Direct Methylation Method for Quantification of Fatty Acids in Lyophilized Human Milk by Gas Chromatography with Flame Ionization Detector

Souza, Patrícia M.; Santos, Patrícia D. S.; Ponhozi, Isadora B.; Cruz, Victor Hugo M.; Pizzo, Jessica S.; Alves, Eloize S.; Santos, Oscar O.; Visentainer, Jesuí V.

doi:10.21577/0103-5053.20240077

Patrícia M. Souza

data curation

formal analysis

investigation

validation

visualization

writing (original draft

review

editing)

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Patrícia D. S. Santos

formal analysis

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Isadora B. Ponhozi

formal analysis

Programa de Pós-Graduação em Ciência de Alimentos, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Victor Hugo M. Cruz

writing (review

editing)

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Jessica S. Pizzo

formal analysis

writing (review

editing)

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Eloize S. Alves

writing (review

editing)

Programa de Pós-Graduação em Ciência de Alimentos, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Oscar O. Santos ^**e-mail: oliveirasantos.oscardeoliveira@gmail.com

data curation

project administration

supervision

visualization

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Programa de Pós-Graduação em Ciência de Alimentos, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Jesuí V. Visentainer

writing (review

editing)

conceptualization

funding acquisition

resources

Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

Programa de Pós-Graduação em Ciência de Alimentos, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá-PR, Brazil

https://orcid.org/0000-0003-3412-897X

*e-mail: oliveirasantos.oscardeoliveira@gmail.com

Editor handled this article: Andrea R. Chaves (Associate)

Experimental run	Basic reaction time / min	Acid reaction time / min	HCl/MeOH content / (mol L^-1)	KOH/MeOH content / (mol L^-1)	Sample mass / mg	Sum of FAs / (mg g^-1 sample)
1	10 (-1)	10 (-1)	0.75	0.75	240	191.3661
2	20 (+1)	10 (-1)	0.75	0.75	100	251.9260
3	10 (-1)	20 (+1)	0.75	0.75	100	213.8109
4	20 (+1)	20 (+1)	0.75	0.75	240	218.5640
5	10 (-1)	10 (-1)	1.5	0.75	100	409.5600
6	20 (+1)	10 (-1)	1.5	0.75	240	329.0885
7	10 (-1)	20 (+1)	1.5	0.75	240	215.9338
8	20 (+1)	20 (+1)	1.5	0.75	100	249.0849
9	10 (-1)	10 (-1)	0.75	1.5	100	246.1021
10	20 (+1)	10 (-1)	0.75	1.5	240	165.6124
11	10 (-1)	20 (+1)	0.75	1.5	240	160.4444
12	20 (+1)	20 (+1)	0.75	1.5	100	147.1313
13	10 (-1)	10 (-1)	1.5	1.5	240	189.7598
14	20 (+1)	10 (-1)	1.5	1.5	100	195.2520
15	10 (-1)	20 (+1)	1.5	1.5	100	175.7692
16	20 (+1)	20 (+1)	1.5	1.5	240	166.2129
17	5 (-α)	15 (0)	1.125	1.125	170	190.6491
18	25 (+α)	15 (0)	1.125	1.125	170	284.3414
19	15 (0)	5 (-α)	1.125	1.125	170	268.1195
20	15 (0)	25 (+α)	1.125	1.125	170	290.1179
21	15 (0)	15 (0)	0.375	1.125	170	222.9148
22	15 (0)	15 (0)	1.875	1.125	170	221.4920
23	15 (0)	15 (0)	1.125	0.375	170	203.7484
24	15 (0)	15 (0)	1.125	1.875	170	142.2655
25	15 (0)	15 (0)	1.125	1.125	30	211.3690
26	15 (0)	15 (0)	1.125	1.125	310	233.4732
27	15 (0)	15 (0)	1.125	1.125	170	263.1749
28	15 (0)	15 (0)	1.125	1.125	170	212.7515
29	15 (0)	15 (0)	1.125	1.125	170	209.9662
30	15 (0)	15 (0)	1.125	1.125	170	275.0098
31	15 (0)	15 (0)	1.125	1.125	170	225.3410
32	15 (0)	15 (0)	1.125	1.125	170	203.2956

Source	Sum of squares	DF	Mean square	F value	Prob. > F	Observation
Model	76871.14	25	3074.85	24.24	0.0004	S
A	3817.63	1	3817.63	30.10	0.0015	S
D	16616.53	1	1616.53	12.74	0.018	S
AE	5724.81	1	5724.81	45.14	0.0005	S
BC	2190.01	1	2190.01	17.27	0.0060	S
BD	1057.71	1	1057.71	8.34	0.0278	S
BE	2317.22	1	2317.22	18.27	0.0052	S
CD	5748.29	1	5748.9	45.32	0.0005	S
B²2 Floris, L. M.; Stahl, B.; Berkeveld, M. A.; Teller, I. C.; Prostaglandins, Leukotrienes Essent. Fatty Acids 2020, 156, 102023. [Crossref] Crossref...	5499.09	1	5499.09	43.36	0.0006	S
D²2 Floris, L. M.; Stahl, B.; Berkeveld, M. A.; Teller, I. C.; Prostaglandins, Leukotrienes Essent. Fatty Acids 2020, 156, 102023. [Crossref] Crossref...	3506.27	1	3506.27	27.64	0.0019	S
A²B	5095.41	1	5095.41	40.17	0.0007	S
A²C	2165.59	1	2165.9	17.07	0.0061	S
A²D	2698.65	1	2698.5	21.28	0.0036	S
A²E	2080.99	1	2080.99	16.41	0.0067	S
AB²2 Floris, L. M.; Stahl, B.; Berkeveld, M. A.; Teller, I. C.; Prostaglandins, Leukotrienes Essent. Fatty Acids 2020, 156, 102023. [Crossref] Crossref...	3763.20	1	3763.20	29.67	0.0016	S
Residual	761.02	6	126.84	-	-	-
Lack-of-fit	353.47	1	353.47	4.34	0.0918	NS
Pure error	407.55	5	81.51	-	-	-
Total	77632.16	31	-	-	-	-

Fatty acid / (mg g^-1 of sample)	Lyophilized human milk
Fatty acid / (mg g^-1 of sample)	Traditional method	Proposed method
4:0	0.3519^a ± 0.0696	0.4654^a ± 0.0152
6:0	0.1821^b ± 0.0090	0.3372^a ± 0.0100
8:0	0.1013^b ± 0.0270	0.3994^a ± 0.0139
10:0	2.2768^b ± 1.8480	3.8474^a ± 0.3923
11:0	0.0526^b ± 0.0667	0.1435^a ± 0.0116
12:0	15.3170^b ± 11.6290	22.7664^a ± 2.4655
14:0	25.8887^a ± 0.4093	27.5870^a ± 3.0020
14:1n-5	0.2510^a ± 0.0048	0.2626^a ± 0.0328
15:0	0.6927^a ± 0.1145	0.8975^a ± 0.0447
15:1n-7	0.2612^a ± 0.0349	0.2292^a ± 0.0393
16:0	44.2994^b ± 1.4295	107.3300^a ± 1.9761
16:1n-7	0.5116^a ± 0.2408	0.8274^a ± 0.1671
16:1n-9	6.4244^b ± 0.4488	8.6474^a ± 0.2680
17:0	0.5712^b ± 0.1573	1.0819^a ± 0.0694
17:1n-9	0.4363^a ± 0.0887	0.5198^a ± 0.0324
18:0	12.7646^b ± 2.3104	25.8741^a ± 0.9993
18:1n-9	109.7036^b ± 6.8992	125.2718^a ± 2.3014
18-:1n-7	3.6595^b ± 0.3195	5.4783^a ± 0.2751
18:2n-6	55.2697^b ± 0.5374	65.6860^a ± 0.8970
18:2n-6 cis9,trans11	0.2326^b ± 0.0428	0.4756^a ± 0.0828
18:2n-6 trans10,cis12	0.6764^a ± 0.4217	0.8875^a ± 0.1314
18:3n-3	2.9131^b ± 0.2148	4.0041^a ± 0.5542
18:3n-6	0.9176^a ± 0.5212	1.1031^a ± 0.1616
20:0	0.2599^a ± 0.1446	0.3440^a ± 0.0587
20:1n-9	1.1128^a ± 0.1359	1.3347^a ± 0.1928
21:0	0.0007^b ± 0.0003	0.0016^a ± 0.0002
20:4n-6	1.8467^a ± 0.3160	2.1332^a ± 0.2585
20:3n-3	0.0698^b ± 0.0089	0.2332^a ± 0.0246
22:0	0.3509^a ± 0.1206	0.4313^a ± 0.0215
20:3n-6	0.0976^b ± 0.0028	0.1415^b ± 0.0175
20:5n-3	0.3221^a ± 0.1104	0.2696^a ± 0.0277
22:1n-9	0.4115^b ± 0.1242	0.6644^b ± 0.0817
24:0	0.0001^a ± 0.0001	0.0002^a ± 0.0001
24:1n-9	0.0004^a ± 0.0001	0.0005^a ± 0.0001
22:6n-3	0.4100^b ± 0.0980	0.6270^b ± 0.0770
∑SFA	102.4488^b ± 14.5898	190.6323^a ± 6.6892
∑MUFA	122.0839^b ± 6.6189	142.2581^a ± 1.7087
∑PUFA	61.6823^b ± 1.6723	75.8363^a ± 1.1489
∑n-6	57.6581^b ± 1.1580	68.8301^a ± 0.6558
∑n-3	3.7054^b ± 0.1953	5.1340^a ± 0.6743
∑(n-6)/(n-3)	15.6238^b ± 0.7712	13.6176^a ± 1.9865
Total sum of fatty acids	277.5035^b ± 8.9380	409.5620^a ± 5.8429

Fatty acid / (mg g^-1 of sample)	Lyophilized human milk		Liquid human milk
Fatty acid / (mg g^-1 of sample)	Colostrum	Transition	Colostrum	Transition	Mature
4:0	0.3994^a ± 0.3520	0.4248^a ± 0.1001	0.6846^a ± 0.2485	0.3898^a ± 0.1846	0.7855^a ± 0.2029
6:0	0.2574^a ± 0.0849	0.3498^a ± 0.0885	0.7864^a ± 0.2501	0.5115^a ± 0.1200	0.9515^a ± 0.2653
8:0	0.3571^a ± 0.0496	0.3845^a ± 0.3675	0.2487^a ± 0.0646	0.1643^a ± 0.0510	0.3019^a ± 0.0748
10:0	3.7091^a ± 0.9514	3.9049^a ± 1.3495	0.3114^a ± 0.0573	0.4478^a ± 0.0734	0.4294^a ± 0.0948
11:0	1.3423^a ± 0.8468	1.5283^a ± 0.3790	0.3660^a ± 0.1162	0.0251^a ± 0.0015	0.5740^a ± 0.2098
12:0	22.3313^a ± 0.4814	25.2226^a ± 0.7840	1.6199^a ± 0.4859	2.3601^a ± 0.3592	2.5481^a ± 0.4278
14:0	23.0962^a ± 0.9281	24.1404^a ± 1.4160	2.4629^a ± 0.4864	3.1146^a ± 0.4166	3.1562^a ± 0.3620
14:1n-5	0.1622^b ± 0.0098	0.1783^b ± 0.0072	0.0158^a ± 0.0044	0.0153^b ± 0.0017	0.0299^a ± 0.0032
15:0	0.7004^a ± 0.0619	0.7485^a ± 0.0322	0.0727^a ± 0.0147	0.0773^b ± 0.0104	0.1130^a ± 0.0111
15:1n-7	0.2505^a ± 0.0072	0.1796^a ± 0.0228	0.0200^a ± 0.0099	0.0217^a ± 0.0027	0.0239^a ± 0.0017
16:0	97.5610^a ± 0.5897	106.0671^a ± 0.7631	8.7901^a ± 1.3885	9.9996^a ± 0.9856	10.3677^a ± 0.6950
16:1n-7	0.8224^a ± 0.0998	0.7195^b ± 0.0955	0.0869^a ± 0.0125	0.0798^a ± 0.0070	0.0768^a ± 0.0034
16:1n-9	6.2970^a ± 0.3752	6.0391^a ± 0.5331	0.6246^a ± 0.0957	0.5645^a ± 0.0640	0.7994^a ± 0.0786
17:0	1.0445^a ± 0.0520	1.0474^a ± 0.0350	0.0981^a ± 0.0143	0.1037^a ± 0.0123	0.1175^a ± 0.0168
17:1n-9	0.4543^a ± 0.0623	0.4606^a ± 0.0647	0.0537^a ± 0.0078	0.0472^a ± 0.0073	0.0668^a ± 0.0004
18:0	20.7420^a ± 0.4862	22.0405^a ± 0.7210	1.7990^a ± 0.2375	2.2289^a ± 0.1763	2.1879^a ± 0.1716
18:1n-9	120.4042^a ± 0.3378	121.7219^a ± 0.3666	11.077^a ± 1.5887	11.8376^a ± 1.2541	13.1275^a ± 0.9043
18:1n-7	0.1838^b ± 0.0167	0.1067^b ± 0.0267	0.0221^b ± 0.0051	0.0239^b ± 0.0104	0.0209^b ± 0.0021
18:2n-6	60.2799^a ± 1.3397	47.0999^b ± 0.9966	4.9607^a ± 0.6307	5.5808^a ± 0.2962	5.9306^a ± 0.2220
18:2n-6 cis9,trans11	0.3594^b ± 0.2106	0.3888^a ± 0.1839	0.0485^a ± 0.0069	0.0568^a ± 0.0141	0.0682^b ± 0.0023
18:2n-6 trans10,cis12	0.5605^a ± 0.0273	0.6679^a ± 0.0644	0.0427^a ± 0.0099	0.0423^a ± 0.0014	0.0410^b ± 0.0008
18:3n-3	2.8701^a ± 0.1773	3.4857^a ± 0.4541	0.2863^a ± 0.0178	0.2807^a ± 0.0335	0.3102^a ± 0.0099
18:3n-6	1.4748^a ± 0.3384	1.3324^a ± 0.1046	0.1845^a ± 0.0076	0.1797^a ± 0.0211	0.1344^a ± 0.0155
20:0	0.2071^a ± 0.0669	0.2139^a ± 0.0212	0.0314^a ± 0.0065	0.0180^a ± 0.0034	0.0190^a ± 0.0067
20:1n-9	2.5517^b ± 0.1510	2.4825^a ± 0.4150	0.2167^a ± 0.0334	0.2184^a ± 0.0287	0.2484^a ± 0.0778
21:0	0.0017^b ± 0.0001	0.0020^a ± 0.0004	0.0001^a ± 0.0001	0.0002^a ± 0.0001	0.0001^a ± 0.0001
20:4n-6	2.1988^a ± 0.1483	2.3886^a ± 0.3436	0.2432^a ± 0.0190	0.2206^a ± 0.0284	0.2514^a ± 0.0338
20:3n-3	0.2804^a ± 0.0337	0.3111^a ± 0.0158	0.0563^a ± 0.0175	0.0309^a ± 0.0030	0.0474^a ± 0.0127
22:0	0.3833^a ± 0.0293	0.3872^a ± 0.0376	0.0411^a ± 0.0050	0.0490^a ± 0.0057	0.0440^a ± 0.0041
20:3n-6	0.3845^a ± 0.0239	0.3670^a ± 0.0812	0.0449^a ± 0.0096	0.0425^a ± 0.0018	0.0394^a ± 0.0018
20:5n-3	0.1353^b ± 0.0564	0.2424^a ± 0.0764	0.0400^a ± 0.0043	0.0047^a ± 0.0208	0.0386^a ± 0.0092
22:1n-9	0.8481^a ± 0.1204	0.7298^a ± 0.0709	0.0832^a ± 0.0089	0.0644^a ± 0.0060	0.0499^a ± 0.0072
24:0	0.0002^a ± 0.0001	0.0002^a ± 0.0001	ND	ND	ND
24:1n-9	0.0004^a ± 0.0001	0.0005^a ± 0.0001	ND	ND	ND
22:6n-3	0.7903^a ± 0.0622	0.7547^a ± 0.0787	0.0661^a ± 0.0127	0.0695^a ± 0.0063	0.0598^a ± 0.0065
∑SFA	170.8478^a ± 2.9443	186.1292^a ± 0.6537	16.7614^a ± 3.1116	19.3865^a ± 2.0080	21.7209^a ± 2.0993
∑MUFA	131.989^a ± 0.8499	132.3168^a ± 1.0545	12.1937^a ± 1.7500	12.8728^a ± 1.3780	14.4507^a ± 1.0615
∑PUFA	69.6408^a ± 1.1149	57.6168^a ± 1.4711	5.9011^a ± 0.7095	6.4752^a ± 0.2251	6.9735^a ± 0.2749
∑(n-6)	62.2775^a ± 1.5096	49.0150^b ± 0.9725	5.1853^a ± 0.6474	5.8052^a ± 0.2744	6.1179^a ± 0.2370
∑(n-3)	3.9014^a ± 0.2682	4.6226^a ± 0.4335	0.4087^a ± 0.0465	0.3811^a ± 0.0256	0.4175^a ± 0.0092
∑(n-6)/(n-3)	16.0437^a ± 1.2836	10.6033^a ± 0.8428	14.3790^a ± 1.2955	15.4895^a ± 1.0889	14.3864^b ± 0.3352
Total sum of fatty acids	373.4417^a ± 0.0001	376.1192^a ± 0.0001	35.4857^a ± 0.0001	38.8711^a ± 0.0001	42.9602^a ± 0.0001

\begin{aligned} Y = 204.17 + 21.84 A - 14.22 D + 18.92 A E - 11.70 B C + \\ 8.13 B C + 12.03 B E - 18.95 C D + 13.69 B^{2} - 10.93 D^{2} - \\ 30.91 A^{2} B + 20.15 A^{2} C - 22.49 A^{2} D - 19.75 A^{2} E - \\ 26.56 {A B}^{2} \end{aligned}

[1] *e-mail: oliveirasantos.oscardeoliveira@gmail.com

Brasil

Brasil

Direct Methylation Method for Quantification of Fatty Acids in Lyophilized Human Milk by Gas Chromatography with Flame Ionization Detector