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 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/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 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/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 microg/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 > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography--fluorescence detection

An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B1 (FB1) and its totally hydrolized metabolite aminopentol-1 (AP1) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o-Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves (r2 was 0.9855 for FB1 and 0.9831 for AP1). Average recoveries from the matrix were 93.6% for FB1 and 95.3% for AP1. The limit of quantification (LOQ) in swine liver was 75 microg/kg for FB1 and 42 microg/kg for AP1.     

Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-beta-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-beta-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid-liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C(18) column (5 microm, 4.6mm i.d. x 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 x 10(-4) to 70.4 x 10(-4) micromol/mL (0.050-2.00 microg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 microg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.     

High-performance liquid chromatographic method with fluorescence detection for the screening and quantification of oxolinic acid,flumequine and sarafloxacin in fish

A previously published liquid chromatographic method for determining residues of nine quinolones in chicken, porcine, bovine and ovine muscle was adapted and applied to fish tissue for simultaneous determination of three quinolones (flumequine, oxolinic acid and sarafloxacin). The analytes were extracted from homogenised muscle using an acetonitrile basic solution. After centrifugation, partial evaporation and cleaning with hexane, direct injection was possible. Separation was achieved on PLRP-S column and detection was performed with a programmable fluorescence detector. Chromatographic conditions were optimised to be compatible with the determination of the three quinolones in a single run. The linearity, recovery, accuracy and precision of the method were evaluated from fortified tissue samples at concentration levels ranging from 15 to 120 microg kg(-1) for sarafloxacin and 75 to 600 microg kg(-1) for oxolinic acid and flumequine according to the EU maximum residue limit of each quinolone. The limits of detection were estimated to be 2, 5 and 7 microg kg(-1), respectively, for sarafloxacin, oxolinic acid and flumequine. The limits of quantification were validated at 15 microg kg(-1) for sarafloxacin and 75 microg kg(-1) for oxolinic acid and flumequine. Mean extraction recoveries of quinolones in fish ranged from 56.9 to 71.0%. This simple and rapid method is suitable for residue control.     

Analysis of alkylphenol and bisphenol A in eggs and milk by matrix solid phase dispersion extraction and liquid chromatography with tandem mass spectrometry

A method based on matrix solid phase dispersion (MSPD) using C18 as dispersant, and a subsequent cleanup step with amino-propyl solid phase extraction cartridges and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed for the simultaneous determination of nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA) in eggs and milk. Recovery studies were performed at different fortification levels. Average recoveries by MSPD varied from 79% of BPA to 98% of NP and relative standard deviations were equal or lower than 15% for egg samples. The average recoveries in milk ranged from 86 to 84% for BPA, 90 to 99% for NP and 82 to 103% for OP and relative standard deviations were equal to or lower than 8%. The limits of detection (LODs) in eggs were 0.10, 0.10 and 0.25 microg/kg for BPA, NP and OP, respectively and LODs for milk were 0.10, 0.05 and 0.10 microg/kg for BPA, NP and OP, respectively. Investigation of the levels in commercial samples indicated that NP was ubiquitous in milk and eggs at levels ranging from 4.24 to 17.60 microg/kg, and the milk samples were more heavily contaminated by NP than were the egg samples.     

Determination of toxaphene specific congeners in fish liver oil and feedingstuff using gas chromatography coupled to high resolution mass spectrometry

A new method for the determination of nine toxaphene specific congeners in fish liver oil and feedingstuff has been developed. The samples were extracted using pressurized liquid extraction followed by a purification on silica and florisil columns. Identification and quantification were conducted using GC-(EI)-HRMS, and comparison with MS/MS detection was performed, using electron ionization and negative chemical ionization. Limits of detection were ranged from 0.01 to 0.22 microg kg(-1) (12% moisture) as required for feed samples. The calibration curves showed a good linearity for all congeners (R(2)>0.99). Repeatability was below 9% for all the congeners and recoveries were in-between 73 and 86%. This analytical method was applied to the quantification of thirteen real samples collected within national monitoring plans for further risk assessment.     

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.     

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for the determination of fluoxetine and norfluoxetine in human plasma

A comparison study on fluoxetine (FL) and norfluoxetine (NORFL) quantitation in human plasma was carried out between the recently developed liquid chromatographic method with fluorescence detection (LC-FLD) and an earlier established liquid chromatography-mass spectrometry (LC-MS) laboratory procedure. Comparative method evaluation was based on the analysis of plasma samples obtained from Parkinsonian patients receiving 20mg of FL per day. The LC-FLD method involves a two-step liquid extraction procedure without any derivatization, followed by direct chromatography on a Zorbax C8 reversed-phase column. The analytical results are discussed in terms of the method validation and the corresponding experimental protocol (r>/=0.998; CV<9%; LOQ 20 microg/l). There was good correlation between FL, as well as NORFL, plasma levels as determined by the LC-MS and LC-FLD techniques (r=0.9597, N=16 and r=0.9852, N=14 for FL and NORFL, respectively). The results confirm that direct FL/NORFL fluorimetric determination is acceptable for routine use in pharmacokinetic and clinical studies.     

Stereospecific high-performance liquid chromatographic assay of isosakuranetin in rat urine

A stereospecific method of analysis of racemic isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study pharmacokinetics. A simple high-performance liquid chromatographic method was developed for the determination of isosakuranetin enantiomers. Separation was achieved on a Chiralpak AD-RH column with ultraviolet (UV)-detection at 286 nm. The standard curves in urine were linear ranging from 0.5 to 100.0 microg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV) and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was <15% and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of isosakuranetin enantiomers in rat urine.     

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.     

Simultaneous determination of three residual barbiturates in pork using accelerated solvent extraction and gas chromatography-mass spectrometry

A new method was developed for the rapid extraction and unequivocal determination of barbital, amobarbital and phenobarbital residues in pork. The isolation of the analytes from pork samples was accomplished by utilizing an accelerated solvent extractor ASE 300. The procedure was automatically carried out in series for fat removing and extraction, respectively with n-hexane and acetonitrile pressurized constantly at 10.3 MPa for 30 min. After evaporation, the extracts were cleaned up on a C(18) solid phase extraction (SPE) cartridge and the barbiturates were eluted with hexane-ethyl acetate (7:3), evaporated on a rotary evaporator and derivatized with CH(3)I. The methylated barbiturates were separated on a HP-5MS capillary column and detected with a mass detector. Electron impact ion source (EI) operating in time program-selected ion monitoring mode (SIM) was used for identification and external standard method was used for quantification. Good linearity was obtained in the range from 0.5 microg/kg to 25 microg/kg. Average recoveries of the three barbiturates spiked in pork ranged from 84.0% to 103.0%, with relative standard deviations from 1.6% to 12%. The limit of detection (LOD) was 0.5 microg/kg for the three barbiturates (S/N>or=3). The quantification limit (LOQ) was 1 microg/kg for the three barbiturates (S/N>or=10).     

Development of an analytical method for organotin compounds in fortified flour samples using microwave-assisted extraction and normal-phase HPLC with UV detection

The normal high-performance liquid chromatography with UV detection was applied for the determination of tributyltin chloride (TBT), triphenyltin chloride (TPhT), tetraphenyltin (TrPhT), triethyltin chloride (TET) and tetraethyltin (TrET) from flour samples. The separation was performed in the isocratic mode on cyanopropyl column with a mobile phase of hexane-acetonitrile-THF (97/1/2). Under the experimental conditions used, quantitative limit of TBT, TPhT, TrPhT, TET and TrET are 0.95, 0.46, 0.97, 0.75 and 0.96 microg/ml, respectively. Microwave-assisted extraction of organotin (OT) compounds at 100 degrees C with an extraction time of 3 min was described. The extraction of organotin can be finished in acetic acid-hexane (20/80) medium. The quantitative extraction of five organotin compounds was achieved with recoveries ranging from 88 to 101% R.S.D. 3-8%.     

Liquid chromatography-mass spectrometry analysis of hydroxylated polycyclic aromatic hydrocarbons, formed in a simulator of the human gastrointestinal tract

Described is a liquid chromatography-mass spectrometry (LC-MS) procedure for the determination of hydroxylated biotransformation products of polycyclic aromatic hydrocarbons (PAH) in the human gastrointestinal tract. The formation of hydroxylated PAHs was monitored upon incubation of PAHs with colon microbiota from the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The analytical method consisted of a biomass removal step followed by a solid phase extraction (SPE) step using C18 packed columns to remove non-digested food compounds and microbial metabolites that interfere with the detection of the target compounds. For quantification, 9-hydroxyphenanthrene (13)C(6)was used as the internal standard. The detection limits of the hydroxylated PAHs were generally in the range 0.36-14.09 microg x l(-1), based on a signal/noise ratio of 3:1. The recovery of hydroxylated PAHs in intestinal suspension was variable ranging from 45 to 107%, with relative standard deviation (R.S.D.) between 5 and 17%. The analytical procedure was used to show the microbial production of 1-hydroxypyrene and 7-hydroxybenzo(a)pyrene, metabolites that may give colon incubated PAHs bioactive properties.     

Technique validation by liquid chromatography for the determination of acyclovir in plasma

In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5'-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250-260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 microg/ml. The detection limit was 0.006 microg/ml with a quantification limit of 0.020 microg/ml. The ACV recuperation percentage for 250 microl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.     

Liquid chromatography-tandem mass spectrometry determination of loperamide and its main metabolite desmethylloperamide in biological specimens and application to forensic cases

A liquid chromatographic mass spectrometric (LC/MS/MS) method has been developed for the determination of loperamide in whole blood and other biological specimens. The procedure involves liquid-liquid extraction of loperamide, desmethylloperamide and methadone-D3 (internal standard) with butyl acetate. Confirmation and quantification was done by positive electrospray ionisation with a triple quadrupole mass spectrometer operating in multiple reaction-monitoring (MRM) mode. Two MRM transitions of each compound were established and identification criteria were set up based on the ratio of the responses between the two MRM transitions of each compound. The standard curves were linear over a working range of 0.1-500 microg/kg for all transitions. The limit of quantification was 0.1 microg/kg in whole blood. The repeatability and reproducibility within the laboratory expressed by relative standard deviation were less than 5 and 11%, respectively, and the accuracy was better than 9%. The method was developed to examine a feces sample from a child whose mother was suspected of Münchausen syndrome by proxy and it proved to be suitable for forensic cases being simple, selective and reproducible. The method was also applied for a case investigation involving a overdose of loperamide.     

Optimization of a matrix solid-phase dispersion method for the determination analysis of carbendazim residue in plant material

The objective of this paper was to prove that matrix solid-phase dispersion (MSPD) coupled with high performance liquid chromatography (HPLC) and column switching could be used for the determination and quantification of carbendazim residue in plant samples. By comparing results obtained after optimization of the extraction conditions on an acidic silica gel column, it was determined that sorption and retention of carbendazim were achieved via specific interactions. The method of standard additions was used for quantitative analysis. Its performance was evaluated and validated: the detection limit (UV-Vis detection at lambda=279 nm) was 0.02 microg/g, the relative standard deviations (R.S.D.) were between 2.7 and 4.1% and the recoveries were ranging from 84.3 to 90.7% at the 0.04, 0.08 and 0.1 microg/g fortification levels. The method was successfully tested on cereal samples, and the results obtained with the present off-line MSPD-HPLC procedure were found to compare well with those obtained with procedure involving LLE.     

Development and validation of an HPLC-UV method for determination of iohexol in human plasma

An HPLC-UV analytical method for estimation of iohexol in human plasma was developed and validated. Protein precipitation and iohexol extraction from plasma (100 microl) was carried out by adding 800 microl perchloric acid (5%, v/v in water) containing iohexol related compound B as the internal standard followed by vortex mixing and centrifugation. The supernatant (90 microl) was then injected onto a microBondapak C(18) column (150 mm x 3.9 mm, 10 microm) maintained at 30 degrees C. The mobile phase comprised of various proportions of acetonitrile and water with a total run time of 12 min and the wavelength of the UV detector was set at 254 nm. The extraction recovery of iohexol from plasma was >95% and the calibration curve was linear (r(2)=0.99) over iohexol concentrations ranging from 10 to 750 microg/ml (n=8). The method had an accuracy of >92% and intra- and inter-day CV of <3.7% and <3.6%, respectively. The method reported is simple, reliable, precise, accurate and has the capability of being used for determination of iohexol in clinical settings.     

Fast detection of fluoroacetamide in body fluid using gas chromatography-mass spectrometry after solid-phase microextraction

A novel method for fast determination of fluoroacetamide, a kind of organic fluorine pesticide, in blood and urine samples was developed with acetamide as an internal standard using gas chromatography/mass spectrometry (GC/MS) after solid-phase microextraction (SPME) technique. The SPME was performed by immersing a PDMS fiber of 100 microm coating thickness in a sample solution for 25 min at 70 degrees C with (CH(3)CH(2))(4)NBr to improve the extraction efficiency. After a GC sample injection, the extracted fluoroacetamide was desorbed from the fiber for 4 min to perform the GC/MS detection with a HP-PLOT Q capillary column. The analytical conditions were optimized by examining systematically, the effects of experimental parameters on the ratio of characteristic ion peak areas of fluoroacetamide to acetamide. Under optimal conditions, the ratio was proportional to the concentration of fluoroacetamide ranging from 5.0 to 90 microg/ml with a detection limit of 1.0 microg/ml. The average recovery of fluoroacetamide in blood sample was 92.2%. The established method could be used for the fast and convenient measurement of fluoroacetamide in poisoned sample.     

Determination of tylosin residues in different animal tissues by high performance liquid chromatography

A HPLC method to determine and quantify tylosin residues from calves, pigs and poultry is reported. This procedure permitted tylosin to be separated from muscle, liver, kidney and fat after a simple extraction with chloroform or ethyl acetate under basic conditions. The analytical methodology showed a high specificity and sensitivity and an adequate precision and accuracy with a limit of quantification of 50 microg/kg. Eight calves were administered 20 mg/kg/day of tylosin for 5 days and slaughtered at 7 and 14 days post-administration. Results showed that at the 14th day tylosin levels were lower than the MRL in all target tissues.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.