Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Recent methods for the determination of volatile and non-volatile organic compounds in natural and purified drinking water

Four analytical protocols for the extraction and preconcentration of organic residues in natural or purified drinking water were investigated and compared: closed loop stripping analysis; simultaneous extraction—distillation; purge and trap analysis; continuous liquid—liquid extraction. Organic extracts were submitted to a variety of separation and identification techniques. Volatiles were determined by conventional capillary column gas chromatography with tandem mass spectrometry, using triple-stage quadrupole instruments. Non-volatile and thermally labile molecules were investigated by several different techniques (high-temperature gas chromatography, capillary column supercritical fluid chromatography, pyrolysis gas chromatography—mass spectrometry, thermospray liquid chromatography with tandem mass spectrometry and conventional fast-atom bombardment with tandem mass spectrometry). Several samples recently examined in the laboratory provide examples of this multitechnique approach for a more complete knowledge of the organic carbon distribution in water-dissolved organic matter, taking into account organic substances with widely different volatilities, polarities and thermal stabilities.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

Estimation of plasma free fatty acids as the trimethylsilyl (TMS) esters

Quantitative estimates of free fatty acids in total lipid extracts of plasma were obtained by glc on nonpolar columns following trimethylsilylation. The presence of other lipid esters in the reaction mixture had no effect upon the yield of the trimethylsilyl (TMS) esters or upon their resolution on the glc column. Routine quantitations by gas-liquid chromatography (glc) were obtained on 2 ft × 1/8 in. o.d. stainless steel columns packed with 3% OV-1 on 100–120 mesh Gas Chrom Q by means of temperature programming in the range 175–350°C with tridecanoin as internal standard. High resolution glc of the TMS esters of fatty acids was done on a 6 ft × 1/8 in. o.d. glass column packed with 1% SE-30 on Gas Chrom Q. In both instances the fatty acids were resolved on the basis of carbon number and by the presence or absence of double bonds. On gas chromatography/mass spectrometry (GC/MS), TMS esters of fatty acids were shown to yield proportionally greater amounts of high mass fragments, including the parent ions, than their methyl or ethyl esters, which has special advantages for the detection and characterization of polyunsaturated fatty acids.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Application of gas chromatography–triple quadrupole mass spectrometry to the determination of sterol components in biological samples in consideration of the ionization mode

The hyphenation of gas chromatography (GC) and triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad mass spectrometry. We present in this paper the application of GC–triple quadrupole mass spectrometry for determination of sterol components in biological samples. Due to the fact that sterols are quite small molecules an appropriate ionization mode has to be found for advantageous exploitation of the triple quad function. Electron ionization (EI), positive and negative chemical ionization (PCI, NCI) have been tested regarding sensitivity improvement in oxysterol and bile acid analysis in plasma samples. Target analytes were 24-, 25- and 27-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 3β,5α,6β-cholestanetriol, cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. In contrast to bile acids, oxysterols could be analyzed with the highest degree of sensitivity by application of PCI in multiple reaction monitoring mode whereas 7β-hydroxycholesterol and 7-ketocholesterol showed even better results with NCI.     

Analysis of taxol and related diterpenoids from cell cultures by liquid chromatography—electrospray mass spectrometry

Authentic taxanes (taxol, 10-deacetyltaxol, cephalomannine, 10-deacetylcephalomannine, baccatin III) and extracts from cell cultures derived from various yew tree species have been analyzed by microbore high-performance liquid chromatography (HPLC)—electrospray mass spectrometry (ESMS). All gave excellent positive-ion ES spectra with dominant protonated molecules at low nozzle-to-skimmer bias value (45 V). By increasing the voltage value to 85 V, fragmentation increased and structurally informative spectra were obtained. The fragments found were both of the C-13 side-chain and of the taxane ring, so their analysis gave important information about the taxane structure and any chemical modifications at different positions of the molecule. When tandem MS was used (argon gas, 25 eV collision energy), fragments similar to those obtained from collision-induced dissociation in the source were detected. The cell culture extracts were analyzed by microbore HPLC—ESMS and excellent spectra were obtained on 5–10 ng of separated compounds; even greater selectivity and sensitivity were obtained through use of selected-ion monitoring (SIM). With SIM, 100 pg of all taxanes could readily be detected. In the HPLC—ESMS mode, only 10% of the eluent was mass-analyzed, so 90% would be available for recovery through fraction collecting.     

High-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry for analysis of oligosaccharides derivatized by reductive amination and N,N-dimethylation

Milk oligosaccharides derivatized by reductive amination with benzylamine followed by N,N-dimethylation (DMBA-oligosaccharides), were analyzed by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-ITMS). Separation of DMBA-oligosaccharides was achieved on a graphitized carbon column eluted with aqueous acetonitrile and the DMBA-oligosaccharides were detected by positive-ion mode ESI-ITMS allowing sample amounts down to approximately 30fmol of single DMBA-oligosaccharides injected on the HPLC column. MS/MS operation of the mass spectrometer resulted in the detection of diagnostic fragments, mainly belonging to the Y-series, allowing differentiation between isomeric milk oligosaccharides. HPLC/ESI-ITMS/MS/MS experiments indicated the migration of fucose residues of the DMBA milk oligosaccharides to the modified reducing end glucose residue during analysis, a migration previously only observed for proton adduct ions.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

斑点叉尾鮰鱼油脂成分分析

采用铁锅炼制提取斑点叉尾鮰内脏鱼油,然后加酸使其甲酯化,以气相色谱/质谱法分析鱼油中的脂肪酸.结果斑点叉尾鮰内脏纯油脂中鱼油达到99%.从鱼油中共鉴定出15种成分,有饱和脂肪酸及不饱和脂肪酸,还有少量烷烃类物质.不饱和脂肪酸含量为76.40%,其中多不饱和脂肪酸为18.03%,以C18∶ 2(16.23%)为主,单不饱和脂肪酸为58.37%,以C18∶ 1(54.88%)为主.饱和脂肪酸含量为20.91%,主要有C16∶ 0 (15.84%)和C18∶ 0(4.29%),多是低于C18以上的中长链脂肪酸.因此斑点叉尾鮰油脂可作功能性脂肪酸的重要膳食来源.     

Screening, analysis and in vitro vasodilatation of effective components from Ligusticum Chuanxiong

Effective components, ligustilide and butylidenephthalide, from Ligusticum Chuanxiong (Ligusticum wallichii Franchat, Umbelliferae) were screened and identified by using a cell membrane chromatography (CMC) and a gas chromatography/mass spectrometry (GC/MS). The components showed the effects of inhibiting vasoconstriction in vitro on rat abdominal aorta segments. The screening procedure was performed in a rat artery CMC column (50 mm x 2.0 mm I.D.) with a sodium phosphate buffer (pH 7.4) as mobile phase at 37 degrees C. The identification was accomplished by a DB-5MS 30 m capillary column (0.25 mm I.D., 0.25 microm film thickness) with helium as carrier gas operating under program control temperature and electron impact ionization mass spectrometer in a scan mode. Results demonstrated that ligustilide and butylidenephthalide can act on rat artery cell membrane similar to verapamil in CMC system. They significantly inhibited the vasoconstrictions induced by norepinephrine bitartrate (NE) and calcium chloride (CaCl2). The relaxing effect of ligustilide on the NE- and CaCl2-induced constrictions is more potent than that of butylidenephthalide. Ligustilide and butylidenephthalide seem to be the two main effective components of Ligusticum Chuanxiong as a traditional Chinese medicine for treating blood vessel diseases.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C₁₈ and C₂₀) and fatty alcohols (C₁₆ to C₂₆) derived from wax esters. Most of these acids and alcohols were unsaturated in the ω-7 position and were accompanied by smaller amounts of the saturated and ω-5 monounsaturated analogues. Glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (ω-5 and ω-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Identification of felodipine metabolites in rat urine

After intragastric administration of 100 mumol kg-1 [14C]felodipine to rats eight urinary metabolites were isolated. Batch extraction at pH 2.2 and semipreparative reversed-phase liquid chromatography were used for trace enrichment of the metabolites. Trimethylsilylation followed by transesterification with diazomethane blocked the carboxylic acid and alcohol groups selectively before gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. Deuterated derivatives of the metabolites and chemical ionization measurements added complementary structural information. All metabolites reported in this study were formed from oxidized felodipine by ester hydrolysis. Hydroxylation of the pyridine methyl group represented an important metabolic pathway and metabolites oxidized to the corresponding carboxylic acids were detected as well. Lactone formation from hydroxy acid metabolites in urine as a possible analytical artefact is discussed.     

Comparison of high-performance liquid chromatography with electrochemical detection and gas chromatography—mass fragmentography for the assay of salsolinol, dopamine and dopamine metabolites in food and beverage samples

High-performance liquid chromatography with electrochemical detection (HPLC—ED) and combined gas chromatograph—mass spectrometry in the single-ion monitoring mode (GC—MS-SIM) have been used for the determination of salsolinol, dopamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydorxyphenylethanol and norepinephrine in a selection of food and beverage samples. The unique specificity of the SIM mode allows a simple one-step extraction to be used even for complex sample matrices. We have been able to demonstrate the quantitative and qualitative advantages offered by GC—MS over HPLC—ED by direct comparison of the chromatographic data obtained. We demostrate that the specificity of SIM and the benefits offered by the incorporation of deuterated internal standards make GC—MS-SIM the method of choice for valid identification and precise quantitation of salsolinol, dopamine and dopamine metabolites in a complex sample matrix.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection

A qualitative and quantitative analysis of the conjugated 1β- and 6α-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C₁₈ silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C₁₈ column (Bile Pak II), with detection by an immobilized 3α-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate—isoluminol—microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Identification of two Thormählen-positive compounds from melanotic urine by gas chromatography—mass spectrometry

The isolation of two Thormählen-positive compounds from the urine of a patient with malignant melanoma and the elucidation of their structure by gas chromatography—mass spectrometry is described. The compounds were isolated using a poly-N-vinylpyrrolidone column and separated by preparative thin-layer chromatography. After elution they were analyzed by gas chromatography and gas chromatography—mass spectrometry as their trimethylsilyl derivatives and after hydrolysis also as their tert.-butyldimethylsilyl derivatives. The results showed the main Thormählen-positive compound A to be the glucuronide of 5-hydroxy-6-methoxyindole, whereas the minor compound AX appeared to be the glucuronide of its isomer 6-hydroxy-5-methoxyindole.     

High-performance liquid chromatography-tandem mass spectrometry for the analysis of bile acid profiles in serum of women with intrahepatic cholestasis of pregnancy

A simple, sensitive, and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the analysis of the bile acid profile has been developed. Fifteen bile acids, including free and conjugated bile acids, were separated and detected by HPLC-MS/MS. The MS detection was performed by electrospray ionization (ESI) in negative ion mode. Quantification was achieved in multiple reaction monitoring (MRM) mode with external standard curve methods. Total analysis time was 15 min for one sample including re-equilibration time of the column. The assay was linear in the range 0.02-100.0 micromol/L with correlation coefficients of standard curves for all bile acids better than 0.999. The detection limits ranged from 0.001 to 0.006 micromol/L for different bile acids. The precisions for each bile acid were CVs<3.8% for within-day and CVs<6.1% for between-day. The average recoveries for all bile acids studied were in the range of 86-110.0%. The developed method was applied to the analysis of clinic samples consisting of 53 women with healthy pregnancies and 43 women with intrahepatic cholestasis of pregnancy (ICP). The results revealed that the bile acid profile was markedly different between women with ICP and women with healthy pregnancies.     

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS) after one single liquid—liquid extraction with chloroform—methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml–5 μg/ml with good accuracy and precision and is used in pharmacokinetic studies.