Mixed-mode Anion Exchange Research Articles

Simultaneous quantification of THC-COOH, OH-THC, and further cannabinoids in human hair by gas chromatography-tandem mass spectrometry with electron ionization applying automated sample preparation.

The detection of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair, for the purpose of identifying cannabis consumption, is conducted in many forensic laboratories. Since external contamination of hair with these cannabis components cannot be excluded, even after hair decontamination, only the detection of THC metabolites such as 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) or 11-hydroxy-Δ9 -tetrahydrocannabinol (OH-THC), is considered to prove cannabis consumption. At present, testing for THC metabolites is not standard practice due to its analytical complexity. For these reasons, we developed a novel method for the detection of THC-COOH and OH-THC as well as THC, CBD, and CBN in one single analytical run using gas chromatography-tandem mass spectrometry (GC-MS/MS) with electron ionization. After manual hair washing and grinding, sample preparation was fully automated, by means of a robotic autosampler. The hair extraction took place by digestion with sodium hydroxide. A solid-phase extraction (SPE) was chosen for sample clean-up, using a mixed-mode anion exchange sorbent. Derivatization of all analytes was by silylation. The method has been fully validated according to guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh), with a limit of detection (LOD) of 0.2pg/mg for THC-COOH and OH-THC and 2pg/mg for THC, CBD and CBN, respectively, thus fulfilling the Society of Hair Testing (SoHT) recommendations. The validated method has been successfully applied to our routine forensic case work and a summary of data from authentic hair samples is given, as well as data from proficiency tests.

Hyperbranched mixed-mode anion-exchange polymeric sorbent for highly selective extraction of nine acidic non-steroidal anti-inflammatory drugs from human urine

This paper describes the poly(divinylbenzene) (PDVB) supported synthesis of quaternized hyperbranched macromolecules (QHMs) and its use as a highly selective, high-capacity mixed-mode anion-exchange (MAX) sorbent. In detail, the aminated PDVB support was firstly synthesized by copolymerization of divinylbenzene and 2-(diethylamino)ethyl methacrylate via Pickering emulsion polymerization. The QHMs were then grafted on PDVB by a divergent synthesis involving consecutive reactions of resorcinol diglycidyl ether with methylamine (N, N-dimethylethanolamine for terminal epoxides), which brought in a high density of quaternary ammonium functionalities. The changes of specific surface area (SBET), pore volume and ion exchange capacity (IEC) with generation number reveal that the QHMs have been grown successfully within the large meso-channels of the porous aminated PDVB. The best compromise between the SBET, pore volume and IEC was obtained at the 4th generation (G4). Due to the highest IEC (0.47 meq/g), the G4-QHMs was successfully applied for mixed-mode solid phase extraction (SPE) of acidic non-steroidal anti-inflammatory drugs (NSAIDs). An efficient approach based on the mixed-mode SPE coupled with HPLC-UV was developed for highly selective extraction and cleanup of nine NSAIDs (tolmetin, TLM; ketoprofen, KEP; naproxen, NAP; flurbiprofen, FLB; diclofenac, DIC; indomethacin, INM; ibuprofen, IBP; mefenamic acid, MFA; tolfenamic acid, TFA) in human urine samples. Under the optimized conditions, the method exhibited satisfactory recoveries ranging from 81.9% to 104.0% with relative standard deviation (RSD) values below 8.5%, good sensitivity (0.004–0.009 μg mL−1 limit of detection) and good linearity (coefficient of determination, R2 > 0.997, 0.01–0.2 μg mL−1 for NAP, 0.05–1.0 μg mL−1 for FLB, DIC, INM, MFA, TFA, 0.1–2.0 μg mL−1 for TLM, KEP, IBP). The hyperbranched MAX sorbent is superior to Oasis HLB and comparable to Oasis MAX in obtaining clean chromatographic profiles. Our results demonstrate the potential application of the hyperbranched MAX for complex sample analysis.

SPE-LC-MS investigations for the isolation and fractionation of acidic oil degradation products

In this study, we focus on isolation and fractionation strategies by solid phase extraction (SPE) for a broad range of environmentally related organic acids. These emerging potential contaminants are primary degradation products of spilled petrogenic compounds but little attention has been given to their environmental analysis and risk assessment. Three mixed-mode anion exchange sorbents possessing various backbone polarities were compared with respect to the extraction and fractionation efficiency. Quantitative analysis was obtained using an optimized and validated LC-MS method. We demonstrated the importance of reasonable ion source settings for highest sensitivity of individual carboxylic acids. Furthermore, fractionation of carboxylic acids into aliphatic and aromatic fractions by SPE was achieved by step-wise elution. Best performance regarding the isolation of low-concentration acids (sub μg L−1 level) in complex samples was attributed to Strata XA and is caused by high secondary hydrophobic interactions in addition to the ionic interactions. Isolation and fractionation of acid spiked diesel-spilled harbor water (22 μg L−1 spike level) revealed recoveries >80% for all tested carboxylic acids and low carry-overs of neutral petroleum compounds. We conclude that the isolation and fractionation of acidic degradation products by SPE is a powerful tool to investigate this new class of contaminants in relation to identification and ecological risk assessments.

Evaluation of polar organic chemical integrative and hollow fibre samplers for the determination of a wide variety of organic polar compounds in seawater

The calibration of two passive samplers for the determination of 20 emerging organic compounds in seawater is described in this work: i) a new version of polar organic chemical integrative sampler (POCIS) containing 100 mg of mixed-mode anion exchanger (Strata X-AW) and 100 mg of polymeric HLB (Plexa) sorbent materials and using a highly porous Nylon membrane (30-μm pore size) and ii) polyethersulfone (PES) hollow fibre. Among the studied contaminants, herbicides, hormones, life style products (stimulants and artificial sweeteners), industrial chemicals (corrosion inhibitor and fluorinated compounds), personal care products and several pharmaceuticals were included. In the case of POCIS, both the sorbents and the Nylon membranes were extracted and analysed independently. The calibration set up consisted on a continuous-flow tank that was fed with a continuous flow of seawater (2 L/h) and a stock mixture of contaminants (20 mL/h), assuring a nominal concentration of ~ 600 ng/L (each analyte) in the tank. The uptake was linear in POCIS sorbent and Nylon membranes but exponential for PES hollow fibres. Furthermore, the highest sampling rates (Rs) values were obtained in POCIS sorbent (between 2.7 for acetaminophen and 491 mL/day for perfluoro-n-octanoic acid, PFOA) followed by Nylon membranes (between 3.6 for OBT and 50 mL/day for telmisartan) and the lowest were those from PES fibres (between 1.7 for bezafibrate and 157 mL/day for butylparaben). Additionally, five deuterated compounds ([2H5]-atrazine, [2H3]-amitriptyline, [2H7]-irbesartan, [2H3]-ketoprofen and [2H9]-progesterone) were studied as candidates for performance reference compounds (PRCs) in both POCIS and PES, and though [2H5]-atrazine, [2H9]-progesterone and [2H3]-amitriptyline showed acceptable results in the case of POCIS, only [2H5]-atrazine provided a good validation. In the case of PES fibres, the PRC corrections did not provide acceptable results due to a low dissipation of the PRCs. Finally, POCIS were deployed in two sites of the low part of the estuary of Bilbao (northern Spain) from where water samples were also taken and analysed. As a result, in addition to the overall good agreement between the passive and active samplings, passive samplers allowed the determination of several compounds that were below the detection limits in the active sampling.

Development of an LC–MS method to quantify coproporphyrin I and III as endogenous biomarkers for drug transporter-mediated drug-drug interactions

Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3μm, 2.1×150mm, maintained at 60°C. A 10mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3→596.3 was selected to monitor CP-I and CP-III, while m/z 659.3→600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02–100ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV<9%) and accuracy (84.3–103.9%) complied with the recommendation of the European Bioanalytical Forum.The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated.

Multifunctionalized mesoporous silica as an efficient reversed-phase/anion exchange mixed-mode sorbent for solid-phase extraction of four acidic nonsteroidal anti-inflammatory drugs in environmental water samples

A mesoporous silica Santa Barbara Amorphous-15 (SBA-15) has been first functionalized with 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (a silane with three amines) and then reacted with an excess of phenyl glycidyl ether to generate a mixed-mode anion-exchanger containing both anion-exchange (three amines) and reversed-phase (multiple ether-linked phenyls) functionalities in a single branched ligand. The resulting material has been characterized by scanning electron microscopy, transmission electron microscopy, nitrogen adsorption-desorption measurements, Fourier-transform infrared spectroscopy, and elemental analysis. The results obtained indicated a BET specific surface area (SBET) of 362.5m2g−1, a pore volume of 0.70cm3g−1 with a narrow pore size distribution centered at 6.6nm, and carbon and nitrogen contents of 28.30% and 2.84%, respectively. The dimensions of these particles (∼5μm diameter, ∼60μm length), their large surface areas, their high-density functionalities and anion-exchange mixed-mode characteristics make them very attractive for highly effective solid phase extraction (SPE) of acidic nonsteroidal anti-inflammatory drugs (NSAIDs). The important parameters on extraction efficiency including sample pH, breakthrough volume, type and volume of eluent were optimized. A simple and sensitive analytical method based on mixed-mode SPE coupled to high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed and successfully applied to the analysis of four NSAIDs (ketoprofen, naproxen, diclofenac, and ibuprofen) in spiked real water samples with satisfactory recoveries (80.6–110.9%) and repeatability (relative standard deviation <11.3%, n=3). The limit of detections of four NSAIDs were 0.006–0.070μgL−1 for tap water, and 0.014–0.16μgL−1 for river water and wastewater, with the enrichment factors of 806–1109-fold.

Preparation of a reversed-phase/anion-exchange mixed-mode spherical sorbent by Pickering emulsion polymerization for highly selective solid-phase extraction of acidic pharmaceuticals from wastewater

The present work represents a simple and effective preparation of a novel mixed-mode anion-exchange (MAX) sorbent based on porous poly[2-(diethylamino)ethyl methacrylate-divinylbenzene] (poly(DEAEMA-DVB)) spherical particles synthesized by one-step Pickering emulsion polymerization. The poly(DEAEMA-DVB) particles were quaternized with 1,4-butanediol diglycidyl ether (BDDE) followed by triethylamine (TEA) via epoxy-amine reaction to offer strong anion exchange properties. The synthesized MAX sorbent was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption-desorption measurements and elemental analysis. The MAX sorbent possessed regular spherical shape and narrow diameter distribution (15–35μm), a high IEC of 0.54meq/g, with carbon and nitrogen contents of 80.3% and 1.62%, respectively. Compared to poly(DEAEMA-DVB), the MAX sorbent exhibited decreased SBET (390.5 vs. 515.3m2g−1), pore volume (0.74 vs. 0.85cm3g−1) and pore size (16.8 vs. 17.3nm). Moreover, changes of N content for producing the MAX sorbent reveal a successful two-step quaternization, which can be highly related to such a high IEC. Finally, the MAX sorbent was successfully evaluated for selective isolation and purification of some selected acidic pharmaceuticals (ketoprofen, KEP; naproxen, NAP; and ibuprofen, IBP) from neutral (hydrocortisone, HYC), basic (carbamazepine, CAZ; amitriptyline, AMT) pharmaceuticals and other interferences in water samples using solid phase extraction (SPE). An efficient analytical method based on the MAX-based mixed-mode SPE coupled with HPLC-UV was developed for highly selective extraction and cleanup of acidic KEP, NAP and IBP in spiked wastewater samples. The developed method exhibited good sensitivity (0.009–0.085μgL−1 limit of detection), satisfactory recoveries (82.1%–105.5%) and repeatabilities (relative standard deviation < 7.9%, n=3).

Determination of propofol glucuronide from hair sample by using mixed mode anion exchange cartridge and liquid chromatography tandem mass spectrometry.

The main objective of this study was to develop and validate a simpler and less time consuming analytical method for determination of propofol glucuronide from hair sample, by using mixed mode anion exchange cartridge and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study uses propofol glucuronide, a major metabolite of propofol, as a marker for propofol abuse. The hair sample was digested in sodium hydroxide solution and loaded in mixed-mode anion cartridge for solid phase extraction. Water and ethyl acetate were used as washing solvents to remove interfering substances from the hair sample. Consequently, 2% formic acid in ethyl acetate was employed to elute propofol glucuronide from the sorbent of mixed-mode anion cartridge, and analyzed by LC-MS/MS. The method validation parameters such as selectivity, specificity, LOD, LLOQ, accuracy, precision, recovery, and matrix effect were also tested. The linearity of calibration curves showed good correlation, with correlation coefficient 0.998. The LOD and LLOQ of the propofol glucuronide were 0.2 pg/mg and 0.5 pg/mg, respectively. The intra and inter-day precision and accuracy were acceptable within 15%. The mean values of recovery and matrix effect were in the range of 91.7-98.7% and 87.5-90.3%, respectively, signifying that the sample preparation, washing and extraction procedure were efficient, and there was low significant hair matrix effect for the extraction of propofol glucuronide from hair sample on the mixed mode anion cartridge. To evaluate the suitability of method, the hair of propofol administered rat was successfully analyzed with this method.

Post-pellet-digestion precipitation and solid phase extraction: A practical and efficient workflow to extract surrogate peptides for ultra-high performance liquid chromatography – tandem mass spectrometry bioanalysis of a therapeutic antibody in the low ng/mL range

The current LC–MS/MS approach for bioanalysis of protein therapeutics requires generating peptides from protein molecules via trypsin digestion, followed by sensitive detection of these surrogate peptides by multiple reaction monitoring (MRM). However, the presence of huge amount of matrix-related interference peptides generated from trypsin digestion often causes substantial matrix effect or isobaric interferences during LC–MS/MS analysis. Solid phase extraction (SPE) exhibits great potential in sample extraction to overcome these challenges due to its characteristic features of high selectivity, reproducibility, cost-effectiveness and potential to be automated. Here, we report an effective SPE methodology for the bioanalysis of protein therapeutics involving post-pellet-digestion precipitation and SPE cleanup prior to UHPLC–MS/MS analysis. Specifically, proteins in serum samples were first precipitated with methanol to enrich the protein analyte in the pellet prior to trypsin digestion of the pellet (pellet-digestion). The trypsin digest was further processed by a post-pellet-digestion precipitation (second precipitation) to remove matrix-related clog-causing components prior to SPE on OASIS™ MAX (Mixed-Mode Anion-Exchange) SPE plate. This methodology successfully overcame SPE clogging issue and enabled extraction of 100μL of monkey serum on SPE with significant sensitivity improvement to achieve a lower limit of quantitation (LLOQ) of 50ng/mL for a human monoclonal antibody of the IgG4 subclass. This UHPLC–MS/MS assay was validated in a concentration range of 50–5000ng/mL with intra- and inter-assay precisions of within 9.6% CV, and assay accuracy of within ±2.9% Dev of their nominal concentrations. To our best knowledge, this is the pellet digestion with SPE method for LC–MS/MS bioanalysis of a monoclonal antibody for the first time to achieve a LLOQ in the low ng/mL concentration range.

Determination of Methamphetamine and Amphetamine in Hair by Mixed-Mode Anion Exchange Solid Phase Extraction and Liquid Chromatography–Tandem Mass Spectrometry

Methamphetamine is one of the most widely used sympathomimetic amines in Asia. To identify cases of methamphetamine abuse and to estimate its abuse, hair segmental analysis is used extensively. However, existing hair analysis has several limitations due to matrix interferences. In an attempt to resolve these issues, a simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry method is reported for the determination of methamphetamine and amphetamine in human hair with anion exchange solid phase extraction. Hair dissolved in sodium hydroxide was loaded on a mixed-mode anion exchange cartridge and amphetamine and methamphetamine were eluted with methanol in less than ten minutes followed by liquid chromatography–tandem mass spectrometry. The recovery of this method was between 93.9 and 102.0 percent. The limits of detection and quantitation were 0.01 and 0.05 nanograms per milligram for both analytes. The accuracy and precision were satisfactory for quantitative analysis. The suitability of the method for practical analysis was investigated using hair from methamphetamine users.

Challenges in optimizing sample preparation and LC-MS/MS conditions for the analysis of carglumic acid, an N-acetyl glutamate derivative in human plasma.

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) - 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) ≥ 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject.

Inactivation of viruses using novel protein A wash buffers.

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities.

Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion–tandem solid phase extraction coupled with high performance liquid chromatography–tandem mass spectrometry

A matrix solid-phase dispersion (MSPD)–tandem mixed mode anion exchange (MAX)–mixed mode cation exchange (MCX) solid phase extraction-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) method was developed for quantification of six endogenous brassinosteroids (BRs) (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, dolichosterone, teasterone and typhasterol) in rice plant tissues. Non-polar interferences were removed effectively by C8 dispersant used in MSPD, while the following tandem MAX–MCX process facilitated the elimination of polar and ionizable compounds. The weak reversed-phase retention feature of MAX–MCX leaded to good compatibility of the elution solvents in the in-line coupled MSPD–MAX–MCX system. This system was optimized for extraction and purification of BRs in plant samples. The effects of the type of solid phase, the elution solvent, the extraction temperature and the clean-up material were studied. Before HPLC separation, BRs purified were derivatized by m-aminophenylboronic acid to enhance the sensitivity of MS/MS to BRs. Compared with traditional liquid–liquid extraction and solid phase extraction (LLE–SPE), the proposed MSPD–MAX–MCX method showed higher extraction efficiency, lower matrix effect, and advantages of easy manipulation and time-saving. The in-line MSPD–MAX–MCX coupled with HPLC–MS/MS method provided a linear response over two orders of magnitude of BRs concentration with correlation coefficients above 0.9982, limits of detection between 0.008 and 0.04ngmL−1, relative standard deviations (RSDs) below 29.4%, and recoveries above 77.8%. The proposed method has been successfully applied to analysis of endogenous BRs in rice plant at booting stage and maturity stage.

Synthetic oligonucleotide separations by mixed-mode reversed-phase/weak anion-exchange liquid chromatography

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a “mixed-mode” reversed-phase/weak anion exchanger stationary phase has been investigated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method development. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution conditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20–23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G)>Adenine (A)/Guanine∼Guanine/Thymine (T)>Adenine/Cytosine∼Cytosine/Thymine>Adenine/Thymine. Selectivities were larger when the modification was at the 3′ terminal-end, declined when it was in the middle of the sequence and was smallest when it was located at the 5′ terminus. Due to the lower surface area of the 200Å pore size mixed-mode stationary phase compared to the corresponding 100Å material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capacities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic oligonucleotide separation and purification.

Optimization of solid phase extraction clean up and validation of quantitative determination of carbazochrome sodium sulfonate in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A mixed-mode anion exchange solid phase extraction (SPE) method for extraction and clean up of carbazochrome sodium sulfonate (CSS) and (1S)-(+)-10-camphorsulfonic acid (IS) was optimized for quantification by high-performance liquid chromatography/negative electrospray ionization mass spectrometry. The analytes were extracted from 1mL of human plasma via SPE on Oasis(®) WAX cartridge. Chromatographic separation was achieved on a Zorbax SB-Aq (4.6×250mm, 5μm) column under an isocratic condition. Detection was performed using electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. The deprotonated precursor to product ion transitions monitored for CSS and IS was at m/z 299.0→256.0 and m/z 230.9→79.8, respectively. The method was fully validated for its selectivity, sensitivity, precision, accuracy, recovery, matrix effect and stability. Linear range was 0.189-37.8ng/mL with a high square regression coefficient (r=0.9995). The intra-and inter-day precision (RSD, %) ranged from 0.95% to 4.17%, and the intra-and inter-day accuracy was between 95.03% and 105.9%. This method was successfully applied to a bioequivalence study of 90mg CSS formulation in 18 healthy Chinese male subjects under fasting condition.

Trace determination of primary nerve agent degradation products in aqueous soil extracts by on-line solid phase extraction–liquid chromatography–mass spectrometry using ZrO2 for enrichment

A method for determination of the primary nerve agent degradation products ethyl-, isopropyl-, isobutyl-, cyclohexyl- and pinacolyl methylphosphonic acid in aqueous soil extracts has been developed utilizing on-line solid phase extraction-liquid chromatography and mass spectrometry (SPE–LC–MS). Four different stationary phases (ZrO2, TiO2, polymeric mixed mode anion exchange and porous graphitic carbon) were investigated for their suitability as SPE materials in the on-line SPE–LC–MS setup. Zirconium dioxide was chosen due to its high affinity for the alkyl methylphosphonic acids (AMPAs), and its compatibility with LC–MS. Aqueous soil extracts were acidified with 0.1% acetic acid and aliquots of 300μL were injected on a 2mm×10mm ZrO2 column. Separation of the analytes was performed on a reversed phase column with acetonitrile/water gradient and 15mM ammonium acetate. Method validation was performed with the analytes added to an aqueous extract of a loam soil, and the AMPAs could be determined at concentrations as low as 0.05–0.5μgL−1. The method was linear (R2>0.995) from the limit of quantification (LOQ) to 100×LOQ, and the within assay repeatability was below 10% and 5% relative standard deviation at LOQ and 50×LOQ, respectively. The developed method was employed for determination of the AMPAs which had been added to the aqueous extracts of five different soil types from cultivated and uncultivated areas. The obtained recoveries showed that the analytes could be determined at the sensitivities achieved in the method validation in four of the extracts. For the first time, we have demonstrated a method capable of detecting primary nerve agent degradation products at sub ppb levels in the aqueous extracts of various soils. The method requires no sample preparation after soil extraction other than pH adjustment of the aqueous extract.

Mixed-mode solid-phase extraction coupled with liquid chromatography tandem mass spectrometry to determine phenoxy acid, sulfonylurea, triazine and other selected herbicides at nanogram per litre levels in environmental waters

The method presented uses a mixed-mode anion exchange SPE and liquid chromatography tandem mass spectrometry to analyze 5 sulfonylurea, 8 phenoxy acid, 12 triazine and 6 other herbicides in environmental waters. The mixed-mode SPE cartridge is able to retain a wide range of herbicides with acidic–neutral–basic characteristics, particularly the highly polar and acidic compounds clopyralid, dicamba and picloram. The neutral and basic herbicides can be effectively eluted with methanol, after which the acidic herbicides can be eluted using acidified methanol. The method has achieved an LOD of 0.7–3ng/L for the sulfonylureas, 4–12ng/L for the phenoxy acids and 0.4–30ng/L for the triazine and additional herbicides, with recoveries in the range 76–107%, 73–126%, and 65–104%, respectively. The precision of the method, calculated as relative standard deviation (RSD), was below 10% for both sulfonylurea and phenoxy acid herbicides, and less than 20% for the remaining herbicides. The developed method was used to determine the concentration of target herbicides in a range of environmental waters, and many of the target herbicides were detected at ng/L level.

In-house-validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for survey of acrylamide in various processed foods from Korean market.

Acrylamide (AA) is a chemical found in starchy foods that have been cooked at high temperatures. The objective of this study is to monitor the levels of AA in a total of 274 samples of potato chips, chips (except potato chips), biscuits, French fries, breakfast cereals, chocolate products, tea, seasoned laver, and nut products sampled in Korean market. These processed foods include (1) potato chips, (2) chips (except potato chips), (3) biscuits, (4) French fries, (5) breakfast cereals, (6) chocolate products, (7) tea, (8) seasoned laver, and (9) nut products. Samples used for this study were cleaned up using HLB Oasis polymeric and Accucat mixed-mode anion and cation exchange solid-phase extraction cartridge. Liquid chromatography–tandem mass spectroscopy (LC-MS/MS) was validated in-house as an efficient analytical method for the routine analysis of AA in various food products. AA was detected with a Fortis dC18 (1.7 μm, 100 mm × 2.1 mm) column using 0.5% methanol/0.1% acetic acid in water as the mobile phase. Good results were obtained with respect to repeatability (RSDs < 5%). The recoveries obtained for a variety of food matrices ranged between 94.5% and 107.6%. Quantification during routine monitoring was sensitive enough to detect AA at a concentration of 10 μg/kg. A total of 274 food samples were analyzed for AA. The AA levels in the food groups were in the following order: potato chips > French fries > biscuits > tea > chips (except potato chips) > seasoned laver > breakfast cereals > chocolate products > nut products. AA was detected at levels ranging from not detectable to 1435 μg/kg.

Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography–tandem mass spectrometry

This paper describes a new liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3′-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC–MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core–shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.

Evaluation and Subsequent Minimization of Matrix Effects Caused by Phospholipids in LC–MS Analysis of Biological Samples

The influence of matrix effects in LC-MS/MS analysis of biological samples can be enormous and has to be evaluated during method development. Phospholipids, which are present in considerable quantities in biological fluids, are supposed to cause matrix effects when co-eluting with analytes. Therefore, the reduction of phospholipids should lead to the minimization of matrix effects. METHODOLOGY & RESULTS: Here, a polymeric reversed-phase (PRP) SPE cartridge was compared with a combination of mixed-mode-anion-exchange (MAX) and mixed-mode-cation-exchange (MCX) SPE cartridges regarding elimination of matrix effects during sample clean-up. For evaluation of matrix effects post-column infusion experiments were performed. Phospholipid amount in the sample extract and matrix effects are enhanced using PRP in contrast to the combination of MAX/MCX. For an efficient elimination of phospholipids during sample preparation and to improve method accuracy and precision it is advisable to use a combination of MAX/MCX SPE cartridges.