Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Determination of hexabromocyclododecane isomers in sewage sludge by LC‐MS/MS

A method has been developed to determine α, β and γ diastereoisomers of hexabromocyclododecane (HBCD), a brominated flame retardant, in sewage sludge, based on the ultrasonic‐assisted extraction (UAE) of samples with dichloromethane–ACN (1:1) and the subsequent clean‐up of extracts by dispersive solid phase extraction with primary–secondary amine. Levels of HBCD diastereoisomers were determined by LC coupled with ESI MS/MS. Evaluation of the matrix effect showed a high ion suppression for all the diastereoisomers studied, which was counteracted by using a mixture of labelled HBCD diastereoisomers as internal standards. This method yielded recoveries in the range of 79.6–112.5% with SDs equal or lower than 9.1 The limits of detection were 0.3 ng/g for α‐ and β‐HBCD and 0.2 ng/g for γ‐HBCD. The developed method was successfully applied to 19 sludge samples collected from the province of Madrid (Spain). In most of the samples, β‐HBCD was below the method detection limit, whereas α‐ and γ‐HBCD were quantified in all of the sludge samples, and γ‐HBCD was the predominant diastereoisomer in 63% of the analyzed samples and α‐HBCD predominated in the rest.     

Chiral amines as reagents for HPLC-MS enantioseparation of chiral carboxylic acids

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.     

Simultaneous determination of dl-lactic acid and dl-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7?min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12?fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.     

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS): synthesis of derivatization reagents for carboxylic acids

The derivatization reagents for carboxylic acids, N‐(Pyridin‐3‐yl)hydrazinecarbothioamide, N‐[4‐(dimethylamino)phenyl]hydrazinecarbothioamide, 1‐(2‐aminoethyl)‐3‐(pyridin‐3‐yl)thiourea, 1‐(2‐aminoethyl)‐3‐[4‐(dimethylamino)phenyl]thiourea and 4‐(2‐aminoethyl)‐N‐phenylpiperazine‐1‐carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed‐phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision‐induced dissociation, and thus they were suitable for MS/MS detection. Copyright © 2010 John Wiley & Sons, Ltd.     

Enantioselective LC‐ESI‐MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective and reliable LC–ESI‐MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 μL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (R_s = 4.45) on a Chiralpak IG‐3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23–2022 ng/mL for each enantiomer. The intra‐ and inter‐day precisions were in the ranges of 3.38–13.6 and 5.11–13.8 for LDP and 4.19–11.8 and 8.89–10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.     

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC‐ESI‐MS/MS

A novel bromoquinolinium reagent, i.e. 1‐(3‐aminopropyl)‐3‐bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC‐MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N‐dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC‐MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI–MS/MS after conversion to MS/MS‐fragmentable derivatives

A liquid chromatography/electrospray ionization–tandem mass spectrometry‐based method was developed for the identification of the conjugation positions of the monoglucuronides of 25‐hydroxyvitamin D₃ [25(OH)D₃] and 24,25‐dihydroxyvitamin D₃ [24,25(OH)₂D₃] in human urine. The method employed derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D₃‐3‐ and ‐25‐glucuronides, or 24,25(OH)₂D₃‐3‐, ‐24‐ and ‐25‐glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D₃ and 24,25(OH)₂D₃, respectively. The 3‐glucuronide was not present for 24,25(OH)₂D₃, unlike 25(OH)D₃, thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH)₂D₃.     

Comparative metabolite profiling of carboxylic acids in rat urine by CE‐ESI MS/MS through positively pre‐charged and 2H‐coded derivatization

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE‐MS and a method for positively pre‐charged and ²H‐coded derivatization. Novel derivatizing reagents, N‐alkyl‐4‐aminomethyl‐pyridinum iodide (alkyl=butyl, butyl‐d9 or hexyl), containing quaternary amine and stable‐isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99–1.32% RSD of migration time) and eliminated problems with capillary coating known in CE‐MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N‐butyl‐4‐aminomethyl‐pyridinum iodide (BAMP) and N‐hexyl‐4‐aminomethyl‐pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE‐MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization–tandem mass spectrometry

The applicability of 3‐pyridyl isothiocyanate, p‐(dimethylamino)phenyl isothiocyanate and m‐nitrophenyl isothiocyanate as the derivatization reagents for amines in high‐performance liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed‐phase column and detected by ESI‐MS/MS. The C–N bond of the generated thiourea structure was efficiently cleaved by collision‐induced dissociation and gave the single and intense product ion. Among the three reagents, 3‐pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantification of D‐84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT,hSERT and hNET

MS Binding Assays represent a label‐free alternative to radioligand binding assays. In this study, we present an LC‐ESI‐MS/MS method for the quantification of (R,R)‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol [(R,R)‐D‐84, (R,R)‐ 1 ], (S,S)‐reboxetine [(S,S)‐ 2 ], and (S)‐citalopram [(S)‐ 3 ] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)‐D‐84], norepinephrine [NET, (S,S)‐reboxetine] and serotonin transporter [SERT, (S)‐citalopram], respectively. The developed LC‐ESI‐MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC‐ESI‐MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration‐based MS Binding Assays were performed for all three monoamine transporters based on this LC‐ESI‐MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)‐D‐84 toward hDAT, for (S,S)‐reboxetine toward hNET, and for (S)‐citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a CE‐MS2 method for the enantiomeric separation of L/D‐carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D /L ‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS² experiments with an ion trap analyser was carried out to confirm the identity of D /L ‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS² method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D ‐carnitine. The determination of L ‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.