Fast Liquid Chromatography-tandem Mass Research Articles

Simultaneous determination of cortisol and cortisone from human serum by liquid chromatography-tandem mass spectrometry.

A fast, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and then the levels of cortisol and cortisone from sera of healthy adults were determined by the LC-MS/MS method. One hundred μL of serum sample was directly extracted by adding 2 mL ethyl acetate, followed by chromatographic separation on a C18 column with a mobile phase consisting of 5 mM ammonium acetate and methanol (25 : 75, v/v). The precision, accuracy, and average recovery of the method were 1.5–5.3%, 95.4–102.5%, and 96.4% for cortisol, and 1.9–6.0%, 89.2–98.8%, and 79.9% for cortisone, respectively. The method was linear from 1.0 to 500.0 ng/mL (r2 = 0.999) for cortisol and 2.5 to 100.0 ng/mL (r2 = 0.998) for cortisone. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 1.0 ng/mL for cortisol, and 1.0 and 2.5 ng/mL for cortisone, respectively. The average cortisol concentration (133.9 ± 63.7 ng/mL) of samples collected between 9:00 and 11:00 a.m. was higher approximately 4.4 times than that of cortisone (30.5 ± 10.7 ng/mL) (P < 0.0001). The average cortisone/cortisol ratio was 0.225. Therefore, the LC-MS/MS method may be useful for the diagnosis of some adrenal diseases and the assessment of 11β-hydroxysteroid dehydrogenase (11β-HSD) activity in clinical laboratories.

Simultaneous determination of benzoylmesaconine and piperine in rat plasma after oral administration of Naru-3 by an ultra fast liquid chromatography-tandem mass spectrometry method and its application in a comparative pharmacokinetic study

A rapid, sensitive and reliable UFLC-MS/MS method has been developed for the simultaneous quantitation of benzoylmesaconine and piperine in rat plasma after oral administration of Naru-3.

Application of nanoring amino-functionalized magnetic polymer dispersive micro-solid-phase extraction and ultra fast liquid chromatography–tandem mass spectrometry in dicyandiamide residue analysis of powdered milk

In this study, a rapid and accurate ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) method combined with dispersive micro-solid-phase extraction (d-µ-SPE) using a core-shell nanoring amino-functionalized magnetic polymer (CS-NR-MP) was established and validated to determine trace dicyandiamide (DCD) in powdered milk. The developed d-µ-SPE cleanup procedure can dramatically reduce the matrix in samples, and lead to a significant reduction in absolute matrix effects. Chromatographic separation was performed on an Acquity UPLC BEH Amide column by using water-acetonitrile (9:91, v/v) as the mobile phase within 2 min. DCD was quantitatively analyzed by using DCD-(15)N2(13)C2 as an internal standard. The results showed that the recoveries were between 99.8 and 105.6% with RSDs in the range of 0.5-4.9%. The target compound had good linearity in the range of 0.1-20.0 µg L(-1) with a correlation coefficient (r) of 0.9996. The limit of quantification (LOQ) was 0.06 µg kg(-1). This method can be used for the rapid and sensitive determination of ultratrace DCD residue in powdered milk samples.

Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1μm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.

Determination of cefuroxime in liver-injured rat plasma by ultra fast liquid chromatography-tandem mass spectrometry via acidified protein precipitation

Determination of cefuroxime in liver-injured rat plasma by ultra fast liquid chromatography-tandem mass spectrometry via acidified protein precipitation

Determination of cefuroxime in liver-injured rat plasma by ultra fast liquid chromatography-tandem mass spectrometry via acidified protein precipitation

In order to investigate the pharmacokinetic profiles of cefuroxime lysine, a new second generation cephalosporins, in liver-injured rat model, an ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the determination of cefuroxime in liver-injured rat plasma was developed and validated. The plasma sample was pretreated by protein precipitation with acidified acetonitrile. The analytes were separated on a Shim-pack XR-ODS column (75 mm x 3.0 mm, 2.2 microm) with acetonitrile-0. 1% formic acid aqueous solution (40:60, v/v) as the mobile phase at a flow rate of 400 microL/min. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with a negative electrospray ionization (ESI) interface. The precursor to product ion transitions of m/z 423.2 --> 206.8 and m/z 454.1 --> 238.4 were selected to determine cefuroxime and cefotaxime (internal standard, IS), respectively. The linearities ranged from 0.01 to 1 mg/L and 1 to 400 mg/L (r > 0.99), and the limit of quantification of cefuroxime was 0.01 mg/L. The relative standard deviations (RSDs) of intra- and inter-day precisions were both less than 11.5%, and the accuracy (relative error) was between -7.1% and 2.2%. The mean extraction recovery was more than 83.5%. The total run time was 3.0 min per sample. The method is simple and fast for the preliminary pharmacokinetic study of cefuroxime lysine in liver-injured rats.

Development of a Stability Indicating UPLC-MS/MS Method for Rapid and Reliable Determination of Fenofibrate in Marketed Product (Lypanthyl 200M) and Human Plasma

A reliable, fast, sensitive and selective Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) method has been developed and validated for the determination of fenofibrate in marketed product (Lipanthyl) and human plasma.

Validation of a rapid liquid chromatography–tandem mass spectrometric assay for the determination of octreotide plasma concentrations

ObjectivesThe aim of this study was to develop and validate a fast liquid chromatography–tandem mass spectrometric (LC–MSMS) assay to determine circulating concentrations of octreotide (a somatostatin analog used in acromegaly and neuroendocrine tumors) in patients' plasma samples. Design and methods500μL of heparin–plasma was used to extract the drug on a cation exchanger SPE column, and the eluate was injected into a LC–MSMS system. Reversed phase chromatography was performed on a phenyl grafted column in isocratic mode. Octreotide and internal standard (triptorelin) were identified in positive electrospray ionization mode using ion transitions of m/z 512>120 and 890>249, respectively. ResultsThe assay was linear in the concentration range of 0.5–25ng/mL, with intra- and inter-assay imprecisions of <10.6% and <12.2%, respectively. The limits of quantification and detection were 0.5 and 0.25ng/mL. The recovery and ion suppression effect ranged between 79.7 and 84.5% and between −8.1 and −21.3%, respectively. A subcutaneous injection of 0.1mg octreotide induced a time- and patient-dependent surge of peptide concentrations peaking at 2h. ConclusionThis fast, sensitive, and selective method for quantification of plasma octreotide by LC–MSMS might be used to investigate the pharmacokinetic–pharmacodynamic relationship, with potential contribution to treatment optimization and therapeutic drug monitoring application.

Aldehyde Oxidase 1 (AOX1) in Human Liver Cytosols: Quantitative Characterization of AOX1 Expression Level and Activity Relationship

Aldehyde oxidase 1 (AOX1) is a cytosolic enzyme highly expressed in liver and plays a key role in metabolizing drugs containing aromatic azaheterocyclic substituents. Rapid metabolism catalyzed by AOX1 can cause a drug to exhibit high clearance, low exposure, and hence decreased efficacy or even increased toxicity (if AOX1 generated metabolites are toxic). There is a need to develop the correlation between AOX1 expression levels and AOX1-substrate clearance. A fast, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify AOX1 in human liver cytosol for the first time. This LC-MS/MS method includes a straightforward ultrafiltration fractionation step and gives great selectivity and wide dynamic range (5.2 pM to 20.7 nM). The AOX1 levels in human liver cytosols of 20 donors were quantified using this method to investigate individual differences in AOX1 expression. No significant individual or gender differences in AOX1 levels were observed, although male donors exhibited a broader distribution than female donors (0.74-2.30 pmol/mg versus 0.74-1.69 pmol/mg, respectively). The AOX1 protein levels measured by LC-MS/MS were consistent with those measured by an enzyme-linked immunosorbent assay. Several donors have a normal AOX1 protein level but low enzyme activity, which might be due to cofactor deficiency, single nucleotide polymorphism, or homodimer dissociation. Cytosols from donors with chronic alcohol consumption had low AOX1-catalyzed carbazeran oxidation activities (<51 µl/min per milligram compared with a median of 455 µl/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value).

A UFLC‐MS/MS method with a switching ionization mode for simultaneous quantitation of polygalaxanthone III, four ginsenosides and tumulosic acid in rat plasma: application to a comparative pharmacokinetic study in normal and Alzheimer's disease rats

A fast, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of polygalaxanthone III (POL), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Re (GRe), ginsenoside Rg1 (GRg1) and tumulosic acid (TUM) in rat plasma after oral administration of Kai-Xin-San, which plays an important role for the treatment of Alzheimer's disease (AD). The plasma samples were extracted by liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) with salidrdoside as internal standard (IS). Good chromatographic separation was achieved using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water. The tandem mass spectrometric detection was performed in multiple reaction monitoring mode on 4000Q UFLC-MS/MS system with turbo ion spray source in a negative and positive switching ionization mode. The lower limits of quantification were 0.2-1.5 ng/ml for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 60.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in normal and AD rat plasma. The results indicated that no significant differences in pharmacokinetic parameters of GRe, GRg1 and TUM were observed between the two groups, while the absorption of POL and GRd in AD group were significantly higher than those in normal group; moreover, the GRb1 absorbed more rapidly in model group. The different characters of pharmacokinetics might be caused by pharmacological effects of the analytes.

Determination of Raloxifene in Urine by Liquid Chromatography–Tandem Mass Spectrometry for Doping

Raloxifene is one of the selective estrogen receptor modulators and is often used to prevent and treat osteoporosis in postmenopausal women. Because of the indirect impact on serum testosterone levels and the potential ability for performance enhancement, it is banned by the World Anti-Doping Agency (WADA). This study established a fast, sensitive and selective liquid chromatography-tandem mass spectrometry method to quantify total raloxifene (unchanged and glucuronidated) in human urine for doping analysis. Urines from six healthy volunteers were collected 240 h after taking a single dose of raloxifene. The concentrations of urinary raloxifene were analyzed by the established method after sample preparation, including hydrolysis with β-glucuronidase. The lowest limit of quantification was 0.5 ng/mL. Linearity was observed for raloxifene concentrations ranging from 0.5 to 100 ng/mL, with a correlation coefficient of 0.999. The recoveries were >92.81%. Inaccuracies were below ±5%, and precisions varied from 2.18 to 5.37%. The results showed that urinary raloxifene was immediately detectable within 4 h after the administration of only a single dose of raloxifene. Such a result indicates a violation of the WADA rules. Furthermore, ingesting raloxifene would be detectable after 6 days in the urine of males or >10 days in the urine of female.

Simultaneous analysis of eight phenolic environmental estrogens in blood using dispersive micro-solid-phase extraction combined with ultra fast liquid chromatography–tandem mass spectrometry

A novel, simple and sensitive method was developed for the simultaneous determination of eight phenolic environmental estrogens in blood by using the dispersive micro-solid-phase extraction (d-µ-SPE) procedure combined with ultra-fast liquid chromatography–tandem quadrupole mass spectrometry (UFLC–MS/MS). The excellent nanomaterials tetraethylenepentamine-functionalized magnetic polymer was used as an adsorbent, and the main factors affecting the extraction efficiency were investigated in detail. All target compounds showed good linearities in the tested range with correlation coefficients (r) higher than 0.999. The mean recoveries were in the range of 85.0–105.0%. The intra-day and inter-day relative standard deviations (RSDs) were lower than 4.9% and 5.2%, respectively. The limits of quantification for the eight phenolic environmental estrogens were between 0.075 and 0.42µgL−1. The developed method can be applied to the routine analyses for the determination of the eight phenolic environmental estrogens in blood samples.

A UFLC–MS/MS method coupled with one-step protein precipitation for determination of docetaxel in rat plasma: Comparative pharmacokinetic study of modified nanostructured lipid carrier

A rapid, simple and sensitive ultra fast liquid chromatography–tandem mass spectrometry (UFLC–MS/MS) method coupled with one-step protein precipitation procedure has been developed and validated for the pharmacokinetic study of docetaxel in rat plasma to investigate the influence of polyethylene glycol (PEG) molecular weights (chain length) using in the modified formulations. Separation was achieved on a Venusil MP C18 column (100mm×2.1mm, 3.0μm) with a mobile phase consisting of methanol–water, and the total running time was 3.5min. The standard curve was linear over the range of 5–5000ng/mL, with lower limits of quantification (LLOQ) of 5ng/mL. The method was shown to be reliable and reproducible with intra-day precision below 10.7%, inter-day precision below 11.2%, accuracy within ±5.2%, and mean extraction recovery of 84.6–90.2%. The validated method was successfully applied to the comparative pharmacokinetic study of docetaxel in rat plasma after intravenous administration of docetaxel-loaded nanostructured lipid carrier modified by copolymers consisting of series of PEG molecular weights (2000, 4000, 10,000Da), respectively. The results indicated that PEG-4000 possessed a better and longer circulation effect, which made the modified formulation one of the promising suspensions for the delivery of docetaxel in cancer.

Determination of naphthalene-derived compounds in apples by ultra-high performance liquid chromatography-tandem mass spectrometry

Naphthylacetic acid, naphthyloxy acetic acid and naphthylacetamide belong to a group of synthetic substances known as “auxin-like” compounds which are used as growth regulators in vegetables and fruits due to their structure similarities with the indoleacetic acid, the most important plant auxin. This paper reports a selective, sensitive and fast ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the determination of naphthylacetamide (NAD) and the isomers (α and β) of naphthylacetic acid (NAA) and naphthyloxy acetic (NOA) acid in apple samples. A baseline separation between the respective isomers was achieved using an RP-Amide column with gradient elution. The UHPLC-MS/MS method developed, using electrospray and selected reaction monitoring (SRM) acquisition mode led to a reliable determination of these family of compounds in apple samples at low quantitation levels, down to 1.0μgkg−1 and 0.25μgkg−1 respectively. For confirmation of NAA accurate mass measurement is proposed giving at these conditions quantitation limits of 10μgkg−1 for this compound. The UHPLC-MS/MS method developed was used for the analysis of apple samples harvested in three different apple fields from Lleida (Spain) during the blooming period. NAD and NAA were found in samples collected during 4–5 weeks after application at concentrations between the quantification limits and 43μgkg−1 and 24μgkg−1, respectively.

A highly sensitive and selective method for the determination of leukotriene B4 (LTB4) in ex vivo stimulated human plasma by ultra fast liquid chromatography–tandem mass spectrometry

Leukotriene B4 (LTB4) is an important inflammatory component in a number of diseases and has been used as a pharmacodynamic (PD) biomarker. In this report, a highly sensitive and selective ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the determination of LTB4 in plasma from ex vivo stimulated human blood, using leukotriene B4-d4 (LTB4-d4, contains four deuterium atoms at the 6, 7, 14, and 15 positions) as the internal standard (IS), was developed and validated. The chromatographic separation of LTB4 from its three isomers and an unknown interference peak from human plasma was crucial to achieve accurate determination of 0.2 ng/mL (LLOQ) of LTB4. LTB4 and the IS were extracted with methyl tertiary butyl ether (MTBE) from 200 μL human plasma. Reversed-phase HPLC separation was carried out with a Phenomenex Synergi Hydro-RP column (100mm×3mm, 2.5 μm). MS/MS detection was set at mass transitions of 335.0→194.9 m/z for LTB4 and 339.0→196.9 m/z for LTB4-d4 in Turbo Ionization Spray (TIS) negative mode. The dynamic range of the method is 0.2-200 ng/mL. LTB4 was found to be stable in human plasma for at least three freeze (-20 °C)/thaw cycles, and on the benchtop (room temperature) for at least 6h. The stock solution storage stability study demonstrated that the LTB4 stock solution, in 50:50 acetonitrile:water, was stable at 4 °C for at least 198 days. The processed samples were found to be stable for at least 72 h at room temperature. The long-term sample storage stability test demonstrated that LTB4 human plasma samples were stable at a storage temperature of -20 °C for at least 198 days. In addition, intraday and interday accuracy and precision, sensitivity, linearity, and recovery were evaluated. An additional partial validation was conducted to decrease the plasma sample volume from 200 to 100 μL. All the data reported in this study fulfilled the requirements and recommendations in the FDA guidance for bioanalytical method validation. Comparison of the validated UFLC-MS/MS method with an ELISA method using ex vivo stimulated samples indicated that although results from the two assays correlated relatively well, the UFLC-MS/MS method has been shown to be superior in selectivity and dynamic range to an ELISA method in our study. The validated UFLC-MS/MS method was successfully used to analyze samples generated from two clinical studies. The excellent assay performance and incurred sample reproducibility (ISR) results obtained from the study sample analysis demonstrated the assay is robust and reliable.

Quantitative Analysis of Acetaminophen and its Primary Metabolites in Small Plasma Volumes by Liquid Chromatography-Tandem Mass Spectrometry

A fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous determination of acetaminophen (APAP) and its glucuronide and sulfate metabolites (APAP-GLU and APAP-SUL) in small plasma volumes. This method included a simple step of sample preparation and a chromatographic separation on an LC-MS-MS system equipped with an electrospray ionization source and a tandem triple quadrupole mass spectrometer in multiple reaction monitoring mode. The analytes and internal standard, APAP deuterated analog, were separated on a C18 column (3.0 µm, 2.1 × 100 mm), using aqueous 1% formic acid and methanol (80:20, v/v) as the mobile phase. The LC-MS-MS method was validated for accuracy, precision, linearity, extraction efficiency, process efficiency and matrix effect. Calibration curves were obtained by fortifying drug-free plasma and ranges of linearity were set between 0.25-20 mg/L. The mean correlation coefficients, r², were >0.99 for APAP and its metabolites. The inter-day and intra-day precision values were less than 11.75 and 13.03%, respectively, at the lower limit of quantification concentration. The usability of the method was demonstrated by studying APAP metabolism in C57BL/6J wild-type and obese ob/ob female mice, in which only small plasma volumes were available. The results showed that APAP glucuronidation was enhanced in obese mice, suggesting that changes in APAP metabolism could modify its toxicity in obesity and related fatty liver disease.

Rapid screening of 176 pesticide residues in vegetables by ultra fast liquid chromatography-tandem mass spectrometry

A multiresidue analytical method for rapid screening of 176 pesticide residues in vegetables was developed by using ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). The vegetable samples were extracted by acetonitrile. It is not necessary for the extract to make a further purification after salting out. Multiple reaction monitoring with information-dependent acquisition of enhanced product ion (MRM-IDA-EPI) was used for the analysis. Based on EPI spectra and chromatographic peak area, identification and quantification of the 176 pesticide residues in vegetables were carried out by using library search technique. All the pesticides had the good linearity within their respective linear ranges (r > 0.99). The average recoveries of the 174 pesticides except for carbosulfan and cyromazine were in the range of 72.4% to 126.4% with the relative standard deviations (RSDs) from 1.0% to 18.7%. The limits of detection and quantification of the method were 0.005 - 2.0 microg/kg and 0.1 - 10 microg/kg, respectively. The results demonstrated that the method has distinct advantages of rapid speed, high sensitivity and good accuracy. Therefore, this method is suitable for the rapid screening of pesticide residues in vegetables.

Determination of 23 Perfluorinated Alkyl Substances in Fishery Products by Ultra Fast Liquid Chromatography-Tandem Mass Spectrometry

A method was proposed for the determination of 23 perfluorinated alkyl substances(PFASs) in fishery products by the QuEChERS method and ultra fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS).The sample was extracted with acidified acetonitrile,cleaned-up by dispersive solid-phase extraction using C18 and graphitized carbon blacks(GCB).The separation of 23 PFASs was performed on a Kinetex XB-C18(100 mm×2.1 mm,2.6 μm) column using gradient elution of 5 mmol/L ammonium acetate and methanol as mobile phase within 12 min.The PFASs were analyzed under the multiple reaction monitoring(MRM) mode with negative electrospay ionization.Under the optimal conditions,the isotope internal standard method was used to determine the content of 23 PFASs,and improved the quantitative accuracy.The calibration curves were linear well with correlation coefficient over 0.995.The limits of detection ranged from 0.002 to 0.02 μg/kg.The average spiked recoveries for 23 PFASs were between 75.6% and 118.2%,with relative standard derivations(RSDs) from 2.7% to 14.5%.The developed method is simple and sensitive,and was suitable for rapid analysis of large quantities of samples.

Novel amide polar-embedded reversed-phase column for the fast liquid chromatography–tandem mass spectrometry method to determine polyether ionophores in environmental waters

A fast chromatographic method has been developed that takes less than 5min per run to determine five polyether ionophores with a novel amide polar-embedded reversed-phase column coupled to a triple quadrupole mass spectrometer. A comparison between Oasis HLB and Oasis MAX sorbents for the solid-phase extraction was done. Oasis HLB sorbent gave recoveries close to 90% and the repeatability (%RSD, 25–100ng/L, n=3) of the method was less than 7% for all compounds in all matrices. The presence of polyether ionophores in environmental waters such as river water and sewage was investigated. Monensin and narasin were frequently determined in influent and effluent sewage at concentrations from 10ng/L to 47ng/L in influents and from 6ng/L to 34ng/L in effluents. In river waters, polyether ionophores were not detected in any sample.

Validation of a fast and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionization method for simultaneous quantitation of voriconazole, itraconazole and its active metabolite hydroxyitraconazole in human plasma

Voriconazole and itraconazole are two broad-spectrum antifungal triazole derivates administered for the prevention and in the treatment of invasive fungal infections. Their broad inter- and intra-individual pharmacokinetic variability and the high probability of drug-drug interactions justify therapeutic drug monitoring. After liquid-liquid extraction with tert-butyl methyl ether, chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column using gradient elution with 10 mM ammonium formate and acetonitrile. Detection was performed by a tandem mass spectrometer coupled to LC via an atmospheric pressure chemical ionization (APCI) and quantification was performed using selected reaction monitoring (SRM) transitions Total run time was 4.5 min. The method was validated for concentrations ranging from 0.05 to 10 μg/mL for voriconazole and from 0.025 to 5 μg/mL for itraconazole and hydroxyitraconazole, respectively. The intra- and inter-day correlation coefficients of variation were <7.7%-<9.2%, respectively. The accuracy ranged from 92.6% to 109%. A rapid and simple liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS/MS) method has been developed and validated to measure voriconazole itraconazole and hydroxyitraconazole in human plasma. This method is successfully applied to samples from patients receiving antifungal treatment.