Analysis of selenium in bovine liver by gas chromatography with mass-selective, electron-capture and nitrogen-phosphorus detection

The concentration of selenium (Se) in liver was determined by gas chromatography (GC) with mass-selective (GC-MS), electron capture (GC-ECD) and nitrogen-phosphorus (GC-NPD) detection. Liver samples were digested in a mixture containing HNO₃ and Mg(NO₃). Se^VI was converted to Se^IV. Se^IV was derivatized with 4-nitrophenylenediamine and then extracted in toluene. A 1-μl volume of the toluene extract was analyzed by the GC-MS, GC-ECD or GC-NPD methods. The detection limits of the GC-ECD, the GC-NPD and the GC-MS methods were 25, 50 and 800 pg, respectively. The GC-NPD method was more selective for the derivatized Se than the GC-ECD method. The GC-MS method had the advantage of using the ⁷⁶Se isotope as the internal standard. Se concentrations in liver samples determined by the three methods were comparable.     

Wet-chemical postcolumn reaction and fluorescence detection analysis of the reference interval of endogenous serum vitamin K1(20)

The reference interval for serum vitamin K1(20) levels was assayed in healthy fasting adults by a method based on high-performance liquid chromatography (HPLC). The isolation procedure involves a solvent extraction of the plasma lipids followed by two chromatographic steps, consisting of a purification of the extract on a semipreparative adsorption column and a final quantitation on a reverse-phase column. Vitamin K1(20) and vitamin K1(25), the internal standard, are monitored by fluorescence detection after postcolumn reduction with a methanolic solution of tetramethylammonium octahydridotriborate. This reaction is performed in an open tubular reaction coil at elevated temperature. The median plasma concentration in 50 healthy fasting adults was 247 pg/ml. The levels showed a skewed distribution with a range of 62 to 980 pg/ml [log x +/- 2 SD (log x)]. The method is linear over the entire physiological range and has a within-run precision of 3.6% (n = 5, mean = 311 pg/ml). The minimum detectable amount in serum is 50 pg/ml. Other extraction procedures resulted in lower recoveries or in interferences in the final measurement. The vitamin K1(20) levels as reported by other research groups are also discussed.     

Sensitive high-performance liquid chromatographic method for profiling phytoestrogens using coulometric electrode array detection: application to plasma analysis.

An HPLC method for profiling 13 phytoestrogens and their metabolites using coulometric electrode array detection was developed. Sensitivity of the method was slightly less than that of our GC-MS method, but significantly higher compared to the HPLC methods using diode-array or UV detection. Detection limits varied from 3.4 (secoisolariciresinol) to 40.3 (genistin) pg on column. Signal linearities ranged from the detection limits to 61 ng on column. Resolution values for the peak pairs varied from 1.1 (O-desmethylangolensin-anhydrosecoisolariciresinol) to 16 (daidzin-genistin). Intra- and interassay retention time variations were negligible and detector response variation was eliminated by frequent calibration. Chromatographic method was applied to plasma analyses and 6 of the 13 compounds were detected. Method accuracy for those six analytes varied from 69% (enterodiol) to 118% (genistein). Intraassay precision CVs ranged from 1.5% (enterolactone, 12.4 nmol/liter) to 14% (genistein, 245 nmol/liter) and interassay precision CVs ranged from 9.9% (daidzein, 67.4 nmol/liter) to 44% (enterodiol, 1.20 nmol/liter).     

Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring

An efficient procedure is described for the simultaneous determination of 9 androgen glucuronides including androsterone, etiocholanolone, 11-ketoandrosterone, 11-ketoetiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, and dehydroepiandrosterone (DHEA) in 3-glucuronide form and dihydrotestosterone (DHT) and testosterone in 17-glucuronide form from urine specimens. The method involves solid-phase extraction of the urinary steroids using Serdolit PAD-1 resin, with subsequent conversion to methyl ester-trimethylsilyl (Me-TMS) ether derivatives for the direct analysis by gas chromatography-mass spectrometry (GC-MS) using high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. Upon split injection of Me-TMS steroids at 330 degrees C into the MXT-1 capillary column initially maintained at 300 degrees C then programmed to 322 degrees C at 2 degrees C/min, each androgen glucuronide was well separated in excellent peak shape. The characteristic ions at m/z 217 constituting the base peaks in the electron-impact (20 eV) mass spectra for most steroids permitted their sensitive detection by GC-MS with selected-ion monitoring (SIM), whereas base peak ion at m/z 271 was used for the SIM of dehydroepiandrosterone-3-glucuronide. The detection limits for SIM of most of the steroids were 15 pg except for the 3-glucuronides of 11-ketoandrosterone and 11-ketoetiocholanolone, which could be detected down to 20 pg. The SIM responses were linear with correlation coefficients varying from 0.981 to 0.993 in the concentration range of 20 to 3000 ng/ml for the androgens studied. When applied to urine samples, the present method allowed rapid screening for the 7 androgens in their glucuro-conjugated forms simultaneously with good overall precision and accuracy within the normal concentration ranges of 15.1 to 3124.6 ng/ml.     

The determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum by high-performance liquid chromatography-electrospray tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method for the determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum has been developed. ED-71 in human serum was extracted using two solid-phase extraction steps on Bond Elut C18 and NH2 cartridge. The separation of ED-71 and preED-71 isomer was attained by LC using 2 mmol/L ammonium acetate-methanol (15:85, v/v) as a mobile phase on a Symmetry C18 column (5 microm, 150 mm x 2.1mm i.d.). ESI-MS/MS analysis was operated using selected reaction monitoring (SRM) in positive ion mode. The method achieved a lower limit of quantitation of 25 pg/mL. The calibration curve (25-3200 pg/mL) gave acceptable linearity (r>0.9964). Intra-assay precision ranged from 2.3 to 9.7%. Inter-assay precision ranged from 1.0 to 3.4%. The accuracy was within 90.8-107.0%. This highly sensitive and reproducible method is able to determine only biologically active ED-71 by separating it from preED-71, which is considered to be applicable for the determination of serum samples from pharmacokinetic studies in human.     

Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry

19-Norandrosterone sulfate (19-NAS) is the sulfoconjugated form of 19-norandrosterone (19-NA), the major metabolite of the steroid nandrolone. A sensitive and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the direct measurement of 19-NAS in human urine samples. The method involved a quaternary amine SPE protocol and subsequently injection of the extract onto an analytical column (Uptisphere ODB, 150 mm x 3.0 mm, 5 microm) for chromatographic separation and mass spectrometry detection in negative electrospray ionisation mode. The sulfoconjugate of 19-NA was identified in urine by comparison of mass spectra and retention time with a reference substance. The limit of detection (LOD) and lowest limit of quantification (LLOQ) of 19-NAS were of 40 pg/mL and 200 pg/mL, respectively. For a nominal concentration of 2 ng/mL, recovery (94%), intra-day precision (2.7%), intra-assay precision (6.6%) and inter-assay precision (14.3%) were determined. Finally, this analytical method was applied for quantifying the concentration of 19-NAS in doping samples, using calibration curves (0.2-20 ng/mL) and the standard-addition method. The results show the feasibility of applying this LC-MS/MS assay as a complementary tool to detect misuse of nandrolone or nandrolone precursors.     

A radioimmunoassay of plasma unconjugated and conjugated estetrol.

A radioimmunoassay for the measurement of both unconjugated and conjugaged estetrol in plasma has been developed. The antiserum obtained after 6 months of immunization with 6-oxoestetrol-6-(O-carboxy-methyl)oxim-BSA was used at a final dilution of 1:90,000 and showed almost no cross reaction with other steroids except for estriol at 1.24%. Esterol-glucosiduronate was synthesized by incubating with adrenalectomized rat liver homogenate and uridine diphosphoglucuronic acid. Then, plasma estetrol-glucosiduronate was measured in the same manner for unconjugated estetrol after hydrolysis with beta-glucuronidase. Sephadex LH-20 column chromatography (7X110 mm, benzene:methanol, 85:15) was employed for accurate assessment. The sensitivity was 10 pg and the smallest amount measurable was 40 pg/sample. The method bland was consistently negligible. The intra and inter assay precision was 11.8% and 14.2% for unconjugated estetrol and that for estetrol-glucosiduronate was 13.5% and 17.1%.     

Simultaneous determination of global DNA methylation and hydroxymethylation levels by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.     

Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes

The F(2)-isoprostanes are products of free-radical-induced oxidation of arachidonic acid (AA) that are stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)). We describe a method for quantitation of several 15-series PGF isomers (15-PGFs) and AA by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Plasma samples were subjected to alkaline hydrolysis and acidified, and total (free + esterified) 15-PGFs and AA were extracted with organic solvents. The analytes were separated by gradient reverse-phase HPLC and detected by multiple reaction monitoring on a triple-quadrupole mass spectrometer, using deuterated internal standards for quantitation. The assay had a linear range of 1-40 pg of 8-iso-PGF(2alpha) on column and can quantify as little as 40 pg/mL (0.11 nM) in plasma. Outcomes significantly correlated (p < 0.0001) with data obtained by gas chromatography-mass spectrometry GC-MS or enzyme-linked immunosorbent assay. All plasma 15-PGF isomers increased over time with in vitro cigarette smoke exposure and correlated (p < 0.0001) with each other. The same strong inter-15-PGF correlations were observed in plasma from healthy young adult subjects. The coefficients of variation of HPLC-MS-MS measurements (24-32%) were smaller than those obtained by GC-MS (53%). Thus, HPLC-MS-MS potentially offers greater precision and allows quantitation of more compounds with simpler sample preparation than existing methods. Ours is the first validated quantitative assay using HPLC-tandem MS applied to plasma total 15-PGFs.     

Determination of propiomazine in rat plasma by direct injection on coupled liquid chromatography columns with electrochemical detection

This method describes the determination of propiomazine by direct injection of rat plasma into a chromatography system based on coupled reversed-phase columns. An extraction column, packed with porous silica particles with covalent-bound ₁-acid glycoprotein (AGP), was used to separate the plasma proteins from the analyte. After isolation the analyte was transferred to the analytical column for separation and detection. Propiomazine was detected by an electrochemical detector and the limit of quantification was 2.0 ng/ml (100 pg injected). The absolute recovery was 80.9±2.4% at 9.0 ng/ml level. The inter-day and intra-day precision was 10.9% (5.6 ng/ml) and 2.8% (9.0 ng/ml), respectively.     

A highly sensitive high-performance liquid chromatography method for the estimation of ascorbic and dehydroascorbic acid in tissues, biological fluids, and foods

A highly sensitive procedure for determining ascorbic acid (AA) and dehydroascorbic acid (DHAA) by high-performance liquid chromatography with electrochemical detection in biological fluids, tissues, and foods is described. AA is separated in a C18 reverse-phase column after extraction from the sample with metaphosphoric acid. An aliquot of 20 microliter of diluted extract is injected into the column for the estimation of AA. DHAA is indirectly estimated by converting it to AA after reduction with DL-homocysteine at pH 7.0-7.2 for 30 min at 25 degrees C. After dilution, a 20-microliter aliquot is injected into the column to obtain total vitamin C (AA + DHAA). The concentration of DHAA is calculated by subtraction. AA can be reproducibly quantified at concentrations as low as 50 pg/20 microliter of sample extract. The method described here used a specially designed mobile phase, gave greater stability and a noiseless baseline, and increased substantially the sensitivity and precision. The procedure is rapid, analysis being completed within 10 min after sample preparation, and has been successfully applied to biological fluids, tissues, and foods.     

Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20-150 microl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C(18) column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ (3)H]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1mg/kg intravenous bolus dose of METH to female Sprague-Dawley rats.     

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptaflourobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m×0.32 m I.D. and a 0.52 μm film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.     

Separation and confirmation of anabolic steroids with quadrupole ion trap tandem mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) is the method of choice for separation and detection of anabolic steroids in urine. Recently, there have been advances in the areas of gas chromatography columns, tandem mass spectrometry using ion traps, and large volume sample injection that have promise for lowering detection limits and extending the utility of GC-MS for steroid analysis. In this work, a Varian Saturn III GC-MS system has been used in its tandem mass spectrometry mode to detect low picogram levels of model steroids in standard solution and the urine matrix. Application of MS-MS-MS provided structurally informative spectra for 3′-hydroxystanozolol at concentrations of 1 ng/ml. In addition, four polysilphenylene-polydimethylsiloxane capillary columns were examined for background and selectivity. The columns had bleed several-fold lower than conventional polysiloxane columns. The columns also exhibited significant differences in selectivity for structurally similar steroids. Finally, a new temperature-programmed split-splitless injector was used to inject as much as 25 μl on column. The resulting limits of detection were 5 pg/ml for norandrosterone.     

Estimation of Enantiomeric Impurity in Piperidin‐3‐Amine by Chiral HPLC With Precolumn Derivatization

A simple, precise, accurate, robust chiral high‐performance liquid chromatographic (chiral HPLC) method was developed for estimation of (S)‐piperidin‐3‐amine (S‐isomer) in (R)‐piperidin‐3‐amine dihydrochloride (R‐AMP). As AMP is a high‐melting solid and nonchromophoric compound, development of a suitable chiral method is a challenging task. The proposed chiral HPLC‐UV method involves a precolumn derivatization technique with para toluene sulphonyl chloride (PTSC) in the presence of a base to introduce chromophore into analytes. It utilizes chiralpak AD‐H column with a simple mobile phase of 0.1% diethyl amine in ethanol with a 0.5 mL/min flow rate. Analytes were monitored by using a UV detector at 228 nm. The resolution between the two enantiomers was more than 4.0. The developed method was validated as per current International Conference on Harmonization (ICH) guidelines. Chirality 26:775–779, 2014. © 2014 Wiley Periodicals, Inc.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Rapid and sensitive LC-MS/MS method for determination of felbamate in mouse plasma and tissues and human plasma

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a second generation antiepileptic drug used to treat seizures refractory to other antiepileptic drugs. With approximately 3500 new patients exposed annually, several important pharmacologic interaction questions remain unanswered necessitating the need for rapid and accurate methods of felbamate analysis in biological matrices. To this end, a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the measurement of felbamate in mouse plasma and tissues and human plasma. Plasma (100 μL) and tissues homogenates (100 μL of 100 mg/mL) were spiked with internal standard (carisoprodol) prior to protein precipitation with acetonitrile. Samples were chromatographed on a XBridge Phenyl, 2.5 μm, 4.6 mm×50 mm column with quantitation by internal standard reference monitoring of the ion transitions m/z 239→117 for felbamate and m/z 261→176 for carisoprodol. Calibration curves were linear from 2.5 to 500 ng/mL in mouse or human plasma and 25-5000 pg/mg in tissue homogenates. Recoveries were greater than 97% for plasma and homogenates with accuracies >92% in any of the mouse matrices and >88% in human plasma. Comparable accuracies and precision were found with and without the use of the internal standard in preparation of the calibration curves and suggest that the internal standard may not be required.     

High-performance liquid chromatography determination of alpha1-acid glycoprotein in small volumes of plasma from neonates

In order to investigate how the alpha1-acid glycoprotein (AGP) concentrations of neonates change in response to surgical stress, a simple high-performance liquid chromatography (HPLC)-assay for the measurement of alpha1-acid glycoprotein levels was developed. A fraction containing alpha1-acid glycoprotein was isolated from the bulk of plasma protein by addition of 0.6M perchloric acid and was then analysed directly on a short PLRP-S 4000A reversed phase column column. The method was validated by analysis of pooled plasma from healthy adults both in comparison with a calibration curve and by standard additions. The procedure was able to isolate alpha1-acid glycoprotein rapidly (<30 min) and required only 50 microl of plasma. The mean extraction recovery was 79.1% (CV 6.4%). The within-run precision for the analysis of three replicates of quality control sample ranged from +/-1.2 to +/-3.8% and the between-run precision was +/-6.1%. The method was linear (r(2)=0.988) over a concentration range from 6 to 100.0 mg/100 ml. The AGP levels in neonatal samples ranged from 25 to 93 mg/100 ml.     

Identification of mycotoxins in keratomycosis-derived Fusarium isolates by gas chromatography—mass spectrometry

The production of mycotoxins from Fusarium species has been demonstrated in isolates cultured from patients suffering from keratomycosis. The method employed a combination of thin-layer chromatography directly performed on gel plugs taken from the growth medium, cartridge column chromatography, silylation and gas chromatography on a non-polar stationary phase capillary column linked to mass spectrometry. The sensitivities of detection obtained for a signal-to-noise ratio of 33:1, were 200 pg for single stage GC-MS and 20 pg using tandem GC-MS-MS. Two mycotoxins, diacetoxyscirpenol and T-2 toxin were identified in three cultures.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.