Original Article COMBINING LIQUID CHROMATOGRAPHY WITH TANDEM MASS SPECTROMETRY (LC-MS/MS) TECHNIQUE, SPECTROPHOTOMETERVALIDATION AND AN IN SILICO STUDY OF 96% ETHANOL EXTRACT OF SPIRULINA PLATENSIS TIWUK SUSANTININGSIH 1,2 , FADILAH FADILAH 3* , ANI RETNO PRIJANTI 4 , NOVI SILVIA HARDIANY 5 1 Doctoral Program of Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia. 2 Department of Biochemistry Faculty of Medicine, UPN Veteran Jakarta, Jakarta-12450, Indonesia. 3 Department of Medical Chemistry, Faculty of Medicine, University of Indonesia, Jakarta-10440, Indonesia. 4 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Indonesia, Jakarta-10430, Indonesia. 5 Magister Program of Biomedical Science, Faculty of Medicine, University of Indonesia, Jakarta- 10430, Indonesia * Corresponding author: Fadilah Fadilah; * Email: fadilah.msi@ui.ac.id Received: 07 May 2024, Revised and Accepted: 20 Jun 2024 ABSTRACT Objective: This study was to analyze the component sofa 96% ethanol extract of Spirulina platensis by the LC-MS/MS technique, then validate them with the spectrophotometer technique using the C-phycocyanin standard and an in silico study approach as an antioxidant property of S. platensis against inflammatory. Methods: Chromatographic resolution was attained with a Phenominex C18 (50 mm×2.6 mm, 3 µm) stationary column technique, validation using C-phycocyanin standard using the spectrophotometer technique, and an in silico study of c-phycocyanin using molecular docking analysis. Results: Tentative active compounds such as flavonoid (Maltol and Morin), peptide (Cyclo Pro-Ala, Cyclo Pro-Pro, and Thymine), and phenol (m- Aminophenol, N-Methyltyramine, and Tyramine) have been identified from a 96% ethanol extract of S. platensis by LCMS/MS analysis. The concentration of c-phycocyanin in the 96% ethanol extract of S. platensis is 229, 2µg/ml. According to our in silico study, c-phycocyanin demonstrates potential as an anti-inflammatory agent. Conclusion: The LC-MS/MS technique can detect flavonoid, peptide, and phenolic components in the 96% ethanol extract of S. platensis. A spectrophotometer can identify the validation equation of c-phycocyanin in a 96% ethanol extract of S. platensis. Based on our in silico study, c- phycocyanin demonstrate the capability to prevent inflammatory activity. Keywords: C-phycocyanin, In silico study, LC-MS/MS, Spectrophotometer, Spirulina platensis © 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2024v16i5.51339 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Spirulina platensis is a cyanobacterium filamentous microalga that contains various flavonoids, among other phytochemicals. These flavonoids enhance antioxidant activity, potentially offer protection against oxidative stress and inflammation [1, 2], and anticancer properties [3, 4]. Spirulina platens scan be used as a preventive supplement since it is high in protein, carbohydrates, polyunsaturated fatty acids, sterols, and other essential components [5, 6]. Spirulina platensis contains 60-70% dry-weight protein [7], essential amino acids, carotenoids, lipids, vitamins E, C, and selenium [8, 9]. It has been reported that the consumption of S. platensis could prevent or manage metabolic syndrome disorders [10-12]. Spirulina platensis has long been utilized for nutrition in Mexico, Africa, and Asia, especially in Indonesia. In Indonesia, S. platensis is cultivated using freshwater aquaculture. Spirulina platensis and its pigments such as c-phycocyanin, carotene, xanthophyll, and chlorophyll, exhibit exogenous antioxidant properties [13]. C-phycocyanin, rich in blue-green microalgae, is known to break the radical chain to inhibit reactive oxygen species and oxidative stress, act as a hepatoprotection, and help reduce blood glucose levels. C-phycocyanin can reduce free radicals, minimize nitrite production, suppress nitric oxide synthase (iNOS) expression, and inhibit lipid peroxidation [2, 14, 15]. Because c- phycocyanin is soluble in water, cells must first be disrupted using physical or chemical methods to extract the pigment [16-18]. C- phycocyanin prevents oxidative stress and cell damage in vitro in the hypoxia model employing the myoblast cell line H9c2 [19, 20]. This study aimed to explore and identify the most potent S. platensis compounds from the Indonesian medicinal algae. Using liquid chromatography coupled with mass spectrophotometry (LC-MS/MS) for precise identification and quantification, the study developed and validated an LC-MS/MS analytical procedure. The validation included a spectrophotometric analysis using a c-phycocyanin standard. Additionally, the in silico research was conducted to evaluate the antioxidant properties of c-phycocyanin from S. plarensis against oxidative stress and inflammation. MATERIALS AND METHODS Extraction of spirulina platensis S. platensis powder from PT Algae park, Klaten-Indonesia (1000 g) was macerated using 96% ethanol and left to stand for 48 h at room temperature. The solution was then filtered to obtain precipitate, which was further macerated using 96% ethanol. A rotary evaporator was used to evaporate the solvent, which was then freeze-dried. Materials The tools used for the in silico study are a laptop (HP ENVY x360 13- AG0023AU). AMD Ryzen 7 2700 8GB 1TB Win 13 (Logical Processors 8) 8GB of memory RAM, a 64-bit operating system, an x64-based processor, and an Autodock 1.5.7 System. Micropipette 1000 µl**, 1.5 ml microtubes, tips, 96-well plate Biologyx, glassware, analytical balance (Sartorius). The chemicals used were Ethanol (BrataChem), Water proinjection (Sigma Aldrich), and a spectrophotometer (Varisocan, Thermo). LCMS/MS analysis of a 96% ethanol extract of Spirulina platensis The liquid chromatography-tandem mass spectrophotometry (LC- MS/MS) was conducted in PT Saraswanti INDO GENETECH, Bogor- Indonesia, under certificate number: SIG. LHP. VII.2023.211434171. the methodology employed was based on protocol 18-16/MU/SMM/- International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 16, Issue 5, 2024