Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Analysis of (1-13C)leucine and (13C)KIC in plasma by capillary gas chromatography/mass spectrometry in protein turnover studies

The methods used for the determination of the concentration and isotope enrichment of (1-13C)leucine and its metabolite (13C) alpha-ketoisocaproic acid (KIC) in plasma for the study of whole-body protein turnover are described. Leucine was analysed as its N-heptafluorobutyryl isobutyl ester and KIC as its quinoxalinol-TMS derivative, both by chemical ionization selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The sensitivity of the leucine assay was improved 30 times by monitoring the negative ions under the conditions described. The coefficient of variation for enrichment and concentration measurements were 0.5% and 2%, respectively, with a minimum detectable enrichment of 0.1 at% excess for both assays.     

A simple and accurate GC/MS method for the quantitative analysis of diacetyl in beer and wine

A GC/MS method for the quantification of diacetyl is described. Diacetyl is derivatized with 4,5-dichloro-1,2-diaminobenzene to form 6,7-dichloro-2,3-dimethylquinoxaline (DCDMQ). The derivative is extracted in benzene and quantified by GC/MS. Formation of DCDMQ is linearly correlated with diacetyl concentration. The method is rapid, sensitive (determination limit 0.0005 g/mL) precise (standard error < 2%), and accurate (recovery of diacetyl 91.5% + 1.5%).     

Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry

We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added. p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(III) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 mumol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Analysis of a range of crown gall and normal plant tissues for Ti plasmid-determined compounds

Summary Twenty-five plant tissues from several species, including thirteen crown gall tissues, were analysed for the full range of unusual compounds (the opines) whose synthesis in crown gall cells and utilization by Agrobacterium tumefaciens are genetically determined by the Ti plasmids found in this bacterial species. A technique for the analysis of the non-guanidino opines by GC and GC/MS is described. None of the opines were detected in any of the various normal tissues analysed. In the crown gall tissues, on the other hand, these compounds were often present at very high levels. The type of opines found in the crown gall tissues was dependent on the strain of initiating bacterium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - HFB heptafluorobutyryl - SIM selected ion monitoring - TLC thin layer chromatography     

Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds.     

Chromatographic and mass spectrometric characteristics of 20-dihydroaldosterone.

The 20 alpha-reduced derivative of aldosterone, 20 alpha-dihydroaldosterone, was needed as reference compound in order to continue the studies on 18-hydroxylation in the Y-1 adrenal cell line. It was obtained by reduction of aldosterone with sodium borohydride. Analysis of the products of the reaction as methoxime trimethylsilyl (MO-TMS) derivatives by gas chromatography (GC) and GC-mass spectrometry (GC-MS) showed three possible forms of the compound. Their identification was confirmed by comparison with the products obtained by stereospecific reduction of aldosterone using 3 alpha,20 beta-hydroxysteroid dehydrogenase. Chromatographic behavior and mass spectra are given for the three forms of 20 alpha-dihydroaldosterone as the MO-TMS derivatives; that is, the 18-aldehyde, the 18,11 beta-hemiacetal, and the 11 beta:18,18:20 alpha-acetal. The possible origin of these different forms is discussed as a function of these results and of the results obtained by complementary analysis on high-performance liquid chromatography.     

Identification of 19-nor-deoxycorticosterone in normal rat serum by enzyme immunoassay and gas chromatography-mass spectrometry

Recent reports on the isolation and identification of 19-nor-deoxycorticosterone from the urine of rats with adrenal regeneration hypertension give rise to the possibility of a role of this steroid in the pathogenesis of low renin essential hypertension. The present study was undertaken to investigate the presence of 19-nor-deoxycorticosterone in normal rat serum both by a sensitive enzyme immunoassay and by gas chromatography-mass spectrometry (GC/MS). 19-Nor-deoxycorticosterone in rat serum was separated from other steroids prior to enzyme immunoassay by using high performance liquid chromatography (HPLC). The average concentration of 19-nor-deoxycorticosterone in normal rat serum was 137 +/- 62 ng/dl (mean +/- SD, n = 32). Pooled normal rat serum (50 ml) was purified by HPLC and the purified sample was acetylated with acetic anhydride for GC/MS measurement. The retention time and m/z ions (358, 285, and 257) on the resulting mass fragmentogram coincided in position with those of authentic 21-acetoxy-19-nor-deoxycorticosterone and acetylated normal rat serum extract. The combined characteristics of HPLC elution, antigen-antibody reaction, GC retention and selected ion responses provided strongly evidence supporting the presence of 19-nor-deoxycorticosterone in normal rat serum.     

Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver

A procedure is described for the extraction of diethylstilbestrol (DES) from animal tissue for quantitative capillary gas chromatography/mass spectrometry (GC/MS). The procedure is based upon use of a strong anion exchange polystyrene divinylbenzene resin for sample purification. The recovery of DES from the resin clean up was 88% in the high parts per trillion (ppt) range. Criteria for identification of DES using selected ion monitoring (SIM) GC/MS are presented. Liquid chromatography/mass spectrometry (LC/MS) was used to investigate altered DES cis/trans ratios observed in biological extracts.     

Determination of platelet-activating factor and alkyl-ether phospholipids by gas chromatography-mass spectrometry via direct derivatization

A mass spectrometric method has been developed for the quantitative analysis of platelet-activating factor (PAF) and lyso-platelet-activating factor (lyso-PAF) based on electron-capture gas chromatography-mass spectrometry using a stable-isotope dilution technique. The cleavage and derivatization was accomplished in a single step by direct reaction of phospholipid with pentafluorobenzoyl chloride at 150 degrees C. Spectroscopic and chromatographic data indicated that PAF and lyso-PAF were converted into derivatives containing a pentafluorobenzoyl group in place of the original phosphocholine group with 95 and 51% yield, respectively. Additionally, in the lyso-PAF derivative, the free hydroxyl group was found to be replaced by chlorine. Phosphatidylcholines containing an arachidonoyl group can be derivatized with a solution of PFBCl/chloroform at 120 degrees C for 18 h, producing 90% derivative. Analysis by GC/MS and LC/MS allowed the detection of 1 or 250 pg derivative, respectively, injected onto the column with S/N greater than 3. Newly available analogues of high isotopic purity containing either three or four deuterium atoms located in the 1-O-hexadecyl chain were used as internal standards. The developed GC/MS assay was used to quantitate PAF and lyso-PAF in rabbit leukocytes before and after stimulation with calcium ionophore. The levels of PAF in unstimulated cells were in the order of 2.27 pmol/10(6) cells and increased about 17-fold during 10-min stimulation with 2 microM ionophore A23187. The lyso-PAF levels in resting cells were in the order of 3.76 pmol/10(6) cells and increased 1.7-fold during stimulation. This assay exhibited satisfactory sensitivity, reproducibility, and accuracy.     

Screening of β-2 agonists and confirmation of fenoterol,orciprenaline, reproterol and terbutaline with gas chromatography–mass spectrometry as tetrahydroisoquinoline derivatives

A new method for a comprehensive screening and confirmation of β-2 agonists in human urine is presented based on gas chromatography–low-resolution mass spectrometry (GC–MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with β-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other β-2 agonists remain unchanged. Liquid–liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four β-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other β-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

OCCURRENCE OF LOROXANTHIN,LOROXANTHIN DECENOATE,AND LOROXANTHIN DODECENOATE IN TETRASELMIS SPECIES (PRASINOPHYCEAE,CHLOROPHYTA)1

The pigment composition of six species of Tetraselmis (Prasinophyceae) was analyzed using improved HPLC methods. All pigment extracts showed three peaks corresponding to unknown carotenoids. The isolated pigments were analyzed using UV–Vis spectroscopy, electrospray ionization–mass spectrometry (ESI–MS), and when carotenoid esters were suspected, gas chromatography–mass spectrometry (GC–MS) of the methyl ester and dimethyloxazoline derivative of the corresponding fatty acid. The new pigments were determined to be loroxanthin, loroxanthin 19‐(2‐decenoate), and loroxanthin 19‐(2‐dodecenoate); this is the first time these pigments have been described in the genus Tetraselmis. Moreover, this is the first report of esterification of 2‐decenoic acid to loroxanthin. The relative contents of these pigments depended on the light regime, with the lowest proportions measured at the highest photon flux density assayed. The implications of the identification of these pigments in the genus Tetraselmis for the pigment types previously described in the class Prasinophyceae are discussed.     

Gas chromatographic–mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance

A method for analysis of veterinary tranquillizers in urine using gas chromatography–mass spectrometry (GC–MS) is described. Detection limits are 5 μg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 μg/l for haloperidol, 20 μg/l for xylazine and 50 μg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC–MS “positive” results is evaluated and discussed.     

Assay of ezlopitant, a substance P receptor antagonist, and metabolites in biological matrices by gas chromatography with mass spectrometric detection: simultaneous analysis of a benzyl alcohol and alkene

A method for the analysis of the substance P antagonist ezlopitant and two active metabolites in serum using solid-phase extraction followed by GC–MS analysis is described. The linear dynamic range was 1.0 to 100 ng/ml and precision and accuracy over this range were within 15%. Upon injection of reconstituted sample extracts into the hot injector port of the gas chromatograph, the benzyl alcohol metabolite undergoes a small amount of spontaneous dehydration to the alkene metabolite. We have incorporated an additional hexadeuterated internal standard of the benzyl alcohol into the assay to permit measurement of the extent of dehydration in each sample. This novel approach should be generally applicable to the simultaneous determination of benzyl alcohols and corresponding alkenes by GC–MS when the possibility exists that the alcohol can undergo spontaneous dehydration to the alkene in the injector port of GC instrumentation.     

GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat

Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolamides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reverse-phase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-2H8) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H8]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamid e as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum.