Determination of 1-aminocyclopropane-1-carboxylic acid and its structural analogue by liquid chromatography and ion spray tandem mass spectrometry

Liquid chromatography coupled to ion spray tandem mass spectrometry was developed as a method for the simultaneous analysis of the amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) and its structural analogue, cyclopropane-1,1-dicarboxylic acid (CDA). ACC and CDA fragmentation as well as optimization of MS parameters were investigated in positive ion mode. In selective reaction monitoring mode the protonated molecule [M+H]+ was selected as parent ion for both ACC and CDA, while the immonium ion from ACC and the [M+H-H2O]+ ion from CDA were selected, respectively, as product ions. In spite of the high selectivity of MS/MS among the 20 protein amino acids potentially present with ACC and CDA in the plant material analyzed, Glu and Thr can interfere with the signal of ACC. As a result, their chromatographic separation is necessary. This was achieved in less than 4 min by ion-pair reversed-phase chromatography with nonafluoropentanoic acid as ion-pair reagent. A linear response within a concentration range of 1-5 mg l(-1) was observed for this LC method and the detection limit was found to be 20 pmol for ACC and 150 pmol for CDA (using a 20-microl loop). This methodology was successfully applied to the detection of ACC in apple tissue.     

Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ-ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20 mM borate buffer (pH 9.35) containing 40 mM sodium dodecyl sulfate and 10 mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 microM with a correlation of 0.9967. The concentration detection limit for ACC was 10 nM (signal-to-noise = 3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.     

Determination of airborne methyl isocyanate as dibutylamine or 1-(2-methoxyphenyl)piperazine derivatives by liquid and gas chromatography

The usefulness of a glass fibre filter method to collect airborne methyl isocyanate (MIC) was studied in laboratory experiments and in a workplace during manufacture of mineral wool insulation material. Filter collection was based on derivatisation in situ with 1-(2-methoxyphenyl)piperazine (2MP). 2MP impinger sampling was also evaluated in the workplace. Impinger sampling with dibutylamine (DBA) was used as an independent method. The samples were analysed by liquid and gas chromatography using various detection techniques: mass spectrometry, ultraviolet and electrochemical detection (LC-MS, LC-UV, LC-EchD and GC-MS). The sampling efficiency of 2MP filters for MIC varied with the origin of the glass fibre filter. Two Whatman filters (diameter 25 mm) with altogether 21 mumol of 2MP collected 100% of 9.8 micrograms of MIC during 30 min at an airflow rate of 1 l min-1. The workplace measurements were performed at two concentration levels, 0.003 and 0.09 mg m-3. The theoretical amounts of derivatisation reagent were 42 mumol (2MP filter), 52 mumol (2MP impinger) and 100 mumol (DBA). MIC concentrations were 20% lower by the 2MP methods compared with the DBA method (statistically significant difference). Breakthrough was 6% for the DBA method and 9% for the 2MP impinger method. To trap both MIC and isocyanic acid, which was also present in the workplace samples, a tenfold molar amount of 2MP reagent was used. The precision of sample preparation, expressed as relative standard deviation, was 3.5% (0.17 microgram ml-1, n = 6). The precision of sampling in the workplace was 15% (0.002 mg m-3, n = 6). The limit of quantification was 0.0006 mg m-3 for 30 l of air by the 2MP impinger method and 0.03-0.05 mg m-3 by the 2MP filter method. Hence, airborne MIC can be determined using 2MP as derivatisation reagent. Impinger sampling is preferable when low concentration levels are expected.     

Separation of Nile Blue-labelled fatty acids by CE with absorbance detection using a red light-emitting diode

The separation of fatty acids derivatised with Nile Blue (NB) by CE with detection using a red light-emitting diode (LED) was examined. NB was selected as the derivatisation agent due to its high molar absorption coefficient of 76,000 M(-1) cm(-1) at 633 nm, making it well suited for sensitive absorbance detection using a red 635 nm LED. NB-labelled fatty acids were separated by both MEKC using SDS micelles, i-PrOH and n-BuOH and by NACE in a number of solvents including MeOH, EtOH and ACN. The sensitivity of NACE was superior to MEKC, with detection limits of 5x10(-7)-7x10(-7) M obtained for each acid, approximately 20 times lower than the MEKC method. The NACE detection limits are approximately 100 times lower than previous reports on the separation of fatty acids by CE using indirect absorbance detection, ten times lower than using indirect fluorescence detection and are inferior only to those obtained using precapillary derivatisation and direct fluorescence detection. The efficiency of the NACE method was also superior to MEKC and allowed the separation of unsaturated fatty acids to be examined, although it was not possible to baseline-resolve linoleic (C18:2) and linolenic (C18:3) acids in a reasonable time. The method was used to analyse the fatty acid profile of two edible oils, namely sunflower and sesame oils, after alkali hydrolysis, where it was possible to identify both the saturated and unsaturated fatty acids in each sample.     

Analysis of trace levels of pesticides in rainwater by SPME and GC-tandem mass spectrometry after derivatisation with PFFBr

Solid-phase microextraction (SPME) was used for the analysis of some pesticides (bromoxynil, chlorotoluron, diuron, isoproturon, 2,4-MCPA, MCPP and 2,4-D) in rainwater after derivatisation with PFBBr and gas chromatography-ion trap mass spectrometry. The derivatisation procedure was optimized by testing different methods: direct derivatisation in the aqueous phase followed by SPME extraction, on-fibre derivatisation and derivatisation in the injector. The best result was obtained by headspace coating the PDMS/DVB fibre with PFBBr for 10 min followed by direct SPME extraction for 60 min at 68 °C (pH 2 and 75% NaCl). Good detection limits were obtained for all the compounds: these ranged between 10 and 1,000 ng L⁻¹ with a relatively high uncertainty due to the combination of derivatisation and SPME extraction steps. The optimized procedure was applied to the analysis of pesticides in rainwater and results obtained shows that this method is a fast and simple technique to assess the spatial and temporal variations of concentrations of pesticides in rainwater.     

Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatography-tandem mass spectrometry.

A LC-MS-MS method is presented to analyse simultaneously the metabolites of four nitrofuran antibacterial agents, furazolidone, furaltadone, nitrofurazone and nitrofurantoin in animal muscle tissue. Sample clean-up and analyte enrichment was performed by solid-phase extraction (SPE) with a polystyrene sorbent following combined hydrolysis of the protein-bound drug metabolites and derivatisation of the homogenised tissue with 2-nitrobenzaldehyde. Limits of detection of 0.5-5 ng g(-1) tissue and limits of determination of 2.5-10 ng g(-1) tissue were achieved using electrospray ionisation in positive mode. Analyte identification and quantification was performed according to EU guidelines, using multiple reaction monitoring (MRM) with one precursor ion and two product ions as identifiers. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analysis method. The yield of the derivatisation reaction was between 66 and 74% and the recovery of SPE reached 92-105% for all values between 10 and 500 ng g(-1). The developed analytical protocol has been applied to contaminated tissue samples of furazolidone- and furaltadone-treated pigs and allowed unequivocal identification and quantification of the metabolites.     

GC-MS determination of parabens,triclosan and methyl triclosan in water by in situ derivatisation and stir-bar sorptive extraction

Stir-bar sorptive extraction in combination with an in situ derivatisation reaction and thermal desorption–gas chromatography–mass spectrometry was successfully applied to determine parabens (methylparaben, isopropylparaben, n-propylparaben, butylparaben and benzylparaben), triclosan and methyltriclosan in water samples. This approach improves both the extraction efficiency and the sensitivity in the GC in a simple way since the derivatisation reaction occurs at the same time as the extraction procedure. The in situ derivatisation reaction was carried out with acetic anhydride under alkaline conditions. Thermal desorption parameters (cryofocusing temperature, desorption flow, desorption time, desorption temperature) were optimised using a Box–Behnken experimental design. All the analytes gave recoveries higher than 79%, except methylparaben (22%). The method afforded detection limits between 0.64 and 4.12 ng/L, with good reproducibility and accuracy values. The feasibility of the method for the determination of analytes in water samples was checked in tap water and untreated and treated wastewater.     

Determination of 1-aminocyclopropane-1-carboxylic Acid in Apple and Peach Extracts by High Performance Liquid Chromatography Coupled to a Fluorescence Detector

A new, sensitive, and simple HPLC method was described for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple and peach extracts. The method was based on the derivatization of ACC with fluorescamine in borate buffer systems of pH 8.0 to yield a highly fluorescent product. The experimental parameters affecting the derivatization reaction efficiency were optimized by fluorimetric analysis. Under optimum derivatization conditions, the derivative product of ACC in apple and peach extracts without extra purification was successfully chromatographed on a C-18 column by HPLC coupled to fluorescence detection. The derivative product of ACC with fluorescamine could be well separated from other concomitant substances or their derivatives that might interfere with the determination of ACC. The linearity of ACC was measured in the range of 23.82–238.82 µg · L^?1 with a good correlation coefficient of 0.9997. Based on signal-to-noise ratio of 3, a low detection limit of 5.0 µg · L^?1 could be reached. The proposed method was applied successfully to the determination of ACC in the crude apple and peach extracts without extra purification with low RSDs of 0.19–1.9% and good recoveries of 90.89–104.4%. The sensitive HPLC quantitative method is of great significance for the investigations of ACC metabolism in plants.     

Trace level determination of phenols as pentafluorobenzyl derivatives by gas chromatography-negative-ion chemical ionization mass spectrometry.

A method for the trace level determination of 11 phenols as pentafluorobenzyl (PFB) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical ionization (NICI) is described. First, the conditions for the PFB derivatisation of phenols were optimized and were found to be reaction temperature 80 degrees C and reaction time 5 h. Second, the detection limits using selected ion monitoring (SIM) were compared between trimethylsilylated (TMS) derivatives in the electron ionization (EI) mode and PFB derivatives in the NICI mode. The responses for the PFB derivatives in the NICI mode were 3.3-61 times higher than those of the TMS derivatives in the EI mode. The instrumental detection limits using NICI-SIM ranged from 2.6 to 290 fg. This method was applied to the analysis of phenols in river water using solid-phase extraction. The recoveries of the phenols from a river water sample spiked with standards at 100 ng l-1 with 2-chlorophenol, 4-chloro-3-methylphenol and pentachlorophenol and at 1000 ng l-1 with phenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 2-nitrophenol, 2,4,6-trichlorophenol and 4-nitrophenol were 81.2-106.3% (RSD 5.1-8.0%), except for 2-methyl-4,6-dinitrophenol and 2,4-dinitrophenol, for which the recoveries were 5.8 and 4.2%, respectively, because water contained in the acetone eluate interfered with the derivatisation of these compounds with two electrophilic nitro groups.     

Isolation of poly(propylene glycol)s from water for quantitative analysis by reversed-phase liquid chromatography

Procedures for the isolation of poly(propylene glycol)s (PPGs) from a water matrix have been developed. Solid-phase extraction with an octadecylsilica cartridge and elution with methanol or with a graphitised carbon black cartridge and elution with a mixture of dichloromethane-methanol (4:1) or liquid-liquid extraction with chloroform were all suitable for model samples. However, only liquid-liquid extraction was suitable both for model and real environmental samples. Methods for reversed-phase liquid chromatographic determination of PPGs based on derivatisation and ultraviolet or fluorescence detection have been developed. Four derivatisation agents [3,5-dinitrobenzoyl chloride, phenyl isocyanate, 1-naphthoyl chloride and 1-naphthyl isocyanate (NIC)] were tested. Only NIC was found to give good reproducibility as well as a satisfactory detection limit. Finally, a method with liquid-liquid extraction with chloroform, derivatisation with NIC and liquid chromatographic separation with fluorescence detection was established. The developed method shows a highly correlated linearity of the analytical signals of particular homologues within a wide concentration range (approximately from 0.01 to 10 mg l(-1)). The precision of measurements is satisfactory for homologues having 5-9 oxypropylene subunits and becomes worse with an increase in the number of oxypropylene subunits. The limit of detection is 2 microg l(-1) for the majority of homologues. The method is suitable for the isolation and quantitative determination of PPGs in river water samples and as a tool for biodegradation testing.     

Injection port silylation of γ-hydroxybutyrate and trans-hydroxycrotonic acid: conditions optimisation and characterisation of the di-tert-butyldimethylsilyl derivatives by GC-MS

Silylation is usually carried out on γ-hydroxybutyrate (GHB) for its analysis by Gas Chromatography/Mass Spectrometry (GCMS) and requires potentially long incubation times before injection during which the derivatisation reagent and derivatives (such as trimethyl-silyl compounds) can hydrolyse. Moreover, alternative internal standards (IS) are often useful depending on sample matrices, extraction/purification procedures, commercial availability and price. This study evaluated the possibility of silylating GHB with an injection port derivatisation procedure using N-methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (TBCS) as the derivatisation reagent, producing di-tert-butyldimethyl-silyl derivatives as a novel means of analyzing GHB. In parallel, trans-hydroxycrotonic acid (t-HCA) was investigated as a potential IS for GHB quantification. Analyses were carried out with a temperature programmable injector and the GHB(t-BDMS)(2) and t-HCA(t-BDMS)(2) derivatives were successfully produced, characterised and derivatisation conditions optimised. t-HCA behaved very similarly to GHB through the derivatisation processes and was used as the IS for the determination of urinary endogenous GHB concentrations in human subjects where the method showed a limit of detection of 0.049 μg mL(-1), a limit of quantification of 0.162 μg mL(-1), and a limit of confirmation of 1.33 μg mL(-1), suitable for toxicological GHB concentration determination.     

Application of ethyl chloroformate derivatization for solid-phase microextraction-gas chromatography-mass spectrometric determination of bisphenol-A in water and milk samples

A simple and rapid analytical method based on in-matrix ethyl chloroformate (ECF) derivatization has been developed for the quantitative determination of bisphenol-A (BPA) in milk and water samples. The samples containing BPA were derivatised with ECF in the presence of pyridine for 20 s at room temperature, and the non-polar derivative thus formed was extracted using polydimethylsiloxane solid-phase microextraction (SPME) fibres with thicknesses of 100 μm followed by analysis using gas chromatography-mass spectrometry. Three alkyl chloroformates (methyl, ethyl and isobutyl chloroformate) were tested for optimum derivatisation yields, and ECF has been found to be optimum for the derivatisation of BPA. Several parameters such as amount of ECF, pyridine and reaction time as well as SPME parameters were studied and optimised in the present work. The limit of detection for BPA in milk and water samples was found to be 0.1 and 0.01 μg L(-1), respectively, with a signal-to-noise ratio of 3:1. The limit of quantitation for BPA in milk and water was found to be 0.38 and 0.052 μg L(-1), respectively, with a signal-to-noise ratio of 10:1. In conclusion, the method developed was found to be rapid, reliable and cost-effective in comparison to silylation and highly suitable for the routine analysis of BPA by various food and environmental laboratories.     

Determination of mercury ion by MEKC with on-column derivatisation and LIF detection

A simple and automatic method for the determination of mercury ion by MEKC with on-column derivatisation and LIF detection is described in the present paper. In this method, solutions of a nonfluorescent rhodamine derivative and mercury ion were injected individually and mixed by applying a short voltage. Subsequently, the mercury ions reacted with the nonfluorescent rhodamine derivative to produce strongly fluorescent product. The resulting product was then removed by EOF and micelles towards the detection window and detected by LIF detector. The experimental conditions in terms of the concentration and injection volume ratios between mercury ion and derivatisation reagent, the mixing time and waiting time for the on-column reaction were optimised. The optimal conditions were determined as follows: the concentration and injection volume ratios between mercury ion and derivatisation reagent were 1:20 and 10:1, respectively; the mixing time was 40 s under the applied voltage of 5 kV; the waiting time was proved unnecessary. The detection limit for mercury ion was 5 x 10(-8) M, and the total analysis time was less than 10 min.     

Determination of phytosiderophores by anion-exchange chromatography with pulsed amperometric detection

Phytosiderophores of the mugineic acid family are separated by anion-exchange HPLC using NaOH gradient elution. Separation of mugineic acid (MA), 2'-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA) and 3-epi-hydroxymugineic acid (epi-HMA) is obtained within 15 min. Detection of the underivatised phytosiderophores is performed using pulsed amperometric detection (PAD) at pH 13. The sensitivity of the detection increases in the order DMA < MA < HMA < epi-HMA and respective detection limits of 5 microM (DMA), 1 microM (MA) and < 0.5 microM (HMA, epi-HMA) are achieved. PAD is discussed in comparison with the well-established fluorimetric detection method after post-column derivatisation with ortho-phthaldialdehyde. The main advantage of PAD is the simplicity of the method (no derivatisation) and the high sensitivity for hydroxylated mugineic acids. The method is used for the determination of phytosiderophores in root washings of iron-deficient and non-deficient wheat and barley plants.     

Determination of urea using high-performance liquid chromatography with fluorescence detection after automated derivatisation with xanthydrol

A high-performance liquid chromatography (HPLC) method for the determination of urea that incorporates automated derivatisation with xanthydrol (9H-xanthen-9-ol) is described. Unlike the classic xanthydrol approach for the determination of urea, which involves the precipitation of dixanthylurea (N,N'-di-9H-xanthen-9-ylurea), the derivatisation procedure employed in this method produces N-9H-xanthen-9-ylurea, which remains in solution and can be quantified using fluorescence detection (lambda(ex)=213 nm; lambda(em)=308 nm) after chromatographic separation from interferences. The limit of detection for urea was 5 x 10(-8) M (0.003 mg L(-1)). This method was applied to the determination of urea in human and animal urine and in wine.     

Application of ethyl chloroformate derivatization for solid-phase microextraction–gas chromatography–mass spectrometric determination of bisphenol-A in water and milk samples

A simple and rapid analytical method based on in-matrix ethyl chloroformate (ECF) derivatization has been developed for the quantitative determination of bisphenol-A (BPA) in milk and water samples. The samples containing BPA were derivatised with ECF in the presence of pyridine for 20 s at room temperature, and the non-polar derivative thus formed was extracted using polydimethylsiloxane solid-phase microextraction (SPME) fibres with thicknesses of 100 μm followed by analysis using gas chromatography–mass spectrometry. Three alkyl chloroformates (methyl, ethyl and isobutyl chloroformate) were tested for optimum derivatisation yields, and ECF has been found to be optimum for the derivatisation of BPA. Several parameters such as amount of ECF, pyridine and reaction time as well as SPME parameters were studied and optimised in the present work. The limit of detection for BPA in milk and water samples was found to be 0.1 and 0.01 μg L⁻¹, respectively, with a signal-to-noise ratio of 3:1. The limit of quantitation for BPA in milk and water was found to be 0.38 and 0.052 μg L⁻¹, respectively, with a signal-to-noise ratio of 10:1. In conclusion, the method developed was found to be rapid, reliable and cost-effective in comparison to silylation and highly suitable for the routine analysis of BPA by various food and environmental laboratories.     

Analysis of acrylamide in food samples by capillary zone electrophoresis

Conditions for the determination of acrylamide (AA) after derivatisation with 2-mercaptobenzoic acid by capillary zone electrophoresis were established. A derivatisation reagent-acrylamide ratio of 35:1 was selected as optimum and the reagent excess was not removed as it did not affect the determination of acrylamide by CZE. The best separation was achieved using a 40 mM phosphate buffer at pH 8.0, working at 25 kV in un-coated fused silica capillaries. Linear calibration curves over the range studied (0.3-100 microg mL(-1)), the limit of detection (0.07 microg mL(-1)), and both run-to-run (RSD values of 5.8 and 2.2% for concentration at low and medium concentration levels, respectively) and day-to-day precisions (up to 11.2 and 6.7% at low and medium concentration levels, respectively) were established. Finally, the applicability of the CZE proposed methodology was demonstrated by analyzing levels of acrylamide present in different foodstuff products such as home made french fries, breakfast cereals and biscuits.     

Determination of carbonyl compounds in beer by derivatisation and headspace solid-phase microextraction in combination with gas chromatography and mass spectrometry

Headspace solid-phase microextraction (SPME) followed by gas chromatography and mass spectrometry was applied for quantification of 41 chemically diverse carbonyl compounds in beer. Therefore, in-solution derivatisation with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) combined with SPME was optimised for fibre selection, PFBHA concentration, extraction temperature and time and ionic strength. Afterwards, the method was calibrated and validated successfully and extraction efficiency was compared to sampling with on-fibre derivatisation. In-solution derivatisation enabled the detection of several compounds that were poorly extracted with on-fibre derivatisation such as 5-hydroxymethylfurfural, acrolein, hydroxyacetone, acetoin, glyoxal and methylglyoxal. Others, especially (E)-2-nonenal, were extracted better with on-fibre derivatisation.     

Solid-phase micro-extraction procedure for the determination of 1,3-dichloro-2-propanol in water by on-fibre derivatisation with bis(trimethylsilyl)trifluoroacetamide

The headspace solid-phase micro-extraction technique with on-fibre derivatisation followed by gas chromatography-tandem mass spectrometry has been evaluated for the analysis of 1,3-dichloro-2-propanol in water. An asymmetric factorial design has been performed to study the influence of five experimental factors: extraction time and temperature, derivatisation time and temperature and pH. The best extraction performance is achieved in the headspace mode, with 5 mL stirred water samples (pH 4) containing 1.3 g of NaCl, equilibrated for 30 min at 25 °C, using divinylbenzene-carboxen-polydimethylsiloxane as the fibre coating. On-fibre derivatisation has been used for the first time with 50 μL of bis(trimethylsilyl)trifluoroacetamide at 25 °C during 15 min, leading to effective yields. The proposed method provides high sensitivity, good linearity and repeatability (relative standard deviation of 5.1% for 10 ng mL⁻¹ and n = 5). The limits of detection and quantification were 0.4 and 1.4 ng mL⁻¹, respectively. Analytical recoveries obtained for different water samples were approx. 100%.     

Characterisation of ethoxylated fatty chains of anionic surfactants and determination of residual ethoxylated fatty alcohols

Bachus et al. [1] recently described a new derivatisation method using 2-furoyl chloride for the characterisation of mixtures of polyethoxylated alcohols and their corresponding sulfates. This paper deals with the control of the derivatisation steps; hydrolysis and extraction conditions were optimised. The method is extended to the characterisation of alkyl sulfosuccinates, alkyl sulfoacetates and alkyl phosphates and to the analysis of residual polyethoxylated alcohols in surfactants. Extraction of non-ionic compounds using solid-phase extraction cartridges was performed before derivatisation. Residual amounts of alcohol were determined in five commercial anionic surfactants. Moreover, direct derivatisation without preliminary SPE in the same anionic surfactants proved to be efficient for dry samples.