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

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

Determination of neonicotinoid insecticides residues in bovine tissues by pressurized solvent extraction and liquid chromatography-tandem mass spectrometry

A rapid, sensitive, and environmental-friendly method has been developed for the simultaneous determination of seven neonicotinoid insecticides residues in bovine muscle and liver. The sample preparation procedure was based on a high automated pressurized solvent extraction (PSE) combined with solid-phase extraction (SPE) clean-up. The target compounds were identified and quantitatively determined by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operated in multiple reaction monitoring mode. Average recoveries of the seven analytes from fortified samples ranged between 83.2% and 101.9%, with relative standard deviations (RSDs) lower than 10.8%. The limits of detection (LODs) and quantification (LOQs) for neonicotinoids were in the ranges of 0.8-1.5 μgkg?1 and 2.5-5.0 μgkg?1, respectively. This validated method was successively applied to the determination of neonicotinoid insecticides in real samples from markets.     

Determination of sodium nifurstyrenate and nitrovin residues in edible food by liquid chromatography-tandem mass spectrometry after ultrasound-assisted extraction

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of nitrovin and sodium nifurstyrenate residues in muscle and liver of swine and chicken and in muscle of fish. Sample preparation procedure includes ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were detected in multiple reaction monitoring (MRM) under negative scan mode acquiring two diagnostic product ions for sodium nifurstyrenate and under positive mode for nitrovin. The averaged decision limits (CCα; α 1%) ranged 0.09-0.26 μg/kg while the detection capability (CCβ; β 5%) was 0.33-0.97 μg/kg in the tissues. Reasonable recoveries (71-110%) spiked in muscle and liver showed excellent relative standard deviation (RSD). The validated method was simple, rapid, sensitive, and complied with the regulations for the determination of nitrovin and sodium nifurstyrenate residues in food matrices.     

Development of a method for determination of the malondialdehyde-deoxyguanosine adduct in urine using liquid chromatography-tandem mass spectrometry

A procedure is described for the quantification of the major malondialdehyde deoxyguanosine adduct, pyrimido[1,2-alpha]purin-10(3H)-one-deoxyribose (M(1)GdR) in urine. M(1)GdR is isolated from urine by a combination of C(18) solid-phase extraction and immunoaffinity chromatography. Sodium borohydride treatment reduces M(1)GdR to the 5,6-dihydro derivative, which is quantified by liquid chromatography-mass spectrometry. Authentic [7,9-15N,8-13C]M(1)GdR is added to urine as an internal standard. A detection limit of 50 fmol M(1)GdR/ml urine is achieved starting with 5 ml of urine. Analysis of urine samples from control rats or rats treated with CCl(4) indicates that the levels of M(1)GdR are below the detection limit of the assay. This method is easily adaptable to the analysis of M(1)GdR in DNA samples or biological fluids.     

Development of a high performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumer's health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.     

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01M EDTA-Na(2)-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5microg g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0microg g(-1) DW, with a limit of quantification (LOQ) of 0.02microg g(-1) DW. The limit of detection (LOD) of the method was 0.007microg g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR.     

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.     

Analysis of tetracycline residues in royal jelly by liquid chromatography-tandem mass spectrometry

A confirmatory method coupling liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed to determine the concentration of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC), which make up the tetracycline (TC) groups present in royal jelly. Sample preparation included deproteination, control of pH, extraction and clean-up on a solid-phase extraction (SPE) cartridge. The analyses were achieved by LC/MS/MS in selected reaction monitoring mode (SRM). The overall recovery of fortified royal jelly at the levels of 5.0, 10.0 and 40.0 microg/kg ranged from 62% to 115%, and the coefficients of variation ranged from 3.4% to 16.3% (n=6). The detection limits for TCs were under 1.0 microg/kg. The transformation between the TCs and its epimers (EpiTCs) was studied in standard solution and during the sample preparation process. This method can be used for the detection of tetracycline residues in royal jelly.     

Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography-tandem mass spectrometry

A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub μg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 μg/kg for MRL compounds and varied from 0.1 to 0.2 μg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3 μg/kg.     

Development of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid-liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M+H](+) at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2-5.0 ng/ml based on 500 microl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.     

Simultaneous determination of triclabendazole and its metabolites in bovine and goat tissues by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous determination of triclabendazole, its main metabolites (triclabendazole sulphone and triclabendazole sulphoxide) and a marker residue (ketotriclabendazole) in bovine and goat muscle, liver, and kidney samples is developed and validated. Analyte extraction from samples is effectively performed using liquid–liquid extraction by acetonitrile. Chromatographic separation is performed on a C₁₈ reversed-phase column with gradient elution. The analytes are detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring. The limits of detection for analytes are found to be 0.25–2.5 μg/kg in muscle tissues and 1–10 μg/kg in liver and kidney tissues, respectively. The recoveries of edible bovine and goat tissues range from 84.9% to 109.5% when spiked at different levels with analytes, with relative standard deviations generally below 12.8%.     

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.     

Development and validation of a high performance liquid chromatography-tandem mass spectrometry for the determination of etodolac in human plasma

A simple and specific method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C(18) column with 65% acetonitrile and 35% water containing 10mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99>172.23 for etodolac and m/z 357.92>139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1microg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.     

Simultaneous determination of six urinary porphyrins using liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method without sample pretreatment was developed and validated for determination of porphyrins in samples of canine urine. Acidified urine samples were directly injected into the LC-MS system and a gradient elution program was applied. The mass spectrometer was operated in the multi-reaction monitoring (MRM) mode and six porphyrins were detected with excellent sensitivity and selectivity. The lower limits of quantification were 0.014 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.029 nmol/mL for uroporphyrin I. Good ln-quadratic responses of calibration standards over the range 0.01 to 1.0 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.02 to 1.0 nmol/mL for uroporphyrin I were demonstrated. This method should be easily adapted through cross-validation for use in determining the effects of chemicals and pharmaceuticals on the urinary excretion profile of porphyrins in preclinical studies with other species, and in assisting the diagnosis of porphyria in clinical studies.     

Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the simultaneous determination of hypericin and hyperforin--the two main active ingredients of St. John's Wort (SJW) extract--in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levosulpiride in human plasma

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levosulpiride in human plasma was developed. Levosulpiride and internal standard, tiapride were extracted from human plasma with ethyl acetate at pH 11 and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile-ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.999) over the concentration range of 1.00-200 ng/ml. The lower limit of quantification for levosulpiride was 1.00 ng/ml using 100 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at three quality control (QC) levels were 3.8-9.1 and -2.9 to -0.1%, respectively. The recoveries of levosulpiride ranged from 80.5 to 87.4%, with that of tiapride (internal standard) being 84.6%. This method was successfully applied to the pharmacokinetic study of levosulpiride in humans.     

Quantitative determination of a novel dual PPAR alpha/gamma agonist using on-line turbulent flow extraction with liquid chromatography-tandem mass spectrometry

Turbulent flow chromatograph (TFC) is a technique for the direct and efficient analysis of drugs and metabolites in biological matrices. We report here TFC on-line with an HPLC-MS/MS assay for the determination of 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, MK-0767, KRP297, Fig. 1) in plasma. Samples were transferred using an automated system followed by the addition of internal standard (II), prepared in 0.1 M ammonium acetate (pH 4.0). The plasma samples were directly injected onto a C18 turbulent flow column on-line with an HPLC-MS/MS system, and the analytical column used was a ThermoHypersil Keystone C18. Detection was achieved by MS/MS, using positive ionization on a TurboIonSpray probe, operated in multiple reaction monitoring (MRM) mode. The linear range was 4-2000 ng/mL for I when using 50 microL of plasma. The method exhibited good linearity and reproducibility. The method also showed good selectivity and ruggedness when applied to clinical samples, and was successfully cross-validated with a conventional off-line SPE, LC-MS/MS method.     

Analysis of urinary aristolactams by on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs), nephrotoxicants and known human carcinogens, are a mixture of structurally related derivatives of nitrophenanthrene carboxylic acids with the major components being aristolochic acid I and aristolochic acid II. People may ingest small amounts of AAs from its natural presence in medicinal plants and herbs of the family Aristolochiaceae, including the genera Aristolochia and Asarum, which have been used worldwide in folk medicine for centuries. In order to assess AA intake, an on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry (on-line SPE-LC/MS/MS) method was developed to analyze their most abundant corresponding metabolites, aristolactams (ALs), in urine to serve as biomarkers. The limits of quantitation were 0.006 ng for aristolactam I (AL-I), and 0.024 ng for aristolactam II (AL-II) on column. Recovery varied from 98.0% to 99.5%, and matrix effects were within 75.3-75.4%. This method was applied to analyze ALs in the urine samples collected on days 1, 2, 4, and 7 from mice treated with 30 mg/kg or 50mg/kg AAs. Their half lives were estimated to be 3.55 h and 4.00 for AL-I, and 4.04 and 4.83 h for AL-II, depending on AAs doses. These results demonstrated that the first simple on-line SPE-LC/MS/MS method was successfully developed to analyze urinary ALs with excellent sensitivity and specificity to serve as biomarkers to assess current AA intake from AAs-containing Chinese herbs.