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Articles |
1
Biochemical Genetics Laboratory, Department of Laboratory Medicine & Pathology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
a Author for correspondence. Fax 507-266-2888; e-mail rinaldo{at}mayo.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We added 1 nmol of the internal standard MMA-d3 to 500 µL of plasma or 100 µL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 °C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d3, respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid.
Results: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 µmol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030x - 0.032 (Sy|x = 1.03 µmol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3.8–8.5% and 1.3–3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 µmol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 µmol/L), 96.0% (0.60 µmol/L), and 94.8% (2.02 µmol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100–150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively.
Conclusions: This method is well suited for large-scale MMA
testing (
100 samples per day) where a shorter analytical time is
highly desirable. Reagents are less expensive than the
anion-exchange/cyclohexanol-HCl method, and sample preparation of
batches up to 100 specimens is completed in less than 8 h and is
automated.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1:48 000 live births. Two disorders
(mut0 and mut-) reflect a
deficiency of the apoenzyme portion of the enzyme methylmalonyl-CoA
mutase; three others (cblA, cblB, and cblH) are associated with
abnormalities of the adenosylcobalamin synthesis pathway. cblC and cblD
are caused by impaired synthesis of both adenosylcobalamin and
methylcobalamin, whereas CblF is a defect of lysosomal cobalamin efflux
and is inconsistently associated with mild accumulation of MMA in
plasma and urine. Acquired causes of methylmalonic acidemia are much more common. Increased MMA in plasma and urine is found in patients with cobalamin (vitamin B12) deficiency as a consequence of intestinal malabsorption, impaired digestion, or poor diet (4). Other conditions, such as renal insufficiency, hypovolemia, and bacterial overgrowth of the small intestine, also are possible causes of mild methylmalonic acidemia or aciduria. Elderly patients with cobalamin deficiency may present with peripheral neuropathy, ataxia, loss of position and vibration senses, memory impairment, depression, and dementia in the absence of anemia (5). Therefore, the determination of MMA in serum and urine is considered an important biochemical screening for the differential diagnosis of signs and symptoms associated with a variety of causes of cobalamin deficiency.
Several studies have suggested that the determination of serum MMA could represent a marker of cobalamin deficiency more reliable than direct cobalamin determination (6). Following our recent development of a method for the determination of total homocysteine (7), we have focused our efforts on developing a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the routine determination of plasma and urine MMA in a high-volume testing laboratory environment, replacing a gas chromatography-mass spectrometry (GC-MS) procedure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sample preparation
A 600-µL serum or plasma sample was placed in a 10 x 75 mm
glass culture tube in a tray on the processor and mixed with 600 µL
of internal standard solution (1.2 nmol
MMA-d3). For pediatric patients,
sample volume could be reduced to a minimum of 50 µL and brought to
volume with distilled water. A 1-mL aliquot of the mixture was
extracted on a Gilson ASPEC automated SPE sample processor. After the
SPE column was preconditioned with 1 mL of methanol followed by 1 mL of
water, 1000 µL of the diluted sample was added to the column, which
was then washed with 1 mL of deionized water. MMA and
MMA-d3 were eluted from the SPE column
in 300 µL of 18 mol/L formic acid in water. The elution tubes were
removed from the processor, and the eluate was evaporated to dryness in
25 min in a water bath at 30 °C under dry nitrogen. The residue was
then transferred to 1-mL glass screw-cap reaction vials using two
200-µL portions of methanol. The methanol was evaporated in 5 min
on a 40 °C dry heating block under a gentle flow of dry nitrogen.
After 200 µL of 3 mol/L HCl in n-butanol was added to each
vial, the reaction vials were capped and heated in a dry heating block
at 65 °C for 15 min to form n-butyl ester derivatives.
The excess reagent was evaporated in 10 min at 40 °C under nitrogen,
and the residue was dissolved in 100 µL of 80:20 (by volume)
acetonitrile:deionized water. Centrifugation for 2 min at
3000g yielded a clear supernatant that was transferred for
analysis to a sealed glass autosampler vial. The preparation for urine
differed from that for plasma/serum only in that 100 µL of sample was
mixed with 500 µL of deionized water and placed in the processor
sample tube. Calibrators were prepared in deionized water by the
addition of working solution (20 µmol/L) corresponding to a blank and
MMA concentrations of 0.25, 0.50, 1.00, 2.00, 5.00, and 8.33 nmol in
0.5 mL.
methods
A triple-quadrupole mass spectrometer API 3000 (Perkin-Elmer
Sciex) operated in ion-evaporation mode (source voltage, 5500 V) was
used. Peripherals included two Perkin-Elmer Series 200 M pumps and an
autosampler. Separation of MMA and
MMA-d3 from the bulk of the specimen
matrix was achieved by use of a short column (LC-18; 33 mm x 4.6
mm; Supelco). Autosampler injections of 10 µL were made, using a
mobile phase composed of acetonitrile in 1 mL/L formic acid (60:40, by
volume). The column was directly connected to the TurboIonSpray
ionization probe operating with the turbo gas on (6 L/min; sensor
temperature, 300 °C) and the LC column effluent flow split to
deliver 100 µL/min to the instrument. MMA and
MMA-d3 coeluted with a retention time
of 2.2 min. Total instrument time was 3 min per sample.
All results were generated in positive-ion mode with the orifice voltage set at 26 V, automatically optimized using the protonated MMA di-n-butyl ester ion. For all MS/MS experiments, mass calibration and resolution adjustments (at 0.7 amu full width half height) on both the resolving quadrupoles were automatically optimized using a poly(propylene) glycol solution introduced via an infusion pump. Collisionally activated decomposition MS/MS was performed through the closed-design Q2 collision cell, operating with nitrogen as collision gas. The 16 eV (lab frame) collision energy was adjusted automatically by the AutoTune algorithm.
MS/MS product ion scans of MMA and succinate standards were collected in continuous flow mode by connecting an infusion pump directly to the TurboIonSpray probe. For MS/MS optimization, a 20 µmol/L MMA di-n-butyl ester solution was prepared in acetonitrile:water (50:50, by volume) containing 2.5 mL/L formic acid and infused at a flow rate of 10 µL/min. In the selected reaction monitoring (SRM) mode, the instrument was optimized automatically by the built-in algorithm to monitor the m/z 231.0 to m/z 119.1 and m/z 234.1 to m/z 122.0 transitions for MMA and MMA-d3, respectively. Data were acquired and processed using the MassChrom software (Ver. 1.1.1; Perkin-Elmer Sciex) including Multiview, Ver. 1.4, for chromatographic and spectral interpretation and TurboQuan for Apple Macintosh (Ver. 1.0; Perkin-Elmer Sciex) for the quantitative processing. For the purpose of method comparison, we compared the MS/MS method with the GC/MS procedure (8)(9), which had been used previously in our laboratory and elsewhere (10). This GS-MS method is based on automated SPE and derivatization of MMA and MMA-d3 with acidified cyclohexanol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Both of these spectra were acquired by
Q1 transmission of the protonated molecular ion,
m/z 231, and scanning the second resolving quadrupole
(Q3) for products resulting from fragmentation in
the collision cell. Loss of the n-butyl moieties from the
protonated molecular ion of MMA (m/z 231) yielded the
fragment at m/z 119, which was not detected in the product
ion scan of 20 µmol/L succinic acid but became apparent at higher
concentrations. These results were used to design the SRM experiment to
sequentially transmit the m/z 231 protonated molecular ion
and the m/z 119 fragment via Q1 and
Q3, respectively. Although a signal for succinic
acid was detected at higher concentrations, the addition of succinic
acid at concentrations up to 2000 µmol/L to serum samples produced no
interference with the MMA peak. The separation of MMA from succinic
acid is shown in Fig. 2
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Four SRM ion chromatograms obtained from serum and urine samples at
normal [serum, 0.25 µmol/L (reference interval, 0–0.4
µmol/L); urine, 2.67 mmol/mol creatinine (reference interval, 0–3.6,
mmol/mol creatinine)] and increased (serum, 18 µmol/L; urine, 36.4
mmol/mol creatinine) concentrations, respectively, are shown in Fig. 3
. Serum specimens with MMA concentrations up to 23 µmol/L have
been analyzed accurately without dilution. At low concentrations, a
serum specimen in which the calculated MMA concentration was 0.12
µmol/L exhibited a signal-to-noise ratio for the MMA-extracted SRM
signal of 40:1 (injected amount equal to 1.2 pmol).
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linearity
Interassay variability of calibration data obtained over
concentrations of 0.25–8.33 nmol/0.5 mL was monitored on 7 consecutive
days. The average slope, intercept, and coefficient of linear
regression (r2) were 0.9050
(95% confidence interval, 0.7991–1.0109), -0.0117 nmol/0.5
mL (-0.0962 to 0.0728 nmol/0.5 mL), and 0.9998
(0.9992–1.0003), respectively.
recovery, precision, and stability
Recovery and precision data are summarized in Table 1
. These experiments were conducted using a pool of human serum
from healthy donors. To 144 aliquots (500-µL) of pooled serum, we
added either 15 µL of a 5 µmol/L MMA solution, or 15 or 60
µL of a 20 µmol/L MMA solution, corresponding to final MMA
concentrations of 0.125, 0.500, and 2.000 µmol/L. Six aliquots of
each set were prepared as described above and analyzed in single
determinations on each of 6 consecutive days. Good quantitative
recovery and precision were obtained, demonstrating the accuracy of the
method for the quantitative determination of MMA.
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The stability of prepared specimens was investigated by repeat
injection of 10 serum samples (selected over a range from 0.09 to 1.95
µmol/L) with interim storage at 4 °C. The data obtained are shown
in Table 2
. ANOVA (single factor) showed variances 
0.007 (1.3% at 1.99
µmol/L), indicating that MMA and
MMA-d3 as di-n-butyl esters
were stable during refrigeration for at least 72 h after sample
preparation.
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method comparison
Unused portions of 106 specimens routinely analyzed by GC-MS were
retested using the new LC-MS/MS method. The correlation between the
MS/MS method and the GC-MS method was: y =
1.030x - 0.032 (n = 106; r =
0.994; Sy|x = 1.03 µmol/L), with an
average 
of 0.01 µmol/L. A Bland-Altman plot indicated equivalence
of MMA results over the concentration range found in routine specimen
analysis (Fig. 4
) (11). A group of urine specimens was
retrospectively analyzed by LC-MS/MS and GC-MS methods, and results
similar to those presented for plasma were obtained: y
= 0.956x - 0.121 (n = 49;
r2 = 0.8981).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first application of MS/MS to the rapid qualitative detection of MMA in urine was reported in 1984 (12). Product ion analysis of m/z 119 [M+H]+ and m/z 101 [M-OH]+ of underivatized MMA calibrator (100 µg) and of a urine extract of a patient with methylmalonic acidemia, collected during an acute episode of metabolic decompensation, showed an excellent match of the two spectra without any apparent interference despite the complexity of the matrix. The excretion of MMA in that sample, however, was extremely high (11 959 mmol/mol creatinine by GC-MS), and there was only an approximate correlation between the MS/MS and GC-MS results. More recently, MMA was included in a study of the application of GC-MS/MS to the screening of organic acidurias (13). Processing of urine specimens included SPE, oximation, and derivatization as trimethylsilyl esters. The chromatographic run was 10 min, and the MS/MS analysis was based on ion-trap (tandem-in-time) configuration where the precursor ion of MMA was m/z 247 [M-CH3]+ and the product ion was m/z 231. According to the authors, the advantages of this method over conventional GC-MS were greater recovery, a shorter run time (10 min), and simpler data analysis. On the other hand, the applicability of this method to plasma was not investigated, low-end sensitivity was not established (the lowest calibration point was equivalent to 0.8 µmol/L), and there was no indication of whether succinic acid could interfere in the determination of MMA. Reference intervals were expressed in arbitrary units, and the extraction efficiency was on average 10% lower than the SPE used in our method. For these reasons, we believe that our method represents an innovative contribution to the determination of MMA in plasma and urine.
To achieve an accurate determination of MMA without GC separation, we had to overcome the interference from succinic acid, an isomer physiologically present in plasma (0–32 µmol/L) and urine (adult reference interval, 0.5–16 mmol/mol creatinine) (14) at concentrations higher than MMA (plasma, 0–0.4 µmol/L; urine, 0–3.6 mmol/mol creatinine). Because satisfactory separation of underivatized calibrators was not achievable, we tested different derivatization procedures. Butylation, which is also the method of choice for derivatization of acylcarnitines (15), amino acids (16), and acylglycines (17) before MS/MS analysis, offered the best combination of chromatographic behavior and fragmentation pattern, allowing complete separation of the two isomers at succinate concentrations up to 2000 µmol/L.
Using our previous experience with the comparison of MS/MS and HPLC methods for the analysis of total homocysteine (7), we compared MMA values obtained by the GC-MS and LC-MS/MS methods, using the technique described by Bland and Altman (11), and found a very favorable agreement between the proposed method and the GC-MS method used in our laboratory (8). The mean difference for MMA values obtained by both methods was 0.01 µmol/L, and acceptable agreement was demonstrated over a concentration range up to 25 µmol/L. Although this GC-MS method is widely utilized, a recent comparative study of 13 laboratories, including ours, showed mean among-laboratory CVs of 19% and 21% for serum and plasma samples, respectively (10). The authors concluded that significant among-laboratory imprecision is an issue of concern and that improvements are needed to reduce analytical imprecision by GC-MS methods. Although we agree that introduction of high-quality reference materials and institution of external quality assessment programs could be beneficial, we propose that the introduction of an alternative method could also have a positive impact, similar to what we have observed at our institution when homocysteine analysis was moved from an HPLC to a MS/MS platform (7).
The advantages of this method over the GC-MS procedure can be summarized as follows: one MS/MS system used only overnight (7.5 h) can replace two GC-MS systems (30 instrument-hours/day) to run 100–150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. Under these circumstances, we have developed an alternative MMA method that not only displays comparable or greater precision of its GC-MS counterpart, but is also faster and overall less expensive.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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