Preparation of 4,4-Dimethyloxazoline and Pyrrolidine Derivatives from Fatty Acid Methyl Esters Using Sodium Borohydride: Mild and Simple One-Pot Derivatization Procedures for a Gas Chromatographic–Mass Spectrometric Analysis of Fatty Acids

Abstract

Procedures for preparing 4,4-dimethyloxazoline (DMOX) and pyrrolidine derivatives from fatty acid methyl esters (FAMEs) were modified to provide milder and simpler conditions for the derivatization reactions widely used in the mass spectrometric analysis of fatty acids. DMOX and pyrrolidine derivatives were obtained overnight at room temperature in the presence of sodium borohydride. The proposed method, involving a low-temperature condensation-cyclization of FAME to DMOX, allows for the direct preparation of DMOX derivatives from non-hydroxy, 2- and 3-hydroxy acid methyl esters. The described simple one-pot procedures are “mild” alternatives to existing methods, which require the use of elevated temperatures.

Introduction

Methyl esters, 4,4-dimethyloxazolines (or more precisely, 4,4-dimethyl-2-oxazolines), and pyrrolidides are among complementary derivatives that are often used in the structural analysis of fatty acids by GC–MS. Although fatty acid methyl esters (FAMEs) are used almost universally for GC separations and often for MS, they provide only limited structural information. 4,4-Dimethyloxazolines and pyrrolidides give much more specific MS data that make it possible to locate double bonds, methyl branches, and other substituents. In spite of having different structures, 4,4-dimethyloxazoline (DMOX) and pyrrolidine derivatives of a given fatty acid have identical molecular weights and give similar fragmentation patterns with electron impact ionization (EI). ¹ These N-containing derivatives can be prepared in a one-pot reaction from FAMEs. In particular, pyrrolidide can be prepared by reaction of FAME with pyrrolidine in the presence of acetic acid (at 100 °C for 0.5‒1 h), ² and the applied reaction conditions are considered to be relatively mild. ³ The typical reaction conditions for the formation of a DMOX derivative from FAME with 2-amino-2-methyl-1-propanol (AMP) involve higher temperature and extended reaction time (overnight at 180 °C), ⁴ but often provide an incomplete reaction of the ester. ³ Expectedly, the prolonged high temperature presents a problem with polyenes and other compounds containing potentially labile functional groups. For some such FAMEs, an alternative two-step procedure may be used. ⁵ It involves preparing hydroxyamide by aminolysis of FAME (with solutions of AMP and sodium methoxide [CH₃ONa] at room temperature, overnight), followed by cyclization to DMOX (with trifluoroacetic anhydride at 50 °C for 45 min). However, an attempt to prepare a DMOX derivative from the methyl ester of a 2-hydroxy fatty acid by the suggested method was unsuccessful. ⁵ At present, DMOX derivatives of 2-hydroxy acids are prepared from free 2-hydroxy acids, as described earlier,^6,7 although even a mild two-step procedure ⁷ does not always provide successful results. ⁸ Additionally, the derivatization of free fatty acids is generally less suitable than the derivatization of the corresponding FAMEs. Indeed, it is often convenient to analyze new samples first as methyl esters before progressing to alternatives, and, therefore, methanolysis is extensively used in lipid analysis.

To develop simpler protocols for preparing DMOX and pyrrolidine derivatives without heating samples, we studied transformations of some monoenoic, polyunsaturated, and hydroxy FAMEs with AMP and pyrrolidine in the presence of sodium borohydride (NaBH₄). NaBH₄ is known to be a mild and inexpensive reagent for applications in a wide range of reduction processes, but it does not reduce esters and amides under ambient conditions. Like CH₃ONa, NaBH₄ catalyzes transesterification ⁹ but, to the authors’ knowledge, borohydride has never been applied to the low-temperature aminolysis of esters. In the present work, we report the use of NaBH₄ for the one-pot conversion of FAMEs into corresponding DMOX or pyrrolidine derivatives under mild conditions with high yields. The optimum conditions found for the transformations of methyl oleate are described. FAMEs, obtained from cod-liver oil and sphingolipids of marine sponges, are used to demonstrate that these conditions are applicable to the preparation of the N-containing derivatives of polyunsaturated and 2-hydroxy acids. Finally, we report the result with synthetic methyl 3-hydroxytetradecanoate.

Results and Discussion

We prepared DMOX and pyrrolidine derivatives from several FAMEs and mixtures of FAMEs using protocols detailed in the Experimental section and illustrated in Figure 1.

Figure 1.

Preparation of DMOX and pyrrolidine derivatives from FAMEs using sodium borohydride NaBH₄.

After a series of initial experiments, it was established that 15:1 was the effective molar ratio of NaBH₄/ester necessary for approximately 100% yield of not only pyrrolidide, but also DMOX derivative. For example, in the aminolysis of methyl oleate by pyrrolidine (secondary amine), NaBH₄/ester ratios required to attain 31%, 57%, 87%, and 100% yields of the corresponding pyrrolidide were 3:1, 7:1, 10:1, and 15:1, respectively. Although the NaBH₄/ester ratio of 3:1 was sufficient for the complete aminolysis of methyl oleate by AMP (primary amine) in pyridine, the higher ratio of 15:1 was necessary to obtain the final DMOX product in high yield.

Table 1 shows the effect of the reaction time on the percent yield of the DMOX and pyrrolidine derivatives of oleic acid. Methyl oleate was converted to the N-containing cyclic derivatives in high yields after 17 h. Amines and ester were used in proportions similar to those for sodium methoxide (AMP) ⁵ and acetic acid (pyrrolidine)^2,3 catalyzed aminolysis.

Table 1.

Reaction Times and Yields of DMOX and Pyrrolidine Derivatives Obtained by Reacting Methyl Oleate with AMP and Pyrrolidine, Respectively, in the Presence of NaBH₄ at Room Temperature.

Time (h)	DMOX yield (%) a	Time (h)	Pyrrolidide yield (%) a
1	6	1	3
3	22	3	13
5	26	5	38
7	37	7	50
17	99	17	100
24	100

^a

Reactions were monitored by GC.

As mentioned above, a solution of CH₃ONa in methanol has been used for the basic catalysis of aminolysis of FAMEs by AMP. ⁵ Another step (cyclodehydration) has been necessary to cyclize the resulting hydroxyamides to 4,4-dimethyl-2-acyl-2-oxazolines, as in other methods for the preparation of DMOX derivatives.^7,10 In the present study, powdered NaBH₄ that was applied as a catalyst (catalyst precursor) for the low-temperature aminolysis of FАМЕs (Figure 1), promoted not only ester aminolysis (in the case of DMOX and pyrrolidine derivatives), but also cyclodehydration (in the case of DMOX derivatives). As a result, we observed one-pot and direct conversion of FAMEs into corresponding oxazolines under mild reaction conditions. The dehydrating effect of NaBH₄ was also revealed in the derivatization reactions of methyl 8-hydroxyelaidate that tended to lose water and, therefore, produce compounds with conjugated double bonds. In particular, methyl 8-hydroxyelaidate reacted with АМP in pyridine in the presence of NaBH₄ to give a mixture of DMOX products containing some conjugated dienes (presumably cis/trans-isomers) and no allylic hydroxy compounds, according to GC–MS data. The loss of water from allylic alcohol also took place when methyl 8-hydroxyelaidate was treated with pyrrolidine in the presence of NaBH₄.

Apparently, the cyclodehydration to DMOX derivatives with NaBH₄ was quite rapid under anhydrous conditions. When AMP–pyridine and NaBH₄, used in the preparation of the DMOX derivative of oleic acid, were completely dry, we did not observe a GC peak for intermediate hydroxyamide. In this case, only two GC peaks, representing methyl oleate (in gradually decreasing amounts) and the corresponding DMOX derivative (in gradually increasing amounts) were seen with an increase in time. A third GC peak (broad), which represented later eluting hydroxyamide of oleic acid, could be observed when the reagents and/or solvents were not fresh. In particular, major hydroxyamide and minor 4,4-dimethyloxazoline were formed from methyl oleate after long-term use of the AMP‒pyridine mixture (about 3 months). The best yields of the DMOX derivative were obtained when commercial AMP and pyridine from freshly opened bottles were used. Therefore, solvents, samples, and reagents should be as dry as possible. To prevent the intense condensation of air moisture on the reagent surface during the weighing of hygroscopic NaBH₄, the total moisture level in the room should be low (approximately 50% or less).

The formation of pyrrolidides (aminolysis) in the presence of NaBH₄ was less sensitive to moisture than the DMOX formation (aminolysis and cyclodehydration). For example, in contrast to acid-catalyzed aminolysis,^2,3 it was not necessary to use freshly distilled pyrrolidine.

DMOX and Pyrrolidine Derivatives of Fatty Acids from Cod-Liver Oil

Cod-liver oil is known to contain relatively high amounts of potentially labile polyunsaturated fatty acids. The proposed protocols (Experimental section, Figure 1) were used to synthesize DMOX and pyrrolidine derivatives from FAMEs obtained from cod-liver oil. It was found that the GC profile of these FAMEs was similar to those of the resulting N-containing derivatives. In particular, GC–MS analyses showed that the 20:5/20:1/22:6 ratios were 1.0/1.7/1.0 for starting FAMEs, 1.0/2.0/1.0 for DMOX derivatives, and 1.0/1.8/1.0 for pyrrolidides. Therefore, the derivatization in the presence of NaBH₄ did not lead to significant degradation of such polyunsaturated components.

We tried to convert triacylglycerols of cod-liver oil directly into pyrrolidine and DMOX derivatives under the reaction conditions used (Figure 1), but these attempts were not successful. Apparently, when the direct transformation of triacylglycerols into the N-containing derivatives is to be conducted, a procedure including CH₃ONa catalyzed aminolysis ⁵ is advantageous over the present procedure.

DMOX and Pyrrolidine Derivatives of 2-Hydroxy Fatty Acids and Their Mass Spectra. 2-Hydroxy Fatty Acid Composition of Ceramides from the Sponge Monanchora clathrata

It is known that 2-hydroxy fatty acids are common lipid components and are especially important constituents of animal sphingolipids. ¹¹ At present, the known DMOX derivatives of 2-hydroxy fatty acids include those of five acids, namely, 2-hydroxy-18:0, 2-hydroxy-18:1Δ9, 2-hydroxy-18:2Δ9,12, 2-hydroxy-9,10-methylene-18:1Δ9, ¹² and 2-hydroxy-18:3Δ9,12,15. ⁸ These derivatives were prepared from free fatty acids. We were able, for the first time, to synthesize DMOX derivatives of 2-hydroxy fatty acids from corresponding FАМЕs. In general, FAMEs obtained from the sphingolipids of the marine sponges Monanchora clathrata (Figure 2a, Supplemental Scheme S1, and Supplemental Table S1) and Aulosaccus sp. ¹³ were used as starting materials. The total fatty acid profile of ceramides from M. clathrata has not been studied previously. In the EI mass spectra of the resulting DMOX derivatives of six saturated 2-hydroxy С₂₁-С₂₆ acids and two monounsaturated 2-hydroxy С₂₂ and C₂₄ acids (Figures 2b and 3, Supplemental Figures S1-S8), the presence of the 2-hydroxy group was indicated by peaks at m/z 129 (base peak) and 142. These signals have already been reported in EI mass spectra for DMOX derivatives of 2-hydroxy acids^8,14 (in contrast, DMOX derivatives of non-hydroxy acids would present characteristic peaks at m/z 113 and 126 ¹ ). However, the early studies indicated that the mass spectra of the DMOX derivatives of 2-hydroxy C₁₈ acids showed m/z 142 as a base peak and m/z 129 as the second most intense peak. The difference in ion intensities between this study and previous ones^8,14 can probably be explained by the different technical characteristics of GC‒MS equipment.

Figure 2.

(A) GC profile of 2-hydroxy FAMEs (Table S1) and total ion chromatograms (GCMS-QP2020) of (B) DMOX and (C) pyrrolidine derivatives of 2-hydroxy fatty acids obtained from ceramides of the sponge M. clathrata. For the DMOX derivatives, ◊ ‒ unreacted 2-hydroxy FAMEs, × ‒ presumably, boron-containing derivative of 2-hydroxy-22:0.

Figure 3.

Mass spectra (Hewlett-Packard HP6890 GC system) of the DMOX derivatives of (A) 2-hydroxydocosanoic and (B) 2-hydroxydocos-15-enoic acids.

A significant peak at m/z 184 was seen in all the mass spectra of the DMOX derivatives of 2-hydroxy acids illustrated here (Figure 3, Supplemental Figures S1-S8). The ion at m/z 184 could be formed by cleavage from the radical on position C-6, as described for the relatively abundant ion at m/z 168 formed from the DMOX derivatives of some non-hydroxy fatty acids. ¹⁵ We also found that the mass spectra contained [M‒C₂H₅O]⁺ ([M–45]⁺) ions, characteristic of the fragmentation patterns of the known DMOX derivatives of 2-hydroxy C₁₈ acids.^14,16 Apparently, such ions were formed via an α-OH-assisted extrusion mechanism that was reported for the fragmentation of the DMOX derivatives of deuterated 2-hydroxy-18:0 in combined in-beam electron impact (IBEI)-B/E-linked scan mass spectrometry. ¹⁷ In addition to the [M‒C₂H₅O]⁺ ions, we observed homologous fragments [M‒C₃H₇O]⁺, [M‒C₄H₉O]⁺, [M‒C₅H₁₁O]⁺, [M‒C₆H₁₃O]⁺, etc, in the spectra of the DMOX derivatives of saturated 2-hydroxy acids (Figure 3a, Supplemental Figures S1-S7). For example, the mass spectrum of the DMOX derivative of 2-hydroxydocosanoic acid (Figure 3а, Supplemental Figure S2: 409 [M]⁺) showed a prominent peak at m/z 364 ([M‒C₂H₅O]⁺/[M–45]⁺), along with homologous peaks of lower intensities, which represented the ions of m/z 350, 336, 322, and 308. Thus, due to the possible expulsions of the different hydroxyl-containing fragments, common even-mass homologous series m/z 142 + 14n could not be clearly observed in the high-mass region of this mass spectrum.

The DMOX derivatives of monoenoic 2-hydroxy fatty acids (Figure 3b, Supplemental Figure S8) tended to give more abundant ions for the loss of successive methylene groups in comparison to 4,4-dimethyl-2-acyl-2-oxazolines derived from saturated 2-hydroxy acids. As a result, the mass spectra of the DMOX derivatives of monoenoic 2-hydroxy acids permitted the facile location of a double bond using a “rule of the gap of 12 amu,” ⁶ as described earlier. ¹⁴ [M‒C₂H₅O]⁺/[M–45]⁺ peaks in the spectra of these compounds were less prominent.

In comparison with DMOX derivatives, pyrrolidides are easier to prepare from 2-hydroxy FAMEs, and a variety of the mass spectra of 2-hydroxy acyl pyrrolidides was published for illustrative purposes. ⁸ Although DMOX derivatives of fatty acids tend to give more abundant diagnostic ions and have better GC properties than fatty acid pyrrolidine derivatives, pyrrolidides are better for the determination of terminal positions of methyl branches. ¹⁸ To identify methyl-branched component(s), 2-hydroxy FAMEs from M. clathrata were converted to their corresponding pyrrolidides (Figure 2c), as described in the Experimental section below. The mass spectra of the pyrrolidides of normal-chain and iso-methyl-branched 2-hydroxy C₂₃ acids (Supplemental Figure S9: 423 [M]⁺) were used to distinguish these isomers. In the spectrum of the pyrrolidine derivative of 2-hydroxy-iso23:0, the diagnostic gap of 28 amu between peaks at m/z 408 ([M‒CH₃]⁺) and 380 ([M‒CH(CH₃)₂]⁺) located the methyl branch at position 21. In the spectrum of the pyrrolidine derivative of unbranched 2-hydroxy-23:0, the last significant ion before the molecular ion was at m/z 406, representing a loss of 17 amu for the hydroxyl moiety, and not m/z 408 from loss of the terminal methyl group.

Mass Spectrum of the DMOX Derivative of 3-Hydroxy Fatty Acid

Earlier, attempts to prepare DMOX derivatives from 3-hydroxy fatty acids, commonly produced by yeasts and bacteria, ¹¹ were unsuccessful. ⁸ Using the “borohydride” method, we were able to prepare a DMOX derivative from methyl 3-hydroxytetradecanoate. The mass spectrum of the resulting DMOX derivative (Figure 4) is characterized by prominent peaks at m/z 98, 113, and 142. These fragment ions have also been detected in the mass spectrum of the corresponding pyrrolidide. ⁸ However, unlike this pyrrolidide, the DMOX derivative of 3-hydroxytetradecanoic acid produces discernible [M–CH₃–H₂O]⁺ and [M–45]⁺ ions. Additionally, its mass spectrum (Figure 4) shows a variety of minor peaks with steps of 14 m/z due to fragmentations of the [M]⁺ ion. In contrast, the mass spectrum of the pyrrolidide of 3-hydroxytetradecanoic acid ⁸ exhibits homologous ions, which are apparently fragments of the [M–H₂O]⁺ ion.

Figure 4.

Mass spectrum (GC-2010 Plus) of the DMOX derivative of 3-hydroxytetradecanoic acid.

By-Products of DMOX Formation

Hydroxyamides, arising during DMOX formation (aminolysis) and during storage of DMOX derivatives (hydrolysis), may interfere with subsequent GC–MS analyses. Earlier, it was reported that the mass spectra of the DMOX derivative and corresponding uncyclized intermediate (hydroxyamide) were almost identical. ³ However, we found that such mass spectra were quite different. For example, the mass spectrum of the hydroxyamide of oleic acid (Supplemental Figure S10; see also Supplemental Figure S11) did not contain prominent ions at m/z 113 and 126, characteristic of DMOX derivatives of non-hydroxy fatty acids. ¹ Therefore, we assume that the MS data reported for an alleged hydroxyamide ³ were inconsistent with the suggested structure. The mass spectra of the hydroxyamides of 2- and 3-hydroxy acids may also be found for comparison purposes in Supplemental Material (Supplemental Figures S12-S17). Other by-products, arising during the reaction between 2-hydroxy FAMEs and AMP in pyridine in the presence of NaBH₄, presumably included minor boron-containing DMOX derivatives (Supplemental Figure S18) and DMOX congeners (Supplemental Figures S19 and S20).

Conclusion

This study described the modifications of procedures used in GC–MS analysis for preparing DMOX and pyrrolidine derivatives from FAMEs. High yields of DMOX and pyrrolidine derivatives were obtained overnight (17-18 h) at room temperature by reactions of FAMEs with AMP and pyrrolidine, respectively, in the presence of NaBH₄. Using the above-described method, we were able, for the first time, to prepare DMOX derivatives from the methyl esters of 2- and 3-hydroxy fatty acids. As examples, DMOX and pyrrolidine derivatives were used to analyze the 2-hydroxy fatty acid composition of ceramides from the sponge M. clathrata. The methods described herein were mild enough to be used with polyunsaturated fatty acids from cod-liver oil. The simple one-pot procedures proposed for preparing the N-containing derivatives from FAMEs with inexpensive and readily available NaBH₄ are “mild” alternatives to the existing methods that require the use of elevated temperatures. The authors believe that, in the future, attempts should be made to use NaBH₄ as a catalyst for transesterification in preparing 3-pyridylcarbinol (picolinyl) esters ¹ from FAMEs and 3-(hydroxymethyl)pyridine.

Experimental

Chemicals

2-Amino-2-methyl-1-propanol, pyridine, and sodium borohydride were purchased from Sigma-Aldrich (Steinheim, Germany), pyrrolidine from Aldrich (Steinheim, Germany), and oleic acid methyl ester from Fluka Chemie (Buchs, Switzerland). Methyl (15Z)-2-hydroxydocos-15-enoate was obtained in the study of cerebrosides from a sponge Aulosaccus sp. ¹³ Methyl 8-hydroxyelaidate was prepared from methyl oleate using SeO₂ (allylic hydroxylation). ¹⁹ Racemic 3-hydroxytetradecanoic acid was received as a gift from Dr V.I. Gorbach (G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Russia). Cod-liver oil was purchased from a local pharmacy.

Instrumentation and Chromatographic Materials

The GC–MS analyses of FAMEs, DMOX, and non-hydroxy pyrrolidine derivatives of fatty acids were carried out on a Hewlett-Packard HP6890 GC System (Hewlett-Packard Company, Palo Alto, CA, USA) with an HP-5MS (J&W Scientific, Folsom, CA, USA) capillary column (30.0 m × 0.25 mm), helium as the carrier gas, and 70 eV ionizing potential. The GC–MS analyses were conducted using an injector temperature of 270 °C and a temperature program of 100 °C (1 min) − 10 °C/min − 280 °C (30 min). The GC–MS analyses of the hydroxyamide of oleic acid and the pyrrolidine and DMOX derivatives of saturated 2- and 3-hydroxy fatty acids were carried out on gas chromatograph–mass spectrometers GCMS-QP2020 (Shimadzu, Kyoto, Japan) with an Rtx-5MS (Restek, Bellefonte, PA, USA) capillary column (30.0 m × 0.25 mm × 0.25 μm) and GC-2010 Plus (Shimadzu, Kyoto, Japan) with an SLB-5MS (Supelco, Bellefonte, PA, USA) capillary column (30.0 m × 0.25 mm × 0.25 um), helium as the carrier gas, and 70 eV ionizing potential. The GC–MS analyses of pyrrolidides were performed using an injector temperature of 280 °C and a temperature program starting from 220 °C to 300 °C, at 2 °C/min. For the GC–MS analyses of DMOX derivatives, an injector temperature of 270 °C was used and the temperature program was from 220 °C to 280 °C, at 2 °C/min. GC analyses were performed using an Agilent 6850 Series GC System chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with either an HP-1 (Agilent Technology, Santa Clara, CA, USA) or DB-1 (J&W Scientific, Folsom, CA, USA) capillary column (30 m × 0.32 mm). For GC, the carrier gas was helium (flow rate 1.7 mL/min), the detector and injector temperatures were 300 °C and 270 °C, respectively, and the temperature program was 100 °C (1 min) − 10 °C/min − 280 °C (30 min). NMR spectra (CDCl₃, C₅D₅N) were recorded on Bruker Avance 300 (¹H, at 300 MHz), Bruker Avance III HD 500 (¹H, ¹³C, HMBC, at 500 MHz), and Bruker Avance III 700 (¹H, at 700 MHz) spectrometers (Bruker BioSpin, Germany). TMS was used as an internal reference standard. Column chromatography was carried out on Sephadex LH-20 (GE Healthcare, Sweden) and silica gel (50/100 μm, Sorbpolimer, Krasnodar, Russia).

Preparation of FAME Samples

A mixture of FAMEs, containing the methyl esters of polyunsaturated acids, was obtained from cod-liver oil using base-catalyzed transesterification with KOH in MeOH, ²⁰ followed by column chromatography (SiO₂: n-hexane–diethyl ether, 95:5, v/v). The methyl esters of 2-hydroxy fatty acids were obtained by methanolysis of the ceramide fraction isolated from the ethanol extract of the sponge Monanchora clathrata (for more details, see Supplemental Material). Methylation of 3-hydroxytetradecanoic acid (with 1% H₂SO₄ in MeOH, 50 °C, 1 h) followed by purification using column chromatography (SiO₂: n-hexane–ethyl acetate, 10:1, v/v) gave methyl 3-hydroxytetradecanoate (for ¹H NMR and MS data, see Supplemental Material).

Preparation of DMOX Derivatives from FAMEs Using NaBH₄

FAMEs (1.0-3.0 mg) and NaBH₄ (2.0-5.0 mg, molar NaBH₄ to ester ratio, 15) were placed in a screw cap vial (4 mL vol), and AMP–pyridine (1:1, v/v, 0.1 mL) was added. The components were carefully mixed, and the reaction mixture (suspension) was maintained for 17‒18 h (overnight) at room temperature (usually 25-26 °C). Then water (0.5-1.0 mL) and n-hexane (0.5-1.0 mL × 3, mainly used for the derivatives of non-hydroxy acids), or n-hexane–diethyl ether (1:1, v/v, 0.5-1.0 ml × 3, for the derivatives of 2- and 3-hydroxy acids), were added. The addition of water caused the evolution of small hydrogen gas bubbles. The layers of organic solvent were collected and evaporated in vacuo to yield a dry residue. The residue was dissolved in either n-hexane or ethyl acetate for GC–MS analysis. Note: DMOX derivatives of 2- and 3-hydroxy acids should be analyzed immediately after their preparation because these compounds were found to be much more labile than DMOX derivatives of non-hydroxy acids.

Preparation of Pyrrolidine Derivatives from FAMEs Using NaBH₄

FAMEs (1.0-3.0 mg), NaBH₄ (2.0-5.0 mg), and pyrrolidine (0.3 mL) were mixed and maintained for 17-18 h (overnight) at room temperature in a screw cap vial (4 mL vol). NaBH₄ swelled in contact with pyrrolidine. Water (0.7 mL), n-hexane (1.0 mL × 3), or n-hexane–diethyl ether (1:1, v/v, 1.0 mL × 3, instead of n-hexane) were added, and pyrrolidides were obtained following the same procedure described for DMOX derivatives. Samples were dissolved in either CHCl₃ or ethyl acetate for GC–MS analyses.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221131408 - Supplemental material for Preparation of 4,4-Dimethyloxazoline and Pyrrolidine Derivatives from Fatty Acid Methyl Esters Using Sodium Borohydride: Mild and Simple One-Pot Derivatization Procedures for a Gas Chromatographic–Mass Spectrometric Analysis of Fatty Acids

Supplemental material, sj-docx-1-npx-10.1177_1934578X221131408 for Preparation of 4,4-Dimethyloxazoline and Pyrrolidine Derivatives from Fatty Acid Methyl Esters Using Sodium Borohydride: Mild and Simple One-Pot Derivatization Procedures for a Gas Chromatographic–Mass Spectrometric Analysis of Fatty Acids by Elena A. Santalova and Vasily I. Svetashev in Natural Product Communications