Method of analyzing dicarboxylic acids

Details Method of analyzing dicarboxylic acids Method of analyzing dicarboxylic acids

2001-04-17

2004-02-17

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Nuclear magnetic resonance, electron spin resonance or other...

C436S086000

Reexamination Certificate

active

06692971

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method of selectively analyzing structural isomers of dicarboxylic acids based on the unique fragmentation of their derivatives using mass spectrometry (MS), and enhancement of selectivity for the analysis of the dicarboxylic acids through choice of the utilized derivative. In particular, the present invention relates to the determination of methylmalonic acid in a biological sample, and to the diagnosis of vitamin B
12
deficiency.
BACKGROUND OF THE INVENTION
Measurement of methylmalonic acid (MMA) became an important diagnostic procedure in clinical chemistry due to accumulated evidence that slightly increased concentrations of MMA was a marker of vitamin B
12
deficiency. MMA is a metabolic intermediate in the conversion of propionic acid to succinic acid (SA). Vitamin B
12
is an essential cofactor of the enzymatic carbon rearrangement of MMA to succinic acid and the lack of vitamin B
12
leads to elevated levels of MMA. Elevated levels of methylmalonic acid were found in the urine of vitamin B
12
-deficient patients [1]. Deficiency of vitamin B
12
also causes serious and often irreversible neurological disorders named as subacute combined degeneration of the spinal cord [2]. A moderately elevated concentration of MMA (above 0.4 &mgr;mol/L in serum or plasma and above 3.6 mmol/mol CRT in urine) is an early indicator of vitamin B
12
deficiency. Frequency of positively tested samples with results consistent with vitamin B
12
deficiency is 1:20 to 1:50 depending on the population tested. Massive elevation of MMA in serum, plasma or urine (100 to 1000 fold above the concentrations characteristic for the vitamin B
12
deficiency) is consistent with methylmalonic acedemia, an inborn metabolic disorder. Frequency of the methylmalonic acedemia disorder in newborns is 1:20,000 [1,3].
Although, serum MMA and serum cobalamin measurements seem to have equal clinical sensitivity in detecting vitamin B
12
deficiency, there are advantages of measuring MMA instead of cobalamin. Firstly, serum or plasma vitamin B
12
levels may not reflect adequately tissue cobalamin status. Secondly, serum MMA level is 1000-fold greater than serum cobalamin level, and an elevation rather than decreased concentration is measured in vitamin B
12
deficiency. Thirdly, MMA is more stable than cobalamin.
Since the 1960s efforts have been directed towards developing a rapid, simple, sensitive, and specific analytical method for methylmalonic acid determination in biological fluids. In general, sample preparation is required which consists of MMA extraction step from a sample matrix, and, most of the time, a subsequent derivatization. To be able to detect vitamin B
12
deficiency the method is required to measure the low concentrations of MMA found in urine and serum (~1 &mgr;mol/L in urine, ~0.1 &mgr;mol/L in serum). The derivatization step is necessary to improve MMA detection by UV or fluorescent detector [4-10], or to convert the organic acid to a volatile derivative for GC separation [11-30].
Solvent [7,10,12,18-20,22-27,33-36,38,39] and solid-phase extractions [9,11,16,28,29,31,37], preparative chromatography [7,13] or solvent extraction and HPLC (combined) [14,17] have been used to separate MMA from biological samples prior to an instrumental analysis. For serum specimens, a protein precipitation step precedes the extraction. For solvent extraction, the preferred solvents have been diethyl ether, ethylacetate, or both. High MMA recovery was required otherwise the analytical method was not sensitive enough to detect MMA. Some authors used multiple extraction and combined the extracts [10,12,19,20,22-24,34-36], while others utilized saturated NaCl to increase ionic strength of the solution [19,20,22-25,34]. In some cases the extracts were dried with MgSO
4
or Na
2
SO
4
in order to eliminate residual water for the subsequent derivatization for GC analysis [10,12,14,20]. Generally, tedious extraction was required to reduce possible interferences and to obtain an extract that was suitable for further analysis.
There are some methods described in the literature which do not include an extraction step [4-6,8]. Among these are paper [4] and thin-layer chromatography [6,8], colorimetry [5], GC-MS [15], and LC-MS [31,32]. Paper and thin-layer chromatography were used only as qualitative screening methods [4,6,8], and positive specimens were subjected to the more specific and quantitative GC or GC-MS analysis [8]. Norman et al. [15] did not use extraction for urine dicarboxylic acid determination. After addition of the internal standard solution, the sample was evaporated to dryness and derivatized for subsequent GC analysis. This method can be used to identify inborn errors of metabolism from urine samples only and cannot be applied to serum specimens because of their high protein content. The two LC-MS based methods [31,32] were developed for urine organic acid analysis in inborn errors of metabolism screening, and were not optimal for determination of even mildly elevated concentrations of MMA. The authors were able to see methylmalonic acid only at very elevated levels. Instrumental analysis time, using any of the methods described above, range from 10-60 minutes per sample. Furthermore, these methods were only suitable to identify patients with methylmalonic acedemia and were not sensitive enough for the vitamin B
12
deficiency screening.
Derivatization schemes that have been used in methods of determining MMA are method dependent In TLC, HPLC, or CE, derivatization of MMA is required for detection purposes. In GC methods, derivatization is required to convert MMA to a volatile derivative. There are few published methods where analysis of MMA did not require derivatization [31,32,34-39]. Mills et al. [31], Buchanan et al, [32], Kajita et al. [37] have used LC-MS to analyze organic acids in urine specimens No derivatization was needed, however, none of these methods were sensitive enough to analyze MMA in normal urine specimens. Frenkel et al. [34] describe a GC method for urinary MMA determination without derivatization of MMA. MMA from urine specimens was extracted and directly injected into a GC. At the injection port temperature of 225° C. MMA decomposed to propionic acid, and was analyzed as such. This result gave the sum of propionic and methylmalonic acid in the specimen. From a second injection with a lower injection port temperature, propionic acid alone was determined. Concentration of MMA was calculated as the difference between the two measurements. Mikasa et al. [35] describe an isotachophoresis method for urine MMA determination which included an extraction but no derivatization step. Although the detection limit using this method was 0.4 &mgr;mol/L MMA in urine samples, it was achieved by extracting 10 mL of urine. This method is clearly not practical and sensitive enough for serum specimens. Rinaldo et al. [36] describe a CAD MIKES (collisionally activated decomposition mass analyzed kinetic energy spectrometry) technique which has been used to identify patients with methylmalonic acedemia. The technique is not quantitative and by no means is sensitive enough to measure normal concentrations of MMA in urine or serum. Nuttall et al. [38,39] reported a capillary electrophoresis method for MMA determination in urine [38] and serum [39]. To avoid derivatization, they used indirect UV detection; however, using this method, sensitivity was limited and there was no specificity. All the above methods were designed to diagnose organic acedemias in urine specimens. Accordingly, none have adequate sensitivity to measure MMA in serum samples for diagnosis of vitamin B
12
deficiency.
The work by Allen et. al [40] on MMA

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