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MS: Analysis of biologically important carboxylic acids
Clinical Biochemistry 38 (2005) 351 – 361
Review
Contemporary clinical usage of LC/MS: Analysis of biologically important carboxylic acids David W. JohnsonT Department of Genetic Medicine, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia Received 16 April 2004; received in revised form 7 January 2005; 17 January 2005
Abstract Objectives: This review summarizes the current role of LC/MS in the diagnosis and screening of clinical conditions involving the analysis of biologically important carboxylic acids. Design and methods: Carboxylic acids are divided into six logical categories of acid size and function. Details of chromatographic separation methods and modes of mass spectrometer operation are described for each category. Results: The use of LC/MS in clinical applications such as the diagnosis of inherited and acquired metabolic disorders, gastrointestinal disorders, cancer and diabetes and therapeutic drug monitoring is discussed. Conclusions: The mild conditions, speed and sensitivity advantages of LC/MS analysis, over alternatives, are highlighted. The sensitivity and specificity afforded by the combination of tertiary and quaternary ammonium derivatives and tandem mass spectrometry for the analysis of carboxylic acids is emphasized. Potential for a greater range of LC/MS carboxylic analyses, including stereoisomeric intermediates, is predicted. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: LC/MS; Fatty acids; Organic acids; Bile acids; Eicosanoids; Therapeutic agents; Chemical derivatization
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . Long chain fatty acids . . . . . . . . . . . . . . . . Very long chain fatty and branched chain fatty acids. Organic acids . . . . . . . . . . . . . . . . . . . . . Bile acids . . . . . . . . . . . . . . . . . . . . . . . Prostaglandins, thromboxanes and leukotrienes. . . . Therapeutic agents . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: LC/MS, liquid chromatography/mass spectrometry; GC/MS, gas chromatography/mass spectrometry; APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; LCFA, long chain fatty acids; BCFA, branched chain fatty acids; VLCFA, very long chain fatty acids; DMAE, dimethylaminoethyl; TMAE, trimethylaminoethyl; NSAID, non-steroidal anti-inflammatory drug. T Fax: +61 8 81617100. E-mail address: [email protected]. 0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.01.007
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Introduction
Discussion
In biological systems, fatty acids exist in a dynamic equilibrium between the free form and incorporated within lipids as esters or amides. The fatty acids, as well, undergo both elongation to provide key biochemical substrates and oxidative degradation to provide cells with energy. Disruption of these processes in humans, resulting in an absence or an excess of particular fatty acids, is a key contributor to many clinical conditions. In clinical laboratories, fatty acids are analyzed to determine nutritional status, diagnose inherited disease, intestinal disorders, cancer and diabetes, to identify bacterial infection and for therapeutic drug monitoring. For many years, the principal methods of fatty acid analysis have been capillary column GC or GC/MS. Only capillary columns possessed the resolution necessary to separate the many isomeric forms of fatty acids. These include unsaturated isomers (conjugated and methylene interrupted), branched chain isomers, functional group positional isomers, cis/trans isomers (in double bond and ring structures), diastereoisomers and stereoisomers. Prior to GC/MS analysis, fatty acids are first released from lipids by chemical or enzymatic means. This is because lipids chromatograph poorly, if at all, by GC. Additionally, fatty acids are dispersed among a mixture of lipid species. Measuring total fatty acids from all species provides the best sensitivity for their analysis. The development of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) interfaces for coupling LC to MS and the diversity of LC packings, solvent mixtures and modifying agents now mean that LC/MS is a practical alternative to GC/MS for the analysis of fatty acids. Analysis of the free acid rather than lipid species is still frequently required, however, because of the complexity of separating individual lipid species. This review is a systematic discussion of the analyses of six categories of carboxylic acids, where LC/MS is employed, that involve clinical diagnosis or screening. The carboxylic acids of each of the six categories have a distinctly different size and/or biological function. The carboxylic acids have further been restricted to those which incorporate within lipid species which is a usual prerequisite for biological function. In addition to fatty acids, they include bile (steroidal) acids and diacids and hydroxyacids often referred to as organic acids. Excluded are amino acids, aromatic acids and heterocyclic acids. Examples of the abnormalities of carboxylic acid metabolism, and the clinical conditions which result, will be provided for each category of carboxylic acid. Details of the LC separation method and the mode of operation of the mass spectrometer will be outlined for each clinical application. Trends and future possibilities for LC/MS analysis of carboxylic acids in the clinical laboratory will be explored.
Long chain fatty acids Monocarboxylic acids, both saturated and unsaturated, with a carbon chain length between 14 and 22 are referred to as long chain fatty acids (LCFA). They constitute the majority of fatty acids found in the lipids of the human diet. The clinical analysis of these fatty acids is important for the diagnoses of essential fatty acid deficiency and mitochondrial fatty acid oxidation disorders. Essential fatty acid deficiency is caused by an inadequate or unbalanced dietary intake of lipids or by intestinal malabsorption [1]. Biochemical indicators are abnormally low linoleic and a-linolenic acids and an elevation in the triene/tetraene (eicosatrienoic/arachidonic acid) ratio. Mitochondrial fatty acid oxidation disorders, such as long chain hydroxyl CoA dehydrogenase deficiency (LCHAD) and very long chain acyl CoA dehydrogenase deficiency (VLCAD), result from an inherited dysfunction of mitochondria [2]. Biochemical indicators are elevated levels of tetradecenoic, tetradecadienoic and 3-hydroxytetradecanoic acids, mostly conjugated as their carnitine esters. Most recent methods for the analysis of this ubiquitous class of fatty acid employ GC or GC/MS after hydrolysis, extraction and derivatization of lipid material. Excellent sensitivity and separation have been achieved with capillary column electron capture negative ion mass spectrometry [3]. The use of LC/MS for the analysis of long chain fatty acids has mostly concentrated on profiling the fatty acids of intact lipid species [4,5]. This avoids the lengthy hydrolysis time and the need to derivatize. Lipid species such as sphingomyelin and the phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, etc) are already charged and ideal for ESI-MS analysis in either positive or negative ion mode. Practical quantitative methods, suitable for a clinical laboratory, for the analysis of LCFA by LC/MS, however, are not yet available. The measurement of intact lipid species has four major problems. (1) Isotope labeled fatty acids can be purchased for the GC/MS analysis of most free LCFA but few isotope labeled lipids are commercially available. ESI-MS and ESI-MS/MS are less tolerant of the use of related internal standards than detectors like ultraviolet, fluorescence and flame ionization. (2) There is often uncertainty in the identification of specific lipid species. The long chain base or alcohol of the lipid containing the fatty acid, as well as the fatty acid, exists in varying chain length, unsaturation and structural isomeric forms. A (C16:0 alcohol/C18:1 fatty acid) lipid is the same molecular weight as a (C18:1 alcohol/C16:0 branched fatty acid) lipid. (3) Unless prior separation of lipid classes is performed, it is difficult to avoid elevation in the apparent level of some specific lipid species by another class of lipids. (4) Those lipid classes most conveniently measured by LC/MS contain at most 10% of the total of a specific LCFA. This
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puts LC/MS at a sensitivity disadvantage compared with GC/MS methods which measure total LCFA. Very long chain fatty and branched chain fatty acids Very long chain fatty acids (VLCFA) are monocarboxylic acids with a carbon chain length greater than 22. Small amounts are obtained from the diet but in humans the majority is biosynthesized from shorter chain fatty acids. A competing oxidative degradation of VLCFA, to prevent their accumulation, occurs only within the subcellular organelle called the peroxisome. Another class of fatty acid exclusively degraded within the peroxisome is a- and h-branched chain fatty acids (BCFA). Large BCFA are not biosynthesized in humans but obtained from plant lipids usually via the ingestion of dairy products. Peroxisomal disorders which result from an absence or impaired function of peroxisomes [6] are thus characterized by elevated levels of VLCFA and/or BCFA in the tissues and body fluids. Two common blood screening tests to identify the majority of peroxisomal disorder patients are the measurement of the hexacosanoic (C26:0) acid to docosanoic (C22:0) acid ratio and the measurement of phytanic acid (C20:0 branched). VLCFA and the BCFA, pristanic acid 1 (see Fig. 1) and phytanic acid 2 are measured in many clinical laboratories by GC/MS as methyl, trimethylsilyl or for best sensitivity as pentafluorobenzyl esters [7]. The methods are time consuming (typically 1 to 1.5 days) and unsuitable for screening on small samples such as 3 mm blood spots on Guthrie card filter paper. LC/MS methods for the analysis of VLCFA and BCFA have only recently become available. Measurement of intact lipid species containing VLCFA has been unsuccessful because of trace levels (b1Amol/L) of hexacosanoic acid in normal plasma and its dilution among many lipid species. One LC/MS method [8] describes the analysis of VLCFA, after 2 h acid hydrolysis of lipid classes, in negative ion mode. Samples are injected into the flow from an HPLC starting at 20 AL/min and increasing to 50 AL/min over 2 min to achieve a sharp injection profile. Stable isotope labeled VLCFA standards are used for quantitation of the peak heights of the molecular anions during ESI-MS analysis. The method is
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faster (4 h) than GC/MS methods and shows good correlation for the analysis of controls, heterozygote and hemizygote Xlinked adrenoleukodystrophy patients (one of the most common forms of peroxisomal disorders) in plasma and cultured fibroblasts. The sample requirements (100 AL plasma) are similar to sensitive GC/MS methods. Anions of saturated fatty acids do not fragment to a product ion with any significant intensity so the sensitivity and specificity advantages of MS/MS analysis cannot be exploited. A second approach [9] also commences with acid hydrolysis of lipids. The isolated free fatty acids are derivatized as trimethylaminoethyl (TMAE) esters. These are quaternary ammonium derivatives with high molecular cation intensity that undergo fragmentation during positive ion ESI-MS/MS analysis to afford a dominant product ion from neutral loss of trimethylamine (59 Da) [10]. These derivatives are separated (22 min run time) using a reversed phase C8 column with acetonitrile/water at acidic pH. Stable isotope labeled VLCFA standards are used for quantitation. ESI-MS/MS analysis is by MRM of the ion pair of the molecular cation and the neutral loss of 59 Da product ion. Fig. 2 is a composite of 5 MRM ion chromatograms from analysis of the TMAE esters of VLCFA isolated from the plasma of a peroxisomal biogenesis defect patient. Baseline separation of the BCFA, pristanic acid (C19:0br) 1 and phytanic acid (C20:0br) 2 from their corresponding straight chain isomers can be seen. Excellent correlations with GC/MS for pristanic acid 1 and phytanic acid 2 and the C26:0/C22:0 ratios for a variety of peroxisomal disorders were demonstrated. Because of the extra sensitivity afforded by the use of a derivative only 10 AL of plasma is required, which can be reduced further by the use of newer MS/MS instruments. Organic acids Organic acids include short to medium chain mono- and dicarboxylic acids and hydroxylated analogs with a carbon chain length up to 12. All large metabolic laboratories in pediatric hospitals perform an organic acid screen on urine samples from children with symptoms indicative of organic
Fig. 1. Branched chain fatty acids and organic acids: (1) pristanic acid, (2) phytanic acid, (3) 3-hydroxyglutaric acid, (4) dibutyl dimethylglycine derivative of 3-hydroxyglutaric acid.
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Fig. 2. Composite ion chromatogram from ESI-MS/MS analysis of the TMAE derivatives of the fatty acids isolated from the plasma of a peroxisomal biogenesis defect patient. The composite is of five ion traces from 59 Da neutral loss MRM transitions for C19:0, C20:0, C22:0, C24:0 and C26:0 fatty acids. C19:0br is pristanic acid and C20:0br is phytanic acid.
acidurias or for any unexplained symptom. Many of these disorders are now detected by the newborn blood spot screening test for acylcarnitines [11] but there are some which can only be detected by this screening method. The most common method of analysis of organic acids is by GC/MS of the trimethylsilyl esters of the ethyl acetate extract of acidified urine. One of the earliest reports of LC/MS analysis of organic acids used a plasmaspray interface [12]. A related thermospray interface [13] was used for the analysis of key organic acids methylmalonic, 3-hydroxy-3-methylglutaric, propionic, isovaleric and arginosuccinic acids. Two methods of LC separation were described. One employed an HPX-87 H column at acidic pH for good separation and the other a C18 reversed phase column in ammonium acetate solution at neutral pH for better sensitivity. This was followed by the use of an APCI source and the analysis of organic acids in negative ion mode [14]. Methylmalonic acid is an organic acid which is a diagnostic metabolite for a group of inherited disorders, with at least eight variants, called methylmalonic acidemias. Methylmalonic acid is also elevated in the urine or plasma of patients with Vitamin B12 (cobalamin) deficiency as a consequence of poor intestinal absorption, digestion and diet. Renal insufficiency, hypovolemia and bacterial overgrowth in the small intestine can also cause abnormal levels of methylmalonic acid. Two recent papers from clinical laboratories describe the LC/MS analysis of methylmalonic acid.
In the first paper [15], urine or plasma containing d3methylmalonic acid is extracted with an automated SPE sample processor and derivatized with butanol/hydrogen chloride. The butyl esters are chromatographed (3 min run time) on a 3.3 cm C18 reversed phase column and analyzed by ESI-MS/MS. A comparison between this fully automated LC/MS method and a GC/MS method shows excellent correlation ( y = 0.95 + 0.51, R 2 = 0.998) and improvements in cost, labor and instrument time. The second paper [16] describes a simpler and faster technique for measuring methylmalonic acid. Urine or plasma containing d3-methylmalonic acid is extracted with methyl tert-butyl ether and derivatized with butanol/hydrogen chloride. The butyl esters are passed through (1 min run time) a 3.0 cm C18 reversed phase column and analyzed by ESI-MS/MS. A computer algorithm is used to compensate for succinic acid present in the sample. 10% of samples are needed to be reanalyzed by a different method because of the presence of excessive succinic acid. Disappointingly, the sample volume of plasma required for LC/MS analysis of methylmalonic acid by both these methods (typically 0.5 mL) is greater than that for sensitive GC/MS methods. This is in part due to the volatility of the dibutyl ester which makes selective evaporation of butanol, after derivatization, difficult. Additionally, butyl esters are poorly ionized by ESI in comparison with derivatives that are charged in aqueous solution. The use of a quaternary ammonium derivative, to provide greater sensitivity, is not possible with
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Fig. 3. Product ion spectra of the protonated molecular ions of the dibutyl, dimethylglycine derivatives of (top) 3-hydroxyglutaric acid and (bottom) 2-hydroxyglutaric acid.
dicarboxylic acids. Doubly charged binary derivatives of small molecules are not observed during ESI-MS analysis [10]. The triple selectivity provided by chromatographic separation, group specific derivatization and unique sets of
ion pairs in LC/ESI-MS/MS analysis has a practical application to the crowded spectrum of organic acids. 3hydroxyglutaric acid 3 is an organic acid which is regarded as the best diagnostic metabolite for glutaric aciduria type I
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[17]. Its measurement by GC/MS requires a large sample (0.5 mL plasma) and is complicated by its co-elution with the more common 2-hydroxyglutaric acid on the majority of capillary columns. An LC/ESI-MS/MS solution to this problem has been found (author, unpublished work). A systematic evaluation of derivatives afforded the dibutyl dimethylglycine derivative of 3-hydroxyglutaric acid 4 from butylation of the carboxylic acid groups and acylation of the alcohol with dimethylglycine [18]. Derivative 4 gives a strong protonated molecular ion intensity and is distinguishable from its 2-hydroxy isomer using unique product ions during ESI-MS/MS analysis. The product ion spectra of (top) the 3-hydroxy isomer (unique ion at m/z 104) and (bottom) the 2-hydroxy isomer (unique ion at m/z 131) are shown in Fig. 3. Direct ESI-MS/MS analysis of 3-hydroxyglutaric acid is prevented by interference from ions generated by derivatized glutamic acid. The interference can, however, be removed by HPLC separation thus providing an LC/ESIMS/MS method for the analysis of 3-hydroxyglutaric acid 3. The analysis of fatty acids from C2 to C18 as acylcarnitines, by ESI-MS/MS, is routinely performed in newborn screening centers for the diagnosis of inherited disorders of fatty acid metabolism [11]. There is only one recent report of an LC/ESI-MS/MS method for the analysis of eight acylcarnitines in urine [19]. The method employs chromatography of underivatized acylcarnitines on a reversed phase C8 column (runtime 17 min) with methanol/water containing an ion pairing reagent. Quantitation is carried out in MS/MS mode with an ion trap. This method allows the separation of acylcarnitines containing isomeric acyl groups such as butyryl and isobutyryl. A popular method of organic acid analysis used by biological and environmental laboratories but not yet by
clinical laboratories is LC/MS using ion exclusion separation [20,21]. Bile acids Bile acids are fatty acids attached to a steroidal backbone. They are biosynthesized from cholesterol 5 as shown in the abbreviated scheme in Fig. 4. N-Oxidation and hydroxylation give C27 bile acids 6 (di- and trihydroxycholestanoic acids) which are metabolized within peroxisomes to the more abundant C24 bile acids 7 (chenodeoxycholic and cholic acids). Hydroxylation is common as is conjugation with glycine, taurine, sulfate or glucuronide. Further degradation through transient C22 bile acids 8, C21 and C20 bile acids then occurs. Their physiological functions include lipid absorption, mediation of cholesterol synthesis and clearance of drugs, vitamins and toxins. Consequently, they are important indicators of hepatobiliary and intestinal function/dysfunction. Quantitative analysis of bile acids by GC/MS is time consuming. It requires extraction, purification, conjugate hydrolysis and derivatization to generate volatile analytes of these large, polar molecules. A comprehensive analysis requires separation and analysis of the bile acid composition of each of the conjugated (and free) species. The advantages of being able to analyze conjugated species without lengthy physical separation and hydrolysis, offered by LC/MS, have long been recognized. Early analysis of bile acids by LC/MS involved the use of a thermospray interface [22]. Improved sensitivity was obtained with the introduction of MS/MS instruments [23] and then with ESI [24]. Much effort has been focused on a screening test for cholestatic hepatobiliary disease which is
Fig. 4. Abbreviated scheme showing the metabolism of cholesterol to bile acids.
D.W. Johnson / Clinical Biochemistry 38 (2005) 351–361
one of the causes of jaundice in newborns. Long-term survival of these infants is significantly improved if surgery to correct it is performed within 60 days. A screening test for the measurement of glycine and taurine conjugates of C24 bile acids by direct ESI-MS/MS analysis, in neonatal blood spots, was developed [25]. The test unfortunately affords a high number of false positives. A problem with direct ESI-MS/MS analysis of bile acids is the presence of isomeric forms of the di- and trihydroxy C24 bile acids. An LC/ESI-MS/MS method [26] was developed for their separation. Plasma (50 AL) is deproteinized and chromatographed on a 5 cm C18 reversed phase column (run time not given) with methanol/water. The eluent is analyzed by an ESI-MS/MS instrument in negative ion MRM mode for glycine and taurine conjugates using isotope labeled internal standards. Unconjugated bile acids are detected using selected ion monitoring of the molecular anions. Several other groups described similar LC/ESI-MS/MS methods for the analysis of C24 bile acids. One group [27] uses a C18 column (run time 4 min) and elution with acetonitrile/water. There is good correlation with GC/MS methods. The other group [28] uses a 10 cm C18 reversed phase column (run time 20 min) with a three step binary gradient of methanol/water at acidic pH. LC/MS methods have also been described for the analysis of C27 bile acids. These bile acids are found in very low levels in the blood of normal people because once formed they are immediately metabolized in the peroxisome. In patients with peroxisomal biogenesis defects, they accumulate in the blood. Unlike C24 bile acids, the majority of the C27 bile acids are found in the unconjugated form. It is difficult to detect unconjugated bile acids, in either positive or negative ion mode, in small samples such as 3 mm blood spots which are the preferred samples for screening for inherited disorders. This led to the development of a specific double derivative, the acetoxy dimethylaminoethyl (DMAE) ester for very sensitive ESI-MS/MS analysis of unconjugated bile acids [29]. No chromatography is required for the analysis of di- and trihydroxy C27 bile acids, by this method, because of the specificity achieved by analyzing the product ion from neutral loss of 60 Da. An LC/ESI-MS/MS method for sensitive analysis of chromatographically separated isomeric bile acids could be developed from this, analogous to the separation of TMAE derivatives of VLCFA [9]. The corresponding acetoxy TMAE derivative of bile acids although affording greater ion intensity during ESI-MS analysis is too stable and does not fragment to an abundant product ion suitable for MS/MS analysis. If LC separation is employed, then this derivative could be used for LC/MS analysis of unconjugated bile acids. An LC/MS method has been developed to separate and measure the (25R)- and (25S)-isomers of the unconjugated and taurine conjugates of C27 bile acids for the diagnosis of a-methylacyl CoA racemase deficiency [30]. a-Methylacyl CoA racemase is a peroxisomal enzyme that catalyses the conversion of (2R)-methyl branched chain fatty acids to
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their (2S)-isomers which can then undergo a-oxidation. aMethylacyl CoA racemase deficiency is a rare peroxisomal disorder that results in an accumulation of the (25R)-isomers of C27 bile acids. For the analysis of the isomers, plasma (50 AL) containing isotope labeled C24 bile acid standards is deproteinized and chromatographed on a 25 cm C18 reversed phase column (22 min run time) in methanol/water. The eluent is sprayed into an ESI-MS/MS instrument. Taurine conjugates are measured by MRM experiments and unconjugated bile acids by single ion monitoring. Patients with a-methylacyl CoA racemase deficiency show an elevation in (25R)-isomers while patients with Zellweger syndrome (a peroxisomal biogenesis defect) show an elevation in both (25R) and (25S) isomers. Baseline separation of the (25R)- and (25S)isomers of the taurine conjugates was demonstrated. In common with the difficulty separating underivatized fatty acids by HPLC, the resolution of the unconjugated isomers is only partial. Prostaglandins, thromboxanes and leukotrienes These three related classes of compounds are substituted C20 fatty acids containing a cyclopentane ring (prostaglandins), tetrahydropyran ring (thromboxanes) and a conjugated triene (leukotrienes). The structures of some are shown in Fig. 5. They are formed biosynthetically from C20 (eicosanoic) essential fatty acid precursors containing 3 to 5 methylene interrupted double bonds. They cause profound physiological effects in mammalian systems especially on smooth muscle. There is a great deal of research probing the identities and functions of these eicosanoids in diverse areas of arthritis, diabetes, endocrinology and cancer. The multiple sites of hydroxylation and unsaturation and complex thioesters in some of these molecules make them chemically and thermally unstable. Despite this, GC/MS has been a powerful tool for their analysis [31]. Prostaglandins have also been analyzed by HPLC with UV detection for over two decades [32,33]. These acids were found to be better ionized by ESI than saturated fatty acids and sensitive LC/ESI-MS and LC/ESI-MS/MS analyses have developed. Prostaglandin E2 9 and 12-hydroxyeicosatetraenoic acid (12-HETE) 10 are important mediators in inflammatory skin disorders. An LC/MS method for their analysis in human dermal fibroblast supernatants was developed [34]. Supernatant is purified on C18 SPE columns and chromatographed on a 15 cm C18 reversed phase column (run time 22 min) with acetonitrile/water at acidic pH. Quantitation is by ESI-MS of the molecular anions using isotope labeled standards. 5,12 and 15-HETE isomers, PGE2 9 and arachidonic acid 11 are separated, with detection limits in the range 5 to 20 pg. A similar method for the analysis of these compounds as well as leukotriene B4 12 in small tissue biopsies has also been described [35]. Four classes of isoprostanes (free radical catalyzed prostaglandin isomers) have been analyzed in urine samples
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Fig. 5. Eicosanoid fatty acids: (9) PGE2, (10) 12-HETE, (11) arachidonic acid, (12) LTB4, (13) LTE4, (14) THB2.
by LC-ESI-MS/MS [36]. They are regarded as indicators of oxidant stress. Urine samples are purified on BondElut LMS polymer cartridges and chromatographed on a 15 cm C18 reversed phase column (run time 22 min) with acetonitrile/ methanol/water at pH 5.7. Quantitation is by ESI-MS/MS using MRM ion pairs unique to each class of isoprostane. Levels of all classes of isoprostanes are elevated in patients with hypercholesterolemia compared to controls. Only some of the isoprostanes are elevated in patients with congestive heart failure. The majority of recent LC/MS analyses for eicosanoids have employed almost identical chromatography and MS/MS measurement methods. These include the analysis of leukotriene E4 13 in the urine of patients with arthritis [37,38], thromboxane B2 14 in cultured synovial cell media [39] and 2- and 3-series prostaglandins in cultured tumor cells [40]. A novel LC/ESI-MS/MS application for the analysis of prostanoids is the chiral analysis of (12S)-HETE [41]. 12HETE 10 is thought to contribute to the pathogenesis of diabetic cardiovascular disease, renal dysfunction and destruction of pancreas cells. Urine is purified on C18 cartridge and chromatographed on 7.5 cm C18 reversed phase column with acetonitrile/water at acid pH. The fraction containing 12-HETE 10 is switched to a 25 cm chiral CD-Ph column between 6.5 and 10.5 min after injection. Column effluent from the chiral column is introduced into the ESI-MS/MS instrument between 40 and 65 min after injection. Quantitative analysis employs an octadeuterated standard and MRM of the ion pair of the
molecular anion and its major product ion. A significant difference is observed in the levels of (12S)-HETE in female patients with diabetes mellitus compared with controls. Diabetic males are indistinguishable from controls indicating a sex difference in 12-HETE 10 production. Therapeutic agents Many therapeutic agents which are routinely quantitated in clinical laboratories contain a fatty acid side chain. The most important group is the non-steroidal anti-inflammatory drugs (NSAIDs) whose major action is to inhibit prostaglandin synthesis. Ibuprofen 15 (Fig. 6), indomethacin 16, diclofenac 17 and ketoprofen 18 are commonly prescribed NSAIDs. Many methods have been developed for clinical studies of the dose/response of NSAIDs and for drug quality control purposes. Despite LC/MS cost and complexity, it provides low levels of detection with high specificity in short time periods. The power of LC/ESI-MS/MS for therapeutic drug monitoring is demonstrated with a quantitative method for indomethacin 16 [42]. Plasma samples (100 AL) containing mefenamic acid standard are purified with a solid phase extractor and chromatographed on a C8 column (4 min run time) in methanol/ammonium acetate buffer at pH 5.1. The indomethacin 16 is analyzed by ESI-MS/MS with an MRM of the protonated molecular ion and the major product ion (139 Da). Linearity is demonstrated in the range 5–2000 Ag/L. The separation and quantitation of four NSAIDs is achieved with an LC-APCI-MS method [43]. The drugs are injected onto a 15 cm CN column and eluted with
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Fig. 6. Therapeutic drugs containing fatty acid side chains: (15) ibuprofen, (16) indomethacin, (17) diclofenac, (18) ketoprofen, (19) valproic acid, (20) mycophenolic acid.
acetonitrile/ammonium acetate at pH 7.4. The molecular anions of the drugs are measured by APCI-MS in single ion monitoring mode. An elegant demonstration of the power of LC/MS analysis is the enanioselective determination of ibuprofen 15 [44]. Although ibuprofen 15 is sold as a racemate, its anti-inflammatory effect comes from the (+)-(S)-enantiomer. Only (2S)-isomers of a-methyl branched fatty acids can be degraded by h-oxidation. Accordingly, HPLC and capillary electrophoresis methods were developed for its enantioselective analysis. Amylose and cellulose derivatives have shown the greatest resolving power for HPLC analysis under both normal and reversed phase conditions. The authors use a new amylose based chiral column Chiralpak AD-RH (a tris-(3,5-dimethylphenylcarbamate)amylose) in their LC/MS method. Plasma (0.5 mL) containing naproxen standard is acidified and extracted with hexane/ethyl acetate. The dried extracts, in mobile phase, are chromatographed on the 15 cm Chiralpak ADRH column (12 min run time) in methanol/water containing phosphoric acid at pH 2.0. The effluent is mixed with 4.5% ammonia solution before introduction into the ESIMS/MS instrument to form anions of the fatty acids. Quantitation is performed by MRM of the ion pair of the molecular anion (205 Da) and the major product ion (161 Da). Baseline resolution of the enantiomers is demonstrated with a detection limit of 1 ng/mL. Another important therapeutic agent which has been analyzed by LC/MS is valproic acid 19. This is an anticonvulsant used to control infantile spasms and prone to
over-use unless monitored. An LC-APCI-MS method for valproic acid 19 has been described [45]. Finally, the antibiotic mycophenolic acid 20, with a heptadecenoic acid side chain, can be analyzed by an LC/APCI-MS/MS method [46] and an LC/ESI-MS/MS method [47].
Summary The introduction of APCI and ESI interfaces, a decade ago, initiated the rapid development of alternative LC/MS methods for the analysis of carboxylic acids for most clinical applications. Only the routine analysis of long chain fatty acids in nutritional studies [1], and for the identification of bacterial strains [48], remains to be converted. Separation of iso- and anteiso- from n-isomer fatty acids can still only be achieved by high resolution capillary GC columns [9]. LC/MS is indispensable for the analysis of thermally and chemically unstable fatty acids using refrigerated columns, helium sparged solvents and appropriate buffers and solvents. The greater the complexity of the fatty acid, the more likely an LC/MS method will be developed. Additional substituents increase the probability of a significant product ion forming from the molecular cation or anion for use in sensitive MS/MS analysis. Best chromatographic separation of free fatty acids for all categories of carboxylic acids is achieved at acidic pH, either in ammonium acetate buffer or with small (0.025– 1%) concentrations of acetic, formic or trifluoroacetic acid. MS analysis of the molecular anions, on the other hand, is
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optimal in basic solution. Most methods opt for good separation at acidic pH with the loss of potential sensitivity. One successful countermeasure is post column addition of ammonia [44]. For some substituted and unsaturated fatty acids which afford a strong negative product ion from the molecular anion, sensitivity can be recovered by employing MS/MS analysis with its superior signal to noise. A significant strategy to improve the sensitivity of analysis of carboxylic acids by LC/MS is the use of chemical derivatization. This involves the esterification of the carboxylic acid with a charged group, or its precursor, that generates a strong ion in the APCI or ESI source. These derivatives include aminobenzenesulfonates [49], TMAE esters [10], DMAE esters [29] and pyridinium compounds [50]. They are all chromatographed and analyzed in acidic solution in positive ion mode and designed to undergo fragmentation, in high yield, during MS/MS analysis. Carboxylic acids that contain other functional groups, such as alcohols, offer multiple derivative combinations. Selective isomer analysis, as demonstrated with long chain hydroxy fatty acids [51], can be achieved with a clever combination of derivatives. As well as sensitivity advantages, derivatives thus fill the additional role of providing compound or even isomer specificity. A perfect derivative is one which eliminates the need to perform chromatographic separation. Most chromatography of free (unconjugated) carboxylic acids and derivatives is performed on standard reversed phase C8, C18 and CN columns and chiral columns for stereoisomer separation. There is a trend towards the use of short columns, such as guard columns, in clinical analysis. This is to improve the speed of analysis and the realization that baseline separation of all peaks is often unnecessary with the added selectivity that the mass spectrometer provides. In some cases, the column achieves no separation at all but functions to separate salts and interfering polar compounds that suppress signal intensity. Further advances in the LC/MS analysis of carboxylic acids for clinical use will continue to appear first in the wider fields of biological, environmental and forensic research. These include new HPLC columns [21], derivatives [50] and MS instruments [52]. Key steps in the biosynthesis of fatty acids involve stereoisomeric intermediates. The development of new chiral stationary phases [44] will result in the analysis of more biologically active fatty acids by chiral LC/MS. Increased availability of isotope labeled lipid standards and faster means of separating lipid classes will increase the range of carboxylic acids measured by LC/MS in intact lipids.
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Review
Contemporary clinical usage of LC/MS: Analysis of biologically important carboxylic acids David W. JohnsonT Department of Genetic Medicine, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia Received 16 April 2004; received in revised form 7 January 2005; 17 January 2005
Abstract Objectives: This review summarizes the current role of LC/MS in the diagnosis and screening of clinical conditions involving the analysis of biologically important carboxylic acids. Design and methods: Carboxylic acids are divided into six logical categories of acid size and function. Details of chromatographic separation methods and modes of mass spectrometer operation are described for each category. Results: The use of LC/MS in clinical applications such as the diagnosis of inherited and acquired metabolic disorders, gastrointestinal disorders, cancer and diabetes and therapeutic drug monitoring is discussed. Conclusions: The mild conditions, speed and sensitivity advantages of LC/MS analysis, over alternatives, are highlighted. The sensitivity and specificity afforded by the combination of tertiary and quaternary ammonium derivatives and tandem mass spectrometry for the analysis of carboxylic acids is emphasized. Potential for a greater range of LC/MS carboxylic analyses, including stereoisomeric intermediates, is predicted. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: LC/MS; Fatty acids; Organic acids; Bile acids; Eicosanoids; Therapeutic agents; Chemical derivatization
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . Long chain fatty acids . . . . . . . . . . . . . . . . Very long chain fatty and branched chain fatty acids. Organic acids . . . . . . . . . . . . . . . . . . . . . Bile acids . . . . . . . . . . . . . . . . . . . . . . . Prostaglandins, thromboxanes and leukotrienes. . . . Therapeutic agents . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: LC/MS, liquid chromatography/mass spectrometry; GC/MS, gas chromatography/mass spectrometry; APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; LCFA, long chain fatty acids; BCFA, branched chain fatty acids; VLCFA, very long chain fatty acids; DMAE, dimethylaminoethyl; TMAE, trimethylaminoethyl; NSAID, non-steroidal anti-inflammatory drug. T Fax: +61 8 81617100. E-mail address: [email protected]. 0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.01.007
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Introduction
Discussion
In biological systems, fatty acids exist in a dynamic equilibrium between the free form and incorporated within lipids as esters or amides. The fatty acids, as well, undergo both elongation to provide key biochemical substrates and oxidative degradation to provide cells with energy. Disruption of these processes in humans, resulting in an absence or an excess of particular fatty acids, is a key contributor to many clinical conditions. In clinical laboratories, fatty acids are analyzed to determine nutritional status, diagnose inherited disease, intestinal disorders, cancer and diabetes, to identify bacterial infection and for therapeutic drug monitoring. For many years, the principal methods of fatty acid analysis have been capillary column GC or GC/MS. Only capillary columns possessed the resolution necessary to separate the many isomeric forms of fatty acids. These include unsaturated isomers (conjugated and methylene interrupted), branched chain isomers, functional group positional isomers, cis/trans isomers (in double bond and ring structures), diastereoisomers and stereoisomers. Prior to GC/MS analysis, fatty acids are first released from lipids by chemical or enzymatic means. This is because lipids chromatograph poorly, if at all, by GC. Additionally, fatty acids are dispersed among a mixture of lipid species. Measuring total fatty acids from all species provides the best sensitivity for their analysis. The development of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) interfaces for coupling LC to MS and the diversity of LC packings, solvent mixtures and modifying agents now mean that LC/MS is a practical alternative to GC/MS for the analysis of fatty acids. Analysis of the free acid rather than lipid species is still frequently required, however, because of the complexity of separating individual lipid species. This review is a systematic discussion of the analyses of six categories of carboxylic acids, where LC/MS is employed, that involve clinical diagnosis or screening. The carboxylic acids of each of the six categories have a distinctly different size and/or biological function. The carboxylic acids have further been restricted to those which incorporate within lipid species which is a usual prerequisite for biological function. In addition to fatty acids, they include bile (steroidal) acids and diacids and hydroxyacids often referred to as organic acids. Excluded are amino acids, aromatic acids and heterocyclic acids. Examples of the abnormalities of carboxylic acid metabolism, and the clinical conditions which result, will be provided for each category of carboxylic acid. Details of the LC separation method and the mode of operation of the mass spectrometer will be outlined for each clinical application. Trends and future possibilities for LC/MS analysis of carboxylic acids in the clinical laboratory will be explored.
Long chain fatty acids Monocarboxylic acids, both saturated and unsaturated, with a carbon chain length between 14 and 22 are referred to as long chain fatty acids (LCFA). They constitute the majority of fatty acids found in the lipids of the human diet. The clinical analysis of these fatty acids is important for the diagnoses of essential fatty acid deficiency and mitochondrial fatty acid oxidation disorders. Essential fatty acid deficiency is caused by an inadequate or unbalanced dietary intake of lipids or by intestinal malabsorption [1]. Biochemical indicators are abnormally low linoleic and a-linolenic acids and an elevation in the triene/tetraene (eicosatrienoic/arachidonic acid) ratio. Mitochondrial fatty acid oxidation disorders, such as long chain hydroxyl CoA dehydrogenase deficiency (LCHAD) and very long chain acyl CoA dehydrogenase deficiency (VLCAD), result from an inherited dysfunction of mitochondria [2]. Biochemical indicators are elevated levels of tetradecenoic, tetradecadienoic and 3-hydroxytetradecanoic acids, mostly conjugated as their carnitine esters. Most recent methods for the analysis of this ubiquitous class of fatty acid employ GC or GC/MS after hydrolysis, extraction and derivatization of lipid material. Excellent sensitivity and separation have been achieved with capillary column electron capture negative ion mass spectrometry [3]. The use of LC/MS for the analysis of long chain fatty acids has mostly concentrated on profiling the fatty acids of intact lipid species [4,5]. This avoids the lengthy hydrolysis time and the need to derivatize. Lipid species such as sphingomyelin and the phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, etc) are already charged and ideal for ESI-MS analysis in either positive or negative ion mode. Practical quantitative methods, suitable for a clinical laboratory, for the analysis of LCFA by LC/MS, however, are not yet available. The measurement of intact lipid species has four major problems. (1) Isotope labeled fatty acids can be purchased for the GC/MS analysis of most free LCFA but few isotope labeled lipids are commercially available. ESI-MS and ESI-MS/MS are less tolerant of the use of related internal standards than detectors like ultraviolet, fluorescence and flame ionization. (2) There is often uncertainty in the identification of specific lipid species. The long chain base or alcohol of the lipid containing the fatty acid, as well as the fatty acid, exists in varying chain length, unsaturation and structural isomeric forms. A (C16:0 alcohol/C18:1 fatty acid) lipid is the same molecular weight as a (C18:1 alcohol/C16:0 branched fatty acid) lipid. (3) Unless prior separation of lipid classes is performed, it is difficult to avoid elevation in the apparent level of some specific lipid species by another class of lipids. (4) Those lipid classes most conveniently measured by LC/MS contain at most 10% of the total of a specific LCFA. This
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puts LC/MS at a sensitivity disadvantage compared with GC/MS methods which measure total LCFA. Very long chain fatty and branched chain fatty acids Very long chain fatty acids (VLCFA) are monocarboxylic acids with a carbon chain length greater than 22. Small amounts are obtained from the diet but in humans the majority is biosynthesized from shorter chain fatty acids. A competing oxidative degradation of VLCFA, to prevent their accumulation, occurs only within the subcellular organelle called the peroxisome. Another class of fatty acid exclusively degraded within the peroxisome is a- and h-branched chain fatty acids (BCFA). Large BCFA are not biosynthesized in humans but obtained from plant lipids usually via the ingestion of dairy products. Peroxisomal disorders which result from an absence or impaired function of peroxisomes [6] are thus characterized by elevated levels of VLCFA and/or BCFA in the tissues and body fluids. Two common blood screening tests to identify the majority of peroxisomal disorder patients are the measurement of the hexacosanoic (C26:0) acid to docosanoic (C22:0) acid ratio and the measurement of phytanic acid (C20:0 branched). VLCFA and the BCFA, pristanic acid 1 (see Fig. 1) and phytanic acid 2 are measured in many clinical laboratories by GC/MS as methyl, trimethylsilyl or for best sensitivity as pentafluorobenzyl esters [7]. The methods are time consuming (typically 1 to 1.5 days) and unsuitable for screening on small samples such as 3 mm blood spots on Guthrie card filter paper. LC/MS methods for the analysis of VLCFA and BCFA have only recently become available. Measurement of intact lipid species containing VLCFA has been unsuccessful because of trace levels (b1Amol/L) of hexacosanoic acid in normal plasma and its dilution among many lipid species. One LC/MS method [8] describes the analysis of VLCFA, after 2 h acid hydrolysis of lipid classes, in negative ion mode. Samples are injected into the flow from an HPLC starting at 20 AL/min and increasing to 50 AL/min over 2 min to achieve a sharp injection profile. Stable isotope labeled VLCFA standards are used for quantitation of the peak heights of the molecular anions during ESI-MS analysis. The method is
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faster (4 h) than GC/MS methods and shows good correlation for the analysis of controls, heterozygote and hemizygote Xlinked adrenoleukodystrophy patients (one of the most common forms of peroxisomal disorders) in plasma and cultured fibroblasts. The sample requirements (100 AL plasma) are similar to sensitive GC/MS methods. Anions of saturated fatty acids do not fragment to a product ion with any significant intensity so the sensitivity and specificity advantages of MS/MS analysis cannot be exploited. A second approach [9] also commences with acid hydrolysis of lipids. The isolated free fatty acids are derivatized as trimethylaminoethyl (TMAE) esters. These are quaternary ammonium derivatives with high molecular cation intensity that undergo fragmentation during positive ion ESI-MS/MS analysis to afford a dominant product ion from neutral loss of trimethylamine (59 Da) [10]. These derivatives are separated (22 min run time) using a reversed phase C8 column with acetonitrile/water at acidic pH. Stable isotope labeled VLCFA standards are used for quantitation. ESI-MS/MS analysis is by MRM of the ion pair of the molecular cation and the neutral loss of 59 Da product ion. Fig. 2 is a composite of 5 MRM ion chromatograms from analysis of the TMAE esters of VLCFA isolated from the plasma of a peroxisomal biogenesis defect patient. Baseline separation of the BCFA, pristanic acid (C19:0br) 1 and phytanic acid (C20:0br) 2 from their corresponding straight chain isomers can be seen. Excellent correlations with GC/MS for pristanic acid 1 and phytanic acid 2 and the C26:0/C22:0 ratios for a variety of peroxisomal disorders were demonstrated. Because of the extra sensitivity afforded by the use of a derivative only 10 AL of plasma is required, which can be reduced further by the use of newer MS/MS instruments. Organic acids Organic acids include short to medium chain mono- and dicarboxylic acids and hydroxylated analogs with a carbon chain length up to 12. All large metabolic laboratories in pediatric hospitals perform an organic acid screen on urine samples from children with symptoms indicative of organic
Fig. 1. Branched chain fatty acids and organic acids: (1) pristanic acid, (2) phytanic acid, (3) 3-hydroxyglutaric acid, (4) dibutyl dimethylglycine derivative of 3-hydroxyglutaric acid.
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Fig. 2. Composite ion chromatogram from ESI-MS/MS analysis of the TMAE derivatives of the fatty acids isolated from the plasma of a peroxisomal biogenesis defect patient. The composite is of five ion traces from 59 Da neutral loss MRM transitions for C19:0, C20:0, C22:0, C24:0 and C26:0 fatty acids. C19:0br is pristanic acid and C20:0br is phytanic acid.
acidurias or for any unexplained symptom. Many of these disorders are now detected by the newborn blood spot screening test for acylcarnitines [11] but there are some which can only be detected by this screening method. The most common method of analysis of organic acids is by GC/MS of the trimethylsilyl esters of the ethyl acetate extract of acidified urine. One of the earliest reports of LC/MS analysis of organic acids used a plasmaspray interface [12]. A related thermospray interface [13] was used for the analysis of key organic acids methylmalonic, 3-hydroxy-3-methylglutaric, propionic, isovaleric and arginosuccinic acids. Two methods of LC separation were described. One employed an HPX-87 H column at acidic pH for good separation and the other a C18 reversed phase column in ammonium acetate solution at neutral pH for better sensitivity. This was followed by the use of an APCI source and the analysis of organic acids in negative ion mode [14]. Methylmalonic acid is an organic acid which is a diagnostic metabolite for a group of inherited disorders, with at least eight variants, called methylmalonic acidemias. Methylmalonic acid is also elevated in the urine or plasma of patients with Vitamin B12 (cobalamin) deficiency as a consequence of poor intestinal absorption, digestion and diet. Renal insufficiency, hypovolemia and bacterial overgrowth in the small intestine can also cause abnormal levels of methylmalonic acid. Two recent papers from clinical laboratories describe the LC/MS analysis of methylmalonic acid.
In the first paper [15], urine or plasma containing d3methylmalonic acid is extracted with an automated SPE sample processor and derivatized with butanol/hydrogen chloride. The butyl esters are chromatographed (3 min run time) on a 3.3 cm C18 reversed phase column and analyzed by ESI-MS/MS. A comparison between this fully automated LC/MS method and a GC/MS method shows excellent correlation ( y = 0.95 + 0.51, R 2 = 0.998) and improvements in cost, labor and instrument time. The second paper [16] describes a simpler and faster technique for measuring methylmalonic acid. Urine or plasma containing d3-methylmalonic acid is extracted with methyl tert-butyl ether and derivatized with butanol/hydrogen chloride. The butyl esters are passed through (1 min run time) a 3.0 cm C18 reversed phase column and analyzed by ESI-MS/MS. A computer algorithm is used to compensate for succinic acid present in the sample. 10% of samples are needed to be reanalyzed by a different method because of the presence of excessive succinic acid. Disappointingly, the sample volume of plasma required for LC/MS analysis of methylmalonic acid by both these methods (typically 0.5 mL) is greater than that for sensitive GC/MS methods. This is in part due to the volatility of the dibutyl ester which makes selective evaporation of butanol, after derivatization, difficult. Additionally, butyl esters are poorly ionized by ESI in comparison with derivatives that are charged in aqueous solution. The use of a quaternary ammonium derivative, to provide greater sensitivity, is not possible with
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Fig. 3. Product ion spectra of the protonated molecular ions of the dibutyl, dimethylglycine derivatives of (top) 3-hydroxyglutaric acid and (bottom) 2-hydroxyglutaric acid.
dicarboxylic acids. Doubly charged binary derivatives of small molecules are not observed during ESI-MS analysis [10]. The triple selectivity provided by chromatographic separation, group specific derivatization and unique sets of
ion pairs in LC/ESI-MS/MS analysis has a practical application to the crowded spectrum of organic acids. 3hydroxyglutaric acid 3 is an organic acid which is regarded as the best diagnostic metabolite for glutaric aciduria type I
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[17]. Its measurement by GC/MS requires a large sample (0.5 mL plasma) and is complicated by its co-elution with the more common 2-hydroxyglutaric acid on the majority of capillary columns. An LC/ESI-MS/MS solution to this problem has been found (author, unpublished work). A systematic evaluation of derivatives afforded the dibutyl dimethylglycine derivative of 3-hydroxyglutaric acid 4 from butylation of the carboxylic acid groups and acylation of the alcohol with dimethylglycine [18]. Derivative 4 gives a strong protonated molecular ion intensity and is distinguishable from its 2-hydroxy isomer using unique product ions during ESI-MS/MS analysis. The product ion spectra of (top) the 3-hydroxy isomer (unique ion at m/z 104) and (bottom) the 2-hydroxy isomer (unique ion at m/z 131) are shown in Fig. 3. Direct ESI-MS/MS analysis of 3-hydroxyglutaric acid is prevented by interference from ions generated by derivatized glutamic acid. The interference can, however, be removed by HPLC separation thus providing an LC/ESIMS/MS method for the analysis of 3-hydroxyglutaric acid 3. The analysis of fatty acids from C2 to C18 as acylcarnitines, by ESI-MS/MS, is routinely performed in newborn screening centers for the diagnosis of inherited disorders of fatty acid metabolism [11]. There is only one recent report of an LC/ESI-MS/MS method for the analysis of eight acylcarnitines in urine [19]. The method employs chromatography of underivatized acylcarnitines on a reversed phase C8 column (runtime 17 min) with methanol/water containing an ion pairing reagent. Quantitation is carried out in MS/MS mode with an ion trap. This method allows the separation of acylcarnitines containing isomeric acyl groups such as butyryl and isobutyryl. A popular method of organic acid analysis used by biological and environmental laboratories but not yet by
clinical laboratories is LC/MS using ion exclusion separation [20,21]. Bile acids Bile acids are fatty acids attached to a steroidal backbone. They are biosynthesized from cholesterol 5 as shown in the abbreviated scheme in Fig. 4. N-Oxidation and hydroxylation give C27 bile acids 6 (di- and trihydroxycholestanoic acids) which are metabolized within peroxisomes to the more abundant C24 bile acids 7 (chenodeoxycholic and cholic acids). Hydroxylation is common as is conjugation with glycine, taurine, sulfate or glucuronide. Further degradation through transient C22 bile acids 8, C21 and C20 bile acids then occurs. Their physiological functions include lipid absorption, mediation of cholesterol synthesis and clearance of drugs, vitamins and toxins. Consequently, they are important indicators of hepatobiliary and intestinal function/dysfunction. Quantitative analysis of bile acids by GC/MS is time consuming. It requires extraction, purification, conjugate hydrolysis and derivatization to generate volatile analytes of these large, polar molecules. A comprehensive analysis requires separation and analysis of the bile acid composition of each of the conjugated (and free) species. The advantages of being able to analyze conjugated species without lengthy physical separation and hydrolysis, offered by LC/MS, have long been recognized. Early analysis of bile acids by LC/MS involved the use of a thermospray interface [22]. Improved sensitivity was obtained with the introduction of MS/MS instruments [23] and then with ESI [24]. Much effort has been focused on a screening test for cholestatic hepatobiliary disease which is
Fig. 4. Abbreviated scheme showing the metabolism of cholesterol to bile acids.
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one of the causes of jaundice in newborns. Long-term survival of these infants is significantly improved if surgery to correct it is performed within 60 days. A screening test for the measurement of glycine and taurine conjugates of C24 bile acids by direct ESI-MS/MS analysis, in neonatal blood spots, was developed [25]. The test unfortunately affords a high number of false positives. A problem with direct ESI-MS/MS analysis of bile acids is the presence of isomeric forms of the di- and trihydroxy C24 bile acids. An LC/ESI-MS/MS method [26] was developed for their separation. Plasma (50 AL) is deproteinized and chromatographed on a 5 cm C18 reversed phase column (run time not given) with methanol/water. The eluent is analyzed by an ESI-MS/MS instrument in negative ion MRM mode for glycine and taurine conjugates using isotope labeled internal standards. Unconjugated bile acids are detected using selected ion monitoring of the molecular anions. Several other groups described similar LC/ESI-MS/MS methods for the analysis of C24 bile acids. One group [27] uses a C18 column (run time 4 min) and elution with acetonitrile/water. There is good correlation with GC/MS methods. The other group [28] uses a 10 cm C18 reversed phase column (run time 20 min) with a three step binary gradient of methanol/water at acidic pH. LC/MS methods have also been described for the analysis of C27 bile acids. These bile acids are found in very low levels in the blood of normal people because once formed they are immediately metabolized in the peroxisome. In patients with peroxisomal biogenesis defects, they accumulate in the blood. Unlike C24 bile acids, the majority of the C27 bile acids are found in the unconjugated form. It is difficult to detect unconjugated bile acids, in either positive or negative ion mode, in small samples such as 3 mm blood spots which are the preferred samples for screening for inherited disorders. This led to the development of a specific double derivative, the acetoxy dimethylaminoethyl (DMAE) ester for very sensitive ESI-MS/MS analysis of unconjugated bile acids [29]. No chromatography is required for the analysis of di- and trihydroxy C27 bile acids, by this method, because of the specificity achieved by analyzing the product ion from neutral loss of 60 Da. An LC/ESI-MS/MS method for sensitive analysis of chromatographically separated isomeric bile acids could be developed from this, analogous to the separation of TMAE derivatives of VLCFA [9]. The corresponding acetoxy TMAE derivative of bile acids although affording greater ion intensity during ESI-MS analysis is too stable and does not fragment to an abundant product ion suitable for MS/MS analysis. If LC separation is employed, then this derivative could be used for LC/MS analysis of unconjugated bile acids. An LC/MS method has been developed to separate and measure the (25R)- and (25S)-isomers of the unconjugated and taurine conjugates of C27 bile acids for the diagnosis of a-methylacyl CoA racemase deficiency [30]. a-Methylacyl CoA racemase is a peroxisomal enzyme that catalyses the conversion of (2R)-methyl branched chain fatty acids to
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their (2S)-isomers which can then undergo a-oxidation. aMethylacyl CoA racemase deficiency is a rare peroxisomal disorder that results in an accumulation of the (25R)-isomers of C27 bile acids. For the analysis of the isomers, plasma (50 AL) containing isotope labeled C24 bile acid standards is deproteinized and chromatographed on a 25 cm C18 reversed phase column (22 min run time) in methanol/water. The eluent is sprayed into an ESI-MS/MS instrument. Taurine conjugates are measured by MRM experiments and unconjugated bile acids by single ion monitoring. Patients with a-methylacyl CoA racemase deficiency show an elevation in (25R)-isomers while patients with Zellweger syndrome (a peroxisomal biogenesis defect) show an elevation in both (25R) and (25S) isomers. Baseline separation of the (25R)- and (25S)isomers of the taurine conjugates was demonstrated. In common with the difficulty separating underivatized fatty acids by HPLC, the resolution of the unconjugated isomers is only partial. Prostaglandins, thromboxanes and leukotrienes These three related classes of compounds are substituted C20 fatty acids containing a cyclopentane ring (prostaglandins), tetrahydropyran ring (thromboxanes) and a conjugated triene (leukotrienes). The structures of some are shown in Fig. 5. They are formed biosynthetically from C20 (eicosanoic) essential fatty acid precursors containing 3 to 5 methylene interrupted double bonds. They cause profound physiological effects in mammalian systems especially on smooth muscle. There is a great deal of research probing the identities and functions of these eicosanoids in diverse areas of arthritis, diabetes, endocrinology and cancer. The multiple sites of hydroxylation and unsaturation and complex thioesters in some of these molecules make them chemically and thermally unstable. Despite this, GC/MS has been a powerful tool for their analysis [31]. Prostaglandins have also been analyzed by HPLC with UV detection for over two decades [32,33]. These acids were found to be better ionized by ESI than saturated fatty acids and sensitive LC/ESI-MS and LC/ESI-MS/MS analyses have developed. Prostaglandin E2 9 and 12-hydroxyeicosatetraenoic acid (12-HETE) 10 are important mediators in inflammatory skin disorders. An LC/MS method for their analysis in human dermal fibroblast supernatants was developed [34]. Supernatant is purified on C18 SPE columns and chromatographed on a 15 cm C18 reversed phase column (run time 22 min) with acetonitrile/water at acidic pH. Quantitation is by ESI-MS of the molecular anions using isotope labeled standards. 5,12 and 15-HETE isomers, PGE2 9 and arachidonic acid 11 are separated, with detection limits in the range 5 to 20 pg. A similar method for the analysis of these compounds as well as leukotriene B4 12 in small tissue biopsies has also been described [35]. Four classes of isoprostanes (free radical catalyzed prostaglandin isomers) have been analyzed in urine samples
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Fig. 5. Eicosanoid fatty acids: (9) PGE2, (10) 12-HETE, (11) arachidonic acid, (12) LTB4, (13) LTE4, (14) THB2.
by LC-ESI-MS/MS [36]. They are regarded as indicators of oxidant stress. Urine samples are purified on BondElut LMS polymer cartridges and chromatographed on a 15 cm C18 reversed phase column (run time 22 min) with acetonitrile/ methanol/water at pH 5.7. Quantitation is by ESI-MS/MS using MRM ion pairs unique to each class of isoprostane. Levels of all classes of isoprostanes are elevated in patients with hypercholesterolemia compared to controls. Only some of the isoprostanes are elevated in patients with congestive heart failure. The majority of recent LC/MS analyses for eicosanoids have employed almost identical chromatography and MS/MS measurement methods. These include the analysis of leukotriene E4 13 in the urine of patients with arthritis [37,38], thromboxane B2 14 in cultured synovial cell media [39] and 2- and 3-series prostaglandins in cultured tumor cells [40]. A novel LC/ESI-MS/MS application for the analysis of prostanoids is the chiral analysis of (12S)-HETE [41]. 12HETE 10 is thought to contribute to the pathogenesis of diabetic cardiovascular disease, renal dysfunction and destruction of pancreas cells. Urine is purified on C18 cartridge and chromatographed on 7.5 cm C18 reversed phase column with acetonitrile/water at acid pH. The fraction containing 12-HETE 10 is switched to a 25 cm chiral CD-Ph column between 6.5 and 10.5 min after injection. Column effluent from the chiral column is introduced into the ESI-MS/MS instrument between 40 and 65 min after injection. Quantitative analysis employs an octadeuterated standard and MRM of the ion pair of the
molecular anion and its major product ion. A significant difference is observed in the levels of (12S)-HETE in female patients with diabetes mellitus compared with controls. Diabetic males are indistinguishable from controls indicating a sex difference in 12-HETE 10 production. Therapeutic agents Many therapeutic agents which are routinely quantitated in clinical laboratories contain a fatty acid side chain. The most important group is the non-steroidal anti-inflammatory drugs (NSAIDs) whose major action is to inhibit prostaglandin synthesis. Ibuprofen 15 (Fig. 6), indomethacin 16, diclofenac 17 and ketoprofen 18 are commonly prescribed NSAIDs. Many methods have been developed for clinical studies of the dose/response of NSAIDs and for drug quality control purposes. Despite LC/MS cost and complexity, it provides low levels of detection with high specificity in short time periods. The power of LC/ESI-MS/MS for therapeutic drug monitoring is demonstrated with a quantitative method for indomethacin 16 [42]. Plasma samples (100 AL) containing mefenamic acid standard are purified with a solid phase extractor and chromatographed on a C8 column (4 min run time) in methanol/ammonium acetate buffer at pH 5.1. The indomethacin 16 is analyzed by ESI-MS/MS with an MRM of the protonated molecular ion and the major product ion (139 Da). Linearity is demonstrated in the range 5–2000 Ag/L. The separation and quantitation of four NSAIDs is achieved with an LC-APCI-MS method [43]. The drugs are injected onto a 15 cm CN column and eluted with
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Fig. 6. Therapeutic drugs containing fatty acid side chains: (15) ibuprofen, (16) indomethacin, (17) diclofenac, (18) ketoprofen, (19) valproic acid, (20) mycophenolic acid.
acetonitrile/ammonium acetate at pH 7.4. The molecular anions of the drugs are measured by APCI-MS in single ion monitoring mode. An elegant demonstration of the power of LC/MS analysis is the enanioselective determination of ibuprofen 15 [44]. Although ibuprofen 15 is sold as a racemate, its anti-inflammatory effect comes from the (+)-(S)-enantiomer. Only (2S)-isomers of a-methyl branched fatty acids can be degraded by h-oxidation. Accordingly, HPLC and capillary electrophoresis methods were developed for its enantioselective analysis. Amylose and cellulose derivatives have shown the greatest resolving power for HPLC analysis under both normal and reversed phase conditions. The authors use a new amylose based chiral column Chiralpak AD-RH (a tris-(3,5-dimethylphenylcarbamate)amylose) in their LC/MS method. Plasma (0.5 mL) containing naproxen standard is acidified and extracted with hexane/ethyl acetate. The dried extracts, in mobile phase, are chromatographed on the 15 cm Chiralpak ADRH column (12 min run time) in methanol/water containing phosphoric acid at pH 2.0. The effluent is mixed with 4.5% ammonia solution before introduction into the ESIMS/MS instrument to form anions of the fatty acids. Quantitation is performed by MRM of the ion pair of the molecular anion (205 Da) and the major product ion (161 Da). Baseline resolution of the enantiomers is demonstrated with a detection limit of 1 ng/mL. Another important therapeutic agent which has been analyzed by LC/MS is valproic acid 19. This is an anticonvulsant used to control infantile spasms and prone to
over-use unless monitored. An LC-APCI-MS method for valproic acid 19 has been described [45]. Finally, the antibiotic mycophenolic acid 20, with a heptadecenoic acid side chain, can be analyzed by an LC/APCI-MS/MS method [46] and an LC/ESI-MS/MS method [47].
Summary The introduction of APCI and ESI interfaces, a decade ago, initiated the rapid development of alternative LC/MS methods for the analysis of carboxylic acids for most clinical applications. Only the routine analysis of long chain fatty acids in nutritional studies [1], and for the identification of bacterial strains [48], remains to be converted. Separation of iso- and anteiso- from n-isomer fatty acids can still only be achieved by high resolution capillary GC columns [9]. LC/MS is indispensable for the analysis of thermally and chemically unstable fatty acids using refrigerated columns, helium sparged solvents and appropriate buffers and solvents. The greater the complexity of the fatty acid, the more likely an LC/MS method will be developed. Additional substituents increase the probability of a significant product ion forming from the molecular cation or anion for use in sensitive MS/MS analysis. Best chromatographic separation of free fatty acids for all categories of carboxylic acids is achieved at acidic pH, either in ammonium acetate buffer or with small (0.025– 1%) concentrations of acetic, formic or trifluoroacetic acid. MS analysis of the molecular anions, on the other hand, is
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optimal in basic solution. Most methods opt for good separation at acidic pH with the loss of potential sensitivity. One successful countermeasure is post column addition of ammonia [44]. For some substituted and unsaturated fatty acids which afford a strong negative product ion from the molecular anion, sensitivity can be recovered by employing MS/MS analysis with its superior signal to noise. A significant strategy to improve the sensitivity of analysis of carboxylic acids by LC/MS is the use of chemical derivatization. This involves the esterification of the carboxylic acid with a charged group, or its precursor, that generates a strong ion in the APCI or ESI source. These derivatives include aminobenzenesulfonates [49], TMAE esters [10], DMAE esters [29] and pyridinium compounds [50]. They are all chromatographed and analyzed in acidic solution in positive ion mode and designed to undergo fragmentation, in high yield, during MS/MS analysis. Carboxylic acids that contain other functional groups, such as alcohols, offer multiple derivative combinations. Selective isomer analysis, as demonstrated with long chain hydroxy fatty acids [51], can be achieved with a clever combination of derivatives. As well as sensitivity advantages, derivatives thus fill the additional role of providing compound or even isomer specificity. A perfect derivative is one which eliminates the need to perform chromatographic separation. Most chromatography of free (unconjugated) carboxylic acids and derivatives is performed on standard reversed phase C8, C18 and CN columns and chiral columns for stereoisomer separation. There is a trend towards the use of short columns, such as guard columns, in clinical analysis. This is to improve the speed of analysis and the realization that baseline separation of all peaks is often unnecessary with the added selectivity that the mass spectrometer provides. In some cases, the column achieves no separation at all but functions to separate salts and interfering polar compounds that suppress signal intensity. Further advances in the LC/MS analysis of carboxylic acids for clinical use will continue to appear first in the wider fields of biological, environmental and forensic research. These include new HPLC columns [21], derivatives [50] and MS instruments [52]. Key steps in the biosynthesis of fatty acids involve stereoisomeric intermediates. The development of new chiral stationary phases [44] will result in the analysis of more biologically active fatty acids by chiral LC/MS. Increased availability of isotope labeled lipid standards and faster means of separating lipid classes will increase the range of carboxylic acids measured by LC/MS in intact lipids.
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