lc-ms identification of derivatized free fatty acids from adipocere in soil samples

Research Article

LC-MS identification of derivatized free fattyacids from adipocere in soil samples

Free fatty acids were derivatized as amides (DFFA) by reaction with (R)-(1)-1-phenyl-

ethylamine, using a simple, fast and robust reaction scheme. A HPLC method with diode

array and ESI MS detection was developed for the analysis of the derivatized substances.

Six fatty acids were used in the method development: myristic, linoleic, palmitic, oleic,

margaric and stearic acids. Under these conditions the elution of the DFFA are well

resolved with retention times raging from 6.9 to 16.0 min. Fatty acids were extracted from

cemetery soil and from adipocere formation experimental soils using a Soxhlet extraction,

using as solvent ether/dichloromethane (1:1). Each DFFA is characterized by three m/zpeaks: molecular weight of the substance; molecular weight of a dimer of the substance;

the molecular weight of the dimer plus the atomic mass of sodium. The analysis of soil

samples detected the six fatty acids used in the method developed plus palmitoleic and

pentadecanoic. Beside this set of eight fatty acids other 13 fatty acids were detected in trace

quantities or only in some soils and some were tentatively assigned as: 10-hydroxystearic,

myristoleic, heptadecenoic and arachidic acids.

Keywords: Adipocere / Fatty acid derivatization / LC-MS / Soils extractionDOI 10.1002/jssc.200900614

1 Introduction

The major constituents of adipocere, defined as a late-stage

post-mortem decomposition product consists of a mixture of

free fatty acids (FFAs). It is produced when triacylglycerides

in the adipose tissue are converted enzymatically to

unsaturated and saturated fatty acids, the former being

reduced further to saturated acids [1, 2]. In a forensic

context, its characterization yields important information

about a body decomposition rate [3, 4]. Due to their

continued interest, an increased attention has received and

a wide research work is performed to characterize the

chemistry of adipocere. In particular, gas chromatography

has provided useful information about the lipid composition

of adipocere in a number of studies [5–10]. The major fatty

acids present in order of abundance are palmitic (C16:0),

stearic (C18:0) and myristic (C14:0) acids [11]. Depending on

the environmental conditions, oxygenated FFAs are recog-

nized as hydroxyl [12, 13] and oxo derivates at trace level [7,

14]. The literature reports showed that adipocere formation

and FFA composition may be a function of the soil

characteristics, burial environment, soil pH, temperature,

moisture and oxygen content [7, 15–17].

LC analysis of the adipocere composition is mainly

focussed in the determination of FFA composition, tagging

derivatized compounds with reagents that afford chromo-

phores having UV and/or Vis absorption bands. Among

those reagents, phenyl and substituted phenyl bromide are

widely used [18–21]. To improve the detection, a-bromo-20-

acetonaphthone [22] and 2-nitrophenylhydrazine [23, 24]

were proposed. Analyses of FFAs as their hydroxamic acids

[25] and monoalkanoylamides [26] have been also reported.

To simplify the separation process, polymeric derivatizing

reagents have been developed for the analysis of FFAs [27].

LC-MS methodologies, coupled to specific extraction tech-

niques, have been proposed for the analysis of FFA for

chocolate quality control [28] and in the inhibition effect of

sterculic acid in the D9-desaturase activity [29].

In this article, a new synthetic route for the reaction of

(R)-(1)-1-phenylethylamine (R-PEA) with FFA is proposed,

allowing a simple, fast and robust derivatization of FFA to

chiral amides (DFFA). DFFA were prepared using a multi-

step procedure that involves a key step, the functionalization

of the corresponding fatty acid via an acylation using a

carboxylic–carbonic anhydride intermediate. The prepara-

tion of carboxylic–carbonic anhydride intermediates from

the corresponding carboxylic acids and alkyl haloformates is

a convenient method in chiral amide synthesis using soft

conditions leading to high product yields without loss of

optical activity. Moreover, a LC method with MS detection

Manuel Algarra1

Jose E. Rodrıguez-Borges2

Joaquim C. G. Esteves daSilva2

1Centro de Geologia do Porto,Faculdade de Ciencias daUniversidade do Porto, Porto,Portugal

2Centro de Investigac- ao emQuımica, Departamento deQuımica, Faculdade de Cienciasda Universidade do Porto, Porto,Portugal

Received September 22, 2009Revised November 1, 2009Accepted November 2, 2009

Abbreviations: DFFA, derivatized FFA; FFA, free fatty acid;

R-PEA, (R)-(1)-1-phenylethylamine; RT, retention times

Correspondence: Professor J. C. G. Esteves da Silva, Centro deInvestigac- ao em Quımica, Departamento de Quımica, Faculdadede Ciencias da Universidade do Porto, R. Campo Alegre 687,4169-007 Porto, PortugalE-mail: [email protected]: 1351-220-402-659

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 143–154 143

(LC-MS) was developed for the identification of FFA after

extraction from soils. The objective of this study was to

develop a forensic methodology for the identification of the

fatty acid composition that resulted in soil as consequence

of adipocere formation. Besides the fundamental extraction

and detection steps, and taking into consideration the

complexity of the samples under investigation, that are

characterized by quite complex trace level mixtures of

Scheme 1. DFFA derivatization reaction.

RT:A

B

0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

uAU

2.87

2.80

3.113.67 6.77

8.1010.59

4.19

9.93

15.8712.45

22.065.68

22.30 23.2 6

C:\Xcalibur\...\JESMistura_090602111556 02-06-2009 11:15:56

RT:0.00 - 24.99

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

10.716.93 8.218.25

10.8210.5910.52

6.85 8.308.10

16.0212.51

12.599.8316.1512.42 12.64

15.8016.21

12.8515.73

9.76

12.90 15.65 16.41

16.5415.58

22.1415.52 16.67

16.99 22.2520.6013.37 19.446.465.8422.42

0.21 24.894.721.21 3.17

0.00 - 25.00

Figure 1. Typical chromatogramsof the six DFFA with diode array(total scan) (A) and mass (TIC) (B)detection.

J. Sep. Sci. 2010, 33, 143–154144 M. Algarra et al.


FFA in a complex background, the proposed methodology

for forensic applications includes a derivatization reaction

because: (i) when UV-Vis detection is used LC resolution

and detection limits of FFA can be improved

because underivatized FFA show absorption in the UV and

relatively small extinction coefficients; (ii) less mild

chromatographic conditions can be employed for the reso-

lution of derivatized FFA without the necessity of complex

chromatographic eluents and gradients and relatively high

column temperatures; (iii) LC-MS detection of a large

number of FFA derivatives in the same sample is feasible

and unequivocal.

225 230 235 240 245 250 255 260 265 270 275 280 285 290 295

wavelength (nm)

0100020003000400050006000700080009000

10000110001200013000140001500016000170001800019000200002100022000

uAU

259.00

280.00

RT:0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

uAU

2.93

3.10

8.09

6.77

10.59

9.92

15.8712.43

3.58

4.21

2.59 5.78

A

B

Figure 2. Typical UV spectraof the derivatized fatty acids(A) and a typical chromato-gram of the mixture of sixDFFA with detection at259 nm (B).

Table 1. LC RT and characteristic m/z peaks of the DFFA clearly

identified in all cemetery soils and in adipocere

formation experimental soils

Fatty acid MW DFFA RT

(min)

Characteristic

m/z peaks

Myristic C14:0 331.3 6.9 332.5 663.5 685.3

Palmitoleica) C16:1 357.3 7.2 358.3 715.2 737.1

Pentadecanoica) C15:0 345.3 7.7 346.3 691.1 713.2

Linoleic C18:2 383.3 8.2 384.4 767.5 789.1

Palmitic C16:0 359.3 10.0 360.5 719.4 741.3

Oleic C18:1 385.3 10.8 386.4 771.5 793.1

Margaric C17:0 373.6 12.5 374.5 747.5 769.2

Stearic C18:0 387.4 16.0 388.4 775.4 797.2

a) These FFA were assigned to the detected m/z.

J. Sep. Sci. 2010, 33, 143–154 Liquid Chromatography 145


2 Materials and methods

2.1 Chemical and reagents

FFAs standards (linoleic, margaric, myristic, oleic, palmitic,

stearic and acid) were supplied by Sigma-Aldrich Quımica

SA (Spain). The derivatizing chiral agent, R-PEA, dichlor-

omethane, methanol (HPLC grade) and ethylic ether were

purchased from Merck (Darmstadt, Germany) and Panreac

(Barcelona, Spain), respectively. All compounds were

analytical-reagent grade and the purities were stated to be

higher than 99%. Methanol stock standard solutions for

HPLC measurements were used. Deionised water was

employed in all of the experiments.

2.2 Soil samples

Some samples of soils from Agramonte and Prado Repouso

cemeteries (Municipal cemeteries of Porto, Portugal) with

known episodes of unskeletalized corpses were analysed.

Soil samples were collected in the soil adjacent to the grave.

JESMistura_090602111556A

B

#551 RT: 10.84 AV: 1 NL: 3.26E8T: + p ESI Full ms [ 250.00-1500.00]

300 350 400 450 500 550 600 650 700 750 800 850m/z

m/z

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

300000000

320000000

Inte

nsity

793.13

771.47

386.40

408.53431.20 809.00671.20 748.27 876.60611.87476.27 521.20 562.93266.67 359.80301.33

JESMistura_090602111556 #647 RT: 12.62 AV: 1 NL: 2.52E8T: + p ESI Full ms [ 250.00-1500.00]

300 350 400 450 500 550 600 650 700 750 800 8500

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

Inte

nsity

769.20

747.47

374.47

396.40784.73433.47 473.13 39.14874.476 895.07708.33612.27552.73270.47 364.27

Figure 3. Mass spectracharacteristic of the deriva-tives of the acids: (A) myris-tic; (B) linoleic; (C) palmitic;(D) oleic; (E) margaric; (F)stearic.



Also, some adipocere formation experimental soil samples

were obtained from the burial using typical soils of small

pieces of pork loin (10.970.5 g) in a plastic hermetic box

(15 cm� 15 cm� 5 cm) the box was closed and kept inside a

closed black plastic bag protected from light for the pre-

selected time of several months.

2.3 Extraction procedure of FFA in soil

Soxhlet extractions were performed using variable amounts of

analytically weighed soils, without prior treatment [30]. Samples

(30–75 g) were extracted under reflux with 1:1 dichloro-

methane/ether solution (100 mL) during 3 h. The samples were

mixed with anhydrous sodium sulphate and filtered, previous

cooling to room temperature. The extracts were dried using a

rotary evaporator at 401C and reduced pressure.

2.4 Derivatization procedure of FFA: Synthesis of

(R)-N-(1-phenylethyl)amides

Both standards and extracts containing FFA were trans-

ferred into a Schlenk tube reactor, equipped with a magnetic


300 350 400 450 500 550 600 650 700 750 800 850m/z

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

300000000

320000000

Inte

nsity

793.13

771.47

386.40

408.53431.20 809.00671.20 748.27 876.60611.87476.27 521.20 562.93266.67 359.80301.33

C

D


300 350 400 450 500 550 600 650 700 750 800 850m/z

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

Inte

nsity

797.20

775.40

388.40433.33 626.40545.67 813.0774.31778.003 855.2035.47672.105383.00 572.53

Figure 3. Continued.



stirrer, and maintained in an inert atmosphere (argon). To

obtain the amides derivates of FFA (linoleic, margaric

(heptanoic acid), myristic, oleic, palmitic and stearic acid)

they were converted into the corresponding carboxylic–

carbonic anhydride intermediate by treatment with ethyl

chloroformate (2 equiv.) and triethylamine (2 equiv.) in

dried dichloromethane (10 mL/4 mmol of fatty acid) under

argon, using a procedure similar to that previously

described in the literature (Scheme 1) [31]. The correspond-

ing anhydride intermediate, which was not isolated, was

concentrated by vacuum and reacted with R-PEA (1.2 equiv.)

under argon using dried dichloromethane as solvent

(20 mL/4 mmol of initial fatty acid), giving the correspond-

ing amide. The resulting organic mixture solution was

washed with 1 M HCl (4� 10 mL), the organic layer was

washed with brine (20 mL) and it was dried with anhydrous

sodium sulphate. Removal of the solvent on rotary

evaporator yielded the corresponding pure amide quantita-

tively. Standard FAA solution was prepared by dissolving

10 mg of synthesized DFFA in 10 mL methanol, stored at

41C and in absence of light. Working solutions were

prepared by dilution with methanol.


300 350 400 450 500 550 600 650 700 750 800 850m/z

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

Inte

nsity

769.20

747.47

374.47

396.40784.73433.47 473.13 39.14874.476 895.07708.33612.27552.73270.47 364.27


300 350 400 450 500 550 600 650 700 750 800 850m/z

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

Inte

nsity

797.20

775.40

388.40433.33 626.40545.67 813.0774.31778.003 855.2035.47672.105383.00 572.53

E

F




The synthesized DFFA were characterized by 1H-NMR

spectrometry (400 MHz, CDCl3) (see Supporting Informa-

tion).

For the soils extracts the same derivatization procedure

was carried out, using dichloromethane (2 mL/50 mg of

isolated sample), ethyl chloroformate (40 mL/50 mg of

sample) and triethylamine (50 mL/50 mg of sample), for 3 h

at room temperature. After that, a gentle vacuum was

applied, to evaporate all solvents and excess of reactants. R-

PEA was added (30 mL/50 mg of sample) in anhydrous

dichloromethane (2 mL/50 mg of isolated sample) to form

DFFA, left overnight. Extracted with dichloromethane and

washed with HCl and brine, as previously described in the

standard FFA. Extracts were dissolved in 4 mL of methanol

and transferred into injection vials for LC analysis.

2.5 LC method

The analysis was performed using a LC-DAD-MS system

with a Finnigan Surveyor series liquid chromatograph

equipped with Finnigan Surveyor autosampler. Double-

online detection was done by a photodiode spectrophot-

ometer and MS. The mass detector was a Finnigan LCQ

RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

75000

80000

85000

90000

uAU

3.22 3.854.89

5.33 11.09

5.70

6.15

9.947.47

8.36

12.41 16.1013.28

16.95 22.9619.13 19.67 20.53

RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

75000

uAU

3.25 3.85 5.66

9.83

4.63

6.13

6.80 7.47

8.2312.31

13.22 15.75 19.5316.65 22.4420.26 23.262.20Figure 4. Typical UV (detectionat 259 nm) chromatograms ofsoils extracts.



DECA XP MAX (Finnigan, San Jose, CA) quadrupole IT

equipped with API source, using ESI interface. The

vaporizer and the capillary voltages were 5 and 4 V,

respectively. The capillary temperature was set at 3001C.

Nitrogen was used as both sheath and auxiliary gas at flow

rates of 60 and 15, respectively (in arbitrary units). Spectra

were recorded in positive ion mode between m/z 250 and

1500. Chromatographic data analysis was done with

Xcalibur 1.4 software from Thermo Electron.

The separation was done with a chromolith RP-18

column (125� 4.6 mm) from Merck KGaA thermostated at

251C. A mobile phase constituted by a mixture of methanol/

water (90:10) with a flow rate of 0.5 mL/min was used and

the injection volume was 25 mL. For the preparation of the

mobile phases, the solvents were filtered through 0.2 mm

nylon membrane filters and degassed during, at least, 1 h.

3 Results and discussion

3.1 Standard FFAs analysis

Figure 1 shows the UV–Vis chromatogram (total scan DAD

in the range 220–750 nm) and MS chromatogram (total ion

count (TIC) in the range m/z 250–1500 Da) of a mixture of

the six known DFFA. The analysis of the chromatograms in

Fig. 1 show that the peaks corresponding to the six DFFA

are well resolved but those due to palmitic and oleic acids

partially overlap.

Table 1 presents the retention times (RT) of the six

DFFAs. As expected, the general trend is the increasing of

RT with the increasing of the FFA chain length but the

degree of unsaturation and position of the terminal double

bound changes that trend [32].

RT:0.00 - 24.98

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

10.0410.15

10.23

9.89 10.69

10.777.317.26 7.467.59

10.907.72 22.20

9.2610.97

22.139.195.149.107.14

11.089.06 16.31 16.416.875.08 16.115.23 11.458.206.6816.4811.715.56 16.00

11.79 14.13 22.3716.6515.92 21.0715.85 16.76 20.92

22.4217.67 20.5122.52

22.8023.14

4.352.861.810.25

RT:0.00 - 24.99

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

10.11

10.26

10.325.4112.7710.847.03 10.04 12.695.75 13.07

11.026.06 9.957.07 13.165.38 7.16 12.64 13.255.23

7.31 9.89 13.2912.6011.32 16.265.13 13.38 16.457.38

16.548.295.1021.1016.1314.079.82 21.19

21.4116.6514.169.70 21.0121.47

16.088.77 22.1416.7622.2017.90 20.86

18.0714.48 20.7718.38 22.2919.20

22.33

22.42

22.6122.79 24.97

1.68 4.261.11 2.33

Figure 5. Typical MS chromato-grams of soils extracts.



Table 2. LC RT and characteristic m/z peaks of the DFFA identified in trace amounts in some cemetery soils and in adipocere formation

experimental soils

Fatty acida) MW DFFA RT (min) Characteristic m/z peaks

10-Hydroxystearic 300 5.5 404.5 807.3 829.2

Myristoleic C14:1 328.4 7.0 329.4 656.7 679.1

Not assigned 283.4 7.8 284.4 567.5 589.4

Not assigned 309.7 8.7 310.7 619.3 641.3

Heptadecenoic C17:1 371.3 9.1 372.3 743.2 765.1

Not assigned 297.4 9.5 298.4 595.5 617.4

Not assigned 335.5 9.6 336.5 671.3 693.2

Not assigned 311.6 12.6 312.6 623.4 645.4

Arachıdic C20:0 411.2 13.6 412.2 823.1 845.2

Not assigned 399.5 14.1 400.5 799.4 821.3

Not assigned 337.7 14.2 338.7 675.4 697.3

Not assigned 339.6 17.3–21.2 340.6 679.4 701.5

Not assigned 365.7 18.1 366.7 731.5 753.3

a) These FFA were assigned to some of the detected m/z.

RT: 0.00 - 24.99A

B

0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

6.79

6.83

6.68

6.62

16.265.17 10.735.23 7.01 12.65 17.237.48 10.56 11.49 15.81 17.6513.715.08 6.07 18.479.19 20.56 20.6714.94 21.9322.31

24.65

RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

10.14

10.07

10.03

10.20

10.38

9.73 10.467.717.675.665.14 16.1715.80 17.6612.63 20.2314.12 18.66 21.81 22.13 23.43

Figure 6. Typical chromato-grams of cemetery soilsobtained for selected m/zranges: (A) myristic (range792–794.5); (B) palmitic(range 740–742.5); (C) oleic(range 684–686).



The UV spectra of all the DFFA are similar with a

maximum wavelength at 259 nm (Fig. 2A). By selecting a

UV detection at 259 nm the chromatogram becomes

simpler (Fig. 2B). However, some overlap of the palmitic

and oleic acids derivatives is still observed that does not

compromise a quantitative analysis.

As shown in Fig. 3, the mass spectra of the six known

DFFA were easily detected confirming that the derivatization

reaction proceeded as expected and the amides of the FFA

were indeed obtained. Besides that m/z of the DFFA mole-

cule, two more characteristic m/z values of each DFFA were

detected with higher sensitivity: the dimer of the DFFA and

the dimer plus sodium ion. Figure 3 and Table 1 shows and

summarizes the characteristic m/z peaks of each DFFA.

The analysis of the results obtained for the six DFFA

shows that the proposed chromatographic methodology allows

the unequivocal separation and identification of FFA based on

RT and MS of the corresponding derivative. Also, at least for

pure FFA solutions, the quantification of the six studied FFAs

is apparently possible using a UV detection at 259 nm.

3.2 Soil samples analysis

In order to assess the perforce of the proposed methodology

in real samples, 24 samples of soils were studied (extracted,

derivatized and analysed).

Figure 4 shows some examples of UV (detection at

259 nm) chromatogram of soil samples. These chromato-

grams are usually characterized by some intense peaks with

RT close to the previously detected DFFA but with incom-

patible UV spectra. This observation shows that the possibility

of a quantitative analysis of the DFFA using a simple UV

detector should be done with caution. Nevertheless, the DFFA

are chiral molecules and, if further confirmatory qualitative

and/or quantitative analysis is required, chiral columns

coupled to chiral and/or MS detector can be used [33, 34].

Figure 5 shows some examples of MS chromatogram of

the soil samples. A preliminary analysis of these chromato-

grams shows, as expected from the analysis of the UV

chromatogram, that they are more complex, with other m/zpeaks, besides those observed for the six known DFFA.

However, from the analysis of the characteristic m/z values

at the expected RT, the derivatives of myristic, linoleic,

palmitic, oleic, margaric and stearic, plus two other DFFA,

were identified in almost all studied soil samples. These

new DFFA were identified as corresponding to the deriva-

tives of palmitoleic (C15:0) and pentadecanoic acids (C16:1)

(Table 1).

Besides this set of eight DFFA that are present in almost

all soil samples, there are a set of 12 DFFA that are detected

in some samples or are only present in trace quantities. As

shown in Table 2 all the DFFA show the characteristic three

m/z values. Some of these new peaks can be tentatively

assigned (comparing the expected m/z values) to a DFFA [5,

35]: myristoleic, heptadecenoic and arachidic acids.

Although some samples show overlapping of the DFFA

when the TIC detection is used, the extracted ion chromato-

grams using one of the characteristic DFFA m/z are highly

improved. Because the mass of the DFFA corresponding to

the dimer plus sodium show the highest sensitivity, it was

used as characteristic detection mass. Figure 6 shows the

examples of the chromatograms of the derivatives of

myristic, palmitic and oleic as detected in real cemeteries

soil samples. These chromatograms, together with the

corresponding MS, unequivocally identify the FFA.

Adipocere detection studies usually refer the existence

of trace amounts of 10-hydroxystearic acid [36], 10-oxopal-

mitic acid [14] and 10-hydroxypalmitic acid [14]. Although in

trace amounts, a DFFA compatible with 10-hydroxystearic

was indeed detected (Fig. 7) confirming its trace presence in

the soil samples. The 10-oxopalmitic acid has a molecular

weight similar to margaric acid (373 g/mol) and in the soil

chromatograms only one peak with characteristic m/z peaks

C RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

10.79

10.68 10.83

10.62

10.53

11.145.08 10.4010.27 11.395.57 7.656.91 16.088.60 13.41 16.3014.22 18.09 22.1718.79 21.55 22.30 24.96




corresponding to that molecular weight was detected at the

RT of margaric acid. Consequently, either 10-oxopalmitic

acid is not present in the analysed soil samples or it is not

possible to unequivocally identify using this method

because it is confounded with margaric acid. The 10-

hydroxypalmitic acid was not detected in the soil samples

under analysis.

4 Concluding remarks

A simple and sensible method for detection of FFAs in soil

samples is proposed. A large number of fatty acids were

detected in cemetery soils and in experimental soils

containing adipocere. The proposed methodology for FFA

analysis in soils can be used for forensic analysis because

several unique characteristics (RT and the set of three m/zvalues) and the absolute mass of the DFFA constitute

an unequivocal identification. The proposed derivatizing

methodology results in chiral derivatives fatty acids that can

be the basis for the development of new chromatographic

methods using chiral chromatographic columns.

The Direction of the ‘‘Divisao Municipal de HigienePublica’’ of the Municipally of Porto is acknowledged toauthorize soil sample collection inside the cemeteries of Agra-monte and Prado Repouso. Financial support from Fundac- aopara a Ciencia e Tecnologia (Lisboa, Portugal) (FSE-FEDER)(Project PTDC/QUI/71001/2006) is acknowledged. Routinefatty acids soil extraction laboratory work is acknowledged toMarcela J. C. Oliveira, Tatiane Mate, Teresa Alves, LilianaJunqueiro. The authors thank the Fundac- ao para a Ciencia e

RT:A

B

0.00 - 24.98

0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

5.38

5.43

5.30

5.14

11.1811.03 11.34 22.05

22.1810.88 11.4715.835.73 16.50 17.7415.707.48 22.429.97 13.17 19.98 21.30 23.24

JESAmostra1A3#263 RT: 5.47 AV: 1 NL: 6.66E7T: + p ESI Full ms [ 250.00-1500.00]

300 350 400 450 500 550 600 650 700 750 800 850m/z

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

50000000

55000000

60000000

65000000

Inte

nsity

829.00

404.53

805.27

518.33454.47 687.47899.00756.07494.07 72.45800.146561.87 586.27 726.40381.33298.33 333.20

Figure 7. Typical chromatogramof cemetery soils obtained m/zrange 828–830 (A) and the massspectra of the DFFA with a RT of5.5 min (B).



Tecnologia (Lisboa, Portugal) under the frame of the Ciencia2007 program.

The authors have declared no conflict of interest.

5 References

[1] Den Dooren de Jong, L. E., Antonie van LeeuwenhoekJ. Microb. Ser. 1961, 27, 337–361.

[2] Rosenfeld, L., Clin. Chem. 2002, 48, 186–197.

[3] Dent, B. B., Forbes, S. L., Stuart, B. H., Environ. Geol.2004, 45, 576–585.

[4] Forbes, S. L., Stuart, B. H., Dadour, I. R., Dent, B. B.,J. Forensic Sci. 2004, 49, 566–574.

[5] Notter, S. J., Stuart, B. H., Dent, B. B., Keegan, J., Eur. J.Lipid Sci. Tech. 2008, 110 73–80.

[6] Nushida, H., Adachi, J., Takeuchi, A., Asano, M., Ueno,Y., Forensic Sci. Int. 2008, 175, 160–165.

[7] Forbes, S. L., Stuart, B. H., Dent, B. B., Forensic Sci. Int.2002, 127, 225–230.

[8] Adachi, J., Ueno, Y., Miwa, A., Asano, M., Nishimura,A., Tatsuno, Y., Lipids 1997, 32, 1155–1160.

[9] Yamaoka, A., Forensic Sci. 1977, 9, 63–73.

[10] Takatori, T., Forensic Sci. Int. 1996, 80, 49–61.

[11] Vane, C. H., Trick, J. K., Forensic Sci. Int. 2005, 154,19–23.

[12] Takatori, T., Terazawa, K., Nakano, K., Matsumiya, H.,Forensic Sci. Int. 1983, 23, 117–122.

[13] Takatori, T., Forensic Sci. Int. 1996, 80, 49–61.

[14] Takatori, T., Leg. Med. 2001, 3, 193–204.

[15] Forbes, S. L., Dent, B. B., Stuart, B. H., Forensic Sci. Int.2005, 154, 35–43.

[16] Fiedler, S., Schneckenberger, K., Graw, M., Arch.Environ. Contam. Toxicol. 2004, 47, 561–568.

[17] Forbes, S. L., Stuart, B. H., Dent, B. B., Forensic Sci. Int.2005, 154, 24–34.

[18] Wood, R., Lee, T., J. Chrom. 1983, 254, 237–246.

[19] Silva, F. O., Ferraz, V., Talanta 2006, 68, 643–645.

[20] Amet, Y., Adas, F., Berthou, F., Anal. Chim. Acta 2002,465, 193–198.

[21] Yang, W. C., Adamec, J., Regnier, F. E., Anal. Chem.2007, 79, 5150–5157.

[22] Zoutendam, P. H., Bowman, P. B., Rumph, J. L., Ryan, T.M., J. Chrom. 1984, 283, 281–287.

[23] Miwa, H., Yamamoto, M., J. Chrom. 1986, 351, 275–282.

[24] Miwa, H., Yamamoto, M., J. Chrom. 1987, 421, 33–41.

[25] Gutnikov, G., Streng, J. R., J. Chrom. 1991, 587, 292–296.

[26] Ben-Bassat, A. A., Wasserman, T., J. Liq. Chrom. Rel.Tech. 1987, 10, 2939–2950.

[27] Yasaka, Y., Tanaka, M., Matsumoto, T., Funazo, K.,Shono, T., Anal. Sci. 1989, 5, 611–612.

[28] Perret, D., Gentili, A., Marchese, S., Sergi, M., Capor-ossi, L., Rapid Commun. Mass Spectrom. 2004, 18,1989–1994.

[29] Dillon, R., Greig, M., Bhat, B., Anal. Chim. Acta 2008,627, 99–104.

[30] Vane, C. H., Trick, J. K., Forensic Sci. Int. 2000, 154,19–23.

[31] Andrade, M., Sousa, C., Borges, J. E., Freire, C., J. Phys.Org. Chem. 2005, 18, 935–940.

[32] Chen, S. H., Chuang, Y. J., Anal. Chim. Acta 2002, 465,145–155.

[33] Kazuaki, A., Ryuta, S., Seiya, S., Hiroshi, O., Chroma-tography 1999, 20, 308–309.

[34] Lingeman, H., in: Detection-Oriented DerivatizationTechniques in Liquid Chromatography, 1st Edn., CRCPress, Boca Raton 1990.

[35] Frund, H., Schoenen, D., Forensic Sci. Int. 2009, 188,18–22.

[36] Saito, K., J. Biochem. 1966, 59, 487–494.



lc-ms identification of derivatized free fatty acids from adipocere in soil samples

Documents