Fig. 1
Product ion mass spectra of molecular ion [M + H]+ of the n-dibutyl esters of (a) MMA, (b) SA, and (c) mixture of MMA and SA, ratio of the concentrations 1:50, which is comparable to physiologically observed concentrations
We describe here a rapid method for the selective analysis of MMA in serum, plasma , and urine that is based on unique fragmentation of di-butyl MMA combined with a mathematical deconvolution approach, which allows quantitation of MMA in the presence of SA without chromatographic separation. The instrumental analysis time of this method is 1 min per sample.
2 Materials
2.1 Samples
1.
Serum (serum or serum separator tube) and plasma (sodium heparin or EDTA). Samples are stable for 4 days if refrigerated or 6 months frozen at or below −20 °C.
2.
Timed (24-h) or random urine. Samples are stable for 4 days refrigerated or 6 months frozen at or below −20 °C, see Note 2 .
2.2 Reagents and Buffers
1.
MMA, SA, ammonium formate, formic acid, phosphoric acid, methanol, 2-propanol, methyl-tert-butyl ether (MTBE), K2HPO4, NaH2PO4, sodium chloride, urea, creatinine (Sigma-Aldrich, St Louis, MO).
2.
Methyl-d3-malonic acid (MMA d3) (Cambridge Isotope, Tewksbury, MA).
4.
Extraction solvent (3 % phosphoric acid in MTBE): To a 4 L bottle of MTBE, add 84 mL concentrated phosphoric acid (85 %, specific gravity 1.685 g/mL). Cap and mix thoroughly. Stable for 2 weeks at ambient temperature.
5.
Dialyzed plasma (MMA and SA free). (Basematrix 53, SeraCare Life Sciences, Milford, MA). Stable for 1 year at or below −20 °C.
6.
Synthetic urine. In a 1 L glass beaker containing 1 L water, dissolve 2.8 g K2HPO4, 0.55 g NaH2PO4, 7 g of NaCl, 20 g of urea, and 1 g of creatinine. Mix solution until completely dissolved. Stable for 2 years at or below −20 °C.
7.
Mobile phase A (5 mM ammonium formate): To a 1 L beaker containing 300 mL water, add 0.315 g ammonium formate. Fill to 1 L with water, mix for 15 min, and filter through a 0.5 μm filter. Stable for 2 weeks refrigerated or 2 days at ambient temperature.
8.
Mobile phase B (5 mM ammonium formate in methanol): To a 1 L beaker containing 300 mL methanol, add 0.315 g ammonium formate. Fill to 1 L with methanol, mix for 15 min, and filter through a 0.5 μm filter. Stable for 5 days at ambient temperature.
9.
Needle wash solution (methanol:2-propanol:water, 60:20:20 (v/v)): Using separate graduated cylinders measure 600 mL methanol, 200 mL 2-propanol, and 200 mL water; transfer into a 1 L glass bottle, mix, and cap. Stable for 10 days at ambient temperature.
2.3 Standards and Calibrators
1.
MMA stock calibration standard, 10 mmol/L: Weigh 0.0118 g methylmalonic acid , transfer to a 10 mL volumetric flask, and fill to volume with methanol. Mix by inversion until the solid is dissolved. Stable for 1 year at or below −20 °C.
2.
MMA working calibration standard, 10 μmol/L: Add 100 μL of 10 mmol/L MMA stock calibration standard into a 100 mL volumetric flask containing approximately 50 mL water. Fill to volume with nanopure water. Aliquot 400 μL in microcentrifuge tubes. Stable for 1 year at or below −20 °C.
3.
MMA d3 stock internal standard, 10 mmol/L: Weigh 0.0121 g (12.1 mg) of methylmalonic acid d3 (MMA d3) to a 10 mL volumetric flask. Fill to volume with methanol. Stable for 2 years at or below −20 °C.
4.
MMA d3 working internal standard, 15 μmol/L: To a 500 mL volumetric flask, add 400 mL nanopure water and 750 μL methylmalonic acid d3 stock internal standard. Fill to volume with nanopure water and mix. Aliquot 5 mL in polypropylene tubes. Stable for 1 year at or below −20 °C.
5.
SA stock standard, 10 mmol/L: Weigh 0.0118 g succinic acid in a 10 mL volumetric flask. Fill to volume with methanol. Stable for 2 years at or below −20 °C.
6.
SA injection standard: 100 μmol/L: Transfer 100 μL of SA stock standard into a 10 mL glass tube and evaporate the solvent at room temperature. Add 40 μL of the derivatizing reagent and incubate at 70 °C for 10 min. Evaporate the derivatizing reagent; reconstitute the residue with 5.0 mL water: methanol 15:85. Stable for 3 months refrigerated.
2.4 Quality Controls (QC)
1.
Dialyzed plasma spiked with succinic acid , 6 μmol/L (used as a matrix for preparation of quality controls): Add 120 μL succinic acid stock standard to 200 mL dialyzed plasma. Mix for 30 min. Stable for 6 months at or below −20 °C.
2.
MMA stock standard for preparation of QC, 10 mmol/L: Weigh 0.0118 g methylmalonic acid , add to a 10 mL volumetric flask, and fill to volume with methanol. Mix by inversion until completely dissolved. Stable for 1 year at or below −20 °C.
3.
MMA working standard for preparation of QC, 1 mmol/L: In a 10 mL volumetric flask add 1000 μL MMA stock standard, add nanopure water to volume, and mix. Stable for 1 week refrigerated.
4.
Control I, Serum/Plasma , 0.4 μmol/L MMA, and 6 μmol/L SA: Add 500 mL dialyzed plasma into a 1 L beaker with a stir bar and begin mixing. Add 200 μL of MMA working standard for preparing QC (1 mmol/L) and stir for 15 min. Add 300 μL succinic acid stock standard (10 mmol/L), cover, and mix for additional 30 min Aliquot in 1.5 mL microcentrifuge tubes. Stable for 1 year at or below −20 °C.
5.
Control II, Serum/Plasma , 1 μmol/L MMA, and 6 μmol/L SA: Add 500 mL dialyzed plasma in a beaker with a stir bar, add 50 μL MMA stock for preparation of QC, and mix for 15 min. Add 300 μL succinic acid stock standard, cover, and mix for additional 30 min. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below −20 °C.
6.
Negative Control. Dialyzed plasma (free of MMA, determined using this method).
7.
Control III, urine, 10 μmol/L MMA, and 6 μmol/L SA: In a 500 mL beaker pour 250 mL of synthetic urine, add 250 μL of MMA stock standard for preparation of QC, and mix for 15 min. Add 150 μL succinic acid stock standard and fill to volume with synthetic urine, cover, and mix for additional 30 min. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below −20 °C.
8.
Control IV, urine, 20 μmol/L MMA, and 6 μmol/L SA: In a 500 mL beaker pour 250 mL of synthetic urine, add 500 μL of MMA stock standard for preparation of QC, and mix for 15 min. Add 150 μL succinic acid stock standard and fill to volume with synthetic urine, cover, and mix for additional 30 min. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below −20 °C.
2.5 Equipment
1.
Triple quadrupole mass spectrometer AB3200 with TurboV ion source (AB Sciex, Foster City, CA) with built-in switching valve.
2.
Binary HPLC pump series 1260 (Agilent Technologies, Santa Clara, CA), vacuum degasser, autosampler CTC PAL (Carrboro, NC) equipped with fast wash station.
3.
Vortex with adaptor for microcentrifuge tubes.
4.
Evaporator for 96-well plates.
5.
Centrifuge for microcentrifuge tubes.
6.
Centrifuge with buckets for 96-well plates.
7.
Shaker for 96-Well Plates.
2.6 Supplies
1.
Microcentrifuge tubes, 2 mL (Eppendorf, Westbury, NY).
2.
Deep 96-well plates (2 mL well volume) and sealing mats for the plates (Phenomenex, Torrance, CA).
3.
Transfer pipettes.
4.
HPLC Column Luna C18 30 mm × 3 mm, 5 μm particles; SecurityGuard cartridge holder and C18 cartridges (Phenomenex, CA).
3 Methods
3.1 Procedure
1.
Label a set of 2 mL microcentrifuge tubes.
2.
Prepare calibrators and negative control by adding working calibration standard and dialyzed plasma to the corresponding tubes:
(a)
Aliquot in the tubes 500 μL of dialyzed plasma .
(b)
Add in the tubes working calibration standard in amounts according to Table 1.
Table 1
Preparation of calibration standards
Concentration of standard, μmol/L | Working calibration standard, μL | Dialyzed plasma, μL |
|---|---|---|
0.20 | 10 | 500 |
0.40 | 20 | 500 |
0.80 | 40 | 500 |
1.00 | 50 | 500 |
1.50 | 75 | 500 |
2.00 | 100 | 500 |
3.
Aliquot patient samples and controls:
(a)
Add 500 μL of serum or plasma patient sample of control to labeled tubes.
(b)
Add 50 μL of urine sample and control to the corresponding tubes.
(c)
Add 450 μL of nanopure water to the urine sample and the urine controls.
4.
Add to each tube 50 μL of working internal standard.
5.
Add to each tube 1 mL extraction solvent (MTBE/3 % phosphoric acid) and close lids.
6.
Set tubes in adaptor of vortex and shake for 5 min.
7.
Centrifuge the tubes at 14,000 rpm for 3 min.
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