Liquid chromatographic-tandem mass spectrometric determination of selected sulphonamides in milk

Liquid chromatography–tandem mass spectrometry is used for the quantitative analysis of selected sulphonamides in milk. Ultrafiltration is the only sample pre-treatment technique which is required. Consequently, sample throughput is much higher than with conventional procedures, and analyte recoveries are high. As for quantification, both external standard and isotope dilution calibration yield satisfactory results. The method is fully validated for five sulphonamides with a maximum residue limit of 100 μg/kg, and which are included in the Dutch control programme on residues. Furthermore, results are presented on the applicability of the method to detect compounds at a much lower concentration level exemplified by a banned sulphonamide, dapsone, which has a provisional action limit of 5 μg/kg. The main conclusion is that the present, novel approach to the trace-level determination of veterinary drugs is simple and straightforward and has a wide-ranging application potential which is briefly exemplified by the analysis of selected benzimidazoles in milk by essentially the same procedure.     

Simple determination of huperzine A in human plasma by liquid chromatographic-tandem mass spectrometric method

Huperzine A is a potent, reversible acetylcholinesterase inhibitor. In the present work, a rapid and sensitive LC-MS-MS method for the determination of huperzine A in human plasma using codeine phosphate as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using ethyl acetate, chromatographed on a C(18) column (5 microm, 150 x 4.6 mm i.d.) with a mobile phase consisting of 1% formic acid-methanol (40:60, v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. Good linearity was achieved in the range 0.126 -25.2 ng/mL and the limit of detection in plasma was 0.064 ng/mL. The average recovery for huperzine A was 83.4% from plasma. The analytical sensitivity and accuracy of this assay is adequate for characterization of huperzine A in human plasma.     

Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the β-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.     

Thermal and mass spectrometric fragmentation of esters of deuterated long chain carboxylic acids

A method combining thermal fragmentation and mass spectrometry for the determination of the position of double bonds in an unsaturated ester is presented. The thermal fragmentation of methyl esters of deuterated long chain carboxylic acids yields a homologous series of olefins plus a homologous series of unsaturated esters. The positions of the deuterium atoms in the original ester are revealed by the deuterium content of its fragments as determined by mass spectrometry. Therefore, the positions of double bonds of a polyunsaturated acid can be determined by pyrolysis after saturation by deuterium. The structures of the unsaturated fragments are ascertained by mass spectrometric method, and the formation of the ion [M – 32] in the mass spectrometric fragmentation of unsaturated methyl esters is studied by means of deuterium labeling.     

Liquid chromatography fractionation with gas chromatography/mass spectrometry and preparative gas chromatography-nuclear magnetic resonance analysis of selected nonylphenol polyethoxylates

Commercial nonylphenol polyethoxylates, designated as NPnEOs, where n is the number of ethoxy groups, comprise a range of ethoxylate groups. According to the starting material nonylphenol, they may also be composed of a complex mix of isomeric nonyl substituents. In order to study more fully the heterogeneity arising from both the ethoxylate and nonyl groups, a mixture of NPnEOs is first fractionated by normal phase liquid chromatography (NPLC) into separate fractions comprising individual ethoxymers, n. Preparative collection of each early elution ethoxymer fraction allows further separation of different isomeric nonyl group components by using analytical gas chromatography/mass spectrometry (GC/MS). The nonyl isomers are not resolved in the NPLC method. The distribution of the isomeric nonyl side chain of different ethoxymers bears close resemblance with each other, and also with the original nonylphenol starting material, although separation efficiency of the nonyl isomers for each ethoxymer decreases with increasing ethoxymer number. Mass spectrometry of the separated isomers display close similarity for presumed equivalent isomers in each fraction, based on elution order of the nonyl isomers. This suggests that each corresponding peak has the same isomer structure. Mass spectra are interpreted based on branching within the nonyl side chain. Preparative GC coupled with MS and nuclear magnetic resonance spectroscopy elucidated the molecular structure of one of the resolved isomers as 4-(1,3-dimethyl-1-propyl-butyl)-phenol diethoxylate.     

Gas chromatographic and mass spectrometric determination of nitroaromatics in water

Several methods for the extraction of nitroaromatic compounds from water were compared. High recoveries were achieved with discontinuous or continuous extraction of water with dichloromethane and by adsorption on Amberlite XAD-2, -4 and -8 resins (1:1:1) and elution with dichloromethane. The recoveries obtained with solid-phase extraction using cyano-, phenyl- or octadecyl-bonded phases varied, depending on the compounds studied, and were often low. Nitroaromatic compounds were determined by gas chromatography using an electron-capture or a chemiluminescence detector (thermal energy analyser) and by mass spectrometry using electron impact and positive- and negative-ion chemical ionization.     

Determination of ranolazine in human plasma by liquid chromatographic-tandem mass spectrometric assay

A highly sensitive liquid chromatographic-tandem mass spectrometric method (LC-MS-MS) is developed to quantitate ranolazine in human plasma. The analyte and internal standard tramadol are extracted from plasma by liquid-liquid extraction using diethyl ether-dichloromethane (60:40 v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (60:40 v/v, pH 4.0) at a flow of 1.0 mL/min. Detection is carried out by multiple reaction monitoring on a QtrapTM LC-MS-MS system with an electrospray ionization interface. The assay is linear over the range 10-5000 ng/mL with a limit of quantitation of 10 ng/mL and a lower limit of detection (S/N > 3) of 1 ng/mL. Intra- and inter-day precision are < 3.1% and < 2.8%, respectively, and the accuracy is in the range 96.7-101.6%. The validated method is successfully used to analyze the drug in samples of human plasma for pharmacokinetic studies.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP(18) (4.6 x 50 mm, 3 microm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 --> m/z 158.2 for CLA and m/z 837.7 --> m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r(2)) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and -10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of bisoprolol in human plasma

An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned up by protein precipitation with acetonitrile, reconstituted in mobile phase and separated by reversed-phase high-performance liquid chromatography (HPLC) using methanol:10 mm ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on an LC-MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5-100 ng/mL with a limit of quantitation (LOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 and 9.95%, respectively, while the accuracy was in the range 89.4-113%. This method has been utilized in a pharmacokinetic study, where healthy volunteers were treated with an oral dose of 5 mg bisoprolol.     

Gas chromatographic-tandem mass spectrometric identification of phenolic compounds in Sicilian olive oils

Phenolic compounds in Sicilian olive oils were analyzed by GC-MS and GC-MS/MS after extraction with methanol:water 80:20 and derivatization with bis(trimethylsilyl)trifluoracetamide and trimethylchlorosilane (BSTFA:TMCS 99:1). Numerous compounds were detected and 23 were identified. Tyrosol, hydroxytyrosol and the decarbomethoxy ligstroside and oleuropein aglycons in the dialdehydic forms were the most abundant compounds. 4-(Acetoxyethyl)-1-hydroxybenzene, 3,4-dihydroxyphenylacetaldehyde, syringaldehyde and the cis form of ferulic acid were identified: these compounds were not found in olive oils before. The presence of the dialdehydic form of elenolic acid (without carbomethoxy group) linked to 3-methoxy-4-hydroxyphenylethanol was hypothesized. There were quantitative differences in oils from the varieties Nocellara del Belice, Santagatese and Cerasuola; these differences could depend on the olive varieties and ripeness.     

Synthesis and determination of dicarboxylic degradation products of nonylphenol polyethoxylates by gas chromatography-mass spectrometry

The synthesis and determination of persistent dicarboxylic metabolites of alkylphenol polyethoxylates (NPEOs), carboxyalkyl phenoxy ethoxy carboxylates (CAPECs), are investigated. The synthesized CAPECs have three and four carbon atoms and a carboxyl group in the alkyl chain side, and a carboxymethoxy acid group in its para-position (expressed as CA(3)P1EC and CA(4)P1EC in their abbreviation). The synthesis was successfully accomplished via a four-step reaction sequence that started from 4-fluoroanisole. After propylation by a propanol/acetyl chloride procedure, the derivatives of synthesized CA(3)P1EC and CA(4)P1EC were separated and identified by GC-MS with electron impact ionization (EI). The most abundant characteristic ions were produced by benzylic cleavages of carboxyalkyl chain to yield [M-87](+), corresponding to ions of m/z 235 for CA(3)P1EC and m/z 249 for CA(4)P1EC. Recoveries of synthesized CA(3)P1EC and CA(4)P1EC in various spiked water samples ranged from 82 to 92% with relative standard deviations (RSD) lesser than 7%. The limits of quantitation (LOQ) of CA(3)P1EC and CA(4)P1EC were estimated to be 0.005 and 0.01 microg/l in 100ml of water samples, respectively. The concentrations of CA(4)P1EC residues were detected in the aquatic environment ranging from n.d. to 3.24 microg/l. The results show that the synthesized CA(4)P1EC has been successfully applied to more accurately determine the concentrations of CA(4)P1EC residues in water samples.     

Liquid chromatographic tandem mass spectrometric determination of trandolapril in human plasma

A liquid chromatographic tandem mass spectrometric method for the determination of trandolapril in human plasma has been developed and fully validated. The article describes, in detail, the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using HLB Oasis solid-phase extraction cartridges. The chromatographic separation was performed on X-Terra C8 MS column (150 mm × 4.6 mm i.d., 5 μm particle size) using a mobile phase consisting of acetic acid 20 mM and triethylamine 4.3 mM/acetonitrile (40:60 (v/v)), pumped isocratically at 0.35 ml/min.

The analytes were detected using a micromass quattro micro triple quadrupole mass spectrometer with positive electrospray ionization in multiple reaction-monitoring (MRM) mode. Tandem mass spectrometric detection enabled the quantitation of trandolapril down to 2.0 ng/ml. Calibration graphs were linear (r better than 0.996, n = 9) in the concentration ranges 2.0–750 ng/ml and the intra- and inter-day R.S.D. values were less than 3.83 and 3.86% for trandolapril.     

Flophemesyl derivatives of alcohols,phenols, amines and carboxylic acids: Derivatives for mass spectrometric ion monitoring and structure determination

The flophemesyl (or pentafluorophenyldimethylsilyl) derivatives of representative alcohols, phenols, carboxylic acids and amines show rather simple spectra with a few abundant ions at high mass, characteristic of the parent compound and a few ions at low mass characteristic of the flophemesyl group. These derivatives are useful in the structure determination of simple compounds, and for the determination by single or multiple ion monitoring of known compounds at low levels.     

Simultaneous determination of degradation products of nonylphenol polyethoxylates and their halogenated derivatives by solid-phase extraction and gas chromatography-tandem mass spectrometry after trimethylsilylation

An efficient method for the simultaneous determination of the degradation products of nonylphenol polyethoxylates (NPnEOs, n = number of ethoxy units), i.e., nonylphenol (NP), NPnEOs (n = 1-3), nonylphenoxy carboxylic acids (NPnECs, n = 1-2, number of ethoxy units plus an acetate) and their halogenated derivatives (XNP, XNP1EO and XNP1EC; X = Br or Cl), in water samples were developed. After trimethylsilylation with N,O-bis(trimethysilyl)acetamide, all the analytes were determined by gas chromatography-tandem mass spectrometry (GC-MS-MS) with electron ionization (EI). The ion peaks of [M - 85]+ of the derivatives were selected as precursor ions and their product ions showing the highest intensities were used for the quantitative analysis. The instrumental detection limits were in the range from 2.1 to 11 pg. The recoveries of the analytes from the water samples were optimized by using solid-phase extraction (SPE). The deuterated reagents of octylphenol, octylphenol monoethoxylate and octylphenoxyacetic acid were used as the surrogates. The method detection limits (500 ml water sample) using C18 SPE were from 2.5 to 18 ng/l. The recoveries from spiked pure water and the environmental water samples were greater than 78%. The method was successfully applied to environmental samples. Remarkably, the concentrations of the halogenated compounds (CINP, CINP1EO and BrNP1EO) were detected at the hundreds of ng/l levels in the Neya river.     

Liquid chromatographic-tandem mass spectrometric determination of amprenavir (agenerase) in serum/plasma of human immunodeficiency virus type-1 infected patients receiving combination antiretroviral therapy

A selective assay method for quantitation of amprenavir (agenerase) in human immunodeficiency virus type-1 infected patient serum or plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS) is described. Amprenavir and an internal standard (reserpine) are extracted by liquid-liquid extraction and chromatographically separated by a reversed-phase C18-analytical column. The triple quadrupole LC-MS-MS system is operated in the positive-ion mode and multiple reaction monitoring is used for drug quantitation. The method has been validated over the range of 0.05-10.0 microg/ml. The RSDs for the intra-day and inter-day determinations ranged from 5.3 to 6.1% and from 4.7 to 6.2%, respectively. The average assay accuracy at two different concentrations ranged from 96.0 to 103.0% and the extraction recovery of amprenavir was 90.8%. The lower limit of quantitation was 0.05 microg/ml. Using a short microbore column, the analysis was completed in less than 5 min.     

Liquid chromatographic-tandem mass spectrometric assay for the simultaneous quantification of Camptosar and its metabolite SN-38 in mouse plasma and tissues

A simple, rapid and sensitive LC-MS/MS bioanalytical method has been developed to simultaneously quantify Camptosar (CPT-11) and its active metabolite, SN-38, in mouse plasma and tissues. A single step protein precipitation with acetonitrile in 96-well plates was used for sample preparation. Camptothecin (CPT) was used as the internal standard. Fast separation of SN-38, CPT-11 and CPT was carried out isocratically on a C18, 2 mm x 50 mm, 5 microm HPLC column with a mobile phase containing acetonitrile and 20 mM ammonium acetate (pH 3.5) and a 2.5 min chromatographic run time. The API 4000 MS/MS system was operated in positive ionization multiple reaction monitoring mode, and the transitions for SN-38, CPT-11 and CPT were 393.4 --> 349.3, 587.6 --> 167.2 and 349.3 --> 305.3, respectively. The SN-38 and CPT-11 concentrations in samples were calculated from a standard curve of peak area ratios of the analyte to that of the internal standard using a 1/chi2 weighted linear regression. The quantitation limit of 0.5 ng/mL was achieved by using a low sample volume (100 microL) of plasma or tissue homogenates. The assay was linear over the concentration range of 0.5-500 ng/mL with acceptable precision and accuracy. The method was used for the quantification of CPT-11 and SN-38 in plasma and tissues to support a preclinical pharmacokinetics and tissue distribution study of CPT-11 in mice.     

Sensitive liquid chromatographic-tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane-isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex Luna C18 (2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.     

Liquid chromatography-particle-beam mass spectrometric determination of β-carotene by negative-ion chemical ionization

Summary The first method for liquid chromatography-particle-beam mass spectrometry (LC-PBMS) of β-carotene is described. It uses a reversed-phase (RP) HPLC column and methanol-dichloromethane, 90:10, as mobile phase. For comparison purposes, UV-visible detection was performed at 436 nm to determine linearity, sensitivity and precision; the absolute limit of detection of the LC-UV method was estimated to be 0.2 ng. The possible modes of ionization of β-carotene were explored for PBMS detection; the best selectivity and sensitivity were obtained by use of electron-capture negative-ion chemical ionization (NCI). When operating in NCI mode with selected ion monitoring (SIM), the detection limit was 0.3 ng for β-carotene, comparable with that of the UV detector. The relative standard deviation (RSD) of the LC-PBMS system was 2.6 % at the 10 ng level (n=10). For the determination of β-carotene in real samples absolute calibration and calibration by the method of standard addition were used for both UV and PBMS detection.     

Mass spectrometric study of some 4-pyrimidine carboxylic acids

Mass spectrometry has been applied in order to study the main fragmentation routes of some 4-pyrimidene carboxylic acids. Differences in fragmentation were caused by the nature of the substituent in position 2 of the pyrimidine ring, while the methyl group in position 1, 3 or 6 did not influence the fragmentation route.     

Liquid chromatographic/mass spectrometric determination of desmedipham and phenmedipham and their metabolites in soil

A simple method is described for the simultaneous determination of residues of 2 carbamate herbicides (phenmedipham and desmedipham) and related metabolites (m-aminophenol, aniline, and m-toluidine) in soil. The analytes are extracted from spiked soils with methanol. The solvent/soil suspension is centrifuged, and the supernatant is directly injected, without any further cleanup, into a reversed-phase liquid chromatography/mass spectrometry apparatus equipped with a TurbolonSpray interface. The method was tested on 5 soils having different physicochemical properties. Recoveries from the soil types, spiked over the range of 50-200 ppb, were essentially quantitative for each analyte. The detection limits of the method are < or = 25 ng/g.