Strong Cation Exchange Solid-phase Extraction Research Articles

Characterisation of a new antihypertensive angiotensin I-converting enzyme inhibitory peptide from Pleurotus cornucopiae

This study describes the characterisation of a new angiotensin I-converting enzyme (ACE) inhibitory peptide from the fruiting body of Pleurotus cornucopiae which could be used as a functional food or nutraceutical compounds. After purification of the ACE inhibitor in an ultrafiltration, Sephadex G-25 column chromatography, successively C18 and SCX solid-phase extraction and reverse-phase HPLC, two types of the purified ACE inhibitors with IC50 values of 0.46 and 1.14mg/ml were obtained. The two purified ACE inhibitors were analysed, showing two types of oligopeptides. The amino acid sequences of the two purified oligopeptides were found to be RLPSEFDLSAFLRA and RLSGQTIEVTSEYLFRH. The molecular mass of the purified ACE inhibitors was estimated to be 1622.85 and 2037.26Da, respectively. Water extracts of P. cornucopiae fruiting body showed a clear antihypertensive effect on spontaneously hypertensive rats at a dosage of 600mg/kg.

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d(6) as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d(6) were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d(6). The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d(6) was to just compensate the variation of the injections. The detection limit was 5 ng g(-1) swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.

Quantitative determination of corticosteroids in bovine milk using mixed-mode polymeric strong cation exchange solid-phase extraction and liquid chromatography–tandem mass spectrometry

A new method was developed to identify and quantify corticosteroids (prednisolone, methylprednisone, flumetasone, dexamethasone, and methylprednisolone) in raw bovine milk by liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing mixed-mode polymeric strong cation exchange and reversed-phase (MCX) solid-phase extraction (SPE) to reduce ion effects in a multimode ion (MMI) source. The main advantage of this method over other commonly used methods includes the use of a single SPE cartridge with a low volume for sample preparation and fast separation on the HPLC system with reduced ion suppression. This study is the first to report the determination of methylprednisone, a metabolite of methylprednisolone, in bovine milk. This method was validated in accordance with the European Union (EU) Commission Decision 2002/657/EC. The recoveries vary between 90% and 105%. The within-laboratory reproducibility (precision) is less than 30%. The decision limits and detection capabilities were calculated along with LODs, which ranged from 0.02 to 0.07 μg/kg. The method was further enhanced by its successful adaptation to other LC–MS/MS systems equipped with the newly developed ion source, Agilent Jet Stream (AJS). After optimization of the AJS ion source and MS parameters, even lower LOD values were achieved (0.001–0.006 μg/kg) for the corticosteroids. Analytical results obtained with the AJS were characterized by an enhanced area response and similar noise level comparable to those obtained with conventional orthogonal atmospheric ionization (API).

Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia

The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography–electrospray positive ionization–tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection ( r 2 > 0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection were established to be between 5 and 20 nM. Relative standard deviations were <9% for within-day variations and <15% for between-day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C 18 bonded silica column with a deionized water–methanol–acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 μg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1–20 μg/kg were in the range of 71.4–99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 μg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 μg/kg were in the range of 6.2–13.9% and 4.0–8.7%, respectively. The intra-day precision ( n = 5) for nitroimidazoles residues in chicken spiked at 20 μg/kg is 6.9%, and the inter-day precision for 5 days ( n = 25) is 11%. The method is capable of identifying nitroimidazole residues at ≥0.7 μg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.

Validation of an ion-pair liquid chromatography–electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods

A method based on ion-pair liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS) is reported for determining heterocyclic aromatic amines (HAAs) in meat-based infant foods. The HAAs encompassed quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP), and carboline derivatives (AαC, Harman, Norharman) with d3-IQ, 13C2-MeIQx, and d3-PhIP used as labelled internal standards. The method used extraction into acetone followed by a clean-up on an SCX solid-phase extraction column. LC separation was performed on a TSKgel ODS-80TS column (250 × 2.0 mm, 5 µm), the mobile phase being an ammonium formate–formic acid buffer (3.03 mM ammonium formate, pH = 2.8) aqueous solution–acetonitrile gradient at a flow rate of 0.2 ml min−1. For unequivocal identification of each analyte, three ions were detected and chosen for selected reaction monitoring (SRM). Validation was carried out on lyophilized meat samples. Mean recoveries ranged between 78 ± 4% and 98 ± 2% for different analytes. Limits of quantification generally lower than 8 ng g−1 were demonstrated in meat samples for the analytes investigated. The method exhibited a good linearity and repeatability. Robustness testing identified those factors which were statistically significant in influencing chromatographic separation and response, and indicated which parameters have to be strictly controlled for a reliable analysis of HAAs. In particular, the mobile-phase flow rate was found to be statistically significant (α = 0.05) for the capacity factor (k′) of all analytes except for AαC peak, whereas the mobile-phase pH resulted to be a critical parameter for the k′ values of IQ, MeIQ, and Norharman. The method was proved to be robust vs. resolution between IQ and MeIQ peaks. Among mass-spectrometric parameters, collision energy was found to significantly affect quantitative response of all analytes except that of IQ. The applicability of the method to the analysis of meat-based infant food samples was demonstrated.

Association between Cigarette Smoking and Urinary Excretion of 1,N2-Ethenoguanine Measured by Isotope Dilution Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry

Levels of the promutagenic 1,N2-ethenoguanine (1,N2-epsilonGua), an etheno DNA adduct derived mainly from lipid peroxidation, in experimental animals are associated with risk of cancer formation. Since 1,N2-epsilonGua can be repaired by human glycosylases, it is possible to use it as a biomarker for cancer risk assessment in humans. In the present study, a highly sensitive and specific stable isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) was developed for accurate quantification of 1,N2-epsilonGua in human urine. The sample pretreatment involved a consecutive strong cation exchange solid-phase extraction (SPE) and reversed phase SPE chromatography. The pretreated sample was analyzed by LC-ESI/MS/MS under multiple reaction monitoring mode (MRM) using a triple quadrupole mass spectrometer. The detection limit of 1,N2-epsilonGua using this LC-ESI/MS/MS assay was 1.0 pg (5.8 fmol) injected on-column. Levels of urine samples collected from healthy volunteers were found to range from 0 to 199 pg/mL, and levels as low as 5.0 pg/mL (29 pM) could be accurately quantified. After adjusting for creatinine levels and body weight, an statistically significant association was observed between urinary levels of 1,N2-epsilonGua and cigarette smoking (p = 0.0006). This highly specific and sensitive assay should be valuable in measuring urinary 1,N2-epsilonGua as a potential noninvasive biomarker for oxidative DNA damage.

Determination of malachite green and leucomalachite green in carp muscle by liquid chromatography with visible and fluorescence detection

A liquid chromatography-VIS/FLD method for the analysis of malachite green (MG) and its major metabolite, leucomalachite green (LMG) in carp muscle has been described. The method consists in an extraction with acetonitrile-buffer mixture followed by partioning with dichloromethane. Clean up and isolation were performed on SCX solid phase extraction (SPE) column. Chromatographic separation was achieved by using phenyl-hexyl column with an isocratic mobile phase consisting of acetonitrile and acetate buffer (0.05 M, pH 4.5) (60:40, v/v). Liquid chromatography with absorbance detector ( λ = 620 nm) was used for the determination of MG while LMG was detected by fluorescence detector ( λ ex = 265 nm and λ em = 360 nm). The both detectors were connected on-line which allowed direct analysis of a sample extract for MG and LMG without the need for any post-column procedure. The whole method has been validated, according to the EU requirements (Commission Decision 2002/657/EC). Specificity, stability, decision limit (CC α), detection capability (CC β), accuracy and precision were determined. Average recoveries of MG and LMG from muscle fortified at three levels (0.5, 1 and 2 μg/kg) were 62% (range from 60.4 to 63.5%) and 90% (range from 89.0 to 91.5%), respectively. Relative standard deviations (RSD) of recoveries at all fortification levels were less than 10.9 and 8.6% for MG and LMG, respectively. The calculated CC α for MG and LMG were 0.15 and 0.13 μg/kg, and CC β were 0.37 and 0.32 μg/kg, complying with the minimum required performance limit (MRPL) of 2 μg/kg (sum of MG and LMG).

A multi-residue cation-exchange clean up procedure for basic drugs in produce of animal origin

There is considerable interest in maximising the amount of information obtained from animal product analyses, when screening for the presence of veterinary drug residues. One of the barriers to effective multi-residue analysis to date has been a lack of effective clean up procedures to isolate a wide range of residues from the potential interferents, which may be present in both simple and complex (including processed) foods. A cation-exchange clean up has, therefore, been developed for use with acetonitrile extracts of foods, when analysing for several basic drug groups (sulfonamides, benzimidazoles, levamisole, nitroimidazoles, tranquillisers and fluroquinolones). The clean up procedure has also been shown to be effective using a modified extraction solvent for malachite green and leucomalachite green in fish. Several of the key parameters that influence analyte recovery have been investigated and in an optimised procedure, tissue/biofluid samples containing sulfonamides, benzimidazoles, levamisole, nitroimidazoles, tranquillisers and fluoroquinolones are first extracted with acetonitrile. The extract is then dried with sodium sulfate and acidified with glacial acetic acid before loading onto a Bond Elut, strong cation-exchange (SCX) solid phase extraction (SPE) cartridge. Extracts from fish containing malachite green and leucomalachite green can be cleaned up using the same SCX SPE procedure following extraction with citrate buffer/acetonitrile. Typical recoveries of drugs from low level fortified tissues using the optimised procedure lie in the range 53–104% with the exception of carazolol from pig kidney (31%), malachite green from trout (42–51%) and ciprofloxacin from chicken muscle (44%) and from egg (21%).

Minimization of ion suppression in LC–MS/MS analysis through the application of strong cation exchange solid-phase extraction (SCX-SPE)

Ion suppression of drug response is a major source of imprecision for bioanalytical analysis using LC–MS/MS. Endogenous phospholipids cause ion suppression in both positive ESI and negative ESI modes and must be removed or resolved chromatographically. Three types of ion-exchange solid-phase extraction mediums were evaluated to determine their abilities to remove phospholipids. It was determined that although mixed mode phases fulfills the requirements of retaining both analytes and diverse metabolites, reverse phase retention mechanisms are detrimental in eliminating ion suppression caused by late eluting phospholipids. If an analyte and its metabolites can be retained using an ion-exchange mechanism alone, mixed mode extraction phases should be avoided.

Development and evaluation of a GC/FID method for the analysis of free amino acids in quince fruit and jam.

A GC/FID methodology for determination of twenty-one free amino acids in quince fruit (pulp and peel) and jam is described. The sample preparation was simple, involving a SCX Solid-Phase Extraction (SPE) purification step and a fast derivatization with ethyl chloroformate for gas chromatographic analysis. The chromatographic separation was achieved using a CP-Sil 19 CB wcot fused-silica capillary column. Under the chosen conditions, with temperature and pressure programming, this capillary column was able to separate all the amino acids not only in a short time but also with good separation. The GC/FID procedure is rapid, sensitive, reproducible and accurate. The detection limit values for amino acids were low, between 0.004 and 0.115 microg/mL, and the method was precise. As a general rule, the recovery values were high. Due to its rapidity and low cost, this technique can be useful in the quality control of quince products.

N-(1-Deoxy-d-fructos-1-yl) Fumonisin B1, the Initial Reaction Product of Fumonisin B1 and d-Glucose

Incubation of fumonisin B(1) and D-glucose in aqueous solutions resulted in the formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) in addition to the previously reported N-(carboxymethyl) fumonisin B(1). N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) is the first stable product formed after the Amadori rearrangement of the Schiff base formed by the reaction of the primary amine of fumonisin B(1) and the aldehyde group of D-glucose. N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) was synthesized by reacting fumonisin B(1) with an excess of D-glucose in methanol and heating for 6 h at 64 degrees C. It was purified using C(18) and strong cation exchange solid-phase extraction cartridges and characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. Subsequently, N,N-dimethylformamide was found to be a better reaction solvent, requiring reaction for only 2-3 h at 64 degrees C and eliminating the formation of methyl esters. Alkaline hydrolysis of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) gave a mixture of hydrolyzed fumonisin B(1) and hydrolyzed N-(carboxymethyl) fumonisin B(1).

Determination of levamisole in livestock products using high performance liquid chromatography

A method is described for the determination of the anthelmintic levamisole in muscle, liver, kidney and fat of cattle, swine and poultry using high performance liquid chromatography with photodiode array detection. Levamisole was extracted from an alkaline sample with ethyl acetate and back-extracted with 0.1 mol/L hydrochloric acid. The extract was applied to an SCX solid-phase extraction column. The column was washed with water and methanol. Levamisole was eluted with a solution of ammonia in methanol. The eluate was evaporated to dryness and the residue was dissolved in the mobile phase and injected into the HPLC system. Mean recoveries from 0.01-0.10 microgram/g fortified muscle, liver, kidney and fat samples ranged from 78.3 to 99.8%. The detection limit for the assay was 0.005 microgram/g.

Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive inherited neurodegenerative disorder caused by the deficiency of the lysosomal enzyme beta-galactosylceramidase. The pathogenesis of the disorder has been proposed to arise from the accumulation of the cytotoxic metabolite galactosylsphingosine (psychosine). The twitcher mouse is a naturally occurring murine model of globoid cell leukodystrophy. We have developed a rapid, sensitive, and specific mass spectrometric method for determining the galactosylsphingosine concentration in the tissues of twitcher mice. Galactosylsphingosine is extracted from the tissues in methanol, isolated using strong cation-exchange and C18 solid-phase extraction chromatography, and then directly analyzed using electrospray ionization-tandem mass spectrometry. A lactosylsphingosine internal standard has been employed for quantification. The assay demonstrated significant accumulation of galactosylsphingosine in the brain, spinal cord, and kidney of twitcher mice. It is anticipated that this method may be of use in the monitoring of experimental therapies for globoid cell leukodystrophy.

3,4-Dihydroxymethamphetamine (HHMA). A major in vivo 3,4-methylenedioxymethamphetamine (MDMA) metabolite in humans.

There is evidence that some heavy users of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) show signs of neurotoxicity (a cognitive dysfunction, a larger incidence of psychopathology). It has been postulated that the catechol intermediates of methylenedioxyamphetamines such as 3,4-dihydroxymethamphetamine (HHMA), a metabolite of MDMA, may play a role in their neurotoxicity by formation of thioether adducts. This study describes the first validated method for HHMA determination in plasma and urine by strong cation-exchange solid-phase extraction high-performance liquid chromatography/electrochemical detection (HPLC/ED) analysis. The method has been applied for the determination of HHMA in plasma and urine samples from a clinical study in healthy volunteers of MDMA and provides preliminary kinetic data on this metabolite. HHMA appeared to be a major MDMA metabolite with plasma concentrations as high as the parent compound. Thus, HHMA C(max) (154.5 microg/L) and AUC(0-24h)(1990.9 microg/L h) were similar to those obtained in previously published reports for MDMA (181.6 microg/L and 1465.9 microg/L h, respectively). The 24-h urinary recovery of HHMA accounted for 17.7% of the MDMA dose administered and increases the total 24 h recovery of MDMA and metabolites to 58% of the 100 mg dose administered. The determination of HHMA in plasma and urine samples is of interest in order to establish its relevance in MDMA metabolism and its possible contribution to MDMA neurotoxicity in humans. Its validation showed appropriate accuracy and precision for its use in pharmacokinetic studies.

LC determination of Z-338, novel gastroprokinetic agent in dog plasma by SCX solid phase extraction.

A simple high-performance liquid chromatographic assay with using UV detection (266 nm) was developed to determine a novel gastroprokinetic agent, Z-338 in dog plasma. The extraction procedure using solid-phase extraction with a Isolute SCX column produces extremely clean eluates and a high recovery. Intra- and inter-day variabilities were lower than 5%. The limit of quantitation of the method was 2.5 ng/ml. This assay was applied to the monitoring of Z-338 concentrations in dogs after oral administration. The method also appeared rapid, simple and suitable for therapeutic Z-338 monitoring.

Analysis of Hydroxylated Atrazine Degradation Products in Soils

Hydroxylated atrazine degradation products (HADPs) have been shown to persist in soils and contaminate surface waters throughout the Midwestern United States, yet expedient analytical methods for their determination in soils are lacking. The developed method employs a mixed-mode extractant [3:1 0.5M KH2PO4, pH 7.5:CH3CN, v/v] designed to disrupt the two primary mechanisms of HADP sorption to soils: cation exchange and hydrophobic interactions. Strong anion exchange solid-phase extraction (SPE) is used for sample clean-up followed by isolation and concentration using strong cation exchange SPE. HADPs were quantitated by LC/MS/MS and LC/UV. Method recoveries were determined by spiking 14C-HADPs into three soils with lengthy atrazine use histories. Recoveries ranged from 74–81% for 14C-hydroxyatrazine (HA), 79–88% for 14C-deethlhydroxyatrazine (DEHA), and 64–77% for 14C-deisopropylhydroxyatrazine (DIHA). HADP soil concentrations ranged from 66.9–178 μ kg−1 for HA, 8.99–40.9 μg kg−1 for DEHA, and 5.27–16.2 μg kg−1 for DIHA. Utilization of the mixed-mode procedure, in conjunction with existing methodologies for analysis of atrazine and its chlorinated metabolites, enables a more complete and accurate quantitation of all the major stable atrazine residues in soiis. HADPs comprised an average of 91% of the total atrazine residues in three agricultural surface soils, with HA the major constituent present in all soils. These data indicate that repeated atrazine use results in HADPs as the predominant atrazine residues in surface soils.

Liquid Chromatographic Preparative Method for Isolating Ergot Alkaloids, Using a Particle-Loaded Membrane Extracting Disk

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha-ergocryptine, and ergocristine are extracted from wheat with methanol-0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 microg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovine and 20-400 ng/g for ergotamine, ergocornine, alpha-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of <5 and <20 ng/g, respectively, for each ergot alkaloid tested.

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).

Selective clean-up applicable to aqueous acetone extracts for the determination of carbendazim and thiabendazole in fruits and vegetables by high-performance liquid chromatography with UV detection

Fungicide residues in vegetables (benomyl, carbendazim, thiabendazole) are analyzed through a clean-up procedure that uses a portion of the aqueous acetone extract prepared for multiresidue methodology. A portion of the aqueous acetone extract (equivalent to 5 g of vegetables) is loaded onto an Extrelut-20 cartridge (the cartridge is filled with a coarse, large-pore diatomaceous material). Then, acetone is partially removed by an upward stream of nitrogen at 2 l/min for 30 min. Benzimidazolic fungicides are recovered by percolating the cartridge with 100 ml of 0.1 M phosphoric acid solution, which also serves to convert benomyl to carbendazim. The percolating acid solution is drained on-line through a strong cation-exchange (SCX) solid-phase extraction cartridge with the aid of a slight vacuum. Benzimidazolic fungicides are retained on the SCX cartridge. The phosphoric acid solution is discarded together with the washings of the SCX cartridge, i.e., water followed by methanol–water (75:25), that remove unwanted coextractives. Finally, benzimidazolic fungicides are recovered by eluting the SCX cartridge with methanol–ammonium formate buffer (75:25). The final extract is then analyzed by reversed-phase HPLC with UV detection. Recoveries from crops such as apples, lettuce, strawberries and citrus fruits are generally greater than 80% and no interferences were observed. The clean-up is simple and straightforward, requires only disposable items, water solutions and a few milliliters of solvent and a minimum number of manipulations, and does not require concentration steps or electrical equipment.