Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100–20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.     

Flavonoid profile in soybeans by high-performance liquid chromatography/tandem mass spectrometry

Flavonoid profiling of a soybean sample has been performed by liquid chromatography/positive electrospray tandem mass spectrometry (LC/ESI(+)-MS/MS) using a quadrupole-linear ion trap (QLIT) instrument with an information-dependent data acquisition (IDA) protocol that looped, in a single run, an enhanced MS scan and an enhanced product ion scan. As compromise between time and obtainable information, spectra acquisition was split into two distinct runs: 220-450 Th and 400-800 Th, respectively. The isoflavones daidzein and genistein were identified as aglycones, monoglycosides, diglycosides, triglycosides, acetylglycosides, malonylglycosides, malonyl diglycosides, and dimalonyl diglycosides, whereas glycitein triglycosides, acetylglycosides, and dimalonyl diglycosides were not detected. Also kaempferol di- and triglycosides, malonylglycosides and malonyl diglycosides, previously reported in soy leaves and pods, and four naringenin malonylglycosides were identified.     

Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry

A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the limit of detection was 0.125 ng/mL. The excretion profile was determined after administration of 500 microg salmeterol (Serevent) to four standard-bred mares via a metered dose inhaler (MDI) with an Equinehaler adapter. Salmeterol was detected from 1 h until 12 h post-administration. Maximum urinary concentrations varied between 2.3 and 14.9 ng/mL.     

Determination of selenosugars in crude human urine using high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

The narrow gap between essentiality and toxicity of selenium requires detailed investigations on selenium metabolism in order to find suitable indicators for the selenium status in the human body. Current methods for quantitative selenium speciation in human urine are based on separation by high-performance liquid chromatography (HPLC) coupled online with elemental mass spectrometry (MS), and the potential of molecular MS detection techniques for the reliable identification and quantification of selenosugars in crude human urine has not been utilized. Now we report the development of an HPLC tandem mass spectrometric (MS/MS) method for the reliable determination in crude human urine of three significant selenium urinary metabolites, collectively termed selenosugars, namely methyl 2-acetamido-2-deoxy-1-seleno-beta-D-galactopyranoside (SeGalNAc), methyl 2-acetamido-2-deoxy-1-seleno-beta-D-glucopyranoside (SeGluNAc) and methyl 2-amino-2-deoxy-1-seleno-beta-D-galactopyranoside (SeGalNH2). Reversed-phase HPLC, with and without cation-exchange guard columns, was applied for the separation of the selenosugars, and atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) were used for selective and sensitive detection. The collision-induced dissociation behaviour of the selenosugars was studied in detail using APCI triple quadrupole MS/MS and electrospray ion trap MS. The developed method was applied to urine samples collected prior to and after selenium supplementation for the quantification of SeGalNAc using both external calibration and the method of standard additions. Additionally, SeGalNH2 was detected in urine samples after Se supplementation. Finally, neutral loss scanning was explored as a possible method for the detection of unknown methyl-selenosugars.     

Analysis of tetracyclines in honey by high-performance liquid chromatography/tandem mass spectrometry

A confirmatory method coupling liquid chromatography to tandem mass spectrometry (LC/MS/MS) is described for the determination of tetracycline, oxytetracycline, doxycycline and chlortetracycline in honey. Demeclocycline, another tetracycline molecule not reported for its usage in honey, was used as internal standard to quantify the four analytes. The sample preparation entails a clean-up on an Oasis HLB solid-phase extraction cartridge and analyses were realised by LC/MS/MS in selected reaction monitoring mode. The stability of tetracyclines was checked under various storage conditions at -20, +4 and +20 degrees C (both under dark and light exposures). Indeed, tetracyclines are not stable molecules and the epimerisation phenomenon was evaluated in this work. Appropriate correction factors of the MS/MS responses of each epimer were studied for each of the four tetracyclines to accurately quantify them. Moreover, the matrix effects encountered during the LC/MS/MS analyses were also studied in spiked experiments from blank honey samples of various geographical origins and different flower types.     

Determination of methotrexate in human urine at trace levels by solid phase extraction and high-performance liquid chromatography/tandem mass spectrometry

For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highly sensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a precise and accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solid phase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol, the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validated by using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than 0.999, and the limit of detection was assessed at 0.2 microg L(-1). High specificity of the HPLC-MS/MS technique assures that no interfering substances are detected rather than the analyte of interest.     

Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry

The review discusses the pitfalls of the matrix effect in mass spectrometry detection hyphenated to liquid chromatography separation. Matrix effect heavily influences both qualitative and quantitative analyses, giving rise to suppression or enhancement of the signal. As generally recognised, the predominant cause is the presence of undesired components that co-elute in the chromatographic separation and alter the ionisation process. The interfering species can be components of the sample, compounds released during the pre-treatment/extraction process or reagents added to the mobile phase to improve chromatographic resolution. The different mechanisms proposed in literature to explain the suppression or the enhancement of the signal both in electrospray and atmospheric pressure chemical ionisations are presented and the results observed in the different experimental conditions are compared and discussed. All data together lead to conclude that the chemical properties of the target analyte, the kind of matrix, the matrix to analyte concentration ratio, the extraction process, the chromatographic conditions as well as the kind of the mass spectrometry instrumentation and the ionisation conditions can play a role. Likely all these potential causes act in a synergic way and the final effect observed is hardly due to only one of them. Depending on an unpredictable combination of conditions, signal suppression or enhancement can be observed. The review discusses the matrix effects observed in HPLC–MS and HPLC–MS/MS analysis proposes hypotheses to explain the observed behaviours and proposes methods and strategies to overcome the matrix effects.     

A direct assessment of mycotoxin biomarkers in human urine samples by liquid chromatography tandem mass spectrometry

Detection of mycotoxin biomarkers in urine of humans and animals provides a direct approach for assessing exposure to these mycotoxins as opposed to the indirect approach of food analysis, which in most cases is affected by the heterogeneity of the toxin in the food samples. Seven (7) mycotoxins and their metabolites (total 18 analytes) were selected and an LC–MS/MS method for their determination in human urine was developed and validated. The method consisted of direct analysis of two mycotoxin conjugates, deoxynivalenol-glucuronide and zearalenone-glucuronide without beta glucuronidase digestion of the urine samples. Since high method sensitivity is of utmost importance in such study, critical factors which could improve the analyte recovery and method sensitivity were investigated by a D-optimal experimental design. Urine samples (10 mL) were first extracted with 15 mL ethyl acetate/formic acid (99/1, v/v) followed by SAX SPE clean-up of the acidified aqueous fraction. Both extracts were combined and analyzed using an LC–MS/MS system operated in the positive ionization mode. A total run time of 28 min was adopted with all the 18 analytes eluting within 15 min.     

超高效液相色谱-串联质谱法测定人尿液中全氟有机化合物

采用超高效液相色谱-串联质谱(UPLC-MS/MS)联用技术,建立了对人尿液中12种全氟有机化合物(PFCs)的分析方法。首先在尿液样品中加入相应的同位素内标,以2%(体积分数)甲酸甲醇溶液超声萃取、离心后,将提取液用弱阴离子交换固相萃取柱净化,采用UPLC-MS/MS测定,内标法定量。12种目标化合物在0.05～50 μg/L质量浓度范围内线性良好,相关系数(r)均大于0.992,检出限在0.44～3.47 ng/L之间。在20、100、500 ng/L添加水平下,平均回收率范围为80.3%～116.2%,相对标准偏差(n=6)在5.5%～13.8%之间。该方法灵敏度高、重现性好、回收率高、操作简单,适合人尿液中PFCs的测定。     

Determination of triforine using high-performance liquid chromatography with tandem mass spectrometry

A method for determination of triforine using high-performance liquid chromatography with electrospray tandem mass spectrometry was developed. A simple ethyl acetate extraction with solvent exchange into water/methanol was used for sample preparation. The method was validated at 0.01 and 0.05 mg kg(-1) levels in apple, tomato and tinned blackcurrants. Recoveries were in the range 56.6-99.8% and no matrix suppression or enhancement effects were observed.     

Direct determination of alkyl phosphates in human urine by liquid chromatography/electrospray tandem mass spectrometry

A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides) in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 microL of urine sample were directly injected into the LC/MS/MS system. The method was validated yielding calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of spiked samples at 10 and 100 microg/L obtaining recoveries between 78 and 119% with coefficients of variation below 12%. Limits of detection of 1 microg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 microg/L for dimethyldithiophosphate (DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in order to improve their identification in real-world samples. The applicability of the LC/MS/MS method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifos-specific metabolite (1,3,5-trichloro-2-pyridinol) was obtained.     

Study of the matrix effect on the determination of nonconjugated xenobiotics in human urine by high-performance liquid chromatography/tandem mass spectrometry

Factors affecting the sensitivity and selectivity of the determination of diuretics, anabolic steroids, central nervous system stimulants, and narcotics in the analysis of human urine extracts by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure electrospray ionization and recording of positive ions were investigated. Mass spectra were obtained for all of the test compounds; the characteristic ions, retention times, detection limits, degree of ionization suppression by the matrix, the extraction of the analytes from human biological fluids were determined for all analytes; the selectivity and specificity of determination were evaluated.     

Congener-specific analysis of hexabromocyclododecane by high-performance liquid chromatography/electrospray tandem mass spectrometry

A congener-specific method based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS) in the negative ion mode was developed for the analysis of hexabromocyclododecane (HBCDD). On a C(18) analytical column, with a methanol/water mobile phase, the alpha-isomer was completely resolved from the beta- and gamma-isomers while the beta- and gamma-isomers were sufficiently resolved at half their peak heights. The ES spray voltage strongly influenced the intensity of the ion signal. For MS, a source temperature of 500 degrees C and a collision energy of 50 eV were found to be optimum for the [M-H](-) to Br(-) transition. Run-to-run and day-to-day (n = 3) variability was minimal, with relative standard deviations of 2.6-4.1 and 2.4-4.4%, respectively. The limit of detection was 4-6 pg on-column. When applied to tissue samples from Lake Winnipeg fish both alpha- and gamma-isomers of HBCDD were found in low-ng/g (lipid corrected) concentrations.