Rapid method for simultaneous determination of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers in milk by GC-FID

doi:10.21203/rs.3.rs-1943118/v1

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Rapid method for simultaneous determination of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers in milk by GC-FID

https://doi.org/10.21203/rs.3.rs-1943118/v1

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Ruminant milk is the main source of conjugated linoleic acid (CLA) in the human diet, with cis-9, trans-11 CLA being the major among CLA isomers in milk fat. Small amounts of trans-10, cis-12 CLA is also found in synthetic supplements, predominantly. As both isomers being biologically active with potential health benefits, there is great interest in quantifying them in both natural and synthetic sources. An alternative method for the analysis of cis-9, trans-11 and trans-10, cis-12 CLA in milk by fast gas chromatography (GC) has been proposed in the present study. Milk fat was extracted and the fatty acid methyl ester mixture obtained by alkaline catalysis was injected into a gas chromatograph equipped with a flame ionization detector (GC-FID) and fitted with an ionic liquid SLB-IL111 chromatographic column (15 m x 0.10 mm x 0.08 µm). Separation of cis-9, trans-11 and trans-10, cis-12 CLA isomers was achieved in less than 5 min using a 168 °C isotherm run. Both CLA isomers were quantified by standard addition and the results were compared with those obtained using the traditional 100-m long CP-SIL88 capillary column, with no significant differences being detected within 95% confidence interval. The proposed method allowed for the rapid quantification of cis-9, trans-11 and trans-10, cis-12 CLA isomers by GC-FID, which could be particularly useful in focused regulatory routines of a large number of samples with greater analytical frequency.

Fast GC. CLA. SLB-IL111. Fatty acids. Milk. GC-FID

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (C18:2 n-6) distinguished by conjugated double bonds, mainly found in dairies produced with milk from ruminants. Although more than 20 CLA isomers were identified in ruminant milk-containing foods, two of them (cis-9, trans-11 and trans-10, cis-12) are particularly interesting due to their potential health benefits observed in several related studies conducted in the last three decades.[1–3] Dairy fat is the major source of CLA in the human diet, with cis-9, trans-11 CLA being the predominant isomer. Additionally, even though trans-10 cis-12 CLA usually represents less than 1% of total CLA in milk fat, it is present at higher concentrations in most synthetic CLA supplements.[4, 5] Therefore, considering the biochemical relevance of both CLA isomers, there is a great interest on quantifying them in milk-derivative foods and CLA supplements for quality control and adulteration traceability purposes.

Gas chromatography with flame ionization detection (GC-FID) is the official technique used for the analysis of fatty acids (FA) composition in foods (AOAC 996.06).[6] High polar capillary columns coated with cyanopropyl siloxane such as SP-2560 or CP-SIL88 (100 m or longer) are usually required for the analysis of CLA isomers within complex matrixes such as fat-containing foods.[7] Although these columns enable good peak resolution for most CLA isomers, long running times (~ 80 minutes) are required under typical GC-FID operating conditions,[8] which makes the analysis very time-consuming.

More recently, new columns with variable lengths and highly polar stationary phases (SP) are available in supplier companies in order to improve the separation of CLA isomers while reducing the running time.[9–11] In the same period, several studies have compared the efficiency of the new GC columns with those traditionally used for the separation of critical FA, as Fatty Acids Methyl Esters (FAME), in different foods, including milk fat.[7, 12–19]

Ionic liquid (IL) GC columns were previously used for the separation of critical CLA isomers in milk fat such as cis-9, trans-11, and trans-7, cis-9 CLA, which co-elute in the most traditional CP-Sil88 and SP-2560 columns. However, 100 m and 200 m long IL columns were needed, which also resulted in long-running times despite achieving resolution ≥ 1.5.[7, 13] An SLB-IL111 column with 15 m length was previously used for biodiesel analysis,[11, 12, 16, 18] however, to our knowledge, it has not been applied yet for milk fatty acid analysis.

The purpose of the present study was to develop a rapid GC-FID-based method to separate and quantify the cis-9, trans-11, and trans-10, cis-12 CLA isomers in raw milk by using an SLB-IL111 column (15 m x 0.10 mm x 0.08 µm) from Supelco (Bellefonte, PA, USA). The proposed method could be particularly useful in the further analysis focused on assessing the CLA content of a large number of samples for regulatory purposes with enhanced analytical throughput.

2.1 Reagents and solutions

The analytical grade reagents and solvents, those being, methanol, hexane, isopropanol, acetic acid, anhydrous sodium sulfate, sodium hydroxide, sodium methoxide solution, and the FAME mix were all purchased from Sigma-Aldrich (St Louis, MO, USA). The mixture of standards from BASF (BA, Brazil), containing methyl palmitate (C16:0 ME), methyl stearate (C18:0 ME), methyl linoleate (C18:2 ME), and CLA isomers (cis-9, trans-11 and trans-10, cis-12) were kindly provided by EMBRAPA (MG, Brazil). Water was purified by deionization using the Milli-Q system with a resistivity of 18.5 MΩ cm. Individual solutions of FAME were prepared by dissolving appropriate amounts in hexane at a concentration of 1.0 mmol L^− 1. All solutions were stored in a freezer at − 4°C until analysis.

2.2 GC-FID instrumentation

The analysis of the FAME was performed on a GC-2010 Plus Gas chromatograph from Shimadzu (Kyoto, Japan), with auto-injector AOC-i-20 and detection by flame ionization (FID). Two fused silica capillary columns were used, one with polar stationary phase (CP-SIL 88 for FAME, 100 x 0.25 mm x 0.2 µm) from Agilent Technologies (Palo Alto, CA, USA) and another with ionic liquid (SLB-IL111; 15 m x 0.10 mm x 0.08 µm) from Supelco (Bellefonte, USA). The chromatographic conditions for CP-SIL 88 column were described by Cruz-Hernandez et al. (2007).[8] For the SLB-IL111 column, the analytical conditions were set as follows: injection volume of 1.0 µL in split mode with a ratio of 1:100; injector and detector to 250°C; flow rate of carrier gas (H₂) of 1.0 mL min^− 1; linear velocity of 26 cm s^− 1 and isotherm at 168°C. The compounds were identified by co-injection of standards and by comparison of the retention time of a standard mixture. The FAME composition was determined by the standard addition of the individual’s standard solutions and are expressed in mg L^− 1.

2.3 Samples preparation

The samples of raw milk were kindly provided by EMBRAPA. The samples were submitted to the lipid extraction and transesterification procedures. In the lipid extraction procedure was used hexane and isopropanol as organic phase.[20] The solvents were evaporated in nitrogen flow until only the lipid phase remained in the test tube. Samples were prepared and analyzed in duplicate. For basic transesterification catalysts was used sodium methoxide in methanol, the samples were then transferred and stored in a capped vial in a freezer at -20°C until analysis.[21]

2.4 Procedure for quantification

Quantification by standard addition method based on Eqs. (1) and (2) was performed in the trials with both chromatographic columns specified in section 2.2.

$${S}_{1}=k\left[{CLA}_{sample}\right]$$

$${S}_{2}=k\left[{CLA}_{sample}\right]+k\left[{CLA}_{standard}\right]$$

S₁ is the analyte signal within the sample chromatogram, k is a constant of proportionality, [CLA_sample] is the concentration of analyte in the sample, S₂ is the analyte signal in the solution containing both sample and standard, and [CLA_standard] is the concentration of the added standard in the sample. Two solutions were prepared, the first one containing only the extracted sample (920 µL of sample + 80 µL of hexane) and the second containing the extracted sample and standard (80 µL of standard + 920 µL of the sample). By isolating k and equating Eqs. (1) and (2) we have Eq. (3):

$$\left[{CLA}_{sample}\right]=\frac{{S}_{1}\left[{CLA}_{standard}\right]}{({S}_{2}-{S}_{1})}$$

When the concentration of CLA standard [CLA_standard] is 2.7 g L^− 1 and the volumes used in the dilution are the above volumes, the Eq. 3 can be rearranged into Eq. 4, used to calculate the concentration of the CLA in each sample [CLA_sample]:

$$\left[{CLA}_{sample}\right]=\frac{{0.23S}_{1}}{({S}_{2}-{S}_{1})}$$

Figure 1A shows the resulting chromatogram of a CLA standard mixture together with a FAME mix analyzed in a 100 m long CPSil-88 column with a programmed heating ramp.[8] Since traditional FAME mix do not include trans-FA, CLA provides complementary information for full FA profile identification considering milk-derivative analysis, therefore, from this analysis, the composition compiling majoritarian FAME and CLA isomers was established. The final mixture was then used as a secondary standard for all subsequent measurements.

Furthermore, it is not possible to perform the measurement by area normalization in this case because some small FAME can coelute with the solvent peak in the proposed method, as will be discussed forward. Figure 1B shows the same CLA-FAME mixture analyzed by the proposed method using SLB-IL111 column with 15 m in length and isotherm at 168°C. The separation profile is maintained for both analyzes, however, the time spent in the first analysis is about 10 times greater than the second column to separate the isomers of CLA.

To verify that the FAME-CLA profile would be found in real samples, a milk sample prepared according to optimized protocol as described in section 2.3, was injected using both columns (Figs. 2A and 2B). The separation profile of the standards was indeed repeated in the analysis of milk. To confirm the identification of the CLA isomer present, the sample was spiked with CLA standards (Figs. 2C and 2D). By comparison of the retention times shown in Fig. 1, the CLA isomer present in the sample is the cis-9, trans-11. It can be seen that the proposed method was selective for the rapid separation of the isomers of CLA of interest.

The method proved to be selective for the CLA isomers cis-9, trans-11 and trans-10, cis-12, however, it still presents a coelution region between 1.0 and 3.5 minutes of analysis. Some peaks coelute, including with the solvent peak, as can be seen in Fig. 2B, which made us rule out the possibility of quantification by area normalization.[22]

The FA C15:0; C19:0 and C19:1 was tested as an internal standard (IS) but they all eluted in the coelution region, and could not be used as IS. Therefore, the quantification by a response factor was also discarded. Thus, the standard addition method was found to be more suitable for the quantification of CLA cis-9, trans-11 isomer in milk samples. The average values and the respective standard deviations (SD) for the quantitative determination of CLA in two milk samples analyzed in duplicate are shown in Table 1.

Table 1

– *Cis*-9, *trans*-11 CLA isomer quantification in milk.
Samples	Average concentration* ± SD
Samples	CPSil-88	SLB-IL111
Milk A	0.18 ± 0.05	0.23 ± 0.05
Milk B	0.36 ± 0.05	0.35 ± 0.05
LOD*	0.04	0.02
LOQ*	0.12	0.07
^{*concentration in g L−1}

After to confirm the normality assumption (p-value > 0.05) in observations, the paired t-test was performed to compare the determinations of both methods. The p-value obtained was 0.14, so that concludes that at a level of 95% confidence do not have statistical differences between these two approaches to quantify CLA isomer cis-9, trans-11 content in milk samples.

From the use of a column with different SP, it was possible to separate and quantify the CLA isomer cis-9, trans-11 in milk samples. The main contribution of this alternative method of CLA proposed is an increased of the throughput when compared to a traditional method, reducing the time to obtain the results as well the spending on gas used in the analyses by GC-FID. Finally, the method proposal in the present work is very attractive and useful to be considered in routine approaching to monitoring CLA isomer cis-9, trans-11, in laboratories dedicated to this type of analysis.

Acknowledgments

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-475055/2011-0, 301689/2011-3, 424032/2018-0, 303867/2020-5); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-CEX-PPM 00398-13, APQ-00585-21); Rede Mineira de Química (CEX.RED- 00010-14); Financiadora de Estudos e Projetos (FINEP) (CT-INFRA 01/2013-REF 0633/13); Instituto Nacional de Ciência e Tecnologia Bioanalítica (INCTBio) (FAPESP 2014/50867-3, CNPq 465389/2014-7, 401256/2020-0); for financial support.

Funding

This work received financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-475055/2011-0, 301689/2011-3, 424032/2018-0, 303867/2020-5); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-CEX-PPM 00398-13, APQ-00585-21); Rede Mineira de Química (CEX.RED- 00010-14); Financiadora de Estudos e Projetos (FINEP) (CT-INFRA 01/2013-REF 0633/13); and Instituto Nacional de Ciência e Tecnologia Bioanalítica (INCTBio) (FAPESP 2014/50867-3, CNPq 465389/2014-7, 401256/2020-0).

Competing Interests

The authors have no competing interests to declare that are relevant to the content of this article.

Author Contributions

Brenda Lee Simas Porto: conceptualization, data curation, formal analysis, investigation, methodology, project administration, visualization, writing – original draft. Isaura Daniele Leite Faria: conceptualization, investigation, visualization, writing – original draft. Jéssica Cordeiro Queiroz de Souza: conceptualization, investigation, visualization, writing – original draft. Olívia Brito de Oliveira Moreira: conceptualization, investigation, visualization, writing – original draft. Marco Antônio Sundfeld da Gama: conceptualization, supervision, visualization, resources and funding acquisition. Marcone Augusto Leal de Oliveira: conceptualization, supervision, visualization, resources and funding acquisition.

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No competing interests reported.

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Rapid method for simultaneous determination of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers in milk by GC-FID

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Abstract

Figures

1. Introduction

2. Material And Methods

2.1 Reagents and solutions

2.2 GC-FID instrumentation

2.3 Samples preparation

2.4 Procedure for quantification

3. Results And Discussion

4. Conclusions

Statements & Declarations

References

Additional Declarations

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