STUDIA UNIVERSITATIS BABEŞ-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003
BIOMEDICAL APPLICATIONS OF GAS CHROMATOGRAPHY-MASS
SPECTROMETRIY
Monica Culea, Onuc Cozar, Dumitru Ristoiu
Univ. Babes-Bolyai, Dept. of Atomic, Nuclear and
Environmental Physics, 1 Kogalniceanu str, 3400 ClujNapoca, Romania e-mail: mculea@phys.ubbcluj.ro
Qualitative and quantitative determinations of some aroma compounds,
lipids, vitamins, drugs, amino acids from biological media by using a gas
chromatography-mass-spectrometric technique (GC/MS) are presented.
Stable isotopic labeled homologous compounds or compounds from the
same chemical class were chosen for the internal standard. Good
validation parameters were obtained for precision, accuracy (C.V.<10%),
linearity in the range of interest (mean r = 0.99), the limit of detection and
quantitation, selectivity and specificity. Chromatography was performed
on a 5% phenyl methylpolysiloxane column (15 or 30 m x 0.25 mm I.D.,
0.25 µm) operated in suitable temperature programs.
Introduction
In the last years the number of procedures using extraction of organic compounds
from different matrices has increased. Trace analyses involve pretreatment: of the
samples, extraction and concentration procedure to increase the analyte
concentration to within instrument sensitivity. The extraction procedure, internal
standard selection and method validation are the steps to follow for quantitative
analyses by using gas chromatography-mass spectrometric technique (GC-MS).
The preconcentration step could be: (1) solvent extraction, (2) headspace analysis,
(3) purge and trap, (4) solid phase extraction (SPE) (column and discs) (5) solid
phase microextraction (SPME) and some other modern technique.
Experimental
Extraction procedure
Liquid-liquid extraction (LLE): The sample is shaken with an immiscible organic
solvent, hexane, light petroleum, oxygenated and chlorinated solvents. The organic
layer is injected into the chromatograph.
Solid-phase extraction (SPE): SPE sequence involves the following steps: (1)
activation (conditioning) of the sorbent (2) sample introduction, (3) washing (4)
elution of the compounds, (5) regeneration of the column. Ethyl acetate, methanol,
acetone and hexane are the most frequently used solvent for desorption. Solid
phase microextractio (SPME), microwave (MWE) extraction, ultrasonic (USE)
extraction, supercritical fluid extraction (SFE) are some new extraction procedures.
Table 1 presents a comparison of different extraction procedure applied to a
mixture of aroma compounds. 30 µl standard mixture in 0.9 ml hydroalcoholic
MONICA CULEA, ONUC COZAR, DUMITRU RISTOIU
solution, 0.9 ml distilled water and 0.3ml solvent* in a screw cap vessel. After
extraction, 1µl 3-hepten-2-one (ES) was added to the supernatant and analyzed.
Table 1
LLE
Comparative extraction procedure for some aroma compounds
SPE
MWE
Sample+ solvent* RP-18 or TSC cartridges; 3ml methanol
2.45Hz,
4
mix
1.5min; 3ml distilled water; sample, wash; dry: sec,
60oC,
centrifuge 2 min
10 min., elution: 3x0.3ml solvent*
solvent*
*Solvent: mixture ethyl acetate: hexane: dichloromethane, 5/1/1
USE
1
min,
60oC, 1ml
solvent*
Table 2
Extraction procedure for amino acids (AA) and drugs from plasma
Theoph. and caffeine
Amino acids extraction
AA dirivatization
1ml serum + drug
cation exchange resin 1.esterification:
2.acetylation
10µg IS1(5ml screw-cap Dowex 50W-X8 100mesh, 500µl butanol: 200µl TFAA3,
vial)2ml
solvent2, 40x2mm column;
acetyl chloride, 60oC, 20 min,
0.5gNaCl,mixed
Elution: 2ml 3M NH4OH;
5:1,v/v,
1h, cool, dry, 1ml
1min,centrif.3min,conc.
Evaporate
100oC
ethyl acetate
1
IS=15N-theophylline; 2solvent=chloroform:isopropanol 20:1,v/v; 3TFAA=trifluotoacetic
anhydride; Resin is kept into refrigerator with distilled water and 1N NaOH; H+ form
made with 1N HCl. Free AA are analyzed by adding 1ml 1N acetic acid to 0.5ml plasma.
Total AA are analyzed after protein hydrolysis: 6NHCl at 110 oC overnight.
Apparatus
A Hewlett Packard GC 5890 couplet with MS engine 5989B in the EI mode was
used for compounds identification. The GC was equipped with a HP-5MS capillary
column 30mx0.25mm, 0,25µm film, in the temperature program: 50 oC (2’) to
250oC or 300 oC with a rate of 8 oC /min, helium flow rate 1ml/min. A Thermo
Finnigan GC-MS equipped with a Rtx-5MS (15mx0.25mm, 0,25µm) column was
also used in similar conditions. The GC/MS interface line and the ion source were
maintained to 200oC or 250 oC. Electron energy was 70eV and electron emission
300µA.
GC-MS analytical method
GC-MS is a high sensitive and specific technique used in organic analysis. In the
selective ion monitoring (SIM) mode, using a few selected ions, the sensitivity is
increased by one or two orders of magnitude SIM-GC/MS is very useful in
quantitative work and is usually achieved by isotopic dilution (ID). Quantitation is
performed by addition of known amounts of internal standards to the sample before
extraction. The method will compensate for sample losses in the clean-up stage,
assuming that the losses of the standard are identical with those of the analyte.
Method validation: The regression curves obtained for some standards in the range
0 – 24ng were: gave good linearity and r>0.99.
Precision gave relative standard deviation (R.S.D.) for LLE and SPE lower than
3% (n=5). Recovery for LLE was 81% (n=3) and for SPE 92% (n=4). Accuracy
for 20ng shows a R.S.D.mean value of 5% and for 24ng of 6%. The sensitivity
was lower than 100pg at a ratio S/N=10 and L.O.D. was 10pg, S/N=10.
BIOMEDICAL APPLICATIONS OF GAS CHROMATOGRAPHY-MASS SPECTROMETRIY
The recovery results (Fig.2) for the standard mixture are the following:
MWE (103%) > USE (101%)>SPE (92%) > LLE (80.8%)
Table 3
Comparative recovery mean values between two extraction procedures (n=4).
Crt. No. Compounds
tR(min) LLE(%) SPE (C-18)(%)
1
3-hepten-2-one
8.33
76.03 85.19
2
fenchone
11.4
79.12 87.55
3
phenyl ethyl alcohol
12
76.13 92.85
4
terpinen-4-ol
12.8
80.97 90.76
5
citronellol
13.6
82.62 89.84
6
carvone
14
83.96 94.01
7
anisaldehyde
14.4
87.50 102.45
8
methyl myristate (ES)
21
mean values
80.91 91.81
matairesinol
lariciresinol
Results
Active principles in herbs
The method was applied to characterize some active principles in a bitter
(Floratonic) extract (12 herbs extract) and different herbs with terapeutic effects.
Bitter shows sedative, antiseptic, antiinflammatory, carminativ, antirheumatic,
antidepresive and tissue regenerating effects. Therpenoid, azulene, coumarin
derivatives and polyphenols are responsible for some of these properties. The
high levels of lignan flavonoids lariciresinol (250µg/ml) and matairesinol in
“Floratonic” bitter may contribute to the protective effect on coronary heart
diseases and arteriosclerosis. The plant lignans, as well as their mammalian
metabolites enterolactone and enterodiol, have antioxidative properties.
The main volatile compounds identified by the GC-MS analysis of Mentha
piperita L. were: menthol, menthone, isomenthone, 1,8 cineole, menthyl acetate,
limonene, -myrcene, carvone. Mentha piperita L. oil had the active principles:
menthol, menthone, isomenthone, menthyl acetate, -pinene, -pinene,
champhor, limonene, linalool, piperitone(Fig.1). Mentha crispa L. showed
carvone as major component. The oil is used for the flavoring of pharmaceutical
and cosmetic preparations and in medicine as a carminative and gastric stimulant.
Vitamin F, E and C in seabuckthorn fruit have been determined in fruit and fruit
oil. Esterification of the fatty acids with methanol:acetyl chloride is similar as
step 1 in table 2. Dry fruits (400mg/ml) were extracted in 70% ethanol overnight
and 0.5mg IS (methylundecenoate) was added to 40mg sample.
.
.
MONICA CULEA, ONUC COZAR, DUMITRU RISTOIU
T IC : 5 7 .D
A bundanc e
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
T im e - - >
0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
1 4 .0 0
1 6 .0 0
1 8 .0 0
Fig. 1 Mentha piperita L. oil chromatogram; menthol is the major compound.
Table 4
Vitamin F determination in seabuckthorn dry fruits
FAME
mg/g mg/g mg/g mg/g Mean mg/g S.D. R.S.D.,%
methyl undecenoate (C11:1)IS 12.50 12.50 12.50 12.00
12.38 0.25
2.02
methyl palmitoleate(C16:1)
9.90 12.37 9.51 11.18
10.74 1.30
12.11
methyl palmitate(C16:0)
15.05 18.42 15.02 16.96
16.36 1.65
10.06
methyl linoleate (C18:2)(9,12) 2.88 3.64 3.01 3.53
3.26 0.38
11.63
methyl oleate (C18:1)(9)
12.15 14.59 12.85 14.73
13.58 1.28
9.42
methyl stearate (C18:0)
3.61 4.41 4.17 4.45
4.16 0.39
9.30
total
43.59 53.44 44.55 50.84
48.11 4.79
9.96
Vitamin F
28.13 28.23 27.77 28.02
28.04 0.20
0.70
The high value of palmitoleic acid in these fruits make the oil very useful in
cosmetics for skin protection. In wheat germs the vitamin F measured was
200mg/g, but palmitoleic acid is very small. Vitamin C (determined by sililation
and GC analysis) in the dry fruits was 24,9mg %. Vitamin E of 0.06% in fruit oil
was measured versus cholesterol as internal standard.
The methods presented are suitable for determination and control of organic
compounds in medicinal herb. No major differences were observed between LLE
and SPE in the plant extracts studied. Terpenic compounds, polyunsaturated fatty
acids and flavonoids are some of the compounds responsible for
antiinflammatory, antioxidant, anticarcinogenic activity of plants used in
traditional medicine.
Sterol profiles measurement by GC/MS could be used for adulteration detection of
the fruit juices. Juice marker for orange and grapefruit (stigmastero, campesterol),
pineapple (ergostanol and stigmastanol markers), passionfruit (beta-sitosterol)
could be easily extracted: ethanol: juice: hexane, 8/10/1.5, mixed 2 minutes,
centrifuged 5 minutes and analyzed (Fig. 3).
RT: 22.96 - 30.29
NL:
1.97E7
TIC MS
portocala0
2
110
105
25.85
100
95
90
beta-sitosterol
85
80
70
65
60
45
40
35
30
alfa-sitosterol
campesterol
50
isofucosterol
55
stigmasterol
Relative Abundance
75
25.97
25
26.76
25.28
20
26.08
15
10
24.07
24.60
27.66
27.76
28.13
28.52
29.65
24.67
5
23
24
25
26
27
Tim e (m in)
28
29
30
Fig.2 Sterol profile for orange juice: cholesterol, campesterol, β-stigmasterol, β –sitosterol
(the major compound), isofucosterol, α-sitosterol.
BIOMEDICAL APPLICATIONS OF GAS CHROMATOGRAPHY-MASS SPECTROMETRIY
Drugs
GC/MS applications for measuring drugs are very important: purity control,
pharmacokinetic studies, metabolic studies, clinical applications for treatment and
diagnosis. Caffeine clearance is a novel approach for assessing hepatic microsomal
function. 10 µg/ml 15N-theophylline has been used as internal standard. The
temperature program was: 200-250oC with a rate of 10oC/min. The method was
validated in the range 0-20µg/ml caffeine. The regression curve obtained in
GC/MS assay gave r=0.95. Precision gave R.S.D values lower than 5% for 5µg/ml
(n=7) and lower 12% for 3µg/ml(n=7). Accuracy shows values lower than 6%.
Sensitivity measured at a signal/noise 4/1 was 0.5µg/ml.
Study population: A dose of 4mg/kg p.o. was used. Blood caffeine concentrations
were measured before dose and timed intervals at 0, 30 min, 1, 3, 6, 9 and 12 h.
Caffeine clearance, measured in patients with cirrhosis and chronic hepatitis, was
reduced and half live time was increased in children with liver disease as compared
with control. The pharmacokinetic parameters were calculated with the formulas:
Kel=(lnC1-lnC2)Δt; t1/2=ln2/kel; Cl=kelxVd
where: kel is the elimination constant; C1 and C2 are the maximum plasma
concentration and minimum plasma concentration of caffeine, t1/2 is the half-live
time and Vd is the distribution volume of 0.6l/kg body weight.Fig.4 presents the ion
chromatograms in the SIM mode for the molecular ions and basic peaks for the
both components, caffeine (m/z 194) and the internal standard (m/z 181).
Abundance
15000
14000
13000
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
Time-->0
Ion 194.00 (193.70 to 194.70): MP138.D
Ion 181.00 (180.70 to 181.70): MP138.D
2.50
3.00
3.50
4.00
4.50
5.00
5.50
Fig.3 Caffeine determination by GC/MS in the SIM mode (first peak)
The method is simple, precise and rapid, useful in the analysis of xantines. Isotopic
labeled internal standard used avoids metabolites overlapping. Significant changes
(p<0.01) were observed in caffeine metabolism in children with decompensated
Liver cirrhosis
Controls
20
Caffeine
(µmol/l)
Caffeine
(µmol/l)
15
10
5
0
0
5 Time(h)10
15
40
30
20
10
0
0
5
10
Time (h)
15
cirrhosis. The clearance values of 0.41±0.56ml min-1 kg-1 and half-life times of
14.34±14 are changed because of the reduction in “functioning hepatocyte mass”.
The control values for clearance and half-life time were 1.5±0.46 and t1/2=5±1.8 in
Fig.4 The elimination curve of caffeine in control and cirrhotic patient
literature and our data of 1.36±0.23 and t1/2=5.13±0.85 (n=10). Patients with
noncirrhotic liver disease showed intermediate values (Cl=1.2±0.46 and
MONICA CULEA, ONUC COZAR, DUMITRU RISTOIU
t1/2=6.62±2.44) but higher values of caffeine plasma concentrations, as shown in
the figure above. (Controlmax=20 µmoles/l ; hepatitic disease max=120 µmoles/l).
Important other clinical or metabolic applications have been developed by GC/MS
technique with precise quantitation of drugs from plasma, saliva or tissues:
theophylline for severe astma or apneea treatment, anaesthetics (procaine,
tetracaine, dibucaine, lidocaine) for pharmacokinetic and metabolic studies.
Amino acids quantitation
Amino acids are components of proteins. They are nonvolatile and for GC-MS
measurements they need to be derivatized as presented in table 2, column 2-4,.
Normally, most living things can synthetize some of the amino acids (nonessential) from other food components and other amino acids, but those that cannot
be produces internally (essential) must be provided in the diet. They can be found
in foods (infants formulas, cheese, beer, wine, grapes, honey, soy flour), in plasma,
blood, urine, tissues. The assessment of amino acids quantity was made for
metabolic studies (Gly and Ser interconvertion), diagnoses (serum AA level for
phenylketoneuria where Phe must be limited or maple syrup urine disease where
Ile, Leu, Val are problematic), drug control (Trofopar), plant and food
characterization, transmembranar transport study.
Amino acids determination by SIM-GC-MS in some leaves ethanol extracts is
presented. By using 15N-Gly as internal standard and by monitoring the peaks m/z
154 (for Gly)and 155 of (for 15N-Gly), the unlabeled Gly was determined and then
all the other separated amino acids, taking in account the response factors obtained
with standards of the amino acids. Free amino acids levels measured in Fagus,
Gingko biloba and Hedera helix leaf extracts are presented in table 5.
Table 5
Quantitative determination of amino acids (µg/g) from plant extracts
M
Fagus
Ginco biloba Hedera helix
Ala (alamine)
89
46.57
88.63
Gly (glycine)
75
37.16
225.91
155.91
Val (valine)
117
97.34
154.23
Leu (leucine)
131
12.88
95.71
100.31
Ileu (isoleucine)
131
14.17
193.43
80.84
Pro (proline)
115
2363.56
3289.03
2441.15
Asp (aspartic acid)
133
82.4
322.08
1891.68
Phe (phenylalanine)
165
151.18
136.34
Glu (glutamic acid)
146
492.8
416.65
Lys (lysine)
146
85.15
Tyr (tyrosine)
181
195.24
Met (methinine)
149
26.37
The major amino acid is proline, which has an imprtant role in protein syntheses
and in tissue regeneration.
AA transmembranr transport
The isotopic dilution GC/MS technique has been used to study 15N-glycine
transmembranar transport. Small influx was detected for glycine in red cells. The
BIOMEDICAL APPLICATIONS OF GAS CHROMATOGRAPHY-MASS SPECTROMETRIY
presence of some ingredients increased the amino acid influx. The washed cells
suspended in the incubated medium contained 15N-Gly in the range 1-10mM.
Incubation of 20 ml suspention was made in a rotary shaker at 37 oC, followed by
centrifugation at 3000xg. 10% trichloroacetic acid was use to hemolyse, then
centrifuge and finally 0.2ml supernatant solution was analyzed. The peaks m/z 154
and 155 of trifluoroacetyl-butyl ester derivatives of Gly and 15N-Gly were
measured for transport calculation (15N-Gly was used also as internal standard),
where p=sample(µg.ml); S=internal standard (µg); V=sample volume (ml); C,
Cs,Cp=mol ratio of 15N Gly, atom%, for the half part of the sample where internal
standard was added and sample, respectively. The quantity of 15N-Gly (µM) was
calculated as pCp. p  S (Cs  C )
V (V  Cp)
The presence of an electric field, incubation temperature, time of incubation,
concentration, drugs, NaCl, could influence the glycine transport. The efflux of
14
N-Gly was calculated together with the influx of 15N-Gly into the cell.
Fig. 5. 15Gly influx versus concentration of 15N-Gly and incubation time
Conclusions
After about half century of applications, GC-MS technique demonstrates that still
remains an important tool for many studies, including biomedical field.
References
1. M. Culea, M. Apetri, C. Gherman, Analysis of Aroma Compounds by Gas
Chromatography and Gas Chromatography/Mass Spectrometry: Comparative Extraction
Methods, Roum.J. Physics. 46 (2001) 7-8..
32. M. Culea, N. Palibroda, P. Panta Chereches, M. Nanulescu,“Comparative of isotopic
dilution methods for determination of heophylline in the plasma and saliva of infants and
children”, Chromatographia, 53 (2001) S387-S-390.
3. M.Culea, S.Neamtu, N.Palibroda, M.Borsa, S.Nicoarã: Study of amino acid
transmembranar transport in human red cell and rat hepatocyte, Journal of Molecular
Structure, 348 (1995) 377-380.