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.     

Characterization of boldione and its metabolites in human urine by liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

Boldione (1,4-androstadiene-3,17-dione) is a direct precursor (prohormone) to the anabolic steroid boldenone (1,4-androstadiene-17beta-ol-3-one). It is advertised as a highly anabolic/androgenic compound promoting muscularity, enhancing strength and overall physical performance, and is available on the Internet and in health stores. This work was undertaken to determine and characterize boldione and its metabolites in human urine, using both liquid chromatography with electrospray ionization mass spectrometry and gas chromatography with mass spectrometry and derivatization. Boldione and its three metabolites were detected in dosed human urine after dosing a healthy volunteer with 100 mg boldione. The excretion studies showed that boldione and its metabolites were detectable in urine for 48 h after oral administration, with maximum excretion rates after 1.8 and 3.6 h (boldenone case). The amounts of boldione and boldenone excreted in urine from this 100 mg dose were 34.45 and 15.95 mg, respectively.     

Quantification of tamoxifen and metabolites and soy isoflavones in human plasma using liquid chromatography with electrospray ionization tandem mass spectrometry

Tamoxifen is an important estrogen receptor antagonist used successfully for the treatment and prevention of breast cancer. The use of complementary and alternative medicines is an increasingly popular means for patients to participate in their own health care, and soy products, which contain phytoestrogens, have been widely promoted for possible beneficial effects on menopausal symptoms. The possibility that soy isoflavones could reduce tamoxifen efficacy has been demonstrated in animal models of post-menopausal breast cancer, but the occurrence of such an effect in women has not been explored. This paper describes the development and validation of a sensitive method using solid-phase extraction and isotope dilution liquid chromatography/tandem mass spectrometry with multiple reaction monitoring for the concurrent analysis of the major soy isoflavones (genistein and daidzein), an important metabolite of daidzein (equol), tamoxifen, and its important metabolites (4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen or "endoxifen") in the serum of rats and women. The limits of quantification achieved are sufficient to determine accurately and precisely the concentrations of all of these analytes in women consuming soy foods and/or therapeutic doses of tamoxifen at levels consistent with modulation of estrogen receptor-mediated functions. These procedures enable future investigations of the possible impact of diet on the outcome of breast cancer therapy.     

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

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

Determination of synthetic hormones in animal urine by high-performance liquid chromatography/mass spectrometry

A method was developed for the extraction of stanozolol, taleranol, zeranol, hexestrol, dienestrol, ethynylestradiol, diethylstilbestrol, and trenbolone from animal urine. The analytes were extracted from the matrix by enzymatic hydrolysis, solid-phase extraction, and liquid-liquid extraction. Detection and quantitation were performed on a high-performance liquid chromatography system coupled to a triple quadrupole mass spectrometer. The identification criteria met the European Union regulations. Validation of this method established a decision limit between 0.2 and 0.9 microg/L and a detection capability between 0.3 and 1.0 microg/L, depending on the analyte.     

Determination of propamocarb in vegetables using polymer-based high-performance liquid chromatography coupled with electrospray mass spectrometry

A method for the determination of propamocarb in vegetables with liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) was developed. The performance of a polymer-based analytical LC column for the separation was investigated. Residues of propamocarb were extracted from the matrix with methanol. Subsequently, the extract was directly injected into the LC-MS system, without any additional concentration or cleanup procedures. Separation of propamocarb from the matrix components was achieved on a polymethacrylate-based analytical column. Propamocarb was concurrently detected with electrospray ionisation mass spectrometry in the selected ion monitoring mode and two-stage full scan MS application. Quantitation was done with matrix-matched calibration standards of propamocarb. Unambiguous confirmation was achieved by comparison of the full scan product ion mass spectrum of the chromatographic peak in the sample with the spectrum of a standard solution of propamocarb at the same retention time. The analytical performance of the method was validated for five relevant matrices, spiking propamocarb at fortification levels from 0.05 to 15.0 mg kg(-1). This covers the range of maximum residue limits in agricultural commodities, stated in the Dutch national legislation. The mean recovery of propamocarb was better than 90% with a precision of less than 10% in both scanning applications. As could be concluded from the calibration curve and matrix background levels, observed in blank control samples, the estimated limit of detection was 25 microg kg(-1) for the two-stage full scan MS application. The method has been applied in a survey of 285 samples of lettuce, radish, leek, and cabbage for the presence of residues of propamocarb. In 50% of the samples analysed, a residue of propamocarb was detected.     

Determination of abamectin in soil samples using high-performance liquid chromatography with tandem mass spectrometry

Abamectin, which is comprised of a mixture of avermectins B1a and B1b, is a natural pesticide used as an anti-parasitic agent in livestock, ornamental, and agricultural crops, which can potentially be transported to aquatic systems. These compounds are highly toxic to both aquatic vertebrates and invertebrates at low concentrations in water. This investigation developed high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) techniques to support automated extraction by an accelerated solvent extraction (ASE) system and chromatographic techniques to measure residues of avermectins in complex soil samples. HPLC along with atmospheric pressure chemical ionization (APCI) MS/MS was used for separation and determination of avermectin isomers in soil samples. Average method recovery for abamectin by UV was 91%, while detection by MS/MS resulted in a 68% recovery for abamectin. Individual method recoveries by MS/MS were 53.6% for avermectin B1a and 36.8% for avermectin B1b. The use of tandem technology eliminated matrix interferences and resulted in an approximately eight-fold increase in sensitivity.     

Determination of denaverine and its metabolites in urine samples by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed for the determination of the neurotropic-musculotropic spasmolytic agent denaverine and five of its metabolites in urine. In a first step beta-glucuronidase was used to cleave glucuronides in the human urine. After that samples containing denaverine and its phase I metabolites were extracted and cleaned up using an automated solid phase extraction method. An external calibration was used. The analytes were measured employing the multiple reaction-monitoring mode (MRM). The linear dynamic range for denaverine and its five metabolites determination was demonstrated from lower limit of quantification (8.0 ng/ml) to at least 500 ng/ml. The presented method is suitable for pharmacokinetic or toxicokinetic studies. With the help of reference substances some additional potential metabolites could be excluded in the urine samples. To look for additional unknown metabolites the LC-MS-MS system operated on one hand in the precursor ion mode using typical product ions of denaverine and of its metabolites and on the other hand in the product ion mode using postulated protonated molecules [M+H](+). With the help of the chromatographic behaviour and typical fragment ions of the unknown metabolites it was possible to elucidate their structures. Nine until now unknown metabolites were found in the urine samples. However, without reference substances a quantification of these analytes was not possible.     

Identification of metabolites of geniposide in rat urine using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Geniposide, an iridoid glycoside, is an important and characteristic compound in the fruits of Gardenia jasminoides Ellis, a commonly used medicinal herb in Chinese traditional and folk medicine for the treatment of inflammation and jaundice. However, few studies have been carried out on the metabolism of geniposide. In this study, we have established a rapid and sensitive method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC/ESI‐QTOF‐MS) for analysis of the metabolic profile of geniposide in rat urine after oral administration. A total of ten metabolites were detected and identified by comparing their fragmentation patterns with that of geniposide using Metabolynx? and MassFragment? software tools. The results revealed that the principal metabolism pathways of geniposide in rat occurred after deglycosylation of the irdoid glycoside take place and this is followed by glucuronidation and the pyran‐ring cleavages. The major metabolite, the glucuronic acid conjugate of genipin as observed in vivo, was further confirmed by the in vitro enzymatic study. The results of this work have demonstrated the feasibility of the UPLC/ESI‐QTOF‐MS approach for rapid and reliable characterization of metabolites from iridoid compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

Enhanced mass detection of oligonucleotides using reverse-phase high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry

A new method providing enhanced sensitivity for the analysis of oligonucleotides using an on-line coupled system of reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization ion-trap mass spectrometry (ESI-MS) has been developed. The presented method allows the use of the standard gradient elution of 0.1 M triethylammonium acetate (TEAA) buffer (adjusted to pH 7.0 with acetic acid) and acetonitrile that is typically used for the separation of oligonucleotides in RP-HPLC. An added feature of this method is the ability to combine and mix additional 0.1 M imidazole in acetonitrile after the separation column for improved ESI-MS performance. This is similar to the post-column reaction method in liquid chromatography (LC) and the liquid sheath flow method in LC/ESI-MS, both of which offer the advantage of not compromising the chromatographic separation conditions. The application of this new method is demonstrated to afford improved sensitivity for the analysis of oligonucleotides (20-50 mer) via on-line coupled HPLC/ESI-MS analysis and purification systems.     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).     

Quantification of cow milk adulteration in goat milk using high-performance liquid chromatography with electrospray ionization mass spectrometry

A method was developed for the quantification of cow milk adulteration in goat milk, based on solvent separation of whey proteins followed by high-performance liquid chromatography with electrospray ionization mass spectrometry (HPLC/ESI-MS). The presence of cow milk was determined using beta-lactoglobulin whey protein as the molecular marker. The adulterants were identified using both retention time and molecular mass derived from multiply charged molecular ions. Standard solutions containing cow and goat milk in different volume ratios were prepared and analyzed. Good linearity covering cow milk content from 5% and above was obtained. The proposed method identifies the adulterants using accurate molecular masses for protein identification and detects the addition of cow milk to goat milk at levels as low as 5%.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Analysis of metal complex azo dyes by high-performance liquid chromatography/electrospray ionization mass spectrometry and multistage mass spectrometry

Five metal complex azo compounds were analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). Mass spectra of all compounds yield intense peaks corresponding to [M - H](-) ions without any fragmentation, where M denotes the neutral compound with a proton as the counterion. Under collision induced dissociation (CID) conditions, structurally important fragment ions were studied using the ion trap analyzer with a multistage mass spectrometry (MS(n) facility. Synthesized compounds with (15)N atoms in the azo group facilitated the fragmentation pattern recognition. A reversed-phase high-performance liquid chromatography (HPLC) method using 5 mM ammonium acetate in 70% aqueous acetonitrile as mobile phase was developed making possible the separation of all complex compounds tested. The lower detection limits of the ESI-MS method are in the range 10-20 ng of each compound. The HPLC/ESI-MS method makes possible the monitoring of ligand exchange in aqueous solutions of metal complex azo dyes, and also investigation of the stabilities of the complexes in solution. Copyright 2000 John Wiley & Sons, Ltd.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.