Separation and identification of corticosterone metabolites by liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography coupled to atmospheric pressure ionization–electrospray ionization mass spectrometry (API–ESI–MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C₁₈ column (eluted with a methanol–water–acetic acid gradient) with identification using positive ion mode API–ESI–MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in avian intestines.     

Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography–mass spectrometry

A sensitive HPLC–MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C₁₈ cartridges followed by HPLC–MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.     

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC–NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile–methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration–time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.     

High-performance liquid chromatography–tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine

A sensitive, selective and accurate high-performance liquid chromatographic–tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C₁₈) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6–5 nmol/l for the studied compounds in plasma samples.     

Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography–electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5–177 ng/ml (r²≥0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3–13.7 to −4.8–3.0%. The inter-day precision and accuracy ranged from 5.5–14.8 to −6.7–3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Analysis of terfenadine in human plasma using microbore high-performance liquid chromatography–electrospray ionisation mass spectrometry

An assay based on combined microbore high-performance liquid chromatography–positive ion electrospray ionisation mass spectrometry with selected ion recording has been developed for the measurement of the antihistamine drug terfenadine in human plasma. A deuterated analogue of terfenadine was synthesised for use as an internal standard and extraction of terfenadine was carried out on C₁₈ solid phase extraction columns. The limit of detection of terfenadine in plasma is 0.1 ng/ml and the intra-assay coefficient of variation at 1 ng/ml is 10.1%. Plasma concentrations of terfenadine measured in six normal subjects following a 120 mg oral dose are reported.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

Quantitative determination of perifosine, a novel alkylphosphocholine anticancer agent, in human plasma by reversed-phase liquid chromatography–electrospray mass spectrometry

A sensitive and selective reversed-phase LC–ESI-MS method to quantitate perifosine in human plasma was developed and validated. Sample preparation utilized simple acetonitrile precipitation without an evaporation step. With a Develosil UG-30 column (10×4 mm I.D.), perifosine and the internal standard hexadecylphosphocholine were baseline separated at retention times of 2.2 and 1.1 min, respectively. The mobile phase consisted of eluent A, 95% 9 mM ammonium formate (pH 8) in acetonitrile–eluent B, 95% acetonitrile in 9 mM ammonium formate (pH 8) (A–B, 40:60, v/v), and the flow-rate was 0.5 ml/min. The detection utilized selected ion monitoring in the positive-mode at m/z 462.4 and 408.4 for the protonated molecular ions of perifosine and the internal standard, respectively. The lower limit of quantitation of perifosine was 4 ng/ml in human plasma, and good linearity was observed in the 4–2000 ng/ml range fitted by linear regression with 1/x weight. The total LC–MS run time was 5 min. The validated LC–MS assay was applied to measure perifosine plasma concentrations from patients enrolled on a phase I clinical trial for pharmacokinetic/pharmacodynamic analyses.     

High-performance liquid chromatography–electrospray ionization mass spectrometry method for the measurement of moclobemide and two metabolites in plasma

A rapid and sensitive liquid chromatography–electrospray ionisation mass spectrometry (HPLC–ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C₁₈ cartridges. A Nova-Pak phenyl column (Waters, 4 μm, 150×2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 μg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.     

Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography–electrospray mass spectrometry

Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC₅₀ values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.     

Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography–electrospray ionization mass spectrometry

A sensitive LC–MS quantitation method of cetrorelix, a novel gonadotropin releasing hormone (GnRH) antagonist, was developed. Plasma and urine samples to which brominated cetrorelix was added as an internal standard (I.S.) were purified by solid-phase extraction with C₈ cartridges. The chromatographic separation was achieved on a C₁₈ reversed-phase column using acetonitrile–water–trifluoroacetic acid (35:65:0.1, v/v/v) as mobile phase. The mass spectrometric analysis was performed by electrospray ionization mode with negative ion detection, and the adduct ions of cetrorelix and I.S. with trifluoroacetic acid were monitored in extremely high mass region of m/z 1543 and 1700, respectively. The lower limit of quantitation was 1.00 ng per 1 ml of plasma and 2.09 ng per 2 ml of urine, and the present method was applied to the analysis of pharmacokinetics of cetrorelix in human during phase 1 clinical trial.     

Liquid chromatography–electrospray tandem mass spectrometry of acidic monoamine metabolites

A new method based on liquid chromatography–tandem mass spectrometry has been developed for the determination of monoamine metabolites, i.e., homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in human urine. Analytes were separated on a C₁₆ amide (5 cm, 5 μm) column and ionized by negative ion electrospray. Operating in the selected-reaction monitoring mode, linearity was established over three-orders of magnitude and limits of detection were in the range 30–70 μg/l. Precision calculated as RSD was within 0.8–5.2% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of monoamine metabolites in 700 urine samples from occupationally (adults) and environmentally (both children and adults) exposed people living in areas with different soil contamination from lead. The urinary excretion of monoamine metabolites was significantly higher (P<0.001) in the subgroup of children living in polluted areas as compared to the control group (HVA, 6.03 vs. 4.57 mg/g creatinine; VMA, 5.33 vs. 4.37 mg/g creatinine; 5-HIAA 3.24 vs. 2.45 mg/g creatinine). In adults belonging to both groups of subjects occupationally and environmentally exposed, no differences were detected in the urinary concentration of monoamine metabolites. However, adults showed lower values of HVA (2.57 mg/g creatinine), VMA (2.17 mg/g creatinine) and 5-HIAA (2.09 mg/g creatinine) as compared to children groups.     

Analysis of corticosteroids in hair by liquid chromatography–electrospray ionization mass spectrometry

The present study describes a confirmatory method for the quantitative determination in hair of the most common corticosteroids illegaly used as doping agents by athletes. Corticosteroids are extracted from 50 mg of powdered hairs by methanolic extraction follows by a solid-phase extraction on C₁₈ cartridge. After extraction, the dried residue is reconstituted with 50 μl acetonitrile and injected in a liquid chromatograph. Liquid chromatography separation is performed on a reversed-phase C₁₈ column with a binary gradient of formiate buffer pH 3-acetonitrile as mobile phase. Detection is performed with an electrospray ionization mass spectrometer in negative ion and selected-ion monitoring mode. The limits of sensitivity achieved is 0.1 ng/mg in hair. Application to hair sample collected during an antidoping control and comparison to results obtain on urines, collected on the same athletes at the same time, shows the interest and the complementarity of both matrices. Hair analysis could allow the detection of corticosteroids on a large period preceding the control, and the detection of natural corticosteroids administered as pro-drug, like hydrocortisone acetate.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

Quantitative determination of dihydroetorphine in rat plasma and brain by liquid chromatography–tandem mass spectrometry

The extraordinarily strong analgesic dihydroetorphine (DHE) was registered as one of the most strictly controlled narcotic drugs by the United Nations in 1999. However, an effective detection method for DHE in biological samples has not yet been established. We developed a quantitative method for assay of DHE in rat plasma and brain by liquid chromatography–tandem mass spectrometry equipped with an ionspray interface. A 0.5-ml volume of plasma and brain homogenate spiked with buprenorphine (internal standard) was purified by the solid-phase extraction column Bond Elute Certify. DHE produced numerous weak fragment ions by collision induced dissociation. Therefore, collision energy was utilized to decompose the interferences, and the protonated molecular ion was used for both precursor and product ion monitoring. As a result of the method validation, the dynamic concentration range was determined as 0.05–10 ng/ml. DHE in these samples was stable for 2 months at −4°C and for 24 h at ambient temperatures. Using the present method, DHE was detected in rat plasma and brain tissue after intravenous injection (0.5 μg/kg).     

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE·2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5 μg/ml of BADGE, 12.5 to 62.5 μg/ml of first BADGE hydrolysis product (BADGE·H₂O), 25.0 to 100.0 μg/ml of second BADGE hydrolysis product (BADGE·2H₂O) and 6.25 to 50.0 μg/ml of BADGE·2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Rapid determination of gemifloxacin in human plasma by high-performance liquid chromatography–tandem mass spectrometry

A method was developed for the determination of gemifloxacin (I) in human plasma using high-performance liquid chromatography–tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with acetonitrile containing [¹³C²H₃] gemifloxacin (II) to act as an internal standard. The supernatant was injected onto a PLRP-S column without any further clean-up. The mass spectrometer was operated in positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring (MRM) mode. The assay requires 50 μl of plasma and is precise and accurate within the range 10–5000 ng/ml. The average within-run and between-run coefficients of variation were <11% at 10 ng/ml and greater concentrations. The average accuracy of validation standards was generally within ±7% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can safely be stored for at least 6 months at −20°C. The method proved very robust and was successfully applied to the analysis of clinical samples from patients dosed with gemifloxacin.