were assessed to optimize the separation and quantification of IPA in CP pledgets. Various column dimensions were tested, but the best separation was achieved on the RTX-1301 column (30 m \u0026times; 0.53 mm, 3 \u0026micro;m). Several chromatographic parameters were systematically optimized to enhance both reproducibility and peak shape. Initially, methanol was used as the diluent at a column temperature of 50\u0026deg;C. Under these conditions, separation was achieved with methanol eluting at approximately 3 minutes and IPA at 5 minutes, as shown in Fig.\u0026nbsp;2. However, the method\u0026apos;s reproducibility was not satisfactory, where inconsistent peak areas were observed across replicate injections.\u003c/p\u003e\n \u003cp\u003eTo improve reproducibility, the sample temperature was increased to 180\u0026deg;C, which resulted in a more consistent separation profile. While the separation improved, carryover of IPA was observed between injections. To mitigate this, the sample temperature was further raised to 240\u0026deg;C, eliminating carryover but causing a significant increase in the response intensity. The split ratio was modified from 1:1 to 1:10 to adjust for the higher response, which did not yield satisfactory reproducibility. The %RSD of the peak area remained above 15%, indicating that the method was not yet stable.\u003c/p\u003e\n \u003cp\u003eFurther trials were conducted by testing various diluents, including DMSO, dimethylacetamide (DMA), dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP), in addition to methanol. DMA, DMF, and NMP resulted in poor peak shapes shown in Fig.\u0026nbsp;3A (DMF), Fig.\u0026nbsp;3B (DMA), and Fig.\u0026nbsp;3C (NMP), whereas DMSO produced acceptable peak shapes shown in Fig.\u0026nbsp;3D. However, reproducibility remained a challenge, with significant variability in response from preparation to preparation, leading to high %RSD values.\u003c/p\u003e\n \u003cp\u003eThe incorporation of an internal standard (ethanol) in the DMSO diluent was found to improve both peak shape and reproducibility, as shown in Fig.\u0026nbsp;4. The presence of the internal standard allowed for more consistent responses across different preparations, and the %RSD was reduced to acceptable levels. Consequently, the method was validated using DMSO as the diluent and ethanol as the internal standard, ensuring reliable and reproducible results.\u003c/p\u003e\n \u003cp\u003eThe optimized chromatographic conditions mentioned in Table 2 were used to validate the developed method for quantification of IPA as per ICH guidelines., which demonstrated excellent precision, accuracy, and reliability, as detailed in subsequent sections of this study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eSystem Suitability (SS):\u003c/h2\u003e\n \u003cp\u003eSS of the optimized GC-FID method, assessed by analyzing the standard solution (six replicates). Chromatographic parameters, including the number of theoretical plates (N), tailing factor (T), and resolution (Rs), were determined. The percent relative standard deviation (RSD) of IPA and other chromatographic performance criteria met the pre-defined acceptance limits outlined by the ICH, as detailed in Table\u0026nbsp;3, confirming the method\u0026apos;s reliability and consistency.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSystem Suitability Results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRetention time\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% RSD of area (6)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRs\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIPA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcceptance Criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN/A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMT 15.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMT 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNLT 2000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNLT 1.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eN/A-Not applicanle, NMT-Not more than, NLT- Not less than\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eLinearity Study:\u003c/h2\u003e\n \u003cp\u003eThe linearity study was assessed across a concentration range of 50%-200% of the specified concentration level of IPA. The results shown in Table\u0026nbsp;4 confirmed linearity between the analyte concentration and peak area ratio of IPA, meeting all predefined acceptance criteria. The correlation coefficient was 1.000, confirming the method\u0026apos;s reliability and suitability across the linearity range. The %Y-intercept was 0.6%, indicating minimal deviation from linearity at the lower concentrations. The slope of the calibration curve shown in Fig.\u0026nbsp;5 is 0.001381, reflecting the sensitivity of the method to changes in IPA concentration. The %RSD for peak area ratios across all linearity levels ranged from 0.2% to 0.9%, depicting the methods precision throughout the tested range.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eLinearity of IPA\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage Level\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConc (\u0026micro;g /mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak Area (mean)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLOQ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eAccuracy Study:\u003c/h2\u003e\n \u003cp\u003eThe analytical method was assessed for the recovery of IPA at three concentration levels (50%, 100%, and 200%) of the target concentration. The percent recovery of IPA at each level is summarized in Table\u0026nbsp;5. The results showed excellent recovery across all the tested concentration levels, with mean recoveries falling within the 99.1% to 101.8% range. This confirms that the method can recover IPA with high reliability, regardless of the concentration tested. The low %RSD values across all concentration levels was found to be in the range of 0.2% to 0.6% indicating that the method is precise and reproducible. Therefore, this method is suitable for routine quantitative analysis of IPA.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAccuracy Results of IPA\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAccuracy Levels\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration of Isopropyl alcohol (\u0026micro;g /mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercent Recovered\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean Percent Recovered\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eR1-50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eR2-100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e100.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eR3-200%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e1570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e101.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Percent Recovered and %RSD Limits\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e95.0%-105.0%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMT 5.0%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003eSpecificity Study:\u003c/h2\u003e\n \u003cp\u003eThe specificity of the test method confirms a precise match in retention times of the standard and the spiked sample matrix, and no interfering peaks of blank and placebo detected at the target solvent retention time, as shown in Figs.\u0026nbsp;6, 7, and 8.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\"\u003e\n \u003ch2\u003ePrecision Study:\u003c/h2\u003e\n \u003cp\u003eThe method and intermediate precision of the analytical method of IPA were assessed by calculating the percent recovery for a series of six replicate sample preparations. The results in Table\u0026nbsp;6 indicate that the method is highly reproducible and reliable. Both the method precision and intermediate precision analyses showed minimal variation in recovery values, with mean recoveries of 50.3% and 51.6%, respectively. Whereas, the overall precision analysis (combining both the method precision and intermediate precision results, the overall %RSD for all twelve samples) was 1.6%, which is well below the acceptable limit of 5.0%. This further confirms the reliability of the method across different conditions.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eResults of precision study of IPA\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIPA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSystem precision (standard solution) (peak area)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRepeatability (intraday) (content ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntermediate precision (interday) (content ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCumulative (intraday and interday) (content ppm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\"\u003e\n \u003ch2\u003eRobustness Study:\u003c/h2\u003e\n \u003cp\u003eThe robustness study determines the method\u0026rsquo;s ability to withstand deliberate variations in analytical parameters. The SS was assessed by analyzing standard solutions, altering column temperature and carrier gas flow rate other than the target method specifications. The results shown in Table\u0026nbsp;7 indicate that the method remains highly consistent and reliable even when subjected to deliberate changes in the initial column temperature. The low %RSD of 0.5% confirms the precision of the method under these conditions, and the resolution indicates good separation between peaks.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 7\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eIPA Robustness Results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConditions\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e%RSD (peak area (n\u0026thinsp;=\u0026thinsp;6))\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRt\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate (\u0026plusmn;0.02) mL/min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eColumn Oven Temperature (\u0026plusmn;5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e55\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22869\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eRt-Retention Time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe optimization process revealed that while methanol initially enabled separation, it failed to provide reproducibility. Increasing temperatures improved reproducibility but introduced issues such as carryover and excessively high response intensities, highlighting the need for a stable diluent. Among the tested solvents, DMSO proved to be superior in maintaining acceptable peak shapes.\u003c/p\u003e\u003cp\u003eThe use of ethanol as an internal standard was critical in addressing variability issues, reducing %RSD, and achieving reliable quantification. This approach enhanced reproducibility by correcting for variability between preparations.\u003c/p\u003e\u003cp\u003eThe validated method fulfilled all ICH requirements for analytical methods. System suitability parameters confirmed the robustness of the chromatographic system. Linearity was excellent across a wide concentration range, with a correlation coefficient of 1.000, and minimal deviation at low concentrations. Accuracy studies demonstrated high recovery rates, indicating the method\u0026rsquo;s suitability for quantitative analysis.\u003c/p\u003e\u003cp\u003eSpecificity studies confirmed the absence of matrix interference, ensuring reliable identification and quantification of IPA. Precision and intermediate precision studies highlighted the method\u0026rsquo;s reproducibility under varying conditions. Robustness results further established the reliability of the method under minor, deliberate variations in operating parameters.\u003c/p\u003e\u003cp\u003eOverall, the developed GC-FID method using DMSO as diluent and ethanol as internal standard proved precise, accurate, specific, robust, and reproducible for the quantification of IPA in CP pledgets.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA robust and reliable GC-FID method was successfully developed and validated to quantify IPA in CP pledgets. The RTX-1301 column and the optimized chromatographic conditions ensured excellent separation and quantification of IPA without interference from the drug matrix or blank with a symmetric peak shape and retention time. The method was validated as per ICH Q2 (R1), ensuring linearity, accuracy, precision, specificity, and robustness. The validated method ensures regulatory compliance and meets stringent quality requirements, applicable to routine quality control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are very much thankful to Glenmark Life Sciences Ltd, Hyderabad, for providing the gift sample of Clindamycin Phosphate, and Nirmala College of Pharmacy, Atmakuru, \u0026nbsp;for their support to complete the work. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding received\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev. 2013 Jul;26(3):526-46. \u003c/li\u003e\n\u003cli\u003eDeotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. Indian J Med Microbiol. 2010 Apr-Jun;28(2):124-6.\u003c/li\u003e\n\u003cli\u003eGrodowska, K., \u0026amp; Parczewski, A. (2010). Analytical methods for residual solvents determination in pharmaceutical products Acta Poloniae Pharmaceutica- Drug Research, Vol. 67 No. 1 pp. 13-26.\u003c/li\u003e\n\u003cli\u003eLiu, S. \u0026amp; Chen, G.-B \u0026amp; Hong, L.-Y. (2014). Determination of residual organic solvents in clindamycin phosphate by headspace gas chromatography. 3. 217-219.\u003c/li\u003e\n\u003cli\u003eHong-Mei, Z. \u0026amp; Zhi-Fang, X. \u0026amp; Ya-Ning, Z. \u0026amp; Quan-Xun, D. (2008). Determination of residual solvents in clindamycin phosphate by headspace gas chromatography. 33. 217-219.\u003c/li\u003e\n\u003cli\u003eJing, F.-m. (2009). \u003cem\u003eDetermination of residual solvents in clindamycin phosphate by headspace GC method\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003eJournal of Pharmaceutical Analysis\u003c/em\u003e, 5(1), 45-50\u003c/li\u003e\n\u003cli\u003eWang Jian, Wang Hongbo, Wang Zhijian. Determination of Nine Residual Organic Solvent in Clindamycin Palmitate Hydrochloride by GC. Chinese Journal of Modern Applied Pharmacy, 2012, 29(9): 829-833.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 2","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Clindamycin phosphate, pledgets, residual solvents, isopropyl alcohol, GC-FID, method validation","lastPublishedDoi":"10.21203/rs.3.rs-7626940/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7626940/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose:\u003c/h2\u003e\u003cp\u003eClindamycin phosphate (CP) is widely prescribed for the treatment of anaerobic and gram-positive bacterial infections. Accurate quantification of residual solvents, particularly isopropyl alcohol (IPA), is essential to ensure product safety and compliance with regulatory standards. This study aimed to formulate CP pledgets and to develop and validate a sensitive Gas Chromatography-Flame Ionization Detector (GC-FID) method for IPA determination.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eChromatographic separation was achieved using an Rtx-1301 column under optimized GC-FID conditions, enabling selective detection of IPA without interference from the matrix. The method was validated in accordance with ICH Q2 (R1) guidelines, evaluating linearity, specificity, accuracy, precision, and robustness.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eThe method demonstrated excellent linearity from the LOQ to 200% of the specified limits, with correlation coefficients (R\u0026sup2;) of 1.000. Recovery studies confirmed high accuracy (99.1\u0026ndash;101.8%), while both method and intermediate precision yielded %RSD values below 15%. Chromatographic performance showed sharp peak resolution and reproducible separation without excipient interference, confirming specificity.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eThe validated GC-FID method provides a reliable, accurate, and robust analytical tool for quantifying IPA in CP pledgets. Its high precision and specificity make it suitable for routine quality control and regulatory compliance, thereby ensuring product safety and efficacy.\u003c/p\u003e","manuscriptTitle":"Optimized Gas Chromatography Method for Quantifying Residual Solvents in Clindamycin Phosphate Pledgets: A Comprehensive ICH Q2(R1) Validation Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 11:33:33","doi":"10.21203/rs.3.rs-7626940/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ecea666-544a-4913-9acf-f816f5d06b41","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T01:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 11:33:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7626940","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7626940","identity":"rs-7626940","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}