Determination of non polar metabolites in human ... - GL Sciences BV

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Determination of non polar metabolites in human plasma using innovative sample preparation strategies coupled with gas chromatography and mass spectrometry Maria Margarida 1Environmental

1 Gonçalves ,

1 Sagredo ,

1 Bernardo ,

1 Mendes ,

Sandra Maria Serrano Benilde 3 4 4 Daniel Etlin , Ana Bragança and Valentina Vassilenko 2Faculty

Maria Cristina

Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, Portugal; of Pharmacy, University of Lisbon, Portugal; 4Centre of Physics and Technological Research, New University of Lisbon, Portugal

2 Marques ,

3Unicam

Sistemas Analíticos, SA, Portugal;

Overview: 









The main objective of this work is to develop simple, fast and selective extraction methods to isolate non polar metabolites or contaminants from human plasma. Solid phase extraction using a novel adsorbant (MonoTrapTM) was tested for direct extration from diluted plasma samples or from the corresponding headspace. Both direct extraction and headspace extraction with MonoTrapTM revealed the presence of terphenyls and partially hydrogenated terphenyls in 39 human plasma samples. Dispersive liquid-liquid microextraction (DLLME) of plasma samples was tested using CCl4 or using heptane. Conventional DLLME procedure was adapted to allow the use of non halogenated solvents. A new method of simultaneous derivatisation, extraction and concentration of plasma fatty acids is proposed. This method couples direct transesterification of plasma samples with methanolic KOH and homogeneous liquid-liquid microextraction (HLLME) with halogenated or non halogenated solvents. All steps take place in a single vial with an extraction time of 15 min or less.

Introduction: 





 

Metabolomics addresses the screening of compounds with medium to low molecular weight, with biological relevance as disease or exposure biomarkers [1]. Sample preparation is a key step of the process, because biological fluids are complex samples often available at limited amounts (1-2 ml in the case of blood plasma). The metabolic profile of plasma samples includes free aminoacids, organic acids, amines, sugar, steroids, nucleic acid bases and other substances [2]. The analysis of these metabolites by GC-MS requires the use of appropriated derivatization reactions. Non polar metabolites like aliphatic hydrocarbons have been determined in biological samples (breath samples) and related to lipid peroxydation processes occurring in cells of cancer patients or of smokers [3,4].

References [1] Harin Kanani, Panagiotis K. Chrysanthopoulos, Maria I. Klapa “Standardizing GC–MS metabolomics” Journal of Chromatography B, 871 (2008) 191–201 [2] Kishore K. Pasikanti, P.C. Ho, E.C.Y. Chan “Gas chromatography/mass spectrometry in metabolic profiling of biological fluids” Journal of Chromatography B, 871 (2008) 202–211 [3] Leiliane Coelho A. Amorim, Zenilda de L. Cardeal “Breath air analysis and its use as a biomarker in biological monitoring of occupational and environmental exposure to chemical agents” Journal of Chromatography B, 853 (2007) 1–9 [4] Michael Phillips, Renee N. Cataneo, Andrew R.C. Cummin, Anthony J., Gagliardi, Kevin Gleeson, Joel Greenberg, Roger A. Maxfield, William N. Rom “Detection of Lung Cancer With Volatile Markers in the Breath” Chest 123 (2003) 2115-2123

Methods : 

Blood samples from 40 volunteers (20 smokers and 20 non smokers) were collected and the corresponding plasma was isolated and kept at -5ºC until analysis.



Plasma deproteinization was performed adding methanol and separating the precipitates by centrifugation.



Solid phase extraction with MonoTrapTM DSC18 disks (GL Sciences, Japan) Plasma samples (500L) were diluted with distilled water and extracted with MonoTrapTM DSC18 disks using: a) Direct extraction (DE)

b) Headspace extraction (HE)

MonoTrapTM disks are placed in direct contact with the plasma sample, at 60 ºC, for 1h, under stirring.

MonoTrapTM disks suspended in the headspace over the plasma sample, at 60 ºC, for 1h, under stirring.

The SPE disks were extracted with dichloromethane, in an ultrasonic bath. The extract was dried, concentrated to 200 µL and analysed by GC-MS. 

Homogeneous liquid-liquid microextraction coupled with direct transesterification . (HLLME-DT): Plasma samples (500L) were diluted with methanolic KOH; a solution of organic solvent CCl4 or heptane (50 - 150 µL) in acetone was added to the reaction mixture and an homogeneous solution was obtained. Phase separation was induced by adding 2 ml of water. After centrifugation the organic phase was recovered and injected in a GC-MS-MS (Focus GC, Polaris Q, Thermounicam).

Results : Detection of terphenyls and hydrogenated terphenyls in human plasma SPE with MonoTrap DSC18 disks (DE and HE) + GC-MS RT: 52.59 - 62.52

DE (Direct Extraction)

105 100 95 90 85

59.31

60.48

80 75

Terphenyls

Extracted Chromatograms with base peak = 230 m/z

NL: 6.01E5 TIC F: MS Soro1MonotrapC 18-1

DE (Direct Extraction)

56.53

70 65 60

HE (Headspace Extraction)

55 50 45

60.58

56.39 53.04

40 35 30 25 57.72

56.89

20 53.84

15

57.88

10 54.58

5

55.25

57.00

58.33

56.03

60.18

60.74

Diphenylcyclohexanes

62.08

0 53

54

55

56

57

58 Time (min)

59

60

61

62

RT: 52.59 - 61.56

100 95 90

HE (Headspace Extraction)

85

Extracted Chromatograms with base peak = 230 m/z

NL: 6.01E5 TIC F: MS Soro1MonotrapC 18-1

RT: 59.31 AA: 2569240

RT: 53.34 - 57.65 100

90 85

RT: 56.53 AA: 1709616

75

NL: 1.39E6 Base Peak m/z= 235.50236.50 F: MS ICIS MQ8366NF1

95

RT: 60.48 AA: 3042690

80

RT: 55.53 AA: 5395533

80

70

RT: 51.73 - 61.14

95 90 85 80

70

70

60

65

65

60

60

60.58

45 RT: 53.04 AA: 959376 40 35 RT: 56.89 MA: 408434 RT: 57.72 AA: 438797

30 25 RT: 53.84 AA: 460400

20 15 10

RT: 55.25 54.58 AA: 33574 56.03

53.20

5

57.00

RT: 56.75 AA: 3780512

Relative Abundance

50

Relative Abundance

65

RT: 56.39 AA: 1073826

55 50 45 40

56

5

58

59

60

54.0

54.5

55.0

61

Relative Abundance

Partially hydrogenated terphenyls are produced in large amounts for industrial use – they are included in the HPV (high production volume) chemicals list by US-EPA. They are mainly used as heat transfer fluids and polymer plasticizers.

These compounds have been found in recycled paper and cardboard, as well as in food products packed with these recycled materials. Our results show the presence of these contaminants in human plasma samples of 39 volunteers, both smokers and non smokers.

51.95

0

0

57 Time (min)

57.72

10

55.5 Time (min)

56.0

56.5

57.0

53.85 52.11 53.10

52

57.5

53.95

53

54

55.11

56.33 56.41

55

58.33

57.54

56 57 Time (min)

58

59.55

58.43 59

60

61

Extracted Chromatograms with base peak = 242 m/z RT: 51.79 - 55.33

RT: 55.17 - 58.72 RT: 52.89 AA: 308491

100

NL: 8.15E4 Base Peak m/z= 241.50242.50 F: MS ICIS MQ8366NF1

95 90 85 80

56.39

100

NL: 2.84E4 Base Peak m/z= 241.50242.50 F: MS Soro1MonotrapC181

95 90 85 80 75

75

70

70

65

65

Relative Abundance

55

15

RT: 54.54 AA: 161266 53.5

54

20

RT: 54.26 AA: 72594

0 53

25

RT: 54.02 AA: 1045938

5

60.74

40

30

10

60.18

45

35

15

60.58

50

30

20

Cyclohexylbiphenyls and diphenylcyclohexanes

55

35

25

RT: 57.88 AA: 258017 RT: 58.33 AA: 41344

NL: 1.06E5 Base Peak m/z= 235.50236.50 F: MS Soro1MonotrapC181

75

75

55

59.31

100

60 55 50 45 40

60 55 50 45 40 35

35

25

RT: 54.28 AA: 80384

25

20

20

15

15

10

10

5

5

0

0 52.0

52.5

53.0

53.5 Time (min)

56.89

30

RT: 53.31 AA: 95245

30

54.0

54.5

55.0

57.87

57.01 56.47 55.69 55.88

55.29 55.5

56.0

56.26 56.5

57.09 57.44 57.73 57.0 Time (min)

57.5

58.00 58.33 58.53 58.0

58.5

Dicyclohexylbenzenes

Results : Extraction of terphenyls and hydrogenated terphenyls using HLLME TIC profile from HLLME of a non smoker plasma

Base peak 230

RT: 51.01 - 62.99 59.07

100

NL: 6.52E6 TIC F: MS dllme_100_ 150CCL4_ MQ8365NF _18_06_02

95 90 85 80

53.76

75 70

Base peak area (m/z= 230)

Peak nº

Name

1 2

o-terphenyl m-terphenyl

MT-DE

MT-HE

HLLME

2429532 333761

241665 -

1200775 115186

65 59.23

60 55 50 45 40

60.01 60.38

35

Base peak area (m/z= 236)

Peak nº

Name

3 4 5 6 7

1,2-diphenylcyclohexane 1,3-diphenylcyclohexane 1,4-diphenylcyclohexane 3-cyclohexylbiphenyl 4-cyclohexylbiphenyl

30 25 52.86

20 15

5

57.52

53.04

10

57.16

52.01 52

56.20 57.69

53.99 55.07 55.75 53

54

55

56

57 Time (min)

58

61.51 61.89 59

60

61

62

Base peak 242

Base peak 236

6000000

DE 164368 1045938 72594 5395533 3780512

HE 17267 45041 5981 457725 237143

HLLME 79147 353403 61418 2872927 1819588

Monotrap - Direct Extraction Monotrap - Headspace HLLME

5000000

Peak area

4000000

3000000

2000000

Peak Name nº 8 9 10

1,3-dicyclohexylbenzene phenyldicyclohexane 1,4-dicyclohexylbenzene

Base peak area (m/z= 242) DE 308491 95245 80384

HE 117248 35084 26736

HLLME 231020 62574 62484

1000000

0 1

2

3

4

5

6

Peak nº

7

8

9

10

Results : direct transesterification + HLLME CCl4 or heptane

Relative Abundance

Tic profile, FFA methyl esters from smoker plasma

RT: 35.08 - 90.01 59.36

100

NL: 5.42E7 TIC F: MS dllme_100_ 150CCL4_ MQC535F_ 23_06_01

95 90 85 54.04

80 75 70

59.50

65 60

64.06

85.15

60.26

55 50 45 40

Relative Concentration (% peak area)

45

Smoker, CCl4

40

Smoker, Heptane

35

Non smoker, CCl4

30

Non smoker, Heptane 25 20 15

35 30 64.55

25

10 69.29

20 60.59

15

5

53.03 10 47.02 5

38.15 40

65.08

49.66

41.96 45

69.66

57.72

50

55

60 65 Time (min)

70

88.60

79.50 71.43 79.96 73.86 76.37 75

80

0 85

90

C12:0

C14:0

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

C20:3

C20:4

C20:5

C22:5

C22:6

Fatty Acid

• Fatty acid profile can be obtained with direct transesterification and HLLME either with heptane or with CCl4. • Comparison of 13 reference fatty acid showed no significant differences between smokers and non smokers. • Although heptane extracts are less concentrated than CCl4 extracts we could identify 26 fatty acid methyl esters. 26 Fatty Acid Methyl Esters Identified (C12:0) Lauric acid C14:0) Myristic acid (C14:0) 12-Methyltetradecanoic acid (C16:1) Palmitoleic acid (C16:0) Palmitic acid (C16:0) 15-methylhexadecanoic acid (C16:0) 14-methylhexadecanoic acid (C17:0), Heptadecanoic acid

(C18:3), Linolenic acid (C18:2), Linoleic acid (C18:1), Oleic acid (C18:1) Elaidic acid (C18:0) Stearic acid (C20:5) Eicosapentaenoic acid C20:4) Eicosatetraenoic acid (3 isomers) (C20:3) Eicosatrienoic acid (2 isomers)

( (C20:2)Eicosadienoic acid (C20:1) Eicosenoic acid (C20:0) Eicosanoic acid (C22:6) Docosahexaenoic (2 isomers) (C22:5) Docosapentaenoic (C22:4) Docosatetraenoic

Conclusions : • •

Terphenyls and partially hydrogenated terphenyls were extracted from human plasma. Extracts of the materials in contact with the plasma samples and blanks of the analytical process revealed no contamination with these compounds.



SPE of deproteinized and diluted plasma with a new monolithic adsorbant (MonoTrapTM), both in direct contact or from the headspace, allows the selective extraction of these plasma contaminants without significant co-extraction of plasma metabolites. The method with direct contact provides better extraction yield than the headspace extraction.



To the best of our knowledge this is the first report on the use of non halogenated solvents for any kind of liquidliquid microextraction. Although showing smaller enrichment factors then halogenated solvents, the non halogenated solvents are generally less toxic and therefore can be a environmentally safer alternative.



When performing DLLME with plasma samples diluted in water the addition of the organic phase (heptane or CCl4), causes the formation of a gel like structure that prevents a good phase separation.



A new method for the direct derivatization (transesterification) of plasma lipids and simultaneous extraction and concentration of the corresponding methyl esters was developed. The method uses organic solvents in the microliter range, is performed in a single vial and has a total derivatization + extraction time of 15 minutes or less.



The homogeneous liquid-liquid microextraction coupled with direct transesterification allows the fast and sensitive determination of up to 40 fatty acid methyl esters derivatives of plasma lipids.



Terphenyls and partially hydrogenated terphenyls were found, at variable concentrations in 39 out of 40 human plasma samples analysed. No significant differences were found between the smokers and the non smokers group in what concerns fatty acid profile or aromatic contaminants level.



Aknowledgements: The authors thank Dr. Etsudo Ikeda (GL Sciences, Japan) for the kind offer of MonoTrapTM samples. Thanks are also due to Dr. Carlos Marques (Nova Era Clinical Analysis Laboratory, Lisbon) for supplying the plasma samples.