Measurement of 19-nortestosterone and its esters in equine plasma by high-performance liquid chromatography with tandem mass spectrometry

A high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS) method for the determination of 19-nortestosterone and its esters (cyclopentanepropionate, phenylpropionate, and decanoate) in equine plasma is achieved using an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. The two internal standards used were 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. The steroids studied were extracted from plasma samples with a mixture of diethyl ether/n-hexane (9:1, v/v). The quantification limits for 19-nortestosterone, 19-nortestosterone cyclopentanepropionate, 19-nortestosterone phenylpropionate, and 19-nortestosterone decanoate were 0.16, 5.0, 0.1, and 2.0 ng/mL, respectively, when 2 mL of plasma were used. The recoveries of most of the steroids were 71.6-101.0% except for the decanoate, which could be recovered to about 39.8%. The responses were linear, with correlation coefficients varying from 0.9897 to 0.9999 in the concentration range of 0.1 to 50.0 ng/mL for the steroids studied. When applied to equine (mare) plasma samples, the present method allowed detection of 19-nortestosterone up to 23 days after an intra-muscular injection of 400 mg as the decanoate.     

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.     

Acrylamide determination in atmospheric particulate matter by high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

A method has been developed for the determination of acrylamide at the picogram per cubic metre level in particulate-phase outdoor aerosol using high-performance liquid chromatography with triple quadrupole tandem mass spectrometric detection. Acrylamide was identified by positive ion electrospray mass spectrometry using m/z 72.00/54.90 as monitoring ion transition. The limit of detection, defined as three times the standard deviation of the procedural blanks, was 0.4?pg?m^?3 (173?pg absolute amount injected); the repeatability was 8% (evaluated as the relative standard deviation of five consecutive measurements on cleaned quartz fibre filters of acrylamide standard spikes) and the recovery was 52?±?4%. The accuracy of the method (evaluated as relative error) has been estimated to be ?2%. This methodology was used to determine acrylamide concentrations in particulate-phase outdoor aerosol in the Venice Lagoon with concentrations ranging between 0.4 and 12.9?pg?m^?3 with an average value of 3.1?pg?m^?3.     

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.     

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.     

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.     

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.     

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.     

Simultaneous determination of 20 beta-agonists in pig muscle and liver by high-performance liquid chromatography/tandem mass spectrometry

A method was developed to determine 20 illegal residual beta-agonists in pork tissues, including muscle and liver simultaneously. The samples were hydrolyzed by beta-glucuronidase, purified by PCX SPE cartridges, and detected by HPLC coupled with electrospray ionization MS/MS operating in the positive ion mode. Matrix-fortified calibration was performed to compensate for the matrix effect and loss in sample preparation. Decision limit ranged from 0.05 to 0.23 microg/kg in muscle and 0.05 to 0.57 microg/kg in liver. Decision capacity ranged from 0.11 to 0.4 microg/kg in muscle and 0.16 to 0.79 microg/kg in liver. In Food Analysis Performance Assessment Scheme proficiency test 0287, a pig liver test material containing 13 beta-agonists was analyzed using the method developed, and clenbuterol and ractopamine were confirmed as being present. Z-scores for clenbuterol and ractopamine were 0.2 and 0.6, respectively.     

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.     

A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma

Artemisinin is a widely used antimalarial drug. To evaluate the pharmacokinetics of artemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of artemisinin in rat plasma. For detection, a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization (ESI) TurboIonSpray inlet in the positive ion multiple reaction monitoring (MRM) mode was used to monitor precursor ([M+NH4]+) --> product ions of m/z 300.4 --> 209.4 for artemisinin and m/z 316.4 --> 163.4 for artemether, the internal standard (IS). The plasma samples were pretreated by a simple liquid-liquid extraction with ether. The standard curve was linear (r > 0.99) over the artemisinin concentration range of 1.0-200.0 ng/mL in plasma. The method had a lower limit of quantification of 1.0 ng/mL for artemisinin in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of artemisinin. The intra- and inter-day precisions were measured to be within +/-5.3% and accuracy between -2.6% and 1.2% for all quality control samples, lower limit of quantification and upper limit of quantification samples. The extraction recoveries of artemisinin and the IS were 95.4 +/- 4.5% and 92.8 +/- 3.9%, respectively. This present method was successfully applied to the characterization of the pharmacokinetic profile of artemisinin in rats after oral administration.     

Differentiation of common diastereoisomeric ursane-type triterpenoids by high-performance liquid chromatography/tandem mass spectrometry

A rapid and stable method consisting of high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for the identification and differentiation of common diastereoisomeric ursane-type triterpenoids at the C-3 position. Two characteristic fragment ions, [M-H-H(2) O-CO(2)](-) and [M-H-H(2)O-HCOOH](-) , exhibited significant stereochemical effects and were utilized to distinguish 3-OH epimers. Based on reference standards, the abundance of the fragment ion [M-H-H(2)O-HCOOH](-) in 3β-OH compounds in the MS(3) experiment was dramatically higher compared to [M-H-H(2) O-CO(2)](-); however, for 3α-OH compounds, the product ion [M-H-H(2) O-CO(2)](-) was noted to be higher than [M-H-H(2)O-HCOOH](-). Energy-resolved mass spectrometric experiments were carried out to support the differentiation of these diastereoisomeric triterpenoids at the C-3 position. Using this method, a total of nine ursane-type triterpenoids from a plant crude extract, including four pairs of epimers at the C-3 position, were identified and distinguished rapidly. Furthermore, offline Fourier transform ion cyclotron resonance tandem mass spectrometry was also performed to assign accurate elemental compositions.     

Quantitative determination of E5880 in rat plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A simple and sensitive method is described for the determination of E5880 in rat plasma. The method is based on high-performance liquid chromatography/electrospray ionization mass spectrometry, using deuterated E5880 as an internal standard. Selected reaction monitoring is employed for selectivity and sensitivity, this in turn enables quantification in a short period of time (within 7 minutes) over the extended range of 0.1-1000 ng/ml with acceptable precision and accuracy. The method demonstrated to be suitable for the quantitative analysis of E5880 in rat plasma. The pharmacokinetic profile of E5880 after a single intravenous administration of E5880 was elucidated.     

Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry

Mass spectrometry of ochratoxin A (OTA) and B (OTB) under electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was studied. ESI offers higher sensitivities and less fragmentation than APCI. A sensitive LC/MS/MS method for the determination of ochratoxin A (OTA) in human plasma samples was developed. The absolute minimum detection limit was around 10-20 pg per injection, corresponding to 0.5 ppb in an injection equivalent to 20-40microg of human plasma. Ochratoxin B (OTB) was used as an internal standard and its absence in real-life samples was carefully checked before samples were spiked with the internal standard. It was found that these two ochratoxins are susceptible to sodium adduct formation. Fragment ions from the [M + H](+) and [M + Na](+) ions of both OTA and OTB were monitored in the multiple reaction monitoring mode. Three quantitative approaches, standard addition method, internal standard method (using ochratoxin B as an internal standard) and external standard method, were compared in the analysis of human blood plasma. Results from the mass spectrometric method were comparable to those from a conventional LC/fluorescence method. The LC/MS/MS method was also applied to the analysis of contaminated coffee samples.     

Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid-liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-500 ng/mL for all analytes. The intra- and inter-day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio-samples including plasma, urine, bile and feces.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Investigations on the degradation of aspartame using high-performance liquid chromatography/tandem mass spectrometry

Aspartame is a widely used sweetener,the long-term safety of which has been controversial ever since it was accepted for human consumption.It is unstable and can produce some harmful degradation products under certain storage conditions.A high-performance liquid chromatography/tandem mass spectrometry method was developed for the simultaneous analysis of aspartame and its four degradation products,including aspartic acid,phenylalanine,aspartyl-phenylalanine and 5-benzyl-3,6-dioxo-2-piperazieacetic acid in water and in diet soft drinks.Aspartame and its four degradation products were quantified by a matrix matched external standard calibration curve with excellent correlation coefficients.The limits of detection were 0.16–5.8 mg/L,which exhibited higher sensitivity than common methods.This method was rapid,sensitive,specific and capable of eliminating matrix interferences.It was also applied to the study of the degradation of aspartame at various pH and temperatures.The results indicated that aspartame was partly degraded under strong acidic or basic conditions and the extent of degradation increased with increasing temperature.     

Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry

Determination of 16 priority pesticides and transformation products specified in the EU Baby Food Directive 2003/13/EC has been compared using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) coupled to tandem quadrupole mass spectrometry (MS/MS). Prior to analysis, co-extractives were removed from acetonitrile extracts using dispersive solid-phase extraction (SPE) with primary secondary amine (50 mg). Extracts spiked with pesticides at 1 microg kg(-1) yielded average recoveries in the range 85-119%, with relative standard deviations less than 17%. The HPLC-MS/MS and UPLC-MS/MS multi-residue methods developed are simple, rapid and suitable for the quantification and confirmation of the 16 priority pesticides in fruit-, potato- and cereal-based baby food at 1 microg kg(-1). The major advantages of UPLC, using 1.7 microm particles, over HPLC are the speed of analysis, the narrower peaks (giving increased signal-to-noise ratio) and improved confirmation for the targeted pesticides in the analyses of baby foods.     

High-throughput quantification for a drug mixture in rat plasma-a comparison of Ultra Performance liquid chromatography/tandem mass spectrometry with high-performance liquid chromatography/tandem mass spectrometry

A quantitative Ultra Performance liquid chromatography/tandem mass spectrometry (UPL/MS/MS) protocol was developed for a five-compound mixture in rat plasma. A similar high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) quantification protocol was developed for comparison purposes. Among the five test compounds, three preferred positive electrospray ionization (ESI) and two preferred negative ESI. As a result, both UPLC/MS/MS and HPLC/MS/MS analyses were performed by having the mass spectrometer collecting ESI multiple reaction monitoring (MRM) data in both positive and negative ion modes during a single injection. Peak widths for most standards were 4.8 s for the HPLC analysis and 2.4 s for the UPLC analysis. There were 17 to 20 data points obtained for each of the LC peaks. Compared with the HPLC/MS/MS method, the UPLC/MS/MS method offered 3-fold decrease in retention time, up to 10-fold increase in detected peak height, with 2-fold decrease in peak width. Limits of quantification (LOQs) for both HPLC and UPLC methods were evaluated. For UPLC/MS/MS analysis, a linear range up to four orders of magnitude was obtained with r2 values ranging from 0.991 to 0.998. The LOQs for the five analytes ranged from 0.08 to 9.85 ng/mL. Three levels of quality control (QC) samples were analyzed. For the UPLC/MS/MS protocol, the percent relative standard deviation (RSD%) for low QC (2 ng/mL) ranged from 3.42 to 8.67% (N = 18). The carryover of the UPLC/MS/MS protocol was negligible and the robustness of the UPLC/MS/MS system was evaluated with up to 963 QC injections.