Anaerobic co-metabolic biodegradation of pharmaceuticals and personal care products driven by glycerol fermentation.

Anaerobic digestion in two sequential phases, acidogenesis and methanogenesis, has been shown to be beneficial for enhancing the biomethane generation from wastewater. In this work, the application of glycerol (GOH) as a fermentation co-substrate during the wastewater treatment was evaluated on the biodegradation of different pharmaceuticals and personal care products (PPCPs). GOH co-digestion during acidogenesis led to a significant increase in the biodegradation of acetaminophen (from 78 to 89%), ciprofloxacin (from 25 to 46%), naproxen (from 73 to 86%), diclofenac (from 36 to 48%), ibuprofen (from 65 to 88%), metoprolol (from 45 to 59%), methylparaben (from 64 to 78%) and propylparaben (from 68 to 74%). The heterotrophic co-metabolism of PPCPs driven by glycerol was confirmed by the biodegradation kinetics, in which kbio (biodegradation kinetics constant) values increased from 0.18 to 2.11 to 0.27-3.60 L g-1-VSS d-1, for the operational phases without and with GOH, respectively. The assessment of metabolic pathways in each phase revealed that the prevalence of aromatic compounds degradation, metabolism of xenobiotics by cytochrome P450, and benzoate degradation routes during acidogenesis are key factors for the enzymatic mechanisms linked to the PPCPs co-metabolism. The phase separation of anaerobic digestion was effective in the PPCPs biodegradation, and the co-fermentation of glycerol provided an increase in the generation potential of biomethane in the system (energetic potential of 5.0 and 6.3 kJ g-1-CODremoved, without and with GOH, respectively). This study showed evidence that glycerol co-fermentation can exert a synergistic effect on the PPCPs removal during anaerobic digestion mediated by heterotrophic co-metabolism.

Modern automated microextraction procedures for bioanalytical, environmental, and food analyses.

Sample preparation frequently is considered the most critical stage of the analytical workflow. It affects the analytical throughput and costs; moreover, it is the primary source of error and possible sample contamination. To increase efficiency, productivity, and reliability, while minimizing costs and environmental impacts, miniaturization and automation of sample preparation are necessary. Nowadays, several types of liquid-phase and solid-phase microextractions are available, as well as different automatization strategies. Thus, this review summarizes recent developments in automated microextractions coupled with liquid chromatography, from 2016 to 2022. Therefore, outstanding technologies and their main outcomes, as well as miniaturization and automation of sample preparation, are critically analyzed. Focus is given to main microextraction automation strategies, such as flow techniques, robotic systems, and column-switching approaches, reviewing their applications to the determination of small organic molecules in biological, environmental, and food/beverage samples.

Two-phase (acidogenic-methanogenic) anaerobic fixed bed biofilm reactor enhances the biological domestic sewage treatment: Perspectives for recovering bioenergy and value-added by-products.

The organic matter bioconversion into methane during anaerobic digestion (AD) comprises different steps, the acidogenic and methanogenic phases being clearly distinct in terms of metabolic activities. In this work, new configurations of anaerobic fixed bed biofilm reactors (AFBBR) were operated under conventional methanogenic conditions (single phase - SP-AFBBR, M1R), and in a sequential two-phase system, acidogenic reactor followed by methanogenic reactor (TP-AFBBR, AcR + M2R), in order to verify the impact of the AD phase separation on the overall system performance in operational, kinetics and microbiological aspects. The results indicated that feeding the methanogenic reactor with the acidogenic effluent stream provided a shorter operating start-up period (11 and 32 days for SP and TP-AFBBR, respectively), a greater alkalinity generation (0.14 and 0.41 g-CaCO3·g-CODremoved-1 for M1R and M2R, respectively), and the optimization of biomethane production (methane yield of 95 and 154 N-mLCH4·g-CODremoved-1 for M1R and M2R, respectively). The COD removal kinetics was also favored in the TP-AFBBR (k1-COD = 1.4 and 2.9 h-1 for M1R and M2R, respectively), since the soluble fermentation products were readily bioavailable to the biomass in the reactor. Hydrogenotrophic methanogenesis was the predominant pathway in the M2R, while the Methanosaeta-driven acetoclastic pathway predominated in the M1R. The greater diversity of Bacteria and Archaea in M2R denotes a better balance between the species that degrade volatile organic acids from AcR (i.e. Syntrophorhabdus, Syntrophus and Syntrophobacter) and the hydrogenotrophic methanogens (Methanoregula, Methanolinea and Methanospirillum) that consume the biodegradation products. The estimated bioenergy generation potential (range of 0.39-0.64 kWh·m-3-sewage considering the COD removed) for full-scale TP-sewage treatment plants evidences the feasibility of energetic recovery in the domestic sewage anaerobic treatment.

On-line solid-phase extraction of pharmaceutical compounds from wastewater treatment plant samples using restricted access media in column-switching liquid chromatography-tandem mass spectrometry.

An on-line solid phase extraction using a lab-made restricted access media (RAM) was developed as sample preparation procedure for determination of the pharmaceutical compounds caffeine (CAF), carbamazepine (CBZ), norfloxacin (NOR), ciprofloxacin (CIP), fluoxetine (FLX) and venlafaxine in wastewater treatment plant samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is suitable for use in routine of analysis, avoiding cross-contamination and requiring only a small sample volume (50 µL), with minimal handling. The method was validated according to international guidelines. The chromatographic efficiency was evaluated using peak resolution and asymmetry parameters. Carryover was also evaluated, in order to ensure reliability of the analysis and the ability to reuse the cartridge. Satisfactory linearity (r2 > 0.99) was obtained for all the compounds. The intra- and inter-day precision values were lower than 5.79 and 14.1%, respectively. The limits of detection ranged from 0.01 to 3 µg L-1 and the limits of quantification were from 0.1 to 5 µg L-1. The method was applied to 20 environmental wastewater samples, with caffeine being the most widely detected compound, at the highest concentration of 392 µg L-1, while other compounds were detected in fewer samples at lower concentrations (up to 9.60 µg L-1). The lab-made modification is a cheaper option for on-line sample preparation, compared to commercially available on-line SPE cartridges and RAM columns. Moreover, a high-throughput procedure was achieved, with an analysis time of 16 min including sample preparation and chromatographic separation. The same RAM column was applied over 200 injections including method optimization, validation and application in wastewater samples without loss of analytical response.

Hybrid constructed wetlands as post-treatment of blackwater: An assessment of the removal of antibiotics.

New sanitation systems have been developed to treat, recover energy and nutrients, and permit reuse processes at the source of generation, minimizing water use and flow segregation. Thus, this study was carried out with the objective of evaluating the potential of hybrid constructed wetlands in the removal of organic matter, nutrients, pathogenic microorganisms, and 12 antibiotics from blackwater previously treated by an upflow anaerobic sludge blanket reactor. A hybrid system of constructed wetlands was used, comprised of a horizontal subsurface flow constructed wetland with a total volume of 0.60 m3 followed by a vertical subsurface flow constructed wetland with a total volume of 0.20 m3. Three different hydraulic retention times were comparatively tested (1.0, 2.0, and 3.0 days for the horizontal subsurface flow constructed wetland, and 1.1, 0.9, and 0.4 days for the vertical subsurface flow constructed wetland) in four distinct stages. The plant species used was Canna x generalis. The results from this study demonstrate the potential of constructed wetlands as a suitable technology for post-treatment of segregated domestic wastewater (anaerobically-digested blackwater). Efficient reduction of COD, BOD5, total nitrogen, and total phosphorus (74, 93, 50, and 61%, respectively) was achieved, with a hydraulic retention time of 3.0 and 1.1 days for horizontal and vertical subsurface flow constructed wetland, respectively (stage IV). The presence of ciprofloxacin was confirmed by chromatographic and mass spectrometric analysis in an average concentration of 442.6 ng.L-1 at the inflow of the horizontal subsurface flow constructed wetland, but was not observed at the outflow.

Influence of organic loading rate on ciprofloxacin and sulfamethoxazole biodegradation in anaerobic fixed bed biofilm reactors.

Antibiotic compounds, notably sulfamethoxazole (SMX) and ciprofloxacin (CIP), are ubiquitous emerging contaminants (ECs), which are often found in domestic sewage. They are associated with the development of antimicrobial resistance. Operational parameters, e.g. organic loading rate (OLR), hydraulic retention time (HRT) and sludge retention time, may influence EC biodegradation in wastewater treatment plants. This study assessed the impact of the OLR variation on the biodegradation of CIP and SMX, applying two configurations of anaerobic fixed bed reactors: anaerobic packed bed biofilm reactor (APBBR) and anaerobic structured bed biofilm reactor (ASBBR). A significant reduction in the biodegradation of SMX (APBBR: 93-69%; ASBBR: 94-81%) and CIP (APBBR: 85-66%; ASBBR: 85-64%) was observed increasing OLR from 0.6 to 2.0 kgCOD m-3 d-1. The decrease in the HRT from 12 to 4 h resulted in higher liquid-phase mass transfer coefficient (APBBR: ks from 0.01 to 0.05 cm h-1; ASBBR: ks from 0.07 to 0.24 cm h-1), but this was not enough to overcome the decrease in the antibiotic-biomass contact time on biofilm, thus reducing the bioreactors' performance. The ASBBR favored biomethane production (from 7 to 17 mLCH4 g-1VSS L-1 d-1) and biodegradation kinetics (kbio from 1.7 to 4.2 and for SMX and from 2.1 to 4.8 L g-1VSS d-1 for CIP) due to the higher relative abundance of the archaea community in the biofilm and the lower liquid-phase mass transfer resistance in the structured bed. CIP and SMX cometabolic biodegradation was associated to the hydrogenotrophic methanogenesis (mainly Methanobacterium genus) in co-culture with fermentative bacteria (notably the genera Clostridium, Bacillus, Lactivibrio, Syntrophobacter and Syntrophorhabdus). The anaerobic fixed bed biofilm reactors proved to be highly efficient in biodegrading the antibiotics, preventing them from spreading to the environment.

Normalizing Untargeted Periconceptional Urinary Metabolomics Data: A Comparison of Approaches.

Metabolomics studies of the early-life exposome often use maternal urine specimens to investigate critical developmental windows, including the periconceptional period and early pregnancy. During these windows changes in kidney function can impact urine concentration. This makes accounting for differential urinary dilution across samples challenging. Because there is no consensus on the ideal normalization approach for urinary metabolomics data, this study's objective was to determine the optimal post-analytical normalization approach for untargeted metabolomics analysis from a periconceptional cohort of 45 women. Urine samples consisted of 90 paired pre- and post-implantation samples. After untargeted mass spectrometry-based metabolomics analysis, we systematically compared the performance of three common approaches to adjust for urinary dilution-creatinine adjustment, specific gravity adjustment, and probabilistic quotient normalization (PQN)-using unsupervised principal components analysis, relative standard deviation (RSD) of pooled quality control samples, and orthogonal partial least-squares discriminant analysis (OPLS-DA). Results showed that creatinine adjustment is not a reliable approach to normalize urinary periconceptional metabolomics data. Either specific gravity or PQN are more reliable methods to adjust for urinary concentration, with tighter quality control sample clustering, lower RSD, and better OPLS-DA performance compared to creatinine adjustment. These findings have implications for metabolomics analyses on urine samples taken around the time of conception and in contexts where kidney function may be altered.

Feasibility of anaerobic packed and structured-bed reactors for sulfamethoxazole and ciprofloxacin removal from domestic sewage.

This study assessed the applicability of fixed bed bioreactors in two configurations - anaerobic structured bed reactor (ASBR) and anaerobic packed bed reactor (APBR) - in the removal of Sulfamethoxazole (SMX) and Ciprofloxacin (CIP), two antibiotics frequently detected in sanitary sewage. The problem of these pharmaceuticals as emerging contaminants in conventional sewage treatment systems is mainly because they encourage the development and spread of resistance genes in bacteria. Both reactors had similar performances, and the antibiotics were highly removed - APBR: 85 ±â€¯10% for SMX and 81 ±â€¯16% for CIP; ASBR: 83 ±â€¯12% for SMX and 81 ±â€¯15% for CIP. The ASBR showed to be potentially more feasible in operating and economic terms compared to the APBR, as the former presents a smaller amount of support material in the bed. SMX was completely biotransformed, while the influence of the sorption mechanism was observed for CIP, as its presence was detected sorbed onto biomass throughout the reaction bed of the reactors, with a partition coefficient (log KD) of around 2.8 L·kg-1TSS. The degradation kinetics of the pharmaceuticals were fitted using a first-order kinetic model, whereby the reactors behaved as plug flow ones, indicating the possibility of optimizing the operation for a hydraulic retention time of 6 h. The removal kinetics was more favorable for CIP (higher apparent constant kinetic - kCIPapp > kSMXapp), since its biodegradation is linked to the biomass, which is more concentrated in the bed bottom layer. The experimental results showed the potential of anaerobic fixed bed reactors in removing environmentally relevant concentrations of SMX and CIP found in sewage.

Evaluation of sulfamethazine removal kinetics using fixed structured bed bioreactor.

The use of anaerobic biomass attached to a support has been recently presented as a good prospect in the treatment of wastewater containing recalcitrant compounds, such as sulfamethazine (SMZ). SMZ has been found in swine wastewater and sewage treatment plants, which motivates assessing their degradation by new wastewater treatment technologies. Thus, this paper describes the use of a continuous fixed structured bed bioreactor for the purpose of evaluating SMZ removal kinetics present in lab-made wastewater. The analysis of SMZ used online solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry (SPE online-LC-MS/MS). Chemical oxygen demand (COD) was also monitored to evaluate the organic matter removal. The bioreactor was operated under mesophilic conditions (30 ∘ C), with a hydraulic retention time of 24 h. In order to evaluate SMZ removal, four different concentration levels were studied: 200, 400, 600, and 800 ng L-1. COD removal efficiency obtained for filtered effluent kept at 91.01% and there was no interference due to the increase of SMZ concentration. For SMZ, the removal efficiencies were of 52.8±12.1% for 200 ng L-1 concentration level; 55.0±8.15% for 400 ng L-1; 53.0±6.14% for 600 ng L-1, and 48.8±5.44% for 800 ng L-1. COD removal kinetics presented a first-order apparent removal rate constant ( kapp ) of 0.281±0.0295 h-1. SMZ also showed a first-order apparent removal rate constant of 0.158±0.0093 h-1 for the following concentrations levels: 200, 400, 600, and 800 ng L-1.

Removal kinetics of sulfamethazine and its transformation products formed during treatment using a horizontal flow-anaerobic immobilized biomass bioreactor.

Sulfamethazine (SMZ) is an antibiotic from sulfonamides class widely used in veterinary medicine and reported in wastewater and sewage. Thus, it is essential to study technologies to reduce SMZ present in the aquatic environment. Anaerobic bioreactors are a low-cost technology applied for wastewater treatment. The objective of this paper is to study kinetics parameters related to SMZ removal using a horizontal flow-anaerobic immobilized biomass reactor (HAIB) and to evaluate its transformation products formed during this treatment. The bioreactor was operated at mesophilic condition with a hydraulic retention time of 12 h. The removal of SMZ was evaluated at three different concentrations: 200 ng L-1 (phase I), 400 ng l-1 (phase II) and 600 ng L-1 (phase III). The apparent first-order removal constant obtained for chemical oxygen demand was 0.885 ± 0.094 h-1 while SMZ showed a removal constant of 0.356 h-1. SMZ was removed with an efficiency of 56.0 ± 13.0 % (phase I); 62.0 ± 12.0 % (phase II) and 62.0 ± 6.00 % (phase III). Seven transformation products were detected and one of these with m/z 233 is reported for the first-time. The HAIB bioreactor has a potential to assist in wastewater treatment to remove contaminants at ng L-1 concentration level.

Sulfamethoxazole and ciprofloxacin removal using a horizontal-flow anaerobic immobilized biomass reactor.

The antibiotics sulfamethoxazole (SMTX) and ciprofloxacin (CIP) are commonly used in human and veterinary medicine, which explains their occurrence in wastewater. Anaerobic reactors are low-cost, simple and suitable technology to wastewater treatment, but there is a lack of studies related to the removal efficiency of antibiotics. To overcome this knowledge gap, the objective of this study was to evaluate the removal kinetics of SMTX and CIP using a horizontal-flow anaerobic immobilized biomass reactor. Two different concentrations were evaluated, for SMTX 20 and 40 µg L(-1); for CIP 2.0 and 5.0 µg L(-1). The affluent and effluent analysis was carried out in liquid chromatography/tandem mass spectrometry (LC-MS/MS) with the sample preparation procedure using an off-line solid-phase extraction. This method was developed, validated and successfully applied for monitoring the affluent and effluent samples. The removal efficiency found for both antibiotics at the two concentrations studied was 97%. Chemical oxygen demand (COD) exhibited kinetic constants that were different from that observed for the antibiotics, indicating the absence of co-metabolism. Also, though the antibiotic concentration was increased, there was no inhibitory effect in the removal of COD and antibiotics.

Rapid determination of 12 antibiotics and caffeine in sewage and bioreactor effluent by online column-switching liquid chromatography/tandem mass spectrometry.

This study presents a column-switching solid-phase extraction online-coupled to a liquid chromatography/tandem mass spectrometry (SPE-LC-MS/MS) method for simultaneous analysis of 12 antibiotics (7 sulfonamides and 5 fluoroquinolones) and caffeine detected in the sewage and effluent of a pilot anaerobic reactor used in sewage treatment. After acidification and filtration, the samples were directly injected into a simple and conventional LC system. Backflush and foreflush modes were compared based on the theoretical plates and peak asymmetry observed. The method was tested in terms of detection (MDL) and quantification limit (MQL), linearity, relative recovery, and precision intra- and inter-day in lab-made sewage samples. The method presented suitable figures of merit in terms of detection, varying from 8.00 × 10(-5) to 6.00 × 10(-2) ng (0.800 up to 600 ng L(-1); caffeine) with direct injection volume of only 100 µL and 13 min of total analysis time (sample preparation and chromatographic run). When the method was applied in the analysis of sewage and effluent of the anaerobic reactor (n = 15), six antibiotics and caffeine were detected in concentrations ranging from 0.018 to 1097 µg L(-1). To guarantee a reliable quantification, standard addition was used to overcome the matrix effect.

Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications.

Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology.

Determination of steroids, caffeine and methylparaben in water using solid phase microextraction-comprehensive two dimensional gas chromatography-time of flight mass spectrometry.

Analysis of several emerging contaminants (steroids, caffeine and methylparaben) in water using automated solid-phase microextraction with comprehensive two dimensional gas chromatography coupled to time of flight mass spectrometry (SPME-GCxGC-ToF/MS) is presented. Experimental design was used to determine the best SPME extraction conditions and the steroids were not derivatized prior to injection. SPME-GCxGC-ToF/MS provided linear ranges from 0.6 to 1200µgL(-1) and limits of detection and quantitation from 0.02 to 100µgL(-1). A series of river water samples obtained locally were subjected to analysis. SPME-GCxGC-ToF/MS is readily automated, straightforward and competitive with other methods for low level analysis of emerging contaminants.

Development of a new stir bar sorptive extraction coating and its application for the determination of six pesticides in sugarcane juice.

In this article, a novel polydimethylsiloxane/activated carbon (PDMS-ACB) material is proposed as a new polymeric phase for stir bar sorptive extraction (SBSE). The PDMS-ACB stir bar, assembled using a simple Teflon(®)/glass capillary mold, demonstrated remarkable stability and resistance to organic solvents for more than 150 extractions. The SBSE bar has a diameter of 2.36 mm and a length of 2.2 cm and is prepared to contain 92 µL of polymer coating. This new PDMS-ACB bar was evaluated for its ability to determine the quantity of pesticides in sugarcane juice samples by performing liquid desorption (LD) in 200 µL of ethyl acetate and analyzing the solvent through gas chromatography coupled with mass spectrometry (GC-MS). A fractional factorial design was used to evaluate the main parameters involved in the extraction procedure. Then, a central composite design with a star configuration was used to optimize the significant extraction parameters. The method used demonstrated a limit of quantification (LOQ) of 0.5-40 µg/L, depending on the analyte detected; the amount of recovery varied from 0.18 to 49.50%, and the intraday precision ranged from 0.072 to 8.40%. The method was used in the analysis of real sugarcane juice samples commercially available in local markets.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system.

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 microm of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 10(3) less sample than using conventional methods).

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system.

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry.

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.

Capillary column switching restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry system for simultaneous and direct analysis of drugs in biofluids.

Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 microm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.