LC-MS/MS Analysis. Liquid Chromatography. Separation of
analytes was carried out on a Luna 3-µm Phenyl-Hexyl 2 × 150
mm column (Phenomenex, Eschborn, Germany) at a flow rate of
0.20 mL/min and column temperature of 45 °C. Eluent A was
MeOH/water (20/80), and B was MeOH/water (95/5), both
containing 1 mM TrBA and 1 mM acetic acid. The gradient was
as follows: 0 min, 25% B; 0.5 min, 40% B; 15 min, 48% B; 16 min,
100% B; 18 min, 100% B; 19 min, 25% B; and 28 min, 25% B.
Mass Spectrometry. Analytes were detected by electrospray
ionization tandem mass spectrometry (ESI-MS/MS) operated in
the negative ion mode with multiple reaction monitoring (MRM).
The capillary voltage was 3.0 KV, source temperature was 120
°C, desolvation temperature was 200 °C, nebulizer gas flow was
100 L/h, and the drying gas flow was set to 700 L/h. Argon
pressure in the collision cell was kept at 1.0 × 10-3 bar for MS/
MS measurements. Individual MS/MS parameters for each
compound are shown in Table 1.
Samples subjected to SPE were quantified by the standard
addition procedure over the extracts: 50 µL of a standard solution
containing increasing concentration of analytes was added to 200-
µL aliquots of each extract.
Exact mass determination with the Q-TOF instrument was
performed with a capillary voltage of 3.0 KV, a cone voltage of 30
V, a source temperature of 120 °C, desolvation temperature of
300 °C, cone gas flow of 50 L/h, and desolvation gas at 500 L/h.
When operated in MS/MS, the collision gas (Ar) pressure was
kept at 1.0 × 10-3, and the product ion spectra were recorded at
collision energies of 10, 20, and 30 eV. Aqueous phosphoric acid
(0.06%) was used as the mass lock reference.
Figure 1. Structures and acronyms of the 13 phosphoric acid mono-
and diesters covered in this study.
two different levels (0.05 and 1.0 µg/L), and WWTP influent and
effluent were spiked at 4 µg/L. Recovery experiments were
performed in triplicate. Another aliquot of each sample type was
extracted, and the same amounts of analytes were added to the
extracts to detect matrix effects.6,16
Biodegradation Tests. Microbial batch degradation tests
using activated sludge of a municipal wastewater treatment plant
as inoculate were performed with three triesters (TiBP, TPhP, or
TEHP) in 2.5-L glass bottles containing 2 L of deionized water,
50 µg/L of one of the triesters, 10 mg/L of fresh sludge, and a
phosphate buffer.17 Additionally, 50 mg/L of powdered milk was
added once a week as an additional carbon source. The open
bottles were stirred in the dark without active aeration. A poisoned
control (1 mg/L of Hg(II)) was performed for each of the trialkyl
phosphates.
Samples (∼40 mL) were taken at variable intervals, filtered
through 0.45-µm membrane filters (cellulose acetate; Sartorius,
Goettingen, Germany), and stored frozen until being analyzed by
LC-MS/MS (without SPE). Phosphoric acid di- and monoesters
were determined as described above, and trialkyl phosphates, as
described previously.6 The activity of the microorganisms was
controlled by determining the removal of powdered milk by
dissolved organic carbon (DOC) detection with a HighTOC
analyzer (Elementar, Hanau, Germany).
Solid-Phase Extraction. Solid-phase extraction (SPE)
was performed with an Autotrace SPE Workstation (Zymark,
Hopkinton, MA). Three SPE cartridges were initially tested: Oasis
HLB (60 mg; Waters, Milford, MA); Lichrolut EN (200 mg);
and Lichrolut RP-18 (500 mg), both from Merck (Darmstadt,
Germany).
RESULTS AND DISCUSSION
Determination by LC-ESI-MS/MS. Phosphoric acid di- and
monoesters (Figure 1) have one or two highly acidic functional
groups (pKa ) 1-27,8) that render these compounds very polar
and difficult to retain in reversed-phase LC. Ion-pair reversed-phase
liquid chromatography with volatile tributylamine (TrBA) has been
shown to be well-suited for the LC-MS determination of highly
acidic aromatic sulfonates13 by providing strong chromatographic
retention of acidic analytes and being sufficiently volatile to enable
coupling with electrospray ionization MS.18 It appeared promising
to use this approach also for the LC-MS analysis of phosphoric
acid mono- and diesters. Indeed, a good separation of the
commercially available three diester standards and the 10 di- and
monoesters synthesized in this work was obtained with 1 mM
TrBA (Figure 2). DCPP exhibits three peaks (one of them hardly
visible in Figure 2), which likely reflect three of the isomers of
TCPP in the technical mixture, from which the DCPP was
synthesized.
Extraction at Acidic pH. Samples were acidified to pH 2, and
SPE cartridges were sequentially conditioned with 5 mL of MeOH
and 5 mL of ultrapure water (pH 2). A 100-mL volume of sample
was then passed through at a flow rate of 10 mL/min and rinsed
with 2.5 mL ultrapure water (pH 2), and the cartridges were dried
for 30 min (N2) and eluted with the appropriate volume of MeOH.
Ion-Pair Extraction. For ion-pair solid-phase extraction (IP-
SPE), 11 mL of a buffer solution (2% MeOH, 50 mM TrBA, pH 5
with formic acid) was added to 100 mL of the samples, agitated,
and allowed to stand for ∼30 min prior to extraction. Cartridges
(Lichrolut RP-18, 500 mg) were conditioned with 5 mL of MeOH,
5 mL of ultrapure water, and 2 × 5 mL of buffer solution. The
sample (100 mL + 11 mL of buffer) was passed through the
cartridge, washed with 2.5 mL of buffer and 1 mL of ultrapure
water, dried for 30 min (N2), and eluted with 2 × 2 mL of MeOH.
The SPE extracts were concentrated in a Turbovap II nitrogen
concentrator (Zymark) to ∼0.3 mL, spiked with 40 µL of IS
solution (5 µg/mL), and diluted to 1 mL with ultrapure water.
Method Testing and Evaluation. Preliminary experiments for
selection of the appropriate SPE cartridge were performed with
treated wastewater at three spike levels (0, 0.4, and 2 µg/L).
For the determination of recoveries and for validation of the
final method, 100-mL aliquots of ultrapure water were spiked at
(16) Quintana, J. B.; Reemtsma, T. Rapid. Commun. Mass Spectrom. 2004, 18,
765-774.
(17) ISO 7827; In German Standard Methods for the Determination of Water,
Wastewater and Sludge; Beuth Verlag: Berlin, 1995.
(18) Reemtsma, T. J. Chromatogr., A 2003, 1000, 477-501.
(19) Herna´ndez, F.; Sancho, J. V.; Pozo, O. J. Rapid Commun. Mass Spectrom.
2002, 16, 1766-1773.
(20) Reemtsma, T. Trends Anal. Chem. 2001, 20, 533-542.
1646 Analytical Chemistry, Vol. 78, No. 5, March 1, 2006