Medical Applications of Mass Spectrometry

Medical Applications of Mass Spectrometry

Medical Applications of Mass Spectrometry Orval A Mamer, McGill University, Montre´al, Que´bec, Canada ã 2017 Elsevier Ltd. All rights reserved. Mass...

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Medical Applications of Mass Spectrometry Orval A Mamer, McGill University, Montre´al, Que´bec, Canada ã 2017 Elsevier Ltd. All rights reserved.

Mass spectrometry has a wide breadth of application in medicine. This is largely by virtue of having the uncommon ability to combine great sensitivity and specificity with near-perfect generality. Among the most common applications of mass spectrometry in medicine are the diagnosis and confirmation of known acquired and inherited metabolic disorders, characterization and investigation of those previously unknown, and the identification of intoxicants, whether inadvertently or deliberately administered. The use of mass spectrometry (MS), coupled with either gas chromatography (GC) or liquid chromatography (LC), as a metabolic profiling tool (metabonomics, metabolomics) is also increasing, with applications in disease diagnosis and drug safety evaluation. While this article will focus on the area of metabolic disease, the techniques described in brief here can be, and are, applied in a myriad other routine clinical situations. Some of these are measurement of serum homocysteine and methylmalonic acid as an indicator of increased risk for cardiovascular disease, and diagnosis of peptic ulcer, gastritis and gastric cancer caused by Helicobacter pylori by measurement of 13C-labelled carbon dioxide released by H. pylori from an oral dose of 13C-labelled urea. Mass spectrometry is the ‘gold standard’ in the clinical laboratory, and is at the heart of many reference methodologies. Serum cholesterol determinations are highly dependent on the natures of the wet chemistries used, and mass spectrometry with gas or liquid chromatographic or capillary electrophoresic inlets provide the only direct and unambiguous measurements available today. Metabolic disease investigation by mass spectrometry, then, is selected only as an example or model of how it may be applied routinely elsewhere in the clinical setting.

Metabolic Diseases The identification of accumulating metabolites characteristic of a metabolic disease employs mass spectrometry in a qualitative manner. Compounds are identified in a fingerprint sense by computer-comparison of the mass spectra of unknowns with a library of reference spectra either purchased with the instrument or accumulated in-house from reference compounds. Mass spectrometry may also be used in a quantitative sense for the purpose of monitoring or evaluating patients, usually as part of a treatment protocol. The most significant metabolic disorders are life threatening in the neonatal period if left untreated and are usually the result of the inherited inability to catabolize normal substrates derived from diet or from normal tissue degradation and recycling. The typical catabolic pathway can be represented by eqn [1],

where A, B, . . ., N are precursors, intermediates and final products, and subscripted Es are the enzymes or enzyme complexes that effect the changes associated with each metabolic step. A metabolic disease is said to occur when one (or more) of these enzyme-mediated steps fails to produce the required transformation, and the blood and tissue concentrations of the substrate or precursor for that step (or earlier steps) increase to levels that are toxic, inhibit other enzyme systems or significantly alter the pH or other characteristics of blood or tissues and adversely affect the patient’s well-being. Other disorders may be the result of the inability to synthesize a required substance or the inability to transport something essential across a membrane. Identification of accumulating or missing substrates is therefore crucial to understanding the nature of the disorder and the eventual identification of the faulty enzyme or enzyme system. The list of inherited metabolic diseases continues to grow, and over 500 distinct disorders have been at least partially characterized, with the sequences of the responsible defective proteins known for many of them. Most of these disorders are very rare; the incidence of a given disorder may vary from 1 live birth in 500 000 to 1 in 750, and is frequently dependent upon the degree to which an isolated population is inbred. A paediatrician may spend a lifetime in practice and not encounter any but the most common of these rare diseases. Diagnosis in the very early postnatal period is critically important if the newborn is to survive and not suffer toxic accumulations of metabolites that frequently lead to retardation of physical and intellectual development. Through correct early diagnosis of many diseases that result from errors of catabolism of dietary components, such as amino acids, diets restricted in these precursors may be instituted to prevent these accumulations. Diagnosis in utero is often achieved by the mass spectral analysis of a small sample of the amniotic fluid for elevations of metabolites excreted by a possibly affected fetus. This invasive procedure is usually only applied when there is a reason to suspect that a fetus may be affected, such as a defect occurring in a prior birth. This provides a basis for an informed decision to be made either to terminate the pregnancy or to prepare for supportive intervention at birth. The failure of an enzyme may be partial or complete, permanent or transitory. A genetic mutation that encodes for an enzyme that is partially or completely inactive will result in a permanent deficit. Some enzyme deficits that are due to mutations that lead to inefficient binding of a cofactor can be stimulated to a useful capacity by administration of large doses of the cofactor on a life-long basis.

Gas Chromatography–Mass Spectrometry ½1 This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.


The use of mass spectrometry in medicine was greatly facilitated by the development of coupled GC-MS. The first commercially successful GC-MS, the LKB-9000, became available

Encyclopedia of Spectroscopy and Spectrometry, Third Edition

Medical Applications of Mass Spectrometry

in the mid-1960s, and it is estimated that fully half of the inherited metabolic disorders initially described in the 1960s and 1970s were discovered and investigated with various versions of this instrument. Direct coupling of fused-silica capillary columns to a proliferation of small and inexpensive bench-top quadrupole and ion trap instruments has now made practical the screening for metabolic disease in every live birth in developed countries. An example of an inherited disorder that is the result of a permanent enzyme deficit is methylmalonic acidaemia, in which methylmalonic acid (MMA), derived in large part from valine and isoleucine, accumulates. In one of the common forms of this disorder, methylmalonyl-coenzyme A mutase, the enzyme responsible for conversion of methylmalonylcoenzyme A (CoA) to succinyl-CoA is partially or completely inactive, and the clinical result is a severe and life-threatening keto acidosis, accompanied by high concentrations of MMA in the blood and in tissues and the urinary excretion of up to several grams of MMA per day. In other forms, mutase activity can be increased by administration of large doses of vitamin B12 or one of its related cobalamins, which are necessary cofactors for the mutase. For diagnostic purposes, a sample of urine is collected from the patient and an extract is prepared containing the carboxylic acids present in the urine. The dried extract is treated with a chemical reagent that produces the trimethylsilyl (TMS) esters of the carboxylic acids, and this mixture is then analysed by capillary GC-MS. Figure 1 is an organic acid profile obtained in this manner, and is an example of the use of GCMS in the diagnosis of a mutase deficiency. Clearly evident is a very large GC peak due to bis(trimethylsilyl) methylmalonate. Other acids present and identified are normally found in human urine. Table 1 lists the identities of the eluting peaks in this figure and in the other profiles presented below. Integrated ion-current peak areas can be related to the quantities of metabolites present in the extract. While relative sensitivities for ionization must be known and taken into account for precise determinations, frequently one is forced


to assume equality for all metabolites identified when these are unknown. The result is usually accurate to within a factor of 2 or 3 and is sufficient for screening of large numbers of samples. When more precise measurements are required, stableisotope dilution techniques are employed. In this approach to quantitation, a measured amount of a stable-isotopelabelled analogue of the compound of interest (internal standard) is added to the fluid to be analysed. The sample is then prepared for analysis and the mass spectrometer is used to report the ratio of the unlabelled to labelled analogues. With relatively few and minor corrections applied, the concentration of the unlabelled metabolite is easily determined. The technique is insensitive to losses in sample separations and variable derivatization yields as these will affect both analogues in the same proportion. As an example, Figure 2 illustrates the use of this technique in the accurate determination of the concentration of MMA in human serum. The internal standard is 20 mg of [Me-2H3]MMA, which was added to 1 mL of serum. The acidic extract was converted to the TMS derivatives and analysed in a GC-MS mode termed ‘selected-ion monitoring’, in which the quadrupole analyser is stepped discontinuously between selected ions and reports only their intensities. In this case, the ions selected have masses 218.2 and 221.2 Da, which are moderately intense, characteristic and distinguishing fragment ions in the spectra of the TMS derivatives of MMA and [Me-2H3]MMA, respectively. The data are obtained as plots of the intensities of these two ions as functions of GC retention time, and resemble independent gas chromatograms for these two fragment ions. The peak areas are integrated and a correction of the 218.2 area is made for isotopic impurity in the internal standard. The 221.2 area is also corrected for natural-abundance heavy isotope inclusion in the light ion that is present as an Mþ3 signal at 221.2. From these calculations one may then conclude that the serum sample was 20.06 mM in MMA, about 10-fold greater than the normal upper limit.

Figure 1 The electron ionization total ion current chromatogram for the trimethylsilyl derivatives of the organic acids extracted from the urine of a patient with an inherited error of methylmalonyl-CoA mutase. The major acidic component is methylmalonic acid. The annotated peaks are identified in Table 1.


Table 1

Medical Applications of Mass Spectrometry

Identities of acid peaks annotated in Figures 1, 3, 4 and 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

Phenol Lactic 2-Hydroxyisobutyric Glycolic Oxalic Glyoxylic oxime 4-Cresol Pyruvic oxime 3-Hydroxyisobutyric 3-Hydroxybutyric 2-Hydroxyisovaleric 2-Methyl-3-hydroxybutyric Methylmalonic (2-TMS)a Benzoic 2-Ethyl-3-hydroxy-propionic 2-Hydroxyisocaproic (2S)-Hydroxy-3(R)-methylvaleric (2S)-Hydroxy-(3S)-methylvaleric Ethylmalonic Succinic 4-Hydroxybenzaldehyde Glutaric Methylmalonic (3-TMS) 2-Methoxybenzoic (IS)b Capric (IS)b 3-Hydroxyoctanoic Mandelic Adipic 3-Methyladipic 3-Phenyllactic Pimelic Hippuric (secondary derivative) 4-Hydroxybenzoic 4-Hydroxyphenylacetic 4-Hydroxybenzaldoxime Phthalic Suberic Vanillic Homovanillic Azelaic Hippuric Citric 3-(3-Hydroxyphenyl)-3-hydroxypropionic Vanillylmandelic Sebacic 4-Hydroxyphenyllactic 3-Indoleacetic 4-Hydroxyphenyl-pyruvic oxime Palmitic 3-Hydroxysebacic 4-Hydroxyhippuric N-Acetyltyrosine 5-Hydroxyindoleacetic Stearic N-Acetyltryptophan

Figure 2 Selected ion monitoring analysis of serum for methylmalonic acid. The m/z 218.2 ion represents the [M – CO2]þ McLafferty fragment of the TMS derivative of endogenous unlabelled methylmalonic acid. The m/z 221.2 ion is the fragment derived by a similar process from the internal standard [Me-2H3] methylmalonic acid. The areas integrated for the ions are related to the relative concentration of the derivatives (see text). The deuterium-labelled analogue has a slightly shorter retention time than the unlabelled analogue, a phenomenon perhaps unexpected but commonly observed in these circumstances.

oxidatively decarboxylate 2-ketoisovaleric, 2-keto-3methylvaleric and 2-ketoisocaproic acids to the corresponding branched-chain CoA esters is defective, and very high concentrations of these keto acids accumulate in the blood and urine of these patients. They also have in their fluids large elevations of the corresponding 2-hydroxy acids made by reduction of the keto acids, probably by lactate dehydrogenase. In sample preparation, addition of sodium borohydride to the urine will reduce the three 2-keto acids to the corresponding three 2hydroxy acids and eliminate (E) and (Z) isomerism in the TMS derivatives of the enols of the 2-keto acids, thereby simplifying the gas chromatogram. If sodium borodeuteride is added instead, the 2-hydroxy acids that are produced will bear single deuterium substitutions on carbon-2 (eqn [2]) and the labelled/unlabelled ratios may be determined easily for each of the 2-hydroxy acids.


TMS¼trimethylsilyl. IS¼internal standard.


Another inherited error of valine, isoleucine and leucine metabolism is branched-chain a-keto aciduria, also known as maple syrup urine disease because the odour of urine of these patients resembles that of maple syrup. Branched-chain a-keto acid dehydrogenase, the enzyme complex that is used to


These three ratios then represent the ratios of the 2-keto acids originally in the sample to their corresponding 2-hydroxy acids. Figure 3 illustrates the urinary organic acid profile of

Medical Applications of Mass Spectrometry


Figure 3 The total ion current chromatogram obtained for the organic acids isolated from the sodium borodeuteride-treated urine of a patient with maple syrup urine disease. The ratios of the 2H-labelled to unlabelled analogues for 2-hydroxyisovaleric acid, 2-hydroxyisocaproic acid and the two 2-hydroxy-3-methylvaleric acid diastereomers are respectively 0.24, 34.5, 30.0 and 3.70.

one of these patients after reduction of the urinary 2-keto acids by sodium borodeuteride to the labelled 2-hydroxy acids. The keto/hydroxy ratios are clinically significant as they are related to the NADþ/NADH status of these patients. (NADþ/ NADH¼the oxidized/reduced forms of nicotinamide-adenine dinucleotide.) A transitory enzyme deficit is one that arises commonly as the result of the ingestion of a toxic material that is or can be metabolized to a suicide substrate for that enzyme, which if depleted must be replaced by de novo synthesis. Another common temporary deficit is the result of an immature enzyme system in the newborn that spontaneously resolves itself within a few days. As an example of the former, Jamaican vomiting sickness presents with severe hypoglycaemia and acidosis that resembles known types of inherited acyl-CoA dehydrogenase deficiencies. The disorder is precipitated by consuming the unripe fruit of the akee plant that grows in the Caribbean area. The protoxin is an amino acid, hypoglycine A (a-amino-b-(2-methylenecyclopropyl)propionic acid), which is metabolically converted to methylenecyclopropylacetic acid that irreversibly binds covalently to the flavin moiety of several acyl-CoA dehydrogenases and acts as a suicide substrate to permanently disable their active sites. Treatment is usually limited to supportive care during the period in which the inactivated enzyme system is replaced by new synthesis. The organic acid profile is dominated by very large concentrations of butyric, isovaleric and 2-methylbutyric acids, whose CoA esters require active dehydrogenases for further catabolism to crotonyl-CoA, 3-methylcrotonyl-CoA and 2methylcrotonyl-CoA esters. Often these acids and other shortchain acids are also esterified to carnitine and excreted in the urine in easily detected amounts. An example of a transient deficit that is the result of an immature enzyme system is a disorder in which the identification of increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid (4-HPPA) in urine of a premature newborn

would suggest reduced activity of 4-HPPA oxidase. Administration of pharmacological doses of ascorbic acid, the cofactor required for activity of this enzyme, may overcome the temporary oxidase deficit and stimulate catabolism of the accumulating 4-HPPA. Figure 4 is an illustration of this disorder, which is termed transient neonatal tyrosinaemia. In addition to increased excretion of 4-HPPA, present here as the oxime, elevations of 4-hydroxyphenyllactic and 4hydroxyphenylacetic acids are noted, and these are metabolites that are not important in normal catabolism and are derived from 4-HPPA by other enzymes. The oximes are made by addition of hydroxylamine hydrochloride to the urine during sample preparation for the purpose of detecting succinylacetone (see below). Tyrosinaemia has an inherited form that is permanent and is due to inactive fumarylacetoacetate (FAA) hydrolase. As a result, FAA accumulates and is enzymatically converted into succinylacetoacetate (SAA), very low levels of which in turn severely inhibit 4-HPPA-oxidase. Many of the same accumulations seen in the transient neonatal form are measured as a result (Figure 5). N-Acetyltyrosine is often noted in these organic acid profiles obtained for the hereditary form of tyrosinaemia, the result of N-acetylation occurring when blood levels of tyrosine rise to very high values. As a further complication, succinylacetone (SA), a spontaneous decomposition product of SAA, markedly inhibits porphobilinogen synthetase and other enzymes, which leads to the large number of clinical presentations seen in this disorder. To distinguish the transient and inherited forms, one needs only to measure the serum concentration of SA. SA is unstable to the usual sample preparation methods and, to avoid losses, the oxime is made by addition of hydroxylamine hydrochloride to the serum or urine sample. Since all of the hydrogen atoms in SA are easily exchanged with aqueous protons, no deuteriumlabelled analogue of SA resistant to back-exchange can be


Medical Applications of Mass Spectrometry

Figure 4 The urinary organic acids excreted by a premature infant with immature 4-hydroxyphenylpyruvic acid oxidase. This disorder, also known as transient neonatal tyrosinaemia, is characterized by excretion of elevated amounts of 4-hydroxyphenylacetic, 4-hydroxyphenyllactic and 4-hydroxyphenylpyruvic acids. The urine was pretreated with hydroxylamine hydrochloride, which converts the keto acids into the respective oximes.

Figure 5 The organic acid profile obtained for a patient with inherited tyrosinaemia. Very large concentrations of 4-hydroxy-phenyllactic acid and N-acetyltyrosine are in evidence.

made for use as an internal standard, and synthesis of a suitably 13C-substituted analogue would be very difficult. This problem can be side-stepped by using 2-methoxybenzoic acid as the internal standard, as this does not occur in human metabolism and it elutes without interference by an endogenous metabolite. Ions selected for monitoring are m/z 209.1 and 212.1, the [M–CH3]þ fragments of the TMS derivatives of 2-methoxybenzoic acid and the positional isomers 3-[5-(3-methylisoxazolyl)] propionic acid and 3-[3(5-methylisoxazolyl)] propionic acid. The latter two compounds are produced in the oximation of SA, and while they are separable on gas chromatography they are summed for quantitation. Figure 6 illustrates an example of such an analysis of a urine sample.

Hence, this example represents the use of an unrelated compound as the internal standard, 2-methoxybenzoic acid in this case. It is necessary in these circumstances to carefully prepare calibration curves with samples of known composition to take into account the relatively different extraction and ionization efficiencies and appearance potentials for the ions selected.

Fast Atom Bombardment and Electrospray Ionization Several diseases are known that result in elevations in tissues and fluids of various esters of carnitine and reduce the availability of free carnitine, which is normally synthesized by

Medical Applications of Mass Spectrometry


Figure 6 Selected-ion monitoring analysis of a urine sample from a patient with the inherited form of tyrosinaemia. Succinyl-acetone, which is distinctly elevated here, is produced from fumarylacetoacetic acid and is measured as an indication of fumarylacetoacetate hydrolase inactivity, the precipitating cause of this form of tyrosinaemia. The ions monitored are the [M–CH3]þ fragments of the TMS derivatives of the internal standard 2-methoxybenzoic acid (m/z 209.1) and the two positional isomers of the oxime derivative of succinylacetone (m/z 212.1).

humans and is necessary for the transport of long-chain fatty acids into mitochondria for oxidation. In several disorders arising from acyl-CoA dehydrogenase deficiencies, the accumulation of the acyl-CoA substrate frequently sequesters coenzyme A and reduces its availability for other unrelated but important and otherwise competent pathways. Carnitine administration can displace and make available much of the coenzyme A that had been isolated, and stimulate the excretion of the accumulating acidic metabolites now esterified to carnitine. Detection of reduced levels of serum or urinary free carnitine and elevations of esterified carnitine is therefore useful for diagnosis of a variety of metabolic disorders, among them a congenital inability to synthesize carnitine. In this disorder, carnitine must be supplied by a carnitine-enriched diet as it is, in effect, a vitamin.


Carnitine and its esters (see [3]) cannot be introduced to the mass spectrometer by gas chromatography, as they incorporate quaternary amine functions and will decompose in the attempt. Fast atom bombardment (FAB) and electrospray ionization (ESI) can use the formal charge on the quaternary

amine function to advantage, as carnitine and its esters are very easily desorbed from glycerol on the FAB probe and from aerosol sprays in ESI. Figure 7a illustrates the use of FAB in the quantitation of carnitine and its esters excreted in the urine of a patient presenting with a severe dicarboxylic aciduria associated with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. 2 2 D,L-[Me1,N- H3]carnitine, D,L-[Ac- H3]acetylcarnitine, 2 and D,L-decanoyl[Me1,N- H3]carnitine are added as internal standards, and the sample is prepared as the trideuteromethyl ester for FAB analysis. Esterification of the carnitine carboxylate function with perdeuteromethanol removes the zwitterionic character of carnitine and its esters, and leaves them with a full positive charge. It also raises their ion mass by 17 Da to avoid confusion between them and their nonesterified homologues. High-resolution (10 000) spectra confirm the presence of the ions of interest free of interference from unrelated ions of the same nominal masses. Quantitations are then simply a matter of applying the usual calculations associated with a stable-isotope dilution assay. The identity of the annotated peaks is given in Table 2, along with the concentrations determined. Large concentrations of acetyl-, propionyl-, nonanoyl-, suberyland sebacylcarnitines are found that reflect elevations of the free acids in the urine. As a second illustration of the technique, urine from a patient with propionic acidaemia


Medical Applications of Mass Spectrometry

Figure 7 Fast atom bombardment mass spectra obtained in glycerol matrix for quantitation of free carnitine and carnitine esters in the urines of a patient with medium-chain acyl-CoA dehydrogenase deficiency (a) and a patient with propionic acidaemia (b). The identities and concentrations of the annotated ions are listed in Table 2. The composition of the ions has been confirmed by measurements at 10 000 resolution.

Table 2

Identities and concentrations of carnitine metabolites annotated in Figure 7


Free carnitine Carnitine ISa Acetylcarnitine Acetylcarnitine ISa Propionylcarnitine Butyrylcarnitine Octenoylcarnitine Octanolylcarnitine Nonanoylcarnitine Adipylcarnitine Decanoylcarnitine Decanolylcarnitine ISa Suberylcarnitine Decenedioylcarnitine Sebacylcarnitine


Ion composition

179.1475 182.1663 221.1581 224.1769 235.1737 249.1894 303.2363 305.2520 319.2676 324.2293 333.2833 336.3021 352.2606 378.2763 380.2919

C8H215H3O3N C8H212H6O3N C10H217H3O4N C10H214H6O4N C11H219H3O4N C12H221H3O4N C16H227H3O4N C16H229H3O4N C17H231H3O4N C15H226H6O6N C18H233H3O4N C18H230H6O4N C17H226H6O6N C19H228H6O6N C19H220H6O6N

Concentration (mmol/24 h) Fig. 7a

Fig. 7b



1625 25

210 4000

29 41 563 67 28 6

19 8 11 16 NDb NDb

50 34 68



Internal standard. Not detected.


(Figure 7b) is shown to have a large concentration of propionylcarnitine (Table 2). ESI has made possible many recent advances in the application of mass spectrometry to diagnostic medicine. Its great sensitivity and applicability to minuscule sample sizes together with its ability to analyse aqueous solutions form the basis of

its utility. Because it is also a desorption technique, it is especially useful and sensitive for compounds that incorporate formal charges or chemical groups that are easily ionized. Trimethylaminuria is an inherited disease related to the inability to convert trimethylamine (TMA) metabolized from dietary sources into trimethylamine N-oxide. The result is a

Medical Applications of Mass Spectrometry

disorder that is not clinically acute but has the unpleasant effect of producing a body odour resembling that of rotting fish in those affected. The social consequences of this are severely debilitating. While it is relatively easy to diagnose this disorder, objective means are needed to evaluate the efficacy of treatment protocols. TMA can be quaternized with [2H3]methyl iodide and, with 15N-labelled TMA as the internal standard, an ESI method can be used to determine TMA concentrations in urine and blood. Figure 8a represents an example of the analysis of a normal urine for TMA. Figure 8b is obtained for a similar analysis of another normal urine spiked with 40 ng mL1 of unlabelled TMA. A known amount of [15N]TMA hydrochloride is added to the urine, the pH of the solution is increased to 12 in an ice bath, an ether extract is made, and an excess of [2H3]methyl iodide is added. The resulting ether solution is added to water, which is warmed to drive off the ether and excess methyl iodide, and the remaining solution is infused by syringe through the ESI probe. Selected-ion monitoring with averaging or short scan modes in multichannel array detection may be


used. Calculations usually associated with stable-isotope dilution analyses are then applied. [2H3]Methyl iodide is used as the quaternizing reagent to avoid interference by endogenous tetramethylammonium ions (mass 74 Da) usually present in urine and blood. A second use of ESI is in the measurement of inorganic sulfate in blood and urine. Sulfate is extruded actively from cells and resides virtually exclusively in the extracellular fluid compartment. Sulfur has four stable isotopes, 32S, 33S, 34S and 36 S (95.02%, 0.75%, 4.21% and 0.02%, respectively, although these values may vary somewhat with the nature of the source), and as sulfate it is an end-metabolite of sulfur-containing amino acids and other physiologically significant organosulfur compounds. Sulfate does not respond well in positive-ion FAB or ESI, but in negative-ion ESI the ion HSO 4 is desorbed very efficiently from aqueous solutions. A stable-isotope dilution assay for sulfate based upon this fact uses highly enriched sodium [34S]sulfate as the internal standard that is added to the biological fluid. The ratio of the H32SO 4 analyte and H34SO reference ions could then be measured directly at 4

Figure 8 Positive ion electrospray spectrum of tetramethylammonium ions produced by quaternization of trimethylamine (TMA) excreted by a patient with trimethylaminuria. When the amine fraction is quaternized with [2H 3]methyl iodide, endogenous TMA is measured at m/z 77, while the [15N] TMA internal standard is measured at m/z 78. The ion signal at m/z 74 is unlabelled tetramethylammonium produced endogenously, which would interfere with the analysis if unlabelled methyl iodide were used for quaternization.

Figure 9 Negative ion electrospray collision-induced neutral loss of 17 Da (hydroxyl radical) analysis of plasma inorganic sulfate as H32SO 4 (m/z 97)  and H34SO 4 (m/z 99). Phosphate, which occurs in plasma in much larger concentrations, also presents an intense ion with mass 97 Da (H2PO4 ),  32 but can be distinguished from H SO4 and excluded from the analysis as it eliminates 18 Da (water) under similar conditions. Heavy isotopes of hydrogen, oxygen and sulfur included in HSO 4 in their natural abundances contribute an ion intensity at m/z 99 of about 5% of the intensity at m/z 97. Here the analysis shows that approximately 12% of the sulfate in the sample is derived from an oral load of 32S-labelled sulfate.


Medical Applications of Mass Spectrometry

m/z 97 and 99 if phosphate were not also present. H2PO 4 also has mass 97 Da, and is present in concentrations much larger than that of sulfate. Collision-induced dissociation of HSO 4  yields SO3 (80 Da) while H2PO 4 yields PO3 (79 Da), which distinguishes them in the tandem mass spectrometer. It is then a simple matter to measure the relative abundances of H32SO 4 and H34SO 4 by neutral loss of an OH radical (17 Da), producing ions at m/z 80 and 82, respectively, as H2PO 4 dissociates exclusively with the loss of water (18 Da) and does not interfere with the sulfate measurement. An adaptation of this technique can be used to monitor sulfate produced from 34 S-containing amino acids in suspected errors of their catabolism. Radioactively-labelled sulfur-containing substrates have the drawback that 35S (the longest-lived radioisotope) has a half-life of only 87 days, and they must be prepared shortly before each use. [34S]sulfate may also be used in tracer studies to follow the excretion of label in the urine following an oral dose of the labelled sulfate. These experiments are usually conducted with radioactive [35S]sulfate for the purpose of determining a patient’s extracellular fluid volume. An example of this last use is presented in Figure 9. While this article presents and concentrates on examples of the common and routine use of mass spectrometry in the diagnosis of metabolic disease, many other applications are important in the identification and quantitation of metabolites in other areas of clinical medicine.

See also: Atomic Spectroscopy, Biomedical Applications; Atomic Spectroscopy, Pharmaceutical Applications; Biological Applications of Hyperpolarized 13C NMR; Biometallics/Metallomics Techniques and Applications; Circular Dichroism and ORD, Biomacromolecular Applications; Counterfeit Drugs Studied by NMR; Drug Metabolism Studied Using NMR Spectroscopy; Electrospray Ionization in Mass Spectrometry; Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry; Fragment-Based Drug Design by NMR; HPLC–NMR, Pharmaceutical Applications; Hyphenated Techniques, Applications of in Mass Spectrometry; Inductively Coupled Plasma Mass Spectrometry Applications; Ion Mobility Spectrometry (IMS) and Mass Spectrometry (MS); Ion Trap Mass Spectrometers; IR, Biological Applications; IR,

Medical Science Applications; Laser Microprobe Mass Spectrometers; Mass Spectrometry in Drug Metabolism: Principles and Common Practice; MRI Applications, Biological; NMR Spectroscopy in the Evaluation of Drug Safety; Overview of Biochemical Applications of Mass Spectrometry; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Spectroscopic Methods in Drug Quality Control and Development; Spectroscopy for Process Analytical Technology (PAT); Spectroscopy in Biotechnology Research and Development; Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; Vibrational Spectroscopy Applications in Drugs Analysis.

Further Reading Blasi F (ed.) (1986) Human Genes and Diseases, vol 8 in Horizons in Biochemistry and Biophysics. Chichester: Wiley. Borum PR (ed.) (1986) Clinical Aspects of Human Carnitine Deficiency. New York: Pergamon Press. Burlingame AL and Carr SA (eds.) (1996) Mass Spectrometry in the Biological Sciences. Totowa, NJ: Humana Press. Chapman TE, Berger R, Reijngoud DJ, and Okken A (eds.) (1990) Stable Isotopes in Paediatric Nutritional and Metabolic Research. Andover, UK: Intercept. Desiderio DM (ed.) (1992) Mass Spectrometry: Clinical and Biomedical Applications, vol 1. New York: Plenum Press. Goodman SI and Markey SP (1981) Diagnosis of Organic Acidemias by Gas Chromatography–Mass Spectrometry. New York: Alan R. Liss. Mamer OA (1994) Metabolic profiling: A dilemma for mass spectrometry. Biological Mass Spectrometry 23: 535–539. Matsumoto I (ed.) (1992) In: Advances in Chemical Diagnosis and Treatment of Metabolic Disorders, vol 1. Chichester: Wiley. Matsumoto I, Kuhara T, Mamer OA, Sweetman L, and Calderhead RG (eds.) (1994) In: Advances in Chemical Diagnosis and Treatment of Metabolic Disorders, vol 2. Kanazawa: Kanazawa Medical University Press. Matsuo T (ed.) (1992) Biological Mass Spectrometry. Kyo to: Sanei Publishing. Nordstrom A, Want E, Northen T, Lehtio J, and Siuzdak G (2008) Multiple ionization mass spectrometry strategy reveals the complexity of metabolomics. Analytical Chemistry 80: 421–429. Weaver DD (1989) Catalog of Prenatally Diagnosed Conditions. Baltimore: Johns Hopkins University Press. Wolfe RR (1984) Tracers in Metabolic Research. New York: Alan R. Liss.