Quantitation of methylmalonic acid and other dicarboxylic acids in normal serum and urine using capillary gas chromatography-mass spectrometry

Methylmalonic acid, succinic acid, and other dicarboxylic acids have been extracted and partially purified from serum and urine using ether extraction and high-performance liquid chromatography. The t-butyldimethylsilyl derivatives were prepared and analyzed using capillary gas chromatography-mass spectrometry with selected ion monitoring. The addition of [methyl-2H3]methylmalonic acid and [1,4-13C2]succinic acid to the starting samples made it possible to quantitate these two dicarboxylic acids. Normal ranges for methylmalonic acid and succinic acid were determined in human and rat serum and in human urine. The utilization of other internal standards would make it possible to quantitate malonic, dimethylmalonic, ethylmalonic, methylsuccinic, glutaric, and other dicarboxylic acids.     

The trace analysis of alkyl alkylphosphonic acids in urine using gas chromatography-ion trap negative ion tandem mass spectrometry

A sensitive method has been developed for the trace analysis of alkyl alkylphosphonic acids, metabolites of nerve agents, in urine using a benchtop ion trap mass spectrometer. The acids were isolated from urine by simple solid phase extraction and converted to their pentafluorobenzyl esters. An ion trap mass spectrometer in selected reaction monitoring mode provided limits of detection of 0.1 ng/ml for isopropyl, isobutyl, pinacolyl and cyclohexyl methylphosphonic acids and for ethyl ethylphosphonic acid. The detection limit for ethyl methylphosphonic acid was higher (0.5 ng/ml) due to a lower recovery.     

Application of high resolution fast atom bombardment and constant B/E ratio linked scanning to the identification and analysis of acylcarnitines in metabolic disease

Acylcarnitines, a biologically important group of metabolites which have thus far eluded characterization by mass spectrometry, exhibit very intense fast atom bombardment mass spectra dominated by parent cations. The combination of high resolution selected ion detection and daughter ion analysis using the linked scan at constant B/E ratio has enabled the unequivocal identification of the acylcarnitines in the urine of children with propionic acidemia, methylmalonic aciduria and Reye's Syndrome. Quantitative analysis of acetylcarnitine and propionylcarnitine in selected samples was accomplished by isotope dilution, utilizing (2H3)acetyl- and (2H5)propionylcarnitines as internal standards. These techniques will allow assessment of the therapeutic use of L-carnitine in these disorders.     

Gas chromatography–mass spectrometric method for quantitative determination in human urine of dicarboxylic (dioic) acids produced in the body as a consequence of cholesterol biosynthesis inhibition

A capillary gas chromatography–mass spectrometric (GC–MS) method in human urine has been developed and validated for the quantitative determination of dicarboxylic acids (dioic acids) which are produced in the body as a consequence of the administration of an inhibitor of the enzyme squalene synthase, which is involved in the biosynthesis of cholesterol. The standards and quality control (QC) samples were prepared by adding dioic acids into human urine. Internal standard (sebacic acid) was added to each urine sample (0.1 ml) and then dried by evaporation under nitrogen. The dried sample was reacted with pentafluorobenzyl (PFB) bromide under conditions that maximized the formation of the di-PFB ester (at the expense of the mono-PFB ester) of the dioic acids. After drying by evaporation, each sample residue was reconstituted in mesitylene and injected into a capillary GC–MS system via a splitless injection. The detection was by negative ion chemical ionization mass spectrometry with selected ion monitoring (SIM) of the [M−PFB]⁻ of the analytes and the internal standard.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

C6-C10-dicarboxylic acids in liver and kidney tissue in normal, diabetic ketotic and clofibrate-treated rats

A combined gas chromatographic-mass spectrometric method (selected ion monitoring) to determine C6-C10-dicarboxylic acids in liver and kidney tissue is reported. Alterations in tissue concentrations of the dicarboxylic acids were reflected in urinary excretions, i.e., diabetic rats with 'ketotic dicarboxylic aciduria' had corresponding elevated concentrations of short-chain dicarboxylic acids in liver and kidney tissue. Stimulation of the enzymes of fatty acid oxidation by clofibrate was, as a sole event, not sufficient to cause elevated tissue concentrations of dicarboxylic acids, nor did it result in dicarboxylic aciduria, probably because of a relative lack in substrate (fatty acids) compared to the diabetic ketotic state, where lipolysis is increased. These results strongly indicate that 'ketotic dicarboxylic aciduria' parallels the activity of the lipid metabolism at cellular level, and that it is not just a matter of renal handling.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

Liquid chromatography/mass spectrometry of fatty acids as their anthrylmethyl esters

The mass spectra of a series of saturated and unsaturated fatty acids have been recorded as their anthrylmethyl esters using a liquid chromatographic mass spectrometric interface. The spectra show an intense peak for the aromatic nucleus, and a molecular ion. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of acetone + acetonitrile. While a complete separation of the fatty acids known to occur in man was not achieved, the recognition of all of these acids is possible using a scanning mode or by ion monitoring.     

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Comparison of electron and chemical ionization mass spectrometry of sialic acids

Sialic acids were analyzed as per-O-trimethylsilylated compounds by gas-liquid chromatography/mass spectrometry either on electron or chemical ionization by isobutane. Electron ionization mass spectra of these derivatives are very similar to those of the corresponding methyl esters described earlier whereas chemical ionization mass spectra are characterized in the high mass range by loss of the C-2 and the C-4 substituents from the M + 1 ion. Together with other fragment ions the seven different sialic acids analyzed could be clearly identified.     

Hydroxyl negative chemical ionization mass spectrometry linked with collisionally activated decomposition. A modern analytical tool in inborn errors of metabolism

Two kinds of inborn errors of metabolism, dicarboxylic aciduria and hyperoxaluria, have been studied by means of hydroxyl negative ion chemical ionization [NICI(OH-)], linked with collisionally activated decomposition experiments on the [M-H]- species of the pathognomonic organic acids. This method has led to non-controversial qualitative determinations of C4-C10 dicarboxylic acids and oxalic, glyceric and glyoxylic acids. NICI(OH-) linked with collisionally activated decomposition mass analysed ion kinetic energy spectrometry (CAD MIKES) is proposed herein for diagnostic purposes, as a valid mass spectrometric alternative to standard gas chromatographic/mass spectrometric analysis. The procedure is characterized by simplified sample treatment and by fast execution.     

1,2-Dimethylimidazole-4-sulfonyl chloride, a novel derivatization reagent for the analysis of phenolic compounds by liquid chromatography electrospray tandem mass spectrometry: application to 1-hydroxypyrene in human urine

A novel derivatization method employing 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) to improve the mass spectrometric response for phenolic compounds in liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (LC-ESI-MS/MS) is described. Several environmentally relevant compounds, including chloro-, aryl- and alkylphenols, steroidal estrogens, and hydroxy-polycyclic aromatic hydrocarbons (OHPAHs), were selected to evaluate this technique. A facile derivatization procedure employing DMISC results in dimethylimidazolesulfonyl (DMIS) derivatives that are stable in aqueous solution. These DMIS derivatives produced intense [M+H](+) ions in positive-ion LC-ESI-MS. The product ion spectra of the [M+H](+) ions of simple phenols were dominated by ions representing the DMIS and dimethylimidazole moieties, whereas product ion spectra of the DMIS derivatives of OHPAHs with three or more fused aromatic rings showed prominent ArO(+) ions, the relative intensity of which increased with the number of rings. The DMIS derivatives of the selected phenolic compounds showed excellent chromatographic properties. To substantiate the utility of derivatization with DMISC, an analytical method employing enzyme hydrolysis, solid phase extraction, derivatization with DMISC, and analysis by LC-ESI-MS/MS with multiple reaction monitoring for determination in human urine of 1-hydroxypyrene, a widely used biomarker for the assessment of human exposure to PAHs, was developed and validated.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Ketonic bile acids in urine of infants during the neonatal period

Ketonic bile acids have been found to be quantitatively important in urine of healthy infants during the neonatal period. In order to determine their structures, the bile acids in urine from 11 healthy infants were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) and three samples with particularly high levels of ketonic bile acids were selected for detailed studies by ion exchange chromatography, fast atom bombardment mass spectrometry, microchemical reactions, and GLC-MS. The major ketonic bile acid was identified as 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-chol-1-enoic acid, not previously described as a naturally occurring bile acid. The positional isomer 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid, recently described as a major urinary bile acid in infants with severe liver diseases, was also excreted by most infants. Three acids related to cholic acid were identified: 7 alpha, 12 alpha-dihydroxy-3-oxo-, 3 alpha, 12 alpha-dihydroxy-7-oxo-, and 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholanoic acids. Five bile acids having one oxo and three hydroxy groups were also present. Based on mass spectra and biological considerations two of these were tentatively given the structures 1 beta, 7 alpha, 12 alpha-trihydroxy-3-oxo- and 1 beta, 3 alpha, 12 alpha-trihydroxy-7-oxo-5 beta-cholanoic acids. Some of the others had a hydroxy group at C-4 or C-2. The levels of ketonic bile acids were higher on the third than on the first day of life, and lower after 1 month. The formation and excretion especially of 3-oxo bile acids is proposed to result from changes of the redox state in the liver in connection with birth.     

The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats

The conversion of radioactive C6-C16-monocarboxylic acids to urinary adipic, suberic, sebacic and 3-hydroxybutyric acids was investigated in vivo in unstarved, starved and diabetic ketotic rats. Hexanoic, octanoic and decanoic acids were converted to C6-, C6-C8- and C6-C10-dicarboxylic acids, respectively, in fed and 72-h-starved rats. Lauric acid was converted to C6-C8-dicarboxylic acids in starved rats but not in unstarved rats. Decanoic and lauric acids were converted to relatively high amounts of C6-C8-dicarboxylic acids compared with myristic acid in myristic acid in ketotic diabetic rats, while radioactivity from [1-14C]-and [16-(14)] palmitic acid was not incorporated into C6-C8-dicarboxylic acids in diabetic ketotic rats. C6-C12-monocarboxylic acids in hydrolysed rat adipose tissue wee determined by gas-liquid chromatography-mass spectrometry (selected ion monitoring). Decanoic and lauric acids were found in amounts of 7.6-9.1 and 85.9-137.5 micrometers/100 mg tissue, respectively, whereas the amounts of hexanoic and octanoic acids were negligible. It is concluded that the biological origin of the C6-C8-dicarboxylic aciduria seen in ketotic rats are C10-C14-monocarboxylic acids, which are initially omega-oxidised solely or partly as free acids and subsequently beta-oxidised to adipic and suberic acids. The in vitro omega-oxidation of C6-C16-monocarboxylic acids to corresponding dicarboxylic acids in the 100,000 Xg supernatant fraction of rat liver homogenate was measured by selected ion monitoring. 0.09, 0.14, 16.1, 5.8, 7.0 and -6.9% of, respectively, hexanoic, octanoic, decanoic, lauric, myristic and palmitic acid were omega-oxidised to dicarboxylic acids of corresponding chain lengths after 90 min of incubation, when correction for the production of dicarboxylic acids in control assays was made. An in vitro production of C12-C16-dicarboxylic acids was detected in all assays ()including control assays), probably formed from"endogenous' monocarboxylic acids preexistent in the homogenate. Ths "endogenous' production of dicarboxylic acids was inhibited by C10-C16-monocarboxylic acids, where palmitic acid had the strongest effect. In fact, palmitic acid inhibited its own omega-oxidation when added in concentrations above 0.6 mM. Starvation of rats for 72 h did not alter the "endogenous' in vitro production of hexadecanedioic acid.     

The microsomal dicarboxylyl-CoA synthetase.

Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo.     

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.     

Determination of serum methylmalonic acid by alkylative extraction and liquid chromatography coupled to tandem mass spectrometry

Despite the new advances in bioanalytical techniques, the analysis of low-molecular-weight organic acids in complex matrices is still a challenge. Although new strategies applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) seem to be promising, sample preparation methodologies hamper its application in most clinical laboratories. The quantitation of methylmalonic acid (MMA) in biological matrices is an emblematic example due to its low concentration, the need for derivatization to increase its molecular weight, and the presence of the physiologically more abundant isomer succinic acid. Here we present a new strategy for rapid and sensitive MMA quantitation by combining alkylative extraction and LC-MS/MS. Alkylative extraction conditions were optimized to allow endogenous detection of MMA using only 50 μL of serum with a short sample preparation procedure. The formation of a unique ion from the MMA dipentafluorobenzyl derivative in negative atmospheric pressure chemical ionization (APCI) allowed its detection with high sensitivity and with no interference from succinic acid, a more abundant physiologically present isomer.     

Quantitation of fourteen urinary alpha-amino acids using isobutane gas chromatography chemical ionization mass spectrometry with 13C amino acids as internal standards

Isobutane chemical ionization gas chromatography mass spectometry of the N-trifluoroacetyl-carboxy-n-butyl ester derivatives of amino acids, using a commercial per-13C-amino acid mixture as internal standards, provided a sensitive and specific method for quantitative analysis of fourteen urinary alpha-amino acids. A computer controlled quadrupole mass spectrometer was used in a selected ion monitoring mode to record the ion current due to the protonated molecular ions of each alpha-amino acid/13C analogue pair. BASIC programmes located peak maxima, and using previously established standard curves, calculated the amino acid content on the bases of both peak height and peak area ratios. Duplicate amino acid analyses are possible on 5 microliter of urine. Instrumental analysis required 25 minutes, automated data processing 10 minutes, and sample preparation 2 hours. Detection limits approached 1 ng with a typical mean standard deviation of 2% for the instrumental analysis.