Analysis of trimethylsilyl O-methyloximes of carbohydrates by combined gas-liquid chromatography-mass spectrometry

We have concluded that the O-methyloxime derivative effectively “labels” the carbonyl portion of the surgar, and probably contributes to the stability of ions containing that part of the molecule. Electron impact-induced cleavage of the carbon-carbon bonds in the sugar, with charge retention on either fragment, provides a mass spectrum that may be used to analyze substituents on any carbon. We have examined the mass spectra of several classes of sugars, as their -O-methyloxime trimethylsilyl ethers, and as their -O-methyloxime peracetates and in most cases ions are found corresponding to the fragmentation pattern proposed here (unpublished results). In addition, the gas-liquid chromatographic retention behavior of these compounds suggests that they may be useful for quantitative determination of carbohydrates.     

Methylation structural analysis of unusual dextrans by combined gas-liquid chromatography-mass spectrometry

Eight bacterial dextrans from NRRL strainsLeuconostoc mesenteroides B-742, B-1299, B-1355, B-1399, and B-1402, and from Streptobacterium dextranicum B-1254 were examined by methylation structural analysis. Methyl ethers of d-glucose that were present in hydrolyzates of permethylated dextrans were analyzed by combined g.l.c.?m.s. as the peracetylated aldononitriles. The various dextrans differed significantly in frequency and type of chain branching.     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

Determination of loratadine and pheniramine from human serum by gas chromatography-mass spectrometry

In this work, a method for the determination of the antihistaminic drugs loratadine and pheniramine from human serum is presented. Serum samples are extracted under basic conditions with hexane-n-amyl alcohol (95:5, v/v), the analytes are reextracted into diluted hydrochloric acid and, after basification, are once again extracted into the organic phase. The samples are measured by GC-MS. The limits of detection of the assay are 0.5 ng/ml for loratadine and 2 ng/ml for pheniramine. The R.S.D.s in the day-to-day precision test for loratadine are 7.0% at 20 ng/ml and 12.4% at 2 ng/ml. For pheniramine, the R.S.D. are 6.4% at 300 ng/ml and 10.2% at 20 ng/ml.     

Quantification of retinoic acid by gas-liquid chromatography-mass spectrometry: total versus all-trans-retinoic acid in human plasma

An assay based on negative ion chemical ionization mass spectrometry has been developed to quantify retinoic acid in plasma or serum. The lower limit of detection is 75 pg (240 fmol); normal values of retinoic acid can be determined on as little as 40 microliters of human plasma. The plasma concentrations of total retinoic acid in 12 healthy male volunteers taking no medication or vitamin supplementation ranged from 2.8 to 6.6 ng/ml; the mean was 4.9 ng/ml. The assay can be manipulated to measure all-trans-retinoic acid alone; about 75% of retinoic acid in human plasma or rat serum is all-trans-retinoic acid. Both retinol and retinoic acid can be quantified on the same 0.1-ml sample; the concentration of retinoic acid in human plasma or rat serum is at least 150-fold less than that of retinol.     

Determination of metaldehyde in human serum by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of metaldehyde in human serum samples. Metaldehyde is extensively used as a molluscicide for the control of slugs and snails, and cases of metaldehyde poisoning have been reported. Metaldehyde was headspace-extracted on a polydimethylsiloxane (PDMS) fiber at 70 degrees C for 25 min, desorbed, and analyzed rapidly by GC-MS. The method was validated for limit of detection (LOD), linearity, precision, and recovery. Although the recovery of the sample was very low, the method itself was rapid with a low detection limit of 0.25 microg/ml, R.S.D. value 12.6%, and linearity range 0.5-25.0 microg/ml (r(2)=0.999). The results demonstrated that the SPME-GC-MS method for the analysis of metaldehyde is simple, rapid, solvent-free, and does not require any pre-analysis conversions.     

Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry.

The need for specific and sensitive methods for the determination of distinct serum folates is of high priority in clinical research settings. A stable-isotope liquid chromatography-mass spectrometry (LC/ESI-MS) assay was developed for the quantitative determination of the monoglutamyl form of 5-methyltetrahydrofolic acid (5-MTHFA) in human serum. Serum samples (0.5 ml) were amended with the internal standard, [5-13C5]MTHFA that had been labeled on the glutamic acid portion of the molecule and allowed to equilibrate. The analyte was trapped onto a solid-phase cartridge and then eluted with the HPLC mobile phase. Forty microliters was taken for LC/ESI-MS analysis using electrospray ionization operated in the positive ion mode. Using the standard method of addition of 5-MTHFA to serum, a linear dilution curve (y = 12.777x - 1.404; range 0.94-97 ng x ml(-1)) was constructed. The precision of the method was 5.3% (CV) based on the analysis of four sample replicates. The mass spectrum produced upon collision induced dissociation of the analyte in serum was used to confirm the identity of the 5-MTHFA. The method was applied to the analysis of a set of serum samples that contained standardized concentrations of 5-MTHFA. The determinations of 5-MTHFA in these samples using the LC/ESI-MS procedure were found to be in good agreement with other folate methods. A highly accurate and specific method for the analysis of 5-MTHFA in serum has been developed utilizing stable isotope dilution mass spectrometry.     

Sterols of scallop. Part II. Structure of unknown sterols by combination gas-liquid chromatography and mass spectrometry.

Four sterols, isolated from the scallop Pacopecten magellanicus have been identified as 24-nor-5alpha-cholest-22-en-3beta-ol; 24-norcholest-5-en-3beta-ol; 5alpha-cholest-22-en-3beta-ol; and (E) -24-propylidenecholest-5-en-3beta-ol. These bring to seventeen the total number of sterols identified in this marine mollusc. A fifth newly detected sterol, closely similar in its mass spectrometric properties is 22-cis and trans-cholesta-5, 22-dien-3beta-ol, was clearly distinguished from these by its shorter retention time by GLC.     

Quantitation of leukotriene B4 in human serum by negative ion gas chromatography-mass spectrometry

Leukotriene B4 (LTB4) is a potent chemotactic agent formed via the 5-lipoxygenase pathway from arachidonic acid. To understand the role LTB4 plays in several pathological processes it is essential that endogenous concentrations of LTB4 be accurately quantitated. We have developed a method based on electron capture negative ion mass spectrometry for the analysis of LTB4 in serum at low picogram per milliliter concentrations. Blood is collected into the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to suppress ex vivo formation. Serum is isolated, equilibrated with the internal standard [2H4]LTB4, and extracted using octadecyl-silica (C-18) cartridges. After conversion of the carboxylic acids to their pentafluorobenzyl esters the extract is purified by straight-phase HPLC. Gas chromatographic-mass spectrometric analysis is accomplished on the tert-butyldimethylsilyl ether derivatives using dual-selected ion monitoring of m/z 431 and 435. These ions correspond to loss of tert-butyldimethylsilanol from the (M-PFB)- ion of endogenous and [2H4]LTB4, respectively. The concentration of LTB4 in human serum samples was 10.0 +/- 4.0 pg/ml (n = 5). The assay exhibited satisfactory precision, with an intraassay coefficient of variation of 17% and a high degree of accuracy. The concentration of LTB4 in serum collected with (NDGA) was less than 10% of that observed in blood collected without the lipoxygenase inhibitor. Ex vivo formation can therefore be a major obstacle in assessing circulating levels of LTB4.     

Determination of copper in urine and serum by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N'-ethylenebis-(trifluoroacetylacetoneimine) [H2(enTFA2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H2(enTFA2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H2(enTFA2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H2(enTFA2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.     

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry

This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.     

Characterization of serum and urinary bile acids in patients with primary biliary cirrhosis by gas-liquid chromatography-mass spectrometry: effect of ursodeoxycholic acid treatment

We have studied the effect of ursodeoxycholic acid on the serum and urinary bile acids in seven patients with moderate to severe primary biliary cirrhosis. Bile acids were characterized by gas-liquid chromatography-mass spectrometry and quantified by capillary gas-liquid chromatography. Serum bile acids were elevated 26-fold over control values, with 2.2 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 22-fold over control values with a cholic acid:chenodeoxycholic acid ratio of 1.6. In addition, lithocholic acid, deoxycholic acid, ursodeoxycholic acid, 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid, and hyocholic acid were identified in both serum and urine; the proportions of the 1- and 6-hydroxylated bile acids were much higher in urine than in serum of the patients (32.1% versus 4.2%). Three months of placebo administration did not change the serum and urinary bile acid composition. In contrast, ursodeoxycholic acid feeding (12-15 mg/kg body weight per day) for 6 months resulted in a 25% decline in the total serum bile acid concentration from the pretreatment values. The proportion of ursodeoxycholic acid increased from 2.1 to 41.2% of total bile acids, so that total fasting serum endogenous bile acid levels decreased 62.4%. Ursodeoxycholic acid feeding substantially increased urinary bile acid output, with ursodeoxycholic acid comprising 58.1%. The proportion of 1- and 6- hydroxylated endogenous bile acids was reduced by 45.5% from pretreatment levels and approximately 4.5% of the urinary bile acids were omega-muricholic acid, 1 beta-hydroxyursodeoxycholic acid, and 21-hydroxyursodeoxycholic acid. These results demonstrate significant changes in the serum and urinary bile acid pattern in primary biliary cirrhosis during ursodeoxycholic acid treatment. The beneficial effect of ursodeoxycholic acid may be due to reduction of the hydroxylated derivatives of endogenous bile acids together with the appearance of hydroxylated derivatives of ursodeoxycholic acid or it may be due to displacement of the more hydrophobic endogenous bile acids by the hydrophilic ursodeoxycholic acid.     

Determination of the terfenadine metabolite azacyclonol in human serum using gas chromatography-mass spectrometry

In this work, a method for the determination of azacyclonol, a primary metabolite of terfenadine, in human serum is described. Sample preparation is carried out by liquid-liquid extraction under basic conditions. For an efficient clean up, the analytes are back-extracted into diluted hydrochloric acid and, after alkalinization, are once again extracted into the organic phase. No derivatisation step is performed. The samples are measured by gas chromatography-mass spectrometry with good selectivity. The limit of detection is 2 ng/ml. The coefficients of variation are 12.6% at 10 ng/ml and 6.44% at 200 ng/ml in the day-to-day control measurements.     

Identification of 6-hydroxymelatonin in normal human urine by gas chromatography-mass spectrometry.

The major melatonin metabolite, 6-hydroxymelatonin, has been identified in normal human urine by gas chromatography-mass spectrometry (gc-ms) after rapid acid hydrolysis, extraction, and derivatization. An estimate of the amount detected (approximately 20 ng/ml) is consistent with calculated rates of melatonin synthesis and turnover.     

Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [¹⁵N,¹³C₂]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine⁻¹ were found in healthy controls, with reduced levels in hemochromatosis (P < 0.00006) and elevated levels in inflammation (P < 0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P < 0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.