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Mass spectrometric quantitative detection of methyl malonic acid and succinic acid using HILIC on a zwitterionic stationary phase
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A technology for detection of methylmalonic acid and mass spectrometry, applied in the field of analyzing small organic molecules, can solve the problems of difficulty in obtaining good separation of MMA and succinic acid, etc.
Inactive Publication Date: 2010-03-24
MERCK PATENT GMBH
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From its published chromatograms, it can be seen that it is difficult to obtain a good separation of MMA and succinic acid
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Embodiment 1
[0037] Experimental part
[0038] Reagents and Medicines
[0039] Acetonitrile (HPLC pure) and analytically pure ammonium acetate were purchased from Merck (Darmstadt, Germany), while formic acid (%, p.a.) was purchased from JT BAKER (Deventer, Netherlands). All water was purified with a Milli-Q water purification system (Millipore, Bedford, MA). Plasma protein precipitation (PPT) solution was prepared by adding 25 mL of acetonitrile, followed by 250 mL of concentrated acetic acid and 43 mL of 196.5 mmol deuteromethylmalonic acid (d3-MMA) stock solution to a 50 mL volumetric flask, and then adding acetonitrile to the mark . The PPT solution contained 99.5% by volume acetonitrile, 0.5% by volume acetic acid and d3-MMA at a concentration of 169 nM.
[0040] Protein precipitation and plasma sample cleanup
[0041] Very efficient and straightforward sample pretreatment of clinical samples can be implemented using HILIC technology. Since acetonitrile is a weak solvent in HIL...
Embodiment 2
[0050] Experimental conditions
[0051] Column: ZIC -HILIC 50x 4.6mm, 5μm
[0052] UV
[0053] Column temperature: RT
[0054] Mobile Phase: Acetonitrile / Ammonium Acetate (pH 6.8, 30
[0055] mM); 70 / 30 (v / v)
[0056] Flow rate: 1.5mL / min
[0057] Detector: UV with a wavelength of 206nm (UFS 1.0V)
[0058] Injection volume: 5 μL of test solution in mobile phase
[0059] MS
[0060] Column temperature: 30°C
[0061] Mobile Phase: Acetonitrile / Ammonium Acetate (pH 6.8, 25
[0062] mM); 75 / 25 (v / v)
[0063] Flow rate: 1.0mL / min
[0064] Split: 100μL / min to MS
[0065] Detector: Agilent 1100 benchtop MS, positive ion mode ESI
[0066] Capillary voltage: 3000V
[0067] Fragmentation Voltage 150V
[0068] (Fragmentor):
[0069] Mass range: 50-200m / z
[0070] Injection volume: 5μL of each compound in 0.1mg / mL mobile phase solution
[0071] thing
[0072] Sample: homocysteine, methylmalonate in order of elution
[0073] ...
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Abstract
The invention provides a method for qualitatively and quantitatively detecting methyl malonic acid in a clinical sample that also may contain succinic acid and homocysteine, said method involving a liquid chromatography separation step followed by a mass spectroscopy detection step, said method comprising the steps of: a) providing a sample that may contain methyl malonic acid and / or succinic acidand optionally homocysteine; b) injecting said sample in a mobile phase comprising a high amount of water-miscible organic solvent; c) eluting said mobile phase containing said sample through a liquid chromatography column containing zwitterionic groups covalently bound to carriers as a stationary phase; d) detecting the possible presence of methyl malonic acid and succinic acid and optionally homocysteine by mass spectroscopy detection; and e) determining the presence and optionally the amounts of said organic molecules using calibration data.
Description
[0001] The present invention relates to methods for the analysis of small organic molecules in a large number of samples. In particular, the present invention provides methods for the large scale determination of the clinically targeted small organic molecules methylmalonate, succinate and optionally homocysteine from biological matrices such as whole blood, plasma, serum and urine. Background technique [0002] All references disclosed herein are hereby incorporated by reference. [0003] Analytical techniques used in clinical laboratories require fast, simple and robust performance, in addition to sensitivity and selectivity. The large number of samples in different matrices (ie urine, plasma, serum, whole blood, etc.) requires a variety of techniques based on which methods can be developed that are simple and used in an automated fashion with little or no interruption. A commonly used technique is enzyme-linked immunosorbent assay (ELISA), which has gained popularity due...
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