A data-independent acquisition approach based on HRMS to explore the biodegradation process of organic micropollutants involved in a biological ion-exchange drinking water filter.

Drinking water producers continuously develop innovative treatment processes to effectively remove organic micropollutants from raw water. Biological ion-exchange (BIEX) water treatment is one of these new techniques under development and showing great potential. In order to investigate if biodegradation is highly involved in such a removal technique, cultures were prepared with microorganisms sampled on the resins of a BIEX filter. Then, organic micropollutants were spiked into these cultures and their (bio)degradation was followed over 30 days by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The purpose of this study was firstly to develop an analytical method using UHPLC-HRMS able to monitor the degradation of three spiked organic micropollutants in culture. Beyond quantification, this method allowed the simultaneous recording of fragmentation information via the use of a data-independent acquisition approach to perform a non-exhaustive search of transformation products related to the spiked micropollutants in culture aliquots. Secondly, a data treatment approach was developed to process raw spectral data generated by aliquots analysis by optimizing the precursor isolation mass windows, the accurate mass tolerance, peak intensity thresholds and choice of database. The use of this new method with a post-data acquisition treatment approach completed by the exhaustive study of fragmentation spectra allowed the tentative identification of 11 transformation products related to the spiked compounds. Finally, 16S rRNA gene amplicon sequencing revealed that bacterial genera known for their ability to degrade the spiked micropollutants were present in the microbial community of the BIEX drinking water filter.

Evaluation of ELISA-based method for total anabaenopeptins determination and comparative analysis with on-line SPE-UHPLC-HRMS in freshwater cyanobacterial blooms.

Anabaenopeptins (APs) are bioactive cyanopeptides of emerging concern produced by cyanobacteria. The research for analytical development has recently gained in importance due to their abundance in toxic cyanobacterial blooms. A new commercial enzyme-linked immunosorbent assay kit for the determination of total APs (APtot ELISA) has been released promising a rapid response with good cost efficiency for the routine monitoring of uncommon cyanopeptides. The present study explores the suitability of this new kit in comparison with a validated quantitative analytical method based on liquid chromatography coupled to mass spectrometry (LC-MS). The validation results were comparable with both methods for accuracy, precision, and calibration. Method detection limits were more sensitive using LC-MS specifically evaluated at 0.011 and 0.013 µg L-1 for AP-A and B respectively, compared to APtot ELISA evaluated at 0.10 µg L-1 for total of the two. For APtot ELISA, results were independent from the matrix; however, a systematic signal response was measured in blanks, requiring a blank subtraction in data treatment. Cross-reactivity of APtot ELISA was investigated by analyzing ten cyanopeptides selected for their abundance and diversity. Cyanopeptolin A (CP-A), nodularin-R (NOD), microcystin (MC)-RR, [Asp3]RR, and HilR showed cross-reactivity with an average overestimation going from 25 to 66%. Considering the contribution of cross-reactive cyanopeptides, thirteen lake samples out of fifteen showed higher concentrations using APtot ELISA with overestimation values up to 2261% compared to LC-MS. In light of this study results, LC-MS should still be preconized for the study and monitoring of APs when sensitivity and specificity are needed.

A Data-Independent Methodology for the Structural Characterization of Microcystins and Anabaenopeptins Leading to the Identification of Four New Congeners.

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu1, Ser7] MC-HtyR, [Asp3]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L-1. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.

Agro-industrial residues as a unique support in a sand filter to enhance the bioactivity to remove microcystin-Leucine aRginine and organics.

In the past, the versatility of a biosand filter has been successfully checked to counter suspended solids, metals, dissolved organic carbon (DOC), coliforms and other water quality parameters (WQPs) from the drinking water sources. In this study, cyanotoxin in the form of microcystin-LR (MC-LR) along with above-mentioned WQPs including nitrate, nitrite, and ammonia are analyzed for their removal using agro-residue based biosand filters (ARSFs) for 49 days (7 cycles). Three different agro-residue materials (ARMs) viz. deinking sludge (DSF), hemp fiber (HFF) and paper-pulp dry sludge (PPF) were used as the support material (top 5 cm) along with sand (49 cm) as the primary filter media to enhance the overall bioactivity. This enhancement in bioactivity is hypothesized to remove more MC-LR, DOC, coliform along with efficient nitrification/denitrification. Native bacterial community isolated from the filtration unit of a drinking water treatment plant (Chryseobacterium sp. and Pseudomonas fragi = X) along with the MC-LR-degrader: Arthrobacter ramosus (which was screened as the best biofilm-former among two other MC-LR-degraders tested) were used to inoculate the filters (all three ARSFs). Overall, DSF performed the best among all the ARSFs when compared to the sand filter (SFI) inoculated with the same bacterial strains (A + X). An increase in the bioactivity for ARSFs, particularly DSF was evident from the DOC removal (44 ±â€¯11%, 15% more than SFI), coliform removal (92.7 ±â€¯12.8%, 24% more than SFI), MC-LR removal (87 ±â€¯14%, 13% more than SFI) and an effective nitrification/denitrification, reducing ammonia, nitrate and nitrite level to below guideline values. Toxic assessment using bioindicator (Rhizobium meliloti) revealed safe filter water only in case of DSF.

Biodegradation of microcystin-LR using acclimatized bacteria isolated from different units of the drinking water treatment plant.

Bacterial community isolated from different units of a Drinking Water Treatment Plant (DWTP) including pre-ozonation unit (POU), the effluent-sludge mixture of the sedimentation unit (ESSU) and top-sand layer water sample from the filtration unit (TSFU) were acclimatized separately in the microcystin-leucine arginine (MC-LR)-rich environment to evaluate MC-LR biodegradation. Maximum biodegradation efficiency of 97.2 ±â€¯8.7% was achieved by the acclimatized-TSFU bacterial community followed by 72.1 ±â€¯6.4% and 86.2 ±â€¯7.3% by acclimatized-POU and acclimatized-ESSU bacterial community, respectively. Likewise, the non-acclimatized bacterial community showed similar biodegradation efficiency of 71.1 ±â€¯7.37%, 86.7 ±â€¯3.19% and 94.35 ±â€¯10.63% for TSFU, ESSU and POU, respectively, when compared to the acclimatized ones. However, the biodegradation rate increased 1.5-folds for acclimatized versus non-acclimatized conditions. The mass spectrometry studies on MC-LR degradation depicted hydrolytic linearization of cyclic MC-LR along with the formation of small peptide fragments including Adda molecule that is linked to the reduced toxicity (qualitative toxicity analysis). This was further confirmed quantitatively by using Rhizobium meliloti as a bioindicator. The acclimatized-TSFU bacterial community comprised of novel MC-LR degrading strains, Chryseobacterium sp. and Pseudomonas fragi as confirmed by 16S rRNA sequencing.

Analysis of individual and total microcystins in surface water by on-line preconcentration and desalting coupled to liquid chromatography tandem mass spectrometry.

A fast and high-throughput method is proposed for the determination of total microcystins (ΣMC) in environmental surface waters. After a 1-h Lemieux-von Rudloff oxidation step to yield the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) moiety, samples were quenched, filtered, and directly analyzed. This was achieved via solid phase extraction (SPE) coupled on-line to ultra-high performance liquid chromatography electrospray ionization triple stage quadrupole mass spectrometry. The choice of on-line SPE settings was conducted using experimental designs. Given the matrix complexity of oxidation extracts, the on-line desalting step was found to be a critical parameter to ensure suitable method robustness. The on-line sample loading volume was 5mL, and the wash volume applied for on-line desalting was 3mL. Instrumental analysis was performed in just 8min. The method limit of quantification was 0.5ngL-1 ΣMC (i.e. 2000 times lower than the current World Health Organization - WHO drinking water guideline). Excellent determination coefficients were observed for matrix-free and matrix-based calibration curves alike, and the linearity range tested spanned∼4 orders of magnitude. Accuracy and intermediate precision did not depend on the spike level and proved satisfactory (in the range of 93-110% and 3-6%, respectively). A thorough assessment of instrumental matrix effects was conducted by comparing standard additions curves in several lake and river oxidation extracts with the matrix-free reference. Regardless of the internal standard used (4-PB or D3-MMPB), instrumental matrix effects were efficiently compensated. The matrix effect that may occur at the earlier sample preparation stage was evaluated separately. While the oxidation step was generally not complete (yield ∼65%), the conversion rates of MCs into MMPB remained within a consistent range of values regardless of matrix type. No significant back-pressure was observed upon consecutive injections of oxidation-based samples, while the instrumental sensitivity remained unaffected. The herein described method could therefore be eligible for future large-scale monitoring surveys. The method was applied to a selection of surface water samples (n=30) collected across the province of Québec, Canada, and the results were compared to those achieved by an individual variant analysis of 8 MC congeners and a commercial ELISA kit.

Detection of Cyanotoxins in Algae Dietary Supplements.

Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and ß-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae. Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and ß-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumer's awareness on the potential risks associated with the consumption of these supplements.

Fractionation and analysis of veterinary antibiotics and their related degradation products in agricultural soils and drainage waters following swine manure amendment.

The fate of antimicrobial active compound residues in the environment, and especially antibiotics used in swine husbandry are of particular interest for their potential toxicity and contribution to antibiotic resistance. The presence of relatively high concentrations of bioactive compounds has been reported in agricultural areas but few information is available on their degradation products. Veterinary antibiotics reach terrestrial environments through many routes, including application of swine manure to soils. The objectives of this project were first, to develop an analytical method able to quantify and identify veterinary antibiotics and their degradation products in manure, soil and water samples; and second, to study the distribution of these target compounds in soils and drainage waters. A brief evaluation of their potential toxicity in the environment was also made. In order to achieve these objectives, liquid chromatography coupled to high-resolution mass spectrometry was used for its ability to quantify contaminants with sensitivity and selectivity, and its capacity to identify degradation products. Samples of manure, soil and water came from a long-term experimental site where swine manure containing veterinary antibiotics has been applied for many years. In this study, tetracycline antibiotics were found at several hundred µg L(-1) in the swine manure slurry used for fertilization, several hundred of ng L(-1) in drainage waters and several ng g(-1) in soils, while degradation products were sometimes found at concentrations higher than the parent compounds.

Development of a suspect and non-target screening approach to detect veterinary antibiotic residues in a complex biological matrix using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Swine manure can contain a wide range of veterinary antibiotics, which could enter the environment via manure spreading on agricultural fields. A suspect and non-target screening method was applied to swine manure samples to attempt to identify veterinary antibiotics and pharmaceutical compounds for a future targeted analysis method. METHODS: A combination of suspect and non-target screening method was developed to identify various veterinary antibiotic families using liquid chromatography coupled with high-resolution mass spectrometry (LC/HRMS). The sample preparation was based on the physicochemical parameters of antibiotics for the wide scope extraction of polar compounds prior to LC/HRMS analysis. The amount of data produced was processed by applying restrictive thresholds and filters to significantly reduce the number of compounds found and eliminate matrix components. RESULTS: The suspect and non-target screening was applied on swine manure samples and revealed the presence of seven common veterinary antibiotics and some of their relative metabolites, including tetracyclines, ß-lactams, sulfonamides and lincosamides. However, one steroid and one analgesic were also identified. The occurrence of the identified compounds was validated by comparing their retention times, isotopic abundance patterns and fragmentation patterns with certified standards. CONCLUSIONS: This identification method could be very useful as an initial step to screen for and identify emerging contaminants such as veterinary antibiotics and pharmaceuticals in environmental and biological matrices prior to quantification.

On-line solid-phase extraction coupled to liquid chromatography tandem mass spectrometry for the analysis of cyanotoxins in algal blooms.

An analytical method based on on-line SPE-LC-HESI-MS/MS has been developed for the detection and quantification of eight selected cyanotoxins in algal bloom waters that include mycrocystins, anatoxin-a and cylindrospermopsin. The injection volume was 2 mL according to the expected concentration of cyanotoxins in matrix. The method provides an analysis time of 7 min per sample, acceptable recovery values (91-101%), good precision (RSD < 13%) and method limits of detection at the sub-microgram per liter levels (0.01-0.02 µg L(-1)). A detailed discussion on optimization parameters that have an impact on the overall performance of the method are presented. In particular, method optimization permitted the chromatographic separation of anatoxin-a and phenylalanine, an isobaric interference with a similar chromatographic characteristics. All optimization and validation experiments for the on-line SPE method and chromatographic separation were performed in environmentally relevant algal bloom water matrices. The applicability of the method was tested on several algal bloom water samples from monitored lakes across the province of Québec (Québec, Canada) known to produce cyanotoxins. All of the targeted cyanotoxins were detected with the exception of cylindrospermopsin. In addition, it was found that total microcystin concentrations in several surface water samples exceeded the proposed guidelines established by the province of Québec in Canada of 1.5 µg L(-1) as well as the World Health Organization of 1 µg L(-1) for both free and cell-bound microcystin-LR equivalent.

Total Analysis of Microcystins in Fish Tissue Using Laser Thermal Desorption-Atmospheric Pressure Chemical Ionization-High-Resolution Mass Spectrometry (LDTD-APCI-HRMS).

Microcystins (MCs) are cyanobacterial toxins encountered in aquatic environments worldwide. Over 100 MC variants have been identified and have the capacity to covalently bind to animal tissue. This study presents a new approach for cell-bound and free microcystin analysis in fish tissue using sodium hydroxide as a digestion agent and Lemieux oxidation to obtain the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) moiety, common to all microcystin congeners. The use of laser diode thermal desorption-atmospheric pressure chemical ionization coupled with Q-Exactive mass spectrometry (LDTD-APCI-HRMS) led to an analysis time of approximately 10 s per sample and high-resolution detection. Digestion/oxidation and solid phase extraction recoveries ranged from 70 to 75% and from 86 to 103%, respectively. Method detection and quantification limits values were 2.7 and 8.2 µg kg(-1), respectively. Fish samples from cyanobacteria-contaminated lakes were analyzed, and concentrations ranging from 2.9 to 13.2 µg kg(-1) were reported.

Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and high-resolution mass spectrometry.

A new analytical method was developed for the detection of alkaloid cyanotoxins in harmful algal blooms. The detection of the nonproteinogenic amino acid ß-N-methylamino-L-alanine (BMAA) and two of its conformation isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG), as well as three alkaloid cyanotoxins, anatoxin-a (ANA-a), cylindrospermopsin (CYN), and saxitoxin (STX), is presented. The use of a chemical derivatization with dansyl chloride (DNS) allows easier separation with reversed phase liquid chromatography. Detection with high-resolution mass spectrometry (HRMS) with the Q-Exactive enables high selectivity with specific fragmentation as well as exact mass detection, reducing considerably the possibilities of isobaric interferences. Previous to analysis, a solid phase extraction (SPE) step is used for purification and preconcentration. After DNS derivatization, samples are submitted to ultra high-performance liquid chromatography coupled with heated electrospray ionisation and the Q-Exactive mass spectrometer (UHPLC-HESI-HRMS). With an internal calibration using isotopically-labeled DAB-D3, the method was validated with good linearity (R (2) > 0.998), and method limits of detection and quantification (MLD and MLQ) for target compounds ranged from 0.007 to 0.01 µg L(-1) and from 0.02 to 0.04 µg L(-1), respectively. Accuracy and within-day/between-days variation coefficients were below 15%. SPE recovery values ranged between 86 and 103%, and matrix effects recovery values ranged between 75 and 96%. The developed analytical method was successfully validated with 12 different lakes samples, and concentrations were found ranging between 0.009 and 0.3 µg L(-1) except for STX which was not found in any sample.

Quantitative performance of liquid chromatography coupled to Q-Exactive high resolution mass spectrometry (HRMS) for the analysis of tetracyclines in a complex matrix.

The presence of antibiotics in the environment is of increased interest and, as modern mass spectrometers become more efficient, we are increasingly aware of traces of pharmaceuticals appearing in a wide range of environmental and biological matrices. The Q-Exactive mass spectrometer is part of these innovative hybrid high-resolution mass spectrometers (HRMS) which is often associated with peptide sequencing or metabolomics but with a limited number of studies focusing on its application to the quantification of small molecules in environmental and biological matrices. It combines the high resolving power (RP) performance of the Orbitrap with the high performance selectivity of the quadrupole. Tetracyclines (TCs) are a family comprising some of the most widely used antibiotics in veterinary medicine. This study presents the quantitative performances of the Q-Exactive by illustrating a new approach to quantify TCs using liquid chromatography coupled to a HRMS in a complex matrix, i.e., swine manure. The Q-Exactive was used at high-resolution in both full scan (FS) and targeted ion fragmentation (tMS(2)) modes. These two modes were optimized and compared to determine the most reliable and efficient approach to quantify TCs with good accuracy. The proposed method was optimized to obtain the best selectivity and sensitivity, thus eliminating false positive and allowing the detection of trace levels of analyte. The TCs were extracted from the matrix by sonication using McIlvaine buffer followed by an off-line solid phase extraction method to concentrate and clean the extracts. Both FS and tMS(2) modes presented good linearity (R(2)>0.991) and repeatability (RSD<15%). Mass accuracy was acceptable with values below 2 ppm. The method detection limits (MLD) calculated from the calibration curves ranged from 2.0 to 12 ng g(-1) for FS mode and from 1.5 to 3.6 ng g(-1) for tMS(2) mode. Accuracy and interday/intraday relative standard deviations were below 21% for both modes studied. TCs were quantified in real samples of swine manure with concentrations ranging from 29 to 75 ng g(-1). This study showed the possibility of using hybrid HRMS for trace detection and quantification of TCs in a complex matrix, thus avoiding false positive while achieving good selectivity and sensitivity.

High resolution/accurate mass (HRMS) detection of anatoxin-a in lake water using LDTD-APCI coupled to a Q-Exactive mass spectrometer.

A new innovative analytical method combining ultra-fast analysis time with high resolution/accurate mass detection was developed to eliminate the misidentification of anatoxin-a (ANA-a), a cyanobacterial toxin, from the natural amino acid phenylalanine (PHE). This was achieved by using the laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) coupled to the Q-Exactive, a high resolution/accurate mass spectrometer (HRMS). This novel combination, the LDTD-APCI-HRMS, allowed for an ultra-fast analysis time (<15 s/sample). A comparison of two different acquisition modes (full scan and targeted ion fragmentation) was made to determine the most rigorous analytical method using the LDTD-APCI interface. Method development focused toward selectivity and sensitivity improvement to reduce the possibility of false positives and to lower detection limits. The Q-Exactive mass spectrometer operates with resolving powers between 17500 and 140000 FWHM (m/z 200). Nevertheless, a resolution of 17500FWHM is enough to dissociate ANA-a and PHE signals. Mass accuracy was satisfactory with values below 1 ppm reaching precision to the fourth decimal. Internal calibration with standard addition was achieved with the isotopically-labeled (D5) phenylalanine with good linearity (R(2)>0.999). Enhancement of signal to noise ratios relative to a standard triple-quadrupole method was demonstrated with lower detection and quantification limit values of 0.2 and 0.6 µg/L using the Q-Exactive. Accuracy and interday/intraday relative standard deviations were below 15%. The new method was applied to 8 different lake water samples with signs of cyanobacterial blooms. This work demonstrates the possibility of using an ultra-fast LDTD-APCI sample introduction system with an HRMS hybrid instrument for quantitative purposes with high selectivity in complex environmental matrices.

Total microcystins analysis in water using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

A new approach for the analysis of the cyanobacterial microcystins (MCs) in environmental water matrices has been developed. It offers a cost efficient alternative method for the fast quantification of total MCs using mass spectrometry. This approach permits the quantification of total MCs concentrations without requiring any derivatization or the use of a suite of MCs standards. The oxidation product 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) was formed through a Lemieux oxidation and represented the total concentration of free and bound MCs in water samples. MMPB was analyzed using laser diode thermal desorption-atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS). LDTD is a robust and reliable sample introduction method with ultra-fast analysis time (<15 s sample(-1)). Several oxidation and LDTD parameters were optimized to improve recoveries and signal intensity. MCs oxidation recovery yield was 103%, showing a complete reaction. Internal calibration with standard addition was achieved with the use of 4-phenylbutyric acid (4-PB) as internal standard and showed good linearity (R(2)>0.999). Limits of detection and quantification were 0.2 and 0.9 µg L(-1), respectively. These values are comparable with the WHO (World Health Organization) guideline of 1 µg L(-1) for total microcystin-LR congener in drinking water. Accuracy and interday/intraday variation coefficients were below 15%. Matrix effect was determined with a recovery of 91%, showing no significant signal suppression. This work demonstrates the use of the LDTD-APCI-MS/MS interface for the screening, detection and quantification of total MCs in complex environmental matrices.

Ultra-fast analysis of anatoxin-A using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry: validation and resolution from phenylalanine.

A novel approach for the analysis of the cyanobacterial toxin, anatoxin-a (ANA-a), in an environmentally relevant matrix, using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry (LDTD-APCI-MS/MS) is presented. The ultra-fast analysis time (15 s/sample) provided by the LDTD-APCI interface is strengthened by its ability to remove interference from phenylalanine (PHE), an isobaric interference in ANA-a analysis by MS/MS. Thus the LDTD-APCI interface avoids the time consuming steps of derivatization, chromatographic separation or solid-phase extraction prior to analysis. Method development and instrumental parameter optimizations were focused toward signal enhancement of ANA-a, and signal removal of a PHE interference as high as 500 µg/L. External calibration in a complex matrix gave detection and quantification limit values of 1 and 3 µg/L respectively, as well as good linearity (R(2) > 0.999) over nearly two orders of magnitude. Internal calibration with clomiphene (CLO) is possible and method performance was similar to that obtained by external calibration. This work demonstrated the utility of the LDTD-APCI source for ultra-fast detection and quantification of ANA-a in environmental aqueous matrices, and confirmed its ability to suppress the interference of PHE without sample preparation or chromatographic separation.