Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Determination of methadone and its metabolites EDDP and EMDP in human hair by headspace solid-phase microextraction and gas chromatography–mass spectrometry

A simple method for analysis of methadone and its two main metabolites EDDP and EMDP in hair was developed using automatic headspace solid-phase microextraction (HS-SPME) at a multipurpose sampler and gas chromatography – mass spectrometry with electron impact ionization and selected ion monitoring (GC–MS-SIM). The washed hair pieces were digested in the closed headspace vial in 1 ml 1 M NaOH containing 0.5 g NaCl and each 10 ng of the internal standards D₉-methadone and D₃-EDDP at 110°C for 20 min. Then the HS-SPME was performed with a 65 μm polydimethylsiloxan/divinylbenzene fiber at the same temperature in the same vial for another 20 min followed by the desorption in the GC injection port. The calibration curves were linear between 0.1 and 3 ng/mg (methadone and EMDP) and 10 ng/mg (EDDP) respectively, at higher concentrations a negative deviation from linearity was found. The detection limits were 0.03 ng/mg (methadone) and 0.05 ng/mg (EDDP and EMDP), and the reproducibility was 9.2% for methadone and 11.2% for EDDP (n=12). The method was applied to hair samples of 26 drug fatalities. 19 cases were positive with 0.36–11.8 ng/mg methadone and 0.19 –10.8 ng/mg EDDP. EMDP was found only in two cases with 0.18 and 0.84 ng/mg. The methadone concentration range was in agreement with previous data, but the EDDP/methadone concentration ratios (0.19–0.67) were definitely higher than those determined by other methods.     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography–mass spectrometry

A method for the determination of clenbuterol (4-amino-3,5-dichloro-α[(tert.-butylamino)methyl]-benzyl alcohol hydrochloride) in hair of living cows has been developed. Hair samples were digested in an alkaline medium. The diphasic dialysis technique is a semi-permeable membrane technology developed for the direct extraction of relatively low-molecular-mass analytes such as clenbuterol. In this case, we used sodium citrate buffer to homogenize the digested hair, dichloromethane was used as the extraction solvent at 37°C, and stirring was applied at 150 rpm for 4 h. The analysis was carried out using gas chromatography–mass spectrometry. The calibration curve for clenbuterol in hair was linear in the range from 12.5 to 400 ng g⁻¹. The detection limit of clenbuterol was 5 ng g⁻¹ and the quantification limit was 12.5 ng g⁻¹, in hair. A good inter-day reproducibility was obtained (R.S.D.=7.08%). The repeatability and intra-day reproducibility (50 ng g⁻¹ of hair, n=10) show R.S.D.s of 7.1 and 9.5%, respectively.     

Detection of illegal clenbuterol use in calves using hair analysis: Application in meat quality control

This study describes a real-life situation involving nine calves, 106 days old, which received oral doses of clenbuterol administered through their milk. Powdered skim milk containing 6.7 mg of clenbuterol was given daily for fifteen days under supervision (i.e. 100 mg per calf for the whole study) to seven calves, and two calves did not receive the drug. Hair samples and urine were taken and subjected to analysis by gas chromatography–mass spectrometry. Hairs were pulverized in a ball mill and 100 mg were incubated in a mildly acidic medium. The sample clean-up procedure involved solid-phase extraction on C₁₈ cartridges. Metoprolol was used as the internal standard for quantitation, after formation of methylboronate derivatives. The calibration curve for clenbuterol in hair was linear in the range 20–5000 pg/mg. The limit of detection of clenbuterol was 16 pg/mg in hair and 0.14 ng/ml in urine. Hair testing was effective after 7–10 days of treatment, and concentrations were in the range of 20 to 4372 pg/mg. Urinalysis can detect clenbuterol for up to two weeks after discontinuation of the drug. Conveniently, this is around the time when the hair samples attain greatest sensitivity. Therefore, the combination of the two matrices appears to be the method of choice for testing for the illegal use of drugs in meat-producing animals.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

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.     

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.     

High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma

A rapid and sensitive high-performance liquid chromatographic method is described for the quantitative analysis of dipotassium clorazepate (CZP) and its major metabolite nordiazepam (ND) in fresh human and dog plasma. The method consists of two separate selective ND extractions from a plasma sample without and with conversion of all the CZP to ND. For quantitation, diazepam (DZP) is used as the internal standard. The chromatographic phase utilized in a reversed-phase Hibar® EC-RT analytical column prepacked with LiChrosolv RP-18 with a solvent system consisting of acetonitrile-0.05 M sodium acetate buffer, pH 5.0 (45:55). The UV absorbance is monitored at 225 nm using a variable-wave-length detector. The mean assay coefficient of variation over a concentration range of 20–400 ng per ml of plasma is less than 3% for the within-day precision. Recoveries of ND, DZP and CZP (as ND) are essentially quantitative at all levels investigated. The calibration curves of ND are rectilinear (r² = 0.99) from the lower limit of sensitivity (2 ng/ml) to at least 2000 ng/ml in plasma. Applicability of the method to CZP and ND disposition studies in the anaesthetized mongrel dog is illustrated. When the two separate selective nordiazepam extractions from plasma cannot be performed immediately after blood sampling, an extrapolation kinetic method is suggested for the estimation of CZP concentration. In all previous in vivo studies, CZP has been determined only with gas-liquid chromatographic methods.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

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.     

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.     

Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS

A method is described for the simultaneous identification and quantification of opiates, amphetamines, cocainics, diazepam and nordiazepam from one hair extract (typically 10-50mg hair). After decontamination by washing with shampoo, dichloromethane, isopropanol and acetone, drugs were extracted using 0.1M HCl followed by SPE clean-up using mixed-mode extraction cartridges. The SPE extracts were submitted to a two-step derivatisation using MBTFA and MSTFA+1% TCMS and analysis was performed by GC-MS using both SIM and scan modes. Four deuterated standards were used to monitor 14 compounds. The limit of quantification was the total drug detected from the sample. This was 5 ng for amphetamines and 10 ng for remaining drugs which is equivalent to 0.1 and 0.2 ng/mg from a 50mg sample. Standard curves for the range 5-400 ng total drug concentration for all drugs had regression coefficients greater than 0.98. An authentic hair sample was used to validate the method and gave R.S.D.s <25% for both inter and intra-day reproducibility. The results of the analysis of hair taken from four patients attending a drug treatment clinic and six hair samples including head hair, pubic hair, axial hair and beard taken at post-mortem are presented.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Liquid chromatographic–mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthiritis. A robust, highly reliable and reproducible liquid chromatographic–mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C₁₈ column and chromatographed with a mobile phase comprised of acetonitrile–1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r²>0.994) over the concentration range 50–1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58–4.0% relative standard deviation and the accuracy is 99.4–107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC–MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.     

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(−)-propranolol from 100-μl rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl- -leucine anhydride to form diastereomeric propranolol- -leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-μl samples of whole rat blood (12.5–750 ng/ml; r=0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(−)-propranolol. Terminal half-lives of R(+)- and S(−)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid—liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate—acetonitrile—methanol—nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C₁₈ reversed-phase column. The standard curves were linear in the range 50–5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2–14%. In the range 50–5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene

A coupled column liquid chromatographic (LC–LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 μm C₁₈ column (50×4.6 mm I.D.) as the first column (C-1) and a 5 μm C₁₈ semi-permeable surface (SPS) column (150×4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol–0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol–0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol–0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC–LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50–4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.