Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5-500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Liquid chromatography tandem mass spectrometry method for the quantification of clonidine with LLOQ of 10 pg/mL in human plasma

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of clonidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 230 to 44 for clonidine and m/z 254 to 44 for the internal standard. The assay exhibited a linear dynamic range of 10-2000 pg/mL for clonidine in human plasma. The lower limit of quantification was 10 pg/mL with a relative standard deviation of less than 6.8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry method for the quantification of rimonabant, a CB1 receptor antagonist, in human plasma

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of rimonabant in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective (M+H)+ ions, m/z 463-363 for rimonabant and m/z 408-235 for the internal standard. The assay exhibited a linear dynamic range of 0.1-100 ng/mL for rimonabant in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6%. With dilution integrity up to 10-fold, the upper limit of quantification was extendable up to 1000 ng/mL. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

Simultaneous quantification of a non-nucleoside reverse transcriptase inhibitor efavirenz, a nucleoside reverse transcriptase inhibitor emtricitabine and a nucleotide reverse transcriptase inhibitor tenofovir in plasma by liquid chromatography positive ion electrospray tandem mass spectrometry

A high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of efavirenz, emtricitabine and tenofovir was developed and validated with 100 microL human plasma. Following solid-phase extraction, the analytes were separated using a gradient mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 316 to 168 for efavirenz, m/z 248-130 for emtricitabine and m/z 288-176 for tenofovir, m/z 482-258 for rosuvastatin (IS), m/z 260-116 for propranolol (IS). The method exhibited a 100-fold linear dynamic range for all the three analytes in human plasma (20-2000, 2-200 and 20-2000 ng/mL for efavirenz, emtricitabine and tenofovir respectively). The lower limit of quantification was 2 ng/mL for emtricitabine and 20 ng/mL for both efavirenz and tenofovir with a relative standard deviation of less than 11%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 4 min for each sample made it possible to analyze more than 250 human plasma samples per day. The method is precise and sensitive enough for its intended purpose. The method is also successfully applied to quantify efavirenz, emtricitabine and tenofovir concentrations in a rodent pharmacokinetic study.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of Quetiapine in plasma

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of quetiapine in rat plasma. Following liquid-liquid extraction, the analyte was separated using a gradient mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 384 to m/z 221 for quetiapine and m/z 327 to m/z 270 for the internal standard. The assay exhibited a linear dynamic range of 0.25-500 ng/mL for quetiapine in rat plasma. The lower limit of quantification was 0.25 ng/mL with a relative standard deviation of less than 7%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated method was successfully used to analyze rat plasma samples for application in pre-clinical pharmacokinetic studies. This method in rodent plasma could be adapted for quetiapine assay in human plasma.     

Nevirapine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify nevirapine in human plasma using dibenzepine as the internal standard (IS) was developed and validated. The method employed a liquid-liquid extraction. The analyte and the IS were chromatographed on a C(18) analytical column, (150 x 4.6 mm i.d. 4 microm) and analyzed by tandem mass spectrometry in the multiple reaction monitoring mode. The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 10-5000 ng ml(-1) (r(2) > 0.9970). The between-run precision, based on the relative standard deviation for replicate quality controls was 1.3% (30 ng ml(-1)), 2.8% (300 ng ml(-1)) and 3.6% (3000 ng ml(-1)). The between-run accuracy was 4.0, 7.0 and 6.2% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two nevirapine tablet formulations (Nevirapina from Far-Manguinhos, Brazil, as a test formulation, and Viramune from Boehringer Ingelheim do Brasil Química e Farmacêutica, as a reference formulation) in 25 healthy volunteers of both sexes who received a single 200 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Nevirapina/Viramune was 96.4-104.5% for AUC((0-last)), 91.4-105.1% for AUC((0-infinity)) and 95.3-111.6% for C(max) (AUC = area under the curve; C(max) = peak plasma concentration). Since both 90% CI for AUC((0-last)) and AUC((0-infinity)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Nevirapina was considered bioequivalent to Viramune according to both the rate and extent of absorption.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil‐derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2‐(3,4‐dihydroxyphenyl)acetic acid, 4‐(2‐hydroxyethyl)‐2‐methoxy‐phenol or homovanillyl alcohol, 2‐(4‐hydroxy‐3‐methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC‐ESI MS/MS prior to and after enzymatic treatment. A solid‐phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra‐ and inter‐day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of three bufadienolides in rat plasma after intravenous administration of bufadienolides extract by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry

A novel, rapid and specific ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed for simultaneous determination and pharmacokinetic studies of three bufadienolides (bufalin, cinobufagin and resibufogenin) in rat plasma. The analytes, bufalin, cinobufagin, resibufogenin and the internal standard, diphenhydramine were extracted from rat plasma samples by a one-step liquid–liquid extraction and separated on an ACQUITY UPLC™ BEH C₁₈ column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL min⁻¹. Detection was carried out on a triple-quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) interface. The three bufadienolides could be simultaneously determined within 3.0 min. Linear calibration curves were obtained over the concentration ranges of 1.0–200 ng mL⁻¹ for all the analytes. The intra- and inter-day precisions (relative standard deviation (R.S.D.)) were less than 11.35 and 10.87%, respectively. The developed method was applied for the first time to the pharmacokinetic studies of bufadienolides in rats following a single intravenous administration of 2.10 mg kg⁻¹ bufadienolides.     

Determination of clindamycin in animal plasma by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

A method for the quantification of clindamycin in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS/MS) is presented. Lincomycin is used as the internal standard. The sample preparation includes a simple deproteinization step with trichloroacetic acid. Chromatographic separation is achieved on an RP-18 Hypersil column using gradient elution with 0.01 M ammonium acetate and acetonitrile as mobile phase. Good linearity was observed in the range 0-10 microg ml(-1). The limit of quantification of the method is 50 ng ml(-1) and the limit of detection is 1.3 ng ml(-1). The method was shown out to be of use for pharmacokinetic studies of clindamycin formulations in dogs.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

液相色谱-电喷雾串联质谱法测定生姜中的215种农药残留

建立了生姜中215种农药多残留测定的液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)方法。样品用1%醋酸-乙腈溶液均质提取,经Sep-Pak Vac固相萃取柱净化,乙腈-甲苯(3:1, v/v)洗脱,旋转蒸发浓缩至约0.5 mL后,于室温氮气吹干,用乙腈-水(3:2, v/v)溶解,以电喷雾电离串联质谱在正离子多反应监测(MRM)模式下进行测定。在定量限水平进行添加回收率实验,方法的回收率范围为68.1%～132.6%,其中回收率在70%～120%的占94.4%,相对标准偏差(RSD)范围为0.4%～25.0%。方法的检出限(S/N=3)和定量限(S/N=10)范围分别为0.01～70.45 μg/L和0.04～234.84 μg/L。该方法操作简便,灵敏度、准确度和精密度均符合农药多残留检测技术要求,适用于生姜中215种农药多残留的快速测定。     

Determination of ivermectin B(1a) in animal plasma by liquid chromatography combined with electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b).The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.     

Determination of apomorphine in canine plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a pharmacokinetic study

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were < 5.9% RSD and < 7.5% bias for apomorphine. Extraction recoveries were > 80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.