Clinical Chemistry 47: 351-354, 2001;
(Clinical Chemistry. 2001;47:351-354.)
© 2001 American Association for Clinical Chemistry, Inc.
Quantification of Benzoic, Phenylacetic, and Phenylbutyric Acids from Filter-Paper Blood Spots by Gas Chromatography–Mass Spectrometry with Stable Isotope Dilution
Xiaolin Yu1,
Mark M. Thompson1,
Dashuang Shi1 and
Mendel Tuchmana,1
1
Children’s National Medical Center and the George Washington University, 111 Michigan Ave. NW, Washington DC 20010-2970
a author for correspondence: fax 202-884-6014, e-mail
mtuchman{at}cnmc.org
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Introduction
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An important treatment of hyperammonemia in urea cycle disorders
is the stimulation of alternative pathways for nitrogen excretion
(1). Salts of benzoic acid (BA), phenylacetic acid (PAA),
and phenylbutyric acid (PBA) have been found to be useful vehicles for
the elimination of nitrogen by alternative pathways (2)(3)(4).
BA is conjugated with glycine to form hippurate, which is efficiently
excreted in the urine. PBA is metabolized in the liver to
phenylacetyl-CoA, which is then conjugated with glutamine to form
phenylacetylglutamine and subsequently excreted in the urine.
Furthermore, benzoate has been used to treat nonketotic hyperglycinemia
(5), salts of PBA and PAA are used in clinical trials as
anticancer agents (6)(7)(8)(9), and PBA has been found to
stimulate fetal hemoglobin production and can potentially be used for
treatment of hemoglobinopathies (10)(11).
However, two patients have died in the US after being given the wrong
doses of sodium benzoate and sodium phenylacetate (12),
documenting the risk of toxicity, which could potentially be reduced by
drug concentration monitoring.
HPLC assays for PAA and PBA in plasma have limited sensitivity
(4). A gas chromatographic–mass spectrometric method had
improved sensitivity (13), but the method did not use stable
isotopes as internal standards and the clinical utility of the method
was not documented. Here, we describe a rapid, precise, and convenient
method to measure blood concentrations of these drugs using dried
filter-paper blood spots.
BA, PAA, and PBA were purchased from Sigma-Aldrich Co.
[ring-d5]-BA,
[ring-d5]-PAA, and
[2,2,3,3-d4]-4-PBA were purchased
from Cambridge Isotope Laboratories. Regisil RC-3
[N,O-bis-(trimethylsilyl)trifluoroacetamide
containing trimethylchlorosilane (10:1 by volume; BSTFA-TMCS)]
was purchased from Regis Chemical Co. Blood collection cards (cat.
no. 10538414) were purchased from Schleicher & Schuell.
Blood was blotted onto absorbent filter paper and dried at room
temperature. The analytical samples consisted of four punched-out discs
(6.3 mm in diameter), each of which contained 
10.6 µL of blood.
When less blood was available, smaller discs were used. The method was
sensitive enough to measure peak drug concentrations in a single 3-mm
disc (2.4 µL of blood). The blood was eluted from the paper discs by
sonication for 5 min in 1 mL of deionized water containing 100 µg of
d5-BA,
d5-PAA, and
d4-PBA, respectively. The blood-spot
extract was filtered through an Amicon Centrifree YM-30 filter
(Millipore), yielding a clear filtrate. The filtrate was saturated with
1 g of NaCl and acidified to pH <1 with 0.2 mL of 5 mol/L HCl.
The acids were extracted twice with 3 mL of ethyl acetate for 5 min on
a mechanical shaker. The organic layers were combined and dried under a
stream of nitrogen at 50 °C. Trimethylsilyl (TMS) derivatives of the
organic acids were made by incubation with 100 µL of BSTFA-TMCS (10:1
by volume) for 20 min at 60 °C. The derivatized extract (2 µL) was
injected into the gas chromatograph (Hewlett Packard Model
5890 II; Agilent Technologies) by an automated injector. The
acids were separated on a cross-linked 5% diphenyl-95%
dimethylsiloxane capillary column [Ultra 2; 30 m x 0.2 mm
(o.d.); film thickness, 0.33 µm; Agilent Technologies].
The column temperature began at 100 °C and was ramped to 270 °C
at 5 °C/min. The injection port and transfer line temperatures were
250 and 280 °C, respectively. The split injection ratio was 1:40.
The flow rate of the helium carrier gas was 1 mL/min, and the linear
velocity was 35 cm/s. Column-head pressure was 22.5 psi. The ions were
detected by a quadrupole mass selective detector (Model 5971A; Hewlett
Packard ) equipped with an electron impact ion source that was
autotuned and run in high-resolution mode with selected-ion monitoring.
The following ions were determined within the 15-min runtime:
m/z 179 for native BA-TMS, m/z 184 for
d5-BA-TMS, m/z 164 for
native PAA-TMS, m/z 169 for
d5-PAA-TMS, m/z 221 for
native PBA-TMS, and m/z 225 for
d4-PBA-TMS. The elution times were
7.37 min for native BA-TMS, 7.34 min for
d5-BA-TMS, 8.50 min for native
PAA-TMS, 8.47 for d5-PAA-TMS, 13.85
min for native PBA-TMS, and 13.80 for
d4-PBA-TMS. Dwell time for each
ion was 50 ms, and the resolution was 0.5 atomic mass units.
Quantification was performed with the calibration curves of
m/z 179/184, m/z 164/169, and m/z
221/225 for BA, PAA, and PBA, respectively, representing the ratios of
known amounts of native acids (0–10 µg) to isotopic acids (100
µg).
The extraction efficiency was calculated from six 100-µg aliquots of
BA, PAA, and PBA spotted on filter paper. Reproducibility was
determined by analysis of blood spots with 4.0 µg of added BA, PAA,
and PBA. The stability of BA, PAA, and PBA on filter-paper blood
samples over time was tested by analyzing nine blood spots, each
containing 4.0 µg of the three acids, stored at room temperature and
subsequently analyzed over a 1-month period. The heat stability of BA,
PAA, and PBA in blood spots at 80 °C was examined by placing the
samples in an oven for 4 and 24 h.
Fifteen patients with urea cycle disorders and 1 with propionic
acidemia treated with one or more of these medications were tested
using this method.
The prominent m/z 179 and 184 ions (native and isotope,
respectively) for BA, m/z 164 and 169 ions for PAA, and
m/z 221 and 225 ions for PBA (Fig. 1
) were selected for quantification. The calibration curves were
linear from 0 to 100 µg. The equations of the response curve were:
y = 0.0131x + 0.0003 for BA;
y = 0.0135x + 0.0002 for PAA; and
y = 0.0121x + 0.0006 for PBA.
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Figure 1. Electron impact mass spectra (A,
B, and C) and typical chromatogram
(D) for the native and isotopic BA-, PAA-, and
PBA-TMS derivatives.
(A), the prominent m/z 179 (native) and
184 (isotopic) ions for BA were selected for quantification and
calculation. (B), the prominent m/z 164
(native) and 169 (isotopic) ions for PAA were selected for
quantification and calculation. (C), the prominent
m/z 221 (native) and 225 (isotopic) ions for PBA were
selected for quantification and calculation. (D), a
typical chromatogram obtained from a blood-spot sample supplemented
with 20.0 µg each of native BA, PAA, and PBA and 100 µg each of
their respective isotopes. The elution times were 7.37 min for native
BA-TMS, 7.34 min for d5-BA-TMS, 8.50 min for
native PAA-TMS, 8.47 for d5-PAA-TMS, 13.85
min for native PBA-TMS, and 13.80 for
d4-PBA-TMS.
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To determine the detection limits for BA, PAA, and PBA from
filter-paper blood spots, samples with 0.01–10 µg of the acids added
were prepared, followed by analysis as described above. The detection
limits (signal-to-noise ratio >10) for the blood-spot samples were
0.01, 0.06, and 0.04 µg for BA, PAA, and PBA, respectively. When four
6.3-mm (diameter) discs (42.4 µL of blood) were used, the acids could
be detected at concentrations as low as 1.9, 10.4, and 5.7 µmol/L for
BA, PAA, and PBA, respectively. This method was sensitive enough to
monitor the concentrations of the three drugs in the blood of the
patients. To monitor the peaks of PAA and PBA (typically 200–500
µmol/L), a single 3-mm disc (2.4 µL of blood) was sufficient.
An analysis of the mass spectra of BA, PAA, and PBA at various
concentrations showed spillover to the stable isotopes of 0.07%,
0.06%, and 0.25% from native BA (m/z 184), PAA
(m/z 169), and PBA (m/z 225), respectively. Even
at peak blood concentrations, these spillovers were inconsequential. To
increase the sensitivity and overcome the potential problems of
spillover in measuring these acids, the column temperature could be
ramped at a slower rate (e.g., 3 °C/min) to better separate the
native and isotopic peaks.
The extraction efficiencies for BA, PAA, and PBA were 74% ± 2%, 76%
± 3%, and 74% ± 3%, respectively (mean ± SD; n = 6).
Repeated analysis of blood supplemented with 4.0 µg of BA, PAA, and
PBA gave results of 3.9 ± 0.2, 4.0 ± 0.1, and 4.0 ±
0.1 µg (mean ± SD; n = 6), respectively. Dried
filter-paper blood samples that were supplemented with 4.0 µg of BA,
PAA, and PBA and stored up to 1 month at room temperature before
analysis gave results of 4.0 ± 0.2, 4.0 ± 0.1 and 3.9
± 0.1 µg (mean ± SD; n = 9), respectively. The results
for blood spots heated at 80 °C for 24 h were within 2.6%
(BA), 2.5% (PAA), and 5.2% (PBA) of those not heated.
We determined the peak (1.5 h postdose) and trough (predose)
concentrations of BA, PAA, and PBA in 16 treated patients (Table 1
). In six patients, PBA and PAA were 220–551 µmol/L
and 307–473 µmol/L, respectively, 1.5 h after 100–125 mg/kg
doses of PBA. In several patients taking similar or higher doses
(e.g., patients 10 and 11), however, PBA and PAA were much lower. In
patient 13, PBA was 1542.4 µmol/L, and PAA was 405.3
µmol/L 1.5 h after 4.6 g of sodium
phenylbutyrate (71 mg/kg) given by gastrostomy. In all patients,
the predose concentration of PBA was <26 µmol/L, and the predose
concentration of PAA was <170 µmol/L. The highest postdose
PBA concentration measured was 1542.4 µmol/L, and
the highest postdose PAA concentration was 664.7 µmol/L. The BA
concentration in patients not taking BA was 12–62 µmol/L,
which likely reflects values in healthy individuals.
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Table 1. Peak and trough concentrations of BA, PAA, and PBA from
dried filter-paper blood specimens of 16 individuals with urea cycle
disorder treated with BA and/or
PBA.
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The method described here is sensitive, specific, and accurate. It
allows easy collection and delivery to the testing site and should
promote good monitoring of therapy. We also documented that the three
analytes in the dried blood were stable to heat, alleviating concerns
of heat stability if the sample was placed in an outside mailbox.
Although extraction efficiency was 75%, the high reproducibility
and the use of stable isotopes as internal standards compensated for
incomplete extraction. In addition, the use of selected-ion monitoring
and the highly reproducible retention times of a capillary column
allowed this method to detect the three acids with virtually no
interference from other compounds.
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Acknowledgments
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This work was supported in part by a grant from Medicis
Pharmaceuticals and by Grant RR 13297 from the National Center for
Research Resources.
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