LC/MS confirmation of ionophores in animal feeds.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.     

Determination of the ionophoric coccidiostats narasin, monensin, lasalocid and salinomycin in eggs by liquid chromatography/tandem mass spectrometry

A sensitive and selective liquid chromatographic tandem mass spectrometric method (LC/MS/MS) for the simultaneous detection of the ionophoric coccidiostats narasin, monensin, lasalocid and salinomycin in whole eggs has been developed. A very simple sample preparation consisting of an extraction with an organic solvent was carried out. Sample extracts were injected into the LC/MS/MS system on a C18 column and an isocratic elution was performed. Nigericin was used as internal standard. The precursor ions produced by electrospray positive ionisation were selected for collisional dissociation with argon into product ions. Validation of the methods was performed based on Commission Decision 2002/657/EC.1 CC(alpha) was found to be 1 microg/kg for all four compounds. Monitoring of Belgian egg samples in 2004 revealed that residues of salinomycin, lasalocid and monensin could be found.     

Evaluation of two liquid chromatography/tandem mass spectrometry platforms for quantification of monensin in animal feed and milk

Monensin is an anticoccidial drug that has been used as an additive in medicated feed. The United States Food and Drug Administration (USFDA) has included monensin in the national surveillance schemes for residues in foodstuff. In this study, two simple, selective and rapid methods were developed to determine monensin content in animal feed and milk. The methods enabled the detection of monensin residues as low as 1 ppb. Moreover, the two methods were used as models to compare two common liquid chromatography/tandem mass spectrometry (LC/MS/MS) platforms; an LC linear ion trap (LC/LIT) and an LC triple quadrupole (LC/QqQ). The two instrument platforms were evaluated for their matrix effect dependence, precision and accuracy. The LC/QqQ presented a lower limit of detection and limit of quantitation (LOD and LOQ) and showed less matrix dependence as compared to the LC/LIT. The LC/QqQ instrument also demonstrated a better intermediate precision. For example, the intermediate precision standard deviation calculated for 27 analyses across three days was 4% and 11% for LC/QqQ and LC/LIT, respectively. Overall, the LC/QqQ represents a better choice for analysis of monensin with respect to LOD, LOQ, matrix interference and precision. Copyright © 2010 John Wiley & Sons, Ltd.     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Determination of aflatoxins in animal feeds by liquid chromatography/tandem mass spectrometry with isotope dilution

The objective of the present study is to develop a simple, fast method for detection of aflatoxins in animal feeds. Simultaneous quantitation of four aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) in animal feeds was achieved in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. The solid-phase extraction cleanup step is eliminated with the stable isotope dilution method. Matrix effects were observed and overcome by isotope dilution. The method was tested in a variety of animal feed matrices and proved to be accurate and reliable. Method ruggedness tests resulted in recoveries of 78% to 122% with an intra-day assay precision of 2% to 15% and an inter-day assay precision of 3% to 17%. These results indicate that this method is suitable for quantitation of aflatoxins in animal feeds.     

High-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry determination of cholesterol uptake by Caco-2 cells

A simple, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method (LC/APCI-MS/MS) was developed and applied to quantitative determination of uptake of cholesterol by Caco-2 human intestine cells. Caco-2 cells were cultured in medium containing cholesterol-3,4-13C2 and phytosterols from nutritional supplements after in vitro digestion. Cellular cholesterol (cholesterol-3,4-13C2) and endogenous cholesterol were extracted using methanol/chloroform (1:2, v/v) and directly analyzed using LC/APCI-MS/MS with selected reaction monitoring (SRM), using cholesterol-2,2,3,4,4,6-d6 as an internal standard. Detection and quantification limits were 2.2 and 7.2 pmol, respectively. This method provides an effective tool for rapid determination of cholesterol uptake by cells with increased selectivity and sensitivity in comparison to previously reported LC/APCI-MS analysis using selected ion monitoring (SIM).     

Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry

Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.     

Determination of lincomycin and tylosin residues in honey by liquid chromatography/tandem mass spectrometry

A simple and rapid analytical method was developed for the determination of lincomycin and tylosin residues in honey as part of field studies examining the efficacy and target animal safety of these antibiotics to control American foulbrood disease in honey bees. Residues of the antibiotics were determined using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Honey samples were diluted and injected directly into the LC/MS/MS system without additional cleanup by solid-phase extraction or liquid-liquid partitioning. A six-port valve system was utilized to selectively route eluant from the LC column into the mass spectrometer only during a relatively short portion of the chromatographic run corresponding to the elution of the analytes of interest. Minimal contamination of the MS source chamber was observed despite the analysis of large numbers of samples. Using internal standard quantitation, excellent accuracy and precision were obtained with no apparent matrix-to-matrix variation. Based on the analysis of fortified replicates, the mean percent deviation from the theoretical concentration and the percent relative standard deviation were both less than 10% for tylosin over an analytical range of 10-1000 microg/kg. Slightly higher mean percent deviations and relative standard deviations were observed for the analysis of lincomycin in fortified replicate samples. The method detection limits were determined to be 5 and 2 microg/kg for lincomycin and tylosin, respectively.     

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.     

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

超高效液相色谱-电喷雾串联四极杆质谱法同时测定配合饲料中29种兽药

建立了同时测定配合饲料中喹诺酮类、磺胺类、大环内酯类和硝基呋喃类共计29种兽药的超高效液相色谱-电喷雾串联四极杆质谱(UPLC-ESI-MS/MS)检测方法。饲料样品用甲醇-乙腈(1:1, v/v)混合溶液提取,提取液经Oasis HLB固相萃取柱净化,采用UPLC-ESI-MS/MS检测。以甲醇和含0.1%(v/v)甲酸的水溶液作为流动相,进行梯度洗脱,用C18色谱柱分离,正离子模式扫描,多反应监测模式检测。29种兽药在0.01～5.0 mg/L范围内线性关系良好,相关系数(r)均大于0.99;在复合预混饲料、全价配合饲料、浓缩饲料中4个添加水平下29种兽药的平均回收率在61.2%～94.3%范围内,相对标准偏差(RSD)为2.2%～15.0%;方法的检出限(以信噪比大于10计)为0.01 mg/kg或0.05 mg/kg。该方法简便、快速、准确,重现性好,灵敏度高,适用于配合饲料中多种兽药的同时检测。     

Reversed-phase liquid chromatographic determination of riboflavin in feeds

A method for determination of riboflavin in animal feeds using liquid chromatography (LC) was developed for feed samples fortified with riboflavin at 1 mg/lb or greater (up to 10,000 mg/lb). Feed samples were extracted in 0.1 N HCl with heating on a steam bath for 30 min, followed immediately by mechanical shaking for 30 min. Sample extracts were diluted to target volume with 2% acetic acid and filtered; riboflavin was determined by LC on a reversed-phase C18 column with 2% acetic acid-acetonitrile (85 + 15) mobile phase for separation and fluorescence detection with excitation at 460 nm and emission at 530 nm. The extraction was compared with that of the AOAC Official Method for riboflavin in food and feed premixes. The 2 method extractions were not significantly different from each other at the 95% confidence level. The developed method also had good linearity over 4 orders of magnitude, recovery of 95-99% from spiked feed samples, a limit of detection of riboflavin at 0.00034 microg/mL in solution, a limit of quantitation of 0.023 mg/lb in feed, and good ruggedness.     

High-throughput determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine carbocysteine in human plasma was developed and fully validated. After methanol-induced protein precipitation of the plasma samples, carbocysteine was subjected to LC/MS/MS analysis using electrospray ionization (ESI). The MS system was operated in the selected ion monitoring (SRM) mode. Chromatographic separation was performed on a Hypurity C18 column (i.d. 2.1 mm x 50 mm, particle size 5 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.1-20 microg/mL for carbocysteine. The lower limit of quantification (LLOQ) of the method was 0.1 microg/mL for carbocysteine. The intra- and inter-day precision was less than 7% for all quality control samples at concentrations of 0.5, 2.0, and 10.0 microg/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for carbocysteine compared with methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used to a bioequivalence study of two tablet formulations of carbocysteine in healthy volunteers.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Comparison of RP, Ion-Pair and IE Chromatography Coupled with UV and Tandem Mass Spectrometry for the Determination of 8-Hydroxyquinoline in Cosmetics

Liquid chromatographic methods based on reversed-phase, ion-pair and ion-exchange chromatography coupled with ultraviolet spectrometry and tandem mass spectrometry have been developed and compared for the determination of 8-hydroxyquinoline in cosmetic samples. The individual parameters of different techniques are systematically optimized. The reversed-phase chromatography using ultrasonic extraction, centrifugation and filtration prior to LC–UV and LC–MS–MS detection was selected to be the general analytical method for the routine analysis of 8-hydroxyquinoline in cosmetics due to its simplicity, column reproducibility and sensitivity. The procedure is rapid, simple and suitable for the quality control of cosmetic preparations. The application of this technique to the assay of commercial products is also presented.     

LC–MS–MS Simultaneous Determination of Paracetamol, Pseudoephedrine and Chlorpheniramine in Human Plasma: Application to a Pharmacokinetic Study

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma. Diphenhydramine was used as the internal standard. Analytes were extracted from alkalized human plasma by liquid–liquid extraction (LLE) using ethyl acetate. After electrospray ionization positive ion fragments were detected in the selected reaction monitoring (SRM) mode with a triple quadrupole tandem mass spectrometer. The method was linear in the concentration range of 20.0–10000.0 ng mL^?1 for paracetamol, 1.0–500.0 ng mL^?1 for pseudoephedrine and 0.1–50.0 ng mL^?1 for chlorpheniramine. The intra- and inter-day precisions were below 14.5% and the bias was between ?7.3 and +2.8% for all analytes. The validated LC–MS–MS method was applied to a pharmacokinetic study in which each healthy Chinese volunteer received a tablet containing 300 mg benorylate, 30 mg pseudoephedrine hydrochloride and 2 mg chlorpheniramine maleate. This is the first assay method described for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma samples.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Development of a qualitative liquid chromatography/tandem mass spectrometric method for the detection of narcotics in urine relevant to doping analysis

A new screening procedure for 18 narcotics in urine for anti-doping purposes has been developed using liquid chromatography/triple quadrupole mass spectrometry (LC/MS). Electrospray ionization (ESI) was used as interface. Infusion experiments were performed for all substances to investigate their mass spectrometric behaviour in terms of selecting product specific ions. These product ions were then used to develop a tandem mass spectrometric method using selected reaction monitoring (SRM). For the LC/MS analysis, chromatography was performed on an octadecylsilane column. The total run time of the chromatographic method was 5.5 min. For the sample preparation prior to LC/MS analysis, the urine samples were liquid-liquid extracted at pH 9.5 after overnight enzymatic hydrolysis. Two extraction solvents were evaluated: dichloromethane/methanol 9/1 (v/v), which is currently used for the extraction of narcotics, and diethyl ether, used for the extraction of steroids. With diethyl ether the detection limits for all compounds ranged between 0.5 and 20 ng/mL and with the mixture containing dichloromethane the detection limits ranged between 0.5 and 10 ng/mL. Taking into account the minimum required performance limits of the World Anti-Doping Agency of 200 ng/mL for narcotics, diethyl ether can also be considered as extraction solvent for narcotics. Finally, the described method was applied to the analysis of urine samples previously found to contain narcotics by our routine gas chromatography/mass spectrometry (GC/MS) method.     

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.