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Articles |
1
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108.
2
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132.
3
MDS SCIEX, Concord, ON, L4K 4V8 Canada.
aAuthor for correspondence. Fax 801-584-5207; e-mail kushnmm{at}aruplab.com.
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Abstract |
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Methods: Dicarboxylic acids were extracted from samples with methyl-tert-butyl ether and derivatized with butanolic HCl to form dibutyl esters. The derivative was injected into the liquid chromatography (LC)-MS/MS system using TurboIonSprayTM (nebulizer-assisted electrospray) ionization and quantified by the multiple reaction monitoring mode of MS/MS.
Results: The assay for MMA was linear up to 150 µmol/L. The total imprecision was 
7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 µmol/L, respectively. The degree of interference from SA could be predicted from the branching ratios of the major product ions.
Conclusions: The method is specific for dicarboxylic acids. The LC-MS/MS analysis for MMA requires minimal chromatographic separation and takes <60 s per sample. The entire analysis, including sample preparation, for a batch of 100 specimens can be performed in <4 h.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although either serum MMA or serum vitamin B12 can be used for detecting vitamin B12 deficiency, there are advantages to measuring MMA instead of vitamin B12. This is related to the facts that (a) serum or plasma vitamin B12 concentrations may not adequately reflect tissue vitamin B12 status, (b) MMA is more stable than vitamin B12, (c) serum MMA concentrations are 1000-fold greater than serum vitamin B12 and therefore easier to measure accurately, and (d) an increase rather than a decrease in concentration is measured in vitamin B12 deficiency. Since the 1960s, efforts have been directed toward developing a rapid, simple, sensitive, and specific method for MMA analysis in biological fluids. Sample preparation for the analysis of MMA usually consists of extraction from the sample matrix and, frequently, subsequent derivatization. To be able to detect vitamin B12 deficiency, the method should be able to accurately measure low concentrations of MMA (
1 µmol/L in urine and 0.1 µmol/L in serum). Derivatization of MMA is needed to improve MMA detection with an ultraviolet or a fluorescence detector in HPLC procedures (6)(7)(8)(9) or to convert it to a derivative amenable to gas chromatography (GC) separation and detection (10)(11)(12)(13)(14)(15)(16).
The major obstacle for MMA analysis in biological fluids is the potential interference from other low-molecular weight organic acids and especially from the naturally occurring structurally related isomer SA, which is a product of MMA degradation and is usually present in samples at a concentration greater than that of MMA. SA interference is difficult to overcome because the chromatographic characteristics and mass spectra of SA are almost identical to those of MMA.
MMA is usually analyzed by GC–mass spectrometry (MS) in the selected-ion monitoring mode. All derivatizing reagents used for GC-MS analysis of MMA produce nearly identical mass spectra for MMA and SA derivatives; thus, for the quantification of MMA, adequate chromatographic separation between the peaks is required. The major disadvantage associated with GC-MS methods in general is relatively low instrument throughput (usually three to six samples per hour). There is, therefore, a need for a high-throughput method of determining MMA in biological samples to effectively detect vitamin B12 deficiency and inherited disorders associated with increased MMA.
Different derivatives can be used to enhance selectivity and specificity to organic acids for liquid chromatography (LC)-MS analysis. Recent work by Johnson (17) involved the determination of long- and very long-chain fatty acids by nebulizer-assisted electrospray (ES). The fatty acids were derivatized to form dimethylaminoethyl esters via a two-step condensation reaction using oxalyl chloride and dimethylaminoethanol as reagents. The reagent produces a strongly basic derivative, which enhances the response of all organic acids in the positive-ion mode without any differences in fragmentation between the structural isomers MMA and SA.
One of the clinically important groups of organic acids (of which MMA and SA are representatives) is dicarboxylic acids. The dicarboxylic acids usually are analyzed by chromatographic methods along with other organic acids. The difficulty in the target analysis for these and other low-molecular weight organic acids relates to potential interference from other acids caused by similar properties that do not allow selective extraction and detection. The analysis usually requires extended instrument run time because of the need to chromatographically separate the acids from each other and to condition the chromatographic column before the following injection to elute other coextracted compounds. To date, there is no method described in the literature that is selective only for dicarboxylic acids.
Recently, Magera et al. (18) published a method for the determination of MMA as the n-butyl ester derivative in plasma and urine by LC–tandem MS (MS/MS). The method is based on chromatographic separation of MMA from SA and selective fragmentation of MMA butyl ester. The analysis time per sample was 3 min, which is significantly faster than conventional GC-MS methods. Among the disadvantages of the method are time-consuming sample preparation involving solid-phase extraction, which includes a two-step separation of the extract from a residue produced during the extraction, and the relatively narrow linear range of the method.
The intent of this work was to develop a rapid method for the selective analysis of dicarboxylic acids (and MMA in particular) by LC-MS/MS, based on the power of the MS/MS analyzer to differentiate between MMA and SA, that does not require chromatographic separation of these isomeric analytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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apparatus
A PE Series 200 HPLC system (Perkin-Elmer Analytical Instruments) was equipped with a Luna C18 column (30 mm x 3.0 mm; 3-µm particles; Phenomenex). The mobile phase consisted of 850 mL/L methanol and 150 mL/L ammonium formate buffer (0.005 mol/L), pH 6.5. The mobile phase flow rate was 750 µL/min, and the LC column effluent split flow was 500–600 µL/min. The column temperature was 40 °C, the injection volume was 3 µL, and the injection interval was 60 s. An API 2000 (Applied Biosystems/MDS SCIEX, Foster City, CA) tandem mass spectrometer was used in the positive-ion mode with TurboIonSprayTM (TIS) interface. Quantitative analysis was performed in the multiple reaction monitoring (MRM) mode. The collision gas was nitrogen with a cell pressure of 1.1 Pa. The TIS capillary voltage was 6.0 kV, the orifice voltage was 21V, and the collision energy was 15 V. The MRM transitions monitored were m/z 231
119 and 231175 for MMA, and 234122 and 234178 for d3-MMA. The product ions m/z 119 and 122 were quantitative, whereas the product ions m/z 175 and 178 were qualitative. Quantitative data analysis was performed with TurboQuanTM (Applied Biosystems/MDS SCIEX) software.
assay procedures
Preparation of calibrators and controls.
Dialyzed plasma was prepared from a pool of human plasma and showed no detectable MMA and SA. Samples for method precision, sensitivity, and linearity studies were prepared in dialyzed human plasma and stored at 2–8 °C. Dialyzed plasma used for the preparation of calibrators and quality-control samples was supplemented with SA to a concentration of 6 µmol/L. Calibrators were at concentrations of 0.2, 0.4, 0.75, 1.0, 1.5, and 2.0 µmol/L. The calibrator containing 0.4 µmol/L MMA was used to establish the qualitative ion mass ratio of the intensities of the product ion fragments m/z 231175/231119 of MMA and m/z 234178/234122 of d3-MMA. The qualitative ion mass ratio acceptability limits for the controls and test samples were established as ± 40% of the values observed in the calibrator.
Organic acid ester derivatives.
The n-butyl esters of organic acids for qualitative mass spectral measurements were prepared by transferring 400 nmol of each acid into glass tubes. The solvent was evaporated and the residue reconstituted with 40 µL of n-butanol containing 3 mol/L HCl. The tubes were incubated at 60 °C for 15 min. Excess derivatizing reagent was evaporated, and the remaining residue was reconstituted with 4 mL of methanol containing 50 mL/L ammonium formate (0.005 mol/L), pH 6.5. For both the MS and MS/MS experiments, the ion source and analyzer conditions were the same as those used for the MMA n-butyl ester derivative. The samples were infused by syringe at a flow rate of 5 µL/min into the TIS ion source.
Relative ionization efficiencies and fragmentation patterns were evaluated for the methyl, propyl, isopropyl, and amyl esters of MMA and SA. The various esters of MMA and SA were prepared by transferring 400 nmol of each acid into glass tubes. The solvent was evaporated, and the residues were reconstituted with 100 µL of the corresponding alcohols and 50 µL of concentrated sulfuric acid. The tubes were incubated at 60 °C for 15 min. The esters were extracted from the mixture with hexane, the tubes were centrifuged, and the organic layers were transferred into new tubes. The solvent was evaporated, and the residues were reconstituted with 4 mL of methanol containing 50 mL/L ammonium formate (0.005 mol/L).
sample preparation
Samples were aliquoted into disposable glass tubes (1 mL for serum/plasma analysis, and 0.1 mL of sample and 0.9 mL of water for urine analysis). To this, 100 µL of the working internal standard solution and 3 mL of MTBE containing 30 mL/L phosphoric acid were added. The tubes were then vortex-mixed for 5 min and centrifuged at 3000g for 10 min. The supernatant was transferred to a second set of tubes, the solvent was evaporated, and 40 µL of n-butanol containing 3 mol/L HCl was added. The mixture was incubated at 50 °C for 5 min. The excess derivatizing reagent was evaporated, and the residues were reconstituted with 75 µL of a mixture of methanol–0.005 mol/L ammonium formate (1:1 by volume) and transferred to labeled autosampler vials.
recovery studies
The experiments to evaluate an absolute extraction recovery were performed with human plasma samples containing MMA at concentrations of 0.4 and 4 µmol/L. The internal standard was added to the first group of samples before extraction, whereas for the second group, the internal standard was added into the extract. The dried extracts were derivatized according to the same procedure and analyzed at the same time. The percentage of recovery was determined by comparing the MMA concentration in the samples to which the internal standard was added after extraction to the results obtained for the samples to which the internal standard was added before extraction.
precision, linearity, and sensitivity studies
Method precision was determined by analyzing three replicates per day of plasma samples containing MMA at concentrations of 0.3, 0.75, and 1.2 µmol/L over a 5-day period. In addition, method precision was determined from the control values observed during routine use of the method (concentrations of 0.3 and 1.0 µmol/L in duplicate within 15 days). Instrument imprecision was determined by repetitive injections of an extracted sample containing 0.4 µmol/L MMA from the same vial.
Linearity was evaluated by analyzing supplemented samples prepared at 1, 50, 100, 125, 150, 200, and 250 µmol/L. Method sensitivity was determined by analyzing supplemented samples containing progressively lower concentrations of MMA. We used a criterion of maintaining accuracy within ± 15%, imprecision (CV) <10%, and a qualitative ion ratio within ± 40% of the value set by the calibration to determine the upper limit of linearity and limit of quantification for the assay. Each sample was analyzed in duplicate over a 2-day period.
patient sample comparison studies
A total of 591 samples were included in the correlation study. A group of 211 samples was analyzed with the in-house GC-MS assay (15): 182 serum and plasma samples from patients, 13 urine samples from patients, and 48 MMA-supplemented samples in dialyzed plasma. A group of 380 serum and plasma samples from patients, with concentrations ranging from 0.1 to 3.5 µmol/L, were correlated to the in-house LC-MS/MS assay similar to one described by Magera et al. (18) that used chromatographic separation of MMA and SA. Sample preparation for the LC-MS/MS procedure was the same as for the evaluated method. The analysis was performed on the same LC column with a mobile phase consisting of 700 mL/L methanol and 300 mL/L ammonium formate buffer (0.005 mol/L), pH 6.5. The MMA-supplemented samples included in the study were used because the number of patient samples with MMA concentrations >10 µmol/L was not sufficient for the correlation. To account for bias in both the reference and the evaluated methods, the results were analyzed by Deming regression (19).
interference studies
Interference was evaluated by analyzing n-butyl esters of clinically important organic acids. The total number of acids included in the study was 77, with a concentration of 10 000 µmol/L for each individual acid. The esterified acids were analyzed by the evaluated method in MRM and product-ion scan (with precursor ion m/z 231) modes.
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Results |
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The fragmentation of various dicarboxylic acids was investigated to determine whether differences exist among the dicarboxylic acids and between the isomers, such as MMA and SA. The MS/MS product ion spectra for MMA and SA n-butyl ester [M + H]+ molecular ions are presented in Fig. 2, C and D
. In addition to n-butyl esters, several other alkyl esters were evaluated for relative ionization efficiencies, fragmentation patterns, and the uniqueness of the product ion m/z 119 of MMA relative to SA (Fig. 3
).
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Because the detection is specific to dicarboxylic acids, LC separation plays a lesser role in the method. A short LC column was used to separate compounds from the solvent front to eliminate possible signal suppression. The chromatograms for the monitored mass ion transitions for the extracted plasma control containing 0.4 µmol/L MMA and 1.5 µmol/L d3-MMA internal standard are presented in Fig. 4
. The calibrator containing MMA at 0.4 µmol/L and SA at 6 µmol/L was used to set a threshold for the branching ratio for the positive samples. The acceptance limit for the branching ratio was established in every run as ± 40% of the value observed in the 0.4 µmol/L calibrator.
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performance characteristics
The absolute extraction recovery of MMA for the method was 77% ± 11%. The values obtained for within-run, between-run, and total imprecision for the results in the experiments for the precision study along with between-run precision determined from the control values observed over 3 weeks for the method in routine testing are presented in Table 1
. The CV for instrument imprecision was determined by re-injecting a sample from the same vial (n = 10) and was 0.9%. The assay was linear up to 150 µmol/L (r2 = 0.998). The linear regression equation at concentrations of 0.1–1.2 µmol/L was: y = 0.997x + 0.012 (r2 = 1.000). The limit of quantification for the method was 0.1 µmol/L with an observed accuracy of 98.5% and imprecision of 7%. The limit of detection was 0.05 µmol/L.
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method comparison
Comparison with the in-house GC-MS method (15) for serum, plasma, and urine samples (Fig. 5
) showed close concordance over the entire range of evaluated concentrations. In addition to the correlation with the GC-MS test, method performance was evaluated relative to a LC-MS/MS method that chromatographically resolved SA and MMA in a way similar to a previously described method (18). Samples included in the study contained MMA at concentrations corresponding to the general population and slightly increased concentrations, characteristic of vitamin B12 deficiency (Fig. 6
). No significant bias was seen between the methods for the serum/plasma and urine samples. Of the 380 samples included in the correlation with the LC-MS/MS method, the m/z 175/119 ion ratio was outside of the acceptable range in 41 samples (10.8%) with MMA concentrations 
0.4 µmol/L.
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An interference study showed that the only acid potentially causing interference with MMA analysis was SA. SA, when present at a concentration of 20 µmol/L, produced a signal equivalent to 0.4 µmol/L MMA with a significantly increased qualitative ion mass ratio (>2, when <0.75 is expected for MMA). Hemolysis did not interfere with the LC-MS/MS assay, whereas lipemic samples produced a substantial amount of precipitate in the final solution. Commonly used serum collection tubes (EDTA, heparin, oxalate, and citrate) were evaluated for compatibility with the method. Interference was observed only with the citrate collection tube. Citric acid at physiologic concentrations did not interfere with MMA, but when present in the collection tube, it interfered with the m/z 175 qualitative ion of MMA and caused a significant increase in the qualitative ion mass ratio m/z 175/119 (>20, when 0.75 was expected). No carryover was observed at up to 1000 µmol/L MMA.
A set of 335 random patient serum and plasma samples submitted for MMA analysis was analyzed for SA to determine the distribution of concentrations usually present in the samples. A histogram of the observed SA concentrations is shown in Fig. 7
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Therefore, negative-ion mode detection requires adequate chromatographic separation of MMA from SA and other low-molecular weight organic acids potentially present in biological samples (20)(21)(22)(23).
To speed up the analysis, we attempted to identify conditions that would produce unique mass ion fragments for MMA. One of the possibilities for obtaining selective fragmentation for MMA and SA is to analyze their derivatives rather than the parent compounds. For LC-MS applications using atmospheric pressure ionization techniques such as TIS, organic acid esterification is counterintuitive because the nonpolar derivative in solution is less likely to be ionized than its more polar underivatized form. Despite this, our experiments showed that dicarboxylic acid diesters produced positively charged molecular ions. The ionization study for the dicarboxylic acids (Fig. 1
) demonstrated that all of the evaluated n-butyl esters of dicarboxylic acids produced [M + H]+ molecular ions and [M + NH4]+ ammonium adducts, if ammonium ions were present in solution. It was observed that neither the number of methylene groups between the carboxyl groups nor additional functional groups present within the structure (e.g., hydroxy or keto groups) affected the ability of the n-butyl diesters to produce stable positively charged molecular ions. This positive-ion formation for dicarboxylic acid diesters can be explained through resonance of the tautomer structures promoted by the delocalization of charge within the molecule (Fig. 2C
) and mobility of the hydrogen atoms attached to active 
carbons (24). The molecules may exist in solution as protonated or ammoniated adducts, both of which were observed in the experiments (Figs. 1
and 3
).
To support the hypothesis that positive-ion formation takes place through a resonance of keto-enol equilibrium within the part of the MMA molecule between the two carboxyl groups, we evaluated, along with other acids, fragmentation of n-butyl diesters of two other structural isomers dicarboxylic acids, DiMMA and EMA acids (Fig. 1
). The results of the experiment demonstrated that in single-MS mode, the [M + H]+ ion at m/z 245 was the major ion of the n-butyl-EMA and the intensity of the [M + H]+ ion of n-butyl-DiMMA at m/z 245 was not higher than the background noise. A similar result was observed for the ammonium adduct ion m/z 262, which was abundant for n-butyl-EMA and completely absent for n-butyl-DiMMA. The difference between the isomers is that the carbon in EMA is tertiary, whereas in DiMMA it is quaternary. The phenomenon that the [M + H]+ ion does not exist for DiMMA can be explained by the fact that the carbon in the DiMMA molecule does not have an active hydrogen that would allow tautomer formation.
The absolute abundances of the [M + H]+ and [M + NH4]+ molecular ions of the evaluated esters of MMA and SA were substantially different from one ester to another, with increased intensities for the fragments from the esters with higher molecular weight (Fig. 3
). Comparison of the results for fragmentation of different diesters of MMA demonstrated that the n-butyl derivative provided the best specificity for MMA vs SA, whereas the n-amyl derivative showed better sensitivity but less specificity than the n-butyl diester (Fig. 3
). All other diesters created less specific and less sensitive analytical conditions for MMA. Because the sensitivity of the n-butyl derivative was adequate, we chose to use this derivatizing reagent.
A study of the collisionally induced dissociation fragmentation mechanism for different dicarboxylic acids revealed that MS/MS fragment ions at m/z 175 and 119 (loss of 56 and 112 Da) in n-butyl-MMA are unique compared with the other evaluated dicarboxylic acids derivatives. For n-butyl-SA the major product ions from the M+1 molecular ion were obtained by the loss of 74 and 130 Da. Fragmentation of the molecular ions of a series of dicarboxylic acid n-butyl diesters (2-hydroxyglutaric, adipic, suberic, sebacic and dodecanedioic acids) was similar to that for SA. Of the evaluated dicarboxylic acids, only MMA and malonic acid showed the neutral losses of 56 and 112 Da in the MS/MS mode; the rest of the acid diesters showed a preferred neutral loss of 74 and 130 Da, similar to SA (Fig. 2
). At the same time we did not observe [M + H]+ and [M + NH4 ]+ molecular ions for any of the evaluated monocarboxylic acid esters (Fig. 1
). The major product ions observed by ES-MS/MS of dicarboxylic acid dibutyl ester [M + H]+ as the precursor ion are presented in Scheme 1
.
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The fragment ions B and D in Scheme 1
are common to all dicarboxylic acids, but the fragment ions A and C are almost completely missing in SA. The observed difference in fragmentation provides the necessary specificity for the method.
The sensitivity of the evaluated method and the fragmentation pattern that were observed for dibutyl MMA differed from the sensitivity and fragmentation pattern described by Magera et al. (18) for their method. It appeared that, under the conditions used in the present method, the product ion spectrum exhibited extra mass ion fragments at m/z 175, 157, and 101 for MMA and SA. Although additional product ions detract from the ultimate achievable sensitivity, they enhance the specificity of the assay by permitting monitoring of the product ion-mass branching ratios.
Sample preparation for the procedure incorporated liquid/liquid extraction with MTBE of a sample acidified with phosphoric acid. As determined previously (15), MTBE is a selective solvent for separating MMA from a sample matrix, leading to a cleaner extract compared with ethyl acetate and other organic solvents typically used for the extraction of organic acids. The selectivity for dicarboxylic acids and the unique fragmentation of the MMA diester form the basis for this method of MMA analysis. Total instrumental analysis time was 60 s from injection to injection, with an MMA retention time of 40 s, one-third of the time required for the recently published LC-MS/MS method (18). There is very little retention of the analyte on the HPLC column, which serves only to separate MMA from the solvent front and to provide an improvement of the chromatographic peak shape. The ratio of the integrated peak response for MMA transition m/z 231119 vs the d3-MMA transition m/z 234122 was used to calculate the concentration of MMA. The MS/MS transitions 231175 and 234178 were used for qualitative confirmation purposes to assure correct identification of MMA and the absence of interference from coextracted sample constituents.
The key features of the present LC-MS/MS method are its selectivity for dicarboxylic acids and the ability to differentiate MMA from SA, a potential endogenous interferent. SA is the final product of the metabolic conversion of propionic acid and is present at concentrations greater than those of MMA. Fortunately, the m/z 231119 and m/z 231175 fragmentation pathways are 100 and 30 times more abundant for MMA than for SA, respectively. The SA n-butyl ester produced a minor amount of the same product fragment ions at m/z 119 and 175 as MMA. The relative abundances of the ions are substantially different between the compounds. The branching ion-mass ratio m/z 175/119 for MMA is 0.3 ± 0.05, whereas the ratio for the same fragments of SA is 2.0 ± 0.1. When both MMA and SA are present in a sample, the branching ratio of the two product ions enables the estimation of the SA contribution to MMA quantification.
To determine the potential extent of SA interference with the method and to establish a baseline for the expected SA concentration, we evaluated SA concentration distribution in patient samples. The mean, mode, and SD for SA distribution in the patient samples were 5.7, 5.1, and 4.9, respectively. Because SA is a typical constituent of samples and the distribution of the concentrations is relatively narrow, to compensate for SA contribution to MMA quantification and to establish acceptability limits for qualitative ion ratios we included SA in the calibrators and controls at a concentration corresponding to the mean value observed in the studied population. From the results of the method evaluation, the branching ion-mass ratios appeared to be highly reproducible and can serve as an accurate predictor of SA interference with MMA quantification. Potential interference from SA was evaluated from the branching ratios only for samples with MMA concentrations >0.4 µmol/L. For samples with MMA concentrations below the cutoff, the branching ion ratio was not evaluated. If the branching ion ratio did not exceed the value set by the calibration, the quantitative results were accepted. Samples with ion ratios outside of the limits should be reanalyzed by a method that chromatographically separates MMA and SA. The branching ion ratio of m/z 178/122 was used to assess potential interference with d3-MMA in all samples (negative and positive). The validity of this approach was evaluated through correlation with a LC-MS/MS method that chromatographically resolved MMA and SA (Fig. 6
). The results showed good agreement between the methods.
Evaluation of the results of the correlation showed that 89.2% of the analyzed samples produced acceptable results, whereas 10.8% of the samples needed to be reanalyzed by a method that chromatographically resolves MMA and SA because of unacceptable qualitative ion ratios. All samples with a MMA concentration >1.5 µmol/L showed m/z 175/119 ion ratios within acceptance limits and a narrow distribution of 0.44–0.56. The qualitative ion mass ratio for the internal standard was outside of the established limits in only two of the analyzed patient samples.
In conclusion, we present a method for the selective detection of dicarboxylic acids based on the formation of positively charged molecular ions and subsequent unique fragmentation. The advantages of the method are its sensitivity to dicarboxylic acids and the ability to selectively quantify the structural isomers MMA and SA. The selectivity of the method is based on the facts that nonderivatized acids are transparent to the detector and that the esters of monocarboxylic acids do not produce stable [M + H]+ and [M + NH4]+ molecular ions. Such selective detection eliminates the necessity for extensive chromatographic separation and shortens the instrument analysis time for MMA to less than one-tenth of the analysis time of a conventional GC-MS method. The instrumental analysis portion for our method is 3 times faster than a recently published method (18) that uses MS/MS. The entire analysis, including sample preparation for a batch of 100 specimens, can be performed in <4 h.
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Acknowledgments |
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Footnotes |
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2 Nonstandard abbreviations: MMA, methylmalonic acid; SA, succinic acid; GC, gas chromatography; MS, mass spectrometry; LC, liquid chromatography; ES, electrospray; MS/MS, tandem MS; MTBE, methyl-tert-butyl ether; TIS, TurboIonSpray; MRM, multiple reaction monitoring; EMA, ethylmalonic acid; and DiMMA, dimethylmalonic acid.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. K. Campbell, J. W. Miller, R. Green, M. N. Haan, and L. H. Allen Plasma Vitamin B-12 Concentrations in an Elderly Latino Population Are Predicted by Serum Gastrin Concentrations and Crystalline Vitamin B-12 Intake J. Nutr., September 1, 2003; 133(9): 2770 - 2776. [Abstract] [Full Text] [PDF]
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J. J. Pitt, M. Eggington, and S. G. Kahler Comprehensive Screening of Urine Samples for Inborn Errors of Metabolism by Electrospray Tandem Mass Spectrometry Clin. Chem., November 1, 2002; 48(11): 1970 - 1980. [Abstract] [Full Text] [PDF]
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M. M. Kushnir, B. Shushan, W. L. Roberts, and M. Pasquali Serum Acylcarnitines and Vitamin B12 Deficiency Clin. Chem., July 1, 2002; 48(7): 1126 - 1128. [Full Text] [PDF]
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) and M+17 (
) mass ion fragments of butyl esters of selected dicarboxylic (columns A–I) and monocarboxylic (columns J–M) acids, measured at quadrupole 1.
