*Abstract* **Idea of Rapid Preparation of Fatty Acid Methyl Ester Using In Situ Derivatization from Fresh Horse Mussel †**

**Siriluck Pojjanapornpun 1,\*, Apinya Cheewaphan 2, Akkaradech Nakornsadet 3, Salisa Chumsantea 3, Piraporn Sombutsuwan 3, Kanit Krisnangkura <sup>3</sup> and Kornkanok Aryusuk <sup>3</sup>**


**Abstract:** The analysis of the fatty acid (FA) profile requires multiple preparation steps, which are lipid extraction followed by derivatization of the FA into a fatty acid methyl ester (FAME). The procedures are time-consuming, and generally require large volumes of sample sizes and solvents. This report proposes a technique for the preparation of FAME from fresh horse mussels without a step of lipid extraction. A rapid in situ derivatization using *N*,*N*-dimethylformamide dimethyl acetal (DMF-DMA) methylation followed by alkali-transesterification was examined. In this method, acylglycerols and free fatty acids (medium to long-chain FA) of the sample are targeted to convert into FAME. Direct alkali-transesterification of the fresh sample gave only 58.7% FAME with 12.4% triglyceride and 21.1% FFA. The alkali in situ method showed low conversion efficiency due to the initial sample containing high contents of moisture and FFA (75.11% of the fresh sample and 14.3% of total oil, respectively). The reaction was developed by using two steps in situ derivatization. A 50 mg sample was methylated with 1 mL of DMF-DMA (100 ◦C, 15 min), followed by transesterified with 10 mL of 1% (*w*/*v*) NaOH in methanol (60 ◦C, 3 min). The conversion into FAME was monitored using size-exclusion HPLC with evaporative light-scattering detection. The column was a 100 Å Phenogel with toluene and 0.25% acetic acid as a mobile phase. The FAME yield of 79.9% with 7.8% triglyceride and 8.5% FFA was obtained. The two steps in situ derivatization gave a promising result with the higher conversion with lower FFA. It is a simple and rapid (less than 20 min) method that requires a low volume of sample and solvent for FAME preparation. However, increasing the conversion efficiency as well as the variety of samples should be further studied.

**Keywords:** fatty acids; fresh sample; in situ transesterification; methylation; *N*,*N*-dimethylformamide dimethyl acetal

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/ASEC2021-11142/s1. S1: Idea of rapid preparation of fatty acid methyl ester using in situ derivatization from fresh horse mussel.

**Funding:** The authors acknowledge King Mongkut s University of Technology Thonburi for funding the KMUTT Research Center of Excellence Project to Lipid Technology Research Group.

**Institutional Review Board Statement:** Not applicable, as commercial samples were studied.

**Informed Consent Statement:** Not applicable.

**Citation:** Pojjanapornpun, S.; Cheewaphan, A.; Nakornsadet, A.; Chumsantea, S.; Sombutsuwan, P.; Krisnangkura, K.; Aryusuk, K. Idea of Rapid Preparation of Fatty Acid Methyl Ester Using In Situ Derivatization from Fresh Horse Mussel. *Eng. Proc.* **2021**, *11*, 7. https://doi.org/10.3390/ ASEC2021-11142

Academic Editor: Samuel Adeloju

Published: 15 December 2021

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**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
