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.     

Quantification of ephedrines in urine by column-switching high-performance liquid chromatography

A method for the quantification of five congener ephedrines in urine samples without sample preparation was developed. The analytes were trapped on a C18 precolumn and separated on a C18 BDS analytical column. Baseline separation was achieved for all analytes. The method meets the requirements of the International Olympic Committee (IOC) medical commission regarding cut-off limits for positive doping cases with ephedrines.     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.     

Rapid method for the simultaneous measurement of nicotine and cotinine in urine and serum by gas chromatography–mass spectrometry

A simple, sensitive, and rapid gas chromatographic–mass spectrometric method is described for the simultaneous detection and quantitation of nicotine and its metabolite, cotinine, in urine and serum. The analytes and their respective deuterated internal standards were extracted by liquid–liquid extraction coupled to centrifugation and evaporation. The detection limit of the assay was 0.16 ng/ml for both nicotine and cotinine. The limit of quantitation for each analyte was 1.25 ng/ml.     

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.     

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography–tandem mass spectrometry

A procedure based on gas chromatography–tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d₅ as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography–tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d₅ derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.     

Monitoring of olanzapine in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of olanzapine with liquid chromatography atmospheric pressure chemical ionization (LC–APCI–MS, positive ions) is described. The drug and internal standard (ethyl derivative of olanzapine) were isolated from serum using a solid-phase extraction procedure (C₁₈ cartridges). The separation was performed on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (25:75). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection (SIM) was applied with the following ions: m/z 313 and 256 for olanzapine and m/z 327 and 270 for the internal standard for quantitation. The limit of quantitation was 1 μg/l, the absolute recovery was above 80% at concentration level of 10 to 100 μg/l. The method tested linear in the range from 1 to 1000 μg/l and was applied for therapeutic monitoring of olanzapine in the serum of patients receiving (Zyprexa™) and in one case of olanzapine overdose. Olanzapine in frozen serum samples and in frozen extracts was stable over at least four weeks. The examinations of urine extracts from patients receiving olanzapine revealed peaks of postulated metabolites (glucuronide and N-desmethylolanzapine).     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Determination of leukotriene E4 in human urine using liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed for the quantitation of urinary leukotriene E₄ (LTE₄). LTE₄ and its internal standard were extracted by solid-phase extraction and analysed using LC–MS–MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE₄ ranged from 97.0% to 108.0% with a mean and SD of 100.6±2.4%. We detected LTE₄ (63.1±18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE₄ in biological samples.     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum

A validated gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum is described. Quantitation of phenylalanine and tyrosine with a non-labelled non-endogenous internal standard, -2-chlorophenylalanine, compared favourably with isotope dilution mass spectrometric quantitation. The 95% reference ranges for phenylalanine, tyrosine and the phenylalanine–tyrosine molar ratio in neonate cord blood serum were determined by isotope dilution mass spectrometry and were found to be 77.1–144.7, 33.3–109.3 μmol/l and 1.1–3.0, respectively.     

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.     

Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.     

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.     

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).     

Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap

This report evaluates the use of a quadrupolar ion trap for quantitation in a bioanalytical laboratory. The evaluation was accomplished with the cross-validation of an LC–MS–MS quantitative method previously validated on a triple quadrupole mass spectrometer. The method was a multi-level determination of the anti-obesity drug, orlistat, in human plasma. The method has been refined previously on a triple quadrupole instrument to provide rapid sample throughput with robust reproducibility at sub-nanogram detection limits. Optimization of the method on the ion trap required improved chromatographic separation of orlistat from interfering plasma matrix components coextracted during the initial liquid–liquid extraction of plasma samples. The ion trap produces full-scan collision-induced dissociation mass spectra containing characteristic orlistat fragment ions that are useful for quantitation. Data collection on the ion trap required a precursor ion isolation width of 3.0 Da and optimal quantitative results were obtained when three fragment ions were monitored with a 1.8 Da window for each ion. Although a direct cross-validation between the ion trap and the tandem triple quadrupole mass spectrometer was not possible, quantitative results for orlistat comparable to those obtained from the triple quadrupole instrument were achieved by the ion trap with the modified method. The limit of quantitation for orlistat in plasma on the ion trap was 0.3 ng ml⁻¹ with a linear dynamic range of 0.3 to 10 ng ml⁻¹. Precision and accuracy varied from 4 to 15% over the quantitation range. The overall results provide an example of the utility of an ion trap in bioanalytical work.     

Determination of roxithromycin residues in the flounder muscle with electrospray liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of roxithromycin in the flounder muscle by LC–MS was developed and validated. A dichloromethane extract of the sample was separated on C₁₈ reversed-phase column with acetonitrile–50 mM ammonium acetate (80:20, v/v) as the mobile phase and analyzed by LC–MS via atmospheric pressure ionization/electrospray ionization interface. The limit of detection and limit of quantitation were 0.01 and 0.1 ng/g, respectively. Mean recoveries from spiked muscles were 81.1% (ranged from 71.0 to 90.3%) for roxithromycin. The method has been successfully applied to determine roxithromycin in the flounder muscle.     

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75–1400 ng/ml for PMZ and 2.5–1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within ±11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett–Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.