Simultaneous determination of thiamethoxam,clothianidin, and metazachlor residues in soil by ultrahigh performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

A rapid pioneering method has been developed to simultaneously determine residues of three pesticides (thiamethoxam, clothianidin, and metazachlor) in soil by ultrahigh performance liquid chromatography coupled to a mass spectrometry detector (quadrupole time‐of‐flight). An efficient extraction procedure (90–105% average analyte recoveries) has also been proposed, involving solid–liquid extraction by a mixture of water and methanol (60:40, v/v), centrifugation, and concentration. A chromatographic analysis of the compounds was achieved in 5.5 min by means of a core–shell technology based column (Kinetex^® EVO C₁₈, 50 × 2.1 mm, 1.7 μm, 100 Å). The mobile phase (0.3 mL/min, gradient elution mode) consisted of 0.1% v/v formic acid in water and 0.1% v/v formic acid in acetonitrile. The method was fully validated in terms of selectivity, detection and quantification limits, matrix effect, linearity, trueness, and precision. Low limits of detection and quantification were obtained, ranging from 0.2 to 3.0 μg/kg, which are similar to those published in previous studies, while the absence of a significant matrix effect allowed quantification of the pesticides with standard calibration curves. The proposed method was applied for an analysis of pesticides in several soil samples from experimental fields dedicated to oilseed rape cultivars.     

Development of a LC‐MS/MS method for simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in rat plasma: application to the herb–drug interaction study of metoprolol and breviscapine

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α‐hydroxymetoprolol (HMT) and O‐desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC‐C₁₈ column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post‐column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42–7000 ng/mL for MET, 2.05‐4200 ng/mL for HMT and 1.95‐4000 ng/mL for DMT. The analytical method was successfully applied to herb–drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Development of an ASE‐GC‐MS/MS method for detecting dinitolmide and its metabolite 3‐ANOT in eggs

An accelerated solvent extraction coupled with gas chromatography‐tandem mass spectrometry (ASE‐GC‐MS/MS) method for detecting dinitolmide residue and its metabolite (3‐amino‐2‐methyl‐5‐nitrobenzamide, 3‐ANOT) in eggs was developed and optimized. The samples were extracted using ASE with acetonitrile as the extractant and were purified by passage through a neutral alumina solid‐phase extraction column. Then, the samples were analyzed using the GC‐MS/MS method. The optimized method parameters were validated according to the requirements set forth by the European Union and the Food and Drug Administration. The average recoveries of dinitolmide and 3‐ANOT from eggs (egg white, egg yolk, and whole egg) at the limit of quantification (LOQ), 0.5 maximum residue limit (MRL), 1 MRL, and 2 MRL were 82.74% to 87.49%, the relative standard deviations (RSDs) were less than 4.63%, and the intra‐day RSDs and the inter‐day RSDs were 2.96% to 5.21% and 3.94% to 6.34%, respectively. The limits of detection and the LOQ were 0.8 to 2.8 μg/kg and 3.0 to 10.0 μg/kg, respectively. The decision limits (CC_α) were 3001.69 to 3006.48 μg/kg, and the detection capabilities (CC_β) were 3001.74 to 3005.22 μg/kg. Finally, the new method was successfully applied to the quantitative determination of dinitolmide and 3‐ANOT in 50 commercial eggs from local supermarkets.     

Determination of Carbon‐based Engineered Nanoparticles in Marketed Fish by Microwave‐assisted Extraction and Liquid Chromatography‐atmospheric Pressure Photoionization‐tandem Mass Spectrometry

An optimized method for the determination of two major carbon‐based engineered nanoparticles (C₆₀ and C₇₀) in marketed fish samples is described. The method involves the use of microwave‐assisted extraction (MAE) coupled with liquid chromatography ‐ tandem mass spectrometry with atmospheric pressure photoionization (LC‐APPI‐MS/MS). Factors affecting the extraction efficiency of the analytes from fish samples were optimized by a central composite design method. The optimal extraction temperature and time for MAE were found to be 233 °C for 22 min, and the extraction solution was composed of toluene and acetone in a ratio of 4.64:1. The limits of quantitation (LOQs) were 0.1 and 0.05 ng/g for C₆₀ and C₇₀, respectively. The precision for these analytes at two spiked levels, as indicated by relative standard deviations (RSDs), were less than 10% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 85 and 98%. The method was further validated based on EU Commission Decision 2002/657/EC, including a decision limit (CCα) and detection capability (CCβ) for marketed fish samples.     

Validated Method for the Simultaneous Determination of Gemifloxacin and H2‐receptor Antagonists in Bulk,Pharmaceutical Formulations and Human Serum by RP‐HPLC; In‐vitro Applications

Simple, isocratic and rapid RP‐HPLC method has been developed for the simultaneous analysis of gemifloxacin and H₂‐receptor antagonists i.e. Cimetidine, Famotidine and Ranitidine, in bulk, pharmaceutical formulation and human serum. Separation was achieved on the RP‐Mediterranea column [C18 (250 × 4.6 mm, 5 μ)] at ambient temperature using mobile phase consisting of acetonitrile: methanol: water (20:28:52 v/v/v pH 2.8 adjusted by phosphoric acid). Flow rate was 1.0 mL/min with an average operating pressure of 180 kg/cm². Gatifloxacin (GATI) was used as an internal standard (IS). Quantitation was achieved with UV detection at 221, 256 and 267 nm, respectively. Linear calibration curves, at concentration ranges of 0.05‐37.5 μgmL^‐L with a correlation coefficient of ±0.9994. The detection and quantification limits were in the ranges of 0.023‐0.250 μgmL^‐L and 0.071‐0.756 μgmL^‐L, respectively. Friedman's and Student's t‐test were applied to correlate these results. Method was validated in terms of selectivity, linearity, precision, robustness, recovery, limits of detection and quantitation and is applicable to the routine analysis of GFX and H₂‐receptor antagonists, alone or in combination.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

The determination of capreomycin in human plasma by LC–MS/MS using ion‐pairing chromatography and solid‐phase extraction

A bioanalytical method was developed and validated for the quantification of capreomycin (Cm) analogs, Cm IA and Cm IB, in human plasma. This implemented ion‐pairing solid phase extraction, followed by ion‐pairing high‐performance liquid chromatography, with tandem mass spectrometry detection. Chromatographic separation was achieved using a Discovery C₁₈, 5 μm, 4.6 × 50 mm analytical column. An isocratic mobile phase consisting of water and acetonitrile with 0.1% formic acid and 4mm heptafluorobutyric acid (80:20; v/v) was used at a flow‐rate of 500 μL/min. An AB Sciex API 3000 mass spectrometer at unit resolution, in multiple reaction monitoring mode, was used for detection. Electrospray ionization was used for ion production. The method was successfully validated for the range 469–30,000 ng/mL for Cm IA and for Cm IB, with cefotaxime as the internal standard. The within‐ and between‐day precision determinations for Cm IA and IB, expressed as the percentage coefficient of variation, were < 20.0% at the lower limit of quantification (LLOQ) and < 8.2% at all other test concentrations. Recovery of both analogs was > 72.3% and reproducible at the low, medium and high end of the calibration range. No significant matrix effects were observed for the analyte. The assay performed well when applied to clinical samples generated from children in a clinical multidrug resistant tuberculosis research study in South Africa.     

Multiresidue LC–MS/MS analysis of cephalosporins and quinolones in milk following ultrasound‐assisted matrix solid‐phase dispersive extraction combined with the quick,easy, cheap,effective, rugged,and safe methodology

A sensitive and selective confirmatory method for milk‐residue analysis of ten quinolones and eight cephalosporins by LC‐MS/MS has been developed herein. For the chromatographic separation of target analytes, a Perfectsil ODS‐2 (250 × 4 mm, 5 μm) analytical column was used and gradient elution was applied, using a mobile phase of 0.1% w/w TFA in water and 0.1% w/w TFA in ACN. Ultrasound‐assisted matrix solid‐phase dispersion procedure was applied for the extraction and clean‐up procedure of antimicrobials agents from milk matrix using a mixture of Bond Elut Plexa sorbent and QuEChERS. The method was validated meeting the European Legislation determining selectivity, linearity response, trueness, precision (repeatability and between‐day reproducibility), decision limit, detection capability, and ruggedness following the Youden approach. Recoveries of all antibiotics ranged from 81.7 to 117.9%, while RSD values were lower than 13.7%. Limits of quantification for all examined compounds ranged from 2.4 to 15.0 μg/kg, substantially lower than the maximum residue limits established by the European Union (30–100 μg/kg).     

A simple and cost‐effective HPLC‐UV method for the detection of levetiracetam in plasma/serum of patients with epilepsy

A simple, fast and cost‐effective method was developed and validated for the determination of levetiracetam (LEV) in plasma/serum of patients using high performance liquid chromatography (HPLC) with ultraviolet detection. The stability of LEV plasma/serum samples over time and in different blood collection tubes was evaluated. Serum/plasma samples were deproteinized by methanol spiked with the internal standard, gabapentin. HPLC was carried out on a Venusil XBP C₁₈, 250 × 4.6 mm, 5 μm column, at a flow rate of 1.0 mL/min and with mobile phase consisting of 50 mm potassium dihydrogen phosphate–acetonitrile at a pH of 5.5. The UV detector was set at 205 nm and 10 μL was injected. Total runtime was 15 min. Calibration curves were linear (correlation coefficient = 0.999) over a concentration range of 1–60 μg/mL. Relative standard deviation values for both the inter‐day and intra‐day precision and accuracy were <5% for the concentration range. The influence of different collection tubes and the effect of time on the stability of LEV was investigated. These factors may cause inaccuracies owing to drug–protein binding and interference in the matrix. This method is simple, fast, cost‐effective, reliable and accurate with minimal sample preparation for daily routine use in therapeutic drug monitoring.     

Measurement of fexofenadine concentration in micro‐sample human plasma by a rapid and sensitive LC‐MS/MS employing protein precipitation: application to a clinical pharmacokinetic study

A simple, rapid and sensitive liquid chromatography/positive ion electro‐spray tandem mass spectrometry method (LC‐MS/MS) was developed and validated for the quantification of fexofenadine with 100 μL human plasma employing glipizide as internal standard (IS). Protein precipitation was used in the sample preparation procedure. Chromatographic separation was achieved on a reversed‐phase C₁₈ column (5 μm, 100 × 2.1 mm) with methanol : buffer (containing 10 mmol/L ammonium acetate and 0.1% formic acid; 70 : 30, v/v) as mobile phase. The total chromatographic runtime was approximately 3.0 min with retention time for fexofenadine and IS at approximately 1.9 and 2.1 min, respectively. Detection of fexofenadine and IS was achieved by LC‐MS/MS in positive ion mode using 502.1 → 466.2 and 446.0 → 321.1 transitions, respectively. The method was proved to be accurate and precise at linearity range of 1–600 ng/mL with a correlation coefficient (r) of ≥0.9976. The validated method was applied to a pharmacokinetic study in human volunteers following oral administration of 60 or 120 mg fexofenadine formulations, successfully. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and sensitive LC–MS/MS method for quantification of quercetin‐3‐O‐β‐d‐glucopyranosyl‐7‐O‐β‐d‐gentiobioside in plasma and its application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method with good accuracy and precision was developed and validated for the pharmacokinetic study of quercetin‐3‐O‐β‐d ‐glucopyranosyl‐7‐O‐β‐d ‐gentiobioside (QGG) in Sprague–Dawley rats. Plasma samples were simply precipitated by methanol and then analyzed by LC–MS/MS. A Venusil® ASB C₁₈ column (2.1 × 50 mm, i.d. 5 μm) was used for separation, with methanol–water (50:50, v/v) as the mobile phase at a flow rate of 300 μL/min. The optimized mass transition ion‐pairs (m/z) for quantitation were 787.3/301.3 for QGG, and 725.3/293.3 for internal standard. The linear range was 7.32–1830 ng/mL with an average correlation coefficient of 0.9992, and the limit of quantification was 7.32 ng/mL. The intra‐ and inter‐day precision and accuracy were less than ±15%. At low, medium and high quality control concentrations, the recovery and matrix effect of the analyte and IS were in the range of 89.06–92.43 and 88.58–97.62%, respectively. The method was applied for the pharmacokinetic study of QGG in Sprague–Dawley rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Sensitive and rapid high‐performance liquid chromatography tandem mass spectrometry method for estimation of fulvestrant in rabbit plasma

A rapid and sensitive high‐performance liquid chromatography and electrospray tandem mass spectrometry method was developed and validated for estimation of fulvestrant in rabbit plasma using liquid–liquid extraction. The separation and quantification of fulvestrant were achieved by reverse‐phase chromatography on a Sunfire C₁₈ column (50 × 2.1. i.d., 3.5 μm) with isocratic elution at a flow rate of 300 μL/min using norethistrone as an internal standard from 500 μL plasma sample. The method was validated over the concentration range from 0.092 to 16.937 ng/mL with a lower limit of detection of 0.023 ng/mL. The intra‐day and inter‐day accuracy and precision were within 10%. The recovery was 85 and 90% for fulvestrant and norethistrone respectively. The chromatographic run time was only 2.5 min. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Vortex‐ and CO2‐gas‐assisted liquid–liquid microextraction with salt addition for the high‐performance liquid chromatographic determination of furanic compounds in concentrated juices and dried fruits

A novel microextraction method based on vortex‐ and CO₂‐assisted liquid–liquid microextraction with salt addition for the isolation of furanic compounds (5‐hydroxymethyl‐2‐furaldehyde, 5‐methyl‐2‐furaldehyde, 2‐furaldehyde, 3‐furaldehyde, 2‐furoic and 3‐furoic acids) was developed. Purging the sample with CO₂ was applied after vortexing to enhance the phase separation and mass transfer of the analytes. The optimum extraction conditions were: extraction solvent (volume), propyl acetate (125 μL); sample pH, 2.4; vortexing time, 45 s; salt concentration, 25% w/v and purging time, 5 min. The analytes were separated using an ODS Hypersil C₁₈ column (250×4.6 mm i.d, 5 μm) under gradient flow. The proposed method showed good linearities (r² >0.999), low detection limits (0.08–1.9 μg/L) and good recoveries (80.7–122%). The validated method was successfully applied for the determination of the furanic compounds in concentrated juice (mango, date, orange, pomegranate, roselle, mangosteen and soursop) and dried fruit (prune, date and apricot paste) samples.     

Application of a sensitive and specific LC‐ESI‐MS/MS method for the simultaneous quantification of twelve bioactive components in dog plasma for an intravenous pharmacokinetic study of Yiqifumai Injection in beagle dogs

Yiqifumai Injection is a lyophilized powder preparation widely used to treat coronary heart disease. However, its in vivo bioactive components and pharmacokinetic behavior remain unknown. Therefore a sensitive and specific LC–MS/MS was developed and validated for the simultaneous quantification of eight saponins and four lignans in beagle dog plasma. The plasma samples were pretreated by protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation of all the 12 analytes and estazolam (internal standard, IS) was successfully accomplished on an Ultimate® XB‐C₈ column (100 × 2.1 mm, 3 μm) with a gradient elution system. The total running time was 8 min with a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed via positive electrospray ionization in multiple reaction monitoring mode. The assay was fully validated in terms of selectivity, linear range, lower limit of quantitation, precision, accuracy, matrix effect, recovery and stability. This validated method was successfully applied to the pharmacokinetics of 12 bioactive components after intravenous administration of Yiqifumai Injection to beagle dogs at a dose of 0.541 g/kg.     

LC of sulfonamide residues in poultry muscle and eggs extracts using fluorescence pre‐column derivatization and monolithic silica column

A new, rapid, sensitive and selective HPLC method with fluorescence detection is described for the simultaneous determination of 12 sulfonamides, in the presence of putrescine as internal standard, after pre‐column derivatization with fluorescamine. The drugs were separated on a Chromolith Performance RP‐18 column (100×4.6 mm), using a gradient elution with a binary mobile phase of methanol/0.05 M acetate buffer (pH 3.4). Linearity of derivatization was obtained for concentrations from 3.0 to 300 μg/L in standard solutions. The whole procedure was evaluated and fully validated, according to the European Union Decision 2002/657/EC, for the determination of sulfonamides in turkey muscle and hen eggs following SPE. The LODs varied from 2 to 17 μg/kg in turkey and 2 to 15 μg/kg in egg samples. The average recoveries ranged between 96.9–108.6% in turkey muscle and 96.0–108.4% in egg samples, respectively.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

Solid‐phase extraction and residue determination of glyphosate in apple by ion‐pairing reverse‐phase liquid chromatography with pre‐column derivatization

A new method for glyphosate residue determination in apple has been developed. A SPE cartridge was used to clean up the samples before derivatization. Glyphosate was derivatized with 4‐chloro‐3,5‐dinitrobenzotrifluoride (CNBF) and quantified by reverse ion‐pair liquid chromatography using cetyltrimethylammonium bromide (CTAB) as ion‐pair reagent. In pH 9.5 H₃BO₃–Na₂B₄O₇ medium, the reaction of glyphosate with CNBF was complete after 30 min at 60°C. The stability of the derivative on exposure to light at room temperature in methanol–water was demonstrated. The labeled glyphosate was separated on a Kromasil C₁₈ column (250×4.6 mm, 5 μm) at room temperature and UV detection was applied at 360 nm. Separation was achieved within 15 min in gradient elution mode. The correlation coefficient for the method was 0.9998 at concentrations ranging from 0.1 to 50 μg/g. The calculated recoveries for glyphosate in apple were from 86.00 to 99.55%, and the relative standard deviations (n = 6) were from 1.43 to 6.32. The limit of detection was 0.01 μg/g for glyphosate in apple.