Clinical Chemistry 43: 2256-2261, 1997;
(Clinical Chemistry. 1997;43:2256-2261.)
© 1997 American Association for Clinical Chemistry, Inc.
GC-MS determination of organic acids with solvent extraction after cation-exchange chromatography
Ja Won Suh1,
Seon Hwa Lee and
Bong Chul Chunga
Doping Control Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul, Korea.
1
Department of Special Chemistry, Seoul Medical Science Institute, Seoul, 140–230, Korea.
a Author for correspondence. Fax +82-2-958-5059; e-mail bcc0319{at}kistmail.kist.re.kr
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Abstract
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We combined column and partition chromatography to isolate, purify, and
quantify biological organic acids in urine and cerebrospinal fluid
(CSF). Urine and CSF samples were introduced onto a preconditioned
cation-exchange column (Dowex 50W x 8 resin) to remove the
biological interferences. The effluent with water was extracted with
ethyl acetate two times (pH 1 and 3) and the organic acids were
quantitatively converted into their trimethylsilyl derivatives for
detection by gas chromatography–mass spectrometry. The good
quality-control data were obtained through precision and accuracy
tests. Inter- and intraassay CVs were 0.01–10.2% and 0.02–12.2%,
respectively. Analytical recoveries compared favorably with results
from the commonly used solvent extraction method. This method was used
for the measurement of the 14 organic acids in the urine and CSF of
healthy volunteers. The values obtained were in the range of the
published data.
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Introduction
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Since the isovaleric acidemia case report by Tanaka et al. in the
1960s, determination of organic acids in biological samples has become
an important tool for detection and diagnosis of organic acidemia
[e.g., methylmalonic acidemia (MMA), medium-chain acyl CoA
dehydrogenase deficiency, etc.] (1)1
.
This is because most of the organic acids are directly or indirectly
related as intermediates in biochemical synthetic pathways and organic
acidemia from specific enzymes (methylmalonyl-CoA, ornithine
transcarbamylase, etc.) or because cofactor (vitamin B12,
5'-deoxyadenosyl cobalamine, etc.) defects lead to the accumulation of
organic acids and their corresponding metabolites (2). In
addition, an organic acid profile enables one to evaluate metabolic
disorders pathobiochemically on the basis of their relations to one
another as precursors or products. Therefore, careful consideration of
small changes in organic acid ratios is essential for more accurate
diagnosis and optimal management for prognosis after treatment in
addition to quantitative determination of organic acids.
Because of their clinical usefulness, many analytical methods have been
developed for the analysis of organic acids in biological samples
(3)(4)(5)(6). In particular, solvent extraction and
anion-exchange methods before gas chromatography (GC) or gas
chromatography–mass spectrometry (GC-MS) analyses have been more
widely used for detection in biological samples. The solvent extraction
method is very simple and fast, but it gives inaccurate quantification
because of a lack of specificity resulting from numerous endogeneous
components (urea, amino acids, etc.) at acid pH. The anion-exchange
method gives better specific isolation from urinary components in
comparison with the solvent extraction method
(7)(8). However, a disadvantage of the
anion-exchange method is that large amounts of amino acids with
amphiphilic ions (
COOH and NH2) can mask some
important organic acids on a GC chromatogram. For these reasons,
Sweetmann and coworkers introduced one modified method by liquid
partition chromatography on a silicic acid column and established the
concentration range of organic acids in several types of biological
samples (urine, plasma, amniotic fluid) (9). As expected,
this method gave specific isolation, but was somewhat laborious and
time consuming for routine analysis in most clinical laboratories.
Therefore, a simple and specific isolation method is needed for more
accurate diagnosis of inherited and acquired metabolic disorders.
In this paper, we describe a combined method for the specific
quantitative analysis of biological organic acids based on the method
of Husek and Liebich (10). This method includes column and
partition chromatography, results in quantitative recoveries of many
acids, and overcomes the critical points (lack of specificity) in the
screening of organic acid-related diseases. We present preliminary
evidence that this efficient procedure can be applied to the detection
of MMA.
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Materials and Methods
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calibrators and reagents
Organic acid calibrators were purchased from Sigma Chemical Co.
[D]Homovanillic acid (HVA) used as internal calibrator was purchased
from MSD Isotope Co. All solvents were of guaranteed grade and used
without further purification. Dowex 50W x 8 (strongly acidic
cation-exchange resin, H+) was purchased from Sigma
Chemical Co. Deionized water was distilled before use. Silylating
reagents,
N-methyl-N-trimethylsilyltrifluoroacetamide
(MSTFA) and trimethylsilyl chloride (TMS-Cl), were purchased from Sigma
Chemical Co. Ethyl acetate was high-purity "HPLC solvent" grade.
samples
Urine specimens were obtained from healthy volunteers, 9 women
(26–32 years of age) and 10 men (30–39 years), all with unremarkable
medical history. No dietary restrictions were applied except for total
abstinence from alcoholic beverages for 24 h; three of the
subjects were smokers. Patient urine specimens were obtained from Y
university and cerebrospinal fluid (CSF) specimens (subjects 1–12
years of age) from K university. All samples were stored at -20 °C
until analysis.
gc-ms
A Hewlett Packard GC-MS system used for this study consisted of a
model 5890A gas chromatograph, a model 5970B mass selective detector, a
HP 5970C MS chemstation, and a HP 7946 disc drive. A fused-silica
capillary column coated with HP-5 cross-linked 5% phenylmethyl
silicone (SE-54, 34 m x 0.2 mm i.d., 0.33 µm film thickness)
was also used. The GC temperature program was as follows: initial
temperature was 100 °C, held for 1 min, increased to 130 °C at a
rate of 2 °C/min, then to 200 °C at a rate of 3 °C/min, and
finally to 280 °C at a rate of 6 °C/min and held for 10 min. The
split ratio was 1:12, injection temperature was 250 °C, transfer
line temperature was 270 °C, and ion source temperature was
200 °C. The mass spectrometer was operated at 70 eV in the electron
impact mode with SCAN or selected ion monitoring (SIM). The selected
ion groups for the identification of 15 organic acids in SIM mode are
listed in Table 1
. The dwell time for each ion was set at 50 ms.
sample preparation
Solvent extraction method.
To adjust the pH to <1, 200
µL of 6 mol/L HCl was added to 2 mL of urine containing 5 mg/L
internal calibrator ([D]HVA), and 1.5 g of sodium chloride was
also added. Organic acids in biological samples were extracted into 5
mL of ethyl acetate by mechanically shaking for 10 min, and the tube
was immediately centrifuged at 800g for 5 min. The organic
layer was transferred to a second tube and solvent was evaporated under
reduced pressure. The residue was dried in a vacuum desiccator over
P2O5-KOH.
Strong cation exchange method.
Into a Pasteur pipet
(i.d. 0.5 cm), preconditioned Dowex 50W x 8 resin was poured up
to 3 cm of height. Urine (2 mL) containing 5 mg/L internal calibrator
solution ([D]HVA) was applied to the column adjusted to pH <1. As
soon as urine and internal calibrator were introduced, the effluent
with 2 mL of water was collected without waste and 200 µL of 6 mol/L
HCl was added to adjust the pH to <1. Then, 1.5 g of sodium
chloride and 5 mL of ethyl acetate were also added. The mixture was
shaken mechanically for 5 min and the two layers were separated after
centrifugation (800g, 5 min). The organic layer was
transferred to another tube and 1 mL of 0.1 mol/L glycine buffer (pH 3)
was added to adjust pH to <3. The mixture was shaken mechanically for
5 min and the two layers were separated after centrifugation
(800g, 5 min). The organic layer was transferred to another
tube and solvent was evaporated under reduced pressure. The residue was
dried in a vacuum desiccator over P2O5-KOH.
Derivatization
The residue was dissolved in
50 µL of TMS reagent mixture (MSTFA/TMS-Cl, 100:1 by vol) and heated
at 60 °C for 15 min. After heating, 2-µL aliquots were injected
onto the GC column with an autosampler.
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Results and Discussion
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extraction and analysis
One of the most critical points for organic acid profiles in
biological samples (urine, CSF) is the specific isolation from amino
acids, urea, and creatinine (11), which interfere with the
quantification of some important organic acids. Therefore, on the basis
of the extraction method of Husek and Liebich (10), a
combined method including column and partition chromatography was
established to remove the interferences derived from biological matrix.
The isolation of the organic acids of interest is based on the use of a
cation-exchange column and moderate pH adjustment (<pH 3) to
neutralize the negative charges of the anions (pH <
pKa - 2). Then, to enhance the specificity on the GC
chromatogram, they were derivatized with the TMS reagent mixture
(MSTFA/TMS-Cl). By using the above method, organic acids were
simultaneously determined by GC-MS and in a single run successfully
separated within 45 min.
gc-ms profiles
Figure 1
shows the effects of a newly modified method on the resulting
GC-MS analysis. Total ion chromatogram of TMS-derivatized standard
organic acids is shown in Fig. 1a
, demonstrating the good GC separation
of 15 organic acids in a single run. Although some organic acids were
coeluted, they could be analyzed because of the specificity of SIM mode
with two or three ions on the basis of characteristic ions and
retention times in Table 1
. Also, all of the subsequent quantified data
were obtained by SIM mode. Fig. 1b
, solvent extraction method, shows
that urea and amino acids severely mask important portions (A, B) of
the organic acids profile for the diagnosis of inherited and acquired
metabolic diseases such as MMA, maple syrup urine disease (MSUD), and
Reye syndrome. For example, in the A region are methylmalonic acid,
ethylmalonic acid, and methylsuccinic acid for the diagnosis of organic
aciduria such as MMA, and in the B region are HVA and azelaic acid for
neuroblastoma and dicarboxylic aciduria (12). In contrast
with the solvent extraction method, however, the considerably improved
organic acids profile by the addition of small column is shown in Fig. 1c
. Namely, the disappearance of huge amounts of urea and amino acids
at 10.2, 12.3, 31.0, and 33.0 min is shown. This demonstrates that the
combined method results in the effective isolation of the organic acids
from several interferences (urea, amino acids, etc.).
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Figure 1. GC-MS chromatograms of (a) standard organic
acids (1.0 mg/L), (b) urinary organic acids from solvent
extraction method, and (c) urinary organic acids from
well-combined extraction method (strong cation-exchange method).
For peak identities, see Table 1
.
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precision and recovery
The intra- and interday reproducibility of the urine profiling is
given in Table 2
. Precision and accuracy data from supplemented artificial
urine, as judged from CV (<10%), were satisfactory. Recovery studies
were performed three times at 1 mg/L concentration for each organic
acid. Table 3
represents the analytical recoveries of organic acids on
liquid–liquid extraction and newly improved (strong cation exchange)
extraction methods. As shown in Table 3
, analytical recoveries
exceeding 60% were mostly found regardless of a wide variety of
nonpolar and polar organic acids. Although the extraction recoveries of
lactic acid, hippuric acid, and citric acid were only 39%, 26%, and
10%, respectively, they could be sufficiently detected in biological
samples.
results in urine and csf
The concentration range of organic acids in control urine and CSF
was simultaneously determined by GC-MS with the above extraction method
and the same quantification method used for the calibrator. As shown in
Table 4
, our results agree with the results that Hoffmann et al.
(9) have previously reported.
Figure 2
displays a urinary organic acid profile of a patient with MMA.
As mentioned above, it shows an effective result of the combined method
applied for confident diagnosis of a metabolic disorder.
In conclusion, with the extraction method described here,
quantitative profiles of organic acids were obtained by GC-MS, and the
competing endogeneous substances (amino acids, urea, creatinine, etc.)
on GC-MS chromatograms were effectively excluded. Also, this method
made it possible to establish a more accurate concentration range of
organic acids in biological fluids (urine, CSF). From the above
results, we suggest that this efficient method will be suitable for the
quantitative analysis of organic acids for diagnosis and follow-up
study of patients with new or ill-defined disorders in most clinical
laboratories.
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Acknowledgments
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This study was supported in part by grant 2E14130 from the Korea
Ministry of Science and Technology.
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Footnotes
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1 Nonstandard abbreviations: MMA, methylmalonic acidemia;
GC-MS, gas chromatography–mass spectrometry; HVA, homovanillic acid;
MSTFA,
N-methyl-N-trimethylsilyltrifluoroacetamide; CSF,
cerebrospinal fluid; TMS-Cl, trimethylsilyl chloride; and SIM, selected
ion monitoring. 
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Abstract
Introduction
