Clinical Chemistry 49, No. 3, 2003
Rapid Determination of Orotic Acid in Urine by Liquid
Chromatography–Electrospray Tandem Mass Spectrometry, Mohamed S. Rashed,1* Minnie Jacob,1 Mohamed AlAmoudi,1 Zuhair Rahbeeni,2 Moeen A.D. Al-Sayed,2 Lujane
Al-Ahaidib,1 Amal A.A. Saadallah,1 and Sharon Legaspi1
(Departments of 1Genetics and 2Medical Genetics, King
Faisal Specialist Hospital and Research Centre, Riyadh
11211, Saudi Arabia; * address correspondence to this
author at: Metabolic Screening Laboratory, King Faisal
Specialist Hospital and Research Centre, PO Box 3354,
Riyadh 11211, Saudi Arabia; fax 966-1-442-4546, e-mail
rashed@kfshrc.edu.sa)
Orotic acid (ORA) is an important biochemical marker for
uridine monophosphate synthase deficiency, an autosomal recessive disease characterized by macrocytic hypochromic megaloblastic anemia, growth retardation, orotic
aciduria, and crystalluria (1 ). In addition, patients with
urea cycle diseases excrete increased amounts of urinary
ORA (2 ). Thus, ORA aciduria is observed in patients with
ornithine carbamoylasetransferase deficiency (OCTD), an
X-linked disorder, and could reveal heterozygosity after a
protein load, and in citrullinemia, argininosuccinic aciduria, and argininemia (2, 3 ).
Currently there are two widely accepted approaches to
the determination of urinary ORA, stable-isotope-dilution
gas chromatography–mass spectrometry (GC/MS), and
ion-exchange HPLC methods with ultraviolet detection,
each of which has difficulties and limitations (4 –7 ). Ito et
al. (8 ) recently described a liquid chromatography– electrospray tandem mass spectrometry (LC-ESI-MS/MS)
method for a large number of urinary metabolites, including ORA. In the present work we describe a more specific,
high-throughput, sensitive stable-isotope-dilution LCESI-MS/MS method for the determination of urinary
ORA.
ORA was purchased from Sigma, and [1,3-15N2]orotic
acid used as internal standard (IS) was obtained from
Cambridge Isotope Laboratories. Acetonitrile and glacial
acetic used for HPLC were purchased from Fisher Scientific. Syringe-driven membrane filter units were of LCR
type (0.45 m pore size) obtained from Millipore. Glass
autosampler vials (2-mL capacity) and 300-L vial inserts
were purchased from Agilent. Urine samples were from
pediatric patients investigated for metabolic diseases.
Adult urine samples were from healthy laboratory personnel. All urine samples were stored at 20 °C until
analysis.
MS/MS analysis was carried out on a Micromass QuattroLC triple quadrupole mass spectrometer interfaced
with a Z-spray ESI source and equipped with a Jasco
HPLC pump Model PU-980 and a Jasco Model AS-950
autosampler. The ESI source was operated in the negative-ion mode with the source kept at 150 °C and a
desolvation temperature of 350 °C. The capillary voltage
was 3.0 kV, cone voltage was 18 V, extraction voltage was
4 V, and collision energy was 10 eV. Nitrogen was used as
the nebulizing gas at a flow rate of 120 L/h and for
499
desolvation at a flow rate of 450 L/h. Argon was used as
collision gas at a pressure of 0.2 Pa.
The collision-induced dissociation spectra of the deprotonated ORA (m/z 155) and its IS (m/z 157) were acquired
in the continuous flow injection analysis mode. Under
collision-induced dissociation conditions, both compounds gave one major fragment corresponding to the
neutral loss of carbon dioxide [M-H CO2].
For LC-MS/MS analysis, a short Supelco ABZ-plus
column [3.3 cm 2.1 mm (i.d.); 3-m particles] was used.
The mobile phase consisted of acetonitrile–water– glacial
acetic acid (30:70:0.15 by volume) in the isocratic mode at
a flow rate of 250 L/min. Chromatography was carried
out at ambient temperature. The effluent from the injector
was passed through an online filter Model A-315 (Upchurch Scientific) that was connected to the column via
PEEK tubing. The column was interfaced directly to the
ESI source, with no splitting.
For selected-reaction monitoring (SRM) analysis, two
transitions were monitored, [M-H] at m/z 155 to product
ion at m/z 111 for ORA and m/z 157 to product ion at m/z
113 for the IS. The autosampler was programmed to inject
a sample every 5 min. The isotopic purity of the IS was
determined by repeated injections of 0.16 mmol/L solution (n 10) under the SRM conditions mentioned above.
Peak areas were measured for both transitions, m/z
1553111 for ORA and 1573113 for IS. The averaged data
showed that the isotopic purity of the IS was 99.584%; it
thus contributed 0.416% to the unlabelled ORA channel,
whereas the unlabelled ORA contribution in the IS channel was 0.28%.
Urine samples were membrane-filtered before use to
remove any particulate material. For samples with creatinine concentrations ⱕ5 mmol/L, we used 25 L, and for
samples with creatinine concentrations 5 mmol/L, we
diluted the samples 1:1 with water and placed 25 L in
microcentrifuge tubes with 20 L of IS solution (1 g) and
300 L of mobile phase. The tubes were vortex-mixed,
and 100 L of each of the mixtures was transferred to
inserts in glass autosampler vials, capped, and injected.
The dilution scheme described was necessary because of
signs of overloading as evidenced by peak tailing and/or
peak splitting in urine samples with high creatinine
concentrations. To prevent overloading of the short, narrow-bore column used, we injected only 3-L volumes
into the mass spectrometer. Under these conditions, the
retention time for ORA was reproducible (CV, 3.4%;
mean, 3.02 min; n 50). Some peak tailing was still
observed at the high concentration points of the calibration curve and in the abnormal urine samples. This did
not affect the quantification of ORA because there were no
interfering peaks and because the isotope-labeled IS
eluted in the exact retention window, allowing accurate
definition of the retention window for peak integration.
Shown in Fig. 1A are two overlaid SRM chromatograms
for ORA in a pooled control urine (4.65 mol/L; 2 ng
injected on column gave a signal-to-noise ratio of 40:1)
and for the IS. Fig. 1B shows two overlaid SRM chromatograms for ORA in urine from a male OCTD patient (1570
500
Technical Briefs
Fig. 1. LC-MS/MS negative-ion SRM chromatograms for the m/z
1553111 transition for ORA and the m/z 1573113 transition for IS
added to urine.
(A), pooled pediatric control urine; (B) urine from a male OCTD patient.
mol/L) and for the IS. The short ABZ-plus column
provided excellent retention of ORA, whereas use of a
relatively high acetonitrile concentration in the mobile
phase was necessary to enhance ESI signal intensity. The
addition of acetic acid to the mobile phase improved peak
shapes but decreased the retention time. The decreased
retention time occurred only after 50 injections, presumably after saturation of binding sites on the column. This
behavior was reported previously for ORA on C18 reversed-phase columns (6 ). No further change was observed over the next 300 injections. To demonstrate the
unique retention properties of the ABZ-plus stationary
phase for ORA, we analyzed several clinically relevant
compounds under the same conditions. Among these
were hippuric acid, homovanillic acid, uracil, 5-fluorouracil, and tryptophan. All five compounds eluted before
1.5 min vs the 3.0-min retention for ORA (data not
shown). We also observed that the behavior of the column, at least for ORA, was not truly reversed-phase
because the retention time was shorter for the ORA and IS
calibration solutions in acidified mobile phase containing
200 mL/L acetonitrile than in acidified mobile phase
containing 300 mL/L acetonitrile, and was the reverse
with 400 mL/L acidified acetonitrile.
Calibrators were prepared in pooled control pediatric
urine (n 20). Serially diluted solutions of ORA were
used to achieve concentrations of 0, 1.282, 3.846, 9.615,
320.50, 961.54, 1282.10, and 2564.20 mol/L. The samples
were then treated as described above. A mobile phase
blank was injected before and after each calibration curve.
The carryover between injections was consistently 0.5%.
The peak-area ratio of ORA to IS for the first point in the
curve was subtracted from all subsequent points to eliminate the contribution of endogenous ORA. The peak-area
ratios were then plotted vs ORA concentration to con-
struct calibration curves (y mx b). The calculation
involved corrections for contribution of IS to the ORA
channel and vice versa. We obtained excellent linear
calibration curves for the determination of ORA in urine
in the range of 1.282–2564 mol/L. Because of the large
calibration range, we constructed two calibration plots,
one covering the reference interval (up to 20 mol/L) and
the other covering exceedingly abnormal ORA values.
The calibration results based on the averages of six
separate experiments for each of the ranges were as
follows: mean (SD) slope, intercept, and correlation coefficient (r2) for the reference interval curve were 0.0036
(0.0002), 0.0006 (0.0004) mol/L, and 0.9996 (range,
0.9991– 0.9998), respectively. The mean (SD) slope, intercept, and correlation coefficient (r2) for the abnormal
range curve were 0.0042 (0.0001), 0.0243 (0.0130)
mol/L, and 0.9998 (range, 0.9996 – 0.9999), respectively.
The intra- and interday accuracy and precision of the
method were determined for the control and three known
concentrations (9.615, 96.15, 961.5 mol/L) from six experiments run on 6 consecutive days, with interim storage
at 4 °C. The average recovery at all concentrations was
101.12% (range, 99.95–101.71%) with intraday CVs of
0.38 –2.5%), and interday CVs of 0.6 –2.4% (n 6 for each
concentration). The ORA concentrations in urine from
randomly selected pediatric patients with negative blood
MS/MS and urine GC/MS results, adult female and male
volunteers, and from male patients with OCTD, citrullinemia, or argininosuccinic aciduria are shown in Table 1.
We also tested urine from nine patients with propionic
acidemia. ORA was detectable in all nine samples from
patients with propionic acidemia, with one sample borderline high at 25.54 mol/mmol creatinine (Table 1).
Despite of the wide calibration range used, it was necessary to further dilute the urine for 3 of 18 abnormal
patient samples because the ORA concentration exceeded
the highest point in the curve (2564 mol/L). Our results
were in excellent agreement with the stable-isotope
GC/MS method reported by McCann et al. (4 ) and with
the column-switching ion-exchange HPLC method reported by Ohba et al. (7 ).
Table 1. Urine concentration of ORA in controls and
patients.
ORA, mol/mmol creatinine
a
Controls
0–1 years
1–12 years
Adultsb
Patients
OCTD
Citrullinemia
Argininosuccinic aciduria
Propionic acidemia
a
b
Number of individuals.
10 males and 10 females.
n
Mean ⴞ SD
40
18
20
1.51
1.15
0.78
3
5
10
9
1.36
1.52
0.33
1519.7 190.8
1071.8 243.5
855.8 993.2
4.75 7.89
Range
0.05–5.88
0.05–5.98
0.31–1.60
1316–1694
657–1250
22–3271
0.783–25.54
501
Clinical Chemistry 49, No. 3, 2003
In conclusion, we have developed and validated a
simple, rapid, and specific method for the determination
of ORA in urine. The method can be used for routine
screening of hyperammonemia or urea cycle patients and
may also be used for heterozygosity testing for OCTD.
References
1. Webster DR, Becroft DMO, Suttle DP. Hereditary orotic aciduria and other
disorders of pyrimidine metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle
D, eds. The metabolic bases of inherited disease, 7th ed. New York:
McGraw-Hill, 1995:1799 – 837.
2. Potter M, Hammond JW, Sim KG, Green AK, Wilcken B. Ornithine carbamoyltransferase deficiency: improved sensitivity of testing for protein tolerance in
the diagnosis of heterozygotes. J Inherit Metab Dis 2001;24:5–14.
3. Brusilow SW, Horwich AL. Urea cycle enzymes. In: Scriver CR, Beaudet AL, Sly
WS, Valle D, eds. The metabolic bases of inherited disease, 7th ed. New
York: McGraw-Hill, 1995:1187–238.
4. McCann MT, Thompson MM, Gueron IC, Tuchman M. Quantification of orotic
acid in dried filter-paper urine samples by stable isotope dilution. Clin Chem
1995;41:739 – 43.
5. Brusilow SW, Huaser E. Simple method of measurement of orotic acid and
orotidine in urine. J Chromatogr 1989;493:388 –91.
6. Seiler N, Grauffel C, Therrien G, Sarhan S, Knoedgen B. Determination of
orotic in urine. J Chromatogr 1994;653:87–91.
7. Ohba S, Kidouchi K, Katoh T, Kibe T, Kobayashi M, Wada Y. Automated
determination of orotic acid, uracil and pseudouridine in urine by highperformance liquid chromatography with column switching. J Chromatogr
1991;568:325–32.
8. Ito T, van Kuilenburg ABP, Bootsma AH, Haasnoot AJ, van Cruchten A, Wada
Y, et al. Rapid screening of high-risk patients for disorders of purine and
pyrimidine metabolism using HPLC-electrospray tandem mass spectrometry
of liquid urine or urine-soaked filter paper strips. Clin Chem 2000;46:445–
52.
Accuracy of the Rapid Fetal Fibronectin TLi System in
Predicting Preterm Delivery, Veronica Luzzi, Kelly
Hankins, and Ann M. Gronowski* (Department of Pathology
and Immunology, Division of Laboratory Medicine,
Washington University School of Medicine, 660 South
Euclid Ave., Box 8118, St Louis, MO 63110; * author for
correspondence: fax 314-362-1461, e-mail gronowski@
pathology.wustl.edu)
Numerous studies have demonstrated that fetal fibronectin (fFN) is an excellent marker of preterm delivery with
a negative predictive value (NPV) 99% for predicting
delivery within 7 or 14 days in symptomatic women
(1–3 ). Interestingly, these studies have been performed by
sending the sample to Adeza Biomedical for testing using
an ELISA microtiter plate or by a rapid colloidal gold test
marketed only outside the US. In the US, however, the
only method available for rapid fFN analysis is the TLiTM
system (Adeza Biomedical). Our objective was to assess
the utility of the TLi rapid fFN system to predict delivery
in symptomatic patients within 7, 14, or 21 days.
We used 501 cervicovaginal samples consecutively received in the Barnes-Jewish Hospital Laboratory for physician-ordered fFN analysis during an 18-month period
for the study (February 2001–August 2002). From this
cohort, charts for 243 patients were available for review.
This constitutes the group of patients who delivered at
Barnes-Jewish Hospital. Study inclusion criteria included
the following: signs and symptoms of preterm labor,
intact membranes, cervical dilation 3 cm, and fFN
collection at 24 –35 weeks. These are all criteria for fFN
specimen collection included in the manufacturer’s package insert. Patients who had delivered by cesarean section
or other forms of induced delivery within 21 days of fFN
were excluded. If fFN measurement was performed more
than once, for simplicity we arbitrarily chose the measurement made closest to delivery to be included in the study.
A total of 15 patients had more than one fFN measurement performed. fFN measurements were performed
according to the manufacturer’s instructions. Briefly, the
patient sample was extracted from the Dacron swab into
an extraction buffer (provided by the manufacturer),
incubated at 37 °C in a water bath for 10 min, and filtered
through a plunger filter (provided by the manufacturer).
A 200-L portion of the filtered sample was applied to the
Rapid fFN cassette, and at 20 min the TLi analyzer
quantified the intensity of the lines. The instrument then
provided a positive or negative result. Institutional Review Board approval was obtained for this study.
Of 133 patients who met the inclusion criteria, 38 were
positive for fFN and 95 were negative. The mean (SD)
maternal ages at delivery for the positive and negative
groups were 25.5 (5.8) and 24.1 (6.1) years, respectively
(age ranges, 16.2– 40.8 and 12.9 – 41.4 years, respectively).
The mean gestational ages at the time of collection for the
positive and the negative groups were 29.6 weeks (SD, 2.4;
range, 24.1–33.3 weeks) and 30.0 weeks (SD, 2.8; range,
25.3–34.4 weeks), respectively. The mean gestational ages
at the time of delivery for the positive and the negative
groups were 35.3 weeks (SD, 3.7; range, 24.7–39.8 weeks)
and 37.4 weeks (SD, 2.1; range, 30.9 – 40 weeks), respectively. The mean times from collection to delivery for the
positive and negative groups were 39.9 days (SD, 22.5;
range, 0 –91 days) and 51.4 days (SD, 22.7; range, 2–107
days), respectively. Unpaired t-tests showed that only the
gestational age at the time of delivery and the time from
collection to delivery were significantly different between
the fFN-positive and -negative groups (P 0.003 and
0.017, respectively).
The NPV, positive predictive value (PPV), specificity,
and sensitivity for predicting delivery within 7, 14, and 21
days of testing are shown in Table 1. We also calculated
the likelihood ratio for a positive result [sensitivity/(1
specificity)]; the likelihood ratio was 2.12 for birth within
Table 1. NPV, PPV, specificity, and sensitivity of TLi Rapid
fFN for predicting delivery in symptomatic patients.
Days to delivery
Positive fFN, n
Negative fFN, n
Total, n
NPV, %
PPV, %
Specificity, %
Sensitivity, %
<7
>7
<14
>14
<21
>21
4
3
7
34
92
126
96.8
10.5
73
57
6
6
12
32
89
121
93.7
15.8
73.6
50
9
6
15
29
89
118
93.7
23.7
75.4
60