Determination of Danofloxacin and Marbofloxacin in Milk Samples by Micellar Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, and sensitive analytical method has been developed for the determination of two fluoroquinolones, danofloxacin and marbofloxacin, in bovine milk samples. Separation and quantification were performed by micellar liquid chromatography with fluorescence detection (MLC?FD), using sodium dodecyl sulfate (SDS) as a surfactant. The influence of the principal factors, namely, the micelle concentration, the amount of organic modifier, tail‐reducing agents, the pH, and the temperature were studied. The suitable condition was found to be 75 mM SDS?10 mM phosphate buffer–18 mM tetrabutylammonium bromide/3% (v/v) 1‐propanol at pH 3.0 for the separation of marbofloxacin, danofloxacin, and tosufloxacin (internal standard) in about 20 min. The linear concentration range of application was 1.8–30.0 ng · mL^?1 for danofloxacin and 16–120 ng · mL^?1 for marbofloxacin, and the relative standard deviation ranged between 4.9 and 2.7%. The limit of detection found for danofloxacin was 0.5 ng · mL^?1 and 5 ng · mL^?1 for marbofloxacin. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in bovine milk. It was applied to check the eventual existence of these compounds above these limits on commercial milk samples. The validation method was completed with spiked milk samples. Recovery levels obtained were 90.3–108.2%.     

Simultaneous Determination of 13 Phenoxy Acid Herbicide Residues in Soybean by GC‐ECD

Abstract

In this study, a gas chromatography method for the multiresidue determination of 13 phenoxy acid herbicide residues (4‐CPA, 4‐CPP, phenoxy butyric acid, dicamba, MCPA, MCPP, MCPB, 2,4‐D, 3,4‐D, 2,4,5‐T, 2,4,5‐TP, 2,4‐DP, and 2,4‐DB) in soybean was developed. After the removal of fat using n‐hexane, the sample was extracted with acetonitrile‐50 mM HCl (v/v 7:3), and then followed by liquid‐liquid partition with n‐hexane (saturated by acetonitrile). The soybean extract was further cleaned up by anion exchange column. Then the residues were derived with pentafluorobenzyl bromide and the resulting pentafluorobenzyl (PFB) esters were analyzed by a gas chromatograph equipped with an electron capture detector (ECD), quantified by the external standard method. Recoveries of spiked samples at two fortified levels (0.01 and 0.1 mg/kg) are all above 70%. And the relative standard deviation was less than 20%. It shows that the limits of determination (LOD) of this method (S/N≥3) can meet the requirement of maximum residue analysis in import/export inspection for soybean.     

Effective Separation and Simultaneous Determination of Four Fluoroquinolones in Milk by CE with SPE

A simple and sensitive capillary electrophoresis method with solid phase extraction was developed for the determination of sarafloxacin, ciprofloxacin, enrofloxacin and flumequine in milk. Solid-phase extraction with Oasis HLB cartridge column was used for the isolation of four fluoroquinolones in raw milk from a farm and fresh milk sample. Separation conditions of CE, including running buffer, voltage and temperature, were investigated and optimized. Baseline separation was achieved for the four fluoroquinolones under the developed conditions. Correlation coefficients greater than 0.9998 were obtained for all fluoroquinolones with a dynamic range from 1 up to 100 mg L^?1. The intra-day precision was less than 5%, and the inter-day precision was less than 6%. The method recoveries of four fluoroquinolones were in the range of 70.9–90.6%. The detection limits for sarafloxacin, ciprofloxacin, enrofloxacin and flumequine was 19.8, 15.2, 13.3 and 15.9 μg kg^?1, respectively, which allows positive detection of the fluoroquinolones at the targeted maximum residue levels in milk samples.     

Signal Derivatization for HPLC Analysis of Fluoroquinolone Antibiotic Residues in Milk Products

A simple signal derivatization based on mathematical derivatization was demonstrated to overcome the resolution problem in liquid chromatographic separation of fluoroquinolone antibiotics. A reversed-phase Symmetry C18 column with gradient mobile phase of acetonitrile and formic acid was used for separation. The studied fluoroquinolones were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 450 nm, respectively. The chromatographic resolution was improved after plotting the first-derivative chromatogram without any further optimization of the experimental conditions. Milk samples were treated by preliminary protein precipitation with an acetonitrile–formic acid mixture followed by solid-phase extraction using an Oasis HLB cartridge. Good recoveries were obtained for all analytes and the limits of detection were 15–20 μg L⁻¹. The results demonstrated adequate sensitivity and selectivity to allow quantification of fluoroquinolones in various milk products.

     

Determination of chlorpyrifos in soils using multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction followed by LC–MS/MS

A simple and rapid method based on multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction was developed for the analysis of chlorpyrifos in soil. First, the chlorpyrifos was extracted with acetonitrile aqueous (50%, v/v) under the action of microwave energy. Then the obtained extract was separated clearly and easily into an acetonitrile phase and an aqueous phase with the assistance of ammonium acetate. The acetonitrile phase containing chlorpyrifos was concentrated and determined by liquid chromatography–tandem mass spectrometry. The effects of parameters on extraction efficiency including microwave power, extraction solvent, volumes and flow rate of extraction solvent, sample pH, types and amount of salt were studied and optimised. To eliminate the matrix effect, validation of the method was carried out using the matrix-based calibration curve. The limits of detection and quantification for chlorpyrifos were 0.17 and 0.5 ng g^?1, respectively. The proposed method was applied to analyse chlorpyrifos in five soil samples and verified by the recovery test. The recoveries of chlorpyrifos at three spiked levels (5, 50, 200 ng g^?1) were in the range of 90.0–100.5%, with relative standard deviations varying from 1.3% to 5.7%. Compared with the methods reported previously, the proposed method can simplify the operation procedure and reduce solvent consumption in sample pretreatment.     

Analysis of Aromatic Amines in Surface Waters Receiving Wastewater from a Textile Industry by Liquid Chromatographic with Electrochemical Detection

Abstract

A high performance liquid chromatography (HPLC) method with electrochemical detection (ED) was developed for the determination of benzidine, 3,3‐dimethylbenzidine, o‐toluidine and 3,3‐dichlorobenzidine in the wastewater of the textile industry. The aromatic amines were eluted on a reversed phase column Shimadzu Shimpack C₁₈ using acetonitrile+ammonium acetate (1×10^?4 mol L^?1) at a ratio 46:54 v/v as mobile phase, pumped at a flow rate of 1.0 mL min^?1. The electrochemical oxidation of the aromatic amines exhibits well‐defined peaks at a potential range of +0.45 to +0.78 V on a glassy carbon electrode. Optimum working potentials for amperometric detection were from 0.70 V to +1.0 V vs. Ag/AgCl. Analytical curves for all the aromatic amines studied using the best experimental conditions present linear relationship from 1×10^?8 mol L^?1 to 1.5×10^?5 mol L^?1, r=0.99965, n=15. Detection limits of 4.5 nM (benzidine), 1.94 nM (o‐toluidine), 7.69 nM (3,3‐dimethylbenzidine), and 5.15 nM (3,3‐dichlorobenzidine) were achieved, respectively. The detection limits were around 10 times lower than that verified for HPLC with ultra violet detection. The applicability of the method was demonstrated by the determination of benzidine in wastewater from the textile industry dealing with an azo dye processing plant.     

Quick and Sensitive Determination of Fluoroquinolones by Capillary Electrophoresis–Potential Gradient Detection

Abstract

A rapid, capillary electrophoresis–potential gradient detection method was developed for determining three fluoroquinolones: rufloxacin, enoxacin, and moxifloxacin. The fluoroquinolones were baseline separated within 3.5 min with background electrolyte composed of 50 mM acetic acid and 6 mM potassium hydroxide at pH 3.7. For the first time, electrokinetic injection was applied in capillary electrophoretic separation of fluoroquinolones, and an average of ca. 10-fold improvement in detection limits was observed. The method was applied to a fortified chicken tissue sample with detection limits (S/N = 3) of 22–24 ng g^?1, suggesting the potential of the method for determining fluoroquinolone residues in real samples.     

Determination of Sugars by LC with On-Line Electrogenerated Cu(HIO6)2]5−–Luminol Chemiluminescence Detection

A novel method was developed for the determination of sugars such as glucose, fructose and lactose by column liquid chromatography coupled with chemiluminescence detection. The LC separation used a Kromasil NH₂ column (250 × 4.6 mm, i.d.: 5 μm, pore size, 100 Å) with a mobile phase consisting of acetonitrile and water. The chemiluminescence detection was based on the enhancement effect of the selected sugars on the chemiluminescence intensity between luminol and [Cu(HIO₆)₂]^5?, which was on-line electrogenerated by constant current electrolysis. The limits of detection for determination of glucose, fructose and lactose were 4, 3 and 20 μg mL^?1, respectively. The proposed method has been successfully applied to the determination of glucose and fructose in grape samples and lactose in milk samples.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

Screening Method for the Determination at Parts Per Trillion Levels of Pesticide Residues in Vegetables Combining Solid‐Phase Microextraction and Gas Chromatography‐Tandem Mass Spectrometry

Abstract

A new analytical method is proposed for determining residues of 70 pesticides of different chemical families at parts per trillion levels in fresh vegetables. For that, only 4 g of the vegetable samples were quickly extracted with 10 ml of ethyl acetate. The method is based on a vanguard/rearguard strategy that reduces the average time required per sample when the method is applied to a high number of vegetable samples in a quality control laboratory. At the beginning, an aliquot of the extract is evaporated and re‐dissolved in a mixture water:acetone (9∶1 v/v). For screening purposes, the pesticides were extracted for only 10 min by direct immersion of a solid‐phase microextraction (SPME) fiber (65 µm polydimethylsiloxane‐divinylbenzene, PDMS‐DVB). The SPME device was automated and on‐line coupled to a gas chromatograph with an ion trap mass spectrometer (GC‐MS) operated in full scan mode for screening in less than 18 min those samples that potentially contain pesticides above 0.01 mg kg^?1 (cut off value). After that, only those potentially non‐negative samples were reanalyzed by a sensitive quantifying/confirming method that re‐extract by SPME the pesticides in 55 min of absorption and determine them by GC with tandem MS (MS/MS). The method has been validated following EU guidelines and compared with a conventional extraction method based on the use of higher amounts of organic solvents. The limits of detection (LOD), confirmation (LOC) and quantitation (LOQ) as well as the calibration curves obtained allowed the determination of the target pesticides at concentrations clearly below the maximum residue levels (MRL) stated by EU being possible the determination of parts per trillion of the pesticides in ecological (green) vegetables. The method has been applied to the analysis of real samples and the results compared with those obtained by a conventional extraction method accredited by ENAC (Spanish Accreditation Body). The proposed method was also evaluated participating in a proficiency test with adequate results (z‐score among±2).     

Validated Assay for the Determination of Mitoxantrone in Pharmaceuticals Using Capillary Zone Electrophoresis

Abstract

The first capillary zone electrophoretic (CZE) method for the determination of mitoxantrone (MTX) in pharmaceutical formulations was developed. The influence of background electrolyte (BGE) species, pH, concentration (c _BGE), organic modifier, capillary temperature, applied voltage, and injection time was investigated. Optimum results were achieved with 25 mM ammonium acetate at an apparent pH value of 5.0 in 50% v/v acetonitrile, applied voltage of +30 kV, and capillary temperature of 25°C. The samples were introduced into the capillary hydrodynamically for 2 s at 33.5 mbar. Mitoxantrone was detected at a wavelength of 242 nm. Mitoxantrone and doxorubicin (DOX) (used as internal standard, ISTD) were completely separated in less than 7 min. The method was suitably validated with respect to linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision, selectivity, and robustness. The proposed method was applied successfully for the determination of MTX in its injectable pharmaceutical formulation.     

Liquid Chromatographic Determination of Glyoxal and Methylglyoxal from Serum of Diabetic Patients using Meso‐Stilbenediamine as Derivatizing Reagent

Abstract

Meso‐stilbenediamine has been used as derivatizing reagent for liquid chromatographic (LC) determination of glyoxal (Go), methylglyoxal (MGo), and dimethylglyoxal (DMGo) at pH 3. Liquid chromatographic elution and separation was carried out from the column Kromasil 100 C‐18, 5 µm (15×0.46 mm i.d.) with methanol: water:acetonitrile (59:40:1, v/v/v) with a flow rate of 1 mL/min and ultraviolet detection at 254 nm. The linear calibration curves were obtained for Go, MGo, and DMGo within 0.97–4.86 µg/mL, 1.52–7.6 µg/mL, and 1.41–7.08 µg/mL with detection limits of 48 ng/mL, 76 ng/mL, and 70.8 ng/mL, respectively. The method was applied for the determination of Go and MGo from serum of patients suffering from diabetes and ketosis. The amounts of Go and MGo found were 0.150–0.260 µg/mL and 0.160–0.270 µg/mL with coefficient of variation (C.V.) 2.6–4.7% and 2.5–4.6%, respectively. The results obtained were compared with normal subjects with Go and MGo contents of 0.025–0.065 µg/mL and 0.030–0.070 µg/mL with C.V 1.5–4.9% and 1.6–4.8% in the serum.     

Quantitative Determination of Nadolol in Tablets by High‐Performance Liquid Chromatography and UV‐Derivative Spectrophotometry

Abstract

High‐performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of nadolol in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 230 and 300 nm, respectively. A mobile phase composed by acetonitrile‐water containing 0.1% triethylamine (15∶85 v/v) and pH adjusted to 4.6 with formic acid was used. The UVDS method was performed taken a signal at 279.5 nm. The correlation coefficient (r) obtained for both methods was 0.9999. The proposed methods are simple, precise, accurate, and can be used in routine analysis.     

Development and Validation of Liquid Chromatography–Tandem Mass Spectrometry for Simultaneous Determination of the Four Active Components of Danhong Freeze‐dried Powder Injection in Human Plasma

Abstract

A liquid chromatography‐tandem mass spectrometry was developed and validated for the simultaneous determination of protocatechuic aldehyde, danshensu, salvianolic acid B, and hydroxysafflor yellow A, with rutin as internal standard. Electrospray ionization patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive electrospray tandem mass spectrometry (ESI‐MS/MS) detection. Plasma samples were extracted by solid‐phase extraction columns, and the analytes were separated on a Dikma Diamonsil C₁₈ (200 mm×4.6 mm i.d., 5 µm) column using a mobile phase comprised of methanol:acetonitrile: 0.5% formic acid (20∶25∶55, v/v/v) at a flow rate of 1 ml/min. Detection was performed on a Finnigan TSQ ripple quadrupole tandem mass spectrometer in negative ion‐selected monitoring mode using electrospray ionization. Good linearity over the range 10–1000 ng/ml for danshensu, salvianolic acid B, and hydroxysafflor yellow A, and 5–500 ng/ml for protocatechuic aldehyde with acceptable accuracy and precision, respectively. All the validation data, such as accuracy, precision, and stability, were within the required limits. It was a potential platform for the pharmacokinetic and absorption, distribution, metabolism, and excretion (ADME) study of multiple constituent traditional Chinese medicine.     

Matrix Solid-Phase Dispersion Extraction for Analysis of Cypermethrin Residue in Cows’ Milk

A method of extraction based on matrix solid-phase dispersion has been developed, optimized, and validated by chromatographic analysis of cypermethrin pesticide residues in samples of cows’ milk. Milk (0.25 g) was fortified with cypermethrin and blended with 1 g each of C₁₈ (octadecylsilane) silica and Na₂SO₄ (anhydrous sodium sulfate), used to trap fats and water, respectively. The homogenized material was transferred to a commercial SPE cartridge containing 1 g activated Florisil with 5 mL acetonitrile. Cypermethrin was eluted under vacuum with 5 × 2 mL acetonitrile and the extract was concentrated to 1 mL and analyzed by gas chromatography–mass spectrometry. The limits of detection and quantification of the method were 0.025 and 0.08 mg kg⁻¹, respectively.

     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

Simultaneous Determination of Avermectin and Milbemycin Residues in Bovine Tissue by Pressurized Solvent Extraction and LC with Fluorescence Detection

A rapid and sensitive method has been developed for the simultaneous determination of four avermectins and one milbemycin residues in bovine tissue. The isolation of the analytes from muscle and liver samples was accomplished utilizing a pressurized solvent extractor. The optimized extraction procedure using acetonitrile/water (40:60, v/v) as extraction solvent, was automatically carried out at 100 °C and 10 MPa, applying two static cycles for 3 min. The extracts were cleaned up on a C₁₈ solid-phase extraction cartridge and analyzed by liquid chromatography with fluorescence detection after derivatization. Mean recoveries of the five analytes from fortified samples were between 84.8 and 101.8%, with relative standard deviations lower than 10.8%. The limit of detection and quantification were in the ranges of 0.1–0.2 and 0.5–0.6 μg kg^–1, respectively. The application of the newly developed method was demonstrated by analyzing bovine meat samples from market.     

Simultaneous Determination of Ten Antibiotic Residues in Milk by UPLC

An analytical method for rapid screening and quantitative determination of ten antibiotics (chloramphenicol, thiamphenicol, tetracycline, oxytetracycline, chlortetracycline, metacycline, doxycycline, cefoperazone, ceftriaxone and cefaclor) residues in milk was developed using ultra performance liquid chromatography with photodiode array detector. After extraction with McIIvaine buffer + methanol (8 + 2), the extract was cleaned up with solid-phase extraction cartridge. The conditions of sample extraction, cleaning and separation were optimized. The average spiked recoveries of milk samples were 52.1–68.0, 70.1–81.0 and 76.2–101.0% at spiked levels of 0.1, 0.5, 2.5 μg g^?1, respectively with precisions of 3.3–15.9%. The limits of detection and quantification were 0.003–0.022 and 0.01–0.08 μg g^?1, respectively. The proposed method has been applied to the determination of antibiotics in actual milk samples with satisfactory results.     

Determination of Ciprofloxacin,Enrofloxacin, and Marbofloxacin in Bovine Urine,Serum, and Milk by Microextraction by a Packed Sorbent Coupled to Ultra-High Performance Liquid Chromatography

This article reports new, easy, and rapid microextraction by packed sorbent (MEPS)–ultra high performance liquid chromatography with photodiode array detection for the simultaneous determination in bovine urine, serum, and milk of three antibiotics belonging to the class of the fluoroquinolones, namely ciprofloxacin, enrofloxacin, and marbofloxacin, approved for veterinary and human use (ciprofloxacin). The chromatographic separation of the analytes and all aspects influencing the MEPS performance were optimized for the extraction from the considered biological samples. The optimized procedure required simple sample pretreatment, a short (<8?min) isocratic elution, and provided sufficient sensitivity for the determination of the analytes at trace levels in compliance with current legislation. Limits of quantitation were in the range from 0.002 (ciprofloxacin, urine) to 0.048?μg/mL (enrofloxacin, milk). Recoveries from 79% (enrofloxacin, milk) to 88% (ciprofloxacin, urine/serum) were obtained on spiked samples. The within-day (n?=?6) and between-day (n?=?6 over 3?days) relative standard deviation percentages in bovine urine, serum, and milk samples ranged from 2.2 (ciprofloxacin, urine) to 2.5 (enrofloxacin, serum) and from 3.1 (ciprofloxacin, urine) to 3.7 (enrofloxacin, milk), respectively, and were not concentration dependent. To the best of our knowledge, this is the first study describing a fast and simple method for the determination of fluoroquinolones in bovine biological samples.     

Rapid analysis of aflatoxin M1 in milk using dispersive liquid–liquid microextraction coupled with ultrahigh pressure liquid chromatography tandem mass spectrometry

A simple, rapid, and sensitive method based on simultaneous protein precipitation and extraction of aflatoxin M₁ (AFM₁) followed by dispersive liquid–liquid microextraction (DLLME) and ultrahigh pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was developed for the determination of AFM₁ in milk samples. In order to precipitate the proteins and extract AFM₁ from milk, a sample pretreatment using acetonitrile and NaCl as the extraction/denaturant solvent and salting-out agent, respectively, was optimised. Subsequently, the acetonitrile (upper) phase, containing AFM₁, was used as the disperser solvent in DLLME, and extractant (chloroform) and water were added in turn to the extract to perform the DLLME process. The main parameters affecting the extraction efficiency of the whole analytical procedure, such as acetonitrile volume, amount of salt, type and volume of extractant and water volume, were carefully optimised by experimental design. Under optimum conditions, the developed method provides an enrichment factor of 33 and detection and quantification limits (0.6 and 2.0 ng kg^?1, respectively) below the maximum levels imposed by current regulations for AFM₁ in milk and infant milk formulae. Recoveries (61.3–75.3 %) and repeatability (RSD?<?10, n?=?3), tested in different types of milk at four AFM₁ levels, met the performance criteria required by EC Regulation No. 401/2006. Moreover, the matrix effect on the signal intensity of the analyte was negligible. The proposed method provides a rapid extraction and an accurate determination of AFM₁ in milk and formula milk using a simple and inexpensive sample preparation procedure.