Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Inhibition of xanthine oxidase by phytic acid and its antioxidative action

We examined if phytic acid inhibits the enzymatic superoxide source xanthine oxidase (XO). Half inhibition of XO by phytic acid (IC50) was about 30 mM in the formation of uric acid from xanthine, but generation of the superoxide was greatly affected by phytic acid; the IC50 was about 6 mM, indicating that the superoxide generating domain of XO is more sensitive to phytic acid. The XO activity in intestinal homogenate was also inhibited by phytic acid. However, it was not observed with intestinal homogenate that superoxide generation was more sensitive to phytic acid compared with the formation of uric acid as observed with XO from butter milk. XO-induced superoxide-dependent lipid peroxidation was inhibited by phytic acid, but not by myo-inositol. Reduction of ADP-Fe3+ caused by XO was inhibited by superoxide dismutase, but not phytic acid. The results suggest that phytic acid interferes with the formation of ADP-iron-oxygen complexes that initiate lipid peroxidation. Both phytic acid and myo-inositol inhibited XO-induced superoxide-dependent DNA damage. Mannitol inhibited the DNA strand break. Myo-inositol may act as a hydroxyl radical scavenger. The antioxidative action of phytic acid may be due to not only inhibiting XO, but also preventing formation of ADP-iron-oxygen complexes.     

Analysis of tert.-butyldimethylsilyl [1-C]palmitic acid in stool samples by gas chromatography–mass spectrometry with electron impact ionisation: comparison with combustion isotope-ratio mass spectrometry

The use of ¹³C-labelled compounds to study lipid metabolism is increasing. Typically less than 40% of the orally administered label is recovered in breath CO₂. The remainder must be either absorbed and not oxidised or not absorbed and remain in the faeces. Two methods of determining how much tracer passes through the body, and is present in the stool, were compared. Compound specific analysis of tert.-butyldimethylsilyl [¹³C]hexadecanoic acid by gas chromatography–mass spectrometry (GC–MS) with electron impact ionisation was compared with bulk analysis of whole stool and lipid extract by continuous flow isotope ratio mass spectrometry (CF–IRMS) with a combustion interface. The mean difference between the IRMS and GC–MS methods was −0.02 mmol ¹³C d⁻¹ with a mean excretion of 14.2 mmol ¹³C d⁻¹. Combustion IRMS is both simpler and cheaper, when the objective is to determine how much administered dose appears in stool, and information about the form of the label is not required.     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

Determination of metabolites of pirimicarb in human urine by gas chromatography–mass spectrometry

The analytical method described permits the determination of 2-dimethylamino-5,6-dimethyl-4-hydroxypyrimidine (DDHP), 2-methylamino-5,6-dimethyl-4-hydroxypyrimidine (MDHP) and 2-amino-5,6-dimethyl-4-hydroxypyrimidine (ADHP) in human urine. These hydroxypyrimidines are metabolites of pirimicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yldimethylcarbamate) which is applied as insecticide. The analytes are extracted into a mixture of diethyl ether and acetonitrile. Pentafluorobenzyl bromide serves as derivatising reagent. The derivatives are analysed using capillary gas chromatography with mass selective detection. 2-Amino-4-hydroxy-6-methylpyrimidine and 4-hydroxy-6-trifluoromethylpyrimidine are used as internal standards. The detection limits are 0.5 μg/l (DDHP), 1 μg/l (MDHP) and 4 μg/l (ADHP), respectively. The method was used for analysing seven urine samples collected from workers who had applied pirimicarb. The three metabolites were found in every sample in concentrations up to 60 μg/l.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C₁₈-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Optimization and performance of a rapid gas chromatography–mass spectrometry analysis for methylmalonic acid determination in serum and plasma

We have developed a rapid and sensitive GC–MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 μmol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 μmol/l methylmalonic acid concentration, respectively. The assay are linear up to 9.0 μmol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Effect of phytic acid on postprandial serum uric acid level in healthy volunteers: a randomized,double-blind,crossover study

Abstract

Phytic acid, a constituent of various plants, has been related to health benefits. Phytic acid has been shown to inhibit purine nucleotide metabolism in vitro and suppress elevation of plasma uric acid levels after purine administration in animal models. This study investigated the effect of phytic acid on postprandial serum uric acid (SUA) in humans. This randomized, double-blind, crossover design study included 48 healthy subjects with normal fasting SUA. Subjects consumed a control drink and a phytic acid drink with purine-rich food, and serum and urine uric acid levels were measured for 360?min after purine loading. Phytic acid lowered the incremental area under the curve (0–360?min) and incremental maximum concentration of SUA after purine loading (p?<?0.05); tended to lower cumulative urinary uric acid excretion (0–360?min) after purine loading (p?<?0.10); and suppressed postprandial SUA in this clinical study. Altogether, our findings suggest that phytic acid may play a beneficial role in controlling postprandial SUA.     

Influence of inositolhexaphosphori acid (phytic acid) on the copper distribution in tissues and the excretion of copper in rats

The aim of the present study was to investigate the effect of the administration of phytic acid on copper (Cu) concentrations in several different rat tissues. The animals used were divided into three groups: Group A (received a diet supplemented with 2% phytic acid), group B (received a diet supplemented with 10% phytic acid) and group C (control). At the end of the experiment, the animals were sacrificed and the concentration of copper was determined in the different tissues.

Phytic acid significantly increased Cu concentration in the duodenum of the animals of both groups as well as in the lungs and blood of the animals of group A. The copper concentration was also increased in the uterus and bone of the animals of group B.

On the other hand, the stomach copper concentration of the animals of both groups, the heart and lung copper concentrations of the animals of group B as well as the jejunum, colon and hair copper concentrations of the animals of group A were significantly decreased. Copper excretion through feces was significantly decreased in the animals of both groups, while the excretion through urine was not significantly affected by the administration of phytic acid.

In conclusion, the administration of phytic acid can produce translocation and/ or elimination of copper in various tissues of rats.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Determination of cyclophosphamide in urine by gas chromatography—mass spectrometry

A sensitive gas chromatographic method for the determination of cyclophosphamide in urine is presented. After liquid—liquid extraction with diethyl ether and derivatization with trifluoroacetic anhydride, cyclophosphamide was identified and quantified with mass spectrometry. The method is suitable for the determination of cyclophosphamide at concentrations of more than 0.25 ng/ml, which enables the uptake of cyclophosphamide during occupational activities, such as the preparation and administration of antineoplastic agents in hospitals, to be measured. Simple preparation makes the method appropriate for routine analysis.     

Determination of perfluorodecalin and perfluoro-N-methylcyclohexylpiperidine in rat blood by gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry method (SIM mode) was developed for the determination of perfluorodecalin (cis and trans isomers, 50% each) (FDC), and perfluoromethylcyclohexylpiperidine (3 isomers) (FMCP) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Analysis was performed by electronic impact ionization. The ions m/z 293 and m/z 181 were selected to quantify FDC and FMCP due to their abundance and to their specificity, respectively. The ion m/z 295 was selected to monitor internal standard. Before extraction, blood samples were stored at −30°C for at least 24 h in order to break the emulsion. The sample preparation procedure involved sample clean-up by liquid–liquid extraction. The bis(F-butyl)ethene was used as the internal standard. For each perfluorochemical compound multiple peaks were observed. The observed retention times were 1.78 and 1.87 min for FDC, and 2.28, 2.34, 2.48 and 2.56 min for FMCP. For each compound, two calibration curves were used; assays showed good linearity in the range 0.0195–0.78 and 0.78–7.8 mg/ml for FDC, and 0.00975–0.39 and 0.39–3.9 mg/ml for FMCP. Recoveries were 90 and 82% for the two compounds, respectively with a coefficient of variation <8%. Precision ranged from 0.07 to 15.6%, and accuracy was between 89.5 and 111.4%. The limits of quantification were 13 and 9 μg/ml for FDC and FMCP, respectively. This method has been used to determine the pharmacokinetic profile of these two perfluorochemical compounds in blood following administration of 1.3 g of FDC and 0.65 g of FMCP per kg body weight, in emulsion form, in rat.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.