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Article
Concentrations, Stability and Isolation of the Furan Fatty Acid 9-(3-Methyl-5-
Pentylfuran-2-yl)-Nonanoic Acid (9M5) from Disposable Latex Gloves
Marco Müller, Melanie Hogg, Kerstin Ulms, and Walter Vetter
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02444 • Publication Date (Web): 18 Aug 2017
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Page 1 of 28 Journal of Agricultural and Food Chemistry
1 Concentrations, Stability and Isolation of the Furan Fatty Acid 9-(3-Methyl-5-
2 Pentylfuran-2-yl)-Nonanoic Acid (9M5) from Disposable Latex Gloves
4 Marco Müller, Melanie Hogg, Kerstin Ulms and Walter Vetter*
7 University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry
8 (170b), D-70593 Stuttgart, Germany
10
11
12 * Corresponding author
13 Phone: +49 711 459 24016
14 Fax: +49 711 459 24377
15 Email: walter.vetter@uni-hohenheim.de
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ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry Page 2 of 28
16 ABSTRACT
17 Because of their antioxidant properties, furan fatty acids (furan-FA) are valuable
18 minor compounds with a widespread occurrence in all living matter. Unfortunately, pure
19 standards are not readily available, as they usually contribute only 1% to the lipid fraction. A
20 known exception of this is the milky fluid of Hevea brasiliensis, commonly known as latex,
21 in which the furan-FA 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5) contributes with
22 about 90% to the triacylglycerides. In this study, we investigated the content of 9M5 in 30
23 different disposable latex gloves, which ranged from 0.7 to 8.2 mg/g glove. The light
24 degradability of 9M5 in latex gloves was investigated and different amounts of 9M5 in
25 disposable latex gloves were attributed to varying exposure time to light. Additionally, over
26 100 mg of the methyl or ethyl ester of 9M5 (purity >98%) could be extracted from disposable
27 latex gloves, employing cold extraction and silver ion chromatography. With this method,
28 standards for the quantitation of furan-FA are obtained easily and rapidly in all laboratories.
29
30 KEYWORDS
31 Furan fatty acids, disposable latex gloves, 9M5, Hevea brasiliensis, GC/MS
2
32 INTRODUCTION
33 Furan fatty acids (furan-FA) are a group of naturally occurring fatty acids which are
34 characterized by a furan ring in the center of the molecule. The furan moiety is substituted
35 with a straight-chain (usually 9, 11 or 13 carbons) saturated acid in α-position and a short
36 alkyl chain (usually 3 or 5 carbons) in α'-position (Figure 1).1 A typical example for furan-FA
37 is 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5), 1, which is substituted with one
38 methyl group at the β-position (M). Other naturally occurring furan-FA possess two methyl
39 groups at the β- and β'-positions (D), while furan-FA without methyl groups (F) are rarely
40 found in nature.2
41 Furan-FA are de novo synthesized by various plants and bacteria and can be
42 incorporated into the lipids of animals.1,3 Hence, they are found widespread in all living
43 matter, albeit at low concentrations.1,3–6 Because of their high radical scavenging activity,
44 they are potent antioxidants.7,8 Due to this property they are thought to protect
45 polyunsaturated fatty acids (PUFA) from oxidative stress and support them in their beneficial
46 effects on the prevention of cardiovascular diseases.1
47 Furan-FA are easily transformed into volatile compounds by exposure to light which
48 were found to cause off-flavor in spinach,9 soybean oil10 as well as dried herbs and
49 vegetables.11 In addition, the potential furan-FA metabolite 3-carboxy-4-methyl-5-propyl-2-
50 furanpropionic acid, 2, was found to be significantly high in blood plasma of patients with
51 type 2 diabetes.12,13 2 was also found to enhance type 2 diabetes by hampering the insulin
52 biosynthesis in β-cells.14 However, high amounts of fish in the diet only moderately increased
53 the plasma concentration of 2 and it could not be linked to harmful glucose metabolism.13
54 To gain further knowledge of the biological function of furan-FA, pure standards are
55 needed to perform tests concerning their biological activity. Unfortunately, isolation of furan-
56 FA from natural sources is non-economic because they usually contribute less than 1% to the
3
57 total fatty acids of plant and animal lipids.1,2,15 Hitherto, the only relevant exception is the
58 milky fluid of Hevea brasiliensis, commonly known as latex. About 90% of the fatty acid
59 pattern of latex originated from the furan-FA 9M5, 1.16,17 Recently, isolation of pure 9M5, 1,
60 from liquid centrifuged latex was achieved by countercurrent chromatography (CCC).17 This
61 isolate is suited for the use as an analytical standard in furan-FA analysis15,18,19 and may also
62 serve as substrate for in vitro investigations of the relevance of furan-FA. However, the
63 isolation procedure is not only time consuming,17 but also CCC instruments and the milky
64 latex fluid are not readily available to all researchers interested in such studies.17
65 The goal of this study was to select a more convenient source and isolation procedure
66 for 9M5, 1, For this purpose, we studied its occurrence in disposable latex gloves, i.e. one
67 readily available industrial product made from latex. Disposable latex gloves are widely
68 distributed in medical and chemical laboratories, and samples from different producers were
69 screened for the presence of 9M5, 1, and possibly other furan-FA. We also studied the
70 stability of 9M5, 1, when disposable latex gloves were removed from the (light-protecting)
71 packaging and exposed to daylight. Finally, we present an easy and fast method for the
72 isolation of methyl and ethyl esters of 9M5, 1, from disposable latex gloves.
73
74 MATERIALS AND METHODS
75 Organic solvents and chemicals. Methanol (>99.8%) and n-hexane (>95%) were
76 from VWR Chemicals (Darmstadt, Germany), while ethanol, diethyl ether (both distilled
77 before use) and sulfuric acid were from Carl Roth (Karlsruhe, Germany). Silica gel 60 and
78 silver nitrate were from Sigma Aldrich (Taufkirchen, Germany). Sodium chloride and the
79 fatty acids palmitic acid (16:0; >98.5%), stearic acid (18:0; >98.5%), oleic acid (18:1∆9;
80 >99%), linoleic acid (18:2∆9,12; 99%) and nonadecanoic acid (19:0; >99%) were from Fluka
81 Chemicals (Taufkirchen, Germany). Myristic acid (14:0; >99%) was from Merck Chemicals
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82 (Darmstadt, Germany). A standard of 9M5, 1, was previously isolated from centrifuged latex
83 with CCC in our laboratory.17
84 Samples and standards. At least one disposable latex glove from 30 different
85 products was obtained from 14 producers (Table 1). The quantitation standard 19:0 (ISTD-1)
86 was prepared at a concentration of 10.1 mg/mL in n-hexane/diethyl ether (1:1, v/v). For the
87 second internal standard (14:0-EE, ISTD-2), 10 mg 14:0 were transesterified as described by
88 Wendlinger et al.15 with 2 mL ethanol containing sulfuric acid (1 vol%) instead of methanol.
89 The resulting ISTD-2 (14:0-EE, c = 5.61 mg/mL) was transferred into an amber 1.5 mL screw
90 cap vial and stored at -20 °C.
91 Gas chromatography with flame ionization detector (GC/FID) or mass
92 spectrometry (GC/MS). Samples were measured on an Autosystem XL GC/FID instrument
93 (Perkin Elmer, Rodgau, Germany) which was equipped with a 25 m x 0.53 mm i.d., 0.5 µm
94 DB 23-Megabore (50% cyanopropyl, 50% dimethyl polysiloxane) column (Agilent,
95 Waldbronn, Germany). Measurements were carried out with N2 (5.0) as carrier gas (constant
96 pressure, 15 kPa). Injections (1 µL) in split mode (split ratio 1:4.7, flow rate: 20 mL/min)
97 were performed at 205 °C. The GC oven program started for 1 min at 60 °C. Then the
98 temperature was first raised to 190 °C with a ramp of 20 °C/min and then to 220 °C with 4
99 °C/min. The final temperature of 230 °C was reached with another ramp of 20 °C/min and
100 held for 10 min.
101 GC/MS measurements were performed on a 6890/5973N GC/MS system (Agilent
102 Technologies, Santa Clara, CA), equipped with a 30 m x 0.25 mm i.d., 0.25 µm HP-5MS
103 (95% methyl, 5% phenyl polysiloxane) column (Agilent, Waldbronn, Germany) as recently
104 described (system 1).20 All measurements in full scan mode (m/z 60 - 600) were done with the
105 following oven program: The initial temperature of 60 °C was held for 1 min and was then
106 raised to 180 °C with a ramp of 13 °C/min and further increased to 250 °C with 3 °C/min. The
107 final temperature of 300 °C was reached with a ramp of 20 °C/min and held for 5 min,
5
108 resulting in a total run time of 41.06 min. For measurements in selected ion monitoring (SIM)
109 mode characteristic ions were divided into time windows according to Wendlinger et al.15
110 Response factors of fatty acid methyl esters (FAME) were also measured on another
111 6890/5973 GC/MS system equipped with a cool on-column inlet (Hewlett-Packard/Agilent,
112 Waldbronn, Germany) and a 15 m x 0.25 mm i.d., 0.1 µm Rtx-1 (100% dimethyl
113 polysiloxane) column (Restek, Bellefonte, PA) as recently described (system 2).20 All
114 measurements were performed in full scan mode (m/z 30 – 600). Additionally, response
115 factors were measured on a 5890 Series II GC/FID instrument (Hewlett Packard) equipped
116 with a 60 m Rtx 2330 column (biscyanopropyl cyanopropylphenyl polysiloxane, 0.25 mm
117 internal diameter, 0.1 µm film thickness) (Restek, Bellefonte, PA). Injections of 1 µL in split
118 mode (1:10) were done with a Hewlett-Packard 7673 autosampler.
119 Liquid extraction and determination of the fat content. Initial tests confirmed that
120 the extraction efficiency of 9M5, 1, with 25 mL n-hexane was >90%, which was considered
121 appropriate. Hence, disposable latex gloves were cut into pieces of ~1 cm2 and 1.5 g of these
122 pieces (~30% of one glove) were supplemented with 25 mL n-hexane and extracted by
123 ultrasonication for 5 min. The hexane extract was filtered and the residue on the filter was
124 washed twice with 5 mL n-hexane. The combined hexane phases were condensed to ~2 mL
125 with a rotary evaporator and transferred into a 10 mL volumetric flask. The volume was
126 adjusted to 10 mL with n-hexane. A 1 mL aliquot of this extract was used for gravimetric
127 determination of the (lipid) extract content, performed in a pre-weighed 1.5 mL screw cap vial
128 once the solvent was removed by a gentle stream of air at 40 °C. Due to logistic reasons, air
129 had to be used in this experiment instead of nitrogen. Because 9M5, 1, is sensitive to
130 oxidation, the variation of the sample weight under the chosen experimental conditions was
131 investigated in additional tests (Figure S1, Supporting Information).
132 Formation of methyl esters. Between 3 and 8 mL latex extract (corresponding with
133 ~10 mg extract content) were placed in a 10 mL vial and 50 µL ISTD-1 (19:0) was added.
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134 The solvent was removed by a gentle stream of nitrogen at 40 °C and the sample was
135 transesterified as previously described.15 In short, 2 mL methanol containing 1 vol% sulfuric
136 acid was added, heated to 80 °C and extracted with 2 mL n-hexane. About 1 mL of the
137 resulting FAME solution was transferred into an amber 1.5 mL screw cap vial and stored at -
138 20 °C until analysis by GC/FID or GC/MS.
139 Determination of the GC/FID response factor of 9M5-ME. The GC/FID response
140 of FAMEs in the transesterified sample was found to be nearly the same for all conventional
141 fatty acids.21 The response factor of 9M5-ME was determined with a solution containing
142 methyl esters of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1∆9), linoleic acid
143 (18:2∆9,12) and 9M5, 1, all at the same concentration of 2 mg/mL. This solution was
144 measured three times on two different GC/FID systems each and on GC/MS system 2
145 operated in full scan mode.
146 Quantitation and quality control. Before GC/FID measurements all FAME solutions
147 of purified samples were spiked with 25 µL of ISTD-2 (14:0-EE) which was used to level out
148 differences in the injection volume. The peak area of 9M5-ME was additionally corrected by
149 its GC/FID response factor. A blank sample containing both internal standards ISTD-1 and
150 ISTD-2 was measured every day and the recovery of ISTD-1 (19:0) was generally >95%.
151 Therefore, the content of 9M5, 1, in the sample was quantified by means of ISTD-1. All peaks
152 in GC/FID chromatograms were verified by means of authentic standards. In addition, the
153 lipid extracts of eight samples #9, #10, #12, #13, #20, #22, #28 and #29 (Table 1) were
154 measured on GC/MS system 1.
155 Exposure of 1 (9M5, 1) to sunlight behind a window (inside). Five gloves from two
156 different producers each #13 and #30 (Table 1) were selected and one glove of each producer
157 was extracted as shown above and measured by GC/FID. The remaining four gloves from
158 each producer (eight total) were exposed to light by placing them on a windowsill inside the
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159 laboratory, facing north (August 2016). Over the course of four days (including night- and
160 daytime), every 24 h one glove from each producer was removed and analyzed.
161 Silver ion mini-column chromatography. FAMEs obtained from the latex extracts
162 were subjected to silver ion mini-column chromatography according to Wendlinger et al.15 In
163 short, ~450 mg silica gel containing 20% silver nitrate deactivated with 1% water was placed
164 into a Pasteur pipette whose conical exit was packed with glass wool. The column was
165 conditioned with ~5 mL n-hexane. Then the lipid extract (obtained from 2.5 g disposable
166 latex glove sample) was placed on the column and fractionated with three solvents. Fraction
167 (i) was collected with 15 mL n-hexane/diethyl ether (99.5:0.5, v/v), fraction (ii) was eluted
168 with 15 mL n-hexane/diethyl ether (97:3, v/v) and fraction (iii) with 15 mL n-hexane/diethyl
169 ether (80:20, v/v). The solvents of all fractions were then removed by a gentle stream of
170 nitrogen and adjusted to exactly 1 mL with n-hexane. All fractions were measured on GC/MS
171 system 1.
172 Isolation of 9M5, 1, from latex gloves. About 50 g disposable latex gloves (sample
173 #30) (Table 1) were cut into pieces of ~1 cm² and placed in an amber 1 L-glass bottle. After
174 the addition of 800 mL n-hexane, extraction was performed by ultrasonication (5 min). The
175 extract was filtered and the residue was washed twice with 50 mL n-hexane. The solvent was
176 removed and the residue was taken up in 50 mL methanol containing 1 vol% sulfuric acid (or
177 ethanol for the extraction of 9M5-EE). The solution was refluxed for 2 h at 80 °C. Then 30
178 mL water and 30 mL saturated NaCl solution were added and fatty acid methyl (ethyl) esters
179 were extracted 3x with 50 mL n-hexane. The solvent was removed by a rotary evaporator at
180 40 °C and 230 mbar and the residue taken up in 1 mL n-hexane. Column chromatography as
181 described before was performed on a large scale. About 5 g silica gel containing 20% silver
182 nitrate deactivated with 1% water was placed in a glass column (inner diameter ~1 cm) and
183 fractionation was done by eluting the sample with 70 mL n-hexane/diethyl ether (99.5:0.5,
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184 v/v) (fraction 1), 50 mL n-hexane/diethyl ether (97:3, v/v) (fraction 2) and 50 mL n-
185 hexane/diethyl ether (80:20, v/v) (fraction 3).
186 Statistical analysis. After verifying normal distribution with the Anderson-Darling-
187 test (p > 0.05), all results were analyzed using univariate ANOVA tests (α = 0.05) as
188 integrated in Excel and statistical significance was assumed for p > 0.05. Statistical outliers
189 were detected with the David-Hartley-Pearson-test (α = 0.05) and not considered for ANOVA
190 tests. Correlation of the lipid extract and the amount of 9M5, 1, in disposable latex gloves was
191 calculated with the Pearson correlation coefficient (PCC) in Excel.
192
193 RESULTS AND DISCUSSION
194 Peak identification in latex gloves. The hexane extracts of disposable latex gloves
195 consisted of 0.6-1.6% of the initial sample weight (mean value: 1.1%) (Table 1). In either
196 case, 9M5, 1, was detected as prominent peak in all samples. In addition to 9M5, 1, the fatty
197 acid pattern of the samples was characterized by 18:2∆9,12; 18:0; 18:1∆9; 16:0; 18:3∆9,12,15
198 and 20:0 as their methyl esters (Figure 2A), which was verified by GC/MS analysis (Figure
199 2B) of eight arbitrarily selected samples #9, #10, #12, #13, #20, #22, #28 and #29 (Table 1).
200 FAMEs were identified by means of the correct retention time and the molecular ions relative
201 to authentic external reference standards. Specifically, m/z 270 (16:0-ME), m/z 292 (18:3n-3-
202 ME), m/z 294 (18:2n-6-ME), m/z 296 (18:1n-9-ME), m/z 298 (18:0-ME) and m/z 326 (20:0-
203 ME). Likewise, 9M5-ME was identified by GC/MS data including the characteristic
204 molecular ion at m/z 322, the McLafferty ion at m/z 109, and the base peak at m/z 165).2
205 Additional GC/MS measurements in SIM mode (Figure 3A) allowed to identify traces of the
206 furan-FA 9M3, 3, 9D5, 4, and 11M5, 5, in two samples #9 and #22 (Table 1) by means of
207 their characteristic MS molecular ion, McLafferty ion, and base peak (Figures 3B-D).2
208 Unfortunately, the producer country of the disposable latex gloves was only listed on four
9
209 samples, but the two samples which featured the minor furan-FA were both produced in
210 Malaysia.
211 Amount of 9M5, 1, in disposable latex gloves. Surprisingly, the GC/FID response of
212 9M5-ME (injected in the same concentration of 2 mg/mL) was considerably lower than those
213 of conventional FAMEs (Figure 4). GC/FID measurements on two instruments showed that
214 the response factor of 9M5-ME was only 40% of the response of conventional FAMEs (16:0-
215 ME, 18:0-ME, 18:1∆9-ME, 18:2∆9,12-ME), which had very similar GC/FID response factors
216 (all >85% compared to 16:0). Standard deviations for the calculated response factor for 9M5-
217 ME were <2.5% and deviation between the two instruments was <2% (Figure S2, Supporting
218 Information). Additional GC/MS measurements (system 2) in full scan mode verified the
219 small response factor for 9M5-ME, although the calculated response factor was slightly
220 higher than in GC/FID (~50%). Consequently, the peak areas of 9M5-ME measured with
221 GC/FID were multiplied by a factor of 2.5 before the quantitation of 9M5, 1, in disposable
222 latex gloves.
223 The concentration of 9M5, 1, in disposable latex gloves ranged from 0.7-8.2 mg/g
224 sample (mean/median value 2.2/2.0 mg/g latex glove) (Table 1). No correlation (PCC = 0.41)
225 was observed between the content of 9M5, 1, and the share of the lipid extract. The
226 contribution of 9M5, 1, to the total fatty acids ranged from 37.9-87% (samples #8 and #15,
227 respectively) (Table 1) with a mean value of 61.4%. Although this relative abundance of
228 9M5, 1, is notable, it is lower than the share of 9M5, 1, reported in fresh crude latex from H.
229 brasiliensis (90%).16 Similar results were reported by Englert et al.17 (>90% 9M5, 1,) in
230 triacylglycerols extracted from centrifuged liquid latex from H. brasiliensis) and
231 Liengprayoon et al.22 (~90% 9M5, 1) in the neutral lipids of the PB235 clone of H.
232 brasiliensis). However, other clones RRIM600 and BPM24 only featured 15-30% of 9M5,
233 1.22 Hence, the variable content of 9M5, 1, could be due to the use of different cultivars of H.
234 brasiliensis along with climatic and geographic factors, variations in the handling of the raw
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235 product and during the manufacturing and/or storing process. However, disposable latex
236 gloves from the same producer varied strongly in the content of 9M5, 1 (e.g. 0.7-2.6 mg/g
237 glove) in eight samples from producer A) (Table 1). Since these were most likely made from
238 the same raw material, it appeared that the lower content of 9M5, 1, was rather due to the
239 instability of 9M5, 1. In agreement with that, the pattern of the most abundant fatty acids
240 (18:2∆9,12, 18:0, 18:1∆9, 16:0) was very similar in all samples with 18:2∆9,12 being the
241 most abundant fatty acid followed by 18:0 (~60% of 18:2∆9,12), 18:1∆9 (~40% of
242 18:2∆9,12) and 16:0 (~25% of 18:2∆9,12) (Figure 5). Statistical significance (p > 0.05,
243 ANOVA) was observed for the ratio of 18:2∆9,12 to 18:0 within samples of producers A (p =
244 0.068), B (p = 0.12), C (p = 0.33) and D/E (p = 0.08), for the ratio of 18:2∆9,12 to 18:1∆9 in
245 samples of producer C (p = 0.16) and for gloves of single producers (p = 0.17) as well as for
246 the ratio of 18:2∆9,12 to 16:0 for producer B (p = 0.69) and for gloves of single producers (p
247 = 0.06). Although no general statistical significance was found, the data strongly indicated
248 that the content of 9M5, 1, in the disposable latex gloves was lower and more variable than in
249 fresh liquid latex.16,17,22
250 Processing of crude latex and/or storage could reduce the content of 9M5, 1, but not
251 the content of the more stable conventional fatty acids. For instance, Englert et al.17 noted that
252 9M5, 1, was instable at pH 12 (~30% transformed in 12 h). Thus, partial post-harvest
253 transformation of 9M5, 1, at pH 10 in technical (stabilized) liquid latex could not be excluded.
254 Liengprayoon et al.23 noted that some lipids (saturated and unsaturated fatty acids) are
255 retained in the dry rubber and varied shares of them may have an effect on the plasticizing
256 properties of the product. Hence, 9M5, 1, could have an effect on the product quality of latex.
257 Next to the pH value, the crucial processes could be varying exposure of 9M5, 1, to light.
258 Further investigations were therefore carried out to study the stability of 9M5, 1, in disposable
259 latex gloves when exposed to natural sunlight.
11
260 Exposure of disposable latex gloves to light. The stability of 9M5, 1, in disposable latex
261 gloves when exposed to daylight inside a room behind a window for 1-4 days (24-96 h) was
262 studied with two types of samples in which 9M5, 1, initially contributed with 68% (sample
263 #30) (Table 1) and 53% to the total fatty acids (sample #13) (Table 1). The contribution of
264 9M5, 1, to the total fatty acids decreased every 24 h (Figure 6). Due to the changing
265 meteorological conditions during the exposure time (Table S1, Supporting Information) a
266 kinetic evaluation was not possible. For instance, higher decomposition rates in the first 24
267 hours (28% and 23%, respectively) (Figures 6A and 6B) were likely due to the higher light
268 intensity on day 1. Nevertheless, our data verifies the sensitivity of 9M5, 1, when exposed to
269 light. Varied exposure to light during production and storage of the disposable latex gloves
270 could also partly explain the varying contribution of 9M5, 1, to the fatty acids in individual
271 samples, which was lower than in the raw material (~90%).16,17,22
272 Additional peaks were not detected in the GC/FID chromatograms of the extracts after
273 exposure to sunlight. Thus, degradation products of 9M5, 1, may either be volatile as
274 suggested previously9,10 for other furan-FA and/or polar compounds that were not extracted or
275 accessible to GC analysis.
276 Isolation of 9M5-ME by silver ion chromatography. Separation of other FAMEs
277 from 9M5-ME in the extract of 2.5 g disposable latex gloves from sample #30 (Table 1) was
278 achieved with silver ion chromatography. Fraction 1 contained the methyl esters of saturated
279 fatty acids (16:0-ME, 18:0-ME and 20:0-ME) (Figure 7A), whereas the methyl esters of
280 unsaturated fatty acids (18:1∆9-ME and 18:2∆9,12-ME, 18:3∆9,12,15-ME) eluted into
281 fraction 3 (Figure 7C). Hence, 9M5-ME in fraction 2 could be obtained with a purity of ~98%
282 (Figure 7B). The yield was ~5 mg (~97% from the calculated content of 9M5, 1, of 5.1 mg in
283 2.5 g disposable latex gloves, Table 1).
284 Functionality of the method was confirmed by the extraction of 50 g disposable latex
285 gloves from sample #30 (Table 1) (corresponding with ~12 gloves) which provided 101 mg
12
286 9M5-ME after column chromatography. The yield was still very high (99% of the calculated
287 content of 9M5, 1, vs. 97% in the analytical batch) and the purity was ~99%.
288 Furan-FA analysis is currently hampered by the lack of authentic reference standards
289 for quantitation and method verification.24 Recently, we suggested the use of the ethyl ester of
290 9M5 (9M5-EE) as internal standard for the quantitative analysis of furan-FA in fish and butter
291 samples.2,15,18 This standard can easily be prepared when transesterification is performed with
292 ethanol instead of methanol. In this way, 82.1 mg 9M5-EE (purity ~99%) were obtained from
293 50 g latex from sample #30 (Table 1).
294 The presented data shows that disposable latex gloves contain high amounts of 9M5,
295 1, which can easily be extracted with n-hexane. For quality assurance reasons, disposable
296 latex gloves should not be used during analyses on furan-FA. Yet, the simple extraction and
297 isolation protocol described in this study allows access to sufficient amounts of 9M5-ME and
298 9M5-EE in high purity which can be used as internal standard for furan-FA quantitation. Only
299 5 mg 9M5-EE extracted and isolated from 2.5 g disposable latex gloves will be enough for the
300 use as internal standard in ~5,000 quantitative analyses on fatty acids.
301
302 AUTHOR INFORMATION
303 Corresponding Author
304 * (W.V.) E-mail: walter.vetter@uni-hohenheim.de Phone: +49 711 459 24016. Fax +49 711
305 459 309 24377.
306 Funding
307 No funding was obtained for performing this study.
308 Notes
309 The authors declare no competing interests.
310
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311 ABBREVIATIONS USED. 9M5, 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid; 16:0,
312 palmitic acid; 18:0, stearic acid; 18:1∆9, oleic acid; 18:2∆9,12, linoleic acid; 18:3∆9,12,15, α-
313 linoleic acid; 20:0, arachidic acid; furan-FA, furan fatty acid; FAME, fatty acid methyl ester;
314 ISTD, internal standard; SIM, selected ion monitoring;
315
316 SUPPORTING INFORMATION. Response factors of stearic acid (18:0), oleic acid
317 (18:1∆9), linoleic acid (18:2∆9,12) and the furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-
318 nonanoic acid (9M5), 1, compared to palmitic acid (16:0) measured on two different GC/FID
319 systems and weight reduction of the lipid extract of disposable latex gloves when evaporated
320 with air over the period of 10 minutes in two samples. This material is available free of charge
321 via the Internet at http://pubs.acs.org.
322
323 REFERENCES
324 (1) Spiteller, G. Furan fatty acids: Occurrence, synthesis, and reactions. Are furan fatty
325 acids responsible for the cardioprotective effects of a fish diet? Lipids, 2005, 40, 755–771
326 (2) Vetter, W.; Laure, S.; Wendlinger, C.; Mattes, A.; Smith, A. W. T.; Knight, D. W.
327 Determination of furan fatty acids in food samples. J. Am. Oil Chem. Soc., 2012, 89,1501–
328 1508
329 (3) Shirasaka, N.; Nishi, K.; Shimizu, S. Biosynthesis of furan fatty acids (F-acids) by a
330 marine bacterium, Shewanella putrefaciens. Biochim. Biophys. Acta, Lipids Lipid Metab.,
331 1997, 1346, 253–260
332 (4) Mawlong, I.; Sujith Kumar, M. S.; Singh, D. Furan fatty acids. Their role in plant
333 systems. Phytochem Rev., 2016, 15, 121–127
14
334 (5) Hannemann, K.; Puchta, V.; Simon, E.; Ziegler, H.; Ziegler, G.; Spiteller, G. The
335 common occurrence of furan fatty acids in plants. Lipids, 1989, 24, 296–298
336 (6) Gorst-Allman, C. P.; Puchta, V.; Spiteller, G. Investigations of the origin of the furan
337 fatty acids (F-acids). Lipids, 1988, 23, 1032–1036
338 (7) Okada, Y.; Kaneko, M.; Okajima, H. Hydroxyl radical scavenging activity of naturally
339 occuring furan fatty acids. Biol. Pharm. Bull., 1996, 19, 1607–1610
340 (8) Okada, Y.; Okajima, H.; Konishi, H.; Terauchi, M.; Ishii, K.; Liu, I.-M.; Watanabe, H.
341 Antioxidant effect of naturally occurring furan fatty acids on oxidation of linoleic acid in
342 aqueous dispersion. J. Am. Oil Chem. Soc., 1990, 67, 858–862
343 (9) Masanetz, C.; Guth, H.; Grosch, W. Fishy and hay-like off-flavours of dry spinach.
344 Z. Lebensm.-Unters. Forsch., 1998, 206, 108–113
345 (10) Guth, H.; Grosch, W. Stability of furanoid fatty acids in soybean oil. J. Am. Oil Chem.
346 Soc., 1997, 74, 323–326
347 (11) Sigrist, I. A.; Manzardo, G. G.; Amadò, R. Furan fatty acid photooxidative
348 degradation products in dried herbs and vegetables. Chimia. 2002, 56, 263–265
349 (12) Liu, Y.; Prentice, K. J.; Eversley, J. A.; Hu, C.; Batchuluun, B.; Leavey, K.; Hansen, J.
350 B.; Wei, D. W.; Cox, B. Rapid elevation in CMPF may act as a tipping point in diabetes
351 development. Cell Rep., 2016, 14, 2889–2900
352 (13) Lankinen, M. A.; Hanhineva, K.; Kolehmainen, M.; Lehtonen, M.; Auriola, S.;
353 Mykkanen, H.; Poutanen, K.; Schwab, U.; Uusitupa, M. CMPF does not associate with
354 impaired glucose metabolism in individuals with features of metabolic syndrome. PLoS One,
355 2015, 10, 1-8
15
356 (14) Prentice, K. J.; Luu, L.; Allister, E. M.; Liu, Y.; Jun, L. S.; Sloop, K. W.; Hardy, A.
357 B.; Wei, L.; Jia, W. The furan fatty acid metabolite CMPF is elevated in diabetes and induces
358 beta cell dysfunction. Cell Metab., 2014, 19, 653–666
359 (15) Wendlinger, C.; Vetter, W. High concentrations of furan fatty acids in organic butter
360 samples from the German market. J. Agric. Food Chem., 2014, 62, 8740–8744
361 (16) Hasma, H.; Subramaniam, A. The occurrence of a furanoid fatty acid in Hevea
362 brasiliensis latex. Lipids, 1978, 13, 905–907
363 (17) Englert, M.; Ulms, K.; Wendlinger, C.; Vetter, W. Isolation of a furan fatty acid from
364 Hevea brasiliensis latex employing the combined use of pH-zone-refining and conventional
365 countercurrent chromatography. J. Sep. Sci., 2016, 39, 490–495
366 (18) Wendlinger, C.; Hammann, S.; Vetter, W. Detailed study of furan fatty acids in total
367 lipids and the cholesteryl ester fraction of fish liver. Food Anal. Methods, 2016, 9, 459–468
368 (19) Vetter, W.; Ulms, K.; Wendlinger, C.; van Rijn, J. Novel non-methylated furan fatty
369 acids in fish from a zero discharge aquaculture system. J. Nutr. Food Sci., 2016, 2, 8–14
370 (20) Krauss, S.; Hammann, S.; Vetter, W. Phytyl fatty acid esters in the pulp of bell pepper
371 (Capsicum annuum). J. Agric. Food Chem., 2016, 64, 6306–6311
372 (21) Kállai, M.; Veres, Z.; Balla, J. Response of flame ionization detectors to different
373 homologous series. Chromatographia, 2001, 54, 511–517
374 (22) Liengprayoon, S.; Sriroth, K.; Dubreucq, E.; Vaysse, L. Glycolipid composition of
375 Hevea brasiliensis latex. Phytochemistry, 2011, 72, 1902–1913
376 (23) Liengprayoon, S.; Bonfils, F.; Sainte-Beuve, J.; Sriroth, K.; Dubreucq, E.; Vaysse, L.
377 Development of a new procedure for lipid extraction from Hevea brasiliensis natural rubber.
378 Eur. J. Lipid Sci. Technol., 2008, 110, 563–569
16
379 (24) Vetter, W.; Wendlinger, C. Furan fatty acids - valuable minor fatty acids in food. Lipid
380 Technol. 2013, 25, 7–10
17
381 CAPTIONS TO FIGURES
382 Figure 1. Chemical structures of (9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5)), 1,
383 (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid), 2, (9-(3-methyl-5-propylfuran-2-yl)-
384 nonanoic (9M3)), 3, (9-(3,4-dimethyl-5-pentylfuran-2-yl)-nonanoic acid (9D5)), 4, and (11-
385 (3-methyl-5-pentylfuran-2-yl)-undecanoic acid (11M5)), 5.
386
387 Figure 2. Extract of (A) the GC/FID chromatogram and (B) the GC/MS chromatogram
388 (system 1) of the lipid extract from disposable latex gloves, #28 (Table 1). All fatty acids
389 were measured as methyl esters.
390
391 Figure 3. Extract of (A) GC/MS-SIM chromatogram (system 1) and (B-D) mass spectra of
392 the furan fatty acids (9-(3-methyl-5-propylfuran-2-yl)-nonanoic (9M3)), 3, (9-(3,4-dimethyl-
393 5-pentylfuran-2-yl)-nonanoic acid (9D5)), 4, and (11-(3-methyl-5-pentylfuran-2-yl)-
394 undecanoic acid (11M5), 5, detected in disposable latex gloves from Malaysia, #9 and #22
395 (Table 1).
396
397 Figure 4. GC/FID chromatogram of a standard mix containing methyl esters of palmitic acid
398 (16:0), stearic acid (18:0), oleic acid (18:1∆9), linoleic acid (18:2∆9,12) and 9-(3-methyl-5-
399 pentylfuran-2-yl)-nonanoic acid (9M5), 1, in the same concentration (c = 2 mg/mL).
400
401 Figure 5. Share of fatty acids stearic acid (18:0), oleic acid (18:1∆9) and palmitic acid (16:0)
402 compared to linoleic acid (18:2∆9,12) in disposable latex gloves from different producers.
403 Producers D and E and those with only one sample (single gloves) were summed up in one
404 group each.

18
405
406 Figure 6. Percentage contribution of 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5), 1,
407 to the total fatty acids in disposable latex gloves exposed to daylight from (A) samples #30
408 and (B) #13 (Table 1) at different time points.
409
410 Figure 7. GC/MS chromatograms (system 1) in full scan mode after silver ion mini
411 chromatography of (A) fraction 1, (B) fraction 2 and (C) fraction 3 of the lipid extract of
412 disposable latex gloves, #30 (Table 1).
19
Table 1: Share of Lipid Extract per Glove, Contribution of (9-(3-methyl-5-pentylfuran-2-yl)-
nonanoic acid (9M5)), 1, of the Total Fatty Acids, and Amount of 9M5 in 30 Different
Disposable Latex Gloves from 14 Different Producers.
Share of lipid Contribution of Amount of

Number Product extract per 9M5 to the total 9M5 [mg/g
glove [%] fatty acids [%] glove]
1 A1 1.4 67.2 2.6

2 A2 1.3 67.2 2.5
3 A3 1.2 65.4 1.9
4 A4 1.2 58.1 1.6
5 A5 0.8 52.3 0.8
6 A6 0.6 57.4 1.1
7 A7 1.6 51.9 2.0
8 A8 1.2 37.9 0.7
9 B1 1.6 57.6 2.9
10 B2 1.2 74.7 3.2
11 B3 1.0 39.4 0.6
12 B4 1.0 70.3 2.9
13 B5 0.9 52.8 0.8
14 C1 1.5 54.9 2.0
15 C2 1.4 87.0 8.2
16 C3 1.1 47.3 1.0
17 C4 0.9 54.7 1.5
18 D1 0.9 85.1 4.4
19 D2 0.9 66.9 1.8
20 E1 1.1 68.2 2.0
21 E2 0.9 68.4 1.7
22 F1 1.6 59.2 3.8
23 G1 1.5 68.7 2.8
24 H1 1.2 76.0 3.8
25 I1 1.2 60.5 1.6
26 J1 1.0 58.3 2.0
27 K1 1.0 57.3 1.7
28 L1 0.9 56.1 2.2
29 M1 0.8 53.0 1.1
30 N1 0.8 67.9 2.1
Mean/Median: 1.1/1.1 61.4/58.8 2.2/2.0
20
Figure 1
′
′
9 M 5
1 2
3 4
21
Figure 2
22
Figure 3
Irel
9M5 A
(1)
9M3
(3)
9D5
(4) 11M5
(5)
18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 [min]
Irel 9M3 137

(3) B
294
109
100 140 180 220 260 300 m/z
Irel 9D5
(4) C
179
123 336
100 140 180 220 260 300 m/z
Irel 11M5 165

(5) D
109 350
100 140 180 220 260 300 340 380 m/z
23
Figure 4
24
Figure 5
share of fatty acid compared to 18:2 9,12 [%]

100
120
18:0
18:1∆9
18:1
16:0
80
60
40
20
0
A B C D/E single gloves
different producers
25
Figure 6
80
A
Contribution of 9M5 to total fatty acids [%]
68
60
49
40
40
34
20
22
0
0 24 48 72 96
hours in daylight [h]
80
B
Contribution of 9M5 to total fatty acids [%]
60
53
40
41
36
31 28
20
0
0 24 48 72 96
hours in daylight [h]
26
Figure 7
Irel Fraction 1 18:0 A
16:0
20:0
14.0 16.0 18.0 20.0 22.0 24.0 [min]
Irel Fraction 2 9M5 B
14.0 16.0 18.0 20.0 22.0 24.0 [min]
Irel Fraction 3 18:2∆9,12 C
18:1∆9
18:3∆9,12,15
14.0 16.0 18.0 20.0 22.0 24.0 [min]
27
TABLE OF CONTENTS GRAPHIC
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Journal of Agricultural and Food Chemistry is published by the American Chemical

1 Concentrations, Stability and Isolation of the Furan Fatty Acid 9-(3-Methyl-5-

2 Pentylfuran-2-yl)-Nonanoic Acid (9M5) from Disposable Latex Gloves

4 Marco Müller, Melanie Hogg, Kerstin Ulms and Walter Vetter*

7 University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry

8 (170b), D-70593 Stuttgart, Germany

13 Phone: +49 711 459 24016

14 Fax: +49 711 459 24377

21 in which the furan-FA 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5) contributes with

35 with a straight-chain (usually 9, 11 or 13 carbons) saturated acid in α-position and a short

37 is 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5), 1, which is substituted with one

46 effects on the prevention of cardiovascular diseases.1

49 vegetables.11 In addition, the potential furan-FA metabolite 3-carboxy-4-methyl-5-propyl-2-

74 MATERIALS AND METHODS

83 with CCC in our laboratory.17

87 second internal standard (14:0-EE, ISTD-2), 10 mg 14:0 were transesterified as described by

90 cap vial and stored at -20 °C.

91 Gas chromatography with flame ionization detector (GC/FID) or mass

92 spectrometry (GC/MS). Samples were measured on an Autosystem XL GC/FID instrument

94 DB 23-Megabore (50% cyanopropyl, 50% dimethyl polysiloxane) column (Agilent,

100 held for 10 min.

101 GC/MS measurements were performed on a 6890/5973N GC/MS system (Agilent

116 with a 60 m Rtx 2330 column (biscyanopropyl cyanopropylphenyl polysiloxane, 0.25 mm

118 mode (1:10) were done with a Hewlett-Packard 7673 autosampler.

131 investigated in additional tests (Figure S1, Supporting Information).

135 transesterified as previously described.15 In short, 2 mL methanol containing 1 vol% sulfuric

138 20 °C until analysis by GC/FID or GC/MS.

145 operated in full scan mode.

154 measured on GC/MS system 1.

185 hexane/diethyl ether (80:20, v/v) (fraction 3).

191 calculated with the Pearson correlation coefficient (PCC) in Excel.

193 RESULTS AND DISCUSSION

222 latex gloves.

249 fresh liquid latex.16,17,22

259 latex gloves when exposed to natural sunlight.

275 accessible to GC analysis.

276 Isolation of 9M5-ME by silver ion chromatography. Separation of other FAMEs

283 2.5 g disposable latex gloves, Table 1).

293 50 g latex from sample #30 (Table 1).

300 use as internal standard in ~5,000 quantitative analyses on fatty acids.

302 AUTHOR INFORMATION

303 Corresponding Author

305 459 309 24377.

307 No funding was obtained for performing this study.

309 The authors declare no competing interests.

311 ABBREVIATIONS USED. 9M5, 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid; 16:0,

314 ISTD, internal standard; SIM, selected ion monitoring;

321 via the Internet at http://pubs.acs.org.

331 1997, 1346, 253–260

333 systems. Phytochem Rev., 2016, 15, 121–127

337 fatty acids (F-acids). Lipids, 1988, 23, 1032–1036

344 Z. Lebensm.-Unters. Forsch., 1998, 206, 108–113

346 Soc., 1997, 74, 323–326

351 development. Cell Rep., 2016, 14, 2889–2900

355 2015, 10, 1-8

358 beta cell dysfunction. Cell Metab., 2014, 19, 653–666

362 brasiliensis latex. Lipids, 1978, 13, 905–907

365 countercurrent chromatography. J. Sep. Sci., 2016, 39, 490–495

371 (Capsicum annuum). J. Agric. Food Chem., 2016, 64, 6306–6311

373 homologous series. Chromatographia, 2001, 54, 511–517

375 Hevea brasiliensis latex. Phytochemistry, 2011, 72, 1902–1913

378 Eur. J. Lipid Sci. Technol., 2008, 110, 563–569

380 Technol. 2013, 25, 7–10