Femtomole detection of amino acids and dipeptides by gas chromatography—negative-ion chemical ionization mass spectrometry following alkylation with pentafluorobenzyl bromide

Amino acids and di- and tripeptides were derivatized by extractive alkylation using pentafluorobenzyl bromide (PFBBr) followed by reaction with heptafluorobutyric anhydride (HFBA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Good chromatographic separation and the formation of intense diagnostic ions were observed for the derivative when examined using gas chromatography—negative-ion chemical ionization mass spectrometry (GC—NICI-MS). Of the 20 amino acids investigated, only Arg and Glu could not be detected by this method. Also, dipeptides which included neutral amino acid residues were derivatized with more success than those containing either acidic or basic residues. Each of the amino acids or dipeptides formed one major derivative with the exception of Asn which formed two derivatives with either one or two HFB groups. This derivatization method was optimized with respect to the reaction temperature, reaction time, and choice of derivatizing reagents. Recoveries of derivatized [³H]-labeled Phe, Lys, and Thr were 76, 55, and 34%, respectively. Linearity was observed from 10 to 2000 pg of Ala per vial; selected-ion monitoring provided a detection limit of less than 150 fg with a signal-to-noise (S/N) ratio of 80 to 1. This method has proven to work well with urine samples and shows great promise for the detection of small peptides at low levels.     

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography—negative-ion chemical ionization mass spectrometry

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic—negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.     

Quantitative gas chromatography—chemical ionization mass spectrometry of 2-ketoglutarate from urine as its O-trimethylsilyl-quinoxalinol derivative

Quantitation of 2-ketoglutarate in urine as its O-trimethylsilyl-quinoxalinol derivative by gas chromatography—chemical ionization mass spectrometry is described. This technique, with ammonia as reactant gas, produces no fragmentation and allows only the detection of the protonated molecular ion. It gives the greatest known sensitivity, and could be applied to the determination of urinary 2-ketoglutarate in normal children and in various metabolic disorders, such as dihydrolipoyl dehydrogenase defect, pyruvate carboxylase deficiency and type I glutaric acidemia.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Combined assay for phenacetin and paracetamol in plasma using capillary column gas chromatography—negative-ion mass spectrometry

A gas chromatographic—mass spectrometric assay has been developed for the measurement of phenacetin and its major metabolite paracetamol in plasma. Phenacetin and unconjugated paracetamol are analysed in a single chromatographic run while total paracetamol is measured separately after enzymatic hydrolysis. The two compounds, and the deuterated analogues used as internal standards, are analysed as their trifluoroacetyl derivatives and the mass spectrometer is operated in the electron-capture negative-ion chemical ionisation mode. The negative-ion mass spectra of the derivatives contain fragment ions, formed by loss of an acetyl group from the respective molecular ions, which are the base peaks in the spectra. When these ions are specifically monitored, amounts of derivative equivalent to 1 pg of parent compound can be detected. This allowed the development of an assay for phenacetin, unconjugated paracetamol and total paracetamol in plasma having a precision of 2.6, 1.4 and 2.4%, respectively, and preliminary results for a subject given a 100-mg oral dose of phenacetin are reported.     

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.     

Analysis of urinary organic acids by liquid chromatography—atmospheric pressure chemical ionization mass spectrometry

We developed a new method for the rapid determination of urinary organic acids using liquid chromatography—atmospheric pressure chemical ionization mass spectrometry. Mass spectra of authentic organic acids obtained in the negative-ion mode showed intense [M − H]⁻ ions with some fragment ions. Urine samples of patients with methylmalonic aciduria, ornithine transcarbamylase deficiency, and phenylketonuria were extracted using anion-exchange columns. The mass chromatograms of the extracts showed some dominant peaks of abnormal metabolites characteristic of each disorder. This is a useful method for the analysis of urinary organic acids for the diagnosis of organic aciduria, because the sample preparation is simple.     

Quantification of dimethindene in plasma by gas chromatography—mass fragmentography using ammonia chemical ionization

A gas chromatographic—mass fragmentographic method using ammonia chemical ionization for the determination of dimethindene in human plasma is described. The drug was isolated from plasma by liquid—liquid extraction with hexane—2-methylbutanol. Plasma components were separated on a capillary column coated with chemically bonded methyl silicone. For detection of dimethindene, its quasi-molecular ion (M + H⁺) was mass fragmentographically monitored after chemical ionization with ammonia as reagent gas. Dimethindene was quantified using methaqualone as the internal standard: the quantification limit in plasma was 0.2 ng/ml, the within-run precision was 8.0% and the inter-run precision 5.6%. The plasma concentration—time profile was established after a single dose of 4 mg of dimethindene with an average maximum concentration of 5.5 ng/ml, detectable up to 48 h post application.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Determination of total body water by deuterium isotope dilution measurements using chemical ionization mass spectrometry

Two chemical ionization mass spectrometric methods were developed for direct determination of deuterium in water in the range of 0.0-0.6% 2H2O. One of them utilizes the batch inlet system, methane as the reagent gas, and the peak matching device of a magnetic sector mass spectrometer. The second one utilizes the directly-coupled gas chromatograph of a quadrupole mass spectrometer and computer control for ion selection and data processing. In this method the water itself serves as the reagent gas. The deuterium concentration is calculated from the ratio of ion intensities at m/z 20 (2HH2O+) and m/z 19 (H3O+). We have used these methods to determine total body water in 350 human subjects, which entailed making 900 measurements over a period of four years. Comparisons were made in 200 subjects of our results with those obtained by the creatinine method. No significant differences were found.     

Analysis of procarbazine and metabolites by gas chromatography—mass spectrometry

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography—mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride.A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma.Studies on the degradation of procarbazine - HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine - HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine - HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.     

Determination of cyclophosphamide in urine by gas chromatography—mass spectrometry

A sensitive gas chromatographic method for the determination of cyclophosphamide in urine is presented. After liquid—liquid extraction with diethyl ether and derivatization with trifluoroacetic anhydride, cyclophosphamide was identified and quantified with mass spectrometry. The method is suitable for the determination of cyclophosphamide at concentrations of more than 0.25 ng/ml, which enables the uptake of cyclophosphamide during occupational activities, such as the preparation and administration of antineoplastic agents in hospitals, to be measured. Simple preparation makes the method appropriate for routine analysis.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

Detection of acyl-coenzyme a thioester intermediates of fatty acid β-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography—Mass spectrometry

An analytical method for the separation and quantitation of acyl-CoA thioesters by gas chromatography—Mass spectrometry is described. The method utilizes glycine aminolysis of the acyl-CoA thiolesters, esterification with pentafluorobenzyl bromide followed by gas chromatographic separation, and detection by negative chemical ionization mass spectrometry of the N-acyl-pentafluorobenzyl glycinates. The glycine aminolysis provides over 100-fold discrimination against oxygen esters and obviates the difficulty of removing trace contaminants of free fatty acids. The limit of detection of the described methodology for palmitoyl-CoA has been found to be 300 fmol, which improves at shorter chain lengths. Baseline separation was obtained for a standard mixture of seven acyl-CoAs (60 pmol injected) containing butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA. The above procedure is also applicable to the α-β unsaturated and 3-hydroxyacyl-CoA derivatives, making it possible to quantify all of the intermediates in fatty acid oxidation, except the 3-ketoacyl-CoAs, in a single procedure.     

Application of atmospheric pressure chemical ionization liquid chromatography–mass spectrometry in identification of lymph triacylglycerols

Atmospheric pressure chemical ionization liquid chromatography–mass spectrometry was used in the identification of triacylglycerol molecular species in lymph samples from rats given either a structured lipid or safflower oil. The structured lipid was MLM-type (M, medium-chain fatty acid; L, long-chain fatty acid) and manufactured from caprylic acid (8:0) and the oil (safflower oil or high-oleic sunflower oil). The triacylglycerol composition of lymph varied significantly between structured triacylglycerols and safflower oil. Diacylglycerol fragment ions were found for all triacylglycerols and we could also observe the ammonium adduct molecular ion [M+NH₄]⁺ for all the triacylglycerols at the selected conditions. Protonated molecular ions were formed from triacylglycerols containing unsaturated fatty acids, and fatty acid fragment ions were also observed in the case of strong fragmentation. The lymph triacylglycerols were identified from their ammonium adduct molecular ions and diacylglycerol fragment ions. In addition to the intact MLM-type structured triacylglycerols, the MLL- and LLL-type triacylglycerols were also identified. The absorption pathway of MLM-type structured triacylglycerols is most likely the same as that of conventional long-chain triacylglycerols, i.e. they were hydrolyzed into 2-monoacylglycerol and medium-chain fatty acids, which were then used for resynthesis of triacylglycerols. The present study thereby also demonstrates the possibility to study the absorption pathway of triacylglycerol via identification of triacylglycerol species in biological samples.