Biomedical applications of gas chromatography-mass spectrometriy
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Monica Culea, Onuc Cozar, Dumitru Ristoiu Univ. Babes-Bolyai, Dept. of Atomic, Nuclear and Environmental Physics, 1 Kogalniceanu str, 3400 Cluj-Napoca, 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.
ExperimentalExtraction procedureLiquid-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 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 Comparative extraction procedure for some aroma compounds
*Solvent: mixture ethyl acetate: hexane: dichloromethane, 5/1/1 Table 2 Extraction procedure for amino acids (AA) and drugs from plasma
1IS=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 110oC overnight. ApparatusA 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. The recovery results (Fig.2) for the standard mixture are the following: MWE (103%) > USE (101%)>SPE (92%) > LLE (80.8%)
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
ResultsActive principles in herbsThe 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. 
Fig. 1 Mentha piperita L. oil chromatogram; menthol is the major compound. Table 4 Vitamin F determination in seabuckthorn dry fruits
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).
Fig.2 Sterol profile for orange juice: cholesterol, campesterol, β-stigmasterol, β –sitosterol (the major compound), isofucosterol, α-sitosterol. 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). 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 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”. T 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 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 (non-essential) 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.
Quantitative determination of amino acids (µg/g) from plant extracts
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 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 37oC, 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. The presence of an electric field, incubation temperature, time of incubation, concentration, drugs, NaCl, could influence the glycine transport. The efflux of 14N-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. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
matairesinol 
lariciresinol
