Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols—7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and β-sitosterol—using a high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4-¹³C₂ as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.     

Metabolic profiling of fifteen amino acids in serum of chemical-induced liver injured rats by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Amino acids (AA) play a crucial role in the metabolic process of animals, plants and microbial cells, which are useful for the diagnosis, follow-up and prognostics of liver disorders affecting AA metabolism. A rapid, simple and sensitive analytical method is in urgent need to investigate the intact metabolic profile and to simultaneously determine individual AA in biological samples. Here, a hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry (MS/MS) analytical method was developed and validated for simultaneous quantification of 15 AA in rat serum using isotope stable-labeled phenylalanine and alanine as internal standards. The 15 AA without derivatization were separated on a hydrophilic interaction silica column (TSK-GEL AMIDE-80), the total analytical time was within 8?min, and the concentrations of the 15 AA were determined using a multiple reaction monitoring (MRM) mode. Limits of detection (LOD) ranged from 0.01 to 0.05???g/ml, and the calibration curve was linear in the range of 0.05?C10???g/ml (r?>?0.99). The HILIC-MS method was employed to the analysis of AA in serum samples obtained from N-acetyl-p-amino-phenol (APAP)- and chloropromazine hydrochloride (CH)- induced liver injured rats. The concentrations of each AA ranged from the low quantification value up to 10???g/ml. Based on metabolic profile of AA, multivariate statistics using principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) could differentiate two distinct groups corresponding to APAP-induced and CH-induced rats. This novel metabolic profile study of AA based on the HILIC-MS analysis and chemometric analysis provided not only an accurate quantitative assay of the serum concentrations of biomarkers, but also a promising methodology for evaluation of chemical-induced hepatotoxicity in reflecting AA metabolic pathway.     

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.     

Determination of 2-hydroxyflutamide in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS): Application to a bioequivalence study on Chinese volunteers

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)–ESI-MS/MS method was developed for the determination of 2-hydroxyflutamide in human plasma using tegafur as the internal standard. The plasma sample was pretreated with methanol for protein precipitation and the analytes were separated on an Ultimate C18 column (5 μm, 2.1 mm × 50 mm, MD, USA) with the mobile phase consisted of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a negative multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 290.90–204.8 for 2-hydroxyflutamide and 198.9–128.8 for tegafur. Linear calibration curves were obtained in the concentration range of 1.742–1452 ng/ml with a lower limit of quantification of 1.742 ng/ml. The intra- and inter-batch precision values were less than 8.1% and 5.6%, respectively. The established method was successfully applied to a bioequivalence study of two flutamide preparations (250 mg) in 20 healthy male volunteers.     

A novel mixed-mode solid phase extraction for simultaneous determination of melamine and cyanuric acid in food by hydrophilic interaction chromatography coupled to tandem mass chromatography

Utilizing a solid phase extraction column (MCT) containing mixed hydrophilic functional gel and cation exchange sorbent, a sensitive and rapid HPLC–MS/MS method for simultaneously determining the residues of melamine (MEL) and cyanuric acid (CYA) in human foodstuffs was developed. MEL and CYA in egg, pork, liver, kidney and pork, shrimp, sausage casing, honey, soybean milk, soybean powder and dairy product were extracted using acetonitrile/water, defatted with hexane and isolated using MCT solid phase extraction column. The residues were separated upon a hydrophilic interaction liquid chromatography (HILIC) column and analyzed by electrospray ionization under negative–positive switched mode on a triplequadrupole mass spectrometry. The selected reaction monitoring was performed on [M+H]⁺ of m/z 127.9 to provide the transition of 127 > 85 and 127 > 68 (MEL) while the [M−H]⁻ of m/z 127.1 was selected as the precursor ion for CYA resulting in product ions m/z 85 and 42. Isotope labeled internal standard (¹⁵N₃-MEL and ¹³C₃-CYA) and matrix-matched calibration were both used to observe the recovery to be 70.0–129.6% and 70.0–128.9% with RSD of 1.4–23.3% and 1.5–21.7% for MEL and CYA, respectively (n = 6). All the LODs and LOQs of MEL and CYA were less than 39.4 and 99.1 μg kg⁻¹, respectively, in 18 matrices, which were sensitive enough for quantitative analysis. This method has been proven effective in simultaneous determination of melamine and cyanuric acid when inspecting unknown and positive samples.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.     

An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C₁₈ column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623 → 161 and m/z 289 → 109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0–50.0 and 50.0–5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20 mg/kg) and oral (100 mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.     

Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography–tandem mass spectrometry (XLC–MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC–MS/MS was compared with liquid chromatography with electrochemical detection (HPLC–ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R² > 0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC–MS/MS correlated well with HPLC–ECD (correlation coefficient >0.98). Reference intervals were 1–10 μmol/mol, 10–50 μmol/mol and 60–225 μmol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC–MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Simultaneous stereoselective high-performance liquid chromatographic determination of 10-hydroxycarbazepine and its metabolite carbamazepine-10,11-trans-dihydrodiol in human urine

An enantioselective HPLC method for the simultaneous determination of the concentration of the enantiomers of the oxcarbazepine metabolites 10-hydroxycarbazepine (MHD) and carbamazepine-10,11-trans-dihydrodiol (DHD) in human urine is described. The method is based on extraction with tert.-butylmethyl ether–dichloromethane (2:1, v/v) under alkaline conditions, separation and evaporation of the organic phase and dissolution of the residue in the mobile phase. Enantiomers are resolved on a Diacel Chiralcel OD column (250 mm×4.6 mm I.D.) under isocratic conditions using as mobile phase n-hexane–ethanol–2-propanol (18:2:1, v/v/v) with addition of glacial acetic acid (0.1%). The enantiomers are detected by UV at 215 nm. The method allows reliable determination of the MHD and DHD enantiomers in human urine with limits of quantification of 0.2 mg/l and 0.4 mg/l, respectively.     

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction method for the determination of benzimidazole residues in edible tissues

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with a pressure liquid extraction (PLE) was developed for the determination of 11 benzimidazole and 10 metabolites of albendazole, fenbendazole and mebendazole in the muscles and livers of swine, cattle, sheep and chicken. For sample preparation, we used an automated technique of PLE method. The optimum extraction conditions were obtained using an 11 ml Accelerated Solvent Extraction (ASE) cells, acetonitrile/hexane as the extraction solvent. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 5 mmol l(-1) formic ammonium as mobile phase. The analytes were detected in the positive ion multiple reaction monitoring (MRM) mode by the LC-ESI-MS/MS analysis. The recoveries of benzimidazole (BZDs) spiked at the levels of 0.5 μg kg(-1) ranged from 70.1% to 92.7%; the between-day relative standard deviations were no more than 10%. The limits of quantification were 0.02-0.5 μg kg(-1). The optimized method was successfully applied to monitor real samples containing BZDs, demonstrating the method to be simple, fast, robust and suitable for identification and quantification of BZDs residues in animal products.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.