Efficient method for the quantitation of urinary leukotriene E4: extraction using an Empore C18 disk cartridge

We describe here an efficient procedure for the precise quantitation of leukotriene E₄ (LTE₄) in a small volume of urine, which was achieved mainly by the use of an Empore extraction disk cartridge. After addition of [³H]LTE₄ to 2 ml of urine, an Empore C₁₈ cartridge was used for initial extraction of the urine, which resulted in the extraction of LTE₄ in a small volume of solvent. The eluate could then be injected onto a high-performance liquid chromatography column without further concentration. After separation by high-performance liquid chromatography, LTE₄ was extracted from the effluent using an Empore C₁₈ cartridge. The concentration of LTE₄ was subsequently quantified by enzyme immunoassay. LTE₄ can be recovered from urine with sufficient efficiency (69.9±4.7%, mean±S.D., n = 101). The coefficient of variation of the assay procedure was less than 10%. When urine was spiked with different amounts of LTE₄, the recovery of LTE₄ added to the urine specimen was less than 120%. The concentration of LTE₄ in urine from normal healthy subjects was 48.0±15.3 pg/mg creatinine (n = 15).     

Simultaneous LC-MS/MS Analysis of Glyphosate,Glufosinate, and Their Metabolic Products in Beer,Barley Tea,and Their Ingredients

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H₂O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Extraction and quantification of nicardipine in human plasma

A novel simple method of extraction, separation, identification and quantification of nicardipine in human plasma samples was completely studied. The human plasma samples were initially purified by solid-phase extraction (SPE) using a C₁₈ cartridge. The extracted samples were separated and nicardipine present in the samples was quantified by high-performance liquid chromatography (HPLC) on a reversed-phase C₁₈ column employing a mobile phase consisting of 60% (v/v) acetonitrile in 0.02 M NaH₂PO₄ with pH of 6.3 and a variable wavelength UV detector set at 254 nm. The recovery of nicardipine from plasma samples using selective SPE was 91±6.0% and had less interfering compounds in the HPLC analysis compared to the use of liquid–liquid (L/L) extraction. In the HPLC analysis, examining the effect of pH values of the mobile phase on the capacity factor (k′) of nicardipine revealed a method for selecting a critical k′ value of nicardipine to eliminate interfering peaks near the peak specific to the analyte. This method for quantification of nicardipine in human plasma samples was suitable for studying the pharmacokinetic profile of nicardipine administered as an intravenous bolus to cardiac surgical patients.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Accelerated solvent extraction and liquid chromatography–tandem mass spectrometry quantitation of corticosteroid residues in bovine liver

A new method for the rapid extraction and unequivocal confirmation of two highly potent fluorinated synthetic corticosteroids, dexamethasone and its β-epimer betamethasone, in bovine liver was developed. Flumethasone was used as internal standard. An extraction procedure using an accelerated solvent extraction system was employed for the isolation of the analytes in liver samples. The procedure was highly automated, including defatting and extraction steps, sequentially carried out under 1.0·10⁴ kPa in about 35 min. The extracts were then directly analysed by tandem mass spectrometry with on-line liquid chromatography. The analytes were ionised in a heated nebulizer interface operating in the negative ion mode where the molecular related ions [M-H-CH₂O]⁻ were generated for each analyte, at m/z 361 for betamethasone and dexamethasone and at m/z 379 for flumethasone. They served as precursor ions for collision-induced dissociation and three diagnostic product ions for the drugs were identified to carry out analyte confirmation by selected reaction monitoring. Assessment of recovery, specificity and precision for betamethasone, dexamethasone and flumethasone proved the method suitable for confirmatory purposes. The limit of quantification of betamethasone and dexamethasone in liver tissue was 1.0 μg/kg.     

Gas chromatographic–tandem mass spectrometric determination of anabolic steroids and their esters in hair: Application in doping control and meat quality control

We have developed a powerful and simple sensitive method for testing hair for anabolic steroids and their esters. A 100-mg amount of powdered hair was treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid-phase extraction on amino and silica cartridges. The residue was derivatized with N-methyl-N(trimethylsilyl)-trifluoracetamide (MSTFA) prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. The generally chosen parent ion was the molecular ion while two daughter ions were selected for each compound with collision energies ranging from −16 to −21 eV. Internal standards were nandrolone d₃ for non-esterified drugs and testosterone phenyl propionate for esters. The limits of detection calculated from an analysis of the blanks (n=30) were 0.08 pg/mg for nandrolone, 6.20 pg/mg for boldenone, 0.07 pg/mg for methyl testosterone, 0.15 pg/mg for ethinyl estradiol, 2.10 pg/mg for metandienone, 0.86 pg/mg for testosterone propionate, 0.95 pg/mg for testosterone cypionate, 1.90 pg/mg for nandrolone decanoate, 3.10 pg/mg for testosterone decanoate and 4.80 pg/mg for testosterone undecanoate. Application to doping control has been demonstrated. In a series of 18 sportsmen, two tested positive for anabolic steroids in hair whereas urinalysis was negative for both of them. The first positive case was nandrolone and the second case concerned the identification of testosterone undecanoate. Measured in 10 white males aged between 22 and 31 years, the testosterone concentration was in the range 1.7–9.2 pg/mg (mean=5.0 pg/mg). The method was also applied in meat quality control. Of the 187 analyses realized based upon hair and urine sampling in slaughter houses, 23 were positive for anabolic steroids in hair: one case for boldenone, one case for metandienone, two cases for testosterone propionate, three cases for nandrolone, five cases for testosterone decanoate and 11 cases for methyl testosterone. In the meantime, urinalysis was always negative for these drugs or their metabolites.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Estimation of immunoreactive testicular androgen metabolites in the urine of saddle-back tamarins

Hydrolysis, extraction, and radioimmunoassay techniques for the estimation of excreted testosterone metabolites in the urine of saddle-back tamarins have been validated and are described. The steroids measured with the testosterone antiserum used are mostly present as glucuronides and sulfates. Immunoreactivity in high-performance liquid chromatography (HPLC) fractions of urinary extracts and in a standard mixture of cortisol, testosterone, and dihydrotestosterone (DHT) were compared. The fractions with the same retention data as testosterone accounted for the major part of the immunoreactivity. Several other immunoreactive compounds of unknown identity were present in low concentrations. These results suggest that testosterone conjugates are the major steroid metabolites measured with this method in Saguinus fuscicollis. Urinary testosterone levels of castrated males were much lower than those of intact males. Testosterone treatment of castrated males resulted in a temporary superphysiological increase in the levels of urinary testosterone and in an individually variable increase in the levels of the minor immunoreactive compounds. These results suggest that estimation of testosterone metabolite levels in urine is a valid method for the assessment of testicular activity in Saguinus fuscicollis.     

High-performance liquid chromatographic assay of mepivacaine enantiomers in human plasma in the nanogram per milliliter range

A method enabling quantification of R-(−)- and S-(+)-mepivacaine in human plasma in the low nanogram per milliliter range is described. The procedure involves extraction from plasma with diethyl ether, centrifugation, back-extraction into an acidified aqueous solution, washing with a mixture of pentane and isoamylalcohol, alkalinisation, followed by extraction with a mixture of n-pentane and isoamylalcohol. After evaporation of the organic phase, the residue is redissolved in the mobile phase used for the HPLC analysis, which consists of a 6.8:93.2 (v/v) isopropanol-sodium hydrogenphosphate buffer solution with the pH adjusted to 6.8 using phosphoric acid. The HPLC method has been described previously. Separation of the enantiomers is achieved with an α₁-AGP column and the UV detection wavelength is 210 nm. The minimal detectable concentration is ca. 3 ng/ml and the lower limit of quantification is 5 ng/ml for each enantiomer. For both enantiomers r is >0.9995 over the plasma enantiomeric concentration range of 10.5–1054 ng/ml.     

Enantioseparation and determination of flumequine enantiomers in multiple food matrices with chiral liquid chromatography coupled with tandem mass spectrometry

The present work firstly described the enantioseparation and determination of flumequine enantiomers in milk, yogurt, chicken, beef, egg, and honey samples by chiral liquid chromatography‐tandem mass spectrometry. The enantioseparation was performed under reversed‐phase conditions on a Chiralpak IC column at 20°C. The effects of chiral stationary phase, mobile phase components, and column temperature on the separation of flumequine enantiomers have been studied in detail. Target compounds were extracted from six different matrices with individual extraction procedure followed by cleanup using Cleanert C18 solid phase extraction cartridge. Good linearity (R²>0.9913) was obtained over the concentration range of 0.125 to 12.5 ng g^‐1 for each enantiomer in matrix‐matched standard calibration curves. The limits of detection and limits of quantification of two flumequine enantiomers were 0.015‐0.024 and 0.045‐0.063 ng g^‐1, respectively. The average recoveries of the targeted compounds varied from 82.3 to 110.5%, with relative standard deviation less than 11.7%. The method was successfully applied to the determination of flumequine enantiomers in multiple food matrices, providing a reliable method for evaluating the potential risk in animal productions.     

Quantification of urine 17-ketosteroid sulfates and glucuronides by high-performance liquid chromatography–ion trap mass spectroscopy

The measurement of androgen steroids has been utilized as a clinical indicator of adrenal function, androgen abuse, and as a prediction of general health or biological aging. An improved high-performance liquid chromatography–ion trap mass spectroscopic method with sonic spray ionization (SSI) technology for the quantification of individual urinary 17-ketosteroid sulfates and glucuronides was developed and validated. Sample preparation was simplified using a C₁₈ cartridge followed by direct injection onto a reversed-phase HPLC column. Individual 17-ketosteroid from 63 urinary specimens collected in a 24-h period was measured. 17-Ketosteroid conjugates, total 17-KS-S and the ratio of total 17-KS-S to creatinine referred to herein as the Anabolic/Catabolic Index (ACI) showed statistically significant negative correlations with age.     

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid–liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R-didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated β-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.     

Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection

Clenbuterol, a beta-agonist, was determined in samples of beef liver and muscle. The method employed an acidic aqueous extraction followed by protein precipitation. The supernatant liquid was passed through a weak cation-exchange cartridge and then through a commercially available immunoaffinity cartridge. Clenbuterol was eluted from the immunoaffinity cartridge with 80% ethanol in water. The eluate was concentrated and analysed directly by reversed-phase liquid chromatography using gradient elution and UV detection at 245 nm. Detection limits were estimated to be 0.3 ng g⁻¹ clenbuterol. A single immunoaffinity cartridge was used for ten sample extracts with no significant loss in capacity. No organic solvents other than ethanol and methanol were employed in the procedure. Recoveries of clenbuterol from samples of beef liver and muscle spiked at 2 and 5 ng g⁻¹ carried through the entire procedure were 63±11% (range, 53–74%) compared to pure standards. Absolute recoveries of pure standards (30 ng clenbuterol) carried through the same analytical steps were 70±5% (n = 6), the losses being primarily due to the ion-exchange step.     

An enzyme-immunoassay of abscisic acid in potato (Solanum commersonii) cultured cells

Estimation of abscisic acid (ABA) content in potato (Solanum commersonii) suspension-cultured cells with an enzyme-immunoassay (EIA) was investigated. In crude extracts of potato cultured cells or even after simple clean-up using C₁₈ cartridge, EIA based on commercial monoclonal antibodies (Idetek, Inc) failed to detect any ABA content. An interference could be removed by partitioning against ethyl acetate after the C₁₈ cartridge so that the EIA yielded an estimate of ABA similar to that determined by high pressure liquid chromatography and gas chromatography-mass spectrometry analysis. These results demonstrate the presence of metabolites in potato cultured cell extract that prevent the binding of ABA to its binding site but not the binding of tracer.Abbreviations ABA abscisic acid - EIA enzyme-immunoassay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry     

Sample purification using a C18-bonded reversed-phase cartridge for the quantitative analysis of corticosteroids in adrenal cell cultures by high-performance liquid chromatography or gas chromatography—mass spectrometry

Quantitative extraction and subsequent purification of small biological samples often involve cumbersome procedures. We have devised a short and efficient method for the quantitative extraction of the corticosteroid and the 20α reduced steroid series from culture medium containing 20% sera in a single, pure fraction with separation from cholesterol. Passage through a C₁₈-bonded reversed-phase Sep-Pak^® cartridge of the acidified culture medium and subsequent extraction of the steroid fraction with methanol yields a single fraction containing all steroids in 90% recovery and reduced quantities of cholesterol down to 30%. The extract can then be used without further purification for quantitative analysis by high-performance liquid chromatography or derivatized and analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Determination of the residues of 50 anabolic hormones in muscle,milk and liver by very-high-pressure liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific method for determination of the residues of 50 anabolic hormones in muscle (pork, beef, shrimp), milk and pig liver was developed. Analytes were separated and acquired by liquid chromatography coupled with an electrospray ionization tandem mass spectrometer (LC–ESI–MS/MS). Target compounds were simultaneously extracted with methanol after enzyme hydrolysis, and purified using a graphitized carbon-black solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. Limits of quantification were 0.04–2.0 μg kg^?1; average recoveries were 76.9–121.3%; and the relative standard deviation was 2.4–21.2%. This method has been successfully applied in real samples.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: II. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction–tandem mass spectrometry (DI/GCE–MS–MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE–MS–MS analysis. Due to the novel use of an extremely short C₁₈ guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE–MS–MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC₅₀ value (0.20 μM) measured by the new method is in good agreement with the literature value (0.22 μM).     

Simultaneous determination of 18 tetrahydrocorticosteroid sulfates in human urine by liquid chromatography/electrospray ionization-tandem mass spectrometry

A liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of eighteen tetrahydrocorticosteroid sulfates in human urine has been developed. The analytes were 3- and 21-monosulfates and 3,21-disulfates of tetrahydrocortisol (THF), tetrahydrocortisone (THE), tetrahydro-11-deoxycortisol (THS), and their corresponding 5α-H stereoisomers. The mass spectrometric behavior of these sulfates in negative-ion ESI-MS/MS revealed the production of intense structure specific product ions within the same group of sulfates and permitted distinction between regioisomeric sulfates by collision-induced fragmentation with the MS/MS technique using a linear ion-trap instrument. For the quantitative analysis, selected reaction monitoring analysis in the negative-ion detection mode using triple-stage quadrupole mass spectrometer was performed by monitoring transitions from [M−H]⁻ to the most abundant product ion of each tetrahydrocorticosteroid sulfate. After addition of 3- and 21-monosulfates of [2,2,3β,4,4-d₅]-THF, -THE, and -THS as internal standards, urine sample was applied to a solid phase extraction using a lipophilic-weak anion exchange cartridge column, and then analyzed by LC/ESI-MS/MS. The method had satisfactory performance in terms of intra- and inter-assay precision (less than 9.7% and 9.6%, respectively), and accuracy (91.2–108.2%). The limit of quantification was lower than 2.5 ng/mL for all sulfates examined. We applied this method to determine the concentration of eighteen tetrahydrocorticosteroid sulfates in the urine of healthy subjects. Thus, we have developed a sensitive, precise and accurate assay for urinary tetrahydrocorticosteroid sulfates that should be useful for clinical and biological studies.     

The antibacterial effect of a polyhydroxylated fucophlorethol from the marine brown alga,Fucus vesiculosus

An antibacterial compound was isolated from the brown alga Fucus vesiculosus. Purification consisted of extraction of plant material with 0.05% trifluoroacetic acid, concentration on a C₁₈ cartridge, and reverse-phase high performance liquid chromatography on a C₁₈ semi-preparative column. The isolated compound exhibited antibacterial activity against both the Gram-positive and the Gram-negative bacteria tested. Killing studies conducted indicated that the activity was bactericidal. The compound showed no haemolytic effect against human red blood cells. Results obtained by electrospray ionization mass spectrometry indicated that the antibacterial activity was caused by a polyhydroxylated fucophlorethol.