a b s t r a c t
The present paper describes an analytical method for the determination of 2 widely administered anti- cancer drugs, ifosfamide and cyclophosphamide, contained in sewage sludge. The method relies on the extraction from the solid matrix by pressurized liquid extraction, sample puriﬁcation by solid-phase extraction and analysis by ultra highperformanceliquidchromatography coupled with tandem massspectrometry. The different parameters affecting the extraction efﬁciency were optimized using an experimental design. Solvent nature was the most decisive factor for the extraction but interactions between some parameters also appeared very inﬂuent. The method was applied to seven different types of sludge for validation. The performances of the analytical method displayed high variability between sludges with limits of detection spanning more than one order of magnitude and conﬁrming the rele- vance of multi-sample validation. Matrix effect has been determined as the most limiting analytical step for quantiﬁcation with different extent depending on analyte and sludge nature. For each analyte, the use of deuterated standard spiked at the very beginning ensured the complete compensation of losses regardless of the sample nature. The suitability of the method between freshly spiked and aged sam- ples has also been veriﬁed. The optimized method was applied to different sludge samples to determine the environmental levels of anticancer drugs. The compounds were detected in some samples reaching 42.5 mg/kg DM in ifosfamide for the most contaminated sample.
a b s t r a c t
The present paper describes an analytical method for the determination of 2 widely administered anti- cancer drugs, ifosfamide and cyclophosphamide, contained in sewage sludge. The method relies on the extraction from the solid matrix by pressurized liquid extraction, sample purification by solid-phase extraction and analysis by ultra highperformanceliquidchromatography coupled with tandem massspectrometry. The different parameters affecting the extraction efficiency were optimized using an experimental design. Solvent nature was the most decisive factor for the extraction but interactions between some parameters also appeared very influent. The method was applied to seven different types of sludge for validation. The performances of the analytical method displayed high variability between sludges with limits of detection spanning more than one order of magnitude and confirming the rele- vance of multi-sample validation. Matrix effect has been determined as the most limiting analytical step for quantification with different extent depending on analyte and sludge nature. For each analyte, the use of deuterated standard spiked at the very beginning ensured the complete compensation of losses regardless of the sample nature. The suitability of the method between freshly spiked and aged sam- ples has also been verified. The optimized method was applied to different sludge samples to determine the environmental levels of anticancer drugs. The compounds were detected in some samples reaching 42.5 mg/kg DM in ifosfamide for the most contaminated sample.
A method based on high-performanceliquidchromatography coupled with diode array detection and electrospray ionization massspectrometry (HPLC-DAD-ESI-MS) following fractionation by chromatog- raphy on a Sephadex LH-20 column has been developed to determine the phenolic composition of fruit of Eucalyptus globulus growing in Algeria. The presence of 18 gallotannins, 26 ellagitannins, and 2 flavonols was established. Tentative identification is provided for these compounds on the basis of UV-visible spectra and massspectrometry data. Most compounds described in this study have not previously detected in fruit of E. globulus. Moreover, this is the first report of methyl digalloyl diglucose, 3,3 0 -O-dimethylellagic acid 4-O-β-glucopyranoside, ellagic acid hexose, methyl ellagic acid pentose, methyltetragalloylglucose, and valoneic acid isomers (sanguisorbic, flavogallic acid dilactone) in the genus Eucalyptus. Quantitatively, ellagic acid and its derivatives, including ellagitannins, are largely predominant.
A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performanceliquidchromatography combined with electrospray ionization massspectrometry is described. The sample preparation includes a liquid–liquid extraction with methyl tert-butylmethyl ether after addition of 2 M sodium hydroxide. Ethylmethylglycinexy- lidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and acetonitrile as mobile phases. Good linearity was observed in the range of 2.5–1000 ng ml −1 for lidocaine in both dog and horse plasma. For MEGX, linear calibration curves were obtained in the range of 5–1000 ng ml −1 and 20–1000 ng ml −1 for dog and horse plasma, respectively. In dog and horse plasma good linearity was observed in the range of 200–1500 ng ml −1 for GX. The limit of quantification (LOQ) in dog plasma for lidocaine, MEGX and GX was set at 2.5 ng ml −1 , 20 ng ml −1 and 200 ng ml −1 , respectively. For horse plasma a limit of quantification of 2.5 ng ml −1 , 5 ng ml −1 and 200 ng ml −1 was achieved for lidocaine, MEGX and GX, respectively. In dog plasma, the limit of detection (LOD) was found to be 0.8 ng ml −1 , 2.3 ng ml −1 and 55 ng ml −1 for lidocaine, MEGX and GX, respectively. In horse plasma the LOD’s found for lidocaine, MEGX and GX, were 1.1 ng ml −1 , 0.5 ng ml −1 and 13 ng ml −1 , respectively. The method was shown to be of use in pharmacokinetic studies after application of a transdermal patch in dogs and after an intravenous infusion in horses.
4 Products and Sectors Department, Walloon Agricultural Research Center, Rue de Liroux 8, 5030 Gembloux,
Cow's milk can be used as a potential source of equol in the human diet. In order to study human intake, however, it is necessary to develop a reliable and sensitive analytical method. This paper reports on the validation of an analytical method using ultra-performanceliquidchromatography coupled with a tandem massspectrometry detector to quantify the equol in commercial milks (raw, whole, semi-skimmed, and skimmed milk). The equol was initially released using enzymatic hydrolysis, and it was then extracted using a double liquid/liquid extraction. The analytical method produced a linear calibration curve with a high correlation coefficient (R 2 ≥0.996) between 5 and 1,000 ng.mL -1 . Good intra- and inter-day precision (≤5.3% and≤5.2%, respectively) and accuracy (≤8.6%) were achieved. The recovery rate differed slightly among the different types of milk, ranging between 60.6± 1.09% and 82.3±5.21%. Good method repeatability was observed (≤15%). There was neither matrix effect nor carry-over effect, and the sample extracts were stable for at least 7 days of storage at -21 °C and 5 °C. The method proved to be specific, sensitive, precise, and accurate and was used for the first time to quantify the equol content in Belgian commercial cow's milk. In all the samples analyzed, equol was present at a concentration ≥10 ng.mL -1 and had a significantly higher content in organic than in conventional milk. The study also found that the mean concentrations of equol were similar for each type of commercial conventional cow's milk.
Due to the high concentration of cannabinoids in matrix and the lack of adequate blank matrices, it is not practical or affordable to spike cannabinoids or internal standards directly into matrix prior to extraction. A common method of quanti- tation is to prepare calibration curves in solvent and dilute matrix extracts into the calibration curve range. Internal stan- dards may then be added to calibration standards, QC sam- ples, and diluted matrix extracts as the last preparation step. We opted for this approach with calibration curves and can- nabis extracts diluted in methanol, and believe it is a reason- able compromise between cost and performance. While only three isotopic derivatives were available commercially at the time of this work, it is recommended that additional internal standards be incorporated into the method as they become available.
al other methods are also available for quantitation of 2.2. Instruments and methods sphingolipid metabolites by liquidchromatography
(LC). Since the ceramides lack a chromophore group The high-performanceliquid chromatograph to allow a sensitive UV measurement, fluorescent or (HPLC) is a HP 1100 series; it is equipped with a radioisotope detection after derivatization were per- binary pump, a vacuum degasser, a thermostated ferred [9–11] All the above described procedures are column compartment and an autosampler, all from time-consuming and tricky derivatization steps might Agilent Technologies (Waldbronn, Germany). The often be source of error hampering the accurate HPLC separations were performed at 708C on a RP determination of the ceramides levels. The use of an C 18 Nucleosil AB column (5 mm, 7032 mm I.D.)
Similar protocols were implemented for derivatization with PHMB. 53
Highperformanceliquidchromatography and massspectrometry
Chromatographic separation was performed using an Agilent series 1100 HPLC system. A Zorbax eclipse XDB-C18 analytical column (4.6 x 150 mm, 5 µm) and a guard column of the same material were used. Mobile phase consisted of A: water containing 0.1% formic acid and B: methanol containing 0.1% formic acid. The gradient was as follows: 5 % solvent B for 1 min; 9 min linear increase up to 40 % solvent B; 10 min linear increase up to 90% solvent B; 7 min linear decrease to 5 % solvent B; 8 min hold at 5% solvent B. Flow rate was 0.2 mL/min. Injection volume was 10 µL.
Selenium (Se) is an essential trace element. Several reports like the NPC  clinical trial support that high dietary intake of organic Se has beneficial effects, notably protective effects against various cancers. Nonetheless the average selenium dietary intake is lower than the recommended intake value. For example the average selenium intake of Belgium people is ranged from 28 to 61 µg.day -1 per person although the recommended intake value is 70
Liquid chro ma tog ra phy–mass spec trom e try was applied to deter mine the action pattern of dif fer ent chon droi tin lyases. Two com mer cial enzymes, chon dro itin ase ABC (Pro teus vul ga ris) and chon dro itin- ase ACII (Arth ro bac ter au res cens), hav ing action pat terns pre vi ously deter mined by vis cos i me try and gel elec tro pho re sis were first exam ined. Next, the action pat terns of recombinant lyases, chon dro itin ase ABC from Bac te roi des theta i ota omi cron (expressed in Esch e richia coli) and chon dro itin ase AC from Fla vo bac te- rium hep ar i num (expressed in its original host), were exam ined. Chon droi tin sul fate A (CS-A, also known as chon droi tin-4-sul fate) was used as the sub strate for these four lyases. Ali quots taken at var i ous time points were ana lyzed. The prod ucts of chon dro itin ase ABC (P. vul ga ris) and chon dro itin ase AC (F. hep- ar i num) con tained unsat u rated oli go sac cha rides of sizes rang ing from disac cha ride to deca sac cha ride, dem on strat ing that both are end o lyt ic enzymes. The prod ucts affor ded by chon dro itin ase ABC (B. theta- i ota omi cron) and chon dro itin ase ACII (A. au res cens) con tained pri mar ily unsat u rated disac cha ride. These two ex o lyt ic enzymes showed dif fer ent minor prod ucts, sug gest ing some sub tle spec i fic ity dif fer ences between the actions of these two ex o lyt ic lyases on chon droi tin sul fate A.
for such difficult matrix as honey [1,6-8]. However, LLE requires large amounts of solvent, is time consuming, laborious and not well suited for automation [1,6]. As an alternative, solid phase extraction (SPE) or matrix solid phase dispersion (MSPD) has been widely developed in the past decades. Their simplicity, robustness, rapidity and low solvent consumption are attractive parameters for the analytical chemist. Whereas SPE is based on the retention of selected analytes on cartridge sorbents and their elution with appropriate solvent, MSPD consists in the dispersion of the matrix on a free-adsorbent and its homogeneous packing on a column prior to elution of compounds with organic solvent allowing the extraction of semi-solid and solid samples [9,10]. The other side of the coin is its poor capability for high sample input . Solid-phase microextraction (SPME) has also been studied for pesticide analysis in honey [2,12] but showed sample input limitations and relatively high limit of detection . Supercritical fluid extraction (SEE) and stir-bar sorptive extraction (SBSE) still remain quite marginal in this area until now [5,13]. In this study, an on-column liquid-liquid extraction (OCLLE) method has been tested as it seemed to combine advantages of LLE, SPE and SPME.
Institute for National Measurement Standards, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R9
Selenomethionine (SeMet) and methionine (Met), liber- ated by acid hydrolysis of selenium-enriched yeast, were quantified by liquidchromatography-massspectrometry (LC/MS) using standard additions calibrations as well as isotope dilution (ID) based on species-specific 13 C-enriched spikes. LC inductively coupled plasma massspectrometry (ICPMS) was also employed for the quan- tification of SeMet, and 74 Se-enriched SeMet was used for ID calibration. The results were evaluated to ascertain the feasibility of using these methods in a campaign to certify selenized yeast. Good agreement was found between the methods, which, when averaged, gave concentrations of 5482.2 ( 101 and 3256.9 ( 217.4 µg/g for Met and SeMet, respectively. This corresponds to a 1.68:1 Met- to-SeMet ratio in the yeast. Quantification by ID LC/MS and LC ICPMS yields the most precise sets of results with relative standard deviations in the range 0.5-1.3% (n ) 6). A total selenium concentration of 2064.6 ( 45.4 µg/g was obtained for this yeast material. The extraction efficiency and a mass balance budget were determined. Acid hydrolysis liberated 81.0% of the total selenium present. SeMet comprised 79.0% of the extracted sele- nium and 63.9% of the total selenium present in the yeast. Since the discovery in 1957 that selenium is an essential trace element for mammals, 1 it has been increasingly implicated in a beneficial role for human health. Selenium is an essential trace element, present in several proteins as selenocysteine, and selenium availability is directly linked to the regulation of sele- noprotein expression. 2 Due to low levels of selenium available in the average human diet in certain geographic areas, 3 it has become increasingly popular to use supplements. As well as maintaining
digestion after a reduction step will help to reduce the complexity of hCG-sample and favor the ionization of hCGβ isoforms.
Nevertheless, more than 30 isoforms were detected for both r-hCGα and u-hCGα, and some of the main hCGα isoforms were identified by mass matching. The next step of this work will be to carry out analysis with a higher resolution MS analyzer, such as a Fourier Transform Ion Cyclotron Resonance (FT-ICR). The higher sensitivity and, above all, the higher accuracy of the MS should allow the identification of much more isoforms, without having errors due to the overlapping of isotopic patterns. Nevertheless, the transfer of the RPLC method to a nanoscale format will be required to facilitate the coupling with the FT-ICR MS and to increase the sensitivity of the most minor isoforms. However, the optimization of the different LC parameters carried out in this study will facilitate this step.
COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY COMBINED WITH HIGH-RESOLUTION MASSSPECTROMETRY: EXPLORING MASS DEFECT INFORMATION TO CHARACTERIZE BASE OIL
Anupam Giri 1 , Marion Courtiade 2 , Amandine Racaud 2 , Jean-François Focant 1
causing shifts in the retention times. The eﬀect of sulfate on the GC/MS/MS method was studied. The derivatization of 6 ng/g Se was carried out in a solution with Na 2 SO 4 . The sulfate
did not interfere signiﬁcantly with the yield of derivatization/ extraction ( Figure S7 ), and no perturbation on the GC/MS/ MS chromatogram was observed. With 2% sulfate, the matrix suppression was only 5%. This result further conﬁrms that the GC/MS/MS method can eﬃciently handle samples with a high salt content. A wastewater sample from a gold mine facility was also analyzed. While no incurred SeCN − was found,
Figure 2. Base-peak chromatograms of positive ion scans (left; m/z 450-1150) and precursor ion scans (right; m/z 135 for Adda-containing compounds or m/z 265 for ADMAdda-containing compounds) for (a) SV-81 (Category 1) and (b) SV-02 (Category 3). See Table 1 for definition of categories.
Table 1. Microbial mat samples from Svalbard, the extracted mass of lyophilized material, their toxin content as determined by enzyme-linked immune sorbent assays (ELISAs), the liquidchromatographymassspectrometry (LC-MS) precursor ion screening method as well as a detailed LC-MS/MS analysis and the detection of genes involved in toxin production. Categories of the LC-MS precursor ion scan: (1) Microcystin (MC) likely to be present in the sample, (2) MC possibly present in the sample, and (3) MC absent from the sample. Genes: Non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), microcystin gene E (mcyE), microcystin gene B (mcyB), and saxitoxin gene A (sxtA). Structures of MC congeners can be found in Figure 1 .
improved by using a larger pool of (carefully selected) ISs. We would recommend to inject at least three ISs per major chemical class and one per minor one. One IS per intensity level (high, medium, or low) and one per retention window (1tR divided into three equal parts). One could also ﬁnd it useful to implement some recently developed procedures that combine various ISs (CCMN96 and NOMIS97). Absolute quantiﬁcation, which would require one IS per compound, seems out of reach for global proﬁling in the actual state of the ﬁeld. Finally, the method developed for the untargeted analysis of 30 μL of serum samples should be adaptable to other matrices common in metabolomics, such as plasma and urine.37,50 QC System. A QC system repeats QC sample injections to monitor the stability of the performances over time, providing assurance of the quality 16 of the data. It can also be used to increase it by correcting for the
Acquisition setup, processing and visualisation of imaging data were performed using High Definition Imaging (HDI) 1.5 (Waters Corporation, Manchester, UK). Data were acquired and mined using MassLynx version 4.2 (Waters Corporation, Manchester, UK). Further data mining was carried out using DriftScope 2.9 (Waters Corporation, Manchester, UK) to visualise the IMS dimension of the data. Datasets were re-arranged by drift time (either bins or milliseconds) to evaluate the different drift time (Dt) of the compounds of interest. Also, using DriftScope, it was possible to extract distinct regions in the m/z vs. Dt 2D-plot corresponding to different classes of molecules present on the surface of the samples.