Weak Cation-exchange Solid-phase Extraction Research Articles

Multi-analyte screening of small peptides by alkaline pre-activated solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry in doping controls

Peptidic drugs with wide spectrum of physiological activity are of interest for cheating athletes and can be misused as doping in sports. A growing number of small peptide drugs capable of enhancing performance are included in the prohibited list issued by World Anti-Doping Agency (WADA), therefore the improvement of the detection methods is constantly needed. In the present study, a screening assay was developed comprising 54 prohibited small peptides and the related substances in urine by means of the alkaline pre-activated weak cation exchange-solid phase extraction (WCX-SPE) with liquid chromatography-high resolution mass spectrometry (LCHRMS). This method performed good enrichment and purification effect of traditional WCX for basic peptides, and also improve the purification power of acidic peptides, which significantly expanded the coverage of detection substances. The method was validated in accordance with WADA relevant criteria and validated with a main focus on qualitative parameters including selectivity, limits of detection (0.02–0.2 ng/mL), linearity (0.1–20 ng/mL for 46 analytes and 0.2–20 ng/mL for 9 analytes), accuracy and precision (RE% and RSD% < 20% at 1, 5 and 10 ng/mL), recovery (39.2%–100.1% except for the TB500(1–2) free acid: 9.2%), matrix effects (ion suppression effect: 0 to 49.4% and ion enhancement effect: 100% and 264.6%), carryover, reliability and sample extract stability. As a proof-of-principle, urine samples from two patients received a single injection of leuprorelin acetate microspheres (3.75 mg) 30 days before were analyzed and the results proved the applicability of the method.

A high throughput approach for determination of dermorphin in human urine using liquid chromatography-mass spectrometry for doping control purposes.

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.

22 CANONICAL CORRELATION ANALYSIS OF ADHD POLYGENIC RISK AND FUNCTIONAL BRAIN CONNECTIVITY

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL−1, 0.62 μg mL−1, 4.15 μg mL−1, 0.58 μg mL−1 and 4.04 μg mL−1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Determination of trace inorganic antimony in PET-bottled soy sauce by ion chromatography-inductively coupled plasma mass spectrometry

In this work, a method for the determination of trace inorganic antimony in polyethylene terephthalate (PET)-bottled soy sauce by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) was developed. Trace inorganic Sb(III) has been discovered in PET-bottled soy sauce but has been difficult to directly quantify by using IC-ICP-MS. To determine trace inorganic Sb(III) in soy sauce, the sample was first filtered through a weak cation exchange solid-phase extraction (SPE) column and then subsequently washed with 3.0 mL of 0.02% l-ascorbic acid and 3.0 mL of methanol. Finally, the SPE column was eluted with 1.0 mL of 0.1% NaOH +2% H2O2. Under such conditions, the enriched inorganic Sb(III) in the SPE column converted into inorganic Sb(V) and was successfully eluted. Finally, the amount of inorganic Sb(V) in the eluate, equal to the amount of inorganic Sb(III) in the sample, was quantitatively analyzed by IC-ICP-MS. Thus, inorganic Sb(III) in soy sauce was detected. In the above method, the limit of detection (LOD) of inorganic Sb(III) was 0.1 μg L−1, and the linear correlation coefficient r was 0.995 when the calibration curve ranged from 0.5 μg L−1 to 5.0 μg L−1.The RSD (n = 6) was 8.8%, and the recoveries (n = 6) were 91%~97%. Comparing the amount of inorganic Sb(III) (0.8 μg L−1–2.3 μg L−1) with the total antimony (1.7 μg L−1–6.6 μg L−1), we found that 25.6% to 50.0% of the total antimony in soy sauce was inorganic Sb(III). In addition, spiked inorganic Sb(III) slowly converted into other forms of antimony in soy sauce over time, but that conversion was negligible within 10 min after spiking and did not affect the recovery experiment.

Enrichment and analysis of quaternary alkaloids from Zanthoxylum simulans using weak cation exchange solid-phase extraction coupled with LC-MS.

Quaternary alkaloids (QAs) are the major alkaloids in several traditional Chinese medicines, especially in Zanthoxylum simulans. However, few studies on enrichment of QAs from Z. simulans were conducted due to their high polarity and low content. To develop a weak cation exchange (WCX) solid-phase extraction (SPE) coupled with liquid chromatography mass spectrometry (LC-MS) method to enrich and identify QAs from Z. simulans. Meanwhile, the qualitative and quantitative analyses of QAs were carried out based on the optimum conditions of the method. Fresh stem bark of Z. simulans was extracted with 70% aqueous methanol and enriched by WCX-SPE. A high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) with an electrospray ionisation (ESI) source was used for the qualitative and quantitative analyses of QAs. Significant improvements were observed in resolution and abundance of the peaks with WCX-SPE. The linearity, limit of detection (LOD) and limit of quantification (LOQ) were determined for this analytical method. The linear relationship (A=338.85C - 187.72, R2 =0.99) was explored in the range of 0.5 to 312.5μg/mL for chelerythrine. The LOD and LOQ for chelerythrine standard solutions were 0.0539μg/mL and 0.1798μg/mL, respectively. In addition, 22 peaks were detected successfully with WCX-SPE and nine of them are undetectable without the processing of WCX-SPE. A highly selective and efficient method for simultaneous enrichment and identification of QAs from crude extract of Z. simulans was developed for the first time by combining WCX-SPE with LC-MS.

Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

In addition to the development of adequate screening methods for multiple compounds, the World Anti-Doping Agency (WADA) requires anti-doping laboratories to analyze prohibited substances and their metabolites from various classes. This task presents a difficult challenge for all agencies and interests involved in the field of doping control. A screening method is reported in which hybrid sample preparation was performed using a combination of weak cation-exchange solid-phase extraction (WCX-SPE) and the 'Dilute and Shoot' strategy in order to take advantage of both the methodologies. Target substances were extracted using a WCX cartridge and reconstituted with a diluted sample aliquot that included 20% of an untreated urine sample. The target substances were further analyzed by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS). The SPE procedure was optimized using a cartridge-washing step, elution conditions, and elution volume. The cartridge-washing step, which was performed using 10% methanol, improved the overall recovery of target substances. Since the recovery was observed to vary according to the pH of the eluting solution, we applied an elution step using both an acid and a basic organic solvent to achieve complementary recovery. Reconstitution of the diluted aliquot sample was performed to recover the polar substances. The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.

Simultaneous determination of ibuprofen and its metabolites in complex equine urine matrices by GC-EI-MS in excretion study in view of doping control

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL−1, 0.62 μg mL−1, 4.15 μg mL−1, 0.58 μg mL−1 and 4.04 μg mL−1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances.

Two-dimensional solid-phase extraction strategy for the selective enrichment of aminoglycosides in milk.

An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C18 ) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk.

Method for determination of streptomycin and streptidine as markers for streptomycin industrial dregs monitoring in pig and poultry compound feeds.

Antibiotic industrial dregs, generated from the production of antibiotics by fermentation, are banned in China as animal feed additives. Official monitoring programs require the analysis of feeds for possible illegal use of the dregs. A rapid and sensitive method was developed for the simultaneous determination of streptomycin and streptidine as markers for streptomycin industrial dregs in pig and poultry compound feeds. After extraction with 20% aqueous trichloroacetic acid and pH adjustment, sample cleanup was performed by weak cation-exchange solid-phase extraction. UPLC-ESI-MS/MS was carried out using a hydrophilic interaction chromatography(HILIC)column to achieve separation. Quantification required matrix-matched calibrations in a linear range of 50-1000μgkg-1; the calibration curves were linear in this range with coefficients of determination of 0.991 and 0.994 for streptomycin and streptidine, respectively. The method validity parameters-LODs (20μgkg-1) and LOQs (50μgkg-1), recoveries (71-78% and 75-84%, respectively), and relative reproducibility (5.4-9.6%)-satisfy the requirements of routine analysis.

Differential Mobility Spectrometry Coupled with Multiple Ion Monitoring in Regulated LC-MS/MS Bioanalysis of a Therapeutic Cyclic Peptide in Human Plasma

A differential mobility spectrometry (DMS) in combination with a multiple ion monitoring (MIM) method was developed and validated for quantitative LC-MS/MS bioanalysis of pasireotide (SOM230) in human plasma. Pasireotide, a therapeutic cyclic peptide, exhibits poor collision-induced dissociation (CID) efficiency for multiple reaction monitoring (MRM) detection. Therefore, in an effort to increase the overall sensitivity of the assay, a DMS-MIM approach was explored. By selecting the most abundant doubly charged precursor ion in both the Q1 and Q3 of the mass analyzer in MIM and combining the DMS capability to significantly reduce the high matrix/chemical background noise, this new LC-DMS-MIM method overcomes the sensitivity challenge in the typical MRM method due to poor CID fragmentation of the analyte. Human plasma was spiked with pasireotide with concentrations in the range 0.01-50 ng/mL. Weak cation-exchange solid-phase extraction was employed for sample preparation. The sample extracts were analyzed with a SCIEX QTRAP 6500 system equipped with an ESI source and DMS device. The separation voltage and compensation voltage of the DMS and other parameters of the MS system were optimized to maximize signal responses. The performance of the LC-DMS-MIM assay for quantitative analysis of pasireotide in human plasma was evaluated and compared to those obtained via LC-MRM and LC-MIM without DMS. Overall, the assay sensitivity with DMS-MIM was approximately 5-fold better than that observed in MRM or MIM without DMS. The assay was validated with accuracy (% bias) and precision (% CV) of the QC results at eight concentration levels (0.01, 0.02, 0.05, 0.15, 0.3, 1.5, 15, and 37.5 ng/mL) evaluated ranging from -4.8 to 5.0% bias and 0.7 to 8.6% CV for the intraday and interday runs. The current LC-DMS-MIM workflow can be expanded to quantitative analysis of other molecules that have poor fragmentation efficiency in CID.

Development and validation of a UHPLC–MS/MS assay for colistin methanesulphonate (CMS) and colistin in human plasma and urine using weak-cation exchange solid-phase extraction

A rapid ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) assay method was developed for determination of CMS and formed colistin in human plasma and urine. After extraction on a 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the eluents were mixed and injected into the UHPLC–MS/MS system directly. A Phonomenex Kinetex XB-C18 analytical column was employed with a mobile phase consisting of solution “A” (acetonitrile:methanol, 1:1, v/v) and solution “B” (0.1% formic acid in water, v/v). The flow rate was 0.4mL/min with gradient elution over 3.5min. Ions were detected in ESI positive ion mode and the precursor-product ion pairs were m/z 390.7/101.3 for colistin A, m/z 386.0/101.2 for colistin B, and m/z 402.3/101.2 for polymyxin B1 (IS), respectively. The lower limit of quantification (LLOQ) was 0.0130 and 0.0251mg/L for colistin A and colistin B in both plasma and urine with accuracy (relative error, %)<±12.6% and precision (relative standard deviation, %)<±10.8%. Stability of CMS was demonstrated in biological samples before and during sample treatment, and in the extract. This new analytical method provides high-throughput treatment and optimized quantification of CMS and colistin, which offers a highly efficient tool for the analysis of a large number of clinical samples as well as routine therapeutic drug monitoring.

LC-MS/MS analysis of pramipexole in mouse plasma and tissues: elimination of lipid matrix effects using weak cation exchange mode based solid-phase extraction.

Intranasal delivery is emerging as a promising alternative for oral or intravenous administration of central nervous system (CNS) drugs, such as pramipexole which is widely used for the treatment of Parkinson's disease. To evaluate the effectiveness of intranasal delivery of pramipexole, preclinical pharmacokinetic and tissue distribution studies following intranasal administration need to be investigated. In this paper, we developed and validated a robust and sensitive LC-MS/MS assay without matrix effect for accurate measurements of pramipexole in mouse plasma and tissue samples. Pramipexole and its stable isotope labeled internal standard (d3-pramipexole) were extracted from biological samples by protein precipitation (PPT) coupled with solid phase extraction (SPE) using weak cation exchange SPE cartridges. Matrix effects were studied using post-column infusion and post-extraction addition experiments by direct monitoring of typical phospholipids including glycerophosphocholines (GPChos) and lysoglycerophosphocholines (Lyso-GPChos). Chromatographic separation was achieved on a Welch Ultimate(®) XB-CN column using isocratic elution with a run time of 3.0 min. The assay was linear in the concentration range of 0.05-100 ng/mL and the intra- and inter-day precision and accuracy met the acceptance criteria. Compared with previous reported assays, the current sample preparation approach exhibited significant reduction of matrix effects due to the dramatically decreased levels of residual matrix components such as GPChos and Lyso-GPChos. This method has been successfully applied to pharmacokinetic and tissue distribution studies of pramipexole in mice following a single intravenous or intranasal dose of 50 μg/kg.

Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for LC-MS/MS analysis of plasma free metanephrines.

LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for quantifying target analytes in complex matrices. However, the technique lacks selectivity when plasma free metanephrines are measured. We propose the use of multistage fragmentation (MRM(3)) to improve the analytical selectivity of plasma free metanephrine measurement. Metanephrines were extracted from plasma with weak cation exchange solid-phase extraction before separation by hydrophilic interaction liquid chromatography. We quantified normetanephrine and metanephrine by either MRM or MRM(3) transitions m/z 166→134→79 and m/z 180→149→121, respectively. Over a 6-month period, approximately 1% (n = 21) of patient samples showed uncharacterized coeluting substances that interfered with the routine assay, resulting in an inability to report results. Quantification with MRM(3) removed these interferences and enabled measurement of the target compounds. For patient samples unaffected by interferences, Deming regression analysis demonstrated a correlation between MRM(3) and MRM methods of y = 1.00x - 0.00 nmol/L for normetanephrine and y = 0.99x + 0.03 nmol/L for metanephrine. Between the MRM(3) method and the median of all LC-MS/MS laboratories enrolled in a quality assurance program, the correlations were y = 0.97x + 0.03 nmol/L for normetanephrine and y = 1.03x - 0.04 nmol/L for metanephrine. Imprecision for the MRM(3) method was 6.2%-7.0% for normetanephrine and 6.1%-9.9% for metanephrine (n = 10). The lower limits of quantification for the MRM(3) method were 0.20 nmol/L for normetanephrine and 0.16 nmol/L for metanephrine. The use of MRM(3) technology improves the analytical selectivity of plasma free metanephrine quantification by LC-MS/MS while demonstrating sufficient analytical sensitivity and imprecision.

Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater

This paper describes a selective and ultra-sensitive analytical method for simultaneous determination of 11 fluoroquinolone (FQ) antibiotics in environmental and wastewater samples. The method employs offline solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography with fluorescence detection (HPLC-FLD). A weak cation exchange SPE protocol was developed with a novel loading volume optimization algorithm and a methanol cleanup step to remove background organic matter. Various parameters were optimized to recover FQs from water/wastewater and analyte recovery was generally greater than 80%. Chromatographic separation of the 11 FQs was achieved on a 150mm pentafluorophenyl column using a gradient elution scheme with methanol, acetonitrile, and 20mM phosphate buffer (pH=2.4). Excitation and emission wavelengths were individually optimized for each FQ using fluorescence spectroscopy; the excitation and emission wavelengths were 276–296nm and 444–506nm, respectively. Instrumental quantitation limits were 20–100pg of mass injected. Of the 11 FQs investigated, seven (i.e., ciprofloxacin, difloxacin, enrofloxacin, fleroxacin, norfloxacin, moxifloxacin, and ofloxacin) were detected during a four-month sampling campaign of wastewater and wastewater-impacted surface water. Concentrations of FQs in raw wastewater, wastewater effluent, and wastewater-impacted surface water were 5–1292, 2–504, and 4–187ng/L, respectively.

Determination of paraquat in vegetables using HPLC-MS-MS.

A simple, sensitive, reliable and economical method was developed for the determination of paraquat (a widely used herbicide) in four edible vegetables (cabbage, lettuce, spinach and Chinese cabbage) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). The samples were extracted with water under sonication and cleaned up by weak cation exchange solid-phase extraction. Chromatographic separation of paraquat was achieved on a hydrophilic interaction liquid chromatography column (2.1 × 100 mm, 3 µm) with a gradient program using 10 mM ammonium acetate in 0.1% formic acid and acetonitrile as mobile phase. The low salt concentration used in the eluting buffer ensured extended LC-MS analysis of paraquat in different matrices without the necessity of frequent source cleaning. The validity of the developed method was evaluated by spiking paraquat in four edible vegetables at 50 and 500 ng g(-1). Recovery ranged from 43.6 to 73.5%. The limit of detection is 0.94 ng g(-1). With the developed method, the kinetic of paraquat entering plant tissue was also evaluated.

A Sensitive and Interference-Free Liquid Chromatography Tandem Mass Spectrometry Method for Measuring Metanephrines in Plasma

Background: Plasma metanephrines are the primary biomarkers used to aid in diagnosing pheochromocytoma. However, the low physiological levels of metanephrines, physicochemical properties, and potential interferences make it challenging to achieve high sensitivity and specificity. In this report, we developed and validated a sensitive and interference-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring plasma metanephrines with simple sample preparation. Methods: Plasma samples were extracted using weak cation exchange solid-phase extraction cartridges, and analyzed by LC-MS/MS with an analytical cycle time less than six minutes. Results: Absolute ion suppression and matrix effect were observed, however, were completely compensated for by the internal standards. Epinephrine, an isobaric interferent of normetanephrine, was chromatographically separated, and no interference was observed from other common interferents. The method was linear from 0.08 to 22.2 nmol/L for normetanephrine and 0.03 to 8.2 nmol/L for metanephrine with accuracy ranging from 81 to 107%. No carryover was observed up to 56.8 nmol/L for normetanephrine and 8.7 nmol/L for metanephrine. Intra-assay and total CVs were within 6.8% for normetanephrine and 5.2% for metanephrine for three levels tested. Based on Deming regression, comparison with a reference LC-MS/MS method using patient specimens (n=40) showed a slope of 0.973, intercept of 0.11 nmol/L and correlation coefficient of 0.9936 for normetanephrine and a slope of 1.039, intercept of -0.014 nmol/L, and correlation coefficient of 0.9914 for metanephrine. The mean difference was 3.5% and -1.6% for normetanephrine and metanephrine, respectively. Conclusion: This LC-MS/MS assay is sensitive and free of interference for quantitation of plasma metanephrines

English

Rapid and accurate screening for toxicants/chemicals in a broad range is an important element in systematic toxicological analysis (STA). Herein, we report a novel method for the rapid screening of 61 central nervous system (CNS) drugs in plasma, using a solid-phase extraction (SPE) column termed, weak cation exchange (WCX) and high performance liquid chromatography with a diode array detector (HPLC-DAD). The SPE column was preconditioned sequentially with 3 ml of acetonitrile, 1 ml of water and, 2 ml of buffer solution. The pretreated plasma was loaded onto the column, which was then washed with 2 ml of water, followed by 2 ml of acetonitrile, and the acetonitrile elution was collected as the neutral/acid fraction. Subsequently, 3 ml of trifluoroacetic acid-acetonitrile (2+98) was then used to elute the column and the elution was collected as basic fraction. The collected fractions were evaporated at 60&deg;C under a nitrogen stream until about 100 &mu;l of solvent remained. The final volume was then adjusted to 1 ml with 5% of acetonitrile. The HPLC separation was accomplished on an Agilent TC-C18 column (250 &times; 4.6 mm, 5 &micro;m) with acetonitrile and phosphate buffer solution as mobile phase, by gradient elution at a flow rate of 1.5 ml/min. The detection wavelength was 210 nm, and the full spectra were recorded from 200 to 364 nm. The absolute recoveries of 55 drugs tested, exceeded 50%; 42 of them exceeded 80%. In conclusion, the WCX SPE preparation combined with HPLC-DAD, is suitable for a broad drug screening for CNS drugs. &nbsp; Key words:&nbsp;Solid phase extraction (SPE), weak cation exchange (WCX), high performance liquid chromatography with diode array detection (HPLC-DAD), systematic toxicological analysis (STA), drug screening.

Determination of methylene blue residues in aquatic products by liquid chromatography‐tandem mass spectrometry

A method for the determination and confirmation of methylene blue (MB) in aquatic products was developed. Residues of MB were extracted from homogenized tissues with acetonitrile/sodium acetate buffer solution, and simply cleaned up with dichloromethane liquid/liquid extraction. After concentration and dissolution, the sample solutions were cleaned up by the neutral alumina and weak cation-exchange solid phase extraction (SPE) cartridge, prior to LC-MS/MS analysis. MB was determined at 1.0-20 microg/kg in eel, toasted eel and shrimp, with a limit of quantification of 0.5 microg/kg. Recovery for MB was between 73.0% and 108.3%. This method is fast, exact and sensitive. It can be applied to determine MB in aquatic products.

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate

A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC–MSn) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC–MSn with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300ngmL−1. Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8ngmL−1 for NEO and 12.8ngmL−1 for total GEN residues.