Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Multi-residue determination of pesticides in tropical fruits using liquid chromatography/tandem mass spectrometry

Monitoring pesticide residues in tropical fruits is of great interest for many countries, e.g., from South America, that base an important part of their economy on the exportation of these products. In this work, a LC-MS/MS multi-residue method using a triple quadrupole analyzer has been developed for around 30 pesticides in seven Colombian tropical fruits of high commercial value for domestic and international markets (uchuva, tamarillo, granadilla, gulupa, maracuya, papaya, and pithaya). After sample extraction with acetonitrile, an aliquot of the extract was diluted with water and directly injected into the HPLC-MS/MS system (electrospray interface) without any cleanup step. The formation of sodium adducts—of poor fragmentation—was minimized using 0.1% formic acid in the mobile phase, which favored the formation of the protonated molecule. However, the addition of ammonium acetate made the formation of the ammonium adducts in some particular cases possible, avoiding the presence of the sodium adducts. The highest sensitivity was observed in positive electrospray ionization for the wide majority of pesticides, with a few exceptions for acidic compounds that gave better response in the negative mode (e.g., 2,4-D, fluazinan). Thus, simultaneous acquisition on the positive/negative mode was applied. Two MS/MS transitions were acquired for each compound to ensure a reliable quantification and identification of the compounds detected in samples, although for malathion a third transition was acquired due to the presence of interfering isobaric compounds in the sample extracts. A detailed study of matrix effects was made by a comparison of standards in solvent and in matrix. Both ionization suppression and ionization enhancement were observed depending on the analyte/matrix combination tested. Correction of matrix effects was made by the application of calibration in matrix. Three matrices were selected (uchuva, maracuya, gulupa) to perform matrix calibration in the analysis of all seven fruit varieties studied. The method was validated by recovery experiments in samples spiked at two levels (0.05 and 0.5 mg/kg). The data were satisfactory for the wide majority of analyte/matrix combinations, with most recoveries between 70% and 110% and the RSD below 15%. Several samples collected from the market were finally analyzed. Positive findings were confirmed by evaluating the experimental Q/q ratios and retention times, and comparing them with those of reference standards.     

Multiresidue analysis of beta-agonists in bovine and porcine urine,feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry

The use of β-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of β-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 β-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCα and CCβ values of less than 0.2 and less than 0.5 μg/l respectively, for all β-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCβ values of less than 10.0 and less than 5.0 μg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC™) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of β-agonists.     

Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method

Quantification of orotic acid (uracil-6-carboxylic acid) in urine is an important tool to diagnose some inherited diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. New rapid analytical methods are necessary to provide high-throughput orotic acid analyses. A new analytical method has been developed for the rapid analysis of orotic acid in urine by liquid chromatography coupled with ion spray tandem mass spectrometry (LC/MS/MS). After a sample dilution 1:20, the analysis was performed in the selected reaction monitoring mode in which orotic acid was detected through the transition m/z 155 to 111. The retention time was 3.9 min in a 4.5-min analysis. Daily calibration between 0.5-5.0 micromol/L of orotic acid, corresponding to 10-100 micromol/L in urine before the 1:20 dilution, offered consistent linearity and reproducibility. Interassay coefficient of variance (c.v.) was 4.97% at a mean concentration of 10.99 micromol/L. The sensitivity and specificity of tandem mass spectrometry permitted a high volume of analyses of orotic acid. The sample preparation is simple, inexpensive and not time demanding.     

Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip‐based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C₁₈ LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C₁₈ chips. On the other hand, hydrophobic glycopeptides were better retained on C₁₈ chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four‐protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N-dechloroethylcyclophosphamide (DCL-CP), 4-ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D(4)-CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple-quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL-CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24-h urine collections of patients treated with a polychemotherapy regimen (3-175 microg/mL for CP, 0.5-27 microg/mL for 4KetoCP and 0.17-9 microg/mL for CarboxyCP and DCL-CP, respectively). The method was validated according to international guidelines of the ICH and the FDA.     

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.     

Multi-residue method for the determination of thyreostats in urine samples using liquid chromatography coupled to tandem mass spectrometry after derivatisation with 3-iodobenzylbromide

Thyreostats are banned compounds in Europe since 1981 (directive 81/602/EC) because of their carcinogenic and teratogenic properties. The control of their illegal use in breeding animals is amongst the most difficult because of their low molecular weight, high polarity and the existence of tautomeric forms. The analytical procedure described in this paper involves, after a derivatisation step with 3-iodobenzylbromide, the detection and identification at low level (microg/L) by LC-ESI(-)-MS/MS of eight thyreostats in various biological matrices (urine, muscle, liver, thyroid, animal feed, faeces and hair). The urine method has been validated according to the EU criteria (2002/657/EC decision): CCalpha and CCbeta were found in the range (0.1-5.2 microg/L) and (2.6-23.2 microg/L), respectively. The performances fulfil the requirements of the EU regarding the provisional minimum required performance limit (MRPL) fixed at 100 microg/L in urine.     

Automated liquid chromatographic/tandem mass spectrometric method for screening beta-blocking drugs in urine

An automated liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method is presented for the screening and confirmation of 16 beta-blocking drugs in clinical and autopsy urine samples. The described method involved C(18) solid phase extraction, LC separation and MS analysis on a triple-stage quadrupole mass analyser. Samples were initially pre-screened for the presence of any beta-blocking drugs using LC/MS with selected ion monitoring. Any compounds tentatively identified as beta-blocking drugs on the basis of their LC retention time and protonated molecular ion were then automatedly subjected to a second analysis in which the relevant MS/MS product ion mass spectra were acquired. These product ion mass spectra were then automatically searched against a 400-substance mass spectral library containing previously acquired beta-blocking drugs. The results demonstrated that library search of beta-blocking drugs in urine with MS/MS product ion mass spectra was more reliable and produced fewer false negatives than library searching with mass spectra derived from single-stage quadrupole MS. The limits of identification in the MS/MS product ion scan ranged from 0.02 mg l(-1) for carvedilol to 1.2 mg l(-1) for pindolol, the majority of the values being below 0.2 mg l(-1).     

Use of liquid chromatography/tandem mass spectrometric molecular fingerprinting for the rapid structural identification of pharmaceutical impurities

Use of liquid chromatography/tandem mass spectrometric (LC/MSⁿ) molecular fingerprinting is systematically demonstrated as a very effective tool for rapid structural elucidation of pharmaceutical impurities through a case study in which three isomers of betamethasone sodium phosphate (BSP) were rapidly identified as degradants formed due to the D‐homoannular ring expansion of the steroid core structure of BSP in the solid state. The rapid structural elucidation of these degradants was achieved by matching or closely matching the UV profiles, molecular weights, and more importantly the fragmentation patterns obtained from the LC/MSⁿ (n = 1 to 3) analysis of their enzyme‐catalyzed hydrolytic products, respectively, with those of a D‐homoannular isomer of betamethasone available in our LC/MSⁿ molecular fingerprint database. This strategy of using LC/MSⁿ molecular fingerprinting to obtain high‐confidence structures of unknown species is then validated by structure verification through one‐ (1D) and two‐dimensional (2D) nuclear magnetic resonance (NMR) experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of tamoxifene metabolites in human urine utilizing different scan parameters on liquid chromatography/tandem mass spectrometry

Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were considered for the characterization of tamoxifene metabolites in human urine for anti‐doping purpose. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 166, 152 and 129) and neutral loss scan (neutral loss of 72 Da and 58 Da) in positive ion mode were assessed to recognize common ions or common losses of tamoxifene metabolites. The applicability of these methods was checked first by infusion and then by the injection of solution of a mixture of reference standards of four tamoxifene metabolites available in our laboratory. The data obtained by the analyses of the mixture of the reference standards showed that the five methods used exhibited satisfactory results for all tamoxifene metabolites considered at a concentration level of 100 ng/mL, whereas the analysis of blank urine samples spiked with the same tamoxifene metabolites at the same concentration showed that the neutral loss scan of 58 Da lacked sufficient specificity and sensitivity. The limit of detection in urine of the compounds studied was in the concentration range 10–100 ng/mL, depending on the compound structure and on the selected product ion. The suitability of these approaches was checked by the analysis of urine samples collected after the administration of a single dose of 20 mg of tamoxifene. Six metabolites were detected: 4‐hydroxytamoxifene, 3,4‐dihydroxytamoxifene, 3‐hydroxy‐4‐methoxytamoxifene, N‐demethyl‐4‐hydroxytamoxifene, tamoxifene‐N‐oxide and N‐demethyl‐3‐hydroxy‐4‐methoxytamoxifene, which is in conformity to our previous work using a time‐of‐flight (TOF) mass spectrometer in full scan acquisition mode. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre-cut arteries within pre-defined masses. A high-throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between -4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were < or =5.3% for the quality controls in blood and < or =9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries.     

Importance of using highly pure internal standards for successful liquid chromatography/tandem mass spectrometric bioanalytical assays

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra‐injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone‐D3, ursodiol‐D5 and atovaquone‐D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone‐D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone‐D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23‐Nordeoxycholic acid was preferred to ursodiol‐D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol‐D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone‐D4 reference standard revealed the non‐negligible presence of atovaquone‐D5 to atovaquone‐D8 that has a large impact on the method validation. Therefore, atovaquone‐D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of amoxicillin in animal feed using liquid chromatography with tandem mass spectrometric detection

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of amoxicillin (AMO) in animal feed was developed and validated. The method was used to examine the quality requirements for products intended for incorporation into animal feedingstuffs (medicated premixes), as documented in the EMEA/CVMP/080/95-Final guideline. After addition of the internal standard (ampicillin), the medicated feed samples were extracted using a 0.01 M potassium dihydrogenphosphate buffer solution (pH 4.5), followed by a centrifugation and filtration step. An appropriately diluted aliquot of the extract was analysed on a PLRP-S polymeric column (150 mm x 2.1 mm i.d., 100 A) using a mixture of 0.1% formic acid in water and acetonitrile as the mobile phase. Gradient elution was performed at a flow-rate of 0.2 mL min(-1). The mass spectrometer was used in the positive electrospray ionization MS/MS mode. The LC-MS/MS method was validated for linearity, trueness, precision, limit of quantification, limit of detection and specificity. The results fell within the ranges specified. The method was used for the homogeneity and stability testing of AMO in a commercial medicated feed. Some extracts were also injected onto a LC-UV and LC-fluorescence instrument (after pre-column derivatization with a formaldehyde reagent). These experiments showed that the LC-MS/MS method was superior with regard to speed of analysis, selectivity and sensitivity.     

Multi-residue determination of plant growth regulators in apples and tomatoes by liquid chromatography/tandem mass spectrometry

A sensitive and rapid multi-residue analytical method for plant growth regulators (PGRs) (i.e., chlormequat, mepiquat, paclobutrazol, uniconazole, ethephon and flumetralin) in apples and tomatoes was developed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). A homogenised sample was extracted with a mixture of methanol/water (90:10, v/v) and adjusted to pH <3 with formic acid. Primary secondary amine (PSA) adsorbent was used to clean up the sample. The determination was performed using electrospray ionisation (ESI) and a triple quadrupole (QqQ) analyser. Under the optimised method, the results showed that, except for ethephon, the recoveries were 81.8-98.1% in apples and tomatoes at the spiked concentrations of 0.005 to 2 mg/kg, with relative standard deviations (RSDs) of less than 11.7%. The limits of quantification (LOQs) were lower than their maximum residue limits (MRLs). The procedure was concluded as a practical method to determine the PGR residues in fruit and vegetables and is also suitable for the simultaneous analysis of the amounts of samples for routine monitoring. The analytical method described herein demonstrates a strong potential for its application in the field of PGR multi-residue analysis to help assure food safety.     

Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry

The confirmatory LC-MS/MS method for the determination of residues for twelve coccidiostats including ionophore antibiotics (lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin) and chemical coccidiostats (clazuril, decoquinate, diclazuril, halofuginone, nicarbazin and robenidine) in poultry liver has been developed. The sample preparation was based on extraction with acetonitrile, defatting with Alumina columns and clean-up on Oasis HLB spe. The separation of analytes was performed on PhenylHexyl column with a gradient of acetonitrile, methanol and the ammonium formate pH 4.0. For all analytes, at least 2 diagnostic fragmentation ions were monitored. The validation, performed according to the CD 2002/657/EC, proved the suitability of the method for the confirmatory analysis of the coccidiostats. For lasalocid, however, low reproducibility was observed and the proper quantification could not be performed with this method.     

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Simultaneous determination of 20 beta-agonists in pig muscle and liver by high-performance liquid chromatography/tandem mass spectrometry

A method was developed to determine 20 illegal residual beta-agonists in pork tissues, including muscle and liver simultaneously. The samples were hydrolyzed by beta-glucuronidase, purified by PCX SPE cartridges, and detected by HPLC coupled with electrospray ionization MS/MS operating in the positive ion mode. Matrix-fortified calibration was performed to compensate for the matrix effect and loss in sample preparation. Decision limit ranged from 0.05 to 0.23 microg/kg in muscle and 0.05 to 0.57 microg/kg in liver. Decision capacity ranged from 0.11 to 0.4 microg/kg in muscle and 0.16 to 0.79 microg/kg in liver. In Food Analysis Performance Assessment Scheme proficiency test 0287, a pig liver test material containing 13 beta-agonists was analyzed using the method developed, and clenbuterol and ractopamine were confirmed as being present. Z-scores for clenbuterol and ractopamine were 0.2 and 0.6, respectively.     

Determination of nitroimidazole residues in porcine urine by liquid chromatography/tandem mass spectrometry

A reliable and sensitive method was developed for determination of nitroimidazoles in porcine urine. Sample preparation involves an extraction with ethyl acetate, followed by a solid-phase extraction cleanup step. The final extract is injected into the liquid chromatography/tandem mass spectrometry system in the positive-ion electrospray ionization mode. A C8 column with water and acetonitrile as the mobile phase is used for chromatographic separation under gradient conditions. Overall average recoveries ranged from 83 to 107%. The limits of detection ranged from 0.03 to 0.05 ng/mL, and the limits of quantitation, from 0.1 to 0.2 ng/mL.