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<p>This research aims to develop and validate a simple, sensitive, and robust reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the estimation of Alpha-Linolenic acid (ALA) isolated from silkworm pupa oil (SPO) using preparative-HPLC (PR-HPLC). Chromatographic separation was performed on a Shimadzu, C-18 column (4.6 × 250&#xa0;mm, 5&#xa0;μm particle size) using acetonitrile/water in a ratio of 80:20 with a flow rate of 1 mL/min and detection at a λ<sub>max</sub> of 242&#xa0;nm, equipped with a UV detector. The method demonstrated excellent linearity (R<sup>2</sup> = 0.9999, <i>p</i> &lt; 0.05) and good accuracy (98.41–102.10%). It also showed high intra-day and interday precision with RSD values ranging from 0.110% to 0.140%. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.622&#xa0;µg/mL and 2.05&#xa0;µg/mL, respectively. System suitability (99.33–100.02%), and robustness evaluation confirmed acceptable recoveries with RSD &lt; 2%. The method was specific for its ability to detect the analytes and their degradation products during forced degradation studies, which also indicated that ALA was highly prone to oxidative degradation (66.55%). The isolation of ALA from SPO was successfully performed based on this developed method using PR-HPLC. Furthermore, the isolated ALA was compared with standard ALA for purity by chromatographic analysis, FTIR and 1H NMR. The results demonstrated that the isolated ALA was pure with negligible interfering impurities within the detection limits of the applied analytical methods. Overall, the results indicated that the developed method was reliable for its accuracy, precision, sensitivity, and specificity for the estimation and isolation of ALA from SPO.</p>

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Alpha-linolenic acid from mulberry silkworm pupa oil: Isolation, method development, validation and quantification by reverse phase-high performance liquid chromatography

  • 期刊论文

  • 发表日期：

    2026年7月28日

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  • Shiv Kumar,
  • Dilip Kumar Arya,
  • Anit Kumar,
  • Shubham Kanaujiya,
  • Prashant Pandey,
  • Giriraj Pandey,
  • Kumar R. Venkatesh,
  • Parasuraman Pavadai,
  • Saurabh Srivastava,
  • P. S. Rajinikanth

摘要

This research aims to develop and validate a simple, sensitive, and robust reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the estimation of Alpha-Linolenic acid (ALA) isolated from silkworm pupa oil (SPO) using preparative-HPLC (PR-HPLC). Chromatographic separation was performed on a Shimadzu, C-18 column (4.6 × 250 mm, 5 μm particle size) using acetonitrile/water in a ratio of 80:20 with a flow rate of 1 mL/min and detection at a λmax of 242 nm, equipped with a UV detector. The method demonstrated excellent linearity (R2 = 0.9999, p < 0.05) and good accuracy (98.41–102.10%). It also showed high intra-day and interday precision with RSD values ranging from 0.110% to 0.140%. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.622 µg/mL and 2.05 µg/mL, respectively. System suitability (99.33–100.02%), and robustness evaluation confirmed acceptable recoveries with RSD < 2%. The method was specific for its ability to detect the analytes and their degradation products during forced degradation studies, which also indicated that ALA was highly prone to oxidative degradation (66.55%). The isolation of ALA from SPO was successfully performed based on this developed method using PR-HPLC. Furthermore, the isolated ALA was compared with standard ALA for purity by chromatographic analysis, FTIR and 1H NMR. The results demonstrated that the isolated ALA was pure with negligible interfering impurities within the detection limits of the applied analytical methods. Overall, the results indicated that the developed method was reliable for its accuracy, precision, sensitivity, and specificity for the estimation and isolation of ALA from SPO.